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ThesisItem Open Access Bacteria associated with respiratory infections in poultry(Department of Veterinery Microbiology, College of Veterinary and Animal Science, Mannuthy, 2007) Jesto, George; KAU; Krishnan Nair, GThis study was undertaken to isolate and identify of bacteria from respiratory tract of poultry and to study the antibiogram of the isolates. Birds showing respiratory signs were sacrificed, postmortem examination was conducted and samples such as nasal, tracheal and air sac swabs and lungs were collected after taking all sterile precautions. A total of 105 samples were collected by sacrificing birds showing clinical signs. Isolation of causative bacteria was made by culturing on brain heart infusion agar, Mac Conkey agar and blood agar. For identification of isolates all the procedures were followed as described by Barrow and Feltham (1993). A total of 31 bacterial isolates were obtained from samples. A total of 12 Escherichia coli isolates were isolated and identified, 4 Pasteurella multocida isolates and 15 Staphylococcus sp. Isolates were isolated and identified biochemically. Out of 15 Staphylococcus sp. isolated and identified 11 isolates (73.33 per cent) were coagulase negative This result indicate that CoNS were more frequently isolated from staphylococcal infections although they do not possess the virulent coagulase activity. So importance must be given to CoNS also, as given to coagulase positive staphylococci and much study need to be diverted to find the virulence factors and role of them in producing bacterial infections in poultry. Multi drug resistance (resistance to at least three antimicrobials) was found among all E. coli isolates obtained in the study. Hence it may be concluded that the high level of resistance observed among poultry E coli isolates obtained in the study may be due to incorporation of antibiotics in feed as growth promoters. As 100 per cent sensitivity is shown to enrofloxacin and chloramphenicol by P. multocida isolates, these two drugs may be used for treating pasteurellois. Amoxycillin clavulanic acid (Ac) and cephalexin (Cp) was found to be the most effective antibiotic against Staphylococcus sp. in the study. The plasmid DNA content of the seven isolates of E. coli was analysed on agarose gel electrophoresis but correlation between the number of plasmids and antibiotic resistance could not be ascertained in this study. In conclusion, the results of this study provide evidence for significant antimicrobial resistance among bacterial isolates from birds. Long term prospective studies involving isolation, identification and antibiogram from more samples are required to identify novel pathogens causing respiratory disease in birds. Such studies provide data on temporal and spatial difference in antibiotic resistance patterns, which in turn helps the scientific community to design better disease control strategies.ThesisItem Open Access Development and evaluation of outer membrane protein vaccine against duck pasteurellosis(Department of Veterinary Microbiology, College of Veterinary and Animal Science, Mannuthy, 2007) Ranjini, A R; KAU; Krishnan, Nair GA research work was undertaken to prepare effective vaccines against P. multocida grown under different conditions and their immunopotency was assessed in one month old ducklings. The purity of the P. multocida A:l strain (DP1) was confirmed as per standard procedure. Pathogenicity of the isolate was assessed in six to eight weeks old mice. The isolate killed the mice with in 8 h intra peritoneally and within 24 h when injected subcutaneously. Whole cell protein of P. multocida (DP1) was extracted and they were subjected to discontinuous system of SDS- PAGE which revealed 20 to 26 visible protein bands of molecular weight ranging from 102 to 19 kDa. After growing P .multocida in iron sufficient and iron restricted medium its OMPs were extracted and they were analysed by SDS PAGE. In iron sufficient medium, 10 protein bands of MW which ranging from 91.84 to 19.02 kDa were revealed. In addition to the above a protein band with MW of 97.8 kDa were detected in iron restricted medium.The protein concentration was estimated by Modified Lowry method,and it was found to be 4mg/ml. The Median Lethal Dose (LDso) of P. multocida when determined in one, month old ducklings was found to be 10 -7.13 which contained 13 cells. Oil adjuvanted formalin inactivated vaccines were prepared from DPl grown under three different conditions viz., TSB, BHIB and BHIB supplemented with 100 u M 2,2' dipyridyl. Sterility, safety and potency test of the vaccines were done as per standard procedures. A total of 120 one month old ducklings were divided in to four groups with 30 birds in each group. The first three groups were vaccinated with ordinary bacterin, OMP vaccine prepared under iron sufficient condition and OMP vaccine prepared under iron restricted condition respectively and the fourth group served as control.The birds were vaccinated with 0.5millilitre of vaccine intramuscularly. The blood was collected from all the ducks on day 0,7, 14,21,28,45 and 60 day PV. Passive haemagglutination was done and the increase in antibody titre was observed from day 7 PV onwards for groups I,.Il and In. The highest antibody titre was obtained at 14th day PV and 21 st day PV for aMP vaccine prepared under iron restricted condition. All the vaccine groups had shown a significant difference from the control group at all stages of study. On homologous challenging OMP vaccine under iron restricted media on 42nd day PV afforded 60 per cent protection. On 60th day PV afforded 50 per cent protection. Though the aMP vaccine prepared under iron restricted condition was found to provide high antibody titre, the protection percentage afforded by it in comparison with ordinary bacterin and aMP vaccine prepared under iron sufficient media was low. Hence, the aMP vaccine prepared under iron restricted condition need to be subjected to further investigation.ThesisItem Open Access Evaluation of pathogenicity and antigenic relationship of salmonella isolates from poultry(Department of Veterinery microbiology, College of Veterinary and Animal Science, Mannuthy, 2007) Sunil, G; KAU; Koshy, John (Guide)In the present study the pathogenicity and the antigenic relationship of three local isolates of S. gallinarum was compared with that of a reference/known strain. The antigenic homogeneity/heterogeneity of these isolates was studied using SDS PAGE and AGID. All the four isolates when subjected to antibiogram revealed similar pattern. The only difference was with isolate X 4 which was susceptible to co-trimoxazole while all the other three were resistant. Except for the reference/known strain BGL, all the other three isolates were found pathogenic to mice. All the four isolates were pathogenic to day-old chicks. Isolates QS 1 and X 4 were most pathogenic. Major clinical signs were somnolence, weakness, inappetance and whitish diarrhea and were more prominent in IM inoculated group. All chicks that died during the experimental period gave positive Salmonella isolation from liver, spleen, heart blood, lungs, yolk and ceca. At the end of experimental period survived birds were sacrificed and they gave positive isolation only from ceca. All the four isolates were pathogenic to layers. The clinical signs observed were listlessness, inappetance, ruffled feathers, shrunken comb, greenish-yellow diarrhea with gradual weight loss and was more prominent in IV challenged group, followed by those inoculated by oral and IC routes. The most pathogenic strain was QS 1. Cloacal swabs examined for Salmonella revealed intermittent shedding in all groups. The highest re-isolation was obtained from liver. The egg production was affected in all test groups and was most severely affected in IV group. No significant difference was noticed between different routes of inoculation and among different isolates in re-isolation from egg. The re-isolates were confirmed as S. gallinarum by standard biochemical reactions. The gross and histopathological changes observed in experimental infection with all the isolates were the same. There was enlargement and necrosis of liver and spleen, with congestion of heart and lungs. Day-old chicks had omphalitis. Histopathologically there was congestion, necrosis and infiltration of inflammatory cells into the internal organs, including submucosa of intestine. Sodium dodecyl sulphate - polyacrylamide gel electrophoresis analysis of the OMPs of all four isolates revealed 13 different polypeptide bands. These bands were of similar molecular weight in all the four isolates, indicating antigenic homogeneity. Agar gel immunodiffusion carried out using hyper immune serum raised against isolates BGL and QS 1 revealed lines of identity between the four isolates, indicating antigenic homogeneity by serology.ThesisItem Open Access Detection of rotavirus in the faeces of diarrhoeic calves by reverse transcriptase- polymerase chain reaction and silver staining(Department of Veterinary and Microbiology, College of Veterinary and Animal Science, Mannuthy, 2007) Ambily, R; KAU; Koshy JohnA study was undertaken to detect the presence of rotavirus in the faeces of diarrhoeic calves by RT-PCR and RNA-PAGE. Agar Gel Immune Diffusion was performed to detect the presence of rotaviral antigens in faecal samples using hyperimmune serum raised in rabbits. The protein profile of BRV was analyzed using SDS-PAGE. Attempts were made to isolate BRV from faecal samples in MDBK cell line. One hundred and twenty four faecal samples of diarrhoeic calves were collected from University Livestock Farm, Mannuthy, Veterinary Hospitals of Kerala Agricultural University, some dairy farms in Thrissur district and also from individual farmers in and around Thrissur. Twenty samples each were collected from adult cattle above one year of age with diarrhoea and normal healthy calves. All these samples were screened for the presence of BRV by RNA-PAGE, RT-PCR and AGID. Among 124 faecal samples collected 29 (23.39 per cent) samples were detected as positive by RNA-PAGE. The clustered arrangement of the 11 segments of the genome showed a 4:2:3:2 migration pattern, typical of group A bovine rotavirus. Reverse Transcriptase – Polymerase Chain Reaction could detect BRV in 35 (28.23 per cent) samples. By using AGID, only 16 (12.90 per cent) samples were found positive. Among the various tests employed, RT-PCR was found to be more sensitive in the diagnosis of BRV infections. All the 20 faecal samples from adult cattle with diarrhoea were tested negative by the three methods. Rotavirus could not be detected in the faeces of healthy calves by any of the tests employed. The protein profile of BRV revealed nine polypeptides having molecular weight in the range of 16.5 to 131 kDa. The age-wise distribution of BRV infection in calves was studied. It was found that the occurrence of infection was most common in zero to two weeks of age (39.39 per cent) followed by two to four weeks of age (37.04 per cent). The faecal samples which were found positive by RNA-PAGE was inoculated into MDBK cell line. But the attempts to isolate BRV were found unsuccessful.ThesisItem Open Access Production and evaluation of vaccines employing Pasteurella multocida A:1 grown under different growth conditions(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2007) Raja Gopal, R; KAU; Krishnan Nair, GA research work was undertaken to prepare effective vaccines against P. multocida grown under different conditions and their immunopotency assessed in one month old ducklings. The purity of the Pasteurella multocida A: 1 strain (DP1) was confirmed as per standard procedures. Pathogenicity of the isolate was assessed in six to eight weeks old mice. The isolate killed the intraperitoneally inoculated mice within eight hours and within 24 h when injected by subcutaneous route. The organism was grown in different media to assess the amount of capsular material produced. The media employed were DSA, DSA supplemented with 10 per cent FBS and DSA supplemented with 10 per cent FBS and 0.5 per cent yeast extract. The capsule enhancement was measured by capsule demonstration using Maneval staining and by characterization of crude capsular extract. The capsules of the organisms grown in capsule enhancement media when demonstrated using Maneval staining were discernibly larger and denser, when compared to the organisms grown in DSA alone. The capsular polysaccharides increased by approximately 1.6 times when the organism was grown in capsule enhancement media containing 10 per cent FBS and by about 2.06 times when grown in media supplemented with FBS and yeast extract. The potential of the organism to form in vitro biofilm was assessed by growing the organism in nutrient restricted conditions. The organism was grown in 0.32 per cent TSB supplemented with an inert substrate called bentonite clay, for the bacteria to attach and colonize. For quantification of biofilm, plate count by spread plate method was employed and the results showed that the biofilm cells reached a peak on the third day of incubation with an average count of 1.54 ×106 CFU/g of bentonite clay while the planktonic cells were found to be maximum on day one post inoculation, with a peak count averaging about 8.10 ×108 CFU/ml of the broth. Maneval staining of late logarithmic phase of three day old biofilm culture revealed heavily capsulated cells of P. multocida attached as large aggregates and appearing as chains of coccobacillary cells. Also they appeared as a meshwork of aggregated and chain forming cells. The capsular polysaccharide estimation of biofilm cells revealed a 3.25 times increase over the planktonic bacteria grown in DSA alone. The biofilm cells on nutrient agar after 24 h at 37°C produced some colony morphotypes characterized by radiating strands from centre to periphery and wavy margin. Median lethal dose (LD50) of P. multocida when determined in one month old ducklings was 23 cells. In the present study, it was unable to arrive at the median lethal dose in six month old ducks as only one duck died after 48 h of virulent challenge. Oil adjuvant formalin inactivated bacterin vaccines were prepared from DP1 grown in TSB, capsule enhancement medium and under biofilm mode and performed the sterility, safety and potency tests of the vaccine employing standard procedures. A total of 160 four week old ducklings were divided into four groups with 40 birds in each group and the first three groups were vaccinated with ordinary bacterin, capsule enhanced bacterin and biofilm vaccine respectively. The fourth group served as control. The birds were vaccinated with 0.5 millilitre of vaccine intramuscularly in the thigh region. Blood was collected from all the ducks pre-vaccination, at weekly intervals upto 28th day post PV and on day 42 PV by cardiac puncture or by jugular venipuncture. Passive haemagglutination using GA-SRBC sensitized with sonicated antigen of DP1 was used to measure the humoral immune response. The IHA titres obtained for biofilm vaccine group on day 14 was very much higher than the other two groups. The antibody titre was observed from day seven onwards for all the groups. All the vaccine groups have shown significant difference from the control group at all the stages of the study. Groups I and II were having no significant difference in their mean titres during the entire study. On homologous challenging, biofilm vaccine gave higher protection rates of 70 and 90 per cent than the 60 and 80 per cent protection rates of ordinary and capsule enhanced bacterins, when challenged with 200 and 100 LD50 doses respectively. Biofilm vaccine was proved to be the best among the three vaccines tried. The capsule enhanced vaccine did not provide any additional advantage over the ordinary bacterin vaccine. Elaborate field trials are to be done before advocating the vaccine for commercial use.ThesisItem Open Access Polymerase chain reaction based screening of bovine mastitis milk to detect leptospira and mycoplasma(Department of Veterinery Microbiology, College of Veterinary and Animal Science, Mannuthy, 2007) Jibi, M G; KAU; Krishnan Nair, GIn the present study an attempt has been made to detect the Leptospira and Mycoplasma from bovine mastitis milk by PCR. Simultaneously trials were also made to isolate these fastidious organisms from these milk samples. Direct screening of milk sample by PCR could not successfully detect the DNA of either of these organisms. All the previous studies had revealed that inhibitory factors present in the milk might interfere with the amplification of target DNA present in the milk. To overcome this difficulty initially all the milk samples were preenriched in the broth media, subsequently the target DNA was extracted. The genus specific primer A and B were used to detect the leptospires in milk samples. Out of fifty haemagalactic milk samples screened by PCR only one sample was positive for Leptospira. Fletcher’s semisolid media that could support the growth of reference strain of Leptospira pomona was used for the isolation study. But all the attempts to recover the organism from milk sample were unsuccessful. For the detection of Mycoplasma, genus specific primers MGSO and GPO3 were used. Out of the fifty samples screened only one sample was positive for Mycoplasma. But the M.bovis specific primers PpMB 920-1 and PpMB 920-2 failed to amplify the DNA that was found positive in the genus specific PCR. Since several species of Mycoplasma was found to be associated with the mastitis, organisms detected in this study might not be M.bovis. The media used for the isolation of Mycoplasma were BHI broth and BHI agar. Media were standardized with the reference strain and were found to be capable of supporting the growth of organisms. But all the attempts to isolate the Mycoplasma from milk samples were unsuccessful. It was concluded that PCR is a rapid and sensitive technique to detect the fastidious organisms that are difficult to isolate in the ordinary laboratory conditions. But one difficulty encountered with the PCR from milk samples was the extraction of good quality template DNA that contained fewer inhibitors. Since the rapidity and ease of PCR is largely dependent upon the extraction procedure adopted in each bio materials, the protocol followed in this study needs some modifications.ThesisItem Open Access Detection of infectious bovine rhinotracheitis virus by immunofluorescence and polymerase chain reaction(Department of Veterinary Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2008) Anupama, K; KAU; Koshy, JohnA study was undertaken to detect the presence of IBR virus in clinical samples by PCR and immunofluorescence and to compare the efficacy of these tests. Attempts were also made to isolate the virus from clinical samples in MDBK cell line. A total of 60 samples from suspected animals and 5 samples from healthy animals were collected from various sources like, University Livestock Farms, Veterinary Hospital, Mannuthy and some private dairy farms in Thrissur district. The samples included nasal and vaginal swabs, aborted materials and semen. All these samples were screened for the presence of IBR virus by PCR and immunofluorescence. The TK gene based primers were used for PCR and the positive control generated an amplicon of size 298bp. No sample was found positive by PCR. Immunofluorescence was performed on smears prepared from clinical samples using FITC conjugated polyclonal antibovine IgG. This test detected viral antigen in one of the samples, indicated by focal, bright yellowish green fluorescence associated with the cytoplasm of epithelial cells. The inability of PCR for detection of viral DNA in sample tested positive by immunofluorescence may be due to the presence of non-specific inhibitors in clinical sample. These results suggest that even though PCR is highly sensitive compared to other diagnostic tests, false negative results cannot be excluded. Immunofluorescence on smears prepared from clinical samples can be considered as a rapid diagnostic method for IBR. Attempts to isolate the virus from clinical samples in MDBK cell line were found unsuccessful.ThesisItem Open Access Development and evaluation of whole cell and membrane protein vaccines against mycoplasma gallisepticum(Department of Veterinary Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2009) Surya, Sankar; KAU; Krishnan Nair, GA study was carried out to develop and evaluate the efficacy of vaccines against M. gallisepticum infections in Gallus domesticus using whole cell and membrane proteins produced under iron sufficient and restricted conditions, incorporating the adjuvants aluminium hydroxide and saponin either alone or in combination. A total of 50 samples were collected in broth and in addition 25 tracheal swabs were streaked onto PPLO agar plates also, from birds showing respiratory ailments. Mycoplasma genus specific PCR revealed twelve positive samples and on subjecting these samples to species specific PCR, a total of four samples showed positive results. From four species specific PCR samples, two isolates could be obtained, named as MG UPF-1 and MG UPF-2, which were sub cultured and subjected to morphological, biochemical and serological characterization. The SDS-PAGE profile of whole cell proteins of the two isolates revealed similar profile, indicating their homogeneity. Whole cell and membrane proteins were isolated from cultures grown under iron sufficient and iron deficient conditions. Both the whole cell and membrane proteins revealed bands ranging from 24 kDa to 200 kDa on SDS-PAGE. The whole cell proteins had two prominent bands at 75 kDa and 35 kDa and eight faint bands, which the membrane proteins lacked. The whole cell and membrane proteins produced under iron restricted conditions had an additional band of 52 kDa, which was not seen in the respective protein groups isolated under iron sufficient condition. Most of the proteins resolved on SDS-PAGE in the four sets of proteins were found to be antigenic as found out by western blot analysis. The optimum concentration of the proteins used for vaccine preparation was 84.2 µg per dose of the vaccine. Formalin inactivated vaccines were formulated with saponin and aluminium hydroxide alone and in combination at a final concentration of 100 µg/dose and 25 per cent v/v respectively, thereby producing eight different sets of vaccines. The sterility and safety testing of the vaccines were carried before vaccination trials. The infective dose of M. gallisepticum cells for challenge studies was found to be 1.1 x 105. For vaccination studies, 240 chicks of five weeks age were divided into 10 groups and were inoculated with eight different sets of vaccines. One group was inoculated with commercial vaccine and other was kept as control. Potency testing for humoral response was conducted with HI test and CMI response with LMIT and Blastogenic calorimetry. The mean HI titre of groups vaccinated with iron sufficient and restricted saponin adjuvanated vaccines showed an increase in their titre at first week post booster vaccination followed by decline of the titre on subsequent collections on third and fifth week post booster vaccination. The mean HI titres of groups vaccinated with iron sufficient and restricted saponin-aluminium hydroxide adjuvanated vaccines maintained significantly high HI titres during the entire study period. Commercial vaccine group also showed a similar response. The control group showed significantly less titre from other vaccinated groups during the entire time period. Leukocyte migration inhibition test and Blastogenic calorimetry were carried out to assess the CMI response evoked by different sets of vaccines. A significant CMI response was shown by all vaccinated groups except commercial vaccine and control group. To assess the protective response evoked by different vaccines, two challenge studies were conducted two weeks apart. All the vaccinated birds were having significantly lower lesion score when compared to the control group. Mycoplasma gallisepticum was re-isolated from all the control birds with lesions during the first challenge study and from 70 per cent of the birds in the second challenge study. A latex agglutination test was developed using iron restricted M. gallisepticum whole cell proteins. When compared with HI test, LAT had a sensitivity of 95 per cent and specificity of 93 per cent. The test was statistically significant as indicated by a kappa value greater than 0.81. The saponin-aluminium hydroxide adjuvanated whole cell and membrane protein vaccines had elicited similar humoral immune response than vaccines adjuvanated with saponin alone. With regard to CMI response, the experimental vaccines produced a significant CMI response whereas, commercial vaccines failed to evoke a protective CMI response. In brief, saponin-aluminium hydroxide adjuvanated whole cell and membrane protein vaccines, especially those produced under iron restricted conditions, could be an effective tool to afford protection against M. gallisepticum infection, as it would stimulate both humoral and cell mediated immune responses in host Latex agglutination test was found to be an effective tool for flock screening of birds for M. gallisepticum antibodies and the test is simple, rapid, and easy to conduct, with applicability under field conditions.ThesisItem Open Access Isolation and characterization of bacteria associated with gastroenteritis in weaned Piglets(College of Veterinary and Animal Sciences, Mannuthy, 2009) Atulya, M; KAU; Koshy, JohnGastroenteritis is a leading cause of morbidity and mortality in piglets. A number of factors are involved in post weaning diarrhoea in piglets. A comprehensive study was performed to examine the incidence, bacterial etiology, drug sensitivity, plasmid profile and pathogenicity of the bacteria isolated from weaned piglets with gastroenteritis prevalent in and around Kerala Agricultural university. Samples were taken only from piglets with diarrhoea that had not been previously treated with antibiotics. Rectal swabs were collected from live diarrhoeic piglets and intestinal contents, pieces of jejunum, ileum, colon, mesenteric lymph nodes, liver, spleen and stomach were collected during post mortem examination after taking all sterile precautions. Isolation of causative bacteria was made by culturing on Brain Heart Infusion Agar, Mac Conkey agar, Mannitol Salt agar, Blood agar, Brucella agar and Cooked Meat medium. The identification of isolates was carried out as per standard protocols. All the procedures of biochemical testing were followed as described by Barrow and Feltham (1993). For classification of Staphylococcus isolates, a system suggested by Baird Parker (1965) was considered. A total of 53 bacterial isolates were identified to species level from 82 samples tested for pathogens. Six different microorganisms were encountered in this study, with Escherichia coli being dominant, followed by Pseudomonas aeruginosa, Staphylococcus subgroups BPIII and BPV, Serratia fonticola, Salmonella typhimurium and Aeromonas hydrophila. Thirteen different serotypes of E. coli were encountered, with O69 being dominant and the others were O38, O5, O84, O132, O80, O79, O56, O41, O25, O109 and O103 and two were untypable. Majority of the isolates exhibited multidrug resistance. Plasmid profile of the Gram negative isolates were determined and 80.43 per cent were found to bear plasmids. Pathogenicity of the isolates was determined by performing in vivo mice pathogenicity test.