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ThesisItem Open Access Isolation and identification of viruses from waterfowls seen in Kerala(Department of microbiology, College of veterinary and animal sciences, Mannuthy, 1997) Bindu, M S; KAU; Krishnan Nair, GIn the recent years, frequent outbreaks of poultry diseases have caused significant economic losses and among the various sources of infection, freeflying migratory birds and waterfowls are reported to be an important source for the introduction of disease to domestic flocks. These freeflying pirds and waterfowls abound in the waterlogged areas of Kerala especially Alleppey, Kottayam, Thrissur and Malappuram districts. Heavy losses were reported each year due to severe outbreaks of duck plague and duck pasturellosis resulting in the wiping out of the duck population in the state. This may be due to the introduction of the disease agents by migratory waterfowls. Hence a study was undertaken to elucidate the role of waterfowls in the spread of diseases to domestic poultry and ducks. A total of 52 waterfowls were caught from different parts of Kerala of which 15 were lesser whistling teals and 37 were gargany. Postmortem examination of these birds were carried out and the required materials were collected. Impression smears from the cut surface of the liver revealed intranuclear inclusion bodies in the hepatic cells in four cases (Bird Nos. 17, 23, 27 and 49) and two haemagglutinating agents were isolated from the cloacal swabs of the bird numbers 18 and 22. The haemagglutinating agents developed lesions in the chicken embryos which died 3 to 5 days after inoculation. the allantoamniotic fluid of the affected embryos agglutinated teal and chicken erythrocytes. The lesions exhibited by the embryos infected with Ta were congestion of the CAM and embryos while in T22 the embryos were stunted, curled and at the same time congested. The liver of the embryo had yellowish brown patches. The haemagglutination activity of both the viral isolates were tested with red cells of various species like cattle, horse, human '0', duck, rabbit and pig but was negative in all cases. Both the isolates lost their infectivity and haemagglutination property at 56°C for 30 min. the infectivity and HA activity of the viral isolates T18 and T22 were retained at pH 7.2 and were completely destroyed at pH 3.2 and pH 9. Both the isolates were sensitive to treatment with chloroform, revealing both as enveloped viruses, wherein the infectivity was completely lost and HA activity was considerably reduced. In the case of Tl8 HA activity was reduced from 512 to 16 and for T22 from 128 to 8. The nucleic acid types of the viral isolates were confirmed by inoculating the isolates to chicken embryo fibroblast cultures pretreated with 100 µg and 200 µg per ml of IudR which led to the conclusion that Ta was a RNA virus and T22 a DNA virus. The ELD50 of Ta and T22 were 107 ELD50/ml and 106 ELD50/ml. The ICPI, MDT and IVPI. were calculated as for NDV. The ICPI for the isolates were 0.83 and 0.30 for T18 and T22 respectively, MDT was 120 hr for both and the IVPI was calculated to be zero in both cases, indicating that both the isolates were nonpathogenic. In cell cultures both the isolates produced CPE which affected the whole monolayer by 96 hrs. In the case of Tl8 the CPE was characterised by rounding and clumping of cells, the infected cells showing a tendency to get separated from the neighbouring cells leaving long cytoplasmic strands, syncytium formation with four or five nuclei and severe cytoplasmic vacuolation. For T22 the CPE was characterised by rounding and cytoplasmic vacuolation and karryorhexis. Intranuclear and intracytoplasmic inclusion bodies could be demonstrated. The pathogenicity studies of both the isolates were carried out in one week old and six week old ducklings and chicken, for which both were nonpathogenic and did not exhibit any clinical signs or mortality. But viral infection was established in six week old chicks by virus isolation till the 14th day of infection from the cloacal swabs for both the isolates. The sera collected from these birds revealed an antibody titre of 16 for T¬18 and 8 for T22 indicating the infection. The antigenic relationship of the isolates was examined with NDV, EDS-76 and fish viruses (FV and F6), of which T22 did not show any antigenic similarity with any of the viruses. But T1B on the contrary exhibited antigenic relationship with the fishviruses FV and F6 but no antigenic similarity with NDV and EDS-76. The antigenic similarity exhibited by T18 with the fish viruses leads to the conclusion that waterfowls may be disseminating the viruses responsible for the outbreak of epizootic ulcerative syndrome in fishes. The morphological features of T18 by electronmicroscopy revealed an enveloped virus with a size of 150-185 nm with pleomorphic forms and peplomers of a length of 18-20 nm. Except the length of the peplomers it similated a paramyxovirus. The morphology of T22 was not studied due to technical defects.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 Production and application of monoclonal antibodies against duck plague virus(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1999) Ravindra Dattatraya, Padalkar; KAU; Jayaprakasan, VMonoclonal antibodies (Mabs) were raised against the vaccine strain of DPV and three strains of DPV Viz, Vaccine (DPV-V), IVRI (DPV-I) and Alleppy strain (DPV-A) were used to raise polyclonal serum in the present investigation. DPV-V was revived in 11 day old chicken embryo and the embryo death was recorded Tour to five days PI- with congestion all over the body and spleen and necrotic foci in liver. The cytopalhy in CEF cell culture observed was rounding and clumping of the cells, syncylium formation and bridge formation with extensive vacuolation in the Cytoplasm. The detachment of the cells was observed at 120 h PI. DPV-I a virulent strain was inoculated in the ducklings, death was recorded in all the inoculated birds with extensive hemorrhages on serous membranes, muscles and visceral organs. Necrotic foci on liver,' enlargement and congestion of liver, and spleen, and white hecrotic foci in the gizzard were evident. The virus was further passaged in DDE and cultivated in bulk in DEF cell culture. The DPV-V and DPV-A were titrated in CEF cell culture and the TC1D J0 was 4.7 X 105 per ml of the inoculum Tor DPV-V and 3.2 X 10"1 for DPV-A. DPV-I cultivated in DEF cell culture had a TCID50 of 1X10 3'per ml of the inoculum All (lie strains were partially purified at 100000 g for 4.5 h at 4" C in Beckman ultra centrifuge and the protein concentration oF the virus was estimated by biuret method and was found to be 11 mg, 8 mg and 7 mg for DPV-V, A and 1 respectively. All the three strains of DPV were inoculated in mice to raise polyclonal serum. Four mice out of five inoculated with DPV-V showed ELISA litres more than 1:12800, one mouse showed a titre of 1:6400. The mice inoculated with DPV-A showed a titre of more than 1,12800 and those inoculated with DPV-I, 1:6400 ELISA ’Was1’ used to test the sera samples of the mice inoculated with DPV strains. The test was found to be highly sensitive, easy to perform and less time consuming. The test therefore can be recommended for routine diagnosis of DPVThesisItem 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 Secretory immunoglobulins of the duck (Anas platyrrhynchos domesticus)(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1996) Hari, Kumar A V; James, P CThe profile and functional properties of the immunoglobulins of serum, bile, mucos of trachea and intestine of ducks were studied. The immunoglobulins were separated by salting out using ammonium sulphate. The various fractions of immunoglobulines were further resolved by Sephadex G-200 gel filteration which gave two peaks for serum and tracheal immunoglobulins and a single peak each for bile and intestinal immunoglobulins. The purity of these fractions were checked by immunodiffusion and immunoelectrophoresis. The different fractions obtained were quantified by single radial immunodiffusion. The level of fraction 1 in the bile ranged between 1718 µg/ml and 1959 µg/ml and that of serum, 1718.06 µg/ml to 2442 µg/ml, in the S.typhimurium treated groups. The level of fraction 1 in the NDV treated groups ranged from 1115 µg/ml to 1597.35 µg/ml in bile, and 1597.35 µg/ml to 1959.34 µg/ml in serum. The level of fraction 2 in serum ranged from 1797 µg/ml to 2591 µg/ml in S.typhimurium treated group and 1400 µg/ml to 1797 µg/ml in the NDV treated group. Fraction 2 was not detectable in bile. The antibody response of ducks to a bacterial and viral antigen (anaculture of S.typhimurium and R2B strain of New Castle Disease virus respectively) was assessed. On conducting standard tube agglutination test, the serum, bile and oviduct washings revealed antibody tires against S.typhimurium in inoculated birds ranging between 1:20 and 1:160 in the case of serum; 1:10 and 1:80 in the case of bile and tire less than 1:10 for oviduct washings. No antibody titre could be detected for tracheal and intestinal washings and testicular extracts. The HI titre ranging from 1:32 to 1:128 could be observed for serum, 1:32 to 1:64 for bile and a titre of 1:16 was observed for oviduct washings of ducks parenterally administered with NDV. The tire was relatively low for serum when NDV was administered intranasaly. Intestinal and tracheal washings and testicular extract failed to reveal any HI antibodies.ThesisItem Open Access Polymerase chain reaction for the detection of canine parvovirus in faeces of dogs(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2004) Josemi, Mathew; KAU; Mini, MA study was undertaken to compare the ability of haemagglutination (HA), polymerase chain reaction (PCR) and seminested PCR for the diagnosis of canine parvovirus (CPV) infection by detecting CPV from faecal samples of clinically suspected dogs. Characterization of vaccine strain and field strains of CPV was performed by restriction enzyme analysis (REA) in this study. Attempts were made to isolate CPV from faecal samples in MDCK cell line. One hundred and twentysix faecal samples were collected from dogs suspected for CPV infection and 40 faecal samples were also collected from normal healthy non-vaccinated as well as vaccinated dogs after 15 days of vaccination, that were brought to veterinary hospitals attached to KAU. All the samples were screened by HA, PCR and seminested PCR to detect CPV. No significant difference in HA titre could be appreciated on comparing the titre using PBS, pH 7.2 and PBS – BSA as diluents. The haemagglutination reaction by CPV was found to be favoured by a slightly acidic pH in the range of 4.0 to 6.0. Chloroform treatment of faecal samples had no influence on HA titres above 5 log2. Among 126 faecal samples screened, 43.65 per cent, 61.11 per cent and 73.81 per cent were tested positive for CPV infection by HA, PCR and seminested PCR respectively. Therefore, seminested PCR was found to be a more sensitive and specific method over HA and PCR for the early diagnosis of CPV infection. All the faecal samples from healthy non-vaccinated and vaccinated dogs after 15 days of vaccination were tested negative by HA, PCR and seminested PCR. Restriction enzyme analysis using HinfI, Rsa I and Sau 961 revealed no difference in the fragment length patterns between CPV vaccine strain and field strain. Attempts to isolate CPV from faecal samples in MDCK cell line were found unsuccessful. The occurrence of CPV infection among non-vaccinated dogs was found to be higher than that of vaccinated dogs. Vaccine failure was also observed after MLV vaccination. The distribution of CPV infection was highest among dogs between two to four months of age. Breed-wise distribution of CPV infection showed highest distribution in German shepherd dogs when compared to other breeds. Most of the cases of CPV were noticed during February and July in the year 2003 and during March to June in the year 2004.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.
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