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Open Access
Enzyme immuno assay based seromonitoring and detection of canine rabies antigen
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1999) Biju, K G; KAU; Krishnan Nair, G
In the present study an attempt was made to measure the serum neutralizing antibodies against rabies virus in the sera of both vaccinated and unvaccinated dogs, employing ELISA. A long with this special emphasis was given to compare the results of diagnostic techniques used for yaJ.i.es namely Seller's staining and IP test. Four hundred and thirty-four sera samples from dogs of different age groups were screened. Enzyme immuno assay of sera of 83 unvaccinated pups revealed that under the age group of 0-3 months 59.1 percent antibody titre of 1: 50 or above indicating the presence of maternal antibodies. Out of 116 sera samples from primarly vaccinated group only eight had antibody titre below 1:50. Among the booster vaccinated 94 sera samples tested only four samples showed antibody titre below 1:50 .. The assay revealed that during diseases the immune systems of the dogs will be suppressed. It was found that the age of first vaccination could be decided as third month. The present study revealed that primary vaccination might not produces higher antibody titres in dogs. The study showed advantages of booster vaccination over primary vaccination. All the rabies suspected cases were subjected as two diagnostic tests namely Seller's staining and IPT. Out of 20 rabies suspected cases, there were detected by demonstration of Negri bodies and four were by IPT. The present study showed plate ELISA is a sensitive method for titration of rabies virus neutralizing antibodies and IPT can be successfully used for the detection of rabies antigen in impression smears taken from hippocampus.
Open Access
Prevalence of yeast and yeast like fungi in bovine mastitis and their in vitro drug sensitivity
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1996) Sukumar, K; KAU; James, P C
A total of 200 milk samples from clinical cases of bovine mastitis were culturally screened during a period of six months. Pathogenic fungal organisms could be isolated only from 26 samples. Out of this 26 positive samples, yeast and yeast like fungal organisms were isolated from 20 samples and mould from six cases. The major pathogen isolated were candida spp namely C. tropicalis, C. parapsilosis and C. guillermondi. The other organisms were Geotrichum candidum, Trichosporon cutaneum, Sacharomyces cerevisiae, Torulopsis spp and Rhodotorula rubra. The filamentous fungi isolated were Sepedonium spp, Aspergillus ochraceous group, Cladosporium carrionii, Penicillium spp and Trichophyton verrucosum. In majority of the cases yeast and yeast like fungi produced chronic mastitis in which hardness of udder and reduction in milk yield with watery milk containing flakes were noticed. In cases of mastitis where in mould was involved, chronic mastitis characterized by hardness of udder and reduction in milk yield with straw yellow coloured milk, viscid in consistency. Sensitivity pattern of the fungal isolates to the commonly employed antifungal chemotherapeutic agents like Amphotericin B, Clotrimazole, Fluconazole, Griseofuivin, Itraconazole, Ketocanazole, Nystin and Pimaricin (Natamycin) was elucidated. Among the above agents Clotrimazole and Itraconazole exhibited maximum inhibitory activity. All the isolates were found to be resistant to Griseofulvin. In vitro drug sensitivity pattern of fungal isolates employing the discs impregnated with essential oils of cinnamon, clove and lemon grass and alksloids of Cassia alata was studied. Cinnamon leaf oil possessed maximum antifungal activity and the extracts of Cassia alata failed to evince the ability to inhibit the growth of fungal isolates. The antifungal activity of plant extracts were compared with the commonly antifungal chemotherapeutic agents.
Open Access
Characterization of Campylobacter jejuni isolated from pigs and man
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1994) Raju, P V; KAU; Madhusoodanan Pillai, R
Twenty six rectal swabs collected from piglets below two months of age with diarrhoea/enteritis when culturally screened yielded 11 isolates of Camplobacter jejuni. Thirty two rectal swabs from children below five years of age with diarrhoea/enteritis when processed for the isolation of etiological agent, resulted in the recovery of one strain of C. jejuni. Sonicated C. jejuni antigen prepared from the selected strains of C. jejuni from porcine and human origin were found suitable for sensitization of gluteraldehyde stabilized sheep RBC for serum antibody monitoring. The sonicated antigen retained its affinity to sheep RBC even after six months of storage at – 600 C. Gluteraldehyde stabilization was found to be a suitable method for the preservation of SRBC. Gluteraldehyde stabilized SRBC stored at 40 C upto a period of three months did not show any reduction in the sensitization property, as evidenced from the PHA titre. One hundred and fifty microliters of C. jejuni antigen whose protein concentration adjusted to 2 mg/ml was found to be the optimum level for sensitisation of a suspension made from three millilitres of packed SRBC. The optimum level of SAPA cells required in the seromonitoring of C. jejuni specific antibodies by SAPA – AMHA test was found to be 0.1 per cent. Cross titration assay employing homologous and heterologous antigens of C. jejuni and the hyperimmune sera indicated that there was antigenic relationship between porcine and human strains. The results also point to the probable prevalence of different antigenic components in varying proportions in different strains from various sources. In the present investigation, 50 serum samples from pigs were monitored for the presence of C. jejuni specific antibodies by PHA and SAPA – AMHA. PHA detected 48 per cent cases as positives compared to 54 per cent by SAPA – AMHA. Statistical analysis clearly indicated that SAPA – AMHA is superior to PHA in screening C. jejuni specific antibodies. The result of the present study indicated that SAPA – AMHA test could advantageously replace the conventional PHA for serological diagnosis of animal and human Campylobacteriosis. Efforts were also made in this study to find out the change in PHA titre with homologus and heterologous antigens employing sera before and after adsorption with unsensitised SRBC to remove the non specific antibodies. Statistical analysis of the results showed that for performing PHA, homologous antigen should be preferred over heterologous antigen and when homologous antigen were used, adsorption of sera with stabilized unsensitised SRBC had no significant effect of PHA titre. Attempts were also made to study the in vitro antimicrobial sensitivity patterns of the 12 C. jejuni isolates by standard disc diffusion technique. The results indicated all the C. jejuni isolates were sensitive to chloramphenicol, gentamicin and nalidixic acid, irrespective of their source/origin. Eventhough none of the C. jejuni isolated in this study was resistant to erythromycin, only 83.3 per cent of the isolates showed a sensitive zone of inhibition, while, 16.7 per cent showed an intermediary zone of inhibition. C. jejuni isolates recorded 83.3 per cent sensitivity to furazolidone, 75 per cent to streptomycin and 58.3 per cent to ampicillin. Fifty per cent of the isolates showed resistance to oxytetracycline. Of the ten antibiotics tested, 16.6 per cent of the organism were sensitive to penicillin. C. jejuni recorded highest resistance to sulphadiazine as only 8.3 per cent of the organisms were sensitive to sulphadiazine.
Open Access
Comparative efficacy of different vaccines against pasteurellosis in ducks
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1998) Jayakumar, P S; KAU; Jayaprakasan, V
Two isolates of Pasteurella mul tocida from ducks viz. DP1 and DP5 isolated from ailing/dead ducks from Kuttanad area and maintained in the virulent form were used separately for the preparation of different vaccines viz., bacterin, bacterin with oil adjuvant and sonicated antigen with oil adjuvant. The biological and biochemical characters of both the isolates were compared and are in confirmity with the characters described for the species by earlier workers. Both the isolates were pathogenic to ducks and mice as all the inoculated ducks and mice were killed with in 72 h. The LDso for the DP1 isolate in one month old ducklings and six month old ducks were determined to be 23 and 32 cells respectively whereas the LDso of DP5 isolate for one month old ducklings was determined to be 17 and for adult ducks LDso was 21 cells. Immunogenic potential of different vaccines prepared using DP1 and DP5 isolate were tested in ducks by giving two doses of vaccine. The first dose was given at four weeks of age and second dose was given 80 days after the first dose. The birds were challenged with 0.1 ml of culture containing 100 LDso of fully encapsulated virulent form of bacteria at 20 days interval till 80th day and then at 90th and 120th day after the first dose of vaccine. A higher percentage of protection was conferred by oil adjuvant vaccines prepared using DP1 and DP5 isolate and sonicated antigen with oil adjuvant prepared using DP1 isolate. The serum samples were collected from vaccinated birds at regular intervals of 0 I 7 I 20 I 40 I 60 I 80 I 90 and 120 for indirect haemagglutination test and the titres obtained were ranging from 8 to 256. More evaluation and elaborated field trials are required before advocating bacterin with oil adjuvant for field use.
Open Access
Plasmid profile of avian strains of Pasteurella multocida
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1998) Balakrishnan, G; KAU; Mini, M
Four isolates of P. multocida (DCl, DC2, FCl and FC2) from avian species (chicken and ducks) were subj ected to various typing methods like biotyping, antibiogram and plasmid profiling. The pathogenicity of the isolates was also ascertained in mice, rabbits chicken and ducks. By biotyping, the isolates were found to belong to two subspecies P. mul tocida subsp. mul tocida and P. mul t.oc i cie subsp. septica. All the isolates fermented xylose and none of them fermented dulcitol. Antibiogram of the isolates was carried out and they wer~ found to be resistant to furazolidone, metronidazole anC nalidixic acid. Some of the isolates are also resistant tc sulpha and trimethoprim. All the isolates were pathogenic to mice on rip injection. Duck isolates (DCl and DC2) were found to be pathogenic to rabbits killing them on fourth and fifth day respectively. The isolates DCl and DC2 were lethal to orally infected chicks within 48 h of administration and P. multocida could e reisolated from these birds. The same isolates killed the SiC injected ducks within 24 and 48 h. 11 The chicken isolates obtained from apparently healthy birds were non pathogenic to rabbits, chicks and ducks. Plasmid profiling revealed the presence of plasmids in three of the four isolates screened. One isolate was plasmid less and another isolate contained five plasmids. The molecular size of the plasmids ranged from 2.69 kb to 7.07 kb. One isolate did not contain plasmids at all. An isolate (FC2) which was not pathogenic to rabbits, chicks and ducks contained plasmids. Isolate FCl which was resistant to six antibiotics did not possess plasmids. Hence plasmids obtained in this study may not be associated with either antibiotic resistance or pathogenicity in experimental animals. A combination of two parameters such as plasmid profile and one of the typing methods, biotyping or antibiogram made it possible to differentiate the isolates. Thus, the plasmid profile analysis with anyone of the typing methods could be used as an epidemiological tool in the differentiation of P. multocida strains of avian origin.
Open Access
Influence of adaptation of the vaccine strain of duck plague virus in chicken embryo fibroblast on its immunogenicity
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1997) Senthil Kumar, K; KAU; Ponnoose, K T
A chicken embryo adapted vaccine strain of duck plague virus was serially passaged in chicken embryo fibroblast cell cultures and its immunogenicity was evaluated at different passage levels. The vaccine strain of DPV received from VBI, Pal ode was revived in 11 day old chicken embryos by CAM route. The infected embryos died in 70 to 120 hr PI with lesions of congestion on the embryo and CAM and enlargement of liver and spleen. This embryo passaged virus was propagated in CEF cell culture, prepared from 12 day old embryonated chicken eggs. The virus produced CPE, characterised by rounding and clumping of cells, syncytium formation, vacuolation of cytoplasm and eosinophilic intranuclear inclusion bodies. The virus was adapted in CEF cultures by serial passage. It was passaged for ten times and the various characters of the fifth and 10th passaged viruses were studied. There was no change in the CPE but the time required for the appearance of CPE and total detachment of the cells decreased as the passages increased. The CPE appeared at 48 hr, 30 hr and 24 hr for first, fifth and 10th passages respectively. Similarly the time required for total detachment of cells also reduced from 120 hr at first passage to 90 hr at fifth passage and 80 hr at 10th passage. The rapid onset of CPE and desquamation of cells indicated the adaptation of the virus in CEF cell culture. The titres of fifth and 10th passage viruses in chicken embryos were 104.75 and 105.77 ELD50/ml respectively. The titres in CEF cultures were slightly higher. The values were 105.67 and 106.77 TCID50/ml respectively for the fifth and 10th passaged samples. The immunogenicity of the fifth and 10th passage viruses were studied by vaccinating six weeks old ducklings. Each duckling received 3.5 log10 TCID50 of either fifth or 10th passaged virus intramuscularly. The birds remained normal till the 20th day and when challenged with virulent virus. Birds that received the fifth passaged virus showed mean antibody titres of 64 and 32 by SNT and PHA respectively. All the birds withstood challenge indicating the effectiveness of fifth CEF passaged virus as a vaccine. In birds that received the 10th passaged virus, the antibody titres were low both by the SNT (1:54) and PHA (1:22). However all the ducks survived without manifesting any clinical signs. All the control ducks developed clinical signs of DP and died in seven to nine days time. The fifth and 10th CEF passaged viruses were sensitive to pH 3 and 11, but stable at pH 7.2. They were completely inactivated at 56°C in 30 min. These indicated that there was no change in the above physical characters of the virus though it was passaged in CEF cultures incubated at 38.5°C. Though the efficacy of the 10th passage virus was slightly lower as it was evident from the low antibody level, a detailed study is required to establish the present findings that an increase in the number of passages would result in decreased immunogenicity of the DPV vaccine strain. However from the results obtained during this study, it is evident that cell culture adapted DP vaccine strain could be recommended for production of vaccine against DP.
Open Access
Evaluation of enzyme immunoassays in the diagnosis of duck plague
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1997) Malmarugan, S; KAU; Sulochana, S
Use of Enzyme immunoassays namely dot ELISA and plate ELISA were evaluated to detect DP viral antibodies in serum samples and in whole blood dried on filter paper strips and their efficacy was compared with standard passive haemagglutination test. Indirect immunoperoxidase test was also used to detect DP viral antigen in paraffin embedded liver and spleen. ASS at 33 per cent level was used for separation of duck globulins and antiduck globulins. The protein concentration of these globulins were 34 mg/ml and 15 mg/ml respectively. The purity of these globulins were tested by IEP and AGPT using antiduck whole serum raised in rabbits. Duck plague hyperimmune serum was raised in healthy ducklings with live attenuated DP vaccine having a virus titre of 3.5 log10 ELD50/0.5 ml. This serum was used as the positive control. A total of 200 serum samples, 35 liver and 30 spleen samples were collected from different localities for the detection of duck plague viral antibodies and antigen. Corresponding blood samples were also collected on filter paper strips and the serum was eluted and ELISA was carried out. The results in this test were then compared with whole serum ELISA. The percentage of positive reaction in PHA, Dot ELISA, plate ELISA and filter paper strip method are 64 per cent, 68 per cent 72.5 per cent and 70.5 per cent respectively. Comparative efficacy of PHA with Dot ELISA, plate ELISA and filter paper strip method were carried out and sensitivity of the tests are 71.87, 67.64 and 77.30 per cent respectively. The specificity of these tests were 38.88, 43.75, 70.90 and 67.79 per cent respectively. The concordance of PHA with these tests were 60, 75.5 and 74.5 per cent respectively. On statistical analysis high degree of association (P<0.05) was observed between PHA and Dot ELISA, plate ELISA and filter paper strip method. Highly significant different (P>0.05) was observed between PHA and plate ELISA, and PHA and filter paper strip method. Based on the results, it was concluded that because of the simplicity, easiness and accuracy, Dot ELISA is suitable for detection of DPV antibodies under field conditions. But plate ELISA was highly sensitive, specific and able to detect low titred sera. Hence this test may be recommended for titration of DPV antibodies in the laboratories, particularly when the potency of the vaccine is to be checked and the immune status of a flock is to be evaluated. Because of various advantages filter paper strip method will serve as an alternative to collection of whole serum for detection of DPV antibodies. For the detection of DPV antigen, IPT was considered as suitable one because of its ability to detect high positive (83%) cases and less non specific reactions.
Open Access
Characterization of structural proteins of duck-plague virus
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1997) Hudson Taylor, J; KAU; Krishnan Nair, G
Two virulent strains of duck plague virus - DPV-I (IVRI) and DPV-A (Alleppey isolate) and a vaccine strain - DPV-V (VBI Palode) were investigated for the differences in clinical manifestations in naturally and experimentally infected ducks, morphological changes in developing duck/chicken embryo (DDE/DCE) and cytopathic effects in duck embryo fibroblast/ chicken embryo fibroblast culture (DEFC/CEFC) and chicken embryo fibroblast culture (CEFC). Typical symptoms and lesions of duck plague were produced by both the virulent strains. However, in DPV-A infection, the level of mortality and severity of lesions like gizzard muscle necrosis and haemorrhagic bands in the small intestine were more pronounced. DPV-V did not produce any symptoms or lesions on experimental inoculation into ducklings. Embryonated duck eggs were used for passaging DPV-l and isolating DPV-A, while embryonated chicken eggs were used for propagating DPV-V. All the three strains produced mortality of embryos with congestion on CAM and body of the embryo. DPV-A produced more congestion on the extremities of the embryos. Duck embryo fibroblast cultures were used for culturing the virulent strains while chicken embryo fibroblast cultures were used for culturing the vaccine strain. All the three strains produced characteristic CPE, with rounding and clumping of cells, syncytium formation, vacuolation of cytoplasm and formation of intranuclear inclusion bodies. The time for the production of CPE decreased on successive passage. Titration of the three strains of DPV was done in embryonated eggs (ELDso) and cell cultures (TCIDso). DPV-I, DPV-A and DPV-V had an ELD50 of 105.27, 104.86 and 104 per ml respectively. TCIDso of DPV-I and A in DEF culture were 105.75 per ml and 105.25 per ml respectively and that of DPV-V in CEFC was 104.5 per ml. The tissue culture system gives the best titre than the embryonating eggs for all the three DPV strains. On electron microscopy, the field isolate of DPV showed particles ranging from 170-190 nm in diameter. Protein analysis of the virulent strains viz. DPV-I and DPV-A by SDS-PAGE revealed sixteen and fourteen proteins respectively. Mild difference of two proteins (VP17 and VP22) was noticed between the two strains. DPV-A lacked the 28KD and 9KD protein bands. The vaccine strain DPV-V on electrophoresis showed a different pattern of protein bands from the virulent strains. Eighteen proteins could be resolved in the vaccine strain. The molecular weight of sixteen proteins of DPV-I ranged from 9KD to 107KD while the fourteen proteins of DPV-A ranged from l4KD to 107KD. The proteins of the vaccine strain also ranged from 9 KD to 107 KD. The possibility of the occurrence of strain variation as indicated by the difference in the protein patterns of the DP viruses under study is discussed.
Open Access
Microbial load in frozen bull semen and antibiogram of the isolates
(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 1996) Roy Mathew; KAU; Madhusoodanan Pillai, R
The mean bacterial load in the frozen semen procured from the three frozen semen production centres viz., Mannuthy (Centre A), Dhoni (Centre B) and Mattupatti (Centre C) were 2260 + 880.65, 825.91 + 186.06 and 80.33 + 18.47 respectively. The mean bacterial load per dose of semen differed significantly between these three centres. The mean fungal load of semen in Centre A and B were significantly greater than that produced in Centre C. The mean coliform counts, pseudomonad count, staphylococci and micrococci count and mycoplasma count were detected in specific media. Mycoplasma sp. were not detected in Centre A while four out of eleven samples from Centre B and two out of fifteen samples from centre C contained Mycoplasma. The total bacterial load of all the samples from Centre A exceeded the ISI specification of 500 organisms per insemination dose of semen while 72.7 per cent of the samples from Centre B exceeded this limit. None of the samples from Centre C exceeded 500 organisms per dose of semen. The mean bacterial load per dose of semen from bulls below four years of age was significantly higher than those from bulls between the age group of four and six years. The mean bacterial and fungal load per dose of semen from crossbred Holstein Friesians were significantly greater than those of crossbred Jerseys. The bacterial isolates from the semen from these centres where characterised. They belonged to the species, Bacillus licheniformis, B. alvei, B. cereus, B. brevis, Corynebacterium bovis, C. pseudodiphtheriticum, C. hofmanii, Staphylococcus ayreus, S. epidermidis, Aerococcus viridans, Micrococcus varians, Kurthia sp. Flavabacterium sp., Alcaligenes faecalis, Pseudomonas alcaligeness, PS. Mendocina, Levine asp., Enterobacter aerogeness, Enterobacter liquifascians, Edwardsiella tarda, Citrobacter koseri, C. intermedius, C. freundii, Providencia rettgeri, Escherichia coli. The fungal isolates from sêmen, which were characterised belonged to the species Candida albicans, C. pseudotropicalis, C. tropicalis and C. krusei. The filamentous fungi isolated were Aspergillus niger, A. fumigatus, Penicillum sp. and Microporum gypseum. Of the 20 antibiotics studied for the sensitivity pattern, pefloxacin, doxycycline, chloramphenicol, ciprofloxacin and norfloxacin were found to be sensitive to majority of isolates. Among the antifungal agents studied, nystatin followed by clotrimazole were sensitive to more than 80 per cent of the fungal isolates. Nystatin may be used in semen extender for checking the fungal contaminants in frozen semen. The study recommends and addition of the new antibiotics to semen extenders after further field studies.

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