The study was carried out for the cultural and molecular diagnosis of caprine mycoplasmosis in Kerala, India and for the development of an antigen detection latex agglutination test (LAT) for its field diagnosis. A total of 126 samples, comprising 100 nasal swabs and 26 lung tissues, were subjected to isolation and polymerase chain reaction (PCR). The PCR assays included Mycoplasma 16S rRNA genus specific PCR, as well as species and cluster specific PCRs targeting the Mycoides cluster, Mycoides group, Mycoplasma mycoides subsp. capri (Mmc), M. capricolum subsp. capripneumoniae (Mccp), M. capricolum subsp. capricolum (Mcc), M. agalactiae, M. ovipneumoniae, M. conjunctivae, and M. arginini. Additionally, a Mmc strain was characterized by whole genome sequencing (WGS) and the same was used for the development of LAT by sensitizing latex beads with hyperimmune serum raised against Mmc for antigen detection. On Mycoplasma 16S rRNA genus specific PCR, 69 samples (64 nasal swabs and five lung tissue) turned positive. Of these, two lung tissues were positive for Mycoides cluster, Mycoides group, and Mycoplasma mycoides subsp. capri (Mmc) specific PCR. On species specific PCRs, 17 nasal swabs were positive for M. agalactiae, 38 for M. ovipneumoniae, and 8 for M. conjunctivae. The two lung tissue samples, which produced typical colonies on modified Hayflick medium, were confirmed as Mmc using species specific PCR. The representative Mmc strain, Ker.TCR. LT used for developing LAT had a genome size of 1,211,756 bp with 945 protein coding genes and is closely related to a goat derived strain, IVB-X as indicated by core genome phylogenetic analysis. Of the 50 nasal swab samples collected from goats with respiratory tract infections for validation of LAT, 17 were positive by PCR and 14 by LAT. In comparison to PCR, the LAT had a sensitivity of 87.5 percent and specificity of 100 percent. The latex agglutination test is a simple, rapid and reliable screening test under field conditions.

Infectious bronchitis (IB), caused by the Infectious bronchitis virus (IBV), remains a formidable challenge to the poultry industry worldwide, owing to its multi-systemic nature and the continual emergence of diverse viral variants that undermine vaccine efficacy. Despite widespread vaccination efforts, controlling IBV in regions like Kerala is particularly challenging due to inconsistent vaccination practices and the documented presence of the disease. This study set out to characterize IBV isolates circulating in Kerala, with a focus on assessing their genetic diversity and pathological impact. Field samples (n = 80) were screened using reverse transcriptase polymerase chain reaction (RT-PCR) targeting the hypervariable region 3 of the S1 subunit, leading to the detection of IBV in 32 samples. Virus isolation was successfully achieved from these positive samples using embryonated eggs, with subsequent confirmation via RT-PCR using the same set of primers. From the confirmed positive samples, we amplified the full S1 subunit of the S gene as well as the M and N genes, and the resulting products were sequenced. Comparative analyses were conducted against the widely used vaccine strain H120 and other Indian IBV isolates deposited in GenBank. Phylogenetic analysis indicated that the Kerala isolates clustered together with other isolates from different parts of India. Histopathological examination of key organs revealed characteristic lesions, offering insights into the virus’s tissue tropism. The findings underscore the significant genetic variability among IBV strains in Kerala and highlight the critical need for region-specific vaccination strategies to achieve effective disease management.

Avian gastric yeast (Macrorhabdus ornithogaster) is an anamorphic ascomycetous yeast which colonises the junction between proventriculus and ventriculus in many species of birds.Although a normal flora, sometimes it causes a subclinical infection and at times lead to gastric infections.The present study confirmed the presence of Macrorhabdus ornithogaster from faecal samples of budgerigar with symptoms like regurgitation and chronic wasting.Gram's staining of the direct smear demonstrates large number of pleomorphic Gram positive organisms.The organism could be cultured on blood agar, Sabouraud's dextrose agar and De Man Rogosa and Sharpe agar but not on MacConkey agar.The organisms were found to be negative for catalase and oxidase and did not reduce nitrate.It produced acid from maltose, sucrose, glucose, galactose and trehalose and not from raffinose and lactose.The cultural and biochemical characters of the isolate along with clinical symptoms confirmed the case as avian gastric yeast infection.

A preliminary study was conducted for the detection of the infectious bronchitis virus circulating among the poultry flocks of Kerala employing a reverse transcriptase polymerase chain reaction, where, a high prevalence of the virus was reported. The present study is the first comprehensive study pertaining to the molecular characterization of hypervariable regions of the S1 gene of IBV in Kerala. The positive samples were sequenced with primers targeting the three hypervariable regions of the S1 subunit of spike gene. It was observed that the isolates of Kerala shared more than 95% similarity with isolates from different parts of India. On phylogenetic analysis of hypervariable regions 1 and 2, all the Kerala isolates clustered with isolates from other parts of the country. Phylogenetic analysis of the hypervariable region 3 revealed that the Kerala isolates branched separately from the isolates belonging to different parts of India. The study advocated the need of an area specific vaccine in Kerala, employing the potential candidate vaccine strain isolated in the study.

Chickens with signs of respiratory infection were screened for the presence of infectious bronchitis virus (IBV). Tissue samples were collected from dead and ailing chickens and were propagated intra-allantoically in embryonated chicken eggs and the allantoic fluid was harvested. The tissue samples and harvested allantoic fluid were probed with reverse transcriptase polymerase chain reaction (RT-PCR) with primers targeting 5'UTR of IBV. A total of 95 samples were tested, by RT-PCR. A Taqman probe labelled real time PCR assay targeting the 5' UTR and Nucleocapsid (N) gene of IBV was standardised for the detection of IBV in all the 95 tissue and allantoic fluid samples to assess its efficiency. The real time PCR could detect IBV in all the 95 allantoic fluid and tissue samples including the samples, which were negative in preliminary detection. Hence, in the present study, the real time PCR assay was found to have equal efficacy on clinical samples and allantoic fluid and with a sensitivity of 100% and specificity of 90.90% in comparison with the conventional RT-PCR assay.

Background: Antibiotic resistance is an emerging concern in the therapy of clinical infections worldwide. Previous studies conducted in our laboratory have confirmed an increase in the prevalence of extended spectrum beta-lactamase (ESBL) among the Gram-negative bacterial pathogens associated with dogs, which could act as a potential source for the transfer of these resistant pathogens or their genetic determinants to human. Since carbapenems are the last resort drugs against these resistant pathogens, the study was aimed to isolate and characterise carbapenem resistance among Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae) and Pseudomonas aeruginosa (P. aeruginosa) associated with common clinical infections in dogs.Methods: A total of 100 samples were collected from lesional skin, urine and anterior vagina of dogs presented to the Veterinary Hospitals of Kerala Veterinary and Animal Sciences University at Mannuthy and Thrissur. The samples were cultured onto Brain Heart Infusion Agar (BHIA), Eosin Methylene Blue (EMB) and Mac Conkey (MAC) for isolation of bacteria. Identification of the isolates was performed based on cultural, morphological and biochemical characteristics. The isolates were subjected to antimicrobial susceptibility test (ABST) against the 12 commonly used beta-lactam and non–beta-lactam group of antibiotics by disc diffusion method and further subjected to screening for ESBL double disc diffusion method. Carbapenem-resistant isolates were subjected to phenotypic confirmatory test for carbapenemase production employing Imipenem-EDTA and Ertapenem-boronic acid minimum inhibitory concentration (MIC) strip method.Result: Forty four Gram-negative bacterial isolates obtained were viz., E. coli (30), K. pneumonia (11) and P. aeruginosa (3) from the 100 samples. Apart from these, other isolates obtained were Staphylococcus spp. (53) and Bacillus spp. (2). All the Gram-negative isolates were subjected to ABST employing 12 common antibiotics belonging to beta-lactam and non-beta-lactam groups. Multidrug resistance (MDR) could be observed in 28 E. coli, 11 K. pneumoniae and three P. aeruginosa isolates. All the 42 MDR isolates showed positive results for ESBL production. A total of 14 isolates out of the 44 Gram-negative bacilli were found to be resistant to carbapenem either to imipenem, meropenem or ertapenem. Among the 14 Gram-negative isolates, nine turned out to be positive for metallo-beta-lactamase (MBL) and none for K. penumoniae carbapenemase (KPC) on phenotypic confirmatory test for detecting major carbapenemase enzymes. The present study documented that Gram- negative bacteria like E. coli, K. pneumoniae and P. aeruginosa isolated from dogs are showing an increase rate of resistance against carbapenems which are the last resort drugs against ESBL producers. Hence, there is an urgent need to curb the irrational and excessive use of antibiotics in veterinary sector.

A study was conducted to determine the antibiogram of Escherichia coli isolated from cases of neonatal calf diarrhoea in Thrissur and Wayanad districts of Kerala. A total of 101 isolates were tested for susceptibility against 12 antibiotics. The antibiotics to which the isolates were most susceptible were Streptomycin (49.51%) and Chloramphenicol (47%). The isolates showed varying degrees of susceptibility to the other antibiotics tested such as Enrofloxacin (8.92%), Ciprofloxacin (15.85%), Gentamicin (23.77%), Tetracycline (20.8%), Amoxicillin/sulbactam (8.92%), Co-trimoxazole (20.8%), and Nitrofuratonin (25.75%). All the isolates were resistant to Cefpodoxime, Penicillin G and Cefotaxime/Clavulanic acid. Many of the isolates were found to be multi-drug resistant which has serious implications for the control of these infections through antibiotic therapy.

Corneal ulcers or ulcerative keratitis is the loss of continuity of corneal epithelium leading to exposure of corneal stroma. It is a highly painful condition in dogs. The common aetiological agents responsible for this condition are bacteria and for treatment, antibiotics are widely used. Indiscriminate and prolonged use of antibiotics can cause antimicrobial resistance (AMR) in bacteria. The present communication deals with the isolation of bacteria from corneal ulcers in dogs and the assessment of quinolone resistance. A total of 15 corneal swab samples were collected from dogs suffering from corneal ulcers, presented to the Department of Veterinary Surgery and Radiology, Teaching Veterinary Clinical Complex, College of Veterinary and Animal Sciences, Mannuthy. They were inoculated on to brain heart infusion agar as well as blood agar and incubated at 37 °C for 24 h for the isolation of aerobic bacteria. Eighteen isolates obtained were then subjected to antibiotic susceptibility test by disc diffusion method using the quinolone group of antibiotics, viz. ciprofloxacin, enrofloxacin, ofloxacin and moxifloxacin. Majority of the isolates exhibited varying resistance to these antibiotics. Polymerase chain reaction was performed to amplify gyrA gene, which is reported to be one of the reasons responsible for quinolone resistance with respect to its mutation. Seven out of the 18 isolates amplified gyrA gene. The amplicon obtained corresponding to a representative sample, which was found resistant by disc diffusion method was sequenced and a single point mutation was detected.

Riemerellosis is an infectious disease that primarily affects young ducklings, although it can infrequently be manifested in a chronic localised form in older ducks. Though an inactivated vaccine has been found to be most effective in preventing the disease in ducklings, its potency in adult breeder ducks has not been assessed yet. To investigate the same, 200 Kuttanad breeder ducks maintained in the University Poultry and Duck Farm (UPDF), Mannuthy were grouped into two, of 100 birds each, T1 being the control group and T2, the treatment (vaccinated) group. The oil-adjuvanted inactivated vaccine, prepared as per the previously standardised protocol, was administered to the T2 group in two doses, one week apart. Blood samples in serum vials, 20 each from T1 and T2, were collected on days 0, 14, 28, 56 and 90 post-immunisation (PI) whereas blood samples in heparin vials, eight each from both the groups, were collected on days 0, 14, and 28 PI. Humoral immunity (HI) and cell mediated immunity (CMI) in the adult ducks were assessed by enzyme-linked immunosorbent assay (ELISA) and lymphocyte proliferation assay (LPA), respectively. The two assays revealed that the inactivated vaccine elicited a good immune response in the adult ducks, with HI noticed until 90th day PI and an increasing CMI that peaked on 28th day PI.

Chicken anaemia virus (CAV) is the aetiological agent of chicken infectious anaemia (CIA), an immunosuppressive disease, which brings a huge economic burden to the poultry industry globally. Concurrent infections and vaccination failures further aggravate the impact of the disease on the sector. The epidemiology of the disease and virulence of the circulating strains is to be known while formulating prevention strategies for any infectious disease. So far, there are no reports regarding the presence of CAV among poultry flocks in Kerala. In this scenario, the present study contemplated the investigation of the chicken anaemia virus using ELISA and PCR techniques. Indirect ELISA revealed the presence of CAV antibodies in 80 samples out of the 92 serum samples screened. Among the 100 samples collected from suspected cases, 29 were found to be positive for CAV infection with detection primers targeting the VP2 gene. This implies widespread distribution of the virus in Kerala.

Veterinarians are the primary sourceof knowledge for farmers on how to use antibiotics responsibly in farm animals and how to decrease the risk of antibiotic resistance.Antimicrobial use and resistance is a multidimensional construct that lacks a specific measurement scale. This paper looks into the development of a scale to assess veterinarian perception about antimicrobial use and resistance. Following a thorough literature review, focus group interviews were conducted with selected key informant veterinarians to identify statements.An initial list of 80 statements reflecting positive and negative perception towards antimicrobial use and resistance was prepared. After editing, a final list of 70 statements were arrivedat. Descriptives of antimicrobial use and resistance, consensus regarding antimicrobial use and resistance indicators were arrived at through relevancy rating by a panel of 40 judges in the field of veterinary science based on which 35 statements were selected for item analysis. These 35 statements were subjected to item analysis among 60 veterinarians selected from a non-sample area. Out of the 60 veterinarians to whom the items were administered for item analysis, 15 veterinarians each with the lowest and highest scores were used as the two criterion groups to evaluate individual items. Individual items were selected based on‘t’ value whereinitems that had a t-value of more than 1.75 were selected and incorporated into the final schedule. Based on the calculated‘t’ values, 14 items were finally selected and includedin the final scale. Reliabilityof the scale was tested using Cronbach’s alpha coefficient of reliability test for which a Cronbachalpha of 0.852 was obtained indicating the internal consistancy of the scale.

Infectious bursal disease (IBD), caused by infectious bursal disease virus (IBDV), is one among the top five infectious diseases of poultry that discernibly affects commercial poultry industry. The mutating viral genome of IBDV accounts for disease outbreaks in fields even after following stringent biosecurity measures and vaccination protocols. The present study is focussed on the characterisation of an IBDV field virus, IBD/CVAS/6, from a vaccinated flock in Kerala, based on the sequence analysis of VP1, VP2 and VP3 genes. Bursa of Fabricius samples collected from a 26 days-old chicken flock from a suspected outbreak in the Thiruvananthapuram district of Kerala formed the subject of the study. Reverse transcriptase-polymerase chain reaction (RT-PCR) targeting VP2 gene confirmed the presence of virus in the sample. The sequence analysis revealed that the deduced amino acid sequence of VP1 gene of IBD/CVAS/6 was 100 per cent homologous with an attenuated very virulent vaccine strain of Israel, mb and the VP2 gene was 100 per cent homologous with mb and Ventri IBDV plus vaccine strain of India. The analysis of VP3 gene also revealed the similarity with vaccine strains except for a single variation S745N in its deduced amino acid sequence. The phylogenetic analysis of IBD/CVAS/6 revealed it’s close relation with mb, Ventri IBDV plus and a very virulent strain of Israel, ks. The characteristic virulent marker amino acid motifs ‘SWSASGS’ and ‘TDN’ were present in the VP2 and VP1 genes, respectively. Hence, the study revealed that the obtained virus has emerged from an attenuated very virulent vaccine strain and hence the present study is a report of involvement of the intermediate plus vaccine strain in field outbreaks in Kerala. The role of S745N in virulence cannot be accounted from the present study, however the involvement of IBD/CVAS/6 in the outbreak might be related to S745N variation or variations in other genes of the virus or due to the inefficiency of the vaccine or vaccination protocol followed, which can be defined only after further studies. The present report is the first characterisation study in Kerala focussing on the analysis of VP1, VP2 and VP3 genes of IBDV.

Campylobacteriosis is one of the leading causes of bacterial gastroenteritis world over, with a self-limiting nature. Nevertheless some rare sequelae like Guillain-Barre syndrome, Reiter syndrome and reactive arthritis are evident. With poultry as a major reservoir, the organism is ubiquitous in nature and has been isolated from various sources like animals and the environment. The present study was undertaken to evaluate the occurrence of Campylobacter spp. in marine fishes (n=48), crustaceans (n=35) and molluscs (n=50) from Puthiyappa, a fish catchment area of coastal Kerala (India) using multiplex polymerase chain reaction (mPCR) in combination with conventional plating technique using Blood-free campylobacter broth and modified Charcoal Cefoperazone Deoxycholate (mCCD) agar. Campylobacter spp. was found to be present in marine fishes, crustaceans and molluscs at a level of 6.3, 8.6 and 28.0 per cent, respectively. The predominant species was Campylobacter jejuni in fishes, while Campylobacter coli was more prevalent in molluscs. Of the seasonally available fishes, the organism was found to be present in ribbon fish, pink perch, pony fish, while among molluscs, it was present in mussel, squid and clam. Only 8.3 and 6.0 per cent of Campylobacter spp. were recoverable from fishes and molluscs, respectively, on mCCDA plates. Campylobacter spp. in these marine fishes and shellfishes indicate that these can act as possible sources for the transmission of food-borne campylobacteriosis.

Bovine tuberculosis (bTB), predominantly caused by Mycobacterium bovis, is one of the most neglected zoonotic diseases of cattle. The lack of documented information on bTB is one of the most important hurdles in controlling the disease. The present study was carried out to detect the presence of Mycobacterium tuberculosis complex (MTBC) from blood samples of cattle presented for slaughter, using polymerase chain reaction. The study revealed presence of MTBC in seven out of 50 animals (fourteen per cent) with symptoms suggestive of bTB. The detection of the organism emphasises the need of further prevalence studies in Kerala, to implement proper control strategies.

Background: Carbapenems are beta-lactam antibiotics that are considered as the last line of therapy against multidrug resistant extended spectrum beta-lactamase. The resistance to carbapenems predominantly through carbapenemase is one of the most important emerging health problems worldwide in the therapy of clinical infections. The objective of the present study is to determine the presence of carbapenemase encoding genes among Gram-negative bacterial spp. associated with clinical infections in dogs.Methods: 30 Escherichia coli, 11 Klebsiella pneumoniae and three Pseudomonas aeruginosa isolated from urine, swabs from lesional skin and anterior vagina of dogs presented with different clinical ailments formed the samples for the study. Polymerase chain reaction was carried out to detect the presence of carbapenemase encoding genes viz., KPC, NDM, OXA, VIM and IMP among the isolates.Result: Out of the 44 Gram-negative isolates tested, 28 (76.3%) were positive for at least one tested carbapenemase gene. The highest frequency of carbapenemase recorded was for NDM followed by OXA-181, KPC, OXA-48 and VIM. Our study identified a high prevalence of carbapenemases among companion animals like dogs which could act as potential source of transmission of these resistance bacteria or their genomes to humans.

Leptospirosis is an emerging zoonotic disease endemic in Kerala and close monitoring of emerging serovars is essential to adopt appropriate control strategies. Multi-Locus Sequence Typing (MLST) was reported to be less expensive compared to other cumbersome methods like whole genome sequencing. The present study was conducted to obtain isolates of Leptospira from infected animals and rats and for the identification of serovars using MLST. A total of 205 blood samples (dog, cat, cattle, goat), 43 urine samples (dog, cattle) and post-mortem kidney samples from various animals such as dog (n=12), cattle (n=2) and rat (n=25) were collected and subjected to polymerase chain reaction (PCR) using G1/G2 primers to identify the pathogenic Leptospira. Fifteen samples were found to be positive, these samples when inoculated in the EllinghausenMcCullough-Johnson-Harris (EMJH) semi-solid medium to obtain ten isolates. These ten isolates were further subjected to secY, icdA and GyraseB PCR and sequenced. The obtained sequences were analysed using BLAST and were fed into specified MLST database of Leptospira scheme-3, the allelic profile and sequence type were generated. The MLST results obtained in the study indicated that the isolates S24 and S33 belonged to serovar Canicola, S40 and 47 were Sejroe and S19, S27, S55, S69 and S71 were Bataviae, Autumnalis, Pomona, Icterohaemorraghiae and Australis, respectively. It was concluded that MLST is a convenient method for identifying leptospiral serovars.