The study was carried out to investigate two important foodborne pathogens, Salmonella and Listeria spp. in five organic farms in Uttarakhand state in the year 2018. The samples from soil, manure, water, and plants/plant parts were collected and screened for pathogens. The covered isolates were assayed for their antimicrobial susceptibility and the presence of representative beta-lactamase antibiotic-resistant gene (ARG) by PCR. A total of 2.2% (11/500) of samples tested positive for the genus Salmonella. However, none of the samples tested positive for Listeria spp. All the Salmonella isolates were recovered from environmental sources. On serotyping, 4 isolates were identified as serogroup Group C1, 3 as Salmonella Miyazaki, 2 as Salmonella Virchow and 1 each as Salmonella Infantis and Salmonella Gabon. Two Salmonella isolates, belonging to S. Group C1 and one S. Miyazaki isolate were pan-susceptible to all the antibiotics tested, while S. Virchow and S. Infantis showed multidrug-resistant. On PCR screening for three β-lactamase resistant genes (blaCTX-M-9, blaTEM, and blaAmpC), only three S. Miyazaki isolates amplified the blaCTX-M-9 gene. The study highlights the presence of MDR Salmonella in the organic farm environment warranting further studies in this direction to ensure safe organic farm produce for consumers.

The objective of this study was to compare rotavirus shedding and performance of piglets from gilts immunized using natural planned exposure (NPE) or an RNA particle vaccine (RPV) prior to farrowing following initial NPE pre-breeding. A final total of 117 farrowed gilts and their piglets were enrolled into 4 groups. All gilts received two administrations of NPE prior to breeding. Gilts in group 1 were later given three NPE administrations at 5, 4, and 3 weeks prior to farrowing (WPF). Group 2 was dosed with an RNA particle vaccine (RPV) at 5 and 3 WPF and group 3 at 1 WPF only. Group 4 (control group) did not receive any NPE or RPV. Fecal samples from gilts and fecal swabs from their piglets were tested for rotavirus A (RVA), rotavirus B (RVB), and rotavirus C (RVC) by qRT-PCR. The 117 gilt samples were tested individually at 5 sampling points from pre-breeding to entry into the farrowing rooms. Piglet samples were pooled by litter (3 piglets sampled per litter) and tested at 3, 7, 14, and 21 days of age. The clinical and production impact of the treatments were assessed by comparing average adjusted weaning weights, the percentage of piglets weighing in the bottom 10% of study piglets at weaning, the percentage of piglets placed in the nursery, and the percentage of litters with the presence of diarrhea. No statistically significant differences were identified but there appeared to be several interesting numerical trends that are both biologically plausible and consistent among treatment groups. The control group, which received no pre-farrow immunization, had the highest percentage of litters with diarrhea at all time points and overall in the farrowing house. A numerical trend was also observed on average cycle threshold (Ct) values for RVC in piglets. Interestingly, the control group had the lowest numerical average Ct value at 7, 14, and 21 days of age. Ct values for RVA were consistently lower than for RVC in the stock solution and natural planned exposure gruel. This likely correlated to a lower level of RVC exposure in all gilts pre-breeding and group 1 gilts pre-farrow. This may have an inverse correlation to the higher levels of shedding of RVC in piglets. The lack of statistical significance between treatment groups with regard to diarrhea and weights in the piglets may be attributed to sample size, lack of sufficient natural rotavirus challenge, pre-breeding NPE in all gilts, or a variety of other reasons. Replication of this study with a larger sample size, using a challenge model, or relocated to a farm with increased natural rotavirus clinical challenges may yield different results. If beneficial under different conditions, the RPV would eliminate many of the risks associated with NPE administration, including continuous introduction of live virus on-farm, potential spread of other pathogens, difficulty of isolating on-farm strains, and labor and cost of producing NPE material. Limitations of the original research include the use of quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) to measure the levels of RVA and RVC in the stock solution and gruel and to monitor RV shedding, rather than techniques with the ability to differentiate live and non-viable virus. Additionally, these studies were performed on an all-gilt breeding farm. It may be more challenging to appreciate differences in treatment groups on a multiparous sow farm due to the higher level of immunity correlated to increasing parity. These immunization protocols were designed to be applicable in a field setting for routine rotavirus immunization, though their feasibility must be carefully considered before implementation on each individual farm.

Natural planned exposure (NPE) remains one of the most common methods in swine herds to boost lactogenic immunity against rotaviruses. However, the efficacy of NPE protocols in generating lactogenic immunity has not been investigated before. A longitudinal study was conducted to investigate the dynamics of genotype-specific antibody responses to different doses (3, 2 and 1) of Rotavirus A (RVA) NPE (genotypes G4, G5, P[7] and P[23]) in gilts and the transfer of lactogenic immunity to their piglets. Group 1 gilts received three doses of NPE at 5, 4 and 3 weeks pre-farrow (WPF), group 2 received two doses at 5 and 3 WPF, group 3 received one dose at 5 WPF, and group 4 received no NPE (control group). VP7 (G4 and G5) and truncated VP4* (P[7] and P[23]) antigens of RVA were expressed in mammalian and bacterial expression systems, respectively, and used to optimize indirect ELISAs to determine antibody levels against RVA in gilts and piglets. In day-0 colostrum samples, group 1 had significantly higher IgG titers compared to the control group for all four antigens, and either significantly or numerically higher IgG titers than groups 2 and 3. Group 1 also had significantly higher colostrum IgA levels than the control group for all antigens (except G4), and either significantly or numerically higher IgA levels compared to groups 2 and 3. In piglet serum, group 1 piglets had higher IgG titers for all four antigens at day 0 than the other groups. Importantly, RVA NPE stimulated antibodies in all groups regardless of the treatment doses and prevented G4, G5, P[7] and P[23] RVA fecal shedding prior to weaning in piglets in the absence of viral challenge. The G11 and P[34] RVA genotypes detected from pre-weaning piglets differed at multiple amino acid positions with parent NPE strains. In conclusion, the results of this study suggest that the group 1 NPE regimen (three doses of NPE) resulted in the highest anti-RVA antibody (IgG and IgA) levels in the colostrum/milk, and the highest IgG levels in piglet serum.

Introduction:African swine fever virus (ASFV) is a pathogen of great economic importance given that continues to threaten the pork industry worldwide, but there is no safe vaccine or treatment available. Development of a vaccine is feasible as immunization of pigs with some live attenuated ASFV vaccine candidates can confer protection, but safety concerns and virus scalability are challenges that must to be addressed. Identification of protective ASFV antigens is needed to inform the development of efficacious subunit vaccines.Methods:In this study, replication-incompetent adenovirus-vectored multicistronic ASFV antigen expression constructs that covered nearly 100% of the ASFV proteome were generated and validated using ASFV convalescent serum. Swine were immunized with a cocktail of the expression constructs, designated Ad5-ASFV, alone or formulated with either Montanide ISA-201™ (ASFV-ISA-201) or BioMize® adjuvant (ASFV-BioMize).Results:These constructs primed strong B cell responses as judged by anti-pp62-specific IgG responses. Notably, the Ad5-ASFV and the Ad5-ASFV ISA-201, but not the Ad5-ASFV BioMize®, immunogens primed significantly (p < 0.0001) higher anti-pp62-specific IgG responses compared with Ad5-Luciferase formulated with Montanide ISA-201™ adjuvant (Luc-ISA-201). The anti-pp62-specific IgG responses underwent significant (p < 0.0001) recall in all the vaccinees after boosting and the induced antibodies strongly recognized ASFV (Georgia 2007/1)-infected primary swine cells. However, following challenge by contact spreaders, only one pig nearly immunized with the Ad5-ASFV cocktail survived. The survivor had no typical clinical symptoms, but had viral loads and lesions consistent with chronic ASF.Discussion:Besides the limited sample size used, the outcome suggests that in vivo antigen expression, but not the antigen content, might be the limitation of this immunization approach as the replication-incompetent adenovirus does not amplify in vivo to effectively prime and expand protective immunity or directly mimic the gene transcription mechanisms of attenuated ASFV. Addressing the in vivo antigen delivery limitations may yield promising outcomes.

Rotaviruses (RVs) are endemic in swine populations, and all swine herds certainly have a history of RV infection and circulation. Rotavirus A (RVA) and C (RVC) are the most common among all RV species reported in swine. RVA was considered most prevalent and pathogenic in swine; however, RVC has been emerging as a significant cause of enteritis in newborn piglets. RV eradication from swine herds is not practically achievable, hence producers’ mainly focus on minimizing the production impact of RV infections by reducing mortality and diarrhea. Since no intra-uterine passage of immunoglobulins occur in swine during gestation, newborn piglets are highly susceptible to RV infection at birth. Boosting lactogenic immunity in gilts by using vaccines and natural planned exposure (NPE) is currently the only way to prevent RV infections in piglets. RVs are highly diverse and multiple RV species have been reported from swine, which also contributes to the difficulties in preventing RV diarrhea in swine herds. Human RV-gut microbiome studies support a link between microbiome composition and oral RV immunogenicity. Such information is completely lacking for RVs in swine. It is not known how RV infection affects the functionality or structure of gut microbiome in swine. In this review, we provide a detailed overview of genotypic diversity of swine RVs, host-ranges, innate and adaptive immune responses to RVs, homotypic and heterotypic immunity to RVs, current methods used for RV management in swine herds, role of maternal immunity in piglet protection, and prospects of investigating swine gut microbiota in providing immunity against rotaviruses.

A longitudinal study was conducted to investigate the dynamics of genotype-specific (G6 and P[5]) antibody response to different doses (3, 2 and 1) of rotavirus C (RVC) natural planned exposure (NPE) in gilt serum, colostrum/milk and piglet serum, and compare with antibody response to rotavirus A NPE (RVA genotypes G4, G5, P[7] and P[23]). G6 and P[5] antigens of RVC were expressed in mammalian and bacterial cells, and used to develop individual indirect ELISAs. For both antigens, group 1 with 3 doses of NPE resulted in significantly higher IgG and IgA levels in colostrum compared to other groups. In piglet serum, group 1 P[5] IgG levels were significantly higher than other study groups at day 0 and 7. Piglet serum had higher IgA levels for group 1 piglets compared to other groups for both antigens. A comparison of colostrum antibody levels to rotavirus A (RVA) and RVC revealed that colostrum RVC IgG and IgA titers were lower than RVA titers irrespective of the G and P-type. Next generation sequencing (NGS) detected same RVC genotypes (G6 and P[5]) circulating in the piglet population under the window of lactogenic immunity. We conclude that the low RVC load in NPE material (real-time PCR Ct-values 32.55, 29.32 and 30.30) failed to induce sufficient maternal immunity in gilts (low colostrum RVC antibody levels) and passively prevent piglets from natural RVC infection in the farrowing room. To the best of our knowledge, this is the first study comparing differences in antibody response to porcine RVA and RVC in a commercial setting.

The complement cascade consists of cell bound and serum proteins acting together to protect the host from pathogens, remove cancerous cells and effectively links innate and adaptive immune responses. Despite its usefulness in microbial neutralization and clearance of cancerous cells, excessive complement activation causes an immune imbalance and tissue damage in the host. Hence, a series of complement regulatory proteins present at a higher concentration in blood plasma and on cell surfaces tightly regulate the cascade. The complement cascade can be initiated by B-1 B cell production of natural antibodies. Natural antibodies arise spontaneously without any known exogenous antigenic or microbial stimulus and protect against invading pathogens, clear apoptotic cells, provide tissue homeostasis, and modulate adaptive immune functions. Natural IgM antibodies recognize microbial and cancer antigens and serve as an activator of complement mediated lysis. This review will discuss advances in complement activation and regulation in bacterial and viral infections, and cancer. We will also explore the crosstalk of natural antibodies with bacterial populations and cancer.

The genus Rotavirus comprises eight species, designated A to H, and two recently identified tentative species I in dogs and J in bats. Species Rotavirus A, B, C and H (RVA, RVB, RVC and RVH) have been detected in humans and animals. While human and animal RVA are well characterized and defined, complete porcine genome sequences in the GenBank are limited compared to human strains. Here, we used a metagenomic approach to sequence the 11 segments of RVA, RVC and RVH strains from piglets in the United States (US) and explore the evolutionary relations of these RV species. Metagenomics identified Astroviridae, Picornaviridae, Caliciviridae, Coronoviridae in samples MN9.65 and OK5.68 while Picobirnaviridae and Arteriviridae were only identified in sample OK5.68. Whole genome sequencing and phylogenetic analyses identified multiple genotypes with the RVA of strain MN9.65 and OK5.68, with the genome constellation of G5/G9-P[7]/P[13]-I5/I5- R1/R1-C1-M1-A8-N1-T7-E1/E1-H1 and G5/G9-P[6]/P[7]-I5-R1/R1-C1-M1-A8-N1-T1/T7-E1/E1-H1, respectively. The RVA strains had a complex evolutionary relationship with other mammalian strains. The RVC strain OK5.68 had a genome constellation of G9-P[6]-I1-R1-C5-M6-A5-N1-T1-E1-H1, and shared an evolutionary relationship with porcine strains from the US. The RVH strains MN9.65 and OK5.68 had the genome constellation of G5-P1-I1-R1-C1-M1-A5-N1-T1-E4-H1 and G5-P1-I1-R1-C1-M1-A5-N1-T1-E1-H1, indicating multiple RVH genome constellations are circulating in the US. These findings allow us to understand the complexity of the enteric virome, develop improved screening methods for RVC and RVH strains, facilitate expanded rotavirus surveillance in pigs, and increase our understanding of the origin and evolution of rotavirus species.

A distinct Russian Mammalian orthorubulavirus 5 (PIV5) was detected in cell culture exhibiting cytopathic effect and hypothesized to be contaminated by a scientist with respiratory symptoms. The identification of the divergent strain indicated a lack of knowledge on the diversity of PIV5 strains and calls for surveillance of global PIV5 strains.

Arcobacter genus is an emerging pathogen, with three main spp. are pathogenic (A. butzleri, A. skirrowii and A. cryaerophilus) in nature. In present study raw sewage water, chicken, mutton and fish market associated environmental samples were screened for presence of Arcobacter spp. from Bareilly city of India. Out of total 170 samples, 50 from sewage water, 40 each of meat shops (chicken, mutton, fish) associated environment such as swab samples from knife (10), chopper (10), wooden block (10) and utensils (10) from each meat shop respectively. 26/170 (15%) samples were positive for Arcobacter spp. by multiplex PCR detection and culture positive samples were 18/170 (10.58%). The 8/50 from sewage water, 8/40 from chicken shop, 4/40 mutton shop and 6/40 from fish market associated environment detected positive by m-PCR. Out of total 170 samples screened 18 were culture positive with isolation rate 6/50 (12%) from sewage water, 5/40 (12.5%) from chicken shop, 3/40 (7.5%) from mutton shop, 4/40 (10%) from fish market associated environments. Out of 18 Culture positive samples Arcobacter butzleri was predominant spp. isolated, than A. skirrowii and A. cryaerophilus and mix infection. The 18 positive strains were selected for disk diffusion, tested against 12 different types of antibiotic discs. High antibiotic resistance were seen as in cephalothin (17/18), co-trimoxazle (16/18), ampicillin (14/18), Vancomycin (14/18), tetracycline (14/18), kanamycin (12/18), Azitromycin (11/18) and other antibiotics shows greater sensitivity to Arcobacter butzleri with resistance pattern as 0/18 by nalidixic acid, Erytromycin (4/18), Ciprofloxacin (4/18), Gentamicin (5/18) and streptomycin (6/18). Multi drug resistance (MDR) was seen in this the study. Which may concerned to treatment failure in human and animal concerned to Arcobacter MDR strains. Sewage water may acts as reservoir of Arcobacter spp. as human, animal excreta are major part in and associated water. Most of the sewage water is recycled by municipal corporation. Hence water processing plant, purification methods must aware of probable public health risk related to sewage water. More cautions are recommended for personnel hygiene in processing, handling and awareness related to meat and meat products. The unnecessary use or misuse of antibiotics should be limited or avoided for public health safety.

The objective of this study was to determine the prevalence of antibiotic resistant nontyphoidal Salmonella in 39 retail chicken meat shops located in 7 towns of North India. Overall, nontyphoidal Salmonella prevalence of 9.43% (70/742) was observed. Highest Salmonella prevalence was observed in Lalkuan (20.99%). Chicken meat samples (40%) showed highest Salmonella prevalence followed by poultry feces (20%). Three serotypes of Salmonella were identified, Salmonella Kentucky (S. Kentucky, 74.29%), Salmonella Virchow (S.Virchow, 17.14%) and Salmonella Typhimurium (S.Typhimurium, 7.14%). All isolates were multidrug resistant (MDR) by disk-diffusion assay, showing resistance to three or more classes of antibiotics. High resistance was observed to tetracycline (100%), erythromycin (100%), nalidixic acid (98.57%), ampicillin (95.71%) and ciprofloxacin (82.86%). Fifty-one (98.08%) S. Kentucky isolates were resistant to ciprofloxacin. Of these, 49 S. Kentucky isolates showed MIC values in the range of 3 to > 256 mu g/ml. Twenty-nine (41.43%) Salmonella isolates were co-resistant to ciprofloxacin and cefotaxime by disk-diffusion test. The tetA gene was detected in all Salmonella isolates and the blaTEM was the most predominant (25.37%) beta-lactam gene. Multiple virulence genes such as sipA, mgtC, sopE1, stn, sopB, fliC, spvC and gipA were also detected. High resistance to quinolone, cephalosporin and tetracycline antibiotics in nontyphoidal Salmonella isolates of poultry origin points towards their extra-label use in poultry farms.

Viruses belonging to the genus Bocaparvovirus (BoV) are a genetically diverse group of DNA viruses known to cause respiratory, enteric, and neurological diseases in animals, including humans. An intestinal sample from an alpaca (Vicugna pacos) herd with reoccurring diarrhea and respiratory disease was submitted for next-generation sequencing, revealing the presence of a BoV strain. The alpaca BoV strain (AlBoV) had a 58.58% whole genome nucleotide percent identity to a camel BoV from Dubai, belonging to a tentative ungulate BoV 8 species (UBoV8). Recombination events were lacking with other UBoV strains. The AlBoV genome was comprised of the NS1, NP1, and VP1 proteins. The NS1 protein had the highest amino acid percent identity range (57.89-67.85%) to the members of UBoV8, which was below the 85% cut-off set by the International Committee on Taxonomy of Viruses. The low NS1 amino acid identity suggests that AlBoV is a tentative new species. The whole genome, NS1, NP1, and VP1 phylogenetic trees illustrated distinct branching of AlBoV, sharing a common ancestor with UBoV8. Walker loop and Phospholipase A2 (PLA2) motifs that are vital for virus infectivity were identified in NS1 and VP1 proteins, respectively. Our study reports a novel BoV strain in an alpaca intestinal sample and highlights the need for additional BoV research.

The philosophy of One Health is growing in concept and clarity. The interdependence of human, animal, and environmental health is the basis for the concept of One Health. One Health is a comprehensive approach to ensure the health of people, animals, and the environment through collaborative efforts. Preharvest food safety issues align with the grand concept of One Health. Imagine any food production system, and immediately, parallel images from One Health emerge: for example, transmission of zoonotic diseases, antibiotic residues, or resistance genes in the environment; environmental and animal host reservoirs of disease; challenges with rearing animals and growing fresh produce on the same farm; application and transport of manure or diseased animals. During a recent celebration of #OneHealthDay, information was shared around the globe concerning scientists dedicated to One Health research systems. An ever-growing trade and global commerce system mixed with our incessant desire for food products during the whole year makes it all the more important to take a global view through the One Health lens to solve these growing challenges. The recent explosion of Zika virus around the globe renewed the need for assessing transmissible diseases through the eyes of One Health. It is not good enough to know how a disease affects the human population without a thorough understanding of the environment and vector reservoirs. If 60 to 75% of infectious diseases affecting humans are of animal origin, the need for better One Health research strategies and overdue solutions is imperative.

Preharvest food safety research and activities have advanced over time with the recognition of the importance and complicated nature of the preharvest phase of food production. In developed nations, implementation of preharvest food safety procedures along with strict monitoring and containment at various postharvest stages such as slaughter, processing, storage, and distribution have remarkably reduced the burden of foodborne pathogens in humans. Early detection and adequate surveillance of pathogens at the preharvest stage is of the utmost importance to ensure a safe meat supply. There is an urgent need to develop rapid, cost-effective, and point-of-care diagnostics which could be used at the preharvest stage and would complement postmortem and other quality checks performed at the postharvest stage. With newer methods and technologies, more efforts need to be directed toward developing rapid, sensitive, and specific methods for detection or screening of foodborne pathogens at the preharvest stage. In this review, we will discuss the molecular methods available for detection and molecular typing of bacterial foodborne pathogens at the farm. Such methods include conventional techniques such as endpoint PCR, real-time PCR, DNA microarray, and more advanced techniques such as matrix-assisted layer desorption ionization-time of flight mass spectrometry and whole-genome sequencing.

The SA44 isolate of Rotavirus A (RVA) was identified from a neonatal Peruvian alpaca presenting with diarrhea, and the full-length genome sequence of the isolate (designated RVA/Alpaca-tc/PER/SA44/2014/G3P[40]) was determined. Phylogenetic analyses showed that the isolate possessed the genotype constellation G3-P[40]-I8-R3-C3-M3-A9-N3-T3-E3-H6, which differs considerably from those of RVA strains isolated from other species of the order Artiodactyla. Overall, the genetic constellation of the SA44 strain was quite similar to those of RVA strains isolated from a bat in Asia (MSLH14 and MYAS33). Nonetheless, phylogenetic analyses of each genome segment identified a distinct combination of genes. Several sequences were closely related to corresponding gene sequences in RVA strains from other species, including human (VP1, VP2, NSP1, and NSP2), simian (VP3 and NSP5), bat (VP6 and NSP4), and equine (NSP3). The VP7 gene sequence was closely related to RVA strains from a Peruvian alpaca (K’ayra/3368-10; 99.0% nucleotide and 99.7% amino acid identity) and from humans (RCH272; 95% nucleotide and 99.0% amino acid identity). The nucleotide sequence of the VP4 gene was distantly related to other VP4 sequences and was designated as the reference strain for the new P[40] genotype. This unique genetic makeup suggests that the SA44 strain emerged from multiple reassortment events between bat-, equine-, and human-like RVA strains.

The deposition of manure originating from food animal farms in the environment can lead to the dissemination of antimicrobial-resistant (AMR) bacterial foodborne pathogens, thereby potentially impacting human health. The objective of our study was to determine the dissemination of multidrug methicillin-resistant Staphylococcus sciuri (MDR-MRSS) in the environment after land application of manure on commercial swine farms. A total of 400 environmental samples (40 manure and 360 soil) were collected after repeated sampling from four commercial swine farms located in North Carolina (n=1) and Iowa (n=3) in the United States. At each farm, we collected 10 manure and 40 soil samples (20 samples before and after 2h of manure application) from four plots (five soil samples/plot) on day 0. Subsequently, 20 soil samples were collected on day 7, 14, and 21 from the same plots. A total of 67 (16.75%) MRSS were isolated from the 400 samples. The prevalence in soil and manure was 13.33% (48/360) and 47.5% (19/40), respectively. Prevalence was highest in the soil samples collected after 2h of manure application on day 0 and decreased subsequently on 7, 14, and 21 days. Antimicrobial susceptibility testing was done against a panel of 12 antibiotics. A majority of S. sciuri isolates exhibited resistance against ampicillin (AMP; 95.5%), penicillin (PEN; 95.5%), clindamycin (CLI; 95.5%), cefoxitin (FOX; 92.5%), ceftiofur (XNL; 92.5%), tetracycline (TET; 86.56%), and erythromycin (ERY; 50.74%). The MDR pattern AMP FOX CLI PEN TET XNL (n=24; 35.8%) was the most commonly observed. We detected multiple AMR genes, including mecA, aac(6), Ie-aph(2)Ia, tetM, tetK, mphC, ermA, ermB, and ermC. Pulsed-field gel electrophoresis clustered isolates from different sample collection days from the same farm into one group. Overall, our study identifies swine manure as an important reservoir of MDR-MRSS and highlights its dissemination in the environment upon spreading of manure.

Telecommunication technologies, such as smartphones and digital cameras, are revolutionising both animal and human healthcare through the use of telehealth. This is particularly useful in developing countries, where there may be a lack of healthcare infrastructures and an increased risk from zoonotic diseases. In this article, Anubha Pathak and Deepak Kumar discuss how telemedicine is being used in zoonotic disease research in India.

Salmonellosis is one of the most frequently reported food-borne diseases world-wide commonly caused by Salmonella Typhimurium and Salmonella Enteritidis serovars. The present study was undertaken to confirm Salmonella isolates by various techniques. All the 94 isolates were confirmed to be S. Typhimurium through various morphological, biochemical, serological and molecular methods.

Authentication of meat assumes significance in view of religious, quality assurance, food safety, public health, conservation and legal concerns. Here, we describe a PCR-RFLP (Polymerase Chain Reaction- Restriction Fragment Length Polymorphism) assay targeting mitochondrial cytochrome-b gene for the identification of meats of five most common food animals namely cattle, buffalo, goat, sheep and pig. A pair of forward and reverse primers (VPH-F & VPH-R) amplifying a conserved region (168-776 bp) of mitochondrial cytochrome-b (cytb) gene for targeted species was designed which yielded a 609 bp PCR amplicon. Further, restriction enzyme digestion of the amplicons with Alu1 and Taq1 restriction enzymes resulted in a distinctive digestion pattern that was able to discriminate each species. The repeatability of the PCR-RFLP assay was validated ten times with consistent results observed. The developed assay can be used in routine diagnostic laboratories to differentiate the meats of closely related domestic livestock species namely cattle from buffalo and sheep from goat.