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Tatenda D. Mushangwe and Paidamwoyo B. Mutowembwa
A thesis submitted in partial fulfilment of the requirements for the degree of BACHELOR OF VETERINARY SCIENCE (BVSc)
Department of Clinical Veterinary Studies Faculty of Veterinary Science University of Zimbabwe
The use of fluorescence polarisation assay (FPA) in the diagnosis of bovine brucellosis in Zimbabwe
Tatenda D. Mushangwe and Paidamwoyo B. Mutowembwa
A thesis submitted in partial fulfilment of the requirements for the degree of BACHELOR OF VETERINARY SCIENCE (BVSc)
Approved as to style and content by:
_________________________ Dr G Matope (Supervisor)
Abstract The use of fluorescence polarisation assay (FPA) in the diagnosis of bovine brucellosis in Zimbabwe By
Tatenda D. Mushangwe and Paidamwoyo B. Mutowembwa
A homogenous fluorescence polarisation assay (FPA) which relies on molecular rotational properties to measure binding of antibody to a fluorescein isothiocyanate (FITC) labelled 20-30kDa lipopolysaccharide antigen prepared from Brucella abortus was used to detect antibodies to Brucella species in serum of cattle from Gokwe, Nharira- Lancashire, Wedza, Chimanimani and Chipinge smallholder dairy farms in Zimbabwe. Fluorescence polarisation was measured using a fluorescence polarisation analyzer, Diachemix ®. The potential use of this assay in the diagnosis of bovine brucellosis was assessed in comparison to the competitive enzyme immunosorbent assay (c-ELISA), rose Bengal (RB) and the serum agglutination test (SAT) using 555 sera. For the FPA, a cut off point of 90 millipolarisation (mP) units, determined using the receiver operating characteristic (ROC) curves was found to give the best performance for identifying positive and negative sera when compared to the c-ELISA. Using the c-ELISA as the gold standard, the calculated relative sensitivities for RB, SAT and FPA were, 86.11, 37.01% and 66.11% respectively, while the relative specificities were 97.32%, 99.28 and 96.54% respectively. The FPA kappa coefficient of agreement with respect to c-ELISA was 0.5818, while SAT and Rose
Based on the findings of the study. iv . respectively. both in clinical laboratories and in the field under Zimbabwean conditions because the test is inexpensive. simple. the FPA could be readily adopted as a diagnostic test for bovine brucellosis.7659 and 0.4750. quick to perform and gives instant results that are easy to interpret.Bengal (with respect to c-ELISA) gave coefficients of 0. The limitations of evaluating a serological test in the absence of a gold standard test are discussed in detail.
Pamhidzai.M Linus. you showed me a gift I had not known existed. today I find myself here.Dedications To my mother Rosemary. You are such an angel with a golden heart. Mildred. and a sense of belonging throughout my studies. for giving me hope and guidance. .P. patience.B. you taught me to dream beyond my limitations.D. my Father. my Mother.M v . for courage. –T.
Dr Matope for his guidance in all aspects of the project that include carrying out the laboratory tests. We are grateful the Norwegian Council for Higher Education and Development (NUFU) Project for provision of material and financial support and resources which saw to this project’s completion. We would also like to thank the laboratory staff from the Paraclinical Veterinary Studies (PAVS) Microbiology section. Special mention to Ms Pawandiwa. statistical analysis and write up of the project. Dr Pfukenyi and the Central Veterinary Laboratories (Harare) Virology Section head and staff for providing reagents and assistance for laboratory work. Dr Bhebhe.Acknowledgements We would like to pass our deepest and most gratified thanks to our supervisor. vi .
Table of contents Introduction……………………………………………………………………… Justification……………………………………………………………… Objectives…………………………………………………………………. Serological Diagnosis of Bovine Brucellosis…………………………… Materials and Methods……………………………………………………… Sera……………………………………………………………………… Serological Tests…………………………………………………… Statistical Analyses……………………………………………………… Results…………………………………………………………………………… Discussion……………………………………………………………………. Literature review……………………………………………………………… Definition………………………………………………………………… Epidemiology of Bovine Brucellosis in Zimbabwe……………………….. Control of Bovine Brucellosis…………………………………………. Conclusion……………………………………………………………………… Appendix 1: Tables of results…………………………………………………… References……………………………………………………………………… 1 2 4 5 5 7 9 10 17 17 17 19 20 21 23 24 27 vii .
the 24 Rose Bengal (RB). Serum Agglutination Test (SAT). RB and SAT 26 with respect to c-ELISA viii .2: Test agreements for the Rose Bengal (RB). Serum Agglutination Test 25 (SAT).List of Tables Table 4. and Competitive Enzyme Immunoabsorbent Assay (C-ELISA) Table 4.1: Test agreements for the Fluorescent Polarisation Assay (FPA) with the. and Competitive Enzyme Immunoabsorbent Assay (C-ELISA) Table 4.3: The Relative Sensitivity and specificity results of the FPA.
Abbreviations Ab: B. abortus: BPAT: c-ELISA: CI: CFT: CVL: ELISA: FPA: i-ELISA: IgM: kDa: MRT: mP: OD: PAVS: RB: RIV: ROC: SAT: Antibody Brucella abortus Brucella Plate Agglutination Test Competitive Enzyme Linked Immunosorbent Assay Confidence Interval Compliment Fixation Test Central Veterinary Laboratories Enzyme Linked Immunosorbent Assay Fluorescence Polarisation Assay Indirect Enzyme Linked Immunosorbent Assay Immunoglobulin M Kilo Daltons Milk Ring Test Millipolarisation (units) optical densities Paraclinical Veterinary Studies Rose Bengal Test Rivanol Agglutination Receiver Operator Characteristic Serum Agglutination Test USDA:United States Department of Agriculture µl: Micro litres ix .
2002). include test cost.. 2002). tests that are prone to give false positive results have tendencies to condemn animals that would have been negative. ease of performance. while tests that give false negative results will prolong any control campaign by their inability to capture all truly positive cattle. is caused by biovars of Brucella abortus (B. in order to get optimal results. The disease has been eradicated in some developed countries through implementation of stringent control measures that include regular serological testing and slaughter of positive reactor animals (Anon..Introduction Bovine brucellosis. Thus the control programmes for brucellosis are heavily dependent on presumptive diagnosis of infection by serological tests and the subsequent recommendation of slaughter of the infected cattle (Anon. melitensis in cattle kept close together with sheep and goats (Anon. serological tests are often used in combination using either parallel or serial testing programmes. The accuracy of the serological tests used has considerable impact on the success of a programme. Consequently. 2002). Therefore. a disease of world-wide economic and public health importance. Justification 1 . abortus) and occasionally by B. and turn around time for results. test precision. interference by antibody to vaccine or cross reacting antigens. It is noteworthy that there is no single serological test that is regarded as a perfect test.. Other factors of importance. Bovine brucellosis is endemic in many countries in the world including most countries in Sub-Saharan Africa (McDermott and Arimi. This necessitates the adoption of newer individual tests with superior sensitivity and specificity values that can be used to achieve accurate diagnosis of bovine brucellosis. 2002).
. Although the complement fixation test (CFT) is similarly regarded as the test of choice for international trade (Anon. The Rose Bengal test is highly sensitive but tends to produce many false positives leading to the unnecessary condemnation of animals. 1996).The competitive enzyme linked immunosorbent assay (c-ELISA) is considered to be a very good test because of its superior specificity and sensitivity and can differentiate antibodies due to B. it has inherent problems of low sensitivity and its omission from the panel of suitable tests has been suggested (Anon. abortus S19 vaccinal antibodies from those produced by field strains of B. In contrast. 2002). SAT has shortcomings in failure to differentiate B. but its advantages are accompanied by numerous disadvantages. 2 . the turn around time is longer since the test is done over two days. 2002).. abortus S19 vaccine from antibodies produced against field strains of B. abortus (Nielsen et al. the fluorescence polarisation assay (FPA) gives a single result that is easily read as positive or negative by using standard cut off values. Although the serum agglutination test (SAT) is commonly used in many brucellosis control programme due to its ease of use. 2002) due to its high specificity. It is recommended as the test of choice for international trade (Anon.. It is cumbersome (it is difficult to perform and time consuming) and expensive. A further disadvantage of the test is in its inability to give easily interpretable data because the results obtained have to be calculated either by computer based software or manually by hand. In addition. Together with the CFT and the Rose Bengal. making it prone to error.. abortus (Nielsen et al. it is cumbersome to perform and often requires experienced laboratory technologists. Besides the difficulties in the sourcing of test reagents and kits the need for an ELISA reader makes the test inaccessible to most laboratories in the third world..
1999. other than serum. There is no requirement for removal of excess reagents hence relatively easy to perform (Nielsen et al. 2003). no single test has satisfied the appropriate criteria for each and all epidemiological situations hence the attempt to use the fluorescence polarisation assay (FPA). The FPA is a homogeneous assay which only requires addition of labelled antigen to appropriately diluted test samples. (Nielsen et al. McGiven et al. In contrast. but up till now. 2000). abortus (99.. the FPA can differentiate B. abortus S19 vaccinal antibodies from antibodies produced against field strains of B.96%. 1996). 2002)... abortus (Nielsen et al. 1999. Dajer et al. 1996).. Because of the reported high sensitivity and specificity values for the FPA for detection of bovine serum antibody to B. 2001) 3 .1996).02% and 99.. The above mentioned tests and several other tests have been used in routine monitoring and screening of infected herds. thereby compromising their specificities. 1996. its speed and ease of performance.. the FPA can utilize whole blood and milk to detect antibodies against Brucella species (Nielsen et al... Samartino et al. In addition. respectively). The test has been used with success for the serological diagnosis of brucellosis in cattle in some countries (Nielsen et al. The FPA for detection of antibody to Brucella species has been recommended as the test of choice for international trade and has been suggested as a suitable replacement for the CFT (Anon. it is an ideal candidate for adaptation to use in the simple laboratory set up as well as under field conditions..
Rose Bengal and Serum Agglutination Test. To evaluate the suitability of FPA as a standard test for bovine brucellosis 4 . RB. To determine the level of agreement between FPA. and competitive ELISA 2.Objectives 1. and SAT relative to the competitive ELISA 3. To establish the specificity and sensitivity of FPA.
Following infection with B.. 5 . 2002). involving one or more leg joints. 1994) and decreased milk production. pregnant adult females develop a placentitis usually resulting in abortion between the fifth and ninth month of pregnancy. Zimbabwe). 1989. al. is a common manifestation of brucellosis in some tropical countries (MacDermott et. 1994).. Bulls may develop epidydimitis and orchitis with a subsequent drop in fertility (Radostits et al.Literature Review Definition Bovine Brucellosis. 1987. is listed as a notifiable disease (Madsen. or Bang’s disease.. abortus). In naïve cattle herds the disease is clinically characterised by “abortion storms” where about 90% of the pregnant animals may abort (Radostits et al. 1996). after which a degree of immunity is attained. 1994). abortus or B. and the predilection sites are the gravid uterus and the reproductive tract of male animals. bovine brucellosis. (Brucellosis Control Regulations. melitensis (Anon. suis infections in cattle have not been reported in Zimbabwe (Madsen. melitensis.. 2002). infection can also be caused by B. Females usually abort only once. Hygroma formation. Mohan et al. Anon.. However. melitensis and B.. is a disease of cattle caused by biovars of a gramnegative bacterium called Brucella abortus (B. abortus has been isolated from bovine foetuses. According to the Animal Health Act. While B. and animals remain infected and can shed the organism in subsequent parturitions (Quinn et al. In countries where cattle are kept in close association with sheep or goats.. 1989). Brucellosis is usually a disease of the sexually mature animals. cases bovine brucellosis due to B.
The use of stamping out policy in Brucella seropositive herds causes further loses in production (Alton. Brucellosis is of major public health significance. respiratory. Infection is often due to occupational exposure and is essentially acquired by the oral. Brucellosis is one of the most easily acquired laboratory infections. 1989). causing acute febrile illness – undulant fever – which may progress to a more chronic form and can also produce serious complications affecting the musculo–skeletal. but ingestion of dairy products constitutes the main risk to the general public. cardiovascular.. Although human brucellosis has been reported in many African countries (McDermott and Arimi. B. abortus. economic loss occurs mainly through abortions that lower the calf crop. the re is a drastic reduction in milk production which can be reduced by about 10 %. 1994). Specific recommendations have been made for the safety precautions to be observed with Brucella-infected materials (Anon. abortus S19 vaccine (Corbel et al.such lesions may be found in animals that have been vaccinated with B. 1975). and central nervous systems (Radostits et al. or conjunctival routes. suis are highly pathogenic for man (Anon. such as products of abortion.. and strict safety precautions should be observed when handling cultures and heavily infected samples. If brucellosis is endemic in a herd. In addition. melitensis and B. There is an occupational risk to veterinarians and farmers who handle infected animals and aborted foetuses or placentae. It could be that a lot of cases remain undiagnosed since brucellosis is difficult to detect clinically (McDermott 6 . 2002) and are readily transmissible to humans. 2002). B.. 2002) there are no reports of human brucellosis in Zimbabwe..
The disease has been eradicated in some developed countries through implementation of stringent control measures that include regular serological testing and slaughter of positive reactor animals (Anon. However. 2002) and cases are often misclassified as malaria. 7 . Alternatively. 1989). There is limited data from communal herds (both dairy and beef). 1996). Brucellosis is reported to be endemic in some commercial farms in Zimbabwe (Mohan et al. 2002). 2002). but a survey conducted in the late 1980s showed low sero-prevalence of Brucella abortus antibodies in beef cattle from various communal areas around the country (Madsen. this may be largely due to rigorous brucellosis control measures that include milk and meat hygiene (Madsen. 2002).. 2002) Brucellosis is widespread in most countries in Africa. while other areas have eradicated the diseases presumably due to the implementation of the Brucellosis accreditation scheme that was legislated for the commercial farming sector in the early 1980s (Madsen. with a tendency of higher prevalence in commingled cattle than those confined (McDermott and Arimi. 1989). Epidemiology of bovine brucellosis Distribution Bovine brucellosis is endemic in many countries in the world including most countries in Sub-Saharan Africa (McDermott and Arimi. 1989). the prevalence and incidence vary from country to country and from place to place within a country depending on the type of cattle farming system (MacDermott and Arimi..and Arimi. Brucellosis has been found to be prevalent in communal cattle in other countries.
bactrianus). elk / wapiti (Cervus elaphus) and also occurs in the African buffalo (Syncerus caffer) and various African antelope species. The main route of infection is by ingestion of food or drinking water contaminated by aborted material or uterine discharges from the aborting animal (Radostits et al. infection may occur in utero. serological evidence of brucellosis was demonstrated in both herbivores and scavenging wildlife species (Condy and Vickers. but their role in spreading infection to domestic livestock or vice versa is not known. the diseases has also been reported in the one-humped camel (Camelus dromedearius) and in the twohumped camel (C. The disease dynamics are largely 8 . 2002). The manifestations of brucellosis in these animals are similar to those in cattle (Anon.. However. 1994)... In addition. bonasus). melitensis (Anon.. Less commonly. 1972. B.. the interaction between domestic livestock and wildlife facilitates bimodal transmission of diseases with both domestic animals and wildlife being important reservoirs (Godfroid et al.Host range Although brucellosis is of major economic importance in cattle farming. 2002). via conjunctiva or by inhalation (Quinn et al. Transmission The transmission of the organisms causing bovine brucellosis is by direct or indirect contact with infective excretors. related to contact with large and small ruminants infected with B. abortus or B. American and European bison (Bison bison. Madsen and Andersen. yak (Bos grunniens). 1995). 1994). 2004). brucellosis has been observed in the domestic buffalo (Bubalus bubalus). In Zimbabwe.
abortus S19 and the implementation of the test and slaughter programme. the control of bovine brucellosis is generally based on calf-hood vaccination using B. 1989. the prevalence rate and intensity of contact between herds and herd level immunity. aimed at control and possible eradication. However. A brucellosis Accreditation scheme. blood and other products used for food. There is a general lack of information on occurrence of brucellosis and the risk factors involved in wildlife –livestock interface areas. Moreover. abortus S19 live attenuated vaccine is practiced. In Zimbabwe. milk. vaccination with B.influenced by herd size. In addition. 1996). has been legislated and implemented for the commercial dairy farming sector (Madsen. Brucellosis is a public risk by virtue of it being readily transmissible to humans through handling infected animals and animal products or by consumption of meat. 9 . vaccination alone does not result in complete eradication of bovine brucellosis.. abortus S19 results in complications of interpreting serological results due to the occurrence of cross-reacting antibodies (Nielsen et al. Control of bovine brucellosis Bovine brucellosis has been controlled and successfully eradicated in some countries through test and slaughter policies. Thus necessitated is stringency in control of the disease and new control strategies are always in demand. vaccination of female calves between three and ten months of age using B. management factors.
and each has its own special applications and limitations (Alton et al. 1989). Several many modifications of the original agglutination test have been made to increase the specificity (Angus and Barton. 1897). therefore unreliable and hence its use should be discontinued as recommended by OIE (Anon. To be accredited as brucellosis free. If a positive test is recorded. 1977) and the buffered plate agglutination test in which antigens are used at low pH. they are culled through slaughter and the whole process repeated again until three negative consecutive tests are obtained. the accreditation certificate is forfeited. To be reaccredited. strict implementation of the Accreditation scheme resulted in eradication of bovine brucellosis (Madsen.. This is similar to the standard tube agglutination test that mainly detects the immunoglobulin M (IgM) antibody.Mohan et al.. Therefore the tube agglutination is prone to false positives. 2002).. If positive animals are detected. 1984). Serological diagnosis of brucellosis was first accomplished using an agglutination test (Wright and Smith.. and among them is the use of an acidified antigen in the Rose Bengal/card test (Nicoletti. Individual animals have to be tested serologically to identify reactors. a farm bleeds and tests all dairy cattle over 18 months of age for three consecutive times at three monthly intervals. Mikolon et al. 1996). 1998). Serological diagnosis of bovine brucellosis A number of serological tests have been developed for the diagnosis of brucellosis. an accreditation certificate is issued and is tenable for one year after which animals will be re-bled and tested. All accredited farms are monitored monthly by milk ring test conducted on bulk milk. the whole process of three monthly serological testing and culling of positive animals is conducted. Both tests 10 . 1975. If no positive animals are recorded. In commercial dairy farms.
. have been identified as suitable screening tests but confirmation using a more specific test is necessary (Anon. the SAT has been used widely in brucellosis control in Zimbabwe. Also.. Rose Bengal (RB) test and the buffered plate agglutination test (BPAT). 11 . and is recommended for screening bovine brucellosis (Madsen. 2002). 1989).are considered suitable for screening individual animals. 2002). only samples with antibody titres of at least 1:160 are treated as positives.. Suitable confirmatory tests are therefore called on to confirm the positives. It is commonly used to monitor brucellosis using bulk tank milk. Because they are highly sensitive tests. 1989) but its sensitivity can easily be affected by pooling of samples. Those with titres of 1:20. (Anon. 1977). as well as the ELISA and the FPA.. although false positives can occur due to prozoning (Nielsen. 2002). 2002). false reactors are frequent in cattle vaccinated four months prior to testing. For the control of brucellosis at the national or local level. It should be stressed that the serum agglutination test (SAT) is generally regarded as being unsatisfactory for the purposes of international trade due to its poor sensitivity (Anon. However. In spite of its shortcomings. in mastitic cows’ milk or if colostrum samples are subjected to the test (Nicoletti. the buffered Brucella antigen tests (BBATs). 1:40 and 1:80 are classified as doubtful reactors (and require further testing using the compliment fixation test (CFT) (Madsen. they can give positive reaction due to the S19 vaccine or due to false-positive serological reactions. 2002) An adaptation of the serum agglutination test was applied to develop the milk ring (MR) test to detect the presence of Brucella antibodies in milk (Anon.
in spite of its inherent problems and also that its specificity is lower than that of the competitive ELISA (c-ELISA). their use may be preferred (Wright et al.. 1995). Among other problems. but this also failed to differentiate vaccinal from field infection antibodies. and as they are technically simpler to perform and more robust. 1963)... 2002). and also has a standardised system of unitage. However. in its ability to differentiate vaccinal from field infection antibody. the c-ELISA. 1989). The CFT is diagnostically more specific than the SAT. Dajer et al.. initially had two forms which later were standardised to one (Hill. the CFT failed to distinguish vaccinal from natural infection antibody and the occasional occurrence of serum samples that activate complement in the absence of antigen (Nielsen. has been shown to be highly specific and sensitive and thus a more reliable test than the iELISA (Nielsen et al.. 1996. To improve test sensitivity. 1999). the indirect ELISA (i-ELISA) test was developed (Nielsen et al. the CFT has been a valuable test for many eradication schemes as a confirmatory test and is recommended by OIE as the prescribed test for international trade (Anon. 2002). The performances of several of these tests have been compared (Nielsen et al.The CFT is more recent to the SAT. 12 .. The diagnostic performance characteristics of some enzyme-linked immunosorbent assays (ELISAs) and the fluorescence polarisation assay (FPA) are comparable with or better than that of the CFT. However. 1993).
Thus a large molecule emits more light in a single plane (more polarised) than a small molecule rotating faster and emitting more depolarised light (Nielsen et al. USDA and Mexican milk ring tests and milk ELISA. 2000) 13 . the time of rotation through an angle of 68. French Rose-Bengal plate test. USDA buffered acid agglutination test. It is a homogeneous assay in which analytes are not separated and it is therefore very rapid. The milk ELISA test was found to offer more sensitivity and specificity than the MRT and also provided ease of interpretation (Mikolon et al. The Fluorescence Polarisation Assay The FPA is a simple technique for measuring antigen/antibody interaction and may be performed in a laboratory setting or in the field. The mechanism of the assay is based on random rotation of molecules in solution. but in practice some impractical in use for routine diagnosis of brucellosis. numerous other antibody detection tests for brucellosis have been evaluated. Molecular size is the main factor influencing the rate of rotation.. Mexican Rose Bengal plate test. Thus a small molecule rotates faster than a large molecule..Recently.5° can be determined by measuring polarised light intensity in vertical and horizontal planes. USDA (United States Department of Agriculture) card test. 1998). These include. USDA standard plate test. USDA and Mexican rivanol tests. which is inversely related. If a molecule is labelled with a fluorochrome. rapid automated presumptive test.
1999). The FPA also agreed almost perfectly with the CFT (Kappa=0. 1996). is labelled with a fluorochrome and added to serum or other fluid to be tested for the presence of antibody.0%.. Dajer et al 1999. Nevertheless. (1999) did a field study in Mexico.96). 2001. Dajer et al. Samartino et al.70 and 0. Rivanol Agglutination (RIV)). the FPA has been found to be a suitable confirmatory test for Bovine brucellosis..For most FPAs. which compared the FPA to tests currently in use in that country. It was recommended that the FPA was a suitable replacement for CFT In the serological diagnosis of B. while RB and RIV gave Kappa values of 0. attachment to the labelled antigen will cause its rotational rate to decrease and this decrease can be measured (Anon. In other Canadian studies.. The sensitivity of the test (using sera from culture positive animals) ranged from 66% to 100% (Nielsen et at.. Performance of FPA under Field Conditions Some work has been done to compare the FPA with existing tests in different regions (Nielsen et al. FPA gave higher relative sensitivity and specificity than the other tests (RB. Elsewhere in Canada. less than 50 kDa. 14 . Using the CFT as a gold standard.5% to 99. whilst serological positivity of cattle from infected premises ranged from 65. If antibody is present.61 respectively with respect to CFT. 2002). an antigen of small molecular weight. 1998). the performance of the FPA was apparently varied in different experiments.0% and 100% and the specificity in both cases was 100% (Nielsen et al. the sensitivity values were 99. 1996. abortus..
. 2001). abortus S19 vaccine and field infection by B. Relative sensitivity and specificity values for the FPA performed in the field. it would be useful to test whole blood rather than serum.. Because of its high relative sensitivity and specificity (Nielsen et-al. Dajer et al. sheep (Minas et al.3 and 97. In addition.3%. 2001). respectively. The FPA was evaluated for use in different animal species. 1999). its speed and ease of performance it is an ideal candidate for adaptation to use both in laboratory and field. based on buffered antigen plate agglutination test and competitive enzyme immunoassay results were 95. To expedite field-testing. 1996... 1999. whole blood) (Nielsen and Gall. abortus.. Samartino et al. 2003) and bison (Gall et al. milk.. It has been used successfully in cattle (Nielsen et al. humans (Lucero et al.. This project was conducted to evaluate the suitability of FPA for the serological diagnosis of bovine brucellosis by comparing its performance relative to the conventional tests (Rose Bengal. 15 . Thus the study aptly demonstrated the usefulness of the FPA for testing whole blood samples in the field.. its ability to detect antibodies to Brucella species using a variety of samples (sera. 2005). abortus) with sera from animals with confirmed (bacteriologically) infection (Nielsen et al. thus producing results similar to the c-ELISA. 1996). the study also validated the FPA’s ability to distinguish between antibodies induced by B.The FPA has also been used to test whole blood samples prepared by mixing blood cells from cattle without exposure to Brucella abortus (B. Serum agglutination test and c-ELISA) that are commonly used in the diagnosis of the disease. 2000).
. All sera were previously collected from smallholder cattle from Gokwe (Gokwe district). Control sera were obtained from the Central Veterinary Laboratory (Weybridge. Nharira. Fluorescence polarisation assay (FPA) The FPA was performed by the method described by Nielsen et al. Serological tests Rose Bengal Test The Rose Bengal test was performed essentially as described by Alton et al. 10µl serum sample were 16 . (1996).Materials and methods Sera Sera (n=555) were obtained from the serum bank from the Department of Paraclinical Veterinary Studies (PAVS) of the University of Zimbabwe. However all animals sampled were over 18 months of age.Lancashire (Chikomba district). RB. 25 µl of serum were mixed with equal volume of stained buffered Rose Bengal antigen (Weybridge) onto standard test plates. The vaccination status of the sampled animals was unknown. Wedza (Wedza district) and Rusitu Valley (Chipinge district). (1988). The individual age. All sera were tested in parallel using c-ELISA. All samples were kept at -20°C until they were tested. and FPA. Test plates were agitated onto shakers for five minutes and results recorded as positive or negative. 1ml of FPA buffer was pipetted into 10x75mm culture tubes (Durex™). Briefly. SAT. sex and parity of the cattle sampled were not recorded. UK).
Serum Agglutination test (SAT) The SAT was performed according to the procedure of Alton et al. the fluorescence polarisation of the tracer was determined (with the reading from the blank subtracted).added and a background measurement was obtained using a fluorescein. A predetermined amount of tracer was added and after mixing and incubation at room temperature for at least 2 min. sera to be tested were equilibrated to room temperature (23-25 0C). Sweden) was used to test the sera. Awareness Technology. (1975). 1:1280 and 1:2560 were used. Double dilutions of sera from 1:20.. 17 . The optical densities (OD) were measured at 450 nm in a micro-plate photometer (Humareader®. Data from this assay was expressed as millipolarisation (mP) units. Illinois.labelled B. SvanovirTM Brucella-Ab c-ELISA kit (Svanova Biotech AB Uppsala. 1:640. Diachemix LLC. 1:80. Competitive ELISA The c-ELISA was performed as described by Nielsen et al. Inc. The antigens and control sera were sourced from the Onderstepoort Research Institute (South Africa). Sera and controls were run in duplicates. Grayslake. abortus O-polysaccharide tracer and a fluorescence-polarization analyzer (Sentry FP 100®. The threshold for determining sero positivity was according to the manufacturer’s recommendations. Model 18500/1. 1:160. USA). (1989). 1:320. Antibody titres were recorded as percentage inhibition equivalents of absorbance readings. 1:40. Briefly. Germany).
results defined as slight (kappa < 0. Using the c-ELISA as a gold standard test. USA).2). substantial (0.0 software 18 .6). moderate (kappa 0.2 to 0. c-ELISA. RB and SAT relative to the c-ELISA (plus 95%confidence limits) were calculated using Win Episcope 2.6 to 0. Statistical analysis The cut-off for FPA which gave optimum values of sensitivity and specificity was determined by receiver operator characteristic (ROC) analysis using STATA 9™ software (STATA Corporation.8) and almost perfect (kappa > 0. Texas. 2002).8) (Dohoo et al. SAT.4 to 0. results from the FPA. RBT and SAT was analysed using the McNemar’s χ2 test for paired data in comparison relative to RB.Only results from samples subjected to all four tests were selected for analyses. The sensitivity and specificity of the FPA.4). SAT and c-ELISA using STATA 9 software. The Kappa measure of agreement was calculated using STATA 9 for the FPA with the RB. fair (kappa 0.
19 .3.Results The significance and level of agreement of the FPA to the RB.1% and 96. SAT.1. and c-ELISA are shown in Table 4. respectively. The sensitivities and specificities of the FPA. The experiment suggested a cut-off value of 90 mP resulting in the highest sensitivity and specificity combination values of 60. whilst those of the other tests’ performance comparisons are shown in Table 4. RB and SAT relative to the c-ELISA is shown in Table 4.2.9%.
The evaluation of the tests showed high specificity for all tests. Gall. and moderate sensitivity for FPA and RB. 1999) but however are on the low end of the range. Thus. As expected.. the results from this study agree with those of previous studies (Nielsen et al. which would be suggestive of serious disagreement with regards to the McNemar.0801). and in general agreement with previous studies. This discrepancy was probably due to the use of less well characterized sera in the study (Nielsen et al. but a slight agreement with respect to the Kappa.6891 (RB with SAT (doubtful reactors) gave a low McNemar’s statistic (0.2659. for example 0. Generally.. 2000. it is necessary to first of all carry out a validation study of the c-ELISA for Zimbabwe prior to using it as in such an instance or otherwise. for further studies. Thus. the SAT was the least sensitive test (Madsen. not validated test as a gold standard. use a different but reliable test protocol such as the CFT as gold standard.0000 but a Kappa of 0. test comparison giving a high Kappa value. which showed a fair agreement. for example. The McNemar’s test seemed to not agree with the Kappa test and in some instances. 2000) but could also have resulted from a closed end testing protocol which utilized a single.Discussion The results from the study show the FPA agreeing substantially with all the other tests with the exception of the SAT. 1996. this 20 . FPA with SAT gave a McNemar’s statistic of 0. Dajer et al. 1989). The McNemar’s χ² test and the Kappa statistic gave conflicting outcomes..
disagreement led to the disregard of the McNemar’s test as it was initially included as an assessment of whether or not there was test bias (Dohoo et al. or otherwise incorporating the RIV (this would require a field trial as the RIV is yet to be validated and used in Zimbabwe). Use of a parallel testing protocol involving at least two tests as gold standard is also advised. 1994).. suggested tests being the CFT and c-ELISA. 21 . and due to its unreliability when dealing with dichotomised data (Kraemer and Bloch. one such being to use samples of known serological status. 2002). Possible improvements can be made if a review is intended.
22 .Conclusion The ability of FPA to agree substantially with RB and c-ELISA. together with its ease to perform under field conditions render it suitable for use in the diagnosis of bovine brucellosis under Zimbabwean conditions. and slightly with SAT and it’s ability to duplicate and better the sensitivity and specificity of tests currently in use.
0000 0.6288) 0.2526-0.1: Test agreements for the fluorescent polarisation assay (FPA) with the rose bengal (RB).5690-0.4439 (0.4217-0.5989 (0.5818 (0.3618 Kappa Value (95% CI) 0.5224 0.2792) 0. serum agglutination test (SAT).4661) 0.7877 0.6148) 23 .5699-0. and competitive enzyme linked immunosorbent assay (c-ELISA) FPA Comparison with Rose Bengal SAT SAT(Doubtful reactors as Positives) C-ELISA McNemar’s Statistic 0.2659 (0.Appendix 1: Tables of Results Table 4.
5141 (0.6445-0.0000 SAT with c-ELISA 0.Table 4.4883-0.4513-0.5398) 0.2: Test agreements for the rose bengal (RB).0801 reactors as Positives) RB with SAT 0.6891 (0.5271) 24 .4986) 0.4750 (0.5066 (0.1435 RB with SAT (Doubtful 0.7236) 0.8041) 0.7276-0.0005 SAT (Doubtful reactors as 0.0336 positives) with c-ELISA Kappa Value (95% CI) 0. and competitive enzyme linked immunosorbent assay (c-ELISA) Comparison of McNemars’s Statistic RB with c-ELISA 0.4761-0.7659 (0. serum agglutination test (SAT).
15-97.65 (49.28-82.83-98.28(98.11 (51.16(92.15) 65.80) 97.98) 94.32 (95. RB and SAT with respect to c-ELISA Test FPA RB SAT SAT (Doubtful reactors as positives) Sensitivity (%) (95% CI) 66.82) 99.Table 4.99-81.44) Specificity (%) (95% CI) 96.11 (78.3: The relative sensitivity and specificity results of the FPA.81-97.23-99.40) 37.54 (94.07) 86.49-96.77) 25 .01 (21.14-53.
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