Immunodiagnosis and seroepidemiology of cysticercus bovis infection in cattle in Kenya
Gathuma, J M
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Cysticercosis and taeniasis in animals and man, respectively are widespread in the world and are of great economic and public health importance in East Af'r-i ce , In Kenya, the prevalence of Cyst~~ercus bovi~ infection an cattle is high. The total 10ss8s incurred each year from condemnation, downgrA~ing and freezing of carcasses are very high. Efforts to rid the parasite by treatment of the people and 'improved mat hot.: ,'+ meat inspection have so far met with little success. A r-e lreb le arrtrmor-t ern diagnostic technique plus a chemotherapeutic drug \tJhichwill kill cysts H. muscles without re::ducingthe quality of meat might pro\lide an answer to +he cont ro l of the cysticercosis/taeniasis problem. The objectives of the present investigation were: (l) to fract Ionet.e crude ant igen pr-epared +rorn t.apeworm proglottides in order to increase specificity in the indirect haemagglutination (IHA) test, (2) to try activated oncospheres as antigen for the indirect fluorescent antibody (IFA) test, (3) to standardize IHA and IFA tests, using sera from experimentAlly infected calves, (4) to evaluate the sensitivity and specificity of the IH.A. and IFA tests using sera collected at slaughter. and (5) to use the IHA test in a seroepiderniologic study of bovine cys t i.cer-cosis in Kenya. Taenia saginata antigens were prepared from mature tapeworm segments and ~vere partially purified by Sephadex G-200 column chromatography. This procedure yielded thre8 protein fractions designated Fl, F2 and F3. The crude antigen, protein fractions FI, F2 and F3 were analysed by imnunodiffusion and immunoelectrophoresis against anticrudeT. saginata. rabbit serum. Thr-ee distinct precipitin bands were observed in .irrmrnod iFf'uaiun of the crude antigen while three distinct precipitin bands and a few indistinct bands were obtained in illmunoelectrophoresis of the crude antigen. In imnunodiffusion, fraction FI gave one sharp distinct precipitin band and two diffuse wide bands while F2 showed several fuzzy bands. Fraction F3 showed a weak precipitin reaction. lr1 imnunoelectrophor8sis, fraction FI ehowed one distinct band, fraction F2 showed two distinct bands and several weak bands while fraction F3 either did not show any precipitin band or when it did, the band was weak and not well defined. When t he crude antigen and fractions Fl, F2 and F3 VJel.~e tested for antigenic reactivity using the IHA test, it was found that the crude antigen and fraction FI showed high reactivity while fractions F2 and F3 showed a low degree of reactivity. Fraction Fl which was found to be highly antigenical1y reactive was purified further by DEAr::Sfophadex A-50 and DEAE cellulose DE 52 ion exchange chromatogr-aphy. The IHA test was standardized with sera from seven experimentally infected and four non-infected bull calves, using both crude and partially purified antigens. Using either crude or fraction Fl antigen, antibodies to C. bovis were detectable from 2 weeks post-i.nfection to the end of the experiment 4 months post-infecbon. For evaluation of sensiti.vity and specificity of the IHA test, sera from naturally infected and non--infect:ed cattle were collected from the Kenya f"leatCorrmiss itin abattGir, Athi River. Post-mortem records of each anirnal were obtained during routine meat inspection. Sera from 285 cattle were tested. The IHA test using fraction Fl antigen showed higher sensitivity and specificity than the IHf\ test using crude antigen. Partial purification of the crude antigen by Sephadex G-200 column chromatography reduced cross-reactivity between sera frornanimals infected with Echin~c..9l'cusgranulosus from 59.6% to 31.9%. Fractions Fla and Flb obtained by DEAE Sephadex A-50 and DEAE cellulose DE 52 ion exchange chromatography of fraction fl, respectively, shcwed a lower level of cross-reactivity with sera Fr-om cattle infected wi th E. granulosus than fraction Fl. The sensitivities obtained with these two antigen fractions (Fla and Flb), however, were very1dw (45% and 60%, respectively). The IFA test was carried out using activated encospheres fixed on gelatin &\ ides as antigen. It was standardized wi t.h sera from the seven 8xperirnentr3lly infected and four non infected bull calves used in the standardization of the IHA test. Titres were found to be slightly higher than for IHA test. For evaluation of sensitivity and specificity of the IFA test, the same 285 sera tested wi t.h the IHA test were screened. A sensitivity of 91.5% anda specificity of 90.2% WETe obtained. Cross-reactivity with I. granulosus was 10%. In the seroepidemiological study, the IHA test using crudeT.saginata antigen revealed that 82 out of 195 sera from Rift Valley Province (42.1%) had significant titres (1:64 or above) while 11 out of 202 (5.4%) sera of cattle from Central Province had significant titres (l: 64 or above). When fraction Fl was used in the IHA test 57 out of the 195 (29.2%) sera of cattle from Rift Valley Province gave significant titres while 12 out of the 202 [5.~%J sera of cattle from Central Province gave significant titres. When compared with results of parasitological findings it was found that for animals coming from Rift Valley Province, IHA test using crude antigen gave a significantly higher percentage of positives than the parasitological survey. On the other hand, for animals coming from Central Provinc8, the percentage with positive titres was significantly lower than that obtained by parasitological examination. 'Comparison of' the percentages obtained by parasitological examination with those obtained bv IH,l\ test using fraction Fl antigen showed that there was no significant difference between the two percentages in the case of errirne Ls coming from Rift Valley Province, \A)hereas the percentE1ge of pos l tivc animals by IHA test using fraction Fl antigen was significantly lower than that obtained by parasitological survey in case of animals coming from Central Province. Based on the results obtained in this study the following observations and conclusions were made:- 1. Under experimental conditions the IHA test showed good sensitivity and specificity even when crude antigen was used. 2. In naturally In+ect sd and non-infected adult slauziter cattle the IHA test with crude antigen showed low sensitivity especially in low grade infections. Crossreactivity with E. granulosuswas high. 3. Partial purification of crude antigen by Sephadex r:--200 column chromatography increased both sensitivity and specificity and reduced cross-reactivity with E. granul_osus. 4. Further purification of fraction Fl antigen by DEAE Sephadex A-50 and DEAE cellulose DE 52 reduced sensitivity o-~ the IHA test. However-, prolonged storage of fraction Fl before further purification may have given rise to this problem. 5. Under experimental conditions the IFA test showed higher titres than t~e IHA test. 6. In naturally infected and non-infected adult slaughter cattle the IFA test showed higher sensitivity and specificity than the IHA test with crude antigen. Both sensitivity and specificity of the IFA test were not significantly different from those of IHA test with fraction Fl antigen. 7. The greatest drawback of the IFA test is that it gwes non-specific fluorescence especially if the oncospheres are not completely freed from the oncospheral membranes. Fresh, mature I. saginata eggs are necessary if good results are to be obtained. 8. Further work especially in preparation and purification of 3ntigens is required before the IHA test can be used as a routine d.iagnns t ic or epidemio1ogical tool in bovine cysticercosis.