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ThesisItem Open Access Cellular and humoran immune responses to corynebacterium presudotuberculosis infection in goats(Department of microbiology, College of veterinary and animal sciences, Mannuthy, 1986) Jayaprakasan, V; KAU; Sulochana, SCaseous lymphadenitis was experimentally produced in cross-bred malabari goats aged 8 to 12 months by bilateral inoculation of 1 x 106 viable C. pseudotuberculosis (ATCC 19410) through intradermal, subcutaneous and submucosal routes. The clinical picture, immune response and pathological features were studied up to a period of 13 weeks. The development of immune response in experimentally infected goats was assessed, by comparing the data with those of the controls, with respect to total serum protein, serum protein fractions, antibody activity of the serum, leukocyte count, counts of lymphocyte subpopulations, leukocyte migration inhibition index and skin hypersensitivity reaction. Pathological features in the lymphnodes and other tissues of infected goats necropsied at 15 days interval were also studied. Initial febrile reaction which lasted for 72 to 96 h, local inflammatory changes caused at the site of inoculations during the first two to three weeks of infection and the development of lesions typical of caseous lymphadenitis in local/regional lymphnodes within 21 days post-inoculation were the main features of clinical manifestations of the disease. aS a result of infection, neutrophilic leukocytosis was maximum during the 2nd week of infection. No appreciable was maximum during the 2nd week of infection. No appreciable change in counts of other cells in terms of their absolute numbers was noted during the entire period of observation. The humoral immune response in infected goats was indicated by the rise in serum protein, antitoxic antibody and B-lymphocyte count in the peripheral blood. The serum protein concentration increased to significant levels from the 5th week onwards and it reached the peak value (11.346 g%) by the 8th week. From the 3rd week onwards haemolysis inhibition test, which detected goats and persisted till the end of the observation period. The peak antibody level was recorded by the 5th week of infection and thereafter there was gradual reduction in the titre. Significantly high percentage of B- lymphocyte was recorded in infected animals from the 2nd to 10th week, except at the 5th week. The percentage of B-cells in infected goats ranged between 12.3 + 0.85 – 17.63 + 1.2 while it was 8.56 + 0.75-12.3 + 1.09 in control goats. This was considered as an indication of stimulation of humoral immune response. The cell-mediated immune response was evidenced by the increased T- lymphocyte count in the peripheral blood, inhibition of leukocyte migration and the development of delayed skin hypersensitivity. The mean percentage of T-lymphocytes in the peripheral blood of infected goats by E-rosette assay recorded an initial reduction at the first week (18.44 + 1.4) followed by an increase which was significant during the 5th, 6th, 7th, 8th, 12th and 13th week of infection. The maximum value was recorded (35.24 + 1.58) at the 13th week. In the case of control goats the percentage values ranged from 24.55 + 3.66 to 26.74 + 1.34. The T-lymphocyte count in the peripheral blood enumerated the ANAE method did not show any significant change even after infection. In the experimentally infected goats, leukocyte migration inhibition index was less than 0.8 during post-infection period while the control goats had the index value above 0.8. Significant reduction in the migration index was noted by 45th day of infection and the maximum reduction was on the 60th day. Intradermal injection of the toxic supernatant of the culture elicited characteristic delayed skin hypersensitivity reaction in all the experimentally infected goats while there was no reaction in the controls. The positive reaction was found to be maximum by the 48th hour post-injection. The pathological changes were characterized by an initial stimulatory hyperplastic reaction in the lymphnodes and this was accompanied by necrobiotic changes typical of caseous lymphadenitis. The hyperplastic stimulatory reactions were characterized by the presence of several active follicles with well developed germinal centres in the cortex, distinct medullary cords densely lined with plasma cells and sinus histiocytosis indicating the early elicitation of humoral immune response to the bacterium or to its in vivo products. The results obtained from the present study revealed the operation of both cell-mediated and humoral immune responses in goats against C. pseudotuberculosis infection. Of the various methods employed to monitor the immune response, leukocyte migration inhibition and delayed skin hypersensitivity tests were found to be of value in assessing the cell-mediated immune response and haemolysis inhibition test for humoral immune response. Leukocyte migration inhibition test and haemolysis inhibition test could be employed for early diagnosis of C. pseudotuberculosis infection in goats. FINDINGS : The immune responses and pathological features in Corynebacterium pseudotuberculosis infection were studied by experimental infection of cross- bred Malabari goats of S-12 months of age. Single cell bacterial suspension in chilled sodium chloride bile salt solution was used for this purpose. Goats were inoculated at both sides of the body by three routes viz., intradermal, subcutaneous and submucosal, with 2 x 106 bacteria per site of injection. The experimentally infected and control goats were observed for clinical manifestations of caseous lymphadenitis for a period of 13 weeks. The development of immune response in experimentally infected goats was assessed by comparing the data with those of the controls with respect to total serum protein, serum protein fractions, antibody activity of the serum, leukocyte counts, counts of lymphocyte sub-populations, leukocyte migration inhibition index and skin hypersensitivity reaction. Gross and histopathological changes in the lymphnodes and other tissues of necropsied goats were studied at 15 days interval for a period of 90 days. All experimentally infected goats exhibited rise in temperature, general weakness, lethargy and impaired appetite which lasted for 72 to 96 h. The sites of inoculations showed varying degree of inflammatory reaction during the first two to three weeks of infection. All experimentally inoculated goats except one developed lesions typical of caseous lymphadenitis in regional/local lymphnodes within 21 days post-inoculation. Route of infection did not influence the ability to set up lesions in lymphnodes. Although massive dose of bacterial (1.2 x 107) was administered, none of the goats had fatal infection indicating that goats are relatively resistant to this infection. Majority of goats did not develop generalized form of caseous lymphadenitis as there was no lesions in visceral/deep seated lymphnodes or organs. The normal serum protein concentration of cross-bred Malabari goats was estimated to range from 7.187 to 9.750 g %. Consequent to experimental infection, serum protein concentration was increased and recorded significant rise from the 5th week onwards reaching the peak value by the 8th week – 11.346 g%. Estimation of quantitative distribution of serum protein fractions was done by agar gel electrophoresis and densitometer tracing of electrophoretogram. Though there was initial increase in globulin content in infected animal followed by a decrease, no significant alteration in the albumin-globulin ration (A:G ratio) was noted compared to the control group. C. psuedotuberculosis was cultivated in lemco proteose broth containing sheep serum and incubated aerobically at 370C for 72 h. Supernatant obtained from the above culture, having maximum haemolysin titre and dermonecrotoxicity was used as the toxin of the bacterium in the present studies. The haemolysin content of the culture supernatant was estimated by the haemolysis test using sheep red cells. A maximum titre of 1:256 was found in the culture aged 72 h. The dermonecrotoxicity of the toxic culture supernatant was tested by intradermal inoculation into the rabbit skin. The inflammatory and necrotic reactions were maximum by 48 h post-injection. Specific antibody activity against toxin of C. pseudotuberculosis in the serum was monitored by haemolysis inhibition test and the test was adjudged as a useful test for detecting humoral immune response to C. pseudotuberculosis infection in goats. In infected goat from the 3rd week of infection onwards MIT was positive while it was negative in control goats during the period of 13 weeks of observation. The peak antibody level was achieved by the 5th week of infection and thereafter the titre was found to dwindle gradually till the 11th week. Towards the end of the observation period (12th week) there was a marginal increase in the antibody titre, which would be considered as secondary immune response against the toxin of the multiplying bacteria. Infected goats showed leukocytosis during the entire period of observation and maximum leukocytosis was observed during the 2nd week of infection. The periodical fluctuation in leukocytosis indicated the recurrent flare up of bacterial invasion in the body. The absolute lymphocyte count obtained both at pre and post-infection periods with experimentally infected goats did not show any change which indicated no deleterious effect on peripheral blood lymphocytes. Throughout the period of observation infected animals showed numerically low lymphocyte percentage in differential counts and with several samples the percentage distribution was significantly low. Absolute counts of neutrophils were consistently high in experimental goats when compared to those of controls and the same was reflected in differential count also. The other blood cells were absolutely without any change in infected as well as control goats. Density gradient centrifugation using Ficoll-paque (1.077 g/ml, centrifuged at 720 x g) was found quite useful for separation of mononuclear leukocytes from the whole blood of goats. Such separated mononuclear cells were found to contain on an average 91.80% lymphocytes and 8.2% monocytes with an average viability of 91.2%. Peripheral blood B-lymphocytes of goats were successfully enumerated by EAC rosette assay employing bovine red cells. The normal percentage of B-cells was estimated to range 8.56 and 12.3. Significantly high percentage of B-cells was recorded in infected animal from the 2nd to 10th week post- infection except at the 5th week. B-cell percentage in infected goats ranged between 12.3 + 0.85-17.63 + 1.2 while it was 8.56 + 0.75 to 12.3 + 1.09 in control goats indicating the operation of humoral immune response to concurrently boost the specific antibody activity in the serum. T-lymphocytes of goats were identified and enumerated by E-rosette assay and ANAE activity. Goat lymphocytes presented several receptors to sheep red cells, as majority of rosettes presented erythrocytes at the entire periphery of lymphocytes. The mean percentage of E-rosette positive lymphocytes in the peripheral blood of control goats ranged from 24.55 + 3.66 to 26.74 + 1.34 during 13 weeks of observation. The E-rosette technique employed in the present study was assumed unaffected by unknown variables as the data recorded in the control goats remained near normal throughout the observation period. During the first two weeks of infection E-rosette positive lymphocyte count was found numerically decreased and the reduction was significant at the first week (18.44 + 1.40). From the third week onwards an increase in the E-rosette forming cells was observed and significant increase was noted during the 5th, 6th, 7th, 8th, 12th and 13th week of infection, the maximum being at the 13th week (35.24 + 1.58). T-cells were also identified and enumerated based on the demonstration on ANAE activity. Fixing of mononuclear cells in acetone-citric acid solution enabled the fixed smears to be stored in dry state without any interference to the enzymic activity for longer periods. T-lymphocytes presented one or two localized red coloured reaction product in the cytoplasm adjacent to the cell membrane. Mean percentage of ANAE positive cells in experimental goats was 28.09 + 1.51 while it was 35.80 + 4.86 for control goats when estimated before the start of the experiment. During infection, the count of ANAE positive cells in the peripheral blood did not show any change as the mean percentage ranged between 28.9 + 2.06-33.78 + 1.99 as against the corresponding values (30.83 + 3.5-36.91 + 3.61) in controls. In infected animals significant hike in E-rosette positive lymphocyte counts was recorded while such a change could not be observed with ANAE positive lymphocytes. Thus the results of T-cell estimation by E-rosette assay and ANAE demonstration indicated that estimation of total rosette forming cells could reflect better the T-cell competence. Cell mediated immune response to C. pseudotuberculosis infection in goats was demonstrated by leukocyte migration inhibition test under agarose. A population density of 1.5 x 108 leukocytes/ml was found suitable for LMIT. Toxic culture supernatant having haemolysin titre 1:16 whose pH adjusted to 7.2 could be successfully used as antigen in the test. In experimentally infected goats leukocyte migration index was less than 0.8 during post-infection period while with control goats it was above 0.8. Significant reduction in LMI index was noted by 45th day of infection through 75 days showing maximum reduction by the 60th day. Intradermal injection of toxic culture supernatant elicited characteristic delayed type skin hypersensitivity reaction in all experimentally infected goats, while a negative reaction in controls. Skin hypersensitivity reaction was found to be maximum by 48 h post-injection. Histopathology of skin biopsy taken from the site of inoculation revealed infiltration of lymphocytes, and macrophages at perifollicular and periglandular areas, congestion of blood vessels with perivascular infiltration of lymphocytes and macrophages and dermal oedema. Tuberculin failed to produce a positive skin hypersensitive reaction in C. pseudotuberculosis infected or control goats. From 15th day onwards, experimentally infected goats which were necropsied presented gross lesions typical of caseous lymphadenitis in lymphnodes. The lesions were found to confine to superficial lymphnodes adjacent to the site of inoculations. The histological changes observed in lymphnodes were basically of two types: hyper-plastic stimulatory reaction and degenerative changes. The changes were hyper-plastic reactive follicles with well distinguished germinal centre, accumulation of lymphocytes and varying degrees of sinus histiocytosis in medullary region, dense lining of medullary cords with plasma cells, depletion of lymphocytes from the cortical area; subcapsular and cortical oedema, congestion of blood vessels, haemorrahage, infiltration of mononuclear cells in lymphatics and blood vessels, accumulation of macrophages and plasma cels in the medulla, dilatation of sinusoids, fibrous tissue proliferation, degenerative and necrotic changes of lymphocytes in the cortex and medulla, fibrous tissue encapsulated focal areas of caseation of calcification surrounded by lymphocytes, macrophages and giant cells and finally conversion of parenchyma to a caseated mass enclosed in fibrous tissue capsule. In brief, the results obtained from the present study revealed the operation of both cell-mediated and humoral immune responses in goats against C. pseudotuberculosis infection. Of the various methods employed to monitor the immune response, leukocyte migration inhibition and delayed skin hypersensitivity tests were suitable for ascertaining the cell-mediated immune response and haemolysis inhibition test for humoral immune response. Leukocyte migration inhibition test and haemolysis inhibition test can be successfully employed for the early diagnosis of C. pseudotuberculosis infection in goats.ThesisItem Open Access Characterization of staphylococci isolated from cases of mastitis and study of their R plasmids(Department of microbiology, College of veterinary and animal sciences, Mannuthy, 1986) Mathew, E S; KAU; Punnoose, K TThe emergence of drug resistant staphylococci causing mastitis deserve serious investigation. The work was intended to characterize staphylococci isolated from cases of bovine mastitis and to study their R plasmid transfer in vitro. The characterization was done using glucose and mannitol fermentation, catalase production, coagulase production, Iysostaphin sensitivity and bacteriophage typing. The antibiogram of the isolates was done by agar diffusion method using 14 chemotherapeutic agents (amoxicillin, ampicillin, bacitracin, chloramphenicol, cloxacillin, erythromycin, gentamicin, methicillin, neomycin, nitrofurantoin, penicillin, streptomycin, sulphamethoxazole and tetracycline) and by agar dilution method using eight antibiotics (ampicillin, chloramphenicol, rifampicin, streptomycin, tetracycline , gentamicin, erythromycin and penicillin) The in vitro transfer of R plasmids was tried using selected S.aureus and coagulase-negative staphylococcal isolates as donors and S.aureus RN 450RF and S.epifermidis 131S as recipients. From 360 milk samples collected from cases of bovine mastitis 17 strains of S.aureus and 35 strains of coagulase negative staphylococci were isolated. Lysostaphin sensitivity test was positive for 90.38% of the isolates, but this could not be used to differentiate between coagulase positive and negative staphylococci. The results of phage typing revealed a predominance of group III phages over the other groups and the possible role of human strains of staphylococci in producing mastitis in animals. None of the strains were resistant to mercuric chloride. From the antibiogram rifampicin , bacitracin, neomycin , methicillin, gentamicin, cloxacillin, nitrofurantoin and chloramphenicol were found to be the drugs of choice in the treatment of bovine mastitis caused by staphylococci. When S. aureus Rn 450RF was used as recipient six of the ten selected S.aureus isolates could transfer either one or more drug resistance markers and the mode of transfer was suspected to be by conjugation. All the four selected streptomycin resistant coagulase –negative staphylococcal donors were found to transfer the R plasmid DNA to the recipient., S.aureus RN 450RF, which was established to be through conjugation. The drug resistant S. aureus as well as coagulase negative staphylococcal strains failed t transfer their resistance to S.epidermidis 131S.