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Open Access
“Molecular Identification and Characterization of Lumpy Skin Disease Virus (LSDV) from Cattle in Jammu”
(Sher-e-Kashmir University of Agricultural Sciences & Technology, Jammu (J&K), 2024-04-11) Akhter, Shaista; Gazal, Sabahat
The present study was undertaken for molecular identification and characterization of Lumpy Skin Disease Virus (LSDV) from cattle in Jammu district and phylogenetic analysis of LSDV. A total of 277 nasal swab samples were received from Animal Husbandary (AH) Department Jammu and were screened for the presence of LSDV by Real Time PCR (qPCR). The viral DNA was extracted by using viral nucleic acid extraction kit as per manufacturer’s protocol and LSDV P32 gene was targeted by using specific primers as recommended by OIE by using qPCR. Out of total samples tested, 99 were found to be positive for LSDV in Jammu district with occurrence of 35.74%. The breed wise occurrence of LSD was found to be highest in Sahiwal (50%) and lowest in Jersey (0%) whereas age wise occurrence of LSD was found to be highest in young animals in the age group of 0-2 years i.e., 58.33% and lowest in the age group of 6-8 years (23.40%). Higher disease occurrence was recorded in male animals i.e., 50% while only 35.63% females were found to be positive for LSD. The χ2 value was found to be non-significant for sex and breed but significant for age group affected with LSD. One of the GPCR gene amplicon was sequenced by Sanger sequencing and submitted to GENBANK under accession no. OR209679. BLAST searches were made on the sequence obtained which revealed 12bp nucleotide deletion in the GPCR gene. The phylogenetic tree was constructed by using Neighbour-Joining tree constructing method in MEGA 11 software and maximum homology was observed with sequences of Russia, Serbia, Greece and Pendik.
Open Access
“Cultural and Metagenomic Based Identification of Microbiome of Bovine Mastitic Milk.”
(Sher-e-Kashmir University of Agricultural Sciences & Technology, Jammu (J&K), 2022-01-24) Tikoo, Mehak; Rashid, Mohd.
Mastitis is an economically important disease of livestock sector, affecting many milking animals, including Bovines. This study was designed to ascertain the microbial diversity of udder of healthy and diseased cows using both culture-dependent as well as culture-independent techniques. In this study, a total of four bacterial species (92 isolates) were isolated from 50 mastitic milk samples. Out of these 92 isolates, Staphylococcus aureus (32 isolates) was the predominant bacteria isolated (34.78%) and was found positive for nuc gene. Staphylococcus epidermidis consisted of eight (8.69%) isolates, E.coli 26 isolates (28.26%). All E.coli isolates were found positive for eco gene. Nine (9.78%) isolates were identified as Bacillus spp. and four (4.34%) as Pseudomonas aeruginosa isolates. In case of healthy milk samples 13 isolates were obtained out of which, two species of bacteria were isolated in a vast majority which included 10 isolates of Non-aureus Staphyloccoci (76.92%) and three isolates (23.07%) of Micrococcus species. The metagenomic results obtained revealed that in case of mastitic milk 10 phyla, 11 classes, 27 order, 54 families, 38 genera, 18 species of bacteria were detected while as in case of of healthy milk samples 12 phyla, 19 classes, 22 order, 57 families, 37 genera and 21 species of bacteria were most abundant. The most predominant species found in mastitic milk were Lactococcus and Streptococcus while as Pseudomonas fragi and Staphylococcus aureus were the least prevalent bacteria found in mastitic milk samples in case of healthy milk samples, the predominant bacterial species found were Aeromonas, Agrobacterium, Acitenobacter iwoffi, Propionibacterium acnes. Streptococcus and Acinetobacter johnsonii were the least prevalent bacteria found in healthy milk samples. This study concluded that apart from significant mastitic bacteria, certain non-specific bacteria also cause mastitis thus proving it difficult to be cured by routine treatment methodology.
Open Access
“Characterization of Escherichia coli from sheep and goat for virulence factors and their antibiotic resistance with special reference to extended spectrum β lactamases from Jammu region.”
(Sher-e-Kashmir University of Agricultural Sciences & Technology, Jammu (J&K), 2021-10-20) Mishra, Shekhar; Mishra, Shekhar; Rashid, Mohd; Rashid, Mohd
The study was aimed at investigating the pathogenic strains of Escherichia coli in sheep and goats and their antibiotic resistance with special reference to extended spectrum beta lactamases. Out of total 120 faecal samples, 200 presumptive E. coli isolates were collected. Out of 200 E. coli isolates 62 (31%) showed the presence of at least one virulence gene studied. Out of these 62 isolates, 10 (16.12%) isolates carried eaeA gene either alone or along with ehxA and were detected as EPEC, 44/62 (70.96%) isolates carried the stx gene and were detected as STEC. 08/62 (12.90%) isolates carried ehxA and were designated as EHEC. Out of 62 isolates which were screened for ESBL genes, 22 (35.48%) isolates were tested positive for presence of ESBL genes and the prevalence of blaTEM, blaCTX-M and blaSHV genes were 54.54%, 27.27% and 36.36% respectively. Antibiotic sensitivity profiling of the isolates positive for virulence genes revealed that all were resistant to cefotaxime, 95.16% to ceftazidime, 88.7% to cefoperazone, 88.2% to cefipime, and 69.3% to ciprofloxacin in the current investigation. All of the isolates were sensitive to chloramphenicol, 91.9% to doxycycline hydrochloride and 88.7% to amoxicillin/clavulanic acid.
Open Access
“Investigation of Extended-Spectrum beta-Lactamaseproducing Escherichia coli in Bovines from Jammu”
(Sher-e-Kashmir University of Agricultural Sciences & Technology, Jammu (J&K), 2021-08-25) Shikha, Deep; Wazir, V.S
The study was conducted to determine the occurrence of extended-spectrum beta-lactamase (ESBL) producing E. coli as well as the genetic diversity of ESBL with respect to blaSHV, blaTEM, blaCTX-M and blaOXAgenes and characterize their antimicrobial resistance and integrons in bovines. Out of total 180 faecal samples collected, 360 presumptive ESBL producing E. coli were isolated (2 from each sample). Of 360 isolates, 154 (42.77%) isolates were found to be resistant against cefotaximeadceftazidime.Resistance to cefotaximeadceftazidime was observed in 94 isolates (61.03%) and 60 isolates (38.96%), respectively while 70 (45.45%) isolates showed resistance to both. All the 154 isolates found to be positive in the screening test were confirmed as ESBL producers by phenotypic confirmatory test, Double disc synergy test or Etest. Present study revealed that the total prevalence of ESBL producing E.coli in bovines of Jammu region is 42.77% and is reported for the first time in Jammu region. All the isolates, which were declared as ESBL producers phenotypically, were tested for the presence of blaSHV, blaTEM, blaCTX-M and blaOXAgenes by multiplex PCR. Out of 154 ESBL isolates, only 120 (77.92%) isolates carried the gene/s for blaSHV, blaTEM andblaCTX-M. Out of 120 isolates, only four (3.33%) isolates carried blaTEM gene alone, 65 (54.16%) isolates carriedblaCTX-M gene alone and six (5.0%) isolates carried blaSHV /blaTEM/blaCTX-M. Of the 116 blaCTX-M positive isolates, 82 (70.68%) belonged to CTX-M-1 group, 09 (7.75%) belonged to CTX-M-9 group and 25 (21.55%) isolates carried both blaCTX-M 1/blaCTX-M 9. One representative amplicon of blaSHV, blaTEM andblaCTX-M were cloned in pGEM-Tand pJET1.2 and got sequenced commercially. The sequence of blaCTX-M was found to match with the database sequence of blaCTX-M -15. However, blaSHV gene sequence did not match 100% with the available database but showed 99.07% resemblance with blaSHV-11. Thus, blaSHV seems to be new variant and blaCTX-M-15is the first report of its kind in India. Antimicrobial sensitivity test of 120 ESBL producing E. coli isolates showed that resistance was most commonly to ampicillin (100%), cefexime (100%),neomycin (100%) followed by enrofloxacin (89.16%), amoxicillin/clavulanic acid (87.5%),aztreonam (81.60%), cefepime (81.60%), kanamycin (83.33%) and ceftriaxone(78.33%). In this study, 75.8% of the isolates show intermediate resistance against gentamicin. Antibiotic chloramphenicol (82.5%) and imipenem (78.33%) show sensitive results. Out of 120 multi drug resistance ESBL positive isolates, only 59 isolates tested positive for class 1 and 2 integrons. Among the 59 integrase-positive isolates, 52 (88.23%) isolates harbourintI 1 gene, while 2 (3.38%) isolates carried intI 2 gene. Five isolates, were found to harbour both intI 1 and intI 2. No class 3 integron was detected.
Open Access
Characterization of Dichelobacter nodosus and Expression of its fimA gene
(Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu (J&K), 2020-01-27) Javid, Faizan; Taku, Dr. Anil
The study was conducted to determine the prevalence of ovine foot rot in Jammu and Kashmir (J&K) and to study the serological diversity of Dichelobacter nodosus (D. nodosus) in the area of study. Further, cloning and expression of the fimbrial subunit gene (fimA) ofthe most prevalent serogroup of D. nodosus in the area of study was undertaken. A total of 104 flocks with 2685 sheep from the six administrative districts of J&K (Jammu, Doda Samba, Anantnag, Pulwama and Shopian) were investigated for ovine footrot. Four hundred and sixty six (466) sheep were found to exhibit clinical footrot. The overall prevalence of ovine footrot was 17.35%, ranging from 14.20% to 19.74%. Of the 352 exudate samples (swabs) collected from sheep affected with severe foot lesions, 111(33.43%) were positive for D. nodosus by polymerase chain reaction (PCR), using species-specific 16S rRNA primers. All the D. nodosus positive samples were subjected to serogrouping by multiplex PCR, using nine (A-I) serogroup specific primers. Eighty four (75.60%) samples belonged to serogroup B of D. nodosus, twenty three (20.70%) samples to serogroup E, mixed infection with the presence of both B and E serogroups in same samples was shown by two samples and two of the D. nodosus positive samples were untypable.The present study revealed that serogroup B is the most prevalent serogroup in the area of study, followed by serogroup E. However, besides fimA gene of serogroup B, fimA gene of serogroup E was also undertaken for cloning and expression, due to occurance of serogroup E in significant proportion from the samples taken from Jammu division. Sequence analysis after cloning of fimA genes of D. nodosus serogroups B and E, revealed that the cloned genes had maximum homology with strains JKS-01B and JKS-O8E respectively. The coding sequence of fimA gene (cdsfimA), encoding fimbrial subunit protein was characterized and successfully expressed in E. coli BL21 (DE3), using pET32a(+) expression vector. However, the target protein was found to be toxic to BL21 cells, as the transformed cells could not grow on LB/ampicillin agar without the addition of 1% glucose in the media. This was substantiated by the sizeable decline in viability and division of recombinant BL21cells post induction of expression (IPTG), as judged by decline in turbidity of induced control as compared to the uninduced control. The recombinant D. nodosus fimbrial subunit proteins did not assemble to mature fimbriae in E. coli, after the observation that the expressed protein could not be released from the host without the use of detergent (SDS).Upon SDS- PAGE analysis, the estimated molecular weight of recombinant D. nodosus fimbrial subunit fusion protein (Thioredoxin- 6x Histidine tag) was approx. 35 kDa, which is very close to expected molecular weight. It was further elucidated that increasing the incubation time post induction of expression as well as repeated freeze thawing of cell lysate, increases the level of expression of recombinant protein.The exact identity of recombinant protein was confirmed by western blotting, using Ni-NTA conjugate antibody directed against polyhistidine tagged recombinant fimbrial protein. The fusion protein was purified using Ni-NTA affinity chromatography where relatively purified fraction of the target protein was obtained in elution fractions (2nd& 3rd). Work needs to be continued to assess the protective immune response to the recombinant protein in laboratory animals and eventually formulate a recombinant vaccine using recombinant fimA protein to assess the immune response in sheep.
Open Access
“Pathogenic Strains of Escherichia coli in Buffaloes and their Antibiotic Resistance with Special Reference to Extended Spectrum Beta Lactamases”
(Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu (J&K), 2018-10-15) Janjua, Salena; Bhat, Dr.Mohd. Altaf
The study aimed at finding theoccurrence ofpathogenic strains of Escherichia coli in buffaloes and their antibiotic resistance with special reference to extended spectrumbeta lactamases.Out of a total of 200E.coli isolates from faecal samplesfrombuffaloes, 43 isolates showed the presence of at least one virulence gene studied .Out of these 43 isolates 15(7.50%) isolates carried eae gene either alone or with ehxA gene and thus were detected as EPEC, while 25 (12.50%) isolates carried the stx gene and were detected as STEC. Among STEC isolates seven isolates carried stx2alone, nine carried stx2along with ehxA gene and four carried both stx1and stx2genesalong with ehxA gene,while five isolates carried eae and stx1gene. Three isolates carried ehxA gene alone.All the 43 E.coli isolates which carried any of the virulence genes were subjected to the screening test for detection of resistance to one or both of the twocephalosporinantibiotics (ceftazidime and cefotaxime).Out of 43 isolates five (11.64%) isolates were found to be resistant. Resistance to cefotaxime and ceftazidime was observed in four isolates (80.0%) each and three (60.0%) isolates showed resistance to both. Out of the five isolates that were positive in the screening test, only four (80.0 %) could be confirmed as ESBL producing by phenotypic confirmatory tests.All the isolates which were declared as ESBLs producers phenotypically were tested genotypicallyfor the presence of blaTEM, blaSHV and blaCTX-M genes by PCR.Three isolates were seen positive for blaCTX-Mand one isolate was found positive for blaTEM.
Open Access
Cultural and Metagenomic Characterization of Lower Respiratory Microbiome of Sheep
(Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu (J&K), 2018-07-03) Sharma, Miss RajniKanta; Taku, Dr. Anil Kumar
Sheep is an important species of livestock that has an immense potential to provide livelihood to a large proportion of small and marginal landless farmers, but respiratory diseases are one of the major hurdles in development of this sector. This study was conducted to explore the microbial diversity in lower respiratory tract of healthy as well as diseased sheep using conventional culture based and metagenomic approach using Illumina miseq platform. In cultural analysis a total of 23 bacterial isolates (8 from healthy and 15 from diseased) were found belonging to 4 Phyla, 7 orders, 8 families, 15 genera and 19 species. However, in metagenomic study the 16S rRNA sequences showed high diversity of reads corresponding to, 8 phyla, 21 classes, 38 order, 62 families, 76 genera, and 43 species in healthy sheep. While in diseased sheep the diversity of reads corresponds to 11 phyla, 22 classes, 51 order, 93 families, 105 genera, and 97 species in diseased sheep. Out of these taxanomic units at phylum level proteobacteria was found to be the most abundant in both healthy as well as in diseased sheep (41.15 % in healthy, 37.54% in diseased), while at order level Pseudomonadales were abundant in both healthy and diseased sheep (61.59% in healthy, 47.12% in diseased). At class level Gamma-proteobacteria was abundant in healthy sheep (79.29%) whereas Actinobacteria (61.23%) was abundant in diseased sheep. At family level Moraxellaceae(69.83%) was abundant in healthy and Sphingomonadaceae(54.12%) was the most abundant in diseased sheep. At genus level Acinetobacter (69.75%) was most abundant in healthy sheep while Brevundimonas (63.25%) was the most abundant in diseased sheep. At species level Uncultured Acinetobacter was most abundant (69.54%) in healthy sheep and Pasteurella multocida (37.15%) was the most abundant in diseased sheep. The study revealed presence of a rich and very diverse microbial flora in the lower respiratory tract of sheep and is probably the first comprehensive study on sheep respiratory tract in India that can serve as a benchmark for further research in respiratory diseases of sheep.
Open Access
CHARACTERIZATION OF ENTEROPATHOGENIC ESCHERICHIA COLI FROM DOMESTIC ANIMALS FOR VIRULENCE AND COLONISATION FACTORS
(Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu, 2017) Shikha, Deep; Bhat, Mohd. Altaf
The study aimed at finding the occurrence of, enteropathogenic Escherichia coli (EPEC) and Shiga toxin-producing E. coli (STEC) in different domestic animal species and to characterize the EPEC isolates with respect to their serogroup, accessary virulence attributes like bfpA, astA and ecpA genes and antibiotic resistance. Out of a total of 200 E. coli isolates (one isolate from each sample) from the faecal samples (calves=39, rabbits=24, dogs=38, pigs=22, sheep and goat=36 and poultry=41), 38 (19.00%) were detected as EPEC and rest 28 (14.00%) were identified as STEC. Five (12.82%) and 17 (43.58%) isolates from calves and 12 (33.33%) and 11 (30.55%) isolates from sheep were found to be EPEC and STEC, respectively. In case of rabbits, pigs and dogs, 6 (25.0%), 10 (45.45%) and 5 (13.15%) isolates of E .coli were detected as EPEC, respectively. None of these animals carried STEC. The most predominant EPEC serogroups were O88 (36.84%) and O118 (18.42%) followed by O149 (7.89%), O22 (5.26%) and O11 (5.26%). All the 38 EPEC (100%) isolates carried ecpA gene and only 18 (47.36%) isolates carried astA gene. None of the rabbit EPEC isolate carried the astA gene. Out of 38 EPEC isolates, only 5 (13.15%) isolates from dog carried the bfpA gene and therefore considered as typical EPEC and the other 33 (86.84%) isolates which didn’t carry the bfpA gene were designated as atypical EPEC. Antimicrobial sensitivity test of 38 EPEC isolates showed that resistance was most common to nalidixic acid (55.26%), kanamycin (42.10%) followed by streptomycin (42.10%), doxycycline hydrochloride (28.94%), ciprofloxacin (13.15%), tetracycline (13.15%). Surprisingly, all the 38 isolates were sensitive to ampicillin.
Open Access
MOLECULAR TYPING AND ANTIBIOGRAM OF FIELD ISOLATES OF PASTEURELLA MULTOCIDA
(Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu, 2017) Manhas, Rajeev; Bhat, Mohd. Altaf
The study was aimed to understand the distribution of various serogroups of Pasteurella multocida in bovines, small ruminants, pig, rabbit and poultry from Jammu and to characterize the isolates with respect to LPS synthesizing genes, dermonecrotic toxin gene (toxA) gene and antibiotic resistance. For isolation, the nasopharyngeal swab procedure appeared to be better than nasal swab procedure, particularly in ovine and swine. Out of 200 samples, isolation of P. multocida could be achieved from pig and sheep (5 each) and from poultry and buffalo (2 each) samples only, which accounted for 14 isolates. Upon molecular serogrouping three isolates from sheep and two isolates from poultry were found as serogroup A, two isolates from buffalo were confirmed as serogroup B and five isolates from pig were found to belong to serogroup D. However, two isolates from sheep could not be typed, hence untypable. All the 14 isolates were subjected to mPCR genotyping. A total of 10 isolates, 5 each from pig and sheep, generated an amplicon specific to genotype L6 and L6 indicates Heddleston serovars 10, 11, 12 and 15. Similarly, 2 isolates from bovines generated an amplicon of genotype L2 which indicates Heddleston serovar 2/5. However, 2 isolates from poultry generated specific amplicon with L1 signifying Heddleston serovar 1, but these isolates also produced multiple bands with primer L5. Only, one isolate of capsular type A from sheep possessed the structural gene, toxA for dermonecrotoxin. There was variability in the antimicrobial susceptibility pattern in sheep isolates but overall the rate of tetracycline resistance was relatively high (64.28%) in our strains while all the isolates were sensitive to streptomycin. Except for the swine isolates and one toxigenic sheep isolate, the P. multocida isolates from this study were sensitive to quinolones. Although the level of resistance to commercial antibiotics was generally low, the use of tetracycline and erythromycin was not recommended.

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