The use of fluorescence polarisation assay (FPA) in the diagnosis of bovine brucellosis in Zimbabwe

By

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

August 2006

The use of fluorescence polarisation assay (FPA) in the diagnosis of bovine brucellosis in Zimbabwe

By

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)

August 2006

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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

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Bengal (with respect to c-ELISA) gave coefficients of 0.7659 and 0.4750, respectively. The limitations of evaluating a serological test in the absence of a gold standard test are discussed in detail. Based on the findings of the study, the FPA could be readily adopted as a diagnostic test for bovine brucellosis, both in clinical laboratories and in the field under Zimbabwean conditions because the test is inexpensive, simple, quick to perform and gives instant results that are easy to interpret.

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Dedications
To my mother Rosemary, for courage, patience, and a sense of belonging throughout my studies. You are such an angel with a golden heart. - P.B.M

Linus, my Father, for giving me hope and guidance, Mildred, my Mother, you taught me to dream beyond my limitations, today I find myself here. Pamhidzai, you showed me a gift I had not known existed. T.D.M

Acknowledgements
We would like to pass our deepest and most gratified thanks to our supervisor, Dr Matope for his guidance in all aspects of the project that include carrying out the laboratory tests, statistical analysis and write up of the project. 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 projects completion. We would also like to thank the laboratory staff from the Paraclinical Veterinary Studies (PAVS) Microbiology section. Special mention to Ms Pawandiwa, Dr Bhebhe, Dr Pfukenyi and the Central Veterinary Laboratories (Harare) Virology Section head and staff for providing reagents and assistance for laboratory work.

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Table of contents
Introduction Justification Objectives. Literature review Definition Epidemiology of Bovine Brucellosis in Zimbabwe. Control of Bovine Brucellosis.. Serological Diagnosis of Bovine Brucellosis Materials and Methods Sera Serological Tests Statistical Analyses Results Discussion. Conclusion Appendix 1: Tables of results References 1 2 4 5 5 7 9 10 17 17 17 19 20 21 23 24 27

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List of Tables
Table 4.1: Test agreements for the Fluorescent Polarisation Assay (FPA) with the, the 24 Rose Bengal (RB), Serum Agglutination Test (SAT), and Competitive Enzyme Immunoabsorbent Assay (C-ELISA) Table 4.2: Test agreements for the Rose Bengal (RB), Serum Agglutination Test 25 (SAT), and Competitive Enzyme Immunoabsorbent Assay (C-ELISA) Table 4.3: The Relative Sensitivity and specificity results of the FPA, RB and SAT 26 with respect to c-ELISA

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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 l: Micro litres

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Introduction
Bovine brucellosis, a disease of world-wide economic and public health importance, is caused by biovars of Brucella abortus (B. abortus) and occasionally by B. melitensis in cattle kept close together with sheep and goats (Anon., 2002). Bovine brucellosis is endemic in many countries in the world including most countries in Sub-Saharan Africa (McDermott and Arimi, 2002). 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., 2002). 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., 2002). The accuracy of the serological tests used has considerable impact on the success of a programme. Therefore, tests that are prone to give false positive results have tendencies to condemn animals that would have been negative, while tests that give false negative results will prolong any control campaign by their inability to capture all truly positive cattle. Other factors of importance, include test cost, ease of performance, test precision, interference by antibody to vaccine or cross reacting antigens, and turn around time for results. It is noteworthy that there is no single serological test that is regarded as a perfect test. Consequently, in order to get optimal results, serological tests are often used in combination using either parallel or serial testing programmes. 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.

Justification 1

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. abortus S19 vaccine from antibodies produced against field strains of B. abortus (Nielsen et al., 1996). It is recommended as the test of choice for international trade (Anon., 2002), but its advantages are accompanied by numerous disadvantages. It is cumbersome (it is difficult to perform and time consuming) and expensive. 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. 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, making it prone to error. In contrast, 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 complement fixation test (CFT) is similarly regarded as the test of choice for international trade (Anon., 2002) due to its high specificity, it is cumbersome to perform and often requires experienced laboratory technologists. In addition, the turn around time is longer since the test is done over two days. The Rose Bengal test is highly sensitive but tends to produce many false positives leading to the unnecessary condemnation of animals. Although the serum agglutination test (SAT) is commonly used in many brucellosis control programme due to its ease of use, it has inherent problems of low sensitivity and its omission from the panel of suitable tests has been suggested (Anon., 2002). Together with the CFT and the Rose Bengal, SAT has shortcomings in failure to differentiate B. abortus S19 vaccinal antibodies from those produced by field strains of B. abortus (Nielsen et al., 2

1996), thereby compromising their specificities. In contrast, the FPA can differentiate B. abortus S19 vaccinal antibodies from antibodies produced against field strains of B. abortus (Nielsen et al., 1996).

The above mentioned tests and several other tests have been used in routine monitoring and screening of infected herds, but up till now, 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 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., 2002). The test has been used with success for the serological diagnosis of brucellosis in cattle in some countries (Nielsen et al., 1996; Dajer et al., 1999; Samartino et al., 1999; McGiven et al., 2003). The FPA is a homogeneous assay which only requires addition of labelled antigen to appropriately diluted test samples. There is no requirement for removal of excess reagents hence relatively easy to perform (Nielsen et al., 2000). Because of the reported high sensitivity and specificity values for the FPA for detection of bovine serum antibody to B. abortus (99.02% and 99.96%, respectively); (Nielsen et al., 1996), its speed and ease of performance, it is an ideal candidate for adaptation to use in the simple laboratory set up as well as under field conditions. In addition, other than serum, the FPA can utilize whole blood and milk to detect antibodies against Brucella species (Nielsen et al., 2001)

Objectives 1. To determine the level of agreement between FPA, Rose Bengal and Serum Agglutination Test, and competitive ELISA

2. To establish the specificity and sensitivity of FPA, RB, and SAT relative to the competitive ELISA

3. To evaluate the suitability of FPA as a standard test for bovine brucellosis

Literature Review
Definition Bovine Brucellosis, or Bangs disease, is a disease of cattle caused by biovars of a gramnegative bacterium called Brucella abortus (B. abortus). In countries where cattle are kept in close association with sheep or goats, infection can also be caused by B. melitensis (Anon., 2002). While B. abortus has been isolated from bovine foetuses, cases bovine brucellosis due to B. melitensis and B. suis infections in cattle have not been reported in Zimbabwe (Madsen, 1989, Mohan et al., 1996). According to the Animal Health Act, (Brucellosis Control Regulations, Zimbabwe), bovine brucellosis, is listed as a notifiable disease (Madsen, 1989).

Brucellosis is usually a disease of the sexually mature animals, and the predilection sites are the gravid uterus and the reproductive tract of male animals. Following infection with B. abortus or B. melitensis, pregnant adult females develop a placentitis usually resulting in abortion between the fifth and ninth month of pregnancy. In nave cattle herds the disease is clinically characterised by abortion storms where about 90% of the pregnant animals may abort (Radostits et al., 1994) and decreased milk production. Females usually abort only once, after which a degree of immunity is attained, and animals remain infected and can shed the organism in subsequent parturitions (Quinn et al., 1994). Bulls may develop epidydimitis and orchitis with a subsequent drop in fertility (Radostits et al., 1994). Hygroma formation, involving one or more leg joints, is a common manifestation of brucellosis in some tropical countries (MacDermott et. al., 1987; Anon., 2002). However,

such lesions may be found in animals that have been vaccinated with B. abortus S19 vaccine (Corbel et al., 1989).

If brucellosis is endemic in a herd, economic loss occurs mainly through abortions that lower the calf crop. In addition, the re is a drastic reduction in milk production which can be reduced by about 10 %. The use of stamping out policy in Brucella seropositive herds causes further loses in production (Alton, 1975).

Brucellosis is of major public health significance. B. abortus, B. melitensis and B. suis are highly pathogenic for man (Anon., 2002) and are readily transmissible to humans, causing acute febrile illness undulant fever which may progress to a more chronic form and can also produce serious complications affecting the musculoskeletal, cardiovascular, and central nervous systems (Radostits et al., 1994). Infection is often due to occupational exposure and is essentially acquired by the oral, respiratory, or conjunctival routes, but ingestion of dairy products constitutes the main risk to the general public. There is an occupational risk to veterinarians and farmers who handle infected animals and aborted foetuses or placentae. Brucellosis is one of the most easily acquired laboratory infections, and strict safety precautions should be observed when handling cultures and heavily infected samples, such as products of abortion. Specific recommendations have been made for the safety precautions to be observed with Brucella-infected materials (Anon., 2002). Although human brucellosis has been reported in many African countries (McDermott and Arimi, 2002) there are no reports of human brucellosis in Zimbabwe. It could be that a lot of cases remain undiagnosed since brucellosis is difficult to detect clinically (McDermott

and Arimi, 2002) and cases are often misclassified as malaria. Alternatively, this may be largely due to rigorous brucellosis control measures that include milk and meat hygiene (Madsen, 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, 2002). 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., 2002) Brucellosis is widespread in most countries in Africa. However, 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, 2002). Brucellosis is reported to be endemic in some commercial farms in Zimbabwe (Mohan et al., 1996), 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, 1989). There is limited data from communal herds (both dairy and beef), 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, 1989). Brucellosis has been found to be prevalent in communal cattle in other countries, with a tendency of higher prevalence in commingled cattle than those confined (McDermott and Arimi, 2002).

Host range Although brucellosis is of major economic importance in cattle farming, the diseases has also been reported in the one-humped camel (Camelus dromedearius) and in the twohumped camel (C. bactrianus), related to contact with large and small ruminants infected with B. abortus or B. melitensis (Anon., 2002). In addition, brucellosis has been observed in the domestic buffalo (Bubalus bubalus), American and European bison (Bison bison, B. bonasus), yak (Bos grunniens), elk / wapiti (Cervus elaphus) and also occurs in the African buffalo (Syncerus caffer) and various African antelope species. The manifestations of brucellosis in these animals are similar to those in cattle (Anon., 2002).

In Zimbabwe, serological evidence of brucellosis was demonstrated in both herbivores and scavenging wildlife species (Condy and Vickers, 1972; Madsen and Andersen, 1995), but their role in spreading infection to domestic livestock or vice versa is not known. However, the interaction between domestic livestock and wildlife facilitates bimodal transmission of diseases with both domestic animals and wildlife being important reservoirs (Godfroid et al., 1994).

Transmission The transmission of the organisms causing bovine brucellosis is by direct or indirect contact with infective excretors. 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., 2004). Less commonly, infection may occur in utero, via conjunctiva or by inhalation (Quinn et al., 1994). The disease dynamics are largely

influenced by herd size, management factors, the prevalence rate and intensity of contact between herds and herd level immunity.

There is a general lack of information on occurrence of brucellosis and the risk factors involved in wildlife livestock interface areas.

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, milk, blood and other products used for food. Thus necessitated is stringency in control of the disease and new control strategies are always in demand.

Control of bovine brucellosis Bovine brucellosis has been controlled and successfully eradicated in some countries through test and slaughter policies. In addition, vaccination of female calves between three and ten months of age using B. abortus S19 live attenuated vaccine is practiced. However, vaccination alone does not result in complete eradication of bovine brucellosis. Moreover, vaccination with B. abortus S19 results in complications of interpreting serological results due to the occurrence of cross-reacting antibodies (Nielsen et al., 1996).

In Zimbabwe, the control of bovine brucellosis is generally based on calf-hood vaccination using B. abortus S19 and the implementation of the test and slaughter programme. A brucellosis Accreditation scheme, aimed at control and possible eradication, has been legislated and implemented for the commercial dairy farming sector (Madsen, 1989;

Mohan et al., 1996). To be accredited as brucellosis free, a farm bleeds and tests all dairy cattle over 18 months of age for three consecutive times at three monthly intervals. If no positive animals are recorded, an accreditation certificate is issued and is tenable for one year after which animals will be re-bled and tested. If positive animals are detected, they are culled through slaughter and the whole process repeated again until three negative consecutive tests are obtained. All accredited farms are monitored monthly by milk ring test conducted on bulk milk. If a positive test is recorded, the accreditation certificate is forfeited. Individual animals have to be tested serologically to identify reactors. To be reaccredited, the whole process of three monthly serological testing and culling of positive animals is conducted. In commercial dairy farms, strict implementation of the Accreditation scheme resulted in eradication of bovine brucellosis (Madsen, 1989).

Serological diagnosis of bovine brucellosis A number of serological tests have been developed for the diagnosis of brucellosis, and each has its own special applications and limitations (Alton et al., 1975; Mikolon et al., 1998). Serological diagnosis of brucellosis was first accomplished using an agglutination test (Wright and Smith, 1897). This is similar to the standard tube agglutination test that mainly detects the immunoglobulin M (IgM) antibody. Therefore the tube agglutination is prone to false positives, therefore unreliable and hence its use should be discontinued as recommended by OIE (Anon., 2002). Several many modifications of the original agglutination test have been made to increase the specificity (Angus and Barton, 1984), and among them is the use of an acidified antigen in the Rose Bengal/card test (Nicoletti, 1977) and the buffered plate agglutination test in which antigens are used at low pH. Both tests

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are considered suitable for screening individual animals, although false positives can occur due to prozoning (Nielsen, 2002). Because they are highly sensitive tests, they can give positive reaction due to the S19 vaccine or due to false-positive serological reactions. Suitable confirmatory tests are therefore called on to confirm the positives. (Anon., 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., 2002). It is commonly used to monitor brucellosis using bulk tank milk, and is recommended for screening bovine brucellosis (Madsen, 1989) but its sensitivity can easily be affected by pooling of samples. Also, false reactors are frequent in cattle vaccinated four months prior to testing, in mastitic cows milk or if colostrum samples are subjected to the test (Nicoletti, 1977).

For the control of brucellosis at the national or local level, the buffered Brucella antigen tests (BBATs), Rose Bengal (RB) test and the buffered plate agglutination test (BPAT), as well as the ELISA and the FPA, have been identified as suitable screening tests but confirmation using a more specific test is necessary (Anon., 2002).

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., 2002). In spite of its shortcomings, the SAT has been used widely in brucellosis control in Zimbabwe. However, only samples with antibody titres of at least 1:160 are treated as positives. Those with titres of 1:20, 1:40 and 1:80 are classified as doubtful reactors (and require further testing using the compliment fixation test (CFT) (Madsen, 1989).

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The CFT is more recent to the SAT, initially had two forms which later were standardised to one (Hill, 1963). The CFT is diagnostically more specific than the SAT, and also has a standardised system of unitage. Among other problems, 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, 2002). However, 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), in spite of its inherent problems and also that its specificity is lower than that of the competitive ELISA (c-ELISA). 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, and as they are technically simpler to perform and more robust, their use may be preferred (Wright et al., 1993). The performances of several of these tests have been compared (Nielsen et al., 1996; Dajer et al., 1999).

To improve test sensitivity, the indirect ELISA (i-ELISA) test was developed (Nielsen et al., 1989), but this also failed to differentiate vaccinal from field infection antibodies. However, the c-ELISA, in its ability to differentiate vaccinal from field infection antibody, has been shown to be highly specific and sensitive and thus a more reliable test than the iELISA (Nielsen et al., 1995).

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Recently, numerous other antibody detection tests for brucellosis have been evaluated, but in practice some impractical in use for routine diagnosis of brucellosis. These include; USDA (United States Department of Agriculture) card test, rapid automated presumptive test, Mexican Rose Bengal plate test, French Rose-Bengal plate test, USDA standard plate test, USDA buffered acid agglutination test, USDA and Mexican rivanol tests, USDA and Mexican milk ring tests and milk ELISA. The milk ELISA test was found to offer more sensitivity and specificity than the MRT and also provided ease of interpretation (Mikolon et al., 1998).

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. It is a homogeneous assay in which analytes are not separated and it is therefore very rapid.

The mechanism of the assay is based on random rotation of molecules in solution. Molecular size is the main factor influencing the rate of rotation, which is inversely related. Thus a small molecule rotates faster than a large molecule. If a molecule is labelled with a fluorochrome, the time of rotation through an angle of 68.5 can be determined by measuring polarised light intensity in vertical and horizontal planes. 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., 2000)

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For most FPAs, an antigen of small molecular weight, less than 50 kDa, is labelled with a fluorochrome and added to serum or other fluid to be tested for the presence of antibody. If antibody is present, attachment to the labelled antigen will cause its rotational rate to decrease and this decrease can be measured (Anon., 2002).

Performance of FPA under Field Conditions Some work has been done to compare the FPA with existing tests in different regions (Nielsen et al., 1996; 2001; Dajer et al 1999; Samartino et al., 1999). Dajer et al. (1999) did a field study in Mexico, which compared the FPA to tests currently in use in that country. Using the CFT as a gold standard, FPA gave higher relative sensitivity and specificity than the other tests (RB, Rivanol Agglutination (RIV)). The FPA also agreed almost perfectly with the CFT (Kappa=0.96), while RB and RIV gave Kappa values of 0.70 and 0.61 respectively with respect to CFT. It was recommended that the FPA was a suitable replacement for CFT In the serological diagnosis of B. abortus.

Elsewhere in Canada, the performance of the FPA was apparently varied in different experiments. The sensitivity of the test (using sera from culture positive animals) ranged from 66% to 100% (Nielsen et at., 1998), whilst serological positivity of cattle from infected premises ranged from 65.5% to 99.0%. In other Canadian studies, the sensitivity values were 99.0% and 100% and the specificity in both cases was 100% (Nielsen et al., 1996). Nevertheless, the FPA has been found to be a suitable confirmatory test for Bovine brucellosis.

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The FPA has also been used to test whole blood samples prepared by mixing blood cells from cattle without exposure to Brucella abortus (B. abortus) with sera from animals with confirmed (bacteriologically) infection (Nielsen et al., 2001). Relative sensitivity and specificity values for the FPA performed in the field, based on buffered antigen plate agglutination test and competitive enzyme immunoassay results were 95.3 and 97.3%, respectively. Thus the study aptly demonstrated the usefulness of the FPA for testing whole blood samples in the field. In addition, the study also validated the FPAs ability to distinguish between antibodies induced by B. abortus S19 vaccine and field infection by B. abortus, thus producing results similar to the c-ELISA.

The FPA was evaluated for use in different animal species. It has been used successfully in cattle (Nielsen et al., 1996; Dajer et al., 1999; Samartino et al., 1999), sheep (Minas et al., 2005), humans (Lucero et al., 2003) and bison (Gall et al., 2000).

Because of its high relative sensitivity and specificity (Nielsen et-al., 1996), its ability to detect antibodies to Brucella species using a variety of samples (sera, milk, whole blood) (Nielsen and Gall, 2001), its speed and ease of performance it is an ideal candidate for adaptation to use both in laboratory and field. To expedite field-testing, it would be useful to test whole blood rather than serum. 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, Serum agglutination test and c-ELISA) that are commonly used in the diagnosis of the disease.

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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. All sera were previously collected from smallholder cattle from Gokwe (Gokwe district), Nharira- Lancashire (Chikomba district), Wedza (Wedza district) and Rusitu Valley (Chipinge district). The individual age, sex and parity of the cattle sampled were not recorded. However all animals sampled were over 18 months of age. The vaccination status of the sampled animals was unknown. All samples were kept at -20C until they were tested. All sera were tested in parallel using c-ELISA, RB, SAT, and FPA.

Serological tests Rose Bengal Test The Rose Bengal test was performed essentially as described by Alton et al., (1988). 25 l of serum were mixed with equal volume of stained buffered Rose Bengal antigen (Weybridge) onto standard test plates. Test plates were agitated onto shakers for five minutes and results recorded as positive or negative. Control sera were obtained from the Central Veterinary Laboratory (Weybridge, UK).

Fluorescence polarisation assay (FPA) The FPA was performed by the method described by Nielsen et al. (1996). Briefly, 1ml of FPA buffer was pipetted into 10x75mm culture tubes (Durex). 10l serum sample were

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added and a background measurement was obtained using a fluorescein- labelled B. abortus O-polysaccharide tracer and a fluorescence-polarization analyzer (Sentry FP 100, Diachemix LLC, Grayslake, Illinois, USA). A predetermined amount of tracer was added and after mixing and incubation at room temperature for at least 2 min, the fluorescence polarisation of the tracer was determined (with the reading from the blank subtracted). Data from this assay was expressed as millipolarisation (mP) units.

Serum Agglutination test (SAT) The SAT was performed according to the procedure of Alton et al., (1975). Double dilutions of sera from 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280 and 1:2560 were used. The antigens and control sera were sourced from the Onderstepoort Research Institute (South Africa).

Competitive ELISA The c-ELISA was performed as described by Nielsen et al. (1989). SvanovirTM Brucella-Ab c-ELISA kit (Svanova Biotech AB Uppsala, Sweden) was used to test the sera. Briefly, sera to be tested were equilibrated to room temperature (23-25 0C). Sera and controls were run in duplicates. The optical densities (OD) were measured at 450 nm in a micro-plate photometer (Humareader, Model 18500/1, Awareness Technology, Inc. Germany). The threshold for determining sero positivity was according to the manufacturers recommendations. Antibody titres were recorded as percentage inhibition equivalents of absorbance readings.

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Only results from samples subjected to all four tests were selected for analyses.

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, Texas, USA).

Using the c-ELISA as a gold standard test, results from the FPA, RBT and SAT was analysed using the McNemars 2 test for paired data in comparison relative to RB, SAT and c-ELISA using STATA 9 software. The Kappa measure of agreement was calculated using STATA 9 for the FPA with the RB, SAT, c-ELISA, results defined as slight (kappa < 0.2), fair (kappa 0.2 to 0.4), moderate (kappa 0.4 to 0.6), substantial (0.6 to 0.8) and almost perfect (kappa > 0.8) (Dohoo et al, 2002). The sensitivity and specificity of the FPA, RB and SAT relative to the c-ELISA (plus 95%confidence limits) were calculated using Win Episcope 2.0 software

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Results
The significance and level of agreement of the FPA to the RB, SAT, and c-ELISA are shown in Table 4.1, whilst those of the other tests performance comparisons are shown in Table 4.2.

The sensitivities and specificities of the FPA, RB and SAT relative to the c-ELISA is shown in Table 4.3. The experiment suggested a cut-off value of 90 mP resulting in the highest sensitivity and specificity combination values of 60.1% and 96.9%, respectively.

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Discussion
The results from the study show the FPA agreeing substantially with all the other tests with the exception of the SAT, which showed a fair agreement.

The evaluation of the tests showed high specificity for all tests, and moderate sensitivity for FPA and RB. As expected, and in general agreement with previous studies, the SAT was the least sensitive test (Madsen, 1989).

Generally, the results from this study agree with those of previous studies (Nielsen et al., 1996; Gall, 2000; Dajer et al., 1999) but however are on the low end of the range. This discrepancy was probably due to the use of less well characterized sera in the study (Nielsen et al., 2000) but could also have resulted from a closed end testing protocol which utilized a single, not validated test as a gold standard. Thus, for further studies, 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, use a different but reliable test protocol such as the CFT as gold standard.

The McNemars test and the Kappa statistic gave conflicting outcomes, for example, FPA with SAT gave a McNemars statistic of 0.0000 but a Kappa of 0.2659, which would be suggestive of serious disagreement with regards to the McNemar, but a slight agreement with respect to the Kappa. The McNemars test seemed to not agree with the Kappa test and in some instances, test comparison giving a high Kappa value, for example 0.6891 (RB with SAT (doubtful reactors) gave a low McNemars statistic (0.0801). Thus, this

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disagreement led to the disregard of the McNemars test as it was initially included as an assessment of whether or not there was test bias (Dohoo et al., 2002), and due to its unreliability when dealing with dichotomised data (Kraemer and Bloch, 1994).

Possible improvements can be made if a review is intended, one such being to use samples of known serological status. Use of a parallel testing protocol involving at least two tests as gold standard is also advised, suggested tests being the CFT and c-ELISA, or otherwise incorporating the RIV (this would require a field trial as the RIV is yet to be validated and used in Zimbabwe).

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Conclusion
The ability of FPA to agree substantially with RB and c-ELISA, and slightly with SAT and its ability to duplicate and better the sensitivity and specificity of tests currently in use, together with its ease to perform under field conditions render it suitable for use in the diagnosis of bovine brucellosis under Zimbabwean conditions.

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Appendix 1: Tables of Results

Table 4.1: Test agreements for the fluorescent polarisation assay (FPA) with the rose bengal (RB), serum agglutination test (SAT), and competitive enzyme linked immunosorbent assay (c-ELISA) FPA Comparison with Rose Bengal SAT SAT(Doubtful reactors as Positives) C-ELISA McNemars Statistic 0.5224 0.0000 0.7877 0.3618 Kappa Value (95% CI) 0.5989 (0.5690-0.6288) 0.2659 (0.2526-0.2792) 0.4439 (0.4217-0.4661) 0.5818 (0.5699-0.6148)

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Table 4.2: Test agreements for the rose bengal (RB), serum agglutination test (SAT), and competitive enzyme linked immunosorbent assay (c-ELISA) Comparison of McNemarss Statistic RB with c-ELISA 0.1435 RB with SAT (Doubtful 0.0801 reactors as Positives) RB with SAT 0.0000 SAT with c-ELISA 0.0005 SAT (Doubtful reactors as 0.0336 positives) with c-ELISA Kappa Value (95% CI) 0.7659 (0.7276-0.8041) 0.6891 (0.6445-0.7236) 0.5141 (0.4883-0.5398) 0.4750 (0.4513-0.4986) 0.5066 (0.4761-0.5271)

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Table 4.3: The relative sensitivity and specificity results of the FPA, RB and SAT with respect to c-ELISA Test FPA RB SAT SAT (Doubtful reactors as positives) Sensitivity (%) (95% CI) 66.11 (51.28-82.07) 86.11 (78.81-97.40) 37.01 (21.14-53.15) 65.65 (49.99-81.44) Specificity (%) (95% CI) 96.54 (94.15-97.80) 97.32 (95.83-98.82) 99.28(98.23-99.98) 94.16(92.49-96.77)

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