GOVPH

Claro N. Mingala

Sex: Male

Education:

Postdoctoral Fellowship, Veterinary Biomedical and Clinical Sciences, 2016

Senior Research Fellow, Department of Agriculture, 2011

Doctor of Philosophy in Infectious Diseases – Molecular Immunology and Microbiology, Hokkaido University, Sapporo, Japan, 2009

Master of Veterinary Studies (Preventive Veterinary Medicine), Central Luzon State University, 2002

Bachelor of Science in Animal Husbandry, Central Luzon State University, 1992

Field of Specialization

Veterinary Immunology, Epidemiology, Virology

Infectious Diseases

Molecular Biology and Diagnostics

Vaccine and Drug Development

Researches:

Article title: Prevalence and Characterization of Quinolone-Resistance Determinants in Escherichia coli Isolated from Food-Producing Animals and Animal-Derived Food in the Philippines

Authors: Lawrence Belotindos, Marvin Villanueva, Joel Miguel, Precious Bwalya, et al.

Publication title: Antibiotics 10(4): 413, 2021

Abstract:

Antimicrobial resistance to quinolones, which constitutes a threat to public health, has been increasing worldwide. In this study, we investigated the prevalence of quinolone-resistant determinants in Escherichia coli not susceptible to quinolones and isolated from food-producing animals and food derived from them, in the Philippines. A total of 791 E. coli strains were isolated in 56.4% of 601 beef, chicken, pork, egg, and milk samples, as well as environmental, cloacal, and rectal swab-collected samples from supermarkets, open markets, abattoirs, and poultry, swine, and buffalo farms. Using the disc diffusion method, it was determined that 78.6% and 55.4% of the isolates were resistant to at least one antimicrobial and multiple drugs, respectively. In 141 isolates not susceptible to quinolones, 115 (81.6%) harbored quinolone-resistant determinants and had mutations predominantly in the quinolone-resistance determining regions (QRDRs) of gyrA and parC. Plasmid-mediated, quinolone resistance (PMQR) and Qnr family (qnrA1, qnrB4, and qnrS1) genes were detected in all isolates. Forty-eight sequence types were identified in isolates harboring mutations in QRDR and/or PMQR genes by multilocus sequence typing analysis. Moreover, 26 isolates harboring mutations in QRDR and/or PMQR genes belonged mostly to phylogroup B1 and Enteroaggregative E. coli. In conclusion, a high prevalence of E. coli was found in food-producing animals and products derived from them, which could potentially spread high-risk clones harboring quinolone-resistance determinants.

Article title: Methicillin-resistant Staphylococcus aureus (MRSA) associated with mastitis among water buffaloes in the Philippines

Authors: Alona T. Badua, Sukolrat Boonyayatra, Nattakarn Awaiwanont, Paula Blanca V. Gaban, et al.

Publication title: Heliyon 6(12): e05663, December 2020

Abstract:

Methicillin-resistant Staphylococcus aureus (MRSA) from dairy animals could pose a public health concern in the population. The study was designed to determine the prevalence of S. aureus and MRSA associated with mastitis among water buffaloes in the central part of Luzon island, the Philippines, and to investigate its associated factors. Three hundred and eighty-four water buffaloes were examined for mastitis using California mastitis test (CMT). Composite milk samples (n = 93) were collected from buffaloes showing positive reaction with CMT. S. aureus was identified from milk samples using biochemical tests. Cefoxitin disk diffusion assay and PCR detecting mecA gene were performed to identify MRSA isolates. Disk diffusion assay was used to investigate the antimicrobial resistance against 9 antibiotics. The prevalence of S. aureus was 41.94% (39/93). MRSA isolates resistant to cefoxitin were at 25.81% (24/93) but only 37.5% (9/24) harbored the mecA gene. All 24 MRSA isolates were resistant to penicillin while the majority were susceptible to clindamycin, trimethoprim-sulfamethoxazole, gentamycin, tetracycline, rifampicin, ciprofloxacin and chloramphenicol with intermediate susceptibility to erythromycin. Furthermore, 37.5% of the isolates were found resistant to two or more antibiotics. Animal-level factor associated with MRSA infection was the history of mastitis (OR = 3.18, CI = 1.03-9.79, p = 0.040). Herd-level factors associated with the detection of MRSA in milk included herd size (OR = 4.24, CI = 1.05-17.07, p = 0.042) and the presence of other animals (OR = 0.15, CI = 0.04-0.58, p = 0.006). High prevalence of intramammary infection with S. aureus and MRSA in dairy buffaloes was observed in the region. This finding raises the concern of preventing zoonotic spread of MRSA.

Article title: Antibiotic resistance and genotyping of mecA-positive methicillin-resistant Staphylococcus aureus (MRSA) from milk and nasal carriage of dairy water buffaloes (Bubalus bubalis) in the Philippines

Authors: Alona T. Badua, Sukolrat Boonyayatra, Nattakarn Awaiwanont, Paula Blanca V. Gaban, et al.

Publication title: Journal of Advanced Veterinary and Animal Research, 7(3):397-406, June 2020

Abstract:

Objective: Mastitis is considered as an economically important disease of dairy buffaloes in Asia. This study examined the mastitis milk and nasal swab samples for the detection and genotyping of methicillin-resistant Staphylococcus aureus (MRSA) in water buffaloes.

Materials and methods: Staphylococcus aureus was identified based on biochemical tests and Polymerase Chain Reaction (PCR) detection of nuc gene, whereas MRSA on mecA gene. The disc diffusion test was used to determine the antibiotic resistance and staphylococcal cassette chromosome mec (SCCmec), spa, and multilocus sequence typing for the genotyping of isolates.

Results: Staphylococcus aureus was detected on 39/93 milk (41.94%) and 27/384 nasal swab (7.03%) samples. However, only nine isolates (23.08%) harbored the mecA gene from milk samples and three isolates (11.11%) from the nasal carriage. All MRSA isolates exhibited resistance to cefoxitin and penicillin, whereas 50% were found resistant to clindamycin. All these isolates were found susceptible to sulfa-trimethoprim and chloramphenicol, whereas the majority of the isolates were susceptible to gentamicin, ciprofloxacin, tetracycline, and rifampicin. The SCCmec types of the MRSA isolates were type IVc (50.00%), type II (8.33%), type I (8.33%), and non-typeable (33.33%). The spa types and sequence type (ST) identified were t019 (ST30), t701 (ST1649), t311 (ST5), t657 (ST1148), t015 (ST508), t1939 (ST12), t800 (ST9), t091 (ST2454), t138 (ST5991), and t1642 (ST5992).

Conclusion: Milk and nasal swab samples from dairy water buffaloes were found positive for MRSA. The MRSA isolates were still susceptible to most antibiotics tested. Moreover, the genotypes of some MRSA isolates were found similar to some human MRSA strains, suggesting a possible human to animal transmission.

Article title: Trypanosoma Evansi and Neospora Caninum among water buffaloes (Bubalus Bubalis) in the Philippines

Authors: Claro N. Mingala, Jaypee A. Abenoja, Christopher V. Rivera, Michelle M. Balbin, et al.

Publication title: Archives of Veterinary Science 25(1): 10-19, 2020

Abstract:

The  study  determined  the  positivity  rate  of  Trypanosoma  evansi and Neospora  caninum  antibodies  in  water  buffaloes  in  the  province  of  Nueva  Ecija, Philippines using Polymerase Chain Reaction (PCR) for  T.  evansi  and  competitive Enzyme-linked Immunosorbent Assay (cELISA) for N. caninum antibodies. A total of 100 whole blood and 100 serum samples were collected to test for T. evansi and N. caninum,  respectively.  Rotat  1.2  VSG  gene  was  target  using  PCR  for  T.  evansi detection. Neospora caninum antibody detection was done from the serum samples using cELISA test kit. Results revealed that the positivity rate of T. evansi in Nueva Ecija was 11% (11/100). The positive animals identified were from the municipalities of  Muñoz  (4/16;  25%),  Sta.  Rosa  (3/13;  23.08%)  and  Talugtug  (4/16;  25%).  The seropositive  rate  of  Nueva  Ecija  for  N.  caninum.  was  46%  (46/100),  seropositive animals  were  identified  in  Cabanatuan  City,  57.14%  (4/7);  Science  City  of  Muñoz, 43.14%  (22/51);  Sta.  Rosa, 40%  (4/10);  Sto.  Domingo,  50%  (6/12);  and  Talugtug 50% (10/20). The seropositivity rate of N. caninum and the presence of T. evansi in Nueva  Ecija  may  contribute  to the  cases of  abortions in  the province  and  further studies  should  be  employed  to  confirm  the  association  of  these  organisms  to abortion cases on water buffaloes.

Article title: Molecular characterization of MHC II DRB3 gene of swamp- and riverine-type water buffaloes

Authors: Noraine P. Medina, Arren Christian M. De Guia, Virginia M. Venturina, Claro N. Mingala

Publication title: Journal of Advanced Veterinary and Animal Research 6(3): 308-314, July 2019

Abstract:

Objective: Major histocompatibility complex (MHC) is a set of molecular proteins on the surface of antigen presenting cells encoded by a large gene family which are important parts of the immune system. This study was conducted to convey information on the genetic characteristics of the MHC II DRB3 gene in riverine and swamp buffaloes.

Materials and methods: Characterization of MHC II DRB3 gene was carried out using polymerase chain reaction (PCR)-based assay. Thirty-milliliter milk samples were collected from 10 swamp-type and 10 riverine-type buffaloes. RNA from milk samples were extracted using Trizol and then followed by reverse transcription-PCR (RT-PCR).

Results: The phylogenetic analysis with 1,000 bootstrap replications clearly showed complex parsimony in MHC II DRB3 gene between 10 riverine- and 10 swamp-type but also confirmed that the samples are similar to Bubalus bubalis. Aligned sequences of the 20 water buffaloes were compared with three other ruminants (Bos taurus, Ovis aries, and Capra hircus) and non-ruminant (Sus scrofa) that serve as an outgroup. MHC sequences from GenBank show that there was an average of 705 identical pairs, with 22 transitional pairs and 30 transversional pairs with a ratio of 0.7.

Conclusion: Based on the molecular data, the current study conforms to other works of literature that this gene is highly polymorphic which can be due to its function in the immune responsiveness and disease resistance. Further study on the immunological response of MHC II DRB3 to infection may elucidate its underlying function and role in the protection against specific disease of animals.

Article title: Screening of the acid meat condition in the rendement napole gene using polymerase chain reaction - restriction fragment length polymorphism 

Authors: Jessica G. Manalaysay, Nathaniel D. Antonio, Ralph Lorenz R. Apilado, Joseph F. Bambico, et al. 

Publication title: Indonesian Journal of Agricultural Sciences 20(1): 29-34, June 2019

Abstract:

A mutation in the rendement napole (RN) gene causes the acid meat condition which results to poor meat quality due to its reduced water holding capacity, low pH, pale color, reduced processing and cooking yield due to increased drip, and strong metallic taste. This study was conducted to detect the mutation in the RN gene in 535 commercial breeder pigs from the Philippines. Blood collection was done then subjected to DNA extraction and genotyping using polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) using the enzyme BsrBI, then validated by DNA sequencing. Results revealed that 97.01% of the breeder pigs did not have the mutation in their RN gene, while 2.69% had at least one copy of the defective allele in their gene. The acid meat condition has only been previously detected in the Hampshire breed whereas this study found the mutations predominantly in Pietrain and Landrace breed they were classified as normal (rn/rn), heterozygous mutants (RN/rn), and homozygous mutants (RN/RN) which allowed breeding systems to be developed ensuring that all offspring are free of the defect. This genetic screening will help in detecting the presence of the defect in a given swine population and reduce the unwanted effects on meat quality thus increasing its market value.

Article title: Phospholipase C zeta 1 mRNA as a marker of Oocyte-Activation and Fertilization Potential of Water Buffalo (Bubalus bubalis) Semen

Authors: E.P. Atabaya, Z.P. Fajardo, R.D.T adeo, E.C. Atabay, E.V. Venturina, et al.

Publication title: Livestock Science 225: 103-108, July 2019

Abstract:

The present study aimed to detect and quantify the expression of buffalo PLCZ1 mRNA (buPLCZ1 mRNA) in buffalo semen and to determine its oocyte-activation and fertilizing ability through IVF. Buffalo semen samples were collected and subjected to standard subjective physical and microscopic evaluation of semen characteristics. Small amount of fresh semen sample was taken for molecular experiment. The rest of the samples were processed into frozen semen. Both fresh and frozen semen were subjected to Reverse Transcriptase quantitative PCR (RT-qPCR) technique to determine and quantify buPLCZ1 mRNA expression. Subsequently, 40 bulls with known buPLCZ1 mRNA content were evaluated for its oocyte-activation activity through IVF using frozen semen. Correlation analysis was done to determine the relationship between the expression of buPLCZ1 mRNA and percentage of IVF, and other semen variables. PLCZ1 mRNA expression in buffalo semen was observed variable among donor bulls, but was not significantly different (P > 0.05) between fresh and frozen semen. The result suggests that the cryopreservation procedure does not affect the expression of buPLCZ1 mRNA in semen. Statistical analysis showed that the expression of buPLCZ1 mRNA in fresh semen was strongly correlated with that of frozen semen (r = 0.992; P < 0.001). Similarly, expression of buPLCZ1 mRNA in frozen semen was positively correlated with percentage of IVF (r = 0.776; P < 0.001), but not with Initial (r = −0718; P > 0.05) nor with Post-Thaw Motility (r = −0.0313; P > 0.05). A strong relationship between buPLCZ1 mRNA concentration and IVF rate indicates that buPLCZ1 mRNA concentration can be used to assess oocyte-activation and fertilizing potential of the buffalo semen. The present study essentially demonstrated buPLCZ1 mRNA as biological marker for male fertility and that molecular technique serves as an objective approach of semen quality evaluation to enhance bull selection for genetic improvement in water buffaloes.

Full text available upon request to the author

Article title: Genome-wide Analysis for Variants in Philippine Trypanosoma evansi Isolates with Varying Drug Resistance Profiles

Authors: Jose Enrico H. Lazaro, Neil Andrew D. Bascos, Francis A. Tablizo, Nancy S. Abes, et al.

Publication title: Philippine Journal of Science 148(S1): 219-233, March 2019

Abstract:

Surra, a parasitic disease transmitted by hematophagous flies and caused by Trypanosoma evansi, affects many domesticated animals – including water buffaloes, camels, horses, pigs, dogs, and other carnivores – throughout the world. When left untreated, this disease can cause anemia, significant loss of weight, abortion, and death in affected animals. Among Philippine isolates of T. evansi, variability has been reported in terms of virulence as well as response to drug treatment. In this study, trypanosoma-positive blood was obtained from 15 Philippine water buffalo samples from different sites in the country. The collected T. evansi strains were propagated in mice then subjected to in vivo virulence, in vitro drug sensitivity testing, and whole genome sequencing.  One strain (O14) was found to be highly virulent in vivo, and was found to be resistant to three commonly used drugs [i.e., isometamidium chloride (IC), diminazene diaceturate (DD), and melarsamine hydrochloride (CY for Cymelarsan®)] in vitro. This highly resistant sample was compared with two less-virulent strains using genome-wide analysis of single nucleotide polymorphisms (SNPs) and short insertions and deletions (indels) relative to the reference strain STIB 805. Variant analysis between O14 and the less virulent strains (M4 and C117) identified a number of distinctive SNPs, many of which corroborate previous data. Genes with relatively high copy numbers were observed in mutation hotspots. These included genes that code for variant surface glycoproteins (VSGs), expression site-associated genes (ESAGs), retrotransposon hot spot (RHS) proteins, and leucine rich repeat proteins. Notable mutations were also predicted from genes coding for membrane transporters and cysteine peptidases, as well as those involved in RNA degradation. The whole genome sequences acquired from the Philippine isolates (O14, M4, and C117) vary greatly from the reference strain (STIB 805). These WGS data serve as a good resource for the discovery of genetic and phenotypic features that may be translated to effective treatment strategies, relevant to the Philippine setting.

Article title: Anthelmintic effect of betel nut (Areca catechu) and neem (Azadirachta indica) extract against liver fluke (Fasciola spp.)

Authors: Elnalyn C. Yamson, Gabriel Alexis S. P. Tubalinal, Victoria V. Viloria, Claro N. Mingala

Publication title: Journal of Advanced Veterinary and Animal Research 6(1): 44-49, January 2019

Abstract:

Objective: This study aimed to measure the anthelmintic effects of betel nut (Areca catechu) and neem (Azadirachta indica) leaf extracts against Fasciola spp. in vitro in comparison with the commercial dewormer, Albendazole, and the negative control, nutrient broth. The study determined the extract concentration that produced the highest efficacy based on the average recorded mean motility time, gross, and microscopic changes of the flukes treated with different concentrations of plant extracts.

Material and methods: The study consisted of eight treatments. Every treatment consisted of 10%, 20%, and 40% concentrations of both betel nut extract (BNE) and neem leaf extracts, positive control treatment (Albendazole-treated) and negative control treatment (25 ml nutrient broth). The motility of the flukes on all treatments was based on the established motility criteria scoring. The flukes subjected to all treatments were processed for histopathological analysis.

Results: The result of the study revealed that after exposure of Fasciola spp. under 10%, 20%, and 40% extract concentrations, betel nut showed higher efficacy having the recorded mean motility time of 0.22, 0.07 min, and no movement upon contact, respectively, than Albendazole which produced mean motility time of 0.38 min. Nevertheless, the flukes treated with 10%, 20%, and 40% neem leaf extracts obtained the average mean motility time of 220, 151, and 98 min, respectively.

Conclusions: The results gathered showed that 40% BNE concentration showed the highest efficacy based on the recorded mean motility time. All treatments of betel nut extract evidently showed marked changes in the gross and microscopic morphology of the flukes. However, the neem extract was ineffective in all concentrations although changes were observed microscopically. Furthermore, the nutrient broth was proven to be effective as a culture medium since the flukes remained active until 8 h of exposure.

Article title: Comparative molecular characterization of Forkhead box protein 3 (FoxP3) gene of swamp-type (Bubalus carabanensis) and riverine-type (Bubalus bubalis) water buffaloes

Authors: Jonifel C. Gamboa, Ryan Bismark C. Padiernos, Mary Rose D, Uy, Elfren F. Celestino Jr, et al. 

Publication title: Comparative Immunology, Microbiology and Infectious Diseases 64: 1-6, June 2019

Abstract:

FoxP3 is a forkhead family member that plays an important role in the development and function of a type of CD4 + T cell called T regulatory cells. Molecular characterization of FoxP3 gene in swamp- and riverine-type water buffaloes was conducted to determine its homology and compare it to the FoxP3 gene of other animal species (cattle, goat, sheep, horse, pig, cat, and dog), determine its unique characteristics in water buffaloes, and provide a reference for future studies to analyze its immunological function. FoxP3 nucleotide sequence of swamp- and riverine-type water buffaloes was 99% identical, whereas its protein translation revealed 97% homology. FoxP3 of swamp- and riverine-type water buffaloes were compared to FoxP3 of other animal species and revealed a high degree of homology which suggests that they may have the same biological properties. This study is the first report that describes the genetic characteristic of FoxP3 gene in water buffalo.

Full text available upon request to the author

Article title: A luminescence-based assay for evaluating bactericidal antibody to Borrelia Burgdorferi in vaccinated horses’ serum

Authors: J. J. Lee, C. L. Hsieh, J. Widman, C. Mingala, et al

Publication title: Equine VeterinaryJournal 51(5): 669-673, September 2019

Abstract:

Background: Current serological tests cannot discriminate between bactericidal Borrelia burgdorferi antibodies from others that are merely a response to Borrelia antigenic stimulation.

Objective: To develop a sensitive and convenient luminescence-based serum bactericidal assay (L-SBA) to identify serum borreliacidal activity.

Study design: Prospective validation study and method comparison.

Methods: Serum samples were obtained either from archives of the Animal Health Diagnostic Center at Cornell University (N = 7) or from a vaccination trial (N = 238). Endogenous complement-inactivated serum sample was incubated with exogenic complement and B. burgdorferi ML23 pBBE22luc, which is able to process luciferin with luciferase and produce luminescence in viable Borrelia. After incubation, a light signal can be detected by using a luminometer to calculate the borreliacidal antibody titre.

Results: Components of the reaction mixture including spirochetes and complement from various sources and concentrations were tested to identify a reliable recipe for our complement-mediated L-SBA. We also applied this L-SBA on measuring bactericidal antibody activities and calculated the half inhibitory concentration (IC50 ) of serum samples from clinical collections. Furthermore, we analysed the L-SBA titres and anti-outer surface protein A (OspA) antibody levels from vaccinated horses using the multiplex assays and found that there is a relationship between results generated using these two different assays. The increases of L-SBA titres correlated with increases of anti-OspA antibody titre in sera (r = 0.423).

Main limitations: Immunoreactivity of commercial complement may differ from different batches. Clinical protection of borreliacidal antibody levels has not been determined.

Conclusions: The L-SBA provided a sensitive and easy-operating platform for the evaluation of bactericidal antibody to B. burgdorferi, and we anticipated L-SBA would function well as an evaluation tool of vaccine efficiency in the future.

Full text available upon request to the author

Article title: Screening of Pig ( Sus scrofa ) Bactericidal Permeability-Increasing Protein (BPI) Gene as Marker for Disease Resistance

Authors: Michelle A. Miguel and Claro N. Mingala 

Publication title: Animal Biotechnology 30(2): 146-150, April 2019

Abstract:

Salmonella infection can cause septicemia, acute or chronic enteritis and wasting in weaned pigs, but may occur in other age groups. The bactericidal/permeability-increasing protein (BPI) gene plays an important role in the natural defense of the host and is found to be associated with resistance/susceptibility to Salmonella infection and identified as a candidate gene for disease resistance breeding in pig. This study was conducted to screen the resistance and/or susceptibility of pigs to Salmonella infection, to determine the genotype and evaluate presence of resistant allele of the BPI gene in population of pigs, and to establish genetic data for pig breeders for the improvement of Philippine pig industry. In this study, 389 blood samples from different pig breeds were collected from pig breeder farms in the Philippines. Genomic DNA was extracted from these samples and genotyping was done by PCR-RFLP analysis using AvaII restriction enzyme. Out of 389 pigs, the genotypic frequency showed that 98.4, 1.3, and 0.3% pigs are resistant (GG), heterozygous type (AG), and susceptible (AA), respectively. The application of BPI gene as marker for disease resistance will provide information to the pig industry to implement strategies for the identification of Salmonella infection-resistant pigs.

Full text available upon request to the author

Article title: Characterization of drug resistance-associated TevAT1 gene of Trypanosoma evansi from Philippine water buffaloes (Bubalus bubalis)

Authors: Claro N. Mingala, Alma Corazon P. Pasag, Marvin Bryan S. Salinas, Michelle M. Balbin, et al.

Publication title: Annals of Parasitology 65(4): 381–386, 2019

Abstract:

This study detected and characterized the TevAT1 gene of Trypanosoma evansi isolates from Philippine water buffaloes (Bubalus bubalis). A total of 68 blood samples from Philippine water buffaloes were subjected to DNA extraction and PCR assay was performed using RoTat 1.2 gene to detect T. evansi. Those samples positive for T. evansi subsequently underwent another PCR assay to detect the presence of TevAT1 gene. Trypanosoma evansi was detected in 26.47% (18/68) blood samples in which distributed throughout the main islands of the country (4 from Luzon, 2 from Visayas and 12 from Mindanao). However, only 10 of these samples were positive for TevAT1 gene. Sequence alignment of the TevAT1 gene from local isolates showed no single nucleotide polymorphisms when compared to other strains in various countries. Those T. evansi without the gene of interest could be possibly resistant to some trypanocidal drugs but this needs to be further investigated in-vitro or in-vivo.

Article title: Molecular Detection of Tetracycline and Sulfonamide Resistance Genes in Respiratory and Gastrointestinal Bacterial Isolates of Ruminants

Authors: Gemerlyn G. Garcia, Allan Jeffrey E. Francia, Kevin B. Costales, Michelle M. Balbin, et al.

Publication title: International Journal of Veterinary Science 8(1): 1-9, 2019

Abstract:

The resistance of respiratory bacterial isolates (Acinetobater schindleri, Bacillus pumilus, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus sciuri and Staphylococcus sporosarcina) and gastrointestinal isolates (Arthrobacter sp., Bacillus megaterium, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis) from ruminants to tetracycline and sulfonamide was evaluated. Antibiotic sensitivity test in agar cup well method that applied different concentrations of tetracycline and sulfonamide demonstrated the resistance of the isolates to different concentrations of tetracycline and sulfonamide. Tetracycline and sulfonamide resistance in antibiotic sensitivity test was further validated by PCR amplification of genes that code for tetracycline and sulfonamide resistance. Methods involved the utilization of primers that recognize efflux pumps (tetB), ribosomal protective proteins (tetM) and enzyme inactivation (tetX) in genes that regulate tetracycline resistance in bacteria while testing for sulfonamide resistance involved the application of primers for dihydropteroate synthase (DHPS) genes (sul1 and sul2). DNA sequencing of amplified products revealed tetracycline resistance in one respiratory bacterial isolate (E. faecalis) out of the 6 isolates tested. The amplicon with the putative tetracycline resistance had a molecular weight of 171 bp and explains the involvement of ribosomal protective proteins encrypted in the tetM gene as a mediator of tetracycline resistance in E. faecalis. Sulfonamide resistance gene was exhibited by the GIT bacterial isolate P. aeruginosa. The DNA amplicon with the reputed sulfonamide resistance is linked with the sul2 genes which had a molecular weight of 721 bp. The detection of the sul2 genes explains the inhibition of DHPS to effect resistance of P. aeruginosa to sulfonamides.

Article title: Serological and molecular evaluation of Mycobacterium avium subspecies paratuberculosis (Johne’s disease) infecting riverine-type water buffaloes (Bubalus bubalis) in the Philippines

Authors: Mary Rose D. Uy, Jeffrey L. Cruz, Michelle A. Miguel, Marvin Bryan S. Salinas, et al.

Publication title: Comparative Immunology, Microbiology and Infectious Diseases 61: 24-29, December 2018

Abstract:

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease and a possible cause of Crohn's disease in humans. A total of 70 blood and fecal samples were collected from water buffaloes in selected municipalities of Nueva Ecija for ELISA and qPCR assay. Results revealed presence of antibodies of MAP in 3 serum samples for ELISA. The qPCR assay was carried out using standard curve method targeting the MAP specific insertion element IS900. Results revealed that 10 of the samples were positive for MAP DNA in qPCR. ELISA was able to detect antibodies for MAP showing 2.48% infection rate among the 70 buffaloes tested using blood serum samples. On the other hand, qPCR was able to detect MAP using IS900 showed 14.28% infection rate among buffaloes tested using fecal samples. Nucleotide sequence of isolated MAP showed high homology (99–100%) among the reported MAP isolates in the GenBank.

Full text available upon request to the author

Article title: Evaluation of LAMP for detection and/or screening of Leptospira spp. infection among domestic animals in the Philippines Anti-Mullerian hormone as a marker of embryo production in ruminants View project A New Loop-Mediated Isothermal Amplification Method for Rapid, Simple, and Sensitive Detection of Leptospira spp. in Urine View project Claro Mingala Evaluation of LAMP for detection and/or screening of Leptospira spp. infection among domestic animals in the Philippines

Authors: Gabriel Alexis SP. Tubalinal, Michelle M. Balbin, Marvin A. Villanueva, Clarissa Yvonne J. Domingo and Claro N. Mingala

Publication title: Journal of Advanced Veterinary and Animal Research 5(4): 459–465, December 2018

Abstract:

Objective:

This study assessed the applicability of loop-mediated isothermal amplification (LAMP) for the detection of leptospirosis among domesticated animals and sewage rats. Specifically, it evaluated the ability of LAMP to amplify Leptospira spp. targeting the 16s rRNA gene in boiled urine samples.

Materials and methods:

A total of 140 samples from different domestic animals were tested for the presence of the antigen. A nested-polymerase chain reaction (nPCR) protocol was used to compare and determine the sensitivity of LAMP in detecting Leptospira spp. The LAMP was also evaluated by comparing its amplification result using agarose gel electrophoresis and color change using dye.

Results:

Positivity rate of Leptospira spp. antigen was 29.0% (40/140) for LAMP and 9.3% (13/140) for nPCR. Also, LAMP results for gel electrophoresis and dye color change varied in some samples that may be due to the interpretation of the result in dye color change.

Conclusion:

Overall, LAMP is a rapid, sensitive, and cost-effective diagnostic method compared with nPCR. Also, LAMP has a potential application as pen-side screening, surveillance, and clinical diagnostic kits of infectious diseases without requiring advanced equipment and skilled personnel.

Full text available upon request to the author

Article title: Gene expression analysis of swine leukocyte antigen (SLA-1 and SLA-2) involved in porcine pre-weaning and post-weaning diarrhea in Nueva Ecija, Philippines

Authors: Mary Rose D. Uy, Gemerlyn G. Garcia, Jeffrey P. Aquino, Joan F. Sampang, et al. 

Publication title: Philippine Journal of Science 147 (3): 473-481, September 2018

Abstract:

The immune responses of two breeds of piglets to diarrhea at pre-weaning and post-weaning were evaluated in terms of the relative quantification of Major Histocompatibility Complex (MHC) glycoproteins represented by the swine leukocyte antigen (SLA) class I. The expression of SLA-1 and SLA-2 genes of diarrheic and non-diarrheic Native and Large White piglets were measured using real time polymerase chain reaction (qPCR). Blood samples from 20 Native and 20 Large White piglets were used in this study. It is comprised of 5 Native piglets with clinical signs of diarrhea and 5 Native piglets with no diarrhea at pre-weaning. Same number of piglets were used for Native piglets at post-weaning and Large White piglets at pre-and post-weaning periods. The cDNA samples were amplified using primers for SLA-1 and SLA-1 alleles having amplicon sizes of 217 bp and 126 bp, respectively. Factors that were considered in the study include breed and status of piglets. Relative quantification was done using comparative threshold cycle (CT) method. Significantly higher levels of SLA-1 were noted in diarrheic pigs compared to those of non-diarrheic piglets (P=0.040) of the Native and Large White breeds at pre-weaning period. This observation was not analogous with the non-significant differences in SLA-2 expression, deduced as SLA-linked immune responses of piglets from the Native and Large White breeds with and without diarrhea observed at pre-weaning and post-weaning stages. The upregulation of SLA-1 in piglets with diarrhea at pre-weaning in the two breeds of swine examined the potential role of SLA-1 in the host’s response to diarrhea. These data associate the significance of the SLA-1 gene as a marker for diarrhea in pre-weaning piglets.

Article title: Characterisation of porcine epidemic diarrhea virus isolates during the 2014–2015 outbreak in the Philippines

Authors: Gemerlyn G. Garcia, Mark Arman D. Aquino, Michelle M. Balbin, Lawrence P. Belotindos, et al.

Publication title: VirusDisease 29(3): 342–348, September 2018

Abstract:

The viral agent of the porcine epidemic diarrhea (PED) was investigated during the reported 2014–2015 outbreaks in commercial farms in Central Luzon, Philippines. The study covered detection of PED virus (PEDV) in fecal and intestinal samples through reverse transcription PCR and sequence analysis of the nucleocapsid (N) gene. Results showed that 10 out of 34 fecal and intestinal samples examined were positive for PEDV. The partial nucleotide sequence of the N gene of the field samples showed 98–99% homologous to PEDV sequences registered in the GenBank. It was also noted that N gene sequences between field samples were 98% homologous. Interestingly, the partial sequences of the N genes of the field samples were genetically similar to the PEDV isolates from USA, China, Mexico, Canada and Japan. The phylogenetic tree analysis revealed that the Philippine samples clustered in group 2–1 of the PEDV, wherein the isolates of this group were responsible for the outbreaks in Asia and the USA. Analysis of the partial nucleotide and amino acid sequences revealed polymorphisms, deletions and insertions in the N-gene of the PEDV. Amino acid sequence alignment also showed deletions and insertion in the PEDV detected in the Philippines.

Article title: Molecular detection of ephemeral fever virus among large ruminants in the Philippines

Authors: John Emmanuel E. Lapira, Michelle M. Balbin, Lawrence P. Belotindos, Victoria V. Viloria, et al.

Publication title: VirusDisease 29(3): 400-404, September 2018

Abstract:

In the Philippines, bovine ephemeral fever (BEF) is currently undetected and considered as an exotic disease of both cattle and water buffaloes. The Philippines until now has no official data regarding the occurrence of BEF. There were no existing control programs or vaccine used for the prevention of the disease. However, there are claims of BEF existence in different water buffalo and cattle farms based on the clinical signs but never confirmed using laboratory test yet. Detection of BEF virus in cattle and water buffalo blood samples was conducted using reverse-transcription PCR targeting the glycoprotein (G) gene, a conserved region in the BEF virus genome. The samples were collected from 22 cattle and 50 water buffaloes with clinical signs suggesting of BEF infection. All water buffalo blood samples were negative while four cattle blood samples turned positive for BEF virus. The G gene partial sequence analysis from two BEF virus positive samples showed close relationship to Australian isolates.

Article title: Outer membrane proteins: Its role in Brucella virulence and immunogenicity

Authors: Joey Marvin C. Carpio and Claro N. Mingala

Publication title: International Journal of Veterinary Science 7(1): 33-37, 2018

Abstract:

Members of the genus Brucella are facultative intracellular bacterial pathogens that have the ability to survive and multiply in the phagocytes and cause abortion in cattle and undulant fever in humans. Brucella spp. particularly Brucella melitensis, Brucella abortus, and Brucella suis represent a significant public health concern. The ability of Brucella to invade and replicate in host cells which is being linked to its outer membrane properties as well as to structures found within the cell envelope continue to be a major challenge with regards to treatment and control of the disease. The Brucella outer membrane proteins (OMPs) has been proposed to be involved in virulence (i.e., resistance to bactericidal cationic peptides and polycations), permeability to hydrophobic agents, resistance to divalent cation chelators, and poor activation of bactericidal mechanisms by LPS. Studies on a molecular level have now highlighted the mechanisms that are involved surrounding the pathogenesis of Brucella particularly involving OMPs.

Article title: Gold nanoparticle-based probes for the colorimetric detection of Mycobacterium avium subspecies paratuberculosis DNA

Authors: Thenor Aristotile Charles S. Ganareal, Michelle M. Balbin, Juvy J. Monserate, Joel R. Salazar, et al.

Publication title: Biochemical and Biophysical Research Communications 496(3): 988-997, February 2018

Abstract:

Gold nanoparticle (AuNP) is considered to be the most stable metal nanoparticle having the ability to be functionalized with biomolecules. Recently, AuNP-based DNA detection methods captured the interest of researchers worldwide. Paratuberculosis or Johne's disease, a chronic gastroenteritis in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), was found to have negative effect in the livestock industry. In this study, AuNP-based probes were evaluated for the specific and sensitive detection of MAP DNA. AuNP-based probe was produced by functionalization of AuNPs with thiol-modified oligonucleotide and was confirmed by Fourier-Transform Infrared (FTIR) spectroscopy. UV-Vis spectroscopy and Scanning Electron Microscopy (SEM) were used to characterize AuNPs. DNA detection was done by hybridization of 10 μL of DNA with 5 μL of probe at 63 °C for 10 min and addition of 3 μL salt solution. The method was specific to MAP with detection limit of 103 ng. UV-Vis and SEM showed dispersion and aggregation of the AuNPs for the positive and negative results, respectively, with no observed particle growth. This study therefore reports an AuNP-based probes which can be used for the specific and sensitive detection of MAP DNA.

Full text available upon request to the author

Article title: Molecular identification of Buxtonella sulcata from associated-diarrhea in water buffaloes (Bubalus bubalis) in the Philippines

Authors: Joseph A. Dianso, Gemerlyn G. Garcia, Lawrence P. Belotindos, Claro N. Mingala

Publication title: Annals of Parasitology 64(2): 93-100, 2018

Abstract:

Sixty suspected protozoan oocysts were demonstrated from 260 fecal samples collected from water buffaloes aged one month to seven years old with clinical signs of diarrhea in four provinces in the Philippines after conventional methods of isolation, sporulation, morphological characteristics and Kinyoun Acid Fast Staining techniques. The recovered protozoan oocysts were subjected to molecular analysis. Amplification of DNA extracted from recovered Eimeria oocysts using universal primers for the ITS-1 region of 18S rRNA revealed PCR products with 348 bp size demonstrated by samples collected from Benguet, La Union and Nueva Ecija provinces in the Philippines while DNA extracted from oocysts of suspected Cryptosporidium spp. samples that applied primers for the SSU of 18S rRNA registered PCR products but no genes were amplified from diarrheic water buffaloes from these provinces. Alignment of the DNA sequences of the suspected Eimeria and Cryptosporidium species revealed sequences for three isolates of Buxtonella sulcata with product lengths that varied from 235 to 252 bp. This is an initial observation on the involvement of B. sulcata in diarrhea condition of water buffaloes in the Philippines. Phylogenetic analysis of the three local isolates of B. sulcata revealed no similarity with other protozoan constructed according to Neighbor-Joining method.

Article title: Detection of quinolone resistance through amplification of the gyrA Gene of mycobacterium species from human and animal sources

Authors: Gemerlyn G. Garcia, Ralph Kevin M. Espinosa, Michelle A. Miguel, Mark Lester Bernardino, et al.

Publication title: International Journal of Veterinary Science 7(4): 190-194, 2018

Abstract:

Ten (10) DNA samples of Mycobacterium tuberculosis (Mtb) isolated from sputum of TB-positive humans, DNA samples from Mycobacterium species isolated from lymph nodes and fecal samples of avians and bubaline animals were analysed by PCR targeting primers for gyrase A (gyrA), quinolone resitance A(qnrA) and topoisomerase IV (parC) genes. Results demonstrated that quinolone resistance recognized by gyrA was seen in one out of 10 DNA samples from human Mtb isolates and that no qnrA and parC genes were detected. The gene for quinolone resistance detected by the primer gyrA had a molecular weight of 333 bp. Resistance to quinolone mediated by gyrA, qnrA and parC genes in avian (M. avium avium) and bubaline (M. avium paratuberculosis) isolates of mycobacteria were not detected after PCR. The non-amplification of genes observed in this study explains the non-existence of quinolone resistance arbitrated by gyrA, qnrA and parC genes in the specified avian and bubaline mycobacterial isolates.

Article title: Detection of Cryptosporidium parvum DNA in fecal samples of infected cattle (Bos indicus) and water buffaloes (Bubalus bubalis) in the Philippines using loop mediated isothermal amplification method

Authors: Clarissa Yvonne J. Domingo, Hamelyn G. Pascual, Claro N. Mingala

Publication title: Annals of Parasitology 64(4): 331-338, 2018

Abstract:

Fecal DNA samples from 17 cattle and 38 water buffaloes found to be infected with Cryptosporidium oocysts using Kinyoun acid fast stain from a previous study, were subjected to loop-mediated isothermal amplification (LAMP) assay using specific primers for Cryptosporidium parvum (C. parvum) from three municipalities, Maria, Baler and San Luis of the province of Aurora in the Philippines. Results of the fecalysis using Kinyoun acid fast stain and LAMP assay were compared with the PCR results of the examined farmer/owner who raised these animals to determine the possible zoonoses of C. parvum between the farmers and their animals. Using LAMP assay, only 41% (7/17) were positive in cattle and 76% (29/38) in water buffaloes. Out of the seven LAMP positive cases in cattle, 86% (6/7) came from Maria and 14% (1/7) from Baler. Out of 29 LAMP positive cases in water buffaloes, 62% (18/29) came from Maria, 24% (7/29) from Baler and 14% (4/29) from San Luis. Comparing with the earlier results for probable zoonoses of C. parvum between the farmers and their animal was determined. Eight farmers that were positive in PCR and with their water buffaloes, positive in LAMP assay were detected to have C. parvum. Only one farmer with his cattle was detected positive of Cryptosporidium spp. in PCR, however, it was negative in LAMP assay hence, a non-parvum species might infected the farmer and the animal.

Article title: Anti-Müllerian hormone as a marker of embryo production in ruminants

Authors: Salvador S. Soquilla and Claro N. Mingala

Publication title: Scientific Annals of Polish Society of Animal Production 13(4): 9-16, 2017

Abstract:

This review describes the role of anti-Müllerian hormone (AMH) in embryo production for assisted reproductive technologies in ruminants. AMH is a marker of healthy follicles and oocytes, a reliable marker of gonadotropin-responsive follicles, and an indicator of longevity and productivity in dairy animals. The best times to measure AMH levels in order to select cows for embryo production is during oestrus and the period after the 12th day of the oestro-us cycle. This allows animals with AMH concentrations below 87 pg/mL at oestrus or less than 74 pg/mL for multiple ovulation embryo transfer to be eliminated. Good oocyte donors, which have higher antral follicle counts, can be identified based on their higher AMH levels. In sheep and goats, the blood AMH level can serve as a marker of the animal's potential to produce high or low numbers of high-quality embryos. A plasma AMH level of 97 pg/mL in sheep has been shown to be the optimum cutoff point to predict fertility and can be useful in selecting replacement ewes.

Article title: Development and validation of a loop-mediated isothermal amplification assay for the detection of Mycoplasma bovis in mastitic milk

Authors: Aqeela Ashraf Muhammad Imran, Tahir Yaqub, Muhammad Tayyab, et al.

Publication title: Folia Microbiologica 63(3): 373-380, May 2018

Abstract:

Mycoplasma mastitis is often difficult to control due to a lack of rapid and accurate diagnostic tools. The aim of the current study was to develop a loop-mediated isothermal amplification (LAMP) assay for the detection of Mycoplasma bovis (M. bovis) in mastitic milk. The assay was developed using primers designed for three different target genes: uvrC, 16S rRNA, and gyrB, and validated using mastitic milk samples previously found positive for the target pathogen. Specificity of the developed assay was determined by testing cross-reactivity of LAMP primers against closely related bovine mastitis bacterial pathogens. The sensitivity was found to be higher compared to conventional polymerase chain reaction (PCR). The LAMP assay was also capable of detecting M. bovis in PCR-negative milk samples of cows with clinical mastitis. The uvrC primers were found to be more sensitive, while gyrB primers were more specific; however, 16S rRNA primers were less specific and sensitive compared to either uvrC or gyrB primers. Cohen's kappa values for uvrC, gyrB, and 16S rRNA primers used in the LAMP assays were 0.940, 0.970, and 0.807, respectively. There was a high level of agreement between the test results and the true-disease status as indicated by the receiver operating characteristic (ROC) curve. Our findings suggest that the newly developed LAMP assays targeting the uvrC and gyrB genes could be a useful tool for rapid and accurate diagnosis of mastitis caused by M. bovis.

Full text available upon request to the author

Article title: Historical review and insights on the livestock tick-borne disease research of a developing country: The Philippine scenario

Authors: Adrian P. Ybañez, Claro N. Mingala, Rochelle Haidee D. Ybañez

Publication title: Parasitology International 67(2): 262-266, April 2018

Abstract:

Tick-borne diseases (TBDs) remain to be a global animal health threat. Developing countries like the Philippines is not exempt to this. Despite the potential impact TBDs can give to these countries, local government initiatives and researches remain to be limited. In the Philippines, most epidemiological studies were confined only to specific areas, and predominantly in the Northern Area. Due to its unique geography and limited studies, the current nationwide status of most TBDs could not be clearly established. This review mainly covered published studies and presented challenges in the conduct of TBD research in the Philippines, which may be similar to other Southeast Asian or developing countries. To date, reported livestock TBD pathogens in the Philippines include Anaplasma, Babesia, Theileria, and Mycoplasma spp. With the ubiquitous presence of the Rhipicephalus microplus ticks in the country, it is highly probable that other pathogens transmitted by these vectors could be present. Despite studies on different TBDs in the livestock sector, the Philippine government has not yet heightened its efforts to implement tick control measures as part of the routine animal health program for local farmers. Further studies might be needed to determine the nationwide prevalence of TBDs and the presence of other possible tick species and TBD pathogens. The Philippine scenario may present situations that are similar to other developing countries.

Full text available upon request to the author

Article title: Molecular detection and phylogenetic analysis of Ehrlichia canis in a Philippine dog

Authors: Naoya Maekawa, Satoru Konnai, Michelle M. Balbin, Claro N. Mingala

Publication title: Ticks and Tick-borne Diseases 9(2): 266-269, February 2018

Abstract:

Canine monocytic ehrlichiosis (CME), caused by a rickettsial bacterium, Ehrlichia canis, is distributed worldwide, particularly in tropical and subtropical regions. Transmission of E. canis is primarily mediated by the vector tick, Rhipicephalus sanguineus sensu lato and the bacteria then infect and replicate in monocytes and macrophages. Many cases are seen in veterinary hospitals and treated routinely; however, the genetic variation of E. canis strains found in the Philippines has been poorly investigated to date. In this study, the 16S rRNA gene and the gp200 gene of E. canis were detected by polymerase chain reaction from an infected dog in the Philippines, and the deduced amino acid sequence of the gp200 gene was subjected to a phylogenetic analysis. The Philippine genotype formed a cluster with the Taiwan genotype, and was somewhat divergent from the USA and Brazil strains. This suggested that E. canis underwent evolution in East and Southeast Asia, confirming the utility of the gp200 gene for the assessment of genetic relationships among strains.

Full text available upon request to the author

Article title: Molecular Characterization and Comparison of Phospholipase C zeta (PLCZ1) Gene Between Swamp ( Bubalus carabanensis ) and Riverine ( Bubalus bubalis ) Buffaloes: Its Implications and Future Perspectives

Authors: Eufrocina P. Atabay, Roseline D. Tadeo, Edwin C. Atabay, Emma V. Venturina, et al.

Publication title: 29(3): 190-198, July 2018

Abstract:

Phospholipase C zeta, a novel sperm-specific protein which is widely known to induce oocyte activation following fertilization, had already been characterized in various mammalian species, but not in water buffaloes thus far. The present study was conducted to initially characterize and compare the sequences of PLCZ1 gene of swamp and riverine buffaloes. Semen samples were collected; total RNA was extracted and reverse-transcribed. PLCZ1 cDNA was then amplified, and submitted for sequencing. Buffalo PLCZ1 gene yielded a sequence of 1905 base pair nucleotides translated into 634 bp amino acids. In general, the buffalo PLCZ1 gene was found to have high sequence identity with cattle and other domestic species. Similarly, significant residues and motifs in PLCZ1 gene sequence are found conserved in water buffaloes. However, there are variations in sequences identified between types of water buffaloes that may play a role in species-specific differences in terms of gene and protein expression, physiological mechanisms, and biological functions. The molecular information on buffalo PLCZ1 gene is highly valuable in subsequent works such as correlation studies on the identified gene variations with semen quality and fertility, and the development of biomarkers for bull fertility.

Full text available upon request to the author

Article title: Gross and Molecular Comparison of Fasciola hepatica and Fasciola gigantica from the Field in the Philippines

Authors: Lara Shinette I. Valino, Virginia M. Venturina, Claro N. Mingala

Publication title: International Journal of Veterinary Science, December 2017

Abstract:

The study established the morphologic and molecular differentiation of Fasciola hepatica and Fasciola gigantica in buffaloes. Specifically, the study described the gross structure and morphometry of F. hepatica and F. gigantica and validated the identification of Fasciola spp. based on gross morphology and PCR results. Sixty (60) samples were evaluated grossly and morphometrically using body length, body width, cone width, and cone length as parameters to differentiate the two species. Ten representative samples from each species identified based on the parameters were subjected to single step-duplex polymerase chain reaction (PCR) for molecular identification of the species identity.

Article title: Prevalence of babesiosis (Babesia bovis and Babesia bigemina) in cattle and water buffalo in Nueva Ecija, Philippines using Nested Polymerase Chain Reaction

Authors: Princess Charmaine T. Herrera, Victoria V. Valoria, Michelle M. Balbin, Claro N. Mingala

Publication title: Annals of Parasitology 63(4): 309-316, 2017

Abstract:

The study was conducted to determine the prevalence of Babesia bovis and Babesia bigemina infection in blood samples of cattle and water buffaloes using nested polymerase chain reaction (nested-PCR). It also aimed to generate a spot map showing areas in Nueva Ecija, the Philippines where B. bovis and B. bigemina were detected. Whole blood samples of cattle (148) and water buffalo (65) were collected for DNA extraction and subsequent nested-PCR to detect B. bovis and B. bigemina. To further confirm and validate the nested-PCR results, three selected positive samples for each B. bovis and B. bigemina were sequenced and examined for homology analysis. The results showed that the prevalence of B. bovis, B. bigemina and mixed infection in cattle were 11.49% (17/148), 10.81% (16/148) and 5.41% (8/148), respectively. Homology analysis of nucleotide sequence of three selected DNA samples for each B. bovis showed two 99% and one 96% (partial sequence analysis) identities with B. bovis Thailand strain, while B. bigemina positive samples showed all 100% identities with B. bigemina Philippine strain. The result did not demonstrate in all water buffalo samples. These findings provide information about the prevalence of B. bovis and B. bigemina in cattle and water buffaloes in Nueva Ecija, which can be beneficial for strategic planning, disease management, and control and prevention.

Article title: Molecular characterization of the lymphocyte activation gene-3 (LAG-3, CD223) of swamp-and riverine-type water buffaloes (Bubalus bubalis)

Authors: Shanemae M. Rivera, Ryan Bismark C. Padiernos, Evaristo A. Abella, Satoru Konnai, et al.

Publication title: Japanese Journal of Veterinary Research 65(2): 65-74, May 2017 

Abstract:

The present study was conducted to characterize LAG-3 of swamp- and riverine-type water buffaloes by DNA sequencing, homology and phylogenetic analysis. Bubaline LAG-3 sequence contained an open reading frame of 1551 nucleotide, encoding a polypeptide of 516 amino acids. Nucleotide and amino acid sequence homology of LAG-3 revealed 76-96% and 61-94% identity in water buffalo to that of other mammals, respectively. LAG-3 protein sequence of water buffalo contained four extracellular domains, a transmembrane domain and different conserved regions. There were three N-glycosylation sites, two sequence motifs: ‘RGD’ and ‘WXC’ motif and five cysteine residues located at different positions of extracellular region. Likewise, the possible serine phosphorylation site and the ‘KTGELE’ inhibitory motif were found in the intracellular region of bubaline LAG-3. However, one highly conserved cysteine residue in mammalian LAG-3 was replaced by tyrosine in both swamp- and riverine-type water buffaloes. Phylogenetic analysis generated high bootstrap value between the two types of water buffalo which further confirmed the degree of relationship between bubaline species. This was the first report that describe the genetic characteristic of LAG-3 in swamp- and riverine-type water bufffaloes.

Article title: Screening of BCL-2 associated X protein gene polymorphism associated with scrotal hernia in domesticated swine using polymerase chain reaction-restriction fragment length polymorphism

Authors: Jessica G. Manalaysay, Nathaniel D. Antonio, Ralph Lorenz R. Apilado Joseph F. Bambico

Publication title: Asian-Australasian Journal of Animal Sciences 30(2) :262-266, February 2017

Abstract:

Objective: This study was conducted to screen scrotal hernia in domesticated swine from selected breeders in the Philippines. This defect is associated with a cytosine to thymine mutation in the BCL-2 associated X protein (BAX) gene of swine.

Methods: Genetic screening was done by DNA extraction followed by amplification and digestion using polymerase chain reaction-restriction fragment length polymorphism, amplifying the 416 bp region of the BAX gene that was subjected to digestion using the Ear I enzyme. Sequencing was also conducted to validate the results.

Results: Results revealed that out of 538 samples tested, 411 (76.4%) of the samples were found to be normal whereas the remaining were carriers of the mutation in which 80 (14.9%) were heterozygous mutants and 47 (8.7%) were homozygous mutants. Pietrain breed was found to have the highest incidence.

Conclusion: Having a scrotal hernia eliminates the chances of using the boar as a breeder stock because the following generations arising from it would most likely exhibit herniation. It is therefore advised to establish a genetic screening method for Scrotal Hernia in the Philippines to eliminate the negative gene from the herd.

Article title: Green synthesis of gold nanoparticles reduced and stabilized by sodium glutamate and sodium dodecyl sulfate

Authors: Gil Felicisimo S. Cabrera, Michelle M. Balbinb, Paul John G. Eugenio, Charleo S. Zapanta, et al.

Publication title: Biochemical and Biophysical Research Communications 484(4): 774-780, March 2017

Abstract:

The Turkevich method has been used for many years in the synthesis of gold nanoparticles. Lately, the use of plant extracts and amino acids has been reported, which is valuable in the field of biotechnology and biomedicine. The AuNPs was synthesized from the reduction of HAuCl4 3H2O by sodium glutamate and stabilized with sodium dodecyl sulfate. The optimum concentrations for sodium glutamate and sodium dodecyl sulfate in the synthesis process were determined. The characteristics of the synthesized AuNPs was analysed through UV–Vis Spectroscopy and SEM. The AuNPs have spherical shape with a mean diameter of approximately 21.62 ± 4.39 nm and is well dispersed. FTIR analysis of the AuNPs reflected that the sulfate head group of sodium dodecyl sulfate is adsorbed at the surface of the AuNPs. Thus, we report herein the synthesis of AuNPs using sodium glutamate and sodium dodecyl sulfate.

Full text available upon request to the author

Article title: Colorimetric detection of caprine arthritis encephalitis virus (CAEV) through loop-mediated isothermal amplification (LAMP) with gold nanoprobes

Authors: Michelle M. Balbina, Benchaporn Lertanantawong, Werasak Suraruengchai, Claro N. Mingala

Publication title: Small Ruminant Research 147: 48-55, February 2017

Abstract:

Infectious diseases in goats, CAE in particular have widely affected the productivity of this animal and greatly affected the farmers and the small ruminant industry. Molecular technique such as LAMP has been applied to detect the CAEV proviral DNA specifically and sensitively but this technique has its own drawback. Gold is the most widely used nanoparticle (NP) which has excellent properties such as high surface area and compatibility with biomolecules. In this study, gold nanoparticle (AuNP) conjugated with modified oligonucleotides or gold nanoprobe (AuPr) was used to detect CAEV proviral DNA in LAMP product. The hybridization of AuPr to a complementary sequence on the LAMP product made the gold resistant to high salt concentration. This resistance or non-resistance of the AuNP is observed as color change in the mixture. This study, reported a simple method of visual detection of CAEV proviral DNA in LAMP product using AuPr.

Full text available upon request to the author

Article title: Genetic Factors Affecting Pork Quality: Halothane and Rendement Napole Genes

Authors: Ramon Cesar D. Salas and Claro N. Mingala

Publication title: Animal Biotechnology 28(2): 148-155, April 2017

Abstract:

The most common pork quality problems are pale, soft, and exudative (PSE) and acid pork (AP). PSE is associated with the expression of recessive halothane (Hal) allele Haln. Recessive Hal pigs (Halnn) have defective Ca2+ release channels (CRC) or Ryanodine Receptors (RYR1) within the sarcoplasmic reticulum that allow uncontrolled release of Ca2+ in response to stress. Abnormal lactic acid metabolism caused by stress prior to slaughter leads to the sudden drop in postmortem muscle pH producing the PSE pork. Conversely, AP is caused by the dominant RN- allele of the Rendement Napole gene. RN- pigs have high glycolytic potential that causes the lower ultimate pHu due to excessive lactic acid production postmortem. Poor water holding capacity of muscle cells in PSE and AP causes excessive drip loss leading to low cooking and processing yields. The conventional methods to evaluate Hal and RN genotypes are less effective compared to the more accurate gene marker tests. Selection against the Haln and RN- alleles by genomic selection can potentially reduce the frequencies of the defective genes with high accuracy in less time. As more quantitative trait loci (QTL) are identified, pig breeders are able to select traits more effectively to increase efficiency of pig production and enhance pork quality.

Full text available upon request to the author

Article title: Molecular epidemiology of pathogenic Leptospira spp. among large ruminants in the Philippines

Authors: Marvin A. Villanueva, Claro N. Mingala, Michelle M. Balbin, Chie Nakajima

Publication title: The Journal of Veterinary Medical Science 78(11): 1649-1655, December 2016

Abstract:

The extent of Leptospira infection in large ruminants resulting to economic problems in livestock industry in a leptospirosis-endemic country like the Philippines has not been extensively explored. Therefore, we determined the prevalence and carrier status of leptospirosis in large ruminants using molecular techniques and assessed the risk factors of acquiring leptospirosis in these animals. Water buffalo and cattle urine samples (n=831) collected from 21 farms during 2013-2015 were subjected to flaB-nested PCR to detect pathogenic Leptospira spp. Leptospiral flaB was detected in both species with a detection rate of 16.1%. Leptospiral DNA was detected only in samples from animals managed in communal farms. Sequence analysis of Leptospira flaB in large ruminants revealed the formation of three major clusters with L. borgpetersenii or L. kirschneri. One farm contained Leptospira flaB sequences from all clusters identified in this study, suggesting this farm was the main source of leptospires for other farms. This study suggested that these large ruminants are infected with various pathogenic Leptospira species causing possible major economic loss in the livestock industry as well as potential Leptospira reservoirs that can transmit infection to humans and other animals in the Philippines.

Article title: Larvicidal activity of nematophagous fungi Dudingtonia flagrans against common strongyle roundworms of buffaloes (Bubalus bubalis)

Authors: Toni Rose M. Barroga, Therese Marie A. Collantes, Claro N. Mingala

Publication title: Philippine Journal of Veterinary and Animal Sciences 42(1), 2016

Abstract:

Infestation of gastrointestinal nematodes is a major problem in grazing animals. Control is achieved through administration of anthelmintics; however, because of indiscriminate use, there have been increased reports of resistance to chemical anthelmintics which led to the failure of parasite control. This study determined the efficacy of the chlamydospore of Duddingtonia flagrans as biological control against common strongyle roundworms of buffaloes. Using corn meal agar assay, strongyle infective larvae were treated with and without D. flagrans. The chlamydospore/ gram (CG) assay tested a dose-dependent concentration wherein feces with 2,100 eggs/ gram (EPG) strongyles were treated with D. flagrans at an increasing doses of CG (100,000, 250,000 and 500,000). Results showed an 84.39% larval reduction after treatment with 500,000 CG. The chlamydspore/ egg assay evaluated increasing ratios of egg to chlamydospore dose (1:0, 1:100, 1:500, 1:1000) using the 2,100 EPG feces. The ratio 1:500 achieved the highest percent larval reduction (78.88%). D. flagrans was directly fed to buffaloes at varying concentrations (50,000, 150,000, 250,000 chlamydospores/kg BW). A 78.77% larval reduction was observed at 50,000 chlamydospore/kg BW oral administration for 5 days. This study showed the efficacy of D. flagrans as a potential alternative for anthelmintics in buffaloes.

Article title: The corollary effect of heavy metal accumulation in freshwater ponds on the hematological profile of Nile Tilapia (Oreochromis niloticus)

Authors: Gemerlyn G. Garcia, Elaine Jean L. Miguel, Mark Anthony L. Gabriel, Claro N. Mingala

Publication title: Environmental and Experimental Biology 14: 69–73, 2016

Abstract:

The status of heavy metal buildup in commercial and non-commercial ponds of Nile tilapia (Oreochromis niloticus) and its effect on fish health was evaluated. Pond water and tilapia meat were examined for Pb, As, cadmium Cd and Cu using flame atomic absorption spectrometry; and Hg through manual cold-vapor atomic absorption spectrometry. Standard methods in hematology were applied to estimate red and white blood cell function of fish in relation to heavy metal accumulation. The results revealed significantly higher Cu content in pond water of a commercial farm compared to the Cu content of water from a non-commercial farm, while similar levels of Hg and Pb were recorded. Tilapia meat from commercial ponds had significantly higher Pb and lower levels of Cu compared to meat from a non-commercial farm. Similar levels of Hg were observed in tilapia meat obtained from farms while tilapia meat from non-commercial ponds had significantly higher Cu. Neither As nor Cd were detected in the farms. Hematological evaluation revealed comparable counts of total red blood cell. Red blood cell indices such as hematocrit and mean corpuscular volume were significantly higher in tilapia from the commercial ponds. The amount of hemoglobin per red blood cell was smilar in tilapia from the farms while mean corpuscular hemoglobin concentration was significantly higher in tilapia from the non-commercial farm. Total white blood cell and eosinophil counts of tilapia were similar in the farms. Tilapia from the commercial ponds had significantly high neutrophil and monocyte counts while tilapia from the non-commercial ponds had significantly high lymphocyte counts. The hematological evaluation indicate relationship of cellular components of fish blood and heavy metal accretion from the aquatic environment by Nile tilapia.

Article title: Molecular comparison of Slc11a1 and Slc11a2 genes of swamp- and riverine-type water buffaloes

Authors: R. B. C. Padiernos and C. N. Mingala

Publication title: International Journal of Immunogenetics 43(3): 171-9, June 2016

Abstract:

Solute-linked carrier 11a and 11a2 (Slc) have been associated with disease resistance and/or susceptibility across animal species. These genes have an important mechanism in the regulation against intracellular infection. This study analysed the genetic characteristic of Slc 11a and 11a2 in swamp-type and riverine-type water buffaloes to understand their immunological distinction. Characterization of Slc11a1 and Slc11a2 genes from swamp- and riverine-type water buffaloes was carried out by molecular cloning, sequencing and phylogenetic analysis. The cloned cDNA of Slc11a1 and Slc11a2 contained an open reading frame of 1647 and 1723 nucleotides, encoding 549 and 574 amino acids, respectively. Nucleotide sequence homology of both Slc11a1 and Slc11a2 had 99% in swamp and riverine type, which gives almost identical polypeptide. However, Slc11a1 and Slc11a2 have substitutions of 5 and 1 amino acid residues, correspondingly. These substitutions suggest as a potential gene markers for resistance and/or susceptibility to intracellular infection. Furthermore, phylogenetic analysis confirmed the degree of relationship between the bubaline species and justifies the distinctness of each breed by the bootstrap value generated.

Full text available upon request to the author

Article title: Transporter protein and drug resistance of Trypanosoma

Authors: Noraine P. Medina and Claro N. Mingala

Publication title: Annals of Parasitology 62(1): 11-5, 2016

Abstract:

Trypanosoma infection is one of the most important infections in livestock and humans. One of the main problems of its therapeutic control and treatment is the resurgence of drug resistance. One of the most studied causes of such resistance is the function of its adenosine transporter gene. A trypanosomal gene TbAT1 from Trypanosoma brucei has been cloned in yeast to demonstrate its function in the transport of adenosine and trypanocidal agents. Drug resistant trypanosomes showed a defective TbAT1 variant; furthermore, deletion of the gene and set point mutations in the transporter gene has been demonstrated from isolates from relapse patients. The molecular understanding of the mechanism of action trypanocidal agents and function of transporter gene can lead to control of drug resistance of Trypanosomes.

Article title: Evidence of Fasciola spp. resistance to albendazole, triclabendazole and bromofenofos in water buffaloes (Bubalus bubalis)

Authors: Virginia M. Venturina, Ma Antonette F. Alejandro, Cyril P. Baltazar, Nancy S. Abes, et al.

Publication title: Annals of Parasitology 61(4): 283-9, 2015

Abstract:

Fasciolosis caused by Fasciola spp. is considered the most important helminth infection of ruminants in tropical countries. Anthelmintic resistance has become a global concern. This study compared the efficacy of the commonly used anthelmintics, determined the toxicity level and any indication of resistance. Thirty two water buffaloes naturally-infected with Fasciola spp. were used to determine the efficacy of triclabendazole (TBZ), albendazole (ABZ), and bromofenofos (BRO) using Fecal Egg Count Reduction Test (FECRT). To test the toxicity of the drugs given, serum glutamic-pyruvic transaminase (SGPT) was evaluated before and within one week after treatment. One dose administration of ABZ registered an efficacy of 79.17%, 73.33% for TBZ and 70.83% for BRO. Efficacy in two dose- treatment group was 83.33% for both BRO and ABZ, and 90.00% for TBZ. Two dose-treatment was effective for TBZ (90%), ineffective for BRO and ABZ. SGPT levels were not significantly different between pre-treatment and post- treatment across all treatments. Giving one or two doses of anthelmintics, at one month interval, does not increase the efficacy of the three drugs tested. The study also implies that anthelmintic resistance may have developed in the animals.

Article title: Molecular Evaluation of Pork, Beef and Poultry Meat Sold in Nueva Ecija, Philippines for the Presence of Horse (Equus caballus) and Rat (Rattus rattus) DNA Using Polymerase Chain Reaction Assay

Authors: Sonny C. Ramos, Claro N. Mingala, Erol Jay Y. Balagan, Leslie M. Domingo, et al.

Publication title: The Philippine Journal of Veterinary Medicine 53(1): 44-41, January 2016

Abstract:

The study aimed to detect traces of horse and rat DNA from meat samples declared as pork, beef and poultry meat being sold in the markets of Nueva Ecija, Philippines. Thirty locally-produced and imported canned and processed meat products were bought from various markets. DNAs were extracted using tissue extraction protocol. The quality and quantity of the extracted DNAs were preliminary evaluated by targeting beta-actin gene through PCR and by nanospectrophotometer, respectively. PCR detection of horse and rat DNA was evaluated using specific primers targeting cyt b gene with an expected amplicon size of 439 and 603 bp, respectively. Results showed no band in 100% of the samples after gel electrophoresis which means that all meat products were tested negative for both horse and rat DNA. This suggests that horse and rat meat were absent in the meat products tested.

Article title: Serological investigation of Leptospira infection and its circulation in one intensive-type water buffalo farm in the Philippines

Authors: Marvin A. Villanueva, Claro N. Mingala, Nina G. Gloriani, Yasutake Yanagihara, et al.

Publication title: The Japanese Journal of Veterinary Research 64(1): 15-24, February 2016

Abstract:

Water buffalo is an indispensable livestock in the Philippines. Leptospirosis is a serious zoonosis that can be fatal to humans and cause reproductive problems in livestock. Leptospirosis has been reported in some countries where water buffaloes are commercially raised, highlighting the Leptospira prevalence in this farming system, but information on leptospirosis in water buffalo farms in the Philippines is limited. In this study, we collected blood samples from rats (n = 21), and water buffaloes (n = 170) from different groups and locations in one intensive-type buffalo farm in the Philippines. Serum was analyzed by microscopic agglutination test (MAT). Anti-Leptospira antibodies reacting with serogroups Canicola, Icterohaemorrhagiae and Pomona were found in sera of 30% tested rats, and 48% of water buffalo sera tested positive for at least one Leptospira strain, in which serogroups Mini, Hebdomadis, Tarassovi and Pyrogenes were predominantly agglutinated. The number of seropositive young water buffaloes (< 1 year-old) was lower than that of older seropositive ones. Furthermore, sera from younger water buffaloes were reactive with single serotypes with low MAT titers, but older animals were reactive with multiple Leptospira strains with variable MAT titers. In addition, antibodies against serogroups Icterohaemorrhagiae and Pomona were detected in both animals. Finally, Leptospira infection was found associated with age and animal grouping, highlighting the impact of management in the persistence of leptospirosis at intensive-type buffalo farm settings in the Philippines. Further investigation and appropriate control strategies are required to prevent leptospirosis from causing risks to public health and economic losses to the water buffalo farming industry.

Article title: A field trial of recombinant Schistosoma japonicum paramyosin as a potential vaccine in naturally-infected water buffaloes

Authors: Mario Antonio L. Jiz II, Claro N. Mingala, Ivy Fe M. Lopez, Mike Chua, et al.

Publication title: Annals of Parasitology 62(4): 295-299, 2016

Abstract:

The overall aims of this project are to assess the safety and immunogenicity of the Schistosoma japonicum vaccine paramyosin among water buffaloes residing in endemic areas. The study was conducted in four villages in Leyte, the Philippines, an area highly endemic for schistosomiasis japonica. One hundred and fifteen (N=115) animals provided baseline stool samples for coprologic examination, with preliminary results using FLOTAC showing a 10% prevalence of schistosomiasis. Forty-nine (N=49) animals consented to treatment with 25 mg/kg Praziquantel, and 40, 36 and 32 animals consented to the first, second and third dose of the paramyosin vaccine, respectively. The safety trial involved the first 20 animals and included skin testing, vaccination, anaphylaxis monitoring, as well as hematology and serum chemistry analysis. Skin tests revealed that only three out of 20 animals exhibited redness at the injection site, with none greater than 1 cm. None of the animals exhibited anaphylaxis, and all hematology and serum chemistry markers were within normal range or were similar to pre-vaccination levels. None of the 40 animals administered with the first dose exhibited anaphylaxis, nor any of the subsequent vaccine doses. Immunogenicity assessment of sera collected prior to every vaccination and one month after the last dose showed that the paramyosin vaccine induced robust antibody responses to all animals, as assessed by ELISA. The cytokine levels of whole blood culture supernatants will be further assessed. Our findings demonstrate that the S. japonicum paramyosin vaccine is a safe, well-tolerated and immunogenic treatment among water buffalos residing in endemic areas.

Full text available upon request to the author

Article title: Molecular characterization of T-cell immunoglobulin mucin domain-3 and Galectin-9 genes of swamp- and riverine-type water buffaloes

Authors: P. L. H. Duran, R. B. C. Padiernos, E. A. Abella, S. Konna, C. N. Mingala 

Publication title: International Journal of Immunogenetics 42(6): 469-78, December 2015

Abstract:

Molecular characterization of T-cell immunoglobulin mucin domain-3 (TIM-3) and Galectin-9 (GAL-9) genes of swamp- and riverine-type water buffaloes was conducted to compare these genes with other species; determine the unique characteristic specific in water buffalo; and provide baseline information for the assessment of disease progression in buffalo species. TIM-3 and GAL-9 genes were amplified, purified, sequenced and characterized. The sequence result of TIM-3 in both types of water buffaloes contained 843 nucleotides encoding to 280 amino acids while GAL-9 of swamp-type and riverine-type water buffaloes contained 1023 and 972 nucleotides encoding to 340 and 323 amino acids, respectively. Meanwhile, the nucleotide and amino sequence of TIM-3 in water buffalo were 83-98% and 94-97% identical with other artiodactyl species, respectively. On the other hand, GAL-9 nucleotide and amino acid sequence in water buffalo were 85-98% and 76-96% identical with other artiodactyl species. The tyrosine-kinase phosphorylation motif and potential glycosylation sites were conserved within the tribe Bovinae. It is imperative to have further studies in the assessment of the role of these genes in disease progression in water buffalo during chronic infection. The study is the first report that describes the genetic characteristic of TIM-3 and GAL-9 genes in water buffalo.

Full text available upon request to the author

Article title: Molecular epidemiological survey and genetic analysis of vector-borne infections of cattle in Luzon Island, the Philippines

Authors: Nyamsuren Ochirkhuu, Satoru Konnai, Claro N. Mingala, Tomohiro Okagawa, et al.

Publication title: Veterinary Parasitology 212(3-4): 161-7, September 2015

Abstract:

In the Philippines, vector-borne disease is one of the important problems in the livestock industry. To elucidate the epidemiology of vector-borne diseases in cattle on Luzon Island, the Philippines, the prevalence of five protozoan agents was assessed by polymerase chain reaction. Out of the 339 samples, 324 (95.5%), 154 (45.4%), 209 (61.6%), 140 (41.3%), and 2 (0.6%) were positive for Anaplasma marginale, Babesia bigemina, Babesia bovis, Theileria spp., and Trypanosoma evansi infections, respectively. Mixed infections were detected in 290 (85.5%) samples, of which 115 (33.9%) had two pathogens, 144 (42.5%) had three pathogens, and 31 (9.1%) had four kinds of pathogens. 16S rRNA gene was 100% identical in A. marginale compared with the same lineage across the world. B. bovis RAP-1 and B. bigemina AMA-1 genes were identical with 92.27%-100% and 97.07%-100% sequences, respectively, in the database (Asian isolates). MPSP genes of Theileria spp. were 83.51%-100% identical with the one another. Phylogenetic analysis showed that they belong to the groups of T. sergenti and T. buffeli. Positive rates of the tick-borne pathogens were extremely high in this area. These findings provide vital information that can be used for the planning and execution of effective control measures for vector-borne diseases in the Philippine cattle industry.

Full text available upon request to the author

Article title: Evaluation of treatment alternatives against respiratory bacterial pathogens of small and large ruminants

Authors: Claro N. Mingala

Publication title: Advances in Environmental Biology 9(8), May 2015

Abstract:

The sensitivity of five bacterial isolates from small and large ruminants with respiratory infections to synthetic and herbal-based anti-infectives was evaluated in vitro. Synthetic drugs such as Rifampicin, Erythromycin, Benzylpenicillin, Chloramphenicol and Tetracycline and herbal-based drugs such as Ascof® Lagundi (Vitex negundo) and Lagundi (Vitex negundo) leaf extract were applied at different concentrations adapting the Agar cup plate method to test responses of isolated pathogens. Evaluation of treatment was based on the lowest concentration of each anti-infective that can inhibit the test pathogens and compared with existing antibiotic susceptibility testing standards. Measurements of the zones of inhibition on pathogens in response to the applied anti-infectives revealed that S. sciuri, B. pumilus and P. aeruginosa are susceptible to benzyl penicillin at a minimum concentration of 1 IU/ml. B. pumilus was receptive to Erythromycin at a minimum concentration of 10μg/ml. A. schindleri was sensitive to Chloramphenicol at a minimum concentration of 25 μg/ml while P. aeruginosa was susceptible to 50 μg/ml Chloramphenicol. A. schindleri was responsive to anti-bacterial effect of 10 μg/ml Tetracycline while P. aeruginosa was receptive to 25 μg/ml tetracycline. S. sciuri and B. pumilus were both susceptible to Ascof commercial Lagundi at 10% minimum concentration while B. pumilus alone is sensitive to Lagundi leaf extract at a minimum concentration of 10%.

Full text available upon request to the author

Article title: Expression of regulatory dendritic cell-related cytokines in cattle experimentally infected with Trypanosoma evansi

Authors: Hirohisa Mekata, Shiro Murata, Claro N. Mingala, Kazuhiko Ohashi, et al.

Publication title: The Journal of Veterinary Medical Science 77(8): 1017-9, August 2015

Abstract:

Trypanosoma evansi causes wasting disease in many livestock. T. evansi infection gives rise to inflammatory immune responses, which contribute to the development of inflammation-associated tissue injury. We previously reported that regulatory dendritic cells (DCs), which act as potential regulators of inflammation, were activated in infected mice and transfer of regulatory DCs to infected mice prolonged their survival. However, the kinetics of regulatory DCs in cattle, which are natural hosts of T. evansi, remained unclear. In this study, we report that the expressions of CCL8 and IL-10, which promote the development of regulatory DCs, were up-regulated in cattle experimentally infected with T. evansi. This finding is potentially useful for studying the control strategy of T. evansi infection in cattle.

Article title: Molecular characterization of the gag gene of caprine arthritis encephalitis virus from goats in the Philippines

Authors: Ryan Bismark C. Padiernos, Michelle M. Balbin, Arman M. Parayao, Claro N. Mingala

Publication title: Archives of Virology 160(4): 969-78, April 2015

Abstract:

Caprine arthritis encephalitis virus (CAEV) causes caprine arthritis encephalitis syndrome, which is an emerging disease of goats in the Philippines. DNA sequence analysis showed homology of 86-93 % between Philippine CAEV and available CAEV sequences in GenBank. CAEV was detected using nested polymerase chain reaction (PCR), and new sets of primers were designed in order to amplify the gag gene, which is a highly conserved region of the viral genome. In addition, the Philippine CAEV isolate clustered in group B with the prototype caprine lentivirus. Based on amino acid sequence alignments, it is possible that the Philippine CAEV isolate is a new strain of CAEV, but it is also possible that it was already present in the country even before the start of goat importation. Molecular characterization of the CAEV gag gene is important for the development of a detection kit specific for the local strain of CAEV and the establishment of small ruminant lentivirus eradication programs in the Philippines. This study is the first report to describe the molecular characteristics of CAEV circulating in the Philippines.

Article title: Conservation of exotic and endangered animals through biotechnology

Authors: Sanny C. Babera and Claro Mingala

Publication title: Global Journal of Bio-Science and Biotechnology 4(2): 220-223, 2015

Abstract:

Maintaining the biodiversity of animals in the ecosystem could lessen the effect of environmental imbalance. Conservation and preservation of endangered animals using different methods in biotechnology could save important genetic materials for future reconstruction of extinct species or even the most endangered one. DNA fingerprinting is important in identifying traits vital to understand breeding peculiarities of wild animals specially birds. Genetic resource bank could facilitate the longer storage of diverse endangered and exotic genetic material.

Article title: Evaluation of treatment alternatives against respiratory bacterial pathogens of small and large ruminants

Authors: Gemarlyn Garcia, Lawrence Belotindos, Claro Mingala

Publication title: Advances in Environmental Biology 9(8): 149-155, January 2015

Abstract:

The sensitivity of five bacterial isolates from small and large ruminants with respiratory infections to synthetic and herbal-based anti-infectives was evaluated in vitro. Synthetic drugs such as Rifampicin, Erythromycin, Benzylpenicillin, Chloramphenicol and Tetracycline and herbal-based drugs such as Ascof® Lagundi (Vitex negundo) and Lagundi (Vitex negundo) leaf extract were applied at different concentrations adapting the Agar cup plate method to test responses of isolated pathogens. Evaluation of treatment was based on the lowest concentration of each anti-infective that can inhibit the test pathogens and compared with existing antibiotic susceptibility testing standards. Measurements of the zones of inhibition on pathogens in response to the applied anti-infectives revealed that S. sciuri, B. pumilus and P. aeruginosa are susceptible to benzyl penicillin at a minimum concentration of 1 IU/ml. B. pumilus was receptive to Erythromycin at a minimum concentration of 10µg/ml. A. schindleri was sensitive to Chloramphenicol at a minimum concentration of 25 µg/ml while P. aeruginosa was susceptible to 50 µg/ml Chloramphenicol. A. schindleri was responsive to anti-bacterial effect of 10 µg/ml Tetracycline while P. aeruginosa was receptive to 25 µg/ml tetracycline. S. sciuri and B. pumilus were both susceptible to Ascof commercial Lagundi at 10% minimum concentration while B. pumilus alone is sensitive to Lagundi leaf extract at a minimum concentration of 10%.

Full text available upon request to the author

Article title: The great diversity of major histocompatibility complex class II genes in Philippine native cattle

Authors: S.N. Takeshima, T. Miyasaka, M. Polata, M. Kikuya, et al.

Publication title: Meta Gene 2: 176-190, December 2014

Abstract:

Bovine leukocyte antigens (BoLA) are extensively used as markers for bovine disease and immunological traits. However, none of the BoLA genes in Southeast Asian breeds have been characterized by polymerase chain reaction (PCR)-sequence-based typing (SBT). Therefore, we sequenced exon 2 of the BoLA class II DRB3 gene from 1120 individual cows belonging to the Holstein, Sahiwal, Simbrah, Jersey, Brahman, and Philippine native breeds using PCR-SBT. Several cross-breeds were also examined. BoLA-DRB3 PCR-SBT identified 78 previously reported alleles and five novel alleles. The number of BoLA-DRB3 alleles identified in each breed from the Philippines was higher (71 in Philippine native cattle, 58 in Brahman, 46 in Holstein × Sahiwal, and 57 in Philippine native × Brahman) than that identified in breeds from other countries (e.g., 23 alleles in Japanese Black and 35 in Bolivian Yacumeño cattle). A phylogenetic tree based on the DA distance calculated from the BoLA-DRB3 allele frequency showed that Philippine native cattle from different Philippine islands are closely related, and all of them are closely similar to Philippine Brahman cattle but not to native Japanese and Latin American breeds. Furthermore, the BoLA-DRB3 allele frequency in Philippine native cattle from Luzon Island, located in the Northern Philippines was different from that in cattle from Iloilo, Bohol, and Leyte Islands, which are located in the Southern Philippines. Therefore, we conclude that Philippine native cattle can be divided into two populations, North and South areas. Moreover, a neutrality test revealed that Philippine native cattle from Leyte showed significantly greater genetic diversity, which may be maintained by balancing selection. This study shows that Asian breeds have high levels of BoLA-DRB3 polymorphism. This finding, especially the identification of five novel BoLA-DRB3 alleles, will be helpful for future SBT studies of BoLA-DRB3 alleles in East Asian cattle.

Article title: Assessment of Swine (Sus scrofa domesticus) Alveolar Macrophage Viability Associated with Heavy Metal Air Pollutants

Authors: Gemerlyn G. Garcia, Eduardo H. Grivialde, Lerma C. Ocampo,  Claro N. Mingala

Publication title: The Thai Veterinary Medicine 44(4): 461-468, December 2014

Abstract:

The impact of air pollution on airway cellular defense of pigs raised under backyard, semi-commercial and commercial conditions was investigated. Flame atomic absorption spectrometry was used to analyze Cadmium (Cd) and Lead (Pb) while manual cold vapor atomic absorption spectrometry was applied to evaluate Mercury (Hg) contents of dust particles from farms. Tests for viable counts of swine alveolar macrophages (SAM) that simulate animal responses to air pollutants, microbiological techniques for the recovery of bacterial cells and evaluation for cellular function were undertaken. Results showed that air pollution was accompanied by significant high levels of Pb and Cd. Exposure of SAM to air pollutants induced significant reduction in cell viability and the reduction was substantially contributed by the duration of exposure and the farm source of SAM and air pollutants. Significant differences in colony counts of P. multocida were recorded as effects attributed by SAM exposure, the duration of exposure to air pollutants and the farm origin of the tested SAM and air pollutants. Data that revealed high recovery counts of P. multocida were taken as a measure for altered microbicidal action of SAM against the bacterium. In vitro interaction of backyard-obtained SAM and air pollutants for 6 h does not modify the killing or microbicidal action of SAM on P. multocida but prolonging the SAM and air pollutant interface beyond 6 h may eventually weaken the microbicidal action of the cells which allows incessant yielding of high bacterial recovery counts and failure of SAM to counteract this process.

Article title: Evaluation of a Portable Somatic Cell Counter in the Diagnosis of Bubaline Subclinical Mastitis

Authors: R.T. Salvador, R.L. Soliven, E.J.Y. Balagan, N.S. Abes

Publication title: Thai Journal of Agricultural Science 47(4): 205-209, 2014

Abstract:

The study aimed to evaluate the performance of portable somatic cell counter (PortaSCC) relative to that of the laboratory-based somatic cell counter (Fossomatic) in the diagnosis of bubaline subclinical mastitis. It determined the sensitivity and specificity of PortaSCC with Fossomatic as the reference. The agreement between the results of the two test equipments was also measured using kappa statistics. Eighty milk samples were collected from different farms. Samples were immediately processed for somatic cell count using the PortaSCC. Same samples were brought to the laboratory for the somatic cell counts. All samples were processed in triplicates. The PortaSCC has 94.12% and 87.30% sensitivity and specificity, respectively. A substantial agreement (k=0.70) between the results of the two tests was also observed. These test properties of the PortaSCC and its capability to rapidity to provide results rationalize its utilization as an alternative for the laboratory-based cell counter in evaluating milk samples from herds in remote areas under Philippine field conditions.

Article title: Detection and molecular characterization of bovine leukemia virus in Philippine cattle

Authors: Meripet Polat, Ayumu Ohno, Shin-Nosuke Takeshima, Jiyun Kim

Publication title: Archives of Virology 160(1): 285-96, November 2014

Abstract:

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle. BLV infects cattle worldwide, imposing a severe economic impact on the dairy cattle industry. However, there are no comprehensive studies on the distribution of BLV in the Philippines, and the genetic characteristics of Philippine BLV strains are unknown. Therefore, the aim of this study was to detect BLV infections in the Philippines and determined their genetic variability. Blood samples were obtained from 1116 cattle from different farms on five Philippine islands, and BLV provirus was detected by BLV-CoCoMo-qPCR-2 and nested PCR targeting BLV long terminal repeats. Out of 1116 samples, 108 (9.7 %) and 54 (4.8 %) were positive for BLV provirus, as determined by BLV-CoCoMo-qPCR-2 and nested PCR, respectively. Of the five islands, Luzon Island showed the highest prevalence of BLV infection (23.1 %). Partial env gp51 genes from 43 samples, which were positive for BLV provirus by both methods, were sequenced for phylogenetic analysis. Phylogenetic analysis based on a 423-bp fragment of the env gene revealed that Philippine BLV strains clustered into either genotype 1 or genotype 6. Substitutions were mainly found in antigenic determinants, such as the CD4(+) T-cell epitope, the CD8(+) T-cell epitope, the second neutralizing domain, B and E epitopes, and these substitutions varied according to genotype. This study provides comprehensive information regarding BLV infection levels in the Philippines and documents the presence of two BLV genotypes, genotypes 1 and 6, in this population.

Article title: Increased expression of the regulatory T cell-associated marker CTLA-4 in bovine leukemia virus infection

Authors: Saori Suzuki, Satoru Konnai, Tomohiro Okagawa, Ryoyo Ikebuchi, et al.

Publication title: Veterinary Immunology and Immunopathology 163(3-4): 115-24, October 2014

Abstract:

Regulatory T cells (Tregs) play a critical role in the maintenance of the host's immune system. Tregs, particularly CD4(+)CD25(+)Foxp3(+) T cells, have been reported to be involved in the immune evasion mechanism of tumors and several pathogens that cause chronic infections. Recent studies showed that a Treg-associated marker, cytotoxic T-lymphocyte antigen 4 (CTLA-4), is closely associated with the progression of several diseases. We recently reported that the proportion of Foxp3(+)CD4(+) cells was positively correlated with the number of lymphocytes, virus titer, and virus load but inversely correlated with IFN-γ expression in cattle infected with bovine leukemia virus (BLV), which causes chronic infection and lymphoma in its host. Here the kinetics of CTLA-4(+) cells were analyzed in BLV-infected cattle. CTLA-4 mRNA was predominantly expressed in CD4(+) T cells in BLV-infected cattle, and the expression was positively correlated with Foxp3 mRNA expression. To test for differences in the protein expression level of CTLA-4, we measured the proportion of CTLA-4-expressing cells by flow cytometry. In cattle with persistent lymphocytosis (PL), mean fluorescence intensities (MFIs) of CTLA-4 on CD4(+) and CD25(+) T cells were significantly increased compared with that in control and aleukemic (AL) cattle. The percentage of CTLA-4(+) cells in the CD4(+) T cell subpopulation was positively correlated with TGF-β mRNA expression, suggesting that CD4(+)CTLA-4(+) T cells have a potentially immunosuppressive function in BLV infection. In the limited number of cattle that were tested, the anti-CTLA-4 antibody enhanced the expression of CD69, IL-2, and IFN-γ mRNA in anti-programmed death ligand 1 (PD-L1) antibody-treated peripheral blood mononuclear cells from BLV-infected cattle. Together with previous findings, the present results indicate that Tregs may be involved in the inhibition of T cell function during BLV infection.

Article title: Molecular detection and characterization of Theileria species in the Philippines

Authors: Lawrence P. Belotindos, Jonathan V. Lazaro, Marvin A. Villanueva, Claro N. Mingala

Publication title: Acta Parasitologica 59(3): 448-53, September 2014

Abstract:

Theileriosis is a tick-borne disease of domestic and wild animals that cause devastating economic loss in livestock in tropical and subtropical regions. Theileriosis is not yet documented in the Philippines as compared to babesiosis and anaplasmosis which are considered major tick-borne diseases that infect livestock in the country and contribute major losses to the livestock industry. The study was aimed to detect Theileria sp. at genus level in blood samples of cattle using polymerase chain reaction (PCR) assay. Specifically, it determined the phylogenetic relationship of Theileria species affecting cattle in the Philippines to other Theileria sp. registered in the GenBank. A total of 292 blood samples of cattle that were collected from various provinces were used. Theileria sp. was detected in 43/292 from the cattle blood samples using PCR assay targeting the major piroplasm surface protein (MPSP) gene. DNA sequence showed high similarity (90-99%) among the reported Theileria sp. isolates in the GenBank and the Philippine isolates of Theileria. Phylogenetic tree construction using nucleotide sequence classified the Philippine isolates of Theileria as benign. However, nucleotide polymorphism was observed in the new isolate based on nucleotide sequence alignment. It revealed that the new isolate can be a new species of Theileria.

Article title: A comparison of FLOTAC and CFF techniques in detecting gastrointestinal parasites in water buffaloes (Bubalus bubalis)

Authors: Roderick T. Salvador, Rogelyn P. Abalos, Angeline M. Ruba, Claro N. Mingala

Publication title: Annals of Parasitology 60(2): 119-25, 2014

Abstract:

The objective of the study was to compare the usefulness of FLOTAC and centrifugal fecal flotation (CFF) techniques. More specifically, the taxonomic classes (Nematoda and Cestoda) of endoparasites present in fecal samples of buffaloes are identified, the sensitivity and specificity of FLOTAC relative to CFF are calculated, and the agreement of both techniques is evaluated using Kappa statistics. Fresh fecal samples from 220 buffaloes in 10 municipalities were collected. Sheather's sugar was used as a flotation solution for both the FLOTAC and CFF techniques. Of the 220 animals, 109 samples were nematode positive and 111 samples were nematode negative according to the FLOTAC technique, while 74 were found to be positive and 146 negative according to the CFF technique. No cestodes were detected by either technique. The calculated sensitivity for FLOTAC is 89.19% and its specificity is 70.55%. Kappa statistics revealed moderate agreement (k = 0.535) between the two techniques in detecting nematodes. The prevalence observed based on FLOTAC and CFF test were 49.54% (109/220; 95% CI: 47.75-56.34) and 33.64% (72/220; 95% CI: 27.42-40.3), respectively.

Article title: Genetic Testing for Porcine Stress Syndrome Using Mutagenically Separated-Polymerase Chain Reaction

Authors: Jessica G. Manalaysay, Claro N. Mingala, Domina Flor L. Gamboa, Rubigilda Paraguison-alili, et al.

Publication title: Philippine Journal of Veterinary Medicine 51(2), 2014

Abstract:

Porcine Stress Syndrome (PSS) is a defect in the Halothane (Hal) gene that produces pale, soft and exudative meat of inferior quality that results to significant losses in the meat industry. This study was conducted to detect PSS in pigs from seven farms in Luzon, Philippines which are used for breeding purposes. They were classified as normal (NN), stress carrier (Nn) and mutant (nn). This classification will help to form a new breeding system to be developed ensuring that all offspring are free of the stress gene. Characterization of the Hal gene was done by collecting blood samples subjected to DNA extraction and genotyping using mutagenically separated-polymerase chairn reaction (MS-PCR) which is an optimized one step process of PSS detection. Out of 427 samples, 22 were found to be mutant, 34 were carrier, and 371 were normal. Results for genotypic frequency showed that 87% pigs are normal (NN); 8% are heterozygotes (Nn) and only 5% are stress-positive (nn). Results were validated through DNA sequencing which showed the same results with MS-PCR. A genetic screening using this developed method for the Philippine setting is recommended to be able to minimize the effect of PSS. 

Article title: Biological activity of the Tiger mushroom (Lentinus tigrinus) with notes on its assessment for therapeutic consideration

Authors: Gemerlyn G. Garcia, Allen M. Veloso, Renato G. Reyes, Sofronio P. Kalaw, et al.

Publication title: Advances in Environmental Biology 8(10): 399-403, June 2014

Abstract:

This study has been initiated to explore the therapeutic potential of L. tigrinus. Evaluation of lethal effects and responses of mice to Lentinus tigrinus were undertaken. Intravenous lethal dose 50 (LD50), safe intravenous dose and other safety indices were also determined to validate its prospective therapeutic value. The experiment used 30 male BALB/c mice randomly distributed into 6 treatments. Each mouse received 200 μl of each dose of L. tigrinus extract (LTE) (3 mg/mouse, 7.50 mg/mouse, 15 mg/mouse, 22.50 mg/mouse, 30 mg/mouse). Observation for mortality, perceptible responses such as piloerection, eye secretions, weakness and anorexia were carried out at 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, 36 h and 48 h post-treatment with LTE wherein median lethal dose, safe IV dose, safety indices and dose-related responses were based from. Results showed that treatment with higher LTE concentrations (15 - 30 mg/mouse) elicited perceptible responses earlier preceding death of significantly higher number of mice compared to delayed onset of perceptible responses with no associated mortality in treatments with lower doses. Data also identified the threshold dose at 3 mg/mouse which triggered observable responses of mice while concentrations below the threshold dose have neither effect on perceptible responses nor on death in mice and linked estimated safe IV dose at 7.50 mg/mouse with zero mortality. Identification of the LD50 at 28.47 mg/mouse demonstrated its potent lethal effect in mice. Computed safety indices showing low therapeutic index (1.99), therapeutic ratio (0.67) and a safety factor (0.02) for LTE confirms its low margin of safety when used for therapeutic purposes.

Full text available upon request to the author

Article title: Caprine arthritis encephalitis virus detection in blood by loop-mediated isothermal amplification (LAMP) assay targeting the proviral gag region

Authors: Michelle M. Balbin, Lawrence P. Belotindos, Nancy S. Abes, Claro N. Mingala

Publication title: Diagnostic Microbiology and Infectious Disease 79(1): 37-42, May 2014

Abstract:

Caprine arthritis encephalitis virus (CAEV), of the genus Lentivirus of the Retroviridae family, causes persistent disease, which is characterized by polyarthritis and mastitis in adult goats and progressive paresis (leukoencephalomyelitis) in kids. A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of CAEV in blood samples. Species-specific primers amplifying the gag gene region in the provirus were used for the detection of CAEV. The LAMP assay result was obtained 30 min after incubation on a constant temperature at 63 °C in a heat block. Resulting amplicons were visualized by addition of SYBR green dye after the reaction and checked by agarose gel electrophoresis. The sensitivity of LAMP assay was evaluated by comparing the result with the nested polymerase chain reaction. Based on the experiments, the result of the assay indicated a rapid and sensitive test for the detection of CAEV.

Full text available upon request to the author

Article title: Identification and characterization of bovine programmed death-ligand 2

Authors: Asami Nishimori, Satoru Konnai, Ryoyo Ikebuchi, Tomohiro Okagawa, et al.

Publication title: Microbiology and Immunology 58(7): 388-97, July 2014

Abstract:

Previous reports from this group have indicated that the immunoinhibitory programmed death (PD)-1 receptor and its ligand, PD-L1, are involved in the mechanism of immune evasion of bovine chronic infection. However, no functional analysis of bovine PD-L2 in cattle has been reported. Thus, in this study, the molecular function of bovine PD-L2 was analyzed in vitro. Recombinant PD-L2 (PD-L2-Ig), which comprises an extracellular domain of bovine PD-L2 fused to the Fc portion of rabbit IgG1, was prepared based on the cloned cDNA sequence for bovine PD-L2. Bovine PD-L2-Ig bound to bovine PD-1-expressing cells and addition of soluble bovine PD-1-Ig clearly inhibited the binding of PD-L2-Ig to membrane PD-1 in a dose-dependent manner. Cell proliferation and IFN-γ production were significantly enhanced in the presence of PD-L2-Ig in peripheral blood mononuclear cells (PBMCs) from cattle. Moreover, PD-L2-Ig significantly enhanced IFN-γ production from virus envelope peptides-stimulated PBMCs derived from bovine leukemia virus-infected cattle. Interestingly, PD-L2-Ig-induced IFN-γ production was further enhanced by treatment with anti-bovine PD-1 antibody. These data suggest potential applications of bovine PD-L2-Ig as a therapy for bovine diseases.

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Article title: The prevalence of Ehrlichia canis, Anaplasma platys and Babesia spp. in dogs in Nueva Ecij., Philippines based on multiplex polymerase chain reaction (mPCR) assay

Authors: Joyce Marielle I. Corales, Victoria V. Viloria, Virginia M. Venturina, Claro N. Mingala

Publication title: Annals of Parasitology 60(4): 267-72, 2014

Abstract:

The aim of the study was to determine the prevalence of Ehrlichia canis, Anaplasma platys and Babesia spp. in dogs. It describes the practice of veterinarians in detecting tick-borne diseases in Nueva Ecija, Philippines. Seventy blood samples were collected and were subjected to multiplex PCR for the detection of E. canis, Babesia spp. and A. platys. The prevalence of babesiosis is the highest in Cabanatuan City (2/10), while a 10% prevalence (1/10) was observed in Science City of Muñoz, Talavera and Sta. Rosa. E. canis were only detected in Cabanatuan City. However, no anaplasmosis was detected in any area. The prevalence of babesiosis and ehrlichiosis in Nueva Ecija is 7.14% (5/70) and 2.85% (2/70) respectively. In addition, 70% (7/10) of the Nueva Ecija veterinary practitioners encountered cases of suspected ehrlichiosis in their practice. The diagnosis of ehrlichiosis is based primarily on presented clinical signs and complete blood counts, which include a platelet count. Of the 10 respondents, half utilized test kits while 90% interpreted blood samples. Meanwhile, only 60% of the respondents used an ELISA test kit for ehrlichiosis. For some practitioners, the main reason for not utilizing a kit is the high cost. None of the respondents had previously attended cases of suspected anaplasmosis. Only one respondent diagnosed a case of babesiosis by blood smear microscopy.

Article title: Evaluation of a Portable Somatic Cell Counter in the Diagnosis of Bubaline Subclinical Mastitis

Authors: R.T. Salvador, R.L. Soliven, E.J.Y. Balagan, N.S. Abes, et al.

Publication title: Thai Journal of Agricultural Science 47(4): 205-209, 2014

Abstract:

The study aimed to evaluate the performance of portable somatic cell counter (PortaSCC) relative to that of the laboratory-based somatic cell counter (Fossomatic) in the diagnosis of bubaline subclinical mastitis. It determined the sensitivity and specificity of PortaSCC with Fossomatic as the reference. The agreement between the results of the two test equipments was also measured using kappa statistics. Eighty milk samples were collected from different farms. Samples were immediately processed for somatic cell count using the PortaSCC. Same samples were brought to the laboratory for the somatic cell counts. All samples were processed in triplicates. The PortaSCC has 94.12% and 87.30% sensitivity and specificity, respectively. A substantial agreement (k=0.70) between the results of the two tests was also observed. These test properties of the PortaSCC and its capability to rapidity to provide results rationalize its utilization as an alternative for the laboratory-based cell counter in evaluating milk samples from herds in remote areas under Philippine field conditions.

Article title: Molecular Characterization of Respiratory Bacterial Pathogens in Large and Small Ruminants

Authors: Gemerlyn Gonzales Garcia, Lawrence Pascual Belotindos, Claro Niegos Mingala

Publication title: The Thai Veterinary Medicine 43(4): 483-489, December 2013

Abstract:

Bacterial isolates from different cases of respiratory infections in small and large ruminants were evaluated using bacterial culture and semi-nested PCR and identification was confirmed by DNA sequencing. Universal primers targeting the bacterial 16S rRNA for bacteria were used. Other sets of primers that were specific for Gram-positive bacteria and combinations of primers specific for Gram-negative organisms were used in the second PCR. The amplified PCR products were subjected to DNA sequence analysis. The DNA sequences of the isolated bacteria were aligned with the DNA sequences of bacteria in the GenBank through BLAST. Results confirmed isolation of three Gram-positive and two Gram-negative organisms. Gene sequence studies demonstrated identification of the Gram-positive microorganisms as Staphylococcus sciuri, Staphylococcus sporosarcinae and Bacillus pumilus while the identified Gram-negative organisms were Acinetobacter schindleri and Pseudomonas aeruginosa from ruminants manifesting clinical signs of respiratory infection.

Article title: Concordance of competitive enzyme linked immunosorbent assay and nested-polymerase chain reaction in the detection of caprine arthritis-encephalitis virus

Authors: Justin Christian V. Gonzales, Clarissa Yvonne J. Domingo, Nancy S. Abes, Charito A. Gutierrez, et al.

Publication title: Small Ruminant Research 115(1-3): 134-139, October 2013

Abstract:

The study detected the presence of caprine arthritis-encephalitis virus (CAEV) in blood samples from 262 goats and compared the results using competitive enzyme linked immunosorbent assay (cELISA) and nested-polymerase chain reaction (nested-PCR) assay. Moreover, it determined the agreement using kappa (κ) statistic, analyze the genetic sequence of CAEV and describe the histopathologic features using carpal joint, brain, lung and mammary gland samples of CAEV positive animals. CAEV antibodies were detected in 15/262 (5.73%) of goat serum samples using cELISA, based on the use of monoclonal antibody binding to CAEV gp135 or SU glycoprotein. In nested-PCR assay targeting the CAE proviral gag region, 9/262 (3.44%) goats were positive which increased the number of positive animals detected to 19 (7.25%). Kappa statistic showed fair agreement between cELISA and nested PCR (κ = 0.39). DNA sequence of PCR product showed 91–98% homology among the reported CAEV genome in the GenBank. Histopathological findings were characterized by varying degrees of mononuclear cell infiltrations that conformed to the typical features of lentivirus infection.

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Article title: Expression analysis of Foxp3 in T cells from bovine leukemia virus infected cattle

Authors: Saori Suzuki, Satoru Konnai, Tomohiro Okagawa, Ryoyo Ikebuchi, et al. 

Publication title: Microbiology and Immunology 57(8): 600-4, August 2013

Abstract:

In the present study, we monitored Foxp3(+) T cells in bovine leukemia virus (BLV)-infected cattle. By flow cytometric analysis, the proportion of Foxp3(+) CD4(+) cells from persistent lymphocytotic cattle was significantly increased compared to control and AL cattle. Interestingly, the proportion of Foxp3(+) CD4(+) cells correlated positively with the increased number of lymphocytes, virus titer and virus load, whereas it inversely correlated with IFN-γ mRNA expression, suggesting that Foxp3(+) CD4(+) T cells in cattle have a potentially immunosuppressive function. Further studies are necessary to elucidate the detailed mechanism behind the increased Treg during BLV infection.

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Article title: Intramammary teat sealant rather induced sub-clinical mastitis in water buffaloes (Bubalus bubalis)

Authors: M. Villamor, N. P. Medina, N. S. Abes, C. Mingala

Publication title: Large Animal Review 19: 195-198, 2013

Abstract:

Introduction - Teat sealing is one preventive tools for mastitis control to prevent the access of the bacteria into the mammary gland. Intramammary teat sealants (ITS) have a high potential as preventive management among animal which do not have history of mastitis. Aim - The general aim of the study was to evaluate the intramammary teat sealant (ITS) for the prevention of subclinical mastitis (SCM). It also aimed to compare the Somatic Cell Count (SCC) and California Mastitis Test (CMT) values of buffaloes infused with ITS device to those buffaloes without ITS, response of primiparous and multiparous buffaloes and determine the adverse reaction based on the changes on teat size and behavior of the treated animals. Materials and methods - Eleven purebreed and healthy female buffaloes were enrolled in the study. Buffaloes were infused with ITS one month before the expected calving date. Milk collection was done every week for one month. Result and discussion - Result showed that there was significant increase on the SCC of buffaloes infused with ITS in comparison to buffaloes without ITS based on SCC and CMT. Higher percentage occurrence of SCM was observed among buffaloes sealed with ITS in comparison to unseal. No significant difference was observed between treated multiparous and primiparous buffaloes. It showed that there was no significant difference on the size of the teat after 4-6 hours and 24 hours post infusion. Changes of behavior upon infusion of ITS were observed. Conclusion - The use of ITS as preventive management in the occurrence of SCM should be carefully employed in lactating buffaloes. Since this product is already available in the market, adjunct use of antiseptic and proper infusion should be observed. The concept of reducing bacterial contaminants thru the teat via teat sealing is a rational one but based on the study it is not an absolute protection to the lactating animals particularly on water buffaloes maybe due to anatomical difference compare to cattle where this ITS is patterned. Employment of proper management such as regular cleaning and disinfection of the pens is still the best way of controlling mastitis in water buffaloes.

Article title: Correlation of California mastitis test and somatic cell count on milk of water buffalo cows in the Philippines

Authors: Roderick T. Salvador, DVSM, MPH, Agnes Alexandria A. Garcia, DVM, Nancy S. Abes, DVM, MS and Claro N. Mingala 

Publication title: Tropical Agriculture 90(3), July 2013

Abstract:

The objective of the study was to determine the correlation of California Mastitis Test (CMT) and somatic cells count (SCC) on milk of Murrah buffalo cows. It aimed to calculate the prevalence of subclinical mastitis (SCM), sensitivity and specificity of CMT using SCC as the basis, determine the predictive values of CMT, calculate the true prevalence of subclinical mastitis based on sensitivity and specificity established, and determine the variations on the correlation of SCC and CMT against several factors: age of animal, parity number and length of lactation. Pearson’s correlation analysis and Kappa statistics were used for statistical analysis. Results showed that Correlation and Kappa statistics had a 23.37% (p <.0001) and 20.20% (p <.0001) respective agreement between the results of SCC and CMT. SCM prevalence were 30.29% and 23.46% based on CMT and SCC, respectively. The CMT has 54.43% sensitivity and 77.10% specificity. The positive predictive value of CMT was 42.11% while the negative predictive value was 84.67%. The calculated true prevalence of the test was 23.33%. Test agreement may change from slight to fair depending on the factors considered such as the age of the animal, parity number and length of lactation.

Article title: In-vivo assessment of the effects of trypanocidal drugs against Trypanosoma evansi isolates from Philippine water buffaloes (Bubalus bubalis)

Authors: Bryan B. Macaraeg, Jonathan V. Lazaro, Nancy S. Abes, and Claro N. Mingala

Publication title: Veterinarski Arhiv 83(4): 381-392, July 2013 

Abstract:

The effects of the trypanocidal drugs against Trypanosoma evansi isolated from Philippine water buffaloes from the three island groups were comparatively evaluated. Specifically, the study determined the duration of efficacy, relapse and death per drug dosage using laboratory mice. A total of 270 inbred Balb/c mice were divided into three groups corresponding to the three trypanosome isolates (Luzon, Visayas, and Mindanao). Each group had three sets corresponding to the three trypanocidal drugs used with five treatment levels and one control group each. Each experimental group was composed of five mice. Each mouse was inoculated with 0.2 ml of T. evansi intraperitoneally and blood was examined under the microscope. Parasitemia level was determined using “Rapid Matching Method”. Effective and curative doses were noted and evaluated through t-test and bio-assay graphical analyses. Results showed that Luzon isolate was sensitive to > 5 mg/kg of diminazene diaceturate and > 10 mg/kg of both isometamidium chloride and quinapyramine sulphate + chloride. The Visayas isolate was sensitive to > 5 mg/kg, > 10 mg/kg, and > 3 mg/kg of diminazene diaceturate, isometamidium chloride and quinapyramine sulphate + chloride, respectively. The Mindanao isolate was sensitive to > 3 mg/kg of diminazene diaceturate and quinapyramine sulphate + chloride and 20 mg/kg of isometamidium chloride. The study suggested diminazene as recommended drug against Luzon isolates, quinapyramine against Visayas isolates and either diminazene or quinapyramine against Mindanao isolates.

Article title: Molecular characterization of Trypanosoma evansi isolates from water buffaloes (Bubalus bubalis) in the Philippines

Authors: Marjo V. Villareal, Claro N. Mingala, Windell L. Rivera

Publication title: Acta Parasitologica 58(1): 6-12, March 2013

Abstract:

Trypanosoma evansi infection in the Philippines is frequently reported to affect the country's livestock, particularly, the buffaloes. To assess the prevalence and intraspecific diversity of T. evansi in the country, blood samples from water buffaloes in different geographical regions were collected during an outbreak. T. evansi was detected in all 79 animals tested using PCR targeting the RoTat 1.2 VSG gene. Sequencing of the rDNA complete internal transcribed spacer (ITS) region including the 5.8S subunit showed high similarity (99-100%) between Philippine isolates and known T. evansi isolates in Genbank. Tree construction based on the same region confirmed the close relationship between Philippine and reported Thai isolates as compared to Egyptian isolates separated by relatively small genetic distances, 47 polymorphisms, despite the clustering in four branches. Overall, the results of this study prove genetic diversity within T. evansi species despite previous reports on limited heterogeneity among isolates worldwide.

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Article title: Isolation, cloning, and pathologic analysis of Trypanosoma evansi field isolates

Authors: Hirohisa Mekata, Satoru Konnai, Claro N. Mingala, Nancy S. Abes, et al.

Publication title: Parasitology Research 112(4): 1513–1521, 2013

Abstract:

In recent years, the emergence of highly pathogenic Trypanosoma evansi strains in the Philippines has resulted in substantial losses in livestock production. In this study, we isolated T. evansi from infected-water buffaloes in the Philippines and analyzed their virulence using mice and cattle. A total of 10 strains of T. evansi were isolated. Evaluation of the virulence of each strain using mice depicted significant differences among the strains in the prepatent period, the level of parasitemia, and the survival time of the infected animals. In mice infected with the highly pathogenic T. evansi, signs of excessive inflammation such as marked splenomegaly and increase more than 6-fold in the number of leukocytes were observed at 8 days post-infection. To study the virulence of the parasite strains in cattle (which are the common T. evansi hosts in Philippines), cattle were infected with the T. evansi isolates that showed high and low virulence in mice. The rate of parasite growth and the length of the prepatent periods were found to be similar to those observed in mice for the respective strains. The cattle infected with the highly pathogenic strain developed anemia and a marked decrease in leukocyte counts. To determine the cause of the pathological changes, we analyzed the expression levels of inflammatory cytokines and observed up-regulation of tumor necrosis factor-α in anemic infected cattle. Our findings suggest that the epidemic of T. evansi in the Philippines is characterized by T. evansi strains with varying virulences from low to very high pathogenicity in cattle.

Article title: Genotyping and Molecular Characterization of NRAMP1/-2 Genes as Location of Markers for Resistance and/or Susceptibility to Mycobacterium bovis in Swamp and Riverine Type Water Buffaloes

Authors: C. N. Mingala, Lawrence P. Belotindos, N. S. Abes, L. Cruz

Publication title: Buffalo Bulletin 32: 730-733, January 2013

Abstract:

Natural resistance-associated macrophage proteins (NRAMPs) have been associated to disease resistance across animal species. It has critical role in innate immunity and influence in adaptive immunity. This study investigated the contribution of NRAMP1 and NRAMP2 gene to the resistance or susceptibility of swamp and riverine buffalo to Mycobacterium bovis infection. Animals were tested for TB by single intradermal tuberculin test (SITT) using Bovine antigen. Reactors to SITT were subjected to comparative intradermal tuberculin test (CITT). Blood samples were collected from the reactors then subjected to DNA extraction to isolate the NRAMP1 and NRAMP2 genes. Isolated NRAMP genes were then examined by single strand conformational polymorphism (SSCP) assay. The 3'UTR were sequenced and then aligned. The SSCP result showed that among the reactor animals to intradermal tuberculin test, four conformational patterns were observed in the 3'UTR of the NRAMP1 gene while two were observed in the 3'UTR of the NRAMP2 gene. SSCP showed that the frequency of four-band pattern were mostly from the reactor animals (66.41%). Sequence alignment clearly established the nucleotide polymorphisms between the conformational patterns. These polymorphisms suggested as a potential markers for resistance or susceptibility to Mycobacterium infection. Allelic patterns will be very useful in future breeding plan for the selection of resistant animals.

Article title: Enhanced expression of LAG-3 on lymphocyte subpopulations from persistently lymphocytotic cattle infected with bovine leukemia virus

Authors: Satoru Konnaia, Saori Suzukia, Tatsuya Shiraia, Ryoyo Ikebuchi, et al.

Publication title: Comparative Immunology, Microbiology and Infectious Diseases 36(1): 63-69, January 2013 

Abstract:

An immunoinhibitory receptor, lymphocyte activation gene-3 (LAG-3), which is mainly expressed in T-cells, is involved in the immune evasion of several pathogens causing chronic infections and tumors. However, unlike human or mouse LAG-3, no functional analysis of LAG-3 has been reported in domestic animals. Thus, in this study, bovine LAG-3 expression was analyzed in bovine leukemia virus (BLV)-infected cattle. In persistent lymphocytotic (PL) cattle, the numbers of LAG-3+CD4+ cells and LAG-3+CD8+ cells were conserved whilst the number of MHC class II+ cells was remarkably higher than in the control animals. In contrast, the mean fluorescence intensity (MFI) for LAG-3 on PBMCs from PL cattle was significantly increased compared to control and asymptomatic (AL) cattle. Specifically, the LAG-3 expression level was significantly increased in both CD4+ and CD8+ T cells from PL cattle. LAG-3 expression correlated positively with increased numbers of lymphocytes and MHC class II+ cells in infected animals. Preliminary results from PD-L1 and LAG-3 blockade assay revealed that IFN-γ and IL-2 expressions were significantly up-regulated by addition of anti- PD-L1 and LAG-3 antibodies in PBMCs from PL cattle. These findings suggest that LAG-3 might be involved in the inhibition of T-cell function through its binding and signaling on MHC class II molecule during BLV infection.

Full text available upon request to the author

Article title: Detection of Enzootic Bovine Leukosis in Cattle usingNested Polymerase Chain Reaction Assay

Authors: Jomelson Alfaro Uera, Jonathan Ventura Lazaro, Claro Niegos Mingala

Publication title: The Thai Veterinary Medicine 42(3): 319-324, September 2012

Abstract:

Enzootic bovine leukosis (EBL) is caused by bovine leukemia virus (BLV) infection. BLV was detected in cattle using nested polymerase chain reaction (PCR) assay and were identified BLV infected cattle farms in five selected provinces in the Philippines. A total of 300 cattle blood samples were used. BLV Proviral DNA was extracted and amplified using nested PCR assay targeting the BLV long terminal repeat (LTR). Results showed that 11 samples (3.67%) of the 300 cattle blood samples used were positive for BLV infection. This study is considered first report of cattle EBL in the Philippines.

Article title: A new loop-mediated isothermal amplification method for rapid, simple, and sensitive detection of Leptospira spp. in urine

Authors: Nobuo Koizumi, Chie Nakajima, Tsunehito Harunari, Tsutomu Tanikawa, et al.

Publication title: Journal of Clinical Microbiology 50(6): 2072–2074, June 2012

Abstract:

We developed a new loop-mediated isothermal amplification (LAMP) method to detect rrs, a 16S rRNA gene of pathogenic Leptospira spp. in urine. The method enables detection of two leptospiral cells per reaction mixture following boiling of urine specimens. The sensitivity of this method is higher than that of culture or of flaB nested PCR.

Article title: Kinetics of regulatory dendritic cells in inflammatory responses during Trypanosoma evansi infection

Authors: H. Mekata, S. Konnai, C. N. Mingala, N. S. Abes, et al.

Publication title: Parasite Immunology 34(6): 318-29, June 2012

Abstract:

Trypanosoma evansi (T. evansi) causes a wasting disease in almost all mammals. Trypanosoma evansi infection gives rise to the inflammatory responses that contribute to the development of inflammation-associated tissue injury. To determine what kinds of inflammatory molecules play roles in the pathogenicity of T. evansi infection, polymerase chain reaction array analysis was performed on samples from the infected and uninfected mice. The inflammatory cytokine and chemokine storm, caused mainly by macrophages, was observed. On the other hand, the expression levels of Ccl8 and Il10 in splenocytes were also markedly increased. These results suggested an augmentation in the number and activity of regulatory dendritic cells (DCs). Therefore, the kinetics of regulatory DCs in T. evansi-infected mice were investigated. During T. evansi infection, the regulatory DCs became prevalent, with reducing the amount of inflammatory DCs. Interestingly, when the regulatory DCs were implanted into T. evansi-infected mice, the survival was prolonged, and the expression levels of inflammatory molecules were suppressed. Taken together, these results showed that a subset of regulatory DCs acted as a potential regulator of the inflammatory responses.

Full text available upon request to the author

Article title: Short communication: Prevalence and risk factors of subclinical mastitis as determined by the California Mastitis Test in water buffaloes (Bubalis bubalis) in Nueva Ecija, Philippines

Authors: R.T. Salvador, J.M.C. Beltran, N.S. Abes, C.A. Gutierrez, et al.

Publication title: Journal of Dairy Science 95(3): 1363-1366, March 2012

Abstract:

A retrospective analysis using records of lactating Bulgarian Murrah buffaloes subjected to the California Mastitis Test in a herd in Nueva Ecija, Philippines was done to determine the prevalence of subclinical mastitis (SCM) and to identify risk factors that may influence its occurrence and recurrence. Results showed that SCM prevalence was 42.76%, whereas its recurrence was 75.03%. Age and lactation length influenced the occurrence of SCM. In contrast to the conclusions for dairy cows, younger buffalo cows were more susceptible compared with those at least 6 yr old. Dams younger than 3 yr have a 76% probability, whereas those age 3 yr have an 82% probability of having SCM.

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Article title: Retrospective study on the treatment of subclinical mastitis in water buffaloes

Authors: N.M. Villanada, N.P. Medina, N.S. Abes, C.N. Mingala

Publication title: Large Animal Review 18: 201-205, 2012

Abstract:

Introduction: Mastitis is one of the most common problems of dairy animals. Subclinical mastitis (SCM) is the most prevalent form of mastitis. It cannot be detected visually but cause great economic loss due to degrading of milk quality and price caused by high bacterial or somatic cell count, costs of drugs, veterinary services and increased labor costs, increased risk of subsequent mastitis, herd replacement, and problems related to antibiotic residues in milk and its products Aim: The present study was conducted to evaluate the response of the dairy buffalo on SCM treatment. Specifically, to determined the prevalence rate, recurrence rate and possible risk factors of SCM in dairy water buffaloes and to assess the efficacy of treatment. Materials and methods: A total of 67 treated animals were subjected for the analysis. The prevalence rate of SCM in a 19-month period, including cure rate, recurrence rate and identification of risk factors that can affect the probability of cure was determined. All information that was obtained was cleaned in a prepared collection sheet. Univariate analysis on the possible association between the reoccurrence of SCM and independent variables (risk factors) was examined using Logistic regression. Odds ratios (OR) were computed to determine the strength of association of different factors to treatment of SCM. Results and discussion: The over-all cure of treatment was 65.67% and the recurrence of cured animals was 76.92%. Cloxacillin benzathine achieved the highest efficacy with 47.56% followed by cephapirin benzathine with 16.42% and procaine pen G in combination with dihydrostreptomycin with 1.49%. Among the risk factors only the number of affected quarters was found with significant association (P= 0.008). It showed that as the number of affected quarters increases the probability of cure decreases (CR= -1.13). The over-all prevalence of subclinical mastitis in a 19 month period was 37%. Conclusion: Aside from good management and other control measures, the treatment intervention contributed to the decrease in prevalence of infection. However, treatment alone cannot guarantee that the animal will be cured because of the complexity of the disease condition. Many factors can hinder the success of treatment such as the treatment options, pathogen involved like staphylococcus which is difficult to treat and the animal itself. To further decrease the prevalence of mastitis, other methods of prevention or control should be done such as the use of teat sealant during the dry period. Recurrence was high and should be investigated to identify the causes. Significant factors for the recurrence of the condition should also be explored. Although various factors have been recorded as risk factors for cure of SCM, only the number of affected quarters of lactating mammary gland was significantly associated with the probability of cure and this must be considered in treatment decision for cost effectiveness consideration.

Article title: Comparative virulence of three Trypanosoma evansi isolates from water buffaloes in the Philippines

Authors: John Christian M. Verdillo, Jonathan V. Lazaro, Nancy S. Abes, Claro N. Mingala

Publication title: Experimental Parasitology 130(2): 130-4, February 2012

Abstract:

The virulence of three Trypanosoma evansi isolates in Luzon, Visayas and Mindanao water buffaloes was compared determining the mortality rate, parasitemia level, clinical signs, and lesions on mice. A total of 51 inbred Balb/c mice (5-6 weeks old) were used and divided into two sets. Set A had three groups corresponding to three trypanosomes isolates (Luzon, Visayas, and Mindanao) with seven mice each whose parasitemia level, clinical signs, and lesions were noted at necropsy. Set B had three groups corresponding to the three isolates with ten mice each whose mortality was monitored. Each infected mouse was inoculated with 0.2 ml of T. evansi intraperitoneally and blood was examined under high power magnification. Their parasitemia level was determined using "Rapid Matching Method". Dead mice were subjected to necropsy and the lungs, liver, spleen, brain and heart were subjected to histopathological processing. Results showed that the mortality rate was highest at Day 3 for the Visayas isolates (70%), while at Day 5 for Luzon (90%) and Mindanao (70%) isolates. The parasitemia level of Visayas isolates (1×10(8.7)) reached the earliest peak at Day 4 while Luzon isolates (1×10(9)) at Day 6 and Mindanao isolates (1×10(8.7)) at Day 8. Statistical analysis using Least significant difference (LSD) revealed significant difference among treatment means at Days 2 and 4. All of the affected mice showed rough hair coat, decreased body weight, and decreased packed cell volume. The most obvious gross lesions observed were pale liver with petechiations and pale muscles. Histopathological examination revealed depletion of the red pulp and extramedullary hematopoiesis in the spleen. Congestion, intralesional trypanosomes in blood vessel and extramedullary hematopoiesis were observed in the liver. In the lungs non-specific lesions observed were pulmonary edema, congestion and hemosiderosis.

Full text available upon request to the author

Article title: Genetic analysis and development of species-specific PCR assays based on ITS-1 region of rRNA in bovine Eimeria parasites

Authors: Fumiya Kawahara, Guohong Zhang, Claro N. Mingala, Yu Tamura, et al.

Publication title: Veterinary Parasitology 174(1-2): 49-57, November 2010

Abstract:

At present, morphological characteristics of oocyst is the only achievable method for the identification of bovine coccidia to the species level. In this study, the internal transcribed spacer 1 (ITS-1) region of ribosomal RNA genes of six bovine Eimeria species; E. alabamensis, E. auburnensis, E. bovis, E. cylindrica, E. ellipsoidalis and E. zuernii, were sequenced and analyzed the phylogenetic relationship among them. In pair-wise alignment, the sequences among the same species had high homology of over 90%. E. bovis and E. zuernii were closely related within the same cluster. This cluster and E. alabamensis were distant from major cluster of bovine coccidia that included E. auburnensis, E. cylindrica and E. ellipsoidalis. Species-specific PCR assays based on the amplification of the ITS-1 region were also developed to identify the 6 pathogens. The ITS-1 region of each Eimeria species had sufficient inter-specific sequence variation enough to design the primer sets that differentially amplified each target species. This PCR assay for the detection and differentiation of Eimeria parasite showed higher sensitivity when compared to the conventional oocyst-morphological examination. This is the first attempt for the identification of 6 bovine Eimeria parasites in the genomic level and may provide as useful methods for diagnosis and epidemiology of bovine coccidial infection.

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