Novel peptide nucleic acid (PNA) probes were designed for direct detection of Mycobacterium avium paratuberculosis (MAP) by the Fluorescent in situ Hybridization technique from fecal samples. For this study, fecal samples (n = 218) were collected from cattle (n = 180) and buffaloes (n = 38) with a history or incidence of cases of chronic intermittent diarrhoea from organized dairy farms in the state of Punjab. Fluorescent in situ Hybridization was done on highly positive (acid-fast positive) fecal samples (n = 54) using a newly designed MAP probe. The designed probe was standardized for the standard culture of Mycobacterium avium subsp. paratuberculosis (ATCC K10) at hybridization conditions of 55 °C and 30

The present study investigated outbreaks of Newcastle disease in two commercial layer farms in Ludhiana district of Punjab during 2024-2025. Clinically, affected birds exhibited lethargy, nasal discharge and greenish-white diarrhoea, with death occurring within 48 hours of onset of clinical manifestations.Six representative birds from the three flocks of both farms underwent necropsy, and samples were collected for histopathological and molecular detection by RT-PCRtargeting the partial and complete fusion (F) gene of NDV. Selected RT-PCR-positive samples were subjected to virus isolation in Vero cells. The identity of the isolates was confirmed through RT-PCR and nucleotide sequencing.The gross lesions revealed haemorrhages in the proventriculus, button shaped intestinal ulcers, splenomegaly, fibrino-necrotic ulcers in the esophagus and tracheal haemorrhages. Histopathological examination revealed massive necrosis of the proventriculus, lymphoid depletion in spleen and cecal tonsils, necrosis of intestinal mucosa, sinusoidal congestion in the liver and multifocal lymphocytic infiltration in the pancreas. Molecular detection using RT-PCR amplified 535 bp and 1662 bp fragments of the partial and complete F gene, respectively. Isolation of representative positive samples in Vero cells produced cytopathic effects such as cell elongation, granulation and syncytium formation. Sequence analysis of the NDV isolates based on F gene revealed the strains as genotype XIII. Further, presence of multi-basic amino acid residues at F protein cleavage site (112RRQKRF117) confirmed the isolates as velogenic strains.The detection of genotype XIII, a highly pathogenic lineage widely circulating in South Asia, underscores its increasing epidemiological significance and threat to commercial poultry.

Bovine Tuberculosis (bTB) is a widespread infectious disease of domestic animals and humans. The study aimed to detect mycobacteria at the molecular level in bovine lung tissue and lymph node samples using conventional and TaqMan qRT-PCR assay. Ninety-nine samples of lung tissue (n = 90) and lymph nodes (n = 9) were collected from cattle and buffaloes. All the collected samples were subjected to microscopic examination, decontaminated with 0.75% hexadecylpyridinium chloride (HPC), and inoculated onto Middlebrook7H10 media, followed by PCR screening. In total, 26 isolates were obtained from 99 tissue samples. Nineteen out of twenty-six tissue isolates tested positive for the hsp65 gene, indicative the genus Mycobacterium. However, of the nineteen-genus positive isolates, six samples yielded M. orygis, and 8 yielded M. kansasii on culturing, which were subsequently identified by species-specific PCR. Subsequently six M. orygis isolates were confirmed by the real-time PCR with Cycle Threshold (CT) values ranging from 18.43 and 34.74. Detection of M. orygis from lung tissue and lymph nodes is reported using conventional and TaqMan qRT-PCR assay. The findings of the current study provide evidence of the presence of non-tuberculous mycobacterial infection in lung tissue, along with tuberculous mycobacteria, which may hamper in diagnosis of bTB reactor-positive animals.

Staphylococcus aureus, a common cause of bovine mastitis, relies on several virulence factors, with biofilm formation being a key contributor to its pathogenicity. The present study investigated the occurrence of S. aureus as etiological agents in bovine mastitis with a focus on the existence of various virulence factors and antibiotic resistance status. Among 120 milk samples collected from West Bengal and Uttar Pradesh, 36 (30%) S. aureus strains were confirmed by conventional methods and PCR. Phenotypic analysis revealed hemolysin (55.55%) and coagulase production (36.11%), while molecular analysis revealed the presence of leukotoxin (luksF, 19.44%), hemolysin (hlb, 58.33%), coagulase (coa, 63.88%), and toxic shock syndrome toxin (tsst-1, 30.55%) genes. Biofilm production ability was detected in 97.22% (crystal violet assay) and 86.11% (Congo red agar assay) strains. Biofilm-associated genes, namely, icaA (80.55%, 29/36), icaB (75%, 27/36), icaC (69.44%, 25/36), icaD (86.11%, 31/36), and MSCRAMMs genes, namely, clfA (58.33%, 21/36), clfB (75%, 27/36), fnbA (75%, 27/36), fnbB (55.55%, 20/36), bap (38.88%, 14/36), bbp (83.33%, 30/36), ebps (69.44%, 25/36), eno (66.66%, 24/36), fib (41.66%, 15/36), and cna (8.33%, 3/36), were also detected. Antimicrobial resistance was observed in 88.88% isolates, with 72.22% exhibiting multidrug resistance (MDR). Among the isolates, 83.33% were methicillin-resistant S. aureus (MRSA), and mecA, femA, and femB genes were present either singly or in combination in 76.66% of the isolates. Efflux pump protein genes, namely, norA, norB, norC, mdeA, mepA, and sepA, were detected either singly or in combination in S. aureus isolates. 61.53% of MDR-MRSA isolates harbored all six efflux pump genes. According to this study, S. aureus of mastitis origin harbors various virulence, antibiotic resistance, biofilm-forming, and efflux pump genes. Bovine mastitis-derived MDR S. aureus isolates can pose a significant public health risk and need urgent attention to formulate strategies for their control and preventing transfer to the human food chain.

Unregulated use of antibiotics has led to selection pressure in many pathogens, leading to the development of antimicrobial resistance (AMR), one of the leading causes of death worldwide. The effect of AMR in humans as well as all the species of animals has been observed. Escherichia coli (E. coli), a gram-negative bacterium, colonizes in the gastrointestinal tract and maintains a mutually beneficial relationship with the host. Most of the E. coli strains are harmless and inhabit the intestinal tract, but there are certain pathogenic strains, which cause infections both in humans and animals, leading to higher chances of transmission of AMR genes. Development of AMR in E. coli strains of poultry has become a major public health concern, as poultry meat and eggs act as a direct source of transmission of these multi-resistant strains to humans. This review focuses on the worldwide emergence of AMR in E. coli strains of poultry, their impact on public health, and the importance of "One health approach" to tackle the current situation.

The study encompassed the isolation of Staphylococcus aureus (S. aureus) from bovine mastitic milk samples and its characterization for antibiotic sensitivity and biofilm production. The milk samples were initially screened for Staphylococcus spp. using cultural and biochemical tests, and were confirmed to species level using biochemical tests, Polymerase Chain Reaction (PCR) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Antibiotic susceptibility was tested using the disc diffusion method, and biofilm formation was evaluated using the Modified Congo Red Agar assay (MCRA assay) and Microtitre based Biofilm Assay (MtBA). Out of 112 samples, 98 showed bacterial growth, of which 18.36

Background: The extensive use of beta-lactam antibiotics in food animals and humans has aggravated the issue of antimicrobial resistance (AMR) threatening global health. The rapid spread of extended-spectrum beta-lactamase (ESBLs) producing resistant bacteria mainly in Enterobacteriaceae has raised alarm worldwide. Methods: In the present study, we investigated the prevalence, antimicrobial resistance pattern and genotypic detection of AMR genes of ESBL-positive Escherichia coli (E-EC) and Klebsiella pneumoniae (E-KP) isolated from pigs and animal handlers. Overall, 140 faecal samples from pigs and 33 hand swabs from pig handlers were screened for ESBL production. Among these samples, 70 (65.42%) E-EC strains were identified from pigs whereas only one E-KP and E-EC from pigs and handlers, respectively. Result: The present study revealed a high level of antibiotic resistance dominated by ceftazidime (81.94%) and cotrimoxazole (80.55%). The presence of multidrug resistance (MDR) was observed in 70 ESBL isolates and the multiple antibiotic resistance (MAR) index of all the isolates was more than 0.21. The genotypic detection of ESBL genes by PCR revealed the dominance of the blaTEM gene (98.59%) in ESBL-E. coli isolates. On analyzing the genotypic results of various tetracycline (tet) and quinolone (qnr) resistance antibiotic genes, tet-A accounted for a significant proportion of 95.77% while qnr-S was detected at a proportion of 21.12%. The findings in our study highlight that the pig farms can act as potential carriers of ESBLs and multidrug-resistant E. coli which eventually culminates in a higher concentration of AMR genes in the ecosystem.

The study was conceived with the objective to isolate and characterize lytic bacteriophages (phages) against Staphylococcus aureus (S. aureus) associated with bovine mastitis. Phages were isolated from enriched slurry and milk samples using the agar overlay technique. Characterization included plaque morphology, Field Emission Scanning Electron Microscopy (FE-SEM), pH and thermal stability, host range (spot assay), adsorption assay and one-step growth curve analysis. Lytic efficacy was assessed using the Efficiency of Plating (EOP) method. Five phages (pS1 to pS5) were isolated, of which pS4 and pS5 were temperate phages as indicated by its lysis pattern. The analysis showed that phages pS2 and pS3 produced clear, pinpoint-sized plaques while pS1 formed large, clear plaques. FE-SEM classified all three phages under the class Caudoviricetes. Adsorption assay and one step growth curve analysis revealed that the phages had an adsorption time ranging from 9 to 15 min, and a latent period between 30 and 50 min and burst sizes of 68 (pS2), 220 (pS1) and 280 (pS3) pfu/cell. Stability tests revealed significant difference in thermal stability at 60 °C and 70 °C (p < 0.0001), and in pH tolerance from pH 2 to 10 (p < 0.0001) among pS1, pS2 and pS3. The spot assay revealed that the complete panel of clinical S. aureus isolates (n = 18), standard cultures (ATCC 12600 and ATCC 29213) and clinical isolates of S. chromogenes and S. haemolyticus were sensitive to pS1. In contrast, pS2 and pS3 exhibited lytic activity exclusively against the S. aureus isolates, showing no activity against S. chromogenes or S. haemolyticus. Phage pS1 demonstrated good lytic effect against 10/21 test isolates compared to pS2 (3/21) and pS3 (3/21). Overall, the study characterized three lytic phages targeting S. aureus implicated in bovine mastitis and showed its in vitro efficacy against planktonic S. aureus.

The present study is focused on isolation of phages against Escherichia coli. The study revealed that out of 30 sewage samples from 12 dairy farms, 11 coliphages were isolated. All the 11 phages were characterised and on the basis of morphology plaque, sizes ranging 1-8 mu m in diameter were observed. All the phages were stable at temperatures of 50 degrees C and 60 degrees C for 48 h but were inviable at 65 degrees C > 6 h of exposure. Upon UV exposure for 30 min, all the phages were stable but with reduced viability. Whole genome sequencing of a phage showed 97.58% identity to Escherichia phage vB EcoM-ECP26, complete genome. Also phage contigs were identical to antibiotic resistance genes for beta-lactamases, tetracyclins, glycopeptides, macrolides, diaminopyrimidines, aminoglycosides and carbapenems in abundance. Thus, it could be concluded from the study that phages harbour various antibiotic resistant genes that could lead to transmission of antibiotic resistance in the host, i.e. bacteria.

Background: The study aimed to investigate the effectiveness of phage in controlling Staphylococcus aureus (S. aureus) biofilm phenotypes associated with mastitis. Methods: The study was carried out with qualitative assessment and quantitative assessment of biofilm producing ability of the S. aureus isolates by using Congo Red Agar Assay (CRA) and Modified Congo Red Agar Assay (MCRA) respectively. The biofilm formed was treated with phage to evaluate its efficacy in removing the biofilm. Result: A total of 18 isolates of S. aureus (S1-S18) were isolated and identified from mastitis cases. Among the 18 isolates of S. aureus, many exhibited strong biofilm producing capabilities (22.22% on CRA and 33.33% on MCRA). Biofilm formed was treated with phage to evaluate its efficacy in removing the biofilm and it was observed that exposure to high phage titre (5x107 PFU/ml) over 24 h significantly reduced biofilm biomass (83.14%) whereas low phage titre (1.5x107 PFU/ml) exhibited significant yet less effectiveness in controlling biofilm. The phage action on biofilm resulted in a decline in bacteria (19.99%) while increasing phage (11.50%). These findings demonstrated the potential of high-concentration phage in treating biofilm phenotype.

Bovine tuberculosis (bTB) is a zoonotic disease that affects domestic animals, humans, and the wild animals. The purpose of the study was to use the comparative intradermal tuberculin test to diagnose bTB in cattle and to find out the prevalence in unorganized, and organized farm with variation according to age, sex, and breed. Eighty three animals were tested with the use of comparative intradermal tuberculin test across two districts of Punjab, India. Additionally, ante-mortem (nasal swab, n=44) samples were examined using molecular methods. The samples were inoculated on Middlebrook7H10 media after decontamination with 4% NaOH. One sample revealed the presence of acid fast bacilli on microscopic examination. Out of 83 animals examined, 12 were bTB positive reactors. Additionally, gender-wise data analysis revealed higher positivity among cows/heifers (16.17%, 11/68) compared to bulls/bullocks (6.66%, 1/15). Out of the 44 nasal swab samples, 3 samples yielded Mycobacterium kansasii isolates on culture which were further confirmed by species specific PCR. The finding of our study indicates animals of indigenous cattle breed in Ludhiana have a higher prevalence of bTB and variation in breed susceptibility. The presence of non-tuberculous mycobacteria may hamper diagnosing bovine tuberculosis in cattle.

Background: Mycobacterium avium subspecies paratuberculosis is a significant pathogen causing Johne’s disease. Rational evaluation of the prevalence of this organism in animals has been restricted by the lack of accurate diagnostic techniques. DNA microarray-based and computational study of Mycobacterium avium subsp. paratuberculosis strain K10 discovered 17 distinct large sequence polymorphisms present. Of these 17 LSPPs, 7 were with a specificity of greater than 98% and in some cases, sensitivity of up to 95% suggesting that the LSPs are ideal for diagnosing M. avium subsp. paratuberculosis. Methods: A total of 218 fecal samples were collected from cattle (n=180) and buffaloes (n=38) for detection of M. avium subsp. paratuberculosis (MAP) using Large Sequence Polymorphisms (LSPs) and IS900 PCR. The fecal samples were subjected to Ziehl-Neelsen staining, isolation and PCR. A total of 90 fecal samples were found ZN staining positive and were subjected to isolation of MAP, growths were seen in two fecal samples after 16 weeks of incubation. Result: Out of 218 fecal samples tested, 7 samples were positive by both LSP PCR viz; LSPP2 (MAP0284), LSPP4 (MAP0865), LSPP11(MAP2154) LSPP12 (MAP2182c), LSPP12 (MAP2188c), LSPP15 (MAP3774), LSPP16 (MAP3815) and IS900 sequence with an amplicon size of 600, 597, 375, 430, 719, 621, 611 and 229 bp. DNA was also extracted from the isolates (n=2) and were confirmed by PCR targeting Large Sequence Polymorphism (LSPs) and IS900. Detection of MAP in all the 7 fecal samples both by IS900 and by 7 LSPPs indicates the detection potential of selected 7 LSPPs (LSPP2, LSPP4, LSPP11, LSPP12 (MAP2182c), LSPP12 (MAP2188c), LSPP15, LSPP16) in fecal samples of cattle and buffaloes suspected for Johne’s disease.

Mastitis an inflammatory condition of mammary glands is a multi-factorial disease of dairy animals. It is characterized by physical, chemical and bacteriological changes in the milk. In the present study prevalence of Enterococcus faecalis (E. faecalis) and Pseudomonas aeruginosa (P. aeruginosa) were studied along with their antibiotic resistance pattern. One hundred and ten milk samples were screened from mastitis cattle and buffaloes in and around Ludhiana from December 2020 till June 2021. The prevalence of E. faecalis and P. aeruginosa was 7.05% and 4.70% respectively. The isolates of E. faecalis were resistant to penicillin G (100%), vancomycin, erythromycin, and tetracycline (83.33%) and sensitive to nalidixic acid (100%), streptomycin (83.33%), oxacillin, gentamicin, ciprofloxacin and ceftriaxone (66.66%) whereas isolates of P. aeruginosa were resistant to ampicillin, ciprofloxacin, ceftriaxone, penicillin G, streptomycin, amikacin, vancomycin (100%), tetracycline and teicoplanin (75%) and sensitive to cefuroxime, gentamicin, and oxacillin (100%). InE. faecalis, vanA, vanB (50%), tetL (83.33%) and mrsA/B (100%) antibiotic genes were amplified whereas in P. aeruginosa, aadA, DHAM (100%), sulI, sulII (100%), gyrA, gyrB (100%) and tetC (50%) antibiotic genes were amplified. Thus, it is concluded that there is an evolving prevalence of environmental pathogens such as E. faecalis and P. aeruginosa in mastitis which is alarming and thus necessary action and plans should be followed for controlling the antibiotic resistance in these pathogens as these are of zoonotic significance.

Aim: Mycobacterial infections due to Mycobacterium avium paratuberculosis (MAP) (Paratuberculosis), and M. bovis (Tuberculosis) in dairy animals are potential public health risk that needs to be diagnosí at the earliest for effective prevention. In addition, M. smegmatis (nontuberculous mycobacteria) is a saprophytic mycobactera that may interfere with the diagnosis of mycobacterial infection besides causing opportunistic infections. This study aims at rapid differential detection of these organisms from faecal samples of cattle and buffaloes using in-house designed multiplex PCR, besides other techniques for aiding in the accuracy of diagnosis. Methodology: Faecal samples (n=268) were collected per-rectum from suspected animals and screened by Ziehl-Neelsen staining and graded as low, medium and high shedders; Then faecal culture in Middlebrook 7H11 media for isolation of MAP from faecal samples of medium to high shedders (n=150). All the samples were subjected to in-house designed multiplex PCR targeting MAP, M. bovis and M. smegmatis; besides using IS900 conventional PCR and TaqMan real-time PCR for detection of MAP. Results: A total of 237 (88.43%) samples were found to be positive for Acid-fast bacilli, out of which 49 (20.68%), 106 (44.73%) and 82 (34.60%) samples were from low, medium and high shedders respectively. Only 4 (2.67%) samples were culture positive for MAP. Whereas, multiplex PCR detected 16 (5.97%) faecal samples as positive for MAP, and none were positive for M. bovis and M. smegmatis. However, IS900 conventional PCR and IS900 TaqMan real-time PCR detected 19 (7.09%) and 29 (10.82%) faecal samples as positive for MAP respectively. Conclusion: Thus, the in-house multiplex PCR can be used for screening mycobacterial infections and also for the rapid detection of MAP like the IS900 PCR. After all, a large proportion of the Acid-fast bacilli in the faecal samples may be from other mycobacteria or non-mycobacterial Acid-fast bacilli that need to be studied further.

Bovine tuberculosis (bTB) is a zoonotic disease that affects domestic and wild animals, including livestock. The purpose of the study was to detect tuberculous and non-tuberculous mycobacterial species from buffaloes and cattle with respiratory signs and positive intradermal tuberculin test. Eighteen trans-tracheal washes (TTW) were obtained from buffaloes (n = 5) and cattle (n = 13) with respiratory distress. All the collected samples were subjected to microscopic examination, direct PCR screening, and bacteriological culture in Middlebrook7H10 media followed by PCR. One sample revealed the presence of acid-fast bacilli on microscopic examination. Six out of eighteen TTW samples tested positive for the hsp65 gene indicative of the Mycobacterium genus. Two samples were positive through direct PCR and were further identified as Mycobacterium orygis and Mycobacterium kansasii. However, out of the 18 TTW samples, two samples yielded Mycobacterium kansasii isolates on culturing, which were confirmed using species-specific PCR. Our study indicates PCR is a better screening test of the TTW samples for the detection of both tuberculous and non-tuberculous mycobacteria compared to microscopic examination and culturing. The presence of non-tuberculous mycobacteria may affect the results of tuberculinization in cattle and PCR screening of the tested animals will assist in validating the tuberculin test results.

Emissions of airborne pollutants from livestock buildings affect indoor air quality, the health and well-being of farmers, animals and the environment. This study aimed to evaluate the microbial count within pig sheds and its relationship with meteorological variables (temperature, relative humidity and air velocity) and particulate matter (PM10 and PM2.5) and microbial diversity. Sampling was conducted both inside and outside of two pig sheds over three seasons (summer, rainy and winter), with regular monitoring at fortnightly intervals. Results showed that the bacterial and fungal counts ranged from 0.07 to 3.98 x 103 cfu/m3 inside the sheds and 0.01 to 1.82 x 103 cfu/m3 outside. Seasonal variations were observed, with higher concentrations of particulate matter detected during the winter season, followed by summer. Climatic variables such as temperature, air velocity and relative humidity demonstrated significant impacts on the abundance of Enterobacteriaceae and fungi, while air velocity specifically influenced the presence of mesophilic bacteria and staphylococci. Importantly, no significant disparities were found between microbial counts and particulate matter levels. Staphylococcaceae emerged as the predominant bacterial family, while Aspergillus and Cladosporium spp. were the dominant fungal species within the pig sheds. The average levels of airborne bacteria and fungi in pig sheds were found to be within the recommended range, which can be attributed to the loose housing design and lower animal population on the farms.