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ThesisItem Open Access Immunological and Pathological studies on Clostridium perfringens Type D infection in mice(SKUAST Kashmir, 2020) Kashoo, Zahid Amin; Wani, Shakil AhmedThe present study was undertaken to establish oral infection of Clostridium perfringens Type D in mice and further investigate on immune response upon infection up to 14days post infection (DPI). In the study, pathogenic isolates of C. perfringens Type D from sheep were used. The isolate were confirmed by multiplex PCR harbouring cpa and etx genes, which is confirmatory for C. perfringens Type D. After oral infection, neurological signs like ophisthotonous, convulsions etc were started appearing in most of the animals from 3h post infection and maintained up to 7 DPI, thereafter, recovered from infection. A total of 86 mice were used in the study. For studying immune response, serum concentrations of Th1/Th2 cytokines such as IL-2, IL-4, IL-6, IL-12, IL-13 and IFN-γ (interferon gamma) were quantitatively determined by the ELISA kits at different time intervals up to 14 DPI. The Th1 cytokine, IL-2 started increasing from 3hr post infection and maintained throughout experiment barring on 24hr, however the increase was significant on 48hrs post infection. IFN-γ increased significantly from 3 hrs and the highest peak was on 48-72 hrs. Both Th2 cytokines IL-4 and IL-13 levels were significantly higher throughout infection. The macrophage cytokine IL-12 showed non-significant increase except on 24 hr post infection, however the other proinflammatory cytokine IL-6 showed highly significant increase on 3hr and maintained peak till 96hrs PI. In histopathology, brain and kidney are more affected in subclinical infection. The neuronal changes were observed throughout experimental period as evidenced by in the form of neuronal degeneration and were appreciated by nuclear pyknosis and condensation of nucleus, satellitosis and neuronophagia. Microvascular and mesengial vascular congestion and dilation of perivacular space, focal necrosis, microglial cells proliferation in cerebral cortex was also prominent. In the kidney, degeneration was more pronounced in distal convoluted tubules than proximal tubules. Kidney showed glomerular congestion, degeneration of tubular epithelium, vacuolation of the medullary tubular epithelium and necrosis of the medullary tubules. In general, liver did not show any changes grossly, however, in histopathology, hepatocellular degeneration and cellular swelling were observed. This is first study pertaining to immune response in C. perfringens Type D infection in mice. As the cytokines have been used in the clinical application in treating infectious diseases, the present study enlightens to use of cytokines in controlling entrotoxemia infection in ruminants.ThesisItem Open Access Detection and Characterization of Newcastle Disease Virus in chicken reared in Kashmir Valley(SKUAST Kashmir, 2016) Rafia Maqbool; Wani, Shakil AhmedThe present study was carried out to detect and characterize the Newcastle disease virus in chicken reared in Kashmir Valley. Out of ten disease outbreaks, suspected for ND, nine were positive for ND which included 3, 4 and 2 from Srinagar, Ganderbal and Pulwama districts, respectively.. The clinical and postmortem lesions were characteristic of ND. The virus was isolated on the CEF cell monolayer. APMV1 was detected using Matrix Protein gene (121bp).The positive samples were run for F gene for further characterization. Restriction digestion analysis of the amplified F yielded ~284bp and ~72bp products in all the outbreaks showing the presence of a single type of strain. The representative samples, on cloning and sequencing showed that of the three NDV strains two were identical up to 99.7% and one was 96.9% at nucleotide level; and 100% and 98.3% at amino-acid levels, respectively. The deduced amino acids in the coding region of cleavage site of F gene confirmed the presence of multiple basic amino acids at position 112-116 and Phenylalanine at position 117 viz 112RRQKR*F117 which corresponded to the virulent fusion cleavage site motif . On phylogenetic analysis all the three isolates clustered within the “Genotype VII”. The isolate 1, 2 and 3 showed maximum nucleotide identity with NDV-Pak/NARC-13-HC81103 (97.7%, 96.6%, &98%), followed by China-2004-EU583503 (92.7%, 92.1% & 92.9%),Sweden-97-GU585907(91.5%,91%.&91.8%)and Chicken/Bareilley/India-HQ589257(91%, 89.8% & 91.2%). The phylogeny also revealed that the vaccine virus strains currently used for control of NDV belong to Genotype II and III, respectively, which are far distant from the isolates of Kashmir valley and might not be effective in control of the disease.ThesisItem Open Access Virulence gene profiling and characterization of innate and adaptive immune responses to Avian Pathogenic Escherichia coli adjuvanted with Toll-like receptor agonists in chickens(SKUAST Kashmir, 2018) Syed Tahreem Yasin; Pandit, FarhatAvian pathogenic Escherichia coli (APEC) is the causative agent of Avian colibacillosis, one of the most important disease condition affecting poultry with significant implications on health and production due to severe respiratory and systemic disease in chickens. Pathogenicity of APEC is not entirely associated with serogroups, however, the virulence factors involved in pathogenesis are important to understand the disease process. Toll-like receptors (TLRs) are a group of conserved proteins that play an important role in pathogen recognition in addition to the initiation and regulation of innate and adaptive immune responses. Several TLRs have been identified in chickens, each recognizing different ligands. TLR stimulation in chickens has been shown to play a role in host-responses to pathogens. In view of the impact of APEC as an invasive pathogen in chickens and the potential of TLR agonists as adjuvants in potentiating immune responses, the present study was undertaken to detect virulence genes associated with APEC and to characterize immune response using TLR agonists as immunomodulators with APEC. In the present study, one hundred twenty isolates were screened for five virulent genes considered to be preferential molecular markers for the identification of APEC strains. Screening of APEC revealed presence of iss in (68.33%), iuuC (50.83%), tsh (63.33%), sitA (40.83%), papC (31.66%) genes among the isolates. Serogroups identified from APEC strains from chickens revealed predominant serogroups as O88, O119 and O11. Kinetics of innate and adaptive immune responses to TLR 4 and 21 ligands in various combinations were studied using parameters associated with innate and adaptive immunity. Higher nitric oxide concentrations after primary immunization and booster were observed in APEC+LPS+CpG-ODN group, in contrast to other groups followed by APEC+CpG combination. The study of cellular immune responses viz lymphoprolferation assay by MTT revealed that APEC+LPS+CpG-ODN immunized groups responded well to the respective antigens and induced higher lymphoproliferation in both primary as well as secondary immunized groups as compared to control groups. In chicken PBMC post-immunization, relative expression of immune cytokine and TLR genes showed significantly higher levels in the expression of pro-inflammatory cytokine such as interleukin (IL-1β) and type I interferon (IFN-β) and IFN-γ in APEC+LPS+CpG group. Chicken TLR4 and 21 gene induction was higher in APEC+LPS+CpG group. However, LPS alone did not show significant expression of its cognate receptor in comparison to CpG that showed significantly higher expression of TLR 21 gene. Transcriptional response of relevant protein iNOS is suggestive of TLR stimulation that significantly increased in APEC+LPS+CpG followed by APEC+CpG. Lower expression of iNOS transcript was observed with LPS stimulation alone. Challenge studies revealed that CpG-ODN treatments alone and in combination with APEC and LPS have immunoprotective effect against lethal challenge of APEC giving protection level of 66.67% in chickens.ThesisItem Open Access Molecular typing of Dichelobacter nodosus from ovine footrot and expression of its pilQ gene in E. coli.(SKUAST Kashmir, 2019) Mir, Umul Qurra; Md. Isfaqul HussainFootrot is one of the economically important diseases of sheep particularly in the valley of Kashmir caused by Dichelobacter nodosus (D.nodosus). Protection through vaccination holds promise, but it is found to be serogroup specific and multivalent vaccine is less effective due to antigenic competition. The present study was undertaken to determine the serological diversity of D.nodosus in footrot affected sheep of organized farms and unorganized household flocks including the sheep of nomadic Bakerwal tribe of Kashmir.The gene encoding the PilQ protein was also cloned and expressed for further exploitation as vaccine candidate against virulent footrot. A total of 5475 sheep from different organized farms (Gowbal, Daksum, Kralpathri, Fakir gujri, Mountain Research Center for Sheep and Goat - Shuhama), 61 flocks comprising 2807 sheep in unorganized sector in four different districts namely Ganderbal (Tullamulla, Wayul, Kangan, Shallabugh, Wahidpora, Saloora, Alusteng), Budgam (Sholipora, Yadipora, Qumero, Wasoo, Ringzabal), Srinagar (Fakir gujri, Khimber) and Pulwama (Awantipora) and 14 Bakerwal sheep flocks consisting of 980 sheep were inspected and footrot was recorded in 0.67%, 6.7% & and 3.36% animals, respectively. A total of 260 swab samples (organised farms=37, unorganized sectors=190 and Bakerwal sheep=33) with lesion score of 2 – 4 were collected as per availability of footrot affected sheep and D.nodosus was detected in 56.3to 60.6% samples by direct PCR using 16s rRNA gene specific primers. The samples that were positive for D. nodosus were further subjected to serogroup specific multiplex PCR to determine the serogroups of D.nodosus. Serogroup B was most predominant (83.17%) followed by serogroup E (13.08%) and mixed infection with B and E (3.7%) in unorganized sector. Similarly serogroup B (95.45%) was most prevalent followed by mixed group B and E (4.5%) in organized farms. Likewise serogroup B and E were detected in 90% and 10% positive samples of Bakerwal sheep. The N-terminal outer membrane domain (1251bp) of pilQ gene from D. nodosus was cloned and expressed in pET28a expression vector. The resultant vector construct showed high level expression of the recombinant PilQ protein in BL21 E.coli and confirmed by Western blot analysis. The recombinant PilQ was successfully purified by Ni-NTA affinity chromatography and confirmed by SDS-PAGE.ThesisItem Open Access Genetic Diversity of FMD Virus from Outbreak Cases in Cattle of Kashmir(SKUAST Kashmir, 2019) Sanmeet Kour; Pandit, FarhatFoot-and-Mouth Virus (FMDV) is a highly variable virus prevalent in the form of several serotypes. Understanding the epidemiology and improving vaccinal protection in the cattle of Kashmir requires the knowledge of currently prevalent serotype of FMDV. In the present study we screened the cattle from the four districts of Kashmir valley for the presence of FMDV and determined the phylogenetic nature of FMDV based on the VP1.Out of 32 samples screened, 27 were detected positive for FMDV serotype O. Phylogenetic analysis based on VP1 gene revealed that Kashmir FMDV serotype ‘O’ showed genetic closeness with the serotype ‘O’ prevalent in United Kingdom & Vietnam reflecting panzootic nature of FMDV. Further, we determined the anti-NSP antibody response in clinically affected animals. The results showed only minority of animals were positive for anti-NSP antibodies reflecting delayed immunological response in clinically affected cattle. Overall, the present study provided the molecular evidence for the presence of FMDV among the cattle of Kashmir valley & determined the phylogenetic relation of FMDV prevalent in Kashmir valley with the FMDV from other parts of the world.ThesisItem Open Access Genetic Variation among Campylobacter fetus subsp.venerealis isolated from Cattle(SKUAST Kashmir, 2019) Amani Ishtifaq; Qureshi, SabiaBovine Genital Campylobacteriosis caused by Campylobacter fetus subsp venerealis is of considerable economic importance to the cattle industry worldwide. Campylobacter fetus subsp venerealis is one of the most important infectious agent causing syndrome of temporary infertility in female cattle, early embryonic mortality, aberrant oestrus cycles, delayed conception abortions and poor calving rates. In recent years there has been a steady increase in Brucella negative abortions in organized and unorganized dairy farms of Kashmir valley. The prevalence of BGC in organized and unorganized sector was 29% A total of 200 samples comprising of 103 vaginal swabs, 32 cervicovaginal mucous, 30 preputial washes and 35 semen straws were collected form MLRI, Mansabal, EthoSci Breeding Farm, Pulwama and other unorganized dairy farms. Multiplex PCR assay revealed 49(47.57%) vaginal swabs, 1(3.33%) preputial washes and 8(25%)cervico vaginal mucous samples positive for Campylobacter fetus subsp venerealis, whereas none of the semen straws were positive for Campylobacter fetus subsp venerealis. A total of eleven isolates of Campylobacter fetus subsp. venerealis were recovered on primary isolation. Four different pulsotypes (I-IV) were found to be circulating in the farms. A common pulsotype circulating among farms could not be established. ISCfe1 gene of CFV from Kashmir revealed homology with Dublin, Austria and USA.ThesisItem Open Access Molecular analysis of Orf virus responsible for disease outbreaks in sheep of Kashmir(SKUAST Kashmir, 2017) Ahanger, Showkat Ahmad; Dar, Pervaiz AhmadOrf, also known as contagious ecthyma, is a highly contagious and zoonotic viral disease of small ruminants. Although the disease has been reported in sheep and goat of Kashmir, yet no attempt has been made to characterize the field strains responsible for the outbreaks. In present study, we collected scab material from the suspected Orf cases at four sheep farms and few cases reported at outdoor clinics of the faculty. The presence of Orf virus in scab material was confirmed by the PCR assay targeting major envelope protein (B2L) gene. The outbreak strains were characterized by sequencing and phylogenetic analysis of the viral interferon resistance (VIR) and theB2L gene. Sequence analysis of VIR genes revealed that the gene fragments were 555 bp in all the strains except one strain SBF/Goabal-02/Sheep which had extra 61bp nucleotide at 3′. The identity of VIR at nucleotide level was in the range of 95 to 99% and at amino acid level in the range 97 to 99%. B2L gene came out to be 1206 bp in both the sheep and goat strain with identity of 98 % and 96% at nucleotide and amino acid level, respectively. Phylogenetic analyses revealed that the Orf viral strains from Kashmir can be divided into two clades in both VIR- and B2L-based phylogenetic trees and show more close relation to Indian and Chinese strains. In summary, the study provides first molecular account of the Orf virus strains in sheep and goat of Kashmir and suggests that the Orf strains in Kashmir may have been introduced from India and/or China. More interestingly, the study highlights that the VIR gene sequence can be used to distinguish the host-specific goat and sheep strain which may be important to trace the circulation and movement of Orf strains in Kashmir.ThesisItem Open Access Isolation and Biological Characterisation of Lytic Bacteriophage of Staphylococcus aureus(SKUAST Kashmir, 2016) Ganaie, Mohd Younis; Qureshi, SabiaThe present study aimed at isolation of a lytic bacteriophage of Staphylococcus aureus with a potential for therapeutic use in future. Out of ten sewage and fourteen mastitis milk samples collected form Cattle Research Station, Manasbal five phages were isolated, one from sewage and four from milk samples. Phage isolated from sewage was designated as (SSP) whereas phages from mastitic milk samples were designated as MSP1, MSP2, MSP3 and MSP4. The phages isolated in present study were found to be S. aureus specific. The optimum phage-bacteria ratio (MOI) to achieve 100% lysis of the indicator strain within the shortest period of time (6-7.30hr) was 1:100 for SSP and and 1:50 MSP1 phages, respectively. The PFU count/ml for SSP and MSP1 phages was 2.8 x 107 and 1.8x 109 respectively. The negative stain transmission electron microscopy of the phages revealed phages belonged to order Caudovirales based on head and tail morphology. SSP phage belonged to Siphoviridae family whereas MSP1 was categorized as a Podoviridae phage. Analysis of genomic DNA revealed that SSP was ~23kbp in size whereas MSP was 19 -20kbp approximately. The phages isolated in present study did not reveal any restriction site for . enzymes AluI, BamH1,EcoR1 and HinD111 in their genomes. SDS-PAGE analysis of structural proteins of purified bacteriophage suspensions revealed 15-20 proteins. SSP revealed three major proteins of 12, 15 and 30 kDa whereas MSP1 revealed 12, 15, 30 and 124kDa as its major proteins. Based on TEM imaging, phage protein and genomic DNA analysis SSP and MSP3 were assumed as one phage., The phage counts tend to decrease with increase in pH extremes as well as storage at 0 and -20 oC, both SSP and MSP1 failed to survive at 60 oC.ThesisItem Open Access Molecular characterization of Clostridium perfringens isolated from Poultry(SKUAST Kashmir, 2016) Dar, Parvaiz Sikander; Wani, Shakil AhmadThe present work was undertaken with a view to study the occurrence and molecular diversity of Clostridium perfringens in different species of poultry. A total of 224 samples (184 from chicken, 29 from turkeys and 11 from ducks), comprising of feacal material and intestinal contents were collected from poultry of different regions of Kashmir. Robertson cooked meat media was found to be effective for isolation and isolates produced appreciable growth on Sulphite Polymixin Sulphadiazine (SPS) agar. The screening of isolates by standard morphological and biochemical assays revealed that 63 of 224 samples possessed C. perfringens like organism. The identity of the isolates as C. perfringens was ascertained using species-specific alpha gene amplification which depicted expected 324 bp product. Highest occurence of C. perfringens was observed in chickens around the age group of 2-6 weeks. All the 63 isolates were screened for six toxin genes viz; cpa, cpb, etx, cpi, cpb2 and cpe using a multiplex PCR. Out of 51 C. perfringens isolates from chickens, all (100%) isolates belonged to toxinotype A. Out of 9 isolates from turkeys and 3 isolates from ducks,, all (100%) isolates belonged to toxinotype A. None of the isolate was found to be C. perfringens toxinotype B, C, D or E. In chicken, among C. perfringens toxinotype A isolates 9 (17.64%) of the isolates carried cpb2-gene and none of the isolates carried cpe-gene in addition to the species specific cpa gene. In turkeys and ducks, among C. perfringens toxinotype A isolates none of the isolates carried cpb2-gene or cpe-gene in addition to the species specific cpa gene. Pulsed field gel electrophoresis (PFGE) of selected isolates from three Government Poultry farms and post mortem samples from Division of Veterinary Pathology revealed similarities and differences at genetic level indicating similar clones in Hariparbath and isolate from Division of Pathology (Pulsotype II) and different clone in Mattan from other Farms (Pulsotype III). The isolates from turkeys represents the different clonal type from that of isolates from chickens (Pulsotype I).
