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  • ThesisItemOpen Access
    SEROEPIDEMIOLOGY AND DETECTION OF PESTE DES PETITS RUMINANTS VIRUS IN SHEEP AND GOATS OF SAURASHTRA REGION OF GUJARAT 3264
    (JAU JUNAGADH, 2021-07) TAJPARA MAHESHKUMAR M.; Dr. N. M. Shah
    Peste des petits ruminants (PPR) is a severe viral disease of goats and sheep with high morbidity and high mortality characterized by fever, erosive stomatitis, conjunctivitis, gastroenteritis and pneumonia. PPR is caused by Peste des petits ruminants virus (PPRV), a Paramyxovirus of the Morbilivirus genus. The virus continues to be reported periodically across India and Gujarat state. The present study was aimed to conduct overall, locationwise, specieswise , sexwise and agewise as well as pre & post vaccination seroprevalence survey by using c-ELISA and to detect PPRV in clinical samples using S-ELISA, N & F gene based RT-PCR and cell culture method from clinical samples. A total of 828 (Prevaccinated ) & 423 (Postvaccinated) serum samples from sheep and goats of Jamnagar, Rajkot, Surendranagar, Amreli and Bhavnagar districts of Saurashtra region of Gujarat were screened for PPR specific antibodies using PPR specific c-ELISA. Overall percent seroprevalence of PPR was found to be 53.86% (446 / 828) & 72.34 % (306 / 423) in prevaccinated & post vaccinated small ruminants respectively . Overall percent prevaccinated/ post vaccinated seroprevalance rates were highest in Bhavnagar district (83.89% / 96.30%), followed by Surendranagar (73.61% / 77.78%), Amreli (56.25% / 83.33%) , Jamnagar (32.22% / 43.33 %) and Rajkot (27.78% / 64.44 %) districts. Pre & Post vaccination seropositivity was noted higher in goats (59.90% / 78.75%) than (35.75% / 52.43 %) in sheep. Males showed higher seroprevalence (60.11% / 89.01%) as compared to their female counterparts (52.09% / 67.77 %).Higher Prevaccinated seroprevalence (63.41%) was recorded among age group of 1 to 2 years, followed by below 1 year age group (53.99%) and above 2 years of age group (44.20%). A total of 119 different clinical samples (nasal swab, conjunctival swabs, oral swabs and tissue samples) from goat and sheep were collected from the area under study for detection of PPR antigen by Sandwich-ELISA. Out of 119 clinical samples, 37 samples were found positive in small ruminants by S-ELISA, giving an overall incidence rate of 31.09 % (37/119). In goats, 30.52 % and sheep 33.33 % samples were detected positive. District wise incidence of PPRV in small ruminants differed non significantly. It was recorded in Bhavnagar (33.90%), Amreli (29.41%) and Rajkot (26.92%) districts. Month wise incidence of PPRV in small ruminants differed non significantly. It was recorded in month of October (29.41%), November (31.58%) and December (33.33%). Age wise incidence of PPRV in small ruminants differed non significantly. It was recorded in below 1 year of age group (39.29%), 1 to 2 year (28.95%) and above 2 years of age (16.00%). Sex wise incidence of PPRV in small ruminants differed non significantly. It was recorded in male (30.43%) and female (31.51%).Breed wise incidence of PPRV in small ruminants differed non significantly. It was recorded (36.62%) in non descript breed and (22.92%) in descript breed. Out of 119 clinical samples, 37 samples including 13 Nasal swabs, 3 conjunctival swabs, 7 oral swabs and 14 tissue were found positive. PPRV antigen was detected by S-ELISA in tissue (66.67%), oral swab (43.75%), nasal swab (20.97%) and conjunctival swab (15.00%). Most suitable sample for virus isolation was tissue and oral sample. 37 representative clinical samples positive for PPRV by S-ELISA were subjected for isolation and propagation of PPR virus in vero cells. Out of 37 S-ELISA positive samples, 31 samples showed CPE on first passage of sample, which was confirmed by RT-PCR. One hundred nineteen clinical samples (40 nasal swabs, 20 conjunctival swabs, 16 oral swabs, and 9 lung, 4 trachea, 3 spleen, 3 intestine tissues from goat along with 22 nasal swabs and 1 lung, 1 intestine tissues of sheep) were tested by N gene & F gene based primers. Out of 119 samples, 35 samples produced 351 bp amplicon with N gene primer and 32 samples produced 372 bp amplicon with F gene primer. Out of the total samples tested PPRV could be detected in 37, 35 and 32 samples by S-ELISA, N gene RT-PCR and F gene RT-PCR respectively. Thirty one samples were positive to all four tests. Two samples negative by N gene RT-PCR were found positive by S-ELISA. Relative to S-ELISA, sensitivity and specificity of N gene based RT-PCR was 94.59 and 100 percent respectively. Overall agreement between the two tests was 98.32 percent. Relative to S-ELISA, sensitivity and specificity of F-gene based RT-PCR was 86.49% and 100% respectively. Overall agreement between the two test was 95.80% . Relative to S-ELISA sensitivity of cell culture was 83.78%. All three PPRV sequence obtained from Bhavnagar, Amreli, Rajkot were belonged to lineage IV (Indian strains). All three PPRV sequence showed 100 % genetic identity with eachother & 98-99 % genetic identity with PPRV strain of Mehsana (North Gujarat), Bhopal (Madhayapradesh), Pune (Maharashtra), Jhansi (Uttarpradesh), Sungri (vaccine strain) based on partial N gene sequence.
  • ThesisItemOpen Access
    SEROEPIDEMIOLOGY, CULTURAL AND MOLECULAR DETECTION OF BRUCELLA INFECTION IN RUMINANTS OF SOUTH SAURASHTRA REGION OF GUJARAT
    (JAU,JUANAGDH, 2020-12) J. B. Kathiriya; Dr. N. M. Shah
    Brucellosis is an important zoonosis and a significant cause of economic losses to farmers. Abortion, placentitis, epididymitis and orchitis are the most common clinical manifestations in animals. In view of the considerable problems related to direct diagnosis of brucellosis in animals, the present study envisaged the appraisal of seroepidemiology of brucellosis in ruminants. For the purpose, three serological tests viz., Rose Bengal Plate Test (RBPT), Lateral Flow Assay (LFA) and Indirect-Enzyme Linked Immunosorbent Assay (iELISA) were employed. The Biochemical and molecular characterization of isolates was carried out for the final confirmation. A total of 770 serum samples from five districts (Junagadh, Rajkot, Amreli, Gir Somnath and Porbandar) of South Saurashtra region of Gujarat were collected to detect the brucella specific antibodies. A total of 304 clinical samples with reproductive disorders comprised of deep vaginal swabs (125), vaginal discharge (76), placenta (33), placental cotyledons (12), aborted foetal organs (lung-11, liver-11, heart blood-18 and stomach contents-18) were collected from ruminants. The clinical samples were cultured on Brucella agar medium (BAM) with selective antibiotic supplements. The Brucella isolates obtained from positive samples were tested by using genus and species specific PCR. Out of 770 ruminants sera screened, 116(15.06 %), 84(10.91%) and 124 (16.10 %) sera samples were detected positive by RBPT, LFA and iELISA, respectively. Species wise seroprevalence reported to be 18.13, 13.13 and 18.75 % in cattle, 12.09, 8.79 and 13.19% in buffaloes, 10.71, 7.14 and 13.39% in sheep and 15.38, 11.54 and 16.03% in goats by RBPT, LFA and iELISA, respectively. While, Sex wise seroprevalence rates of brucellosis were 14.37, 8.98 and 13.17% in males (167) and 15.26, 11.44 and 16.92% in females (167) by RBPT, LFA and iELISA, respectively. Higher seroprevalence was observed in females than in males. Location wise analysis revealed, seroprevalance in Junagadh district 14.33, 10.75 and 15.96 %, Rajkot district 16.88, 11.045 and 18.33 %, Amreli district 17.82, 12.61 and 14.41 %, Gir Somnath district 17.82, 11.88 and 13.86 % and in Porbandar district 10.31, 8.25 and 16.49 % by RBPT, LFA and i-ELISA, respectively. The overall higher rate of Abstract ii seroprevalence was noticed in Rajkot district (18.33%) followed by Amreli/Gir Sonmath districts (17.82 %) and 16.49% in Porbandar district as compared to other districts of South Saurashtra region of Gujarat. Overall sensitivity of RBPT and LFA was found to be of 83.06% and 65.32%, while specificity was 97.99% and 99.54%, respectively, considering iELISA as a gold standard test. Thus, RBPT was found to be more sensitive and less specific than that of LFA. The overall positive and negative likelihood ratio was 41.28 & 140.66 and 0.17 & 0.35 for RBPT and LFA respectively as compared to iELISA. The positive predictive value was 88.79% and 96.43%, while negative predictive value was 96.79%, and 93.73% for RBPT and LFA, respectively as compared to iELISA. McNemar chi-square test for independent data revealed non-significant difference for RBPT, while significant difference for LFA considering iELISA as gold standard technique. The concordance of RBPT and LFA was good (k=0.832 and k=0.746, respectively) considering iELISA as gold standard test. The clinical samples (304) subjected to cultural isolation on Brucella agar medium (BAM) with selective antibiotic supplements. Out of these, 17(5.59%) samples (cattle-8, buffaloes-4, goats-4 and sheep-1) yielded bacterial isolates. Which were confirmed as B. abortus by biochemical and molecular characterization. 17 isolates were recovered from vaginal swab-3 (2.4%), vaginal discharge-3 (3.95), placenta-6 (18.18%), placental cotyledons-3 (25%) and aborted foetal stomach content-2 (11.11%) from clinical samples of ruminants. For confirmation of isolates, colony duplex-PCR were carried out targeting Brucella genus specific BCSP-31 and IS-711genes to get 223bp and 350bp PCR products and all isolates were confirmed as Brucella spp. While, all 17 isolates were identified as B. abortus using desired amplified product of 498bp through multiplex PCR technique. The nucleic acid sequences obtained by sequencing of studied genes (BCSP31, IS711 and alkB) of Brucella isolates were aligned with published sequences available at GenBank (NCBI, USA). The nucleotide sequence alignment of the BCSP31, IS711 and alkB genes of Brucella showed as 97.9 to 100%, 98.3-100% and 95.3-100% homology with Brucella species, respectively. It shows that either BCSP31or IS711 gene is the best to study the genetic homogeneity in Brucella. Phylogenetically, the BCSP31, IS711 and alkB genes of B. abortus sequences obtained from the Junagadh isolate coming within the broader clade of different common species of Brucella. In conclusion, serology coupled with biochemical and molecular tests confirmed the prevalence of B. abortus in South Saurashtra region of Gujarat.
  • ThesisItemOpen Access
    ISOLATION AND CHARACTERISATION OF BACTERIOPHAGE AGAINST MULTI DRUG RESISTANT STAPHYLOCOCCI OF ANIMAL ORIGIN
    (JAU,JUNAGADH, 2018-06) ANIRUDDHSINH M. ZALA; Dr. B. S. MATHAPATI
  • ThesisItemOpen Access
    PHENOTYPIC AND MOLECULAR CHARACTERIZATION OF STAPHYLOCOCCAL CASSETTE CHROMOSOME MEC (SCCMEC) TYPES OF METHICILLIN-RESISTANT STAPHYLOCOCCI FROM ANIMAL AND HUMAN ORIGIN 2513
    (JAU,JUNAGADH, 2018-03) SANJAY N. GHODASARA; DR. J. H. PUROHIT
    The present study was carried out with the objectives of isolation, identification and molecular characterization of Staphylococcal Cassette Chromosome mec (SCCmec) types along with antimicrobial resistance patterns and presence of virulent genes (toxic genes) in methicillin-resistant staphylococci from animals and humans. Out of 202 animals and 100 human nasal swabs, 86 (42.57%) and 62 (62%) isolates were Staphylococcus spp., respectively based on biochemical and molecular based identification. The antibiogram study revealed higher rates of methicillin, gentamicin, ofloxacin and levofloxacin sensitivity to human isolates, whereas higher susceptibility to amikacin and rifampicin followed by oxytetracyclin and chloramphenicol were observed in animal isolates. Out of total 86 and 62 staphylococci isolates, 74 and 50 isolates were Coagulase Negative Staphylococci (CoNS), 12 (from each) were Coagulase Positive Staphylococci (CoPS). Of these total Staphylococci isolated from both the species, 9 and 20 isolates were identified as Methicillin-Resistant Staphylococci (MRS) from animal and humans, respectively. Out of these MRS isolates, 8 and 18 were Methicillin-Resistant Coagulase Negative Staphylococci (MRCoNS), 1 and 2 isolates were Methicillin-Resistant Coagulase Positive Staphylococci (MRCoPS) from animal and humans, respectively. One isolate was identified as Methicillin-Resistant Coagulase Negative Staphylococcus aureus (MRCoNSA) from the animals and 2 isolates were identified as MRSA from humans. Of these 2 isolates, one isolate was Methicillin-Resistant Coagulase Negative Staphylococcus aureus (MRCoNSA) and one isolate was Methicillin-Resistant Coagulase Positive Staphylococcus aureus (MRCoPSA). The study conducted for presence of virulence genes and their SCCmec typing on total 235 Staphylococcus spp. including departmental isolates which included, 148 Staphylococcus spp. (86 from animal milk/pus samples and 62 from human nasal swabs) from present study and 87 Staphylococcus spp. (47 from animal milk/pus samples and 40 from human nasal swabs) from departmental isolates. Out of these, 16 (12.03%) from animals and 40 (39.21%) from humans were having mecA gene which were classified as Methicillin-resistant staphylococci. Of these, 3 (2.26%) different isolates were found positive for all these virulence genes i.e. PVL, hla and icaA from Abstract… animals, whereas 7 (6.86%), 6 (5.88%) and 4 (3.92%) isolates were found positive for PVL, hla and icaA gene from humans, respectively. The SCCmec typing of MRS isolates were studied from 16 animals MRS isolates, of these, 14 isolates having one of the SCCmec types (SCCmec type I, 2; SCCmec type II, 0; SCCmec type III, 1; SCCmec type IV, 5; SCCmec type V, 6), whereas 2 isolates were Untypable. Out of total 40 humans MRS isolates, 28 isolates having one of the SCCmec types (SCCmec type I, 7; SCCmec type II, 0; SCCmec type III, 3; SCCmec type IV, 9; SCCmec type V, 9), whereas 12 isolates were Untypable. Based on SCCmec typing, 18.75% (3/16) and 25% (10/40) isolates were classified as hospital associated methicillin-resistant staphylococci (HA-MRS), whereas 68.75% (11/16) and 45% (18/40) isolates were classified as community associated methicillin resistant staphylococci (CA-MRS) from animal and humans, respectively. The overall percentage of CA-MRS (63.04%) was higher as compare to HA-MRS (28.26%) among both the species. The detection of SCCmec types IV and V suggested the prevailed of CA-MRSA strains in this geographical area and occurrence of SCCmec I and II alleles indicated a possible transmission of MRSA from human to animals. The prevailed of same SCCmec types among animal and humans attribute to transmission of MRS from animal to human or vice versa indicating potential zoonotic pathogen prevalence in farm and farm workers.
  • ThesisItemOpen Access
    BACTERIOLOGICAL STUDIES AND MOLECULAR DETECTION OF MAJOR PATHOGENS FROM SUBCLINICAL AND CLINICAL BOVINE MASTITIS 2511
    (JAU,JUNAGADH, 2018-03) BHAVESHKUMAR B. JAVIA; Dr. J. H. PUROHIT
    The present study was carried out with an objective to screen bovine milk samples for status of subclinical mastitis (SCM) by somatic cell count (SCC), to isolate and identify major mastitis pathogens viz: S. aureus, E. coli and predominant Streptococcal species, to evaluate the antimicrobial resistance patterns and to optimize multiplex PCR for rapid and simultaneous detection of these pathogens in the milk samples. Total 390 bovine milk samples (180 from clinical cases of mastitis and 210 from apparently healthy animals) were collected from in and around Junagadh district. According to the measurement of SCC in 180 milk samples, 34.29% prevalence of SCM was observed. A primary culture isolation of 252 milk samples (72 SCM and 180 clinical mastitis milk samples) revealed 60.26% prevalence of bovine mastitis. The prevalence of mastitis caused by S. agalactiae, S. dysgalactiae and S. uberis was found 13.59%, 2.31% and 0.77%, respectively. The overall prevalence of mastitis caused by Staphylococcus spp. was 38.72% which is highest among all other isolated bacteria, while of S. aureus and CoNS were 15.64% and 23.08% respectively. The prevalence of mastitis caused by E. coli and other bacteria were noted 10.77% and 3.59%, respectively. The prevalence of mastitis caused by mixed bacterial infection was 9.49 %. In the present study high resistance of Staphylococcus spp. Streptococcus spp. and E. coli observed against ceftriaxone and amoxicillin-sulbactam which displays antibiotic usage pattern in this region. Likewise bacterial isolates studied were highly sensitive to levofloxacin which suggest judicious use of this antibiotic in treatment of bovine mastitis. The molecular detection of major mastitis causing organisms was also carried out by standardizing PCR. The tuf gene and 16S rRNA was targeted to detect Abstract… Streptococci and Staphylococci at genus level. Further, sip, 16S rRNA and pauA gene were targeted to detect S. agalactiae, S. dysgalactiae and S. uberis respectively. The screening of 65 Streptococcal isolates revealed 53, 9 and 3 isolates as S. agalactiae, S. dysgalactiae and S. uberis respectively. S. aureus was detected by targeting nuc gene and out of 151 isolates of Staphylococci screened, 61 isolates revealed the presence of nuc gene indicating S. aureus. E. coli were detected by targeting alr gene in 42 isolates, which signifies the superiority of molecular diagnostic tools. The two tube mPCR for simultaneous detection of five major mastitis pathogens from milk samples was optimized in this study to reduce the time and labour involved in individual detection. The efficiency of this assay was compared with conventional microbiological methods. The optimized mPCR showed promising results with 100% diagnostic sensitivity and 83.87% diagnostic specificity. The mPCR assay optimized in the present study is very rapid and better option to choose as diagnostic tool for detection of major mastitis pathogens directly from milk samples as compare to conventional methods and can be useful for formulating the strategy for prevention and control of bovine mastitis.