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Open Access
BUBALINE NEUTROPHILS FUNCTIONAL ACTIVITY AND LYMPHOCYTES BLASTOGENESIS RESPONSES IN Escherichia coli INFECTION, in vitro: EFFECT OF IMMUNOMODULATION
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-11) VIJAYA KAMESWARI, VEDULA; ANAND KUMAR, P (MAJOR); RAMANI PUSHPA, R.N.; BINDU KIRANMAYI, CH.
The present study was conducted to investigate the immunomodulatory activity of phytochemicals thymol and quercetin, on innate and adaptive immune responses of buffaloes in correlation to in vitro phagocytic activity of neutrophils and lymphocytes blastogenesis responses against Gram negative bacteria Escherichia coli. Neutrophils eliminate pathogens by phagocytosis which is an important host defense mechanism. An important activity of phagocytes is their ability to respond to stimuli by activation of the respiratory burst. Therefore, the phagocytic activity of neutrophils is estimated by nitro blue tetrazolium (NBT) assay. Also, the effect of phytochemicals quercetin and thymol on phagocytic activity of bubaline neutrophils during in vitro E. coli infection is studied. The phagocytic activity of neutrophils treated with different concentrations of quercetin (25 μM and 50 μM) and thymol (15 μg/ ml and 30 μg/ ml) separately, and incubated with live E. coli/ opsonized E. coli/ zymosan is found to be increased compared to their normal counterparts (without immunomodulators) with significant difference (p<0.05). The immunomodulators quercetin and thymol increased the activity of superoxide anion in neutrophils, which is reflected interms of increased absorbance that is an indicator of increased phagocytic activity. Adaptive immunity is very important as it is specific immune response directed against a targeted antigen. Among the peripheral blood mononuclear cells (PBMC), lymphocytes account for major population. Since, the proliferation is a fundamental characteristic of the lymphocytes, induction of lymphocyte proliferative responses by antigen/ mitogen in vitro with PBMC culture is referred as a representative index for cellular immunocompetence. Therefore in the present study lymphocyte transformation test or lymphocyte proliferation assay is performed by stimulating the PBMC’s of buffaloes with mitogen Concanavalin A (Con A) and antigen E. coli (heat inactivated), separately, using a tetrazolium salt XTT (2, 3-Bis-(2-methoxy-4-nitro-5-sulfophenyl]- 2H-tetrazolium-5-carboxyanilide salt) assay. In this study stimulation of immunomodulators treated PBMC with heat inactivated E. coli resulted in more stimulation index (S.I) compared to stimulation of immunomodulators treated PBMC with Con A. Antigenic stimulation appeared to stimulate the PBMC with more intensity than that of Con A stimulation. These results provided leads about the immunostimulatory role of the phytochemicals in increasing the phagocytic activity of neutrophils and proliferative responses of PBMC in in vitro cultures. Further research is required to establish the mechanism of action underlying it.
Open Access
BUBALINE NEUTROPHILS FUNCTIONAL ACTIVITY AND LYMPHOCYTES BLASTOGENESIS RESPONSES IN Staphylococcus aureus INFECTION, in vitro: EFFECT OF IMMUNOMODULATION
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-11) ASHOK KUMAR, K; ANAND KUMAR, P (MAJOR); LAKSHMI KAVITHA, K; ASWANI KUMAR, K
Buffaloes are important dairy animals of Andhra Pradesh (64.33 lakhs population) that contribute 69% of the total milk produced in the state. Staphylococcus aureus is one of the the most important Gram-positive bacterial pathogen that causes pneumonia, mastitis, phlebitis, meningitis, urinary tract infections, osteomyelitis, endocarditis and superficial skin lesions such as furunculosis etc in animals. Immune response against invading pathogens is crucial for keeping up the host’s health status. The desired optimal immune response in a host may be achieved through immunomodulation. Many synthetic, microbial and natural products are reported to act as immunomodulators. Phytochemicals are naturally occurring plant derived compounds with bioactive potentials and are used as immunomodulators in the treatment and management of infections. Phagocytic activity of neutrophils and lymphocytes proliferation assay or lymphocyte transformation test with PBMC which are considered as indicators of innate and adaptive immune responses, respectively, are included in this study to investigate the immunomodulatory activity of phytochemicals curcumin and quercetin. In this study blood samples were collected from apparently clinically healthy she buffaloes (4 to 5 years of age with similar physiological status) and infected in vitro with S. aureus isolated from clinical mastitic milk sample. Phagocytic activity of the neutrophils was measured by semi quantitative nitroblue tetrazolium assay (NBT). The reduction of NBT to formazan by the superoxide anion generated in the respiratory burst is measured at 540 nm. Bubaline neutrophils are treated with different concentrations of curcumin and quercetin, separately and incubated with live S. aureus / opsonized S. aureus / zymosan. The phagocytic activity of bubaline neutrophils treated with 25 μM curcumin and incubated with opsonized S. aureus is significantly different (p<0.05) with phagocytic activity of neutrophils treated with 50 μM curcumin and incubated with opsonized S. aureus. The phagocytic activity of bubaline neutrophils treated with 50 μM curcumin and incubated with opsonized S. aureus is significantly different (p<0.05) with phagocytic activity of neutrophils treated with 50 μM of quercetin and incubated with opsonized S. aureus. The in vitro lymphocyte proliferation assay is routinely used as a measure to assess the functional status of lymphocytes. Though initially radioactive isotopes were used in cell proliferation assays due to radiation hazards associated with radio isotope assays the tetrazolium salt-based assays are employed for the proliferation assay. In the present study XTT {sodium3'-[1-[(phenylamino)-carbonyl]-3, 4-tetrazolium]-bis (4-methoxy-6- nitro) benzene-sulfonic acid hydrate} assay was used in cell proliferation assays. The stimulation index (S.I) of PBMC stimulated with Con A is significantly different (p<0.05) with the S.I of PBMC treated with 25 μM & 50 μM of quercetin and stimulated with Con A. The S.I of PBMC stimulated with Con A is significantly different (p<0.05) with the S.I of PBMC treated with 25 μM & 50 μM of quercetin and stimulated with heat inactivated S. aureus. Similarly, the S.I of PBMC treated with different concentrations of immunomodulators is significantly different (p<0.05) with other related groups. Therefore it is concluded that the phytochemicals curcumin and quercetin exhibited immunomodulatory activity on innate and adaptive immune response of buffaloes. Further research is required to establish the mechanisms of action underlying it.
Open Access
MOLECULAR EPIDEMIOLOGY OF CANINE PARVOVIRUS IN ANDHRA PRADESH
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-08) DEEPIKA KUMARI, G; Ramani Pushpa, R.N(MAJOR); Subramanyam, K.V.; Srinivasa Rao, T; Satheesh, K
Canine parvovirus-2 (CPV-2) is one of the most pathogenic viral etiologic agents causing hemorrhagic enteritis in dogs. CPV is a small, non-enveloped, single-stranded DNA virus of approximately 5 kb genome and belongs to the genus Protoparvovirus, in the family Parvoviridae.The objective of the present work is to detect, isolate and characterize circulating CPV in different locations of Andhra Pradesh from fecal samples of diarrhoeic dogs and its phylogenetic characterization. A total of 342 fecal samples were obtained from diarrhoeic dogs and preliminary screening of the samples was carried out by haemagglutination assay (HA) using 0.8 % swine RBC, out of which 71 were positive with a titre ranging from 1 in 32 to 1 in 512 (20.76%). The results of HA was confirmed by haemagglutination inhibition assay (HI) using hyperimmune serum raised in rabbits. All 342 fecal samples were screened by PCR using CPV-2ab primers and 233 samples produced an amplicon size of 681 bp. Genotyping of CPV was done by employing multiplex PCR using CPV-2ab and CPV-2b primer pairs. Out of 233 positive samples, 216 (92.70%) samples produced an amplicon size of 681 bp characteristic of CPV-2a and 17 samples (7.29%) yielded two specific amplicons of 681 bp and 427 bp and thus categorized as CPV-2b. The fecal samples not reacted with CPV-2ab and CPV-2b primers were further analysed by PCR-RFLP using restriction enzyme MboII for the detection of CPV-2c strain. Only one sample reacted with 555 primers produced an amplicon size of 583 bp but remain undigested with restriction enzyme. The overall prevalence rate of CPV in Andhra Pradesh was found to be 68.42%. Virus isolation studies from the PCR positive fecal samples were carried out using CRFK and MDCK cell lines. Successful isolation was observed in both the cell lines with varied cytopathic effects. Sixteen out of thirty four processed fecal samples in CRFK cell lines could produce a mild CPE after 72h post infection (PI) at fifth passage level which include increased granularity, rounding of cells and degenerative changes. Five out of nine CPV positive samples produced marked CPE like increased granularity, rounding of cells and complete detachment of cells in MDCK cell lines after 72h PI at third passage. The presence of virus at each passage level was confirmed by PCR assay using H primers. Transmission electron microscopic studies revealed spherical virus like particles of approximately 20 nm in diameter. The Partial VP2 gene sequencing of eighteen CPV field isolates along with the vaccine strain was performed in AB13730 DNA Analyzer and with Chromas lite software. The VP2 gene sequences were compared with CPV reference strains available in the GenBank by BLASTn analysis and were found to be highly specific revealing a maximum identity of 99.84% to 100% with CPV-2a and 99.35 with CPV-2b. One sample which only reacted with 555 primer on sequence analysis, exhibited a nucleotide homology of 99.66 % with CPV-2a. Multiple sequence alignment for the 18 CPV field isolates along with the vaccine strain was done using Clustal W 1.8 program and compared with the reference strains of CPV. The Partial VP2 nucleotide sequences identity of the field isolates when compared with the reference strains was 99.99 % to 100% and a similar identity was also observed among the field isolates. The nucleotide sequences for local CPV-2a isolates exhibited 99.99 % of homology with those of CPV-2b isolates. The nucleotide homology levels between the vaccine strain (CPV-2) and the analysed CPV-2a, CPV-2b isolates was 99.98 and 99.97 %, respectively. The antigenic types CPV-2a and CPV-2b differed from the original CPV-2 in atleast four amino acid positions of the VP2 capsid protein. Amino acid sequence analysis was done using MEGA 7.0 and revealed that thirteen out of 18 found to be new CPV-2a, one as a variant of new CPV-2a and four were characterised as new CPV-2b. Based on the typing systems using key amino acid positions, 77.77% were new CPV-2a and 22.22% were new CPV-2b with no CPV-2c or original CPV-2 found. The phylogenetic analysis revealed that the 14 new CPV-2a isolates were having close ancestral relationship with the Indian and Chinese strains of CPV-2a whereas CPV-2b strains formed a separate clade with the Indian CPV-2b strains. Seroprevalence of CPV was done by Indirect ELISA and HI for a total of 542 sera samples. Indirect ELISA recorded 94.65% in vaccinated and 72.50% in unvaccinated dogs whereas HI detected 56.10% in vaccinated and 16.42% in unvaccinated dogs. The variation in the detection of CPV antibodies by Indirect ELISA and HI from vaccinated and unvaccinated dogs was statistically significant (P < 0.05).
Open Access
GENETIC DIVERSITY OF BOVINE, OVINE AND PORCINE ISOLATES OF Pasteurella multocida
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-04) JAGADESH, J; Lakshmi Kavitha, K(MAJOR); Ramani Pushpa, R.N.; Srinivasa Rao, T
The Gram-negative bacterium, Pasteurella multocida, is an important worldwide primary and opportunistic pathogen as well as the common commensal of many wild and domesticated animals. The present work was taken up to characterize and observe genetic diversity of bovine, ovine and porcine isolates of P. multocida. A total of 283 samples were collected from apparently healthy slaughtered animals (276) and clinical cases (7). Primarily the suspected samples were streaked directly on Brain heart infusion (BHI) agar. Further when all the suspected samples subjected to PMPCR, 86 (30.3%) were found positive. Out of 86 PM-PCR positive samples, 31 were identified (bovine 8, ovine 11 and porcine 12) as pure cultures of P. multocida by morphological tests, different cultural and biochemical tests. Further the 31 isolates were finally confirmed by employing PM-PCR using a primer set KMT1T6-KMT1SP6 with 460 bp amplicon and all the isolates were found positive in PM-PCR. The total isolation percentage of 10.9% was observed. The capsular typing was done by Uniplex PCR targeting Cap A and Cap D capsule specific gene primers, yielded 20 Cap A isolates, 8 Cap D isolates and 3 untypable isolates. The virulence gene profile of isolates showed high prevalence of hgbA gene (77.41%) among the three iron binding proteins, followed by hgbB gene in 41.9% of isolates, among these lowest prevalence was observed in bovine isolates. Gene tbpA has lowest prevalence (19.35%) among the three iron binding proteins. Moreover, the tbpA gene was highly associated with ovine isolates. The higher prevalence of adhesion related gene such as ptfA (76.66%) was observed. This gene was detected 100% in porcine isolates. Dermonecrotxin gene was noticed in high percentage in ovine isolates (27.27%), while only one isolate of bovine harbored this gene. The pfhA gene revealed clear association to Cap A strains (50%), and none of the other capsule types of P. multocida harbored this gene. The dermonecrotoxin toxA was found in 25% of Cap D strains, followed by 10% of Cap A. Moreover, P. multocida strains of capsule type A, and D showed high prevalence of the ompH, ptfA and hgbA compared to cap-negative strains. Molecular typing techniques (REP-PCR, ERIC-PCR and (GTG)5-PCR) differentiated all 49 strains (isolates from this study and previous isolates) into different profiles. All the isolates were found genetically distinct from standard P. multocida strain P52 (Vaccine strain of India). Genetic diversity among the isolates of P. multocida and their genetic dissimilarity with P52 vaccine strain showed an indication about vaccine strategy. This study provided a clear evidence of presence of more than one isolate type in host species and also provided indication of high genetic variation among field isolates of P. multocida and may be the reason of vaccine failure and outbreaks. Furthermore the isolation of different capsular types from single host species warrens the need for the use of polyvalent vaccine against Pasteurella infections in their respective hosts.
Open Access
MOLECULAR CHARACTERIZATION AND PHYLOGENETIC ANALYSIS OF CANINE DISTEMPER VIRUS IN DOMESTIC DOGS
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-03) KEERTHI, BANDARU; RAMANI PUSHPA, R.N.(MAJOR); LAKSHMI KAVITHA, K; SRINIVASA RAO, T
Canine distemper is a highly contagious viral disease caused by Canine distemper virus (CDV) affecting dogs and other wild carnivores throughout the world. CDV is a morbillivirus under Paramyxoviridae family causing highly systemic infection with prominent respiratory, gastrointestinal and nervous signs in dogs and other wild carnivores. The objective of the present work is to isolate and characterize CDV in domestic dogs and its Phylogenetic analysis. The study was aimed to detect CDV from clinical samples of the suspected dogs by diagnostic RT-PCR. The positive samples were subjected to virus isolation in MDCK cells and the presence of viral RNA was confirmed by RT-PCR. All the clinical samples including brain tissue were confirmed for the presence of viral RNA with the partial L gene with an expected amplicon size of 268 bp and the positive L gene cDNA samples were further amplified for H and F genes. All the three genes were sequenced and phylogenetically analyzed with the CDV strains available in GenBank. Different clinical samples viz. ocular, nasal, and fecal swabs, blood, urine and brain tissue were collected from 48 CD suspected dogs and then screened by RT-PCR. Out of 48 suspected samples, 34 (70%) were found positive. The most common clinical signs recorded in the positive cases were pyrexia (102.8-105°F), mucopurulent ocular and nasal discharges, hyperkeratosis of digital pads, vesiculo-pustular dermatitis and nervous disorders. MDCK cell line was successfully used to isolate CDV. Three number of RT-PCR positive ocular samples were propagated in MDCK cells for CDV isolation and all the three initially did not produce characteristic CPE but from third passage onwards characteristic CPE like rounding, aggregation, ballooning and clumping of chromatin material was observed. The presence of virus in all the cell culture harvests was confirmed by RT-PCR using L gene based diagnostic primers. The cell culture harvests were ultracentrifuged and the virus pellet showed amplification of L and H genes. Purification of virus was done by discontinuous sucrose density gradient centrifugation and the purified virus was subjected to SDS-PAGE for protein profiling. The bands of sizes approximately 180KDa (L), 76KDa (H), 66KDa (P), 58KDa (N) were visualized. Brain and spleen tissues were processed for histopathological examination. Lymphoid depletion and focal areas of necrosis in the white pulp of spleen was observed. Histopathology section of brain revealed infiltration of neutrophills, lymphocytes, perivascular cuffing and necrosis of neurons. The partial L, H and F gene sequencing was performed to compare the genetic variation among the field isolates and vaccine strain. All the field isolates of CDV partial L, H and F genes showed 94.6-98% of nucleotide sequence identity with that of the other CDV reference strains available in GenBank. The Onderstepoort vaccine showed 95%, 92% and 90% homology with the partial nucleotide sequences of L, H and F genes of CDV field isolates respectively. The nucleotide sequences identity of L gene among the field isolates in the present study was 98 to 99%. The CDV partial L gene field isolates showed highest similarity with the strains of Ludhiana and Japan. Whereas the partial H and F gene field isolates showed highest similarity with the strains of UP and France isolates respectively. All the field isolates showed lowest similarity with commercially available vaccine strains. The obtained sequences were also compared with the reference CDV sequences available in GenBank to establish the phylogenetic lineage. The results generated with the obtained sequences of the field isolates for L, H and F genes made a distinct clade in phylogenetic tree which was clearly separated from the commercial CDV vaccine strains and grouped with the Ludhiana, Japan, UP and France isolates. The phylogenetic tree based on partial L gene sequence revealed that the two local isolates were grouped together with the Ludhiana strain and another isolate with the Japan strain
Open Access
MOLECULAR CHARACTERIZATION OF Pasteurella multocida AND Mannheimia haemolytica FROM SHEEP PNEUMONIA
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-01) VIDYA SUSMITHA, K; LAKSHMI KAVITHA, K(MAJOR); SRIVANI, M; RAMADEVI, V
Pasteurella multocida and Mannheimia haemolytica are responsible for major economic problems in ruminant production worldwide. These organisms cause respiratory diseases and shipping fever under stress in sheep. The objective of the present study is molecular characterization of Pasteurella multocida and Mannheimia haemolytica from sheep pneumonia. A total of 195 lung samples were collected and were streaked on brain heart infusion agar. The suspected colonies were subjected to P. multocida species specific PCR (PM- PCR) and M. haemolytica species specific PCR using PHSSA and Rpt2 genes by multiplex PCR and found 12 (6.15%) and 15 (7.69%) positives, respectively. Out of 12 PM-PCR positive samples, 7 pure cultures of P. multocida were obtained. Further the isolates were confirmed by conducting PM-PCR and all gave positive amplification of 460 bp product. The total isolation percentage 3.59% was observed. Among the 15 PHSSA and Rpt2 positive samples of M. haemolytica 10 were isolated as pure cultures. Later the cultures were confirmed by PCR. The isolation percentage of M. haemolytica observed was 5.12%. P. multocida capsule biosynthesis gene showed Cap A and Cap D with 28.57% and 57.14%, respectively. One isolate was untypable. The distribution of virulence genes showed pfhA (14.28%), tbpA (71.41%), ompH (57.14%), ptfA (0%), hgbA (57.14%), hgbB (0%) and toxA (57.14%). It was observed Cap D association with toxA gene and Cap A with pfhA gene. The virulence genotyping of M. haemolytica was conducted using lktA gene in which 30% of the isolates possesed this gene. The isolates of P. multocida revealed antibiotic sensitivity to gentamicin, cotrimoxazole, ceftriaxone, ampicillin, sulfamethoxazole, streptomycin, amoxicillin-clavulanic acid, nalidixic acid (100%), followed by chloramphenicol and colistin (71.42%), amoxicillin (57.1%), enrofloxacin (4.85%), clindamycin (28.57%). However the isolates were resistant or intermediately resistant to tetracycline (100%). The distribution of antibiotic resistance genes were catA1 (100%), strA (57.14%), strB (71.42%) and tetB (42.85%). The results of sensitive antibiogram pattern of M. haemolytica revealed gentamicin, cotrimoxazole, ceftriaxone (100%), ampicillin, sulfamethoxazole (90%), chloramphenicol, nalidixic acid (80%), amoxicillin-clavulanic acid (60%), tetracycline (40%), amoxicillin (3%), colistin, enrofloxacin (1%). The distribution of antibiotic resistance genes showed tetB (40%) and absence of other genes studied. The gross pathology of the infected lungs showed cranioventral consolidation and pleural adhesions. The histopathological changes observed in the affected tissues were infiltration of neutrophils and mononuclear cells into the bronchial lumen, alveoli and bronchopneumonia was evident. The research work paved way for development of conventional vaccines using different capsular types. It also paved way for future development of rapid diagnostic kits and probable recombinant vaccines using the properties of virulence genes.
Open Access
STUDIES ON ANTIMICROBIAL RESISTANCE OF Escherichia coli ISOLATES FROM LAMB DIARRHOEA CASES
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2018-12) SUJATHA, T; Srivani, M(MAJOR); Subramanyam, K.V.; Srinivasa Rao, T
A study was carried out on the isolation, molecular characterization, and antimicrobial resistance of E. coli isolates from 1-7, 8-30, 31-60 and 61-90 day-old diarrhoeic lambs from Vizianagaram, West Godavari and Krishna districts of Andhra Pradesh. A total of 212 samples were collected, from which 170(80.18%) E. coli were isolated. Highest prevalence of E. coli was observed in West Godavari (86.41%) and Vizianagaram (82%) districts, while lowest prevalence was found in Krishna district (72.83%). Among different age groups, highest prevalence of E. coli was observed in 1-7 day-old diarrhoeic lambs (84%) while lowest prevalence was detected in 61-90 day-old diarrhoeic lambs (72.72%). Among the E. coli isolates, 87.05% were shiga toxigenic (STEC) and none of the isolates belonged to enterotoxigenic (ETEC). Among the virulence genes of STEC, eaeA &hlyA genes were highest (35.13%) followed by 12.83, 11.48, 10.13, 8.10, 5.4, 4.72 and 2.70% isolates carried stx1; hlyA; stx2;stx2&eaeA; stx1&eaeA; stx2&hlyAand stx1&hlyA and all STEC gens (stx1, stx2, eaeA&hlyA),respectively. Out of 96 hlyA carrying E. coli isolates, only seven isolates did not show any haemolysis on sheep blood agar. Highest antibiotic resistance was observed for the E.coli isolates against colistin (98.82%) and sulphamethizole (89.41%) while enrofloxacin (5.88%), gentamicin (5.33%), and chloramphenicol (1.17%) were effective. Among the STEC isolates, highest antimicrobial resistance (100%) was observed to colistin followed by sulphamethizole (95.94%), while chloramphenicol (1.35%) was effective. An ESBL phenotype was confirmed in a total of 72 STEC isolates. β lactamase genes were detected in 91.21% of STEC isolates with blaTEM being the predominant gene detected (91.21%) followed by blaCTX-M group 1 (77.70%,), blaCTX-M group 2 (10.13%) blaOXA (4.72%,), blaSHV, blaTEM+OXA, and blaCTX-9 (3.37%,), CTX-1+CTX-9 (2.02%) and SHV+OXA (1.35%), respectively. Clove oil was able to inhibit 70% and 50% of multidrug resistant E. coli by well and disc diffusion methods while cinnamic acid did not show any antibacterial activity by both the methods. The present study provides an insight on prevalence of multidrug resistant E. coli against which herbal extracts like clove oil may be effective in treating lamb diarrhoea cases.
Open Access
STUDIES ON BIOFILM INHIBITION AND ANTIMICROBIAL RESISTANCE IN S. aureus AND STREPTOCOCCUS SPECIES CAUSING BOVINE MASTITIS
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2018-12) JOSEPH, GREESHMA ANN; RAMANI PUSHPA, R.N.(MAJOR); LAKSHMI KAVITHA, K; SRINIVASA RAO, T
Bovine mastitis is recognised as the most economically important disease affecting Dairy industry in India and all over the world. Most prevalent bacterial etiological agents identified in bovine mastitis are Staphylococcus aureus, Streptococcus species, E. coli and other Gram-negative organisms. The preferred treatment regime during the past decades for mastitis is antibiotic therapy. The associated antimicrobial resistance and recurrent infections caused by slime producing bacteria is the major constraint. The time has elapsed to think about new treatment methods and agents. Hence the present study is on the biofilm forming S. aureus and Streptococcus species causing bovine mastitis and the effect of antibiofilm agents on the antimicrobial resistance of the microorganisms. A total of 91 Bovine mastitic milk samples were examined and out of this 75 (82.41 %) samples were positive for S. aureus and Streptococcus species. A total of 110 isolates were obtained, the prevalence observed was S. aureus 62 (56.36%), other Staphylococci 2 (1.81%), S. uberis 44 (40%) and other Streptococci 2 (1.81%). All S. aureus and Streptococcus species isolates were confirmed by genus specific PCR followed by species specific PCR. Two Streptococcus isolates did not react to any of the specific primers used for S. uberis, S. agalactiae and S. dysgalactiae. Biofilm formation was detected using qualitative Congo red agar method (CRA), quantitative microtiter plate (MTP) assay and biofilm gene was detected using PCR. On CRA method 88.71% S. aureus isolates and on MTP assay 98.39 % isolates were biofilm producers. The ica locus was detected in 93.54% of the S. aureus isolates. The icaA gene was detected in 87.09% isolates and icaD was detected in 40 isolates. Fifty eight percent were carrying both icaA and icaD genes. On CRA 80.43% Streptococcus species isolates and on MTP assay 84.78% were biofilm producers. Among S. uberis isolates 34.09% were positive for the biofilm gene luxS by PCR. Twenty four isolates without luxS gene also produced very weak or moderate biofilm. On antibiotic sensitivity test the S. aureus isolates were resistant to Penicillin G, Ampicillin, Erythromycin, Methicillin, Clindamycin, Amoxycillin/Clavulanic acid, Streptomycin and were least resistant to Gentamicin, Cotrimoxazole, Chloramphenicol, Tetracycline and Vancomycin. All MRSA isolates were found to be biofilm producers while 96.87 % of MSSA were biofilm producers. MRSA was showing 2 to 4 times more resistance to all tested antibiotics than MSSA. The Streptococcus species showed high resistance to Ceftriaxone followed by Streptomycin, Erythromycin, Penicillin G, Tetracycline, Enrofloxacin, Amoxycillin/Clavulanic acid and least resistance to Gentamicin, Chloramphenicol and Ampicillin/ Sulbactam. Biofilm forming isolates were highly resistant to Ceftriaxone (66.66%), Streptomycin (41.02%), Erythromycin (38.46%), Tetracycline (33.33%) and Penicillin G (30.76%), whereas non biofilm formers showed considerably low resistance to Ceftriaxone (28.57%), Penicillin G (14.28%), Streptomycin (14.28%) and were 100% sensitive to all other antibiotics used. Biofilm inhibition studies were conducted in biofilm producing S. aureus and Streptococcus species identified by MTP. The mean±SE values of inhibition rates by 30 μg /ml Ursolic acid (UA), 100 μg /ml UA, 30 μg /ml resveratrol and 100 μg/ml resveratrol on 26 S. aureus isolates were found to be 40.67±4.64%, 62.03±3.61%, 37.38±4.86% and 53.66±4.25% respectively. Inhibition rates of antibiofilm agents on MSSA were found to be higher than MRSA except for the isolates treated with resveratrol 100 μg /ml concentration. Biofilm inhibition studies were conducted in 31 Streptococcus isolates. The mean±SE values of inhibition rates by 30 μg /ml UA, 100 μg /ml UA, 30 μg /ml resveratrol, 100 μg /ml resveratrol was 33.96±3.17%, 57.40±2.8%, 31.35±3.12% and 46.28±3.47%, respectively. Also, antimicrobial resistance of the isolates treated with antibiofilm agents at concentrations of 100 μg /ml UA and 100 μg /ml resveratrol for 18h was found to be decreased by at least 50% for each antibiotic.
Open Access
MOLECULAR DETECTION AND CHARACTERIZATION OF CLOSTRIDIUM PERFRINGENS TOXIN GENES RESPONSIBLE FOR NECROTIC ENTERITIS IN POULTRY OF ANDHRA PRADESH
(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2008-04) PRAVEEN KUMAR, N; VINOD KUMAR, N(MAJOR); RANI PRAMEELA, D; SUJATHA, K
ABSTRACT: The present study was carried out to investigate the prevalence of Clostridium perfringens toxin genes responsible for necrotic enteritis in poultry of Andhra Pradesh. A total of 691 faecal samples (396 necrotic enteritis suspected and 295 apparently healthy) comprising of cloacal swabs from live birds and intestinal scrapings from dead birds were collected from different districts of Andhra Pradesh viz., Chittoor, Guntur, Nellore, Krishna, East Godavari and West Godavari. Gross pathological studies of affected birds revealed necrosis of the small intestinal mucosa and submucosa with fibrin deposition resulting in pseudo membrane formation and turkish towel appearance was noticed in the small intestine. Microscopically lumen of intestine with fibrinonecrotic material which forms a visible pseudo membrane composed of cell debris, necrotic/distorted villi, inflammatory cells and clumps of bacteria were observed. The samples were inoculated in to fluid thyoglycollate broth and incubated overnight. DNA extracted from 24 h broth cultures by boiling method were screened by multiplex PCR for simultaneous detection of α, β and β2 toxin genes. Out of 396 (broiler 282 & layer 114) necrotic enteritis suspected samples 337/396 (85.1%) were positive for α toxin gene of which 189/337 (56.08%) were β2 toxin gene positive. Out of 295 (broiler 182 & layer 113) apparently healthy samples 61/295 (20.67%) were positive for α of which 4/61 (6.55%) were β2 positive. None of the sample showed amplification of β toxin gene indicating the absence of C. perfringens type C. As some recent studies focused the involvement of NetB toxin in pathogenesis, therefore, uniplex PCR amplification of NetB gene was done from alpha toxin gene positive samples (C. perfringens type A) yielded no positives for NetB toxin gene. From chi square analysis a significant difference (p<0.01) in the prevalence of toxin genes (cpa & cpb2) between necrotic enteritis suspected and apparently healthy at 99% level of confidence with an increased number of positives from necrotic enteritis suspected group. The present research indicates C. perfringens type A along with β2 toxin gene might be responsible for causing necrotic enteritis in broilers and layers in Andhra Pradesh. The multiple sequence analysis of the amplified partial cpa and cpb2 gene sequences revealed 100% homology between the present isolates, and with selected published sequences from GenBank were found to be 98-99% and 94-99% homology respectively. The phylogenetic analysis of the cpa gene of the present C. perfringens isolate (MG600591) with the selected published sequences of cpa revealed the close segregation in distinct clade with cpa gene of broiler isolate of C. perfringens (GU581186) from Iran. The phylogenetic analysis of three cpb2 sequences of present isolates (MF471364; MF471366; KX001813) segregated into close group of poultry originated sequences with exception of MF471365 which segregated in distinct clade with noporcine originated C. perfringens sequence (AY609173) from USA. Since alpha toxin gene (cpa) is considered as signature toxin gene for C. perfringens, amplification of cpa by PCR is considered as confirmative diagnosis of C. perfringens. Hence, in the present study all the PCR positives for cpa (n=398) were isolated by culturing revealed only 221/398 (55.52%) isolates indicating PCR is more sensitive in detecting C. perfringens when compared with isolation by culturing. In the present study culture supernatant of B. subtilis isolated from healthy intestinal contents of birds successfully inhibited C. perfringens by disk diffusion method indicating its importance as a probiotic in controlling necrotic enteritis in poultry.

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