Este estudo comparou o desempenho de quatro protocolos de extração de DNA a partir de homogeneizados de diferentes órgãos provenientes de vacas infectadas experimentalmente com a B. abortus 2308. Os protocolos de extração comparados foram o método de GT (lise com isotiocianato de guanidina), Boom (lise com GT e tratamento com suspensão carreadora Diatomaceous earth), PK (lise com proteinase K) e Santos (lise por fervura e congelamento com nitrogênio líquido). Foram constituídos os grupos padrão ouro positivo e negativo baseados na bacteriologia clássica e compostos por: 54 cotilédones (27 pos. e 27 neg.), 39 linfonodos supra mamários (12 pos. e 27 neg.), 44 pré-escapulares (17 pos. e 27 neg.), 33 fígados (6 pos. e 27 neg.), 37 baços (10 pos. e 27 neg.), e 34 úberes (7 pos. e 27 neg.). Todas as amostras foram submetidas aos quatro protocolos de extração e a um mesmo processo de amplificação com os primers B4 e B5. Nos resultados consolidados por órgãos, a proporção de positivos no cotilédone foi maior do que a encontrada no linfonodo supramamário (p=0,0001), linfonodo pré-escapular (p<0,0001), fígado (p=0,0006), baço (p<0,0001) ou úbere (p=0,0019). Os resultados acumulados para os métodos de extração mostraram que o protocolo de Santos teve maior sensibilidade relativa do que o método de Boom (p=0,003) e GT (p=0,0506), e foi igual ao PK (p=0,2969). As demais comparações de proporções não resultaram em diferenças estatisticamente significantes. No estudo verificou-se amostas positivas a PCR e negativas ao isolamento e viceversa. Assim, apesar das desvantagens do método bacteriológico clássico, a melhor estratégia para o diagnóstico direto da infecção de vacas por B. abortus em homogeneizado de órgãos é a utilização conjunta do isolamento e da PCR, examinando os cotilédones e utilizando os métodos de extração de DNA Santos ou PK. / This study compared the performance of four protocols of DNA extraction from suspensions of different tissues from experimentally infected cows with 2308 strain. The compared extraction protocols were GT protocol (lyse with guanidine isotiocianate), Boom (lyse with GT and treated with the carrying suspension Diatomaceous earth), PK (lise with proteinase K) and Santos (lyse by boiling and freezing with liquid nitrogen). Based on classical bacteriology, positive and negative gold standard groups were built and consisted of 54 cotyledons (27 pos. and 27 neg.), 39 supramammary lymph nodes (12 pos. and 27 neg.), 44 prescapulars (17 pos. and 27 neg.), 33 livers (6 pos. and 27 neg.), 37 spleens (10 pos. and 27 neg.), and 34 udders (7 pos. and 27 neg.). All the samples were submitted to the four DNA extraction protocols and the same amplification process using the primers B4 and B5. According to consolidated results by tissue, the proportion of positives in cotyledon was bigger than supramammary lymph node (p=0,0001), prescapular lymph node (p<0,0001), liver (p=0,0006), spleen (p<0,0001) and udder (p=0,0019). The consolidated results for the extraction methods show that Santos protocol had bigger relative sensitivity than Boom method (p=0,003) and GT (p=0,0506), and was equal than PK (p=0,2969). There were not significant statistical differences in the others comparisons of proportions. In the study, PCR-positive and isolation-negative samples and vice-versa were verified. However, the disadvantages of the classic bacteriological method, the best strategy for direct diagnosis of the infection of cows for B. abortus in homogenized of tissues is combined use of isolation and PCR, examining the cotyledons and using the methods of DNA extraction from Santos or PK.

Este estudo comparou o desempenho de quatro protocolos de extração de DNA a partir de homogeneizados de diferentes órgãos provenientes de vacas infectadas experimentalmente com a B. abortus 2308. Os protocolos de extração comparados foram o método de GT (lise com isotiocianato de guanidina), Boom (lise com GT e tratamento com suspensão carreadora Diatomaceous earth), PK (lise com proteinase K) e Santos (lise por fervura e congelamento com nitrogênio líquido). Foram constituídos os grupos padrão ouro positivo e negativo baseados na bacteriologia clássica e compostos por: 54 cotilédones (27 pos. e 27 neg.), 39 linfonodos supra mamários (12 pos. e 27 neg.), 44 pré-escapulares (17 pos. e 27 neg.), 33 fígados (6 pos. e 27 neg.), 37 baços (10 pos. e 27 neg.), e 34 úberes (7 pos. e 27 neg.). Todas as amostras foram submetidas aos quatro protocolos de extração e a um mesmo processo de amplificação com os primers B4 e B5. Nos resultados consolidados por órgãos, a proporção de positivos no cotilédone foi maior do que a encontrada no linfonodo supramamário (p=0,0001), linfonodo pré-escapular (p<0,0001), fígado (p=0,0006), baço (p<0,0001) ou úbere (p=0,0019). Os resultados acumulados para os métodos de extração mostraram que o protocolo de Santos teve maior sensibilidade relativa do que o método de Boom (p=0,003) e GT (p=0,0506), e foi igual ao PK (p=0,2969). As demais comparações de proporções não resultaram em diferenças estatisticamente significantes. No estudo verificou-se amostas positivas a PCR e negativas ao isolamento e viceversa. Assim, apesar das desvantagens do método bacteriológico clássico, a melhor estratégia para o diagnóstico direto da infecção de vacas por B. abortus em homogeneizado de órgãos é a utilização conjunta do isolamento e da PCR, examinando os cotilédones e utilizando os métodos de extração de DNA Santos ou PK. / This study compared the performance of four protocols of DNA extraction from suspensions of different tissues from experimentally infected cows with 2308 strain. The compared extraction protocols were GT protocol (lyse with guanidine isotiocianate), Boom (lyse with GT and treated with the carrying suspension Diatomaceous earth), PK (lise with proteinase K) and Santos (lyse by boiling and freezing with liquid nitrogen). Based on classical bacteriology, positive and negative gold standard groups were built and consisted of 54 cotyledons (27 pos. and 27 neg.), 39 supramammary lymph nodes (12 pos. and 27 neg.), 44 prescapulars (17 pos. and 27 neg.), 33 livers (6 pos. and 27 neg.), 37 spleens (10 pos. and 27 neg.), and 34 udders (7 pos. and 27 neg.). All the samples were submitted to the four DNA extraction protocols and the same amplification process using the primers B4 and B5. According to consolidated results by tissue, the proportion of positives in cotyledon was bigger than supramammary lymph node (p=0,0001), prescapular lymph node (p<0,0001), liver (p=0,0006), spleen (p<0,0001) and udder (p=0,0019). The consolidated results for the extraction methods show that Santos protocol had bigger relative sensitivity than Boom method (p=0,003) and GT (p=0,0506), and was equal than PK (p=0,2969). There were not significant statistical differences in the others comparisons of proportions. In the study, PCR-positive and isolation-negative samples and vice-versa were verified. However, the disadvantages of the classic bacteriological method, the best strategy for direct diagnosis of the infection of cows for B. abortus in homogenized of tissues is combined use of isolation and PCR, examining the cotyledons and using the methods of DNA extraction from Santos or PK.

Abstracts in English and Zulu / Fluorescence polarization assay (FPA), a serological assay, was validated as an alternative test for the rapid and cost-effective diagnosis of bovine brucellosis, with the aim of improving the control of brucellosis in South Africa. The FPA is anticipated to distinguish between vaccinated and infected cattle, circumventing the challenge associated with the tests that are currently used. Positive cattle serum samples (n =420) confirmed by Complement Fixation Test were tested in conjunction with serum samples (n = 446) from non-infected cattle initially tested on Rose Bengal Test, CFT and compared with FPA. The optimum cut-off value that offers the highest diagnostic sensitivity (Dsn) and diagnostic specificity (Dsp) was determined as 87 mP with the use of ROC analysis. The Dsn and Dsp of FPA using this cut-off value was calculated at 99.09% - 100% and 68.09%- 76.61% respectively with a 95% confidence interval (cl). The area under curve (AUC) was calculated at 0.9842 with a 95% standard error (S.E) of 0.005532 with positive and negative likelihood ratio (+LR) (-LR) at 3.643 and 1.002, respectively. The FPA was found to be as effective as CFT and should be considered because of its accuracy and other advantages such as speed, high throughput and the objectivity of the interpretation of results that can be obtained electronically by the (PHERAstar) machine. The test should be included in routine serological diagnosis for brucellosis. / I- Fluorescence polarization assay (i-FPA) ukuhlolwa kwe-serological okuqinisekiswe njengenye indlela yokuhlola ukuxilongwa okusheshayo nengabizi kwe-bovine brucellosis, okuzokwenza ngcono ukulawulwa kwe-brucellosis eNingizimu Afrika. Ngaphezu kwalokho, i-FPA kulindeleke ukuthi yehlukanise phakathi kwezinkomo ezigonyiwe nezithelelekile futhi lokhu kuzonciphisa inselelo ehambisana nokuhlolwa esetshenziswa njengamanje. Amasampula amahle avumayo we-serum ezinkomo (n = 420) aqinisekiswa yi-CFT ahlolwe ngokuhlangana namasampula e-serum (n = 446) avela ezinkomeni ezingathelelekile ezahlolwa kuqala ku-RBT, CFT futhi kuqhathaniswa ne-FPA. Inani elinqunyiwe elikhulu elinikezela ukuzwela okuphezulu kokuxilonga (i-Dsn) kanye nokucaciswa kokuxilongwa (i-Dsp) kunqunywe njenge-87 mP kusetshenziswa ukuhlaziywa kwe-ROC. I-Dsn ne-Dsp ye-FPA esebenzisa leli nani elisikiwe libalwe ngama-99.09% - 100% no-68.09% - 76.61% ngokulandelana kwesikhathi sokuzethemba esingu-95% (cl). Indawo engaphansi kwe-Curve noma ijika thizeni (i-AUC) ibalwe ku-0.9842 enephutha elingu-95% elijwayelekile (SE) lika- 0.005532 elinezilinganiso ezinhle nezimbi ze-likehood (+ LR) (-LR) ngo-3.643 no- 1.002, ngokulandelana. I-FPA isebenza njenge-CFT futhi kufanele ibhekwe ngenxa yokunemba eneqiniso kwayo nezinye izinzuzo ezifana nejubane lokuthola imiphumela kanye nenhloso yokuchazwa kwemiphumela engatholakala ngomshini wekhomphuyitha (PHERAstar), i-FPA kufanele ifakwe ekuhlolweni okuvamile ngokujwayelekile kwe-serological ye-brucellosis. / Agriculture and  Animal Health / M. Sc. (Agriculture)

Brucellosis is one of the neglected diseases in Nigeria. In Lagos, the commercial capital of Nigeria with about twenty one million people, a descriptive cross-sectional study was carried out in order to determine the sero-prevalence of brucellosis among people at risk in some selected abattoirs and secondary health care facilities (hospitals) in the state. Mixed sampling method was employed at the abattoir while convenient sampling method was used in sampling the respondents at the hospitals. Sera samples from three hundred and one (n=301) abattoir-based workers and traders; and one hundred and twenty one (n=121) hospital-based individuals which include people with febrile illnesses and blood donors were tested for brucellosis using Rose Bengal Plate test (RBPT), with indirect ELISA being used as a confirmatory test. Of the 301 abattoir-based workers and traders, 27 (8.97%) were sero-positive to the infection when Rose Bengal Plate test antigen was used. The twenty seven individuals consists of fifteen (15) butchers; four (4) veterinarians; two (2) meat transporters and bone/cow horn dealers each as well as one each of blood meal producer, abattoir engineer, water seller and meat supplier. When blood samples from the sero-positive individuals were subjected to ELISA, 3 (11.1%) were sero-positive to the brucellosis, while one is equivocal. These results confirm that agglutination observed on RBPT might be related to unknown cross-reactions and confirmation with a different test was necessary. None of the hospital-based respondents is sero-positive to the infection. The clinical signs significant for the infection in this study were fever, joint pain, lower backache, regular headache and miscarriage. Brucellosis awareness level among the respondents was very low. Data was analysed using (SPSS) version 20.0 at α0.05 significant level. The significant risk factors for human brucellosis according to this research are consumption of fura (unpasteurized milk) and wara (fresh cheese). The study revealed that brucellosis is not only an occupational disease but can also affect people who trade or live in proximity with infected animals. / Agriculture, Animal Health and Human Ecology / M. Sc. (Agriculture)