Impact of mycobacterial culture among HIV-infected adults with presumed TB in Uganda: a prospective cohort study.

BACKGROUND: Implementation of new tuberculosis (TB) diagnostic strategies in resource-constrained settings is challenging. We measured the impact of solid and liquid mycobacterial cultures on treatment practices for patients undergoing TB evaluation in Kampala, Uganda. METHODS: We enrolled consecutive smear-negative, human immunodeficiency virus positive adults with cough of ⩾2 weeks from September 2009 to April 2010. Laboratory technicians performed mycobacterial cultures on solid and liquid media. We compared empiric treatment decisions with solid and liquid culture in terms of diagnostic yield and time to results, and assessed impact on patient management. RESULTS: Of 200 patients enrolled, 26 (13%) had culture-confirmed TB: 22 (85%) on solid culture alone, 2 (8%) on liquid culture alone, and 2 (8%) on both solid and liquid culture. Thirty-four patients received empiric anti-tuberculosis treatment, but only 10 (29%) were culture-positive. Median time to a positive result on solid culture was 92 days (interquartile range [IQR] 69-148) compared to 106 days (IQR 66-157) for liquid culture. No patients initiated treatment following a positive result on liquid culture. CONCLUSION: The introduction of mycobacterial culture did not influence care for patients undergoing evaluation for TB in Kampala, Uganda. Attention to contextual factors surrounding implementation is needed to ensure the effective introduction of new testing strategies in low-income countries.

Contexte : La mise en œuvre de nouvelles stratégies de diagnostic de la tuberculose (TB) dans les contextes de ressources limitées constitue un défi. Nous avons mesuré l'impact des cultures mycobactériennes en milieu solide et liquide sur les pratiques de traitement des patients ayant une évaluation de la TB à Kampala, Ouganda.Méthodes : Nous avons enrôlé des patients adultes consécutifs à frottis négatif, positifs pour le virus de l'immunodéficience humaine avec toux de ⩾2 semaines, de septembre 2009 à avril 2010. Les techniciens de laboratoire ont réalisé des cultures mycobactériennes en milieu solide et liquide. Nous avons comparé les décisions de traitement empirique aux cultures en milieu solide et liquide en termes de rendement diagnostique et de délai d'obtention des résultats et nous avons évalué l'impact sur la gestion des patients.Résultats : Des 200 patients enrôlés, 26 (13%) avaient une TB confirmée par culture, 22 (85%) par culture en milieu solide seule, 2 (8%) par culture en milieu liquide seul et 2 (8%) par culture à la fois en milieu solide et liquide. Trente-quatre patients ont reçu un traitement de TB empirique, mais seulement 10 (29%) ont eu une TB à culture positive. Le délai médian d'obtention d'un résultat de culture positive en milieu solide a été de 92 jours (IQR 69­148). Le délai médian d'obtention d'un résultat de culture positive en milieu liquide a été de 106 jours (IQR 66­157). Aucun patient n'a commencé un traitement à la suite d'un résultat de culture positive en milieu liquide.Conclusion : L'introduction de la culture mycobactérienne n'a pas influencé les soins aux patients bénéficiant d'une évaluation de TB à Kampala, Ouganda. Il est nécessaire d'être attentif aux facteurs contextuels entourant la mise en œuvre afin d'assurer une introduction effective de nouvelles stratégies de tests dans les pays à faible revenu.

Marco de referencia: La ejecución de nuevas estrategias de diagnóstico de la tuberculosis (TB) en los entornos con limitación de recursos es problemática. En el presente estudio se midió la repercusión del uso del cultivo de micobacterias en medio sólido o liquido sobre las prácticas de tratamiento de los pacientes en curso de investigación diagnóstica de la TB en Kampala, Uganda.Métodos: Se incluyeron de manera consecutiva en el estudio los pacientes adultos, seronegativos frente al virus de la inmunodeficiencia humana, que consultaban por tos de ⩾2 semanas de duración y presentaban baciloscopia negativa, de septiembre del 2009 a abril del 2010. Los auxiliares de laboratorio practicaron el cultivo de micobacterias en medio sólido y medio líquido. Se compararon las decisiones empíricas de tratamiento y los tipos de cultivo, con respecto al rendimiento diagnóstico y al lapso hasta obtener los resultados y se evaluó su repercusión en el manejo de los pacientes.Resultados: De los 200 pacientes que participaron en el estudio, 26 obtuvieron confirmación del diagnóstico de TB mediante el cultivo (13%), 22 de ellos con el cultivo en medio sólido únicamente (85%), dos con el cultivo en medio líquido exclusivamente y dos con ambos tipos de cultivo (8%). Treinta y cuatro pacientes recibieron tratamiento antituberculoso empírico, pero solo 10 de ellos obtuvieron un cultivo positivo (29%). La mediana del lapso hasta obtener el resultado del cultivo en medio sólido fue 92 días (IQR 69­148). La mediana de este lapso con los cultivos en medio líquido fue 106 días (IQR 66­157). Ningún paciente inició el tratamiento antituberculoso después de haber obtenido el resultado positivo del cultivo en medio líquido.Conclusión: La introducción del cultivo para micobacterias no tiene ninguna influencia en la atención que reciben los pacientes en quienes se investiga la TB en Kampala, Uganda. Es importante prestar atención a los factores contextuales que rodean la ejecución, a fin de lograr una introducción eficaz de las nuevas estrategias diagnósticas en los países con recursos limitados.

Prevalence of occupational exposure to HIV among health workers in Northern Uganda.

BACKGROUND: The prevalence of HIV in Gulu district is 10.3%. This poses a high risk of occupational exposure and transmission to health workers in hospitals attending to these patients. The risk of HIV transmission from a patient to a health worker has been shown to be between 0.3% and 0.09% following percutaneous and mucocutaneous exposure respectively. OBJECTIVES: This research aimed at determining the prevalence of occupational exposure to HIV. METHOD: A cross sectional study of health workers in Gulu Regional Referral Hospital and St. Mary's Hospital Lacor, in northern Uganda was conducted to establish the frequency of occupational exposures to human immunodeficiency virus (HIV)-infected body fluids. RESULTS: 108 (46%) respondents were found to have been exposed to potentially infectious body fluids. Needle stick injuries was the commonest route of exposure, with a prevalence of 27.7%, followed by mucosal exposure 19.1%, contact with broken skin (5.5%) and lastly by a cut with sharp objects (5.1%). There is therefore need for more sensitization of health workers on infection control and post exposure prophylaxis for health workers.

Community engagement in health research: two decades of experience from a research project on HIV in rural Uganda.

OBJECTIVES: To describe how a research project on HIV epidemiology in rural Uganda has engaged the community over the past two decades, describing activities, opportunities and challenges that have arisen. METHOD: The review draws on the experience of the authors as investigators involved in the project at various times since its inception in 1989, and on project documents and peer-reviewed publications. RESULTS: The project attracts community interest, participation and support mostly through community groups. The three main areas of activity are: health care and promotion, HIV/AIDS prevention and care, and community development aimed at poverty reduction. Key opportunities arise from the long-term joint commitment of the project and the community over nearly 20 years, and the potential to accommodate research beyond HIV. Challenges arise from participation fatigue, countered by innovations for the community and investment in capacity development for staff, and from the need to balance community development expectations and the project focus on HIV research. CONCLUSIONS: Judged by criteria of longevity, acceptance, and scientific output, community engagement in this HIV research project in rural Uganda has been successful. The experience from this project contributes to the collective documentation and analysis of case studies from various research projects in developing countries which identify good practices from multiple stakeholder perspectives.

The persistence of Theileria parva infection in cattle immunized using two stocks which differ in their ability to induce a carrier state: analysis using a novel blood spot PCR assay.

An improved Theileria parva DNA detection assay based on the polymerase chain reaction (PCR) using primers derived from the 104 kDa antigen (p104) gene was developed to detect parasite DNA in blood spots on filter paper. The specificity of the assay was validated using DNA from a wide range of cattle-derived and buffalo-derived stocks of T. parva. DNA of T. annulata, T. buffeli, T. lestoquardi, T. mutans and T. taurotragi was not amplified using the p104 primers. The detection threshold of the assay was approximately 1-2 parasites/microl of infected blood. PCR amplification using the p104 primers was applied to sequential samples from groups of cattle experimentally infected with either the T. parva Marikebuni stock that induces a long-term carrier state or the Muguga stock, which does not induce a carrier state. The study extended for up to 487 days post-infection and PCR data from defined time points were compared with parasitological microscopy and serological data, together with xenodiagnosis by experimental application of ticks. Microscopy first detected piroplasms between days 13 and 16 after infection whereas all cattle became PCR +ve between days 9 and 13. Animals infected with the Muguga stock of T. parva had parasite DNA in the peripheral blood, which could be detected by PCR, for between 33 and 129 days post-infection in different animals. By contrast parasite DNA in the blood of cattle infected with the Marikebuni stock could be detected consistently from day 9 up to 487 days, when the study terminated. The data suggest that the nature and persistence of the carrier state may differ markedly between different T. parva parasite stocks.

Cloning, characterization, and expression of a 200-kilodalton diagnostic antigen of Babesia bigemina.

Current serological tests for Babesia bigemina use semipurified merozoite antigens derived from infected erythrocytes. One of the major drawbacks of these tests is that antigen quality can vary from batch to batch. Since the quality of the antigen contributes to the sensitivity and specificity of serological tests, the use of standardized recombinant antigens should ensure consistency in assay quality. Previously, a 200-kDa merozoite antigen (p200) was identified as a candidate diagnostic antigen for use in a serological assay for the detection of B. bigemina antibodies in infected cattle. In this study, we have cloned, characterized, and expressed p200. A 3.5-kbp cDNA clone encoding p200 was isolated and shown to be almost full length, lacking approximately 300 bp at the 5' end. The predicted amino acid sequence shows that p200 consists of a long, highly charged central repeat region of an uninterrupted alpha helix, indicative of a fibrous protein. Immunoelectron microscopy localized p200 to the merozoite cytoplasm, suggesting that the antigen may be a structural protein involved in forming filament structures within the cytoskeleton. The 3.5-kbp cDNA was expressed in bacteria as a fusion protein with glutathione S-transferase (GST), but the yield was poor. To improve the yield, cDNA fragments encoding antigenic domains of p200 were expressed as fusions with GST. One of these fusion proteins, C1A-GST, is composed of a 7-kDa fragment of the p200 repeat region and contains epitopes that react strongly with sera from cattle experimentally infected with B. bigemina. Recombinant C1A-GST should permit the development of an improved enzyme-linked immunosorbent assay for the detection of antibodies against B. bigemina.

Differences in the epidemiology of theileriosis on smallholder dairy farms in contrasting agro-ecological and grazing strata of highland Kenya.

A prospective cohort study was conducted in five purposively-sampled agro-ecological zone (AEZ)-grazing system strata in Murang'a District, Kenya, between March 1995 and June 1996. The study strata were selected based on a preliminary characterization study to represent the widest range of risks to East Coast fever (ECF) in the District and included zero-grazing and open-grazing farms. In total, 225 calves from 188 smallholder farms were examined from birth to 6 months of age and visited within the first 2 weeks of life and thereafter at bi-weekly intervals for up to 14 visits. The purpose of the study was to characterize the differences in epidemiology (risks of infection, morbidity and mortality) and potential control of ECF between the selected strata. Evidence of Theileria parva infection was assessed by increased antibody levels as measured in an indirect ELISA assay by the percent positivity (PP) of serum samples relative to a strong positive reference serum. Sero-conversion risks of T. parva were highest in the open-grazing strata. Antibody prevalence in adult cattle and ECF morbidity and mortality risks were also highest in open-grazing strata. While different, all five AEZ-grazing strata were considered to be endemically unstable for ECF. East Coast fever challenge was low in all zero-grazing strata and this challenge is likely to remain low due to continuing intensification of smallholder farming in the central highlands. In the open-grazing strata, there was higher challenge and a greater impact of ECF.

The epidemiology of Theileria parva infections on smallholder dairy farms in Kenya.

The purpose of the study was to characterize the differences in epidemiology (risks of infection, morbidity, mortality) and potential control of East Coast fever (ECF) between the selected strata. Evidence of Theileria parva infection was assessed by increased antibody levels as measured in an indirect ELISA test by the percent positivity (PP) of serum samples relative to a strong positive reference serum. A prospective cohort study was conducted in five purposively sampled agroecological zone (AEZ)-grazing system strata in Murang'a District, Kenya, between March 1995 and June 1996. The study strata were selected to represent the widest range of ECF risks in the district and included, zero-grazing and open-grazing farms in the Upper Midlands (UM) one and four AEZs and zero-grazing farms in the UM2 AEZ. In total, 225 calves from 188 smallholder farms were examined from birth to age six months. Calves were recruited into the study at birth and visited within the first two weeks of life and thereafter at biweekly intervals for up to 14 visits. Important differences were observed between the different AEZ-grazing strata. Seroconversion risks of T. parva were highest in the UM4-open grazing stratum. Antibody prevalence in adult cattle and ECF morbidity and mortality risks were also highest in this stratum. In the open-grazing strata, particularly in the lower elevation AEZ, UM4, there was stronger challenge and a greater impact of ECF. There is likely to be an expansion of smallholder dairy farming into this area so that it is likely to be the most important target production system for ECF control in the central highlands of Kenya.

Development of sero-diagnostic and molecular tools for the control of important tick-borne pathogens of cattle in Africa.

Tick-borne diseases (TBDs) are a major economic constraint to livestock production in sub-Saharan Africa. ILRI is focussing on developing a range of products, such as vaccines, diagnostics and decision support services to underpin improved control programmes against these diseases. We have developed three highly sensitive and specific enzyme linked immuno-assays (ELISAs), which allow precise diagnosis of Theileria parva, Babesia bigemina and Anaplasma marginale. These tests have been standardised and validated using defined experimental and field infection sera. Parasite specific recombinant antigens and monoclonal antibodies against bovine immunoglobulins as secondary antibodies have played an important role in in enhancing the sensitivity and specificity of the assays. They have been further evaluated in on-farm longitudinal sero-epidemiological studies to define infection dynamics and disease risks in various farming systems in Kenya and Uganda. In addition, DNA-based tests for differentiation of Theileria species and characterisation of Theileria parva stocks have been developed. These tests have been derived through physical mapping and sequencing of key elements of the T. parva genome, which include repetitive and telomeric regions, minisatellite sequences, antigen genes and a number of random DNA sequences. These tools are currently being deployed in conjunction with field immunisation programmes to determine the biological impact of introducing live vaccines of T. parva on population dynamics.

An enzyme-linked immunosorbent assay for detection of Theileria parva antibodies in cattle using a recombinant polymorphic immunodominant molecule.

Field and experimental bovine infection sera were used in immunoblots of sporozoite and schizont lysates of Theileria parva to identify candidate diagnostic antigens. Four parasite antigens of Mr 67,000 (p67), 85,000 (the polymorphic immunodominant molecule, PIM), 104,000 (p104), and 150,000 (p150) were selected for a more detailed analysis. The p67 and p104 antigens were present only in the sporozoite lysates, whereas PIM and p150 were found in both sporozoite and schizont lysates. The four antigens were expressed as recombinant fusion proteins and were compared with each other in an enzyme-linked immunosorbent assay (ELISA) and in the whole-schizont-based indirect fluorescent antibody test (IFAT) in terms of their ability to detect antibodies in sera of experimentally infected cattle. The PIM-based ELISA provided a higher degree of sensitivity and specificity than did the ELISA using the other three recombinant antigens or the IFAT. Further evaluation of the PIM-ELISA using experimental sera derived from cattle infected with different hemoparasites and field sera from endemic and nonendemic T. parva areas showed that the assay had a sensitivity of > 99% and a specificity of between 94% and 98%.

Evaluation of an ELISA for detection of antibodies to Babesia bovis in cattle in Australia and Zimbabwe.

An enzyme-linked immunosorbent assay (ELISA) for antibodies to Babesia bovis was evaluated in comparison with the indirect fluorescent antibody test (IFAT) in Australia and Zimbabwe. Positive and negative threshold values for the ELISA were set using sera from cattle of known infection status. Sensitivity and specificity estimates for the ELISA based on 158 positive sera from cattle experimentally infected with Australian isolates of B. bovis and 318 negative sera collected from B. bovis-free herds in Australia were 100% and 99.4%, respectively. The specificity of the assay in Africa, based on 328 sera from B. bovis-free herds in Kenya and South Africa, was 99.7%. The ELISA was compared with the IFAT using sequential sera from 16 calves experiencing primary B. bovis infections, and a total of 777 field sera collected from B. bovis-endemic herds in Australia and Zimbabwe. In primary infections, the ELISA and IFAT detected antibodies at or about the same time. With sera from endemic herds, the performance of the ELISA was at least comparable with that of the IFAT. Two hundred and fourteen of 221 sera that were negative by IFAT, were negative by ELISA, and 428 of 439 sera that were clearly positive by IFAT were positive by ELISA. Of 117 sera that gave equivocal (suspect or weak positive) results in the IFAT, 20 were positive by ELISA, 7 were suspect and 90 were negative. We conclude that the ELISA will be useful for epidemiological studies on B. bovis in Australia and Zimbabwe, and probably elsewhere.

The prevalence of serum antibodies to tick-borne infections in cattle in smallholder dairy farms in Murang'a District, Kenya; a cross-sectional study.

The most important tick-borne disease of cattle in eastern, central and southern Africa is East Coast fever (ECF) caused by Theileria parva and transmitted by the tick Rhipicephalus appendiculatus. Other less-important tick-borne diseases in cattle are benign theileriosis caused by Theileria mutans, babesiosis caused by Babesia bigemina, anaplasmosis caused by Anaplasma marginale and cowdriosis caused by Cowdria ruminatum. In Murang's District, Central Province of Kenya, five agroecological zones (AEZs) are defined according to climate, altitude and agricultural activities. A cross-sectional serological study was conducted on 750 smallholder dairy farms in Murang's District, selected in a stratified random sampling method. The farms had a total of 362 calves. One hundred and fifty farms were studied from three administrative sublocations in each of the five AEZs. Prevalence of serum antibodies to three tick-borne parasites, that is T. parva, T. mutans and B. bigemina, were determined using the enzyme-linked immunosorbent assay (ELISA) technique. Antibody prevalence values differed across the AEZs. The ranges of means for the prevalences were: T. parva (18-72%), T. mutans (1.5-28%) and B. bigemina (12-49%). The above results serve as indicators of the possible existence of endemic stability in some AEZs for some parasites.

Generation of monoclonal antibodies to the anti-trypanosomal drug isometamidium.

Mice were immunized with either an isometamidium-human serum albumin (HSA) conjugate or an isometamidium-porcine thyroglobulin conjugate (PTG). Thereafter, monoclonal antibodies (MAbs) IL-A 1001, IL-A 1002, IL-A 1003, 5F7.B7, and 5F7.C9 were generated and selected on the basis that they recognized conjugated and unconjugated isometamidium, but lacked cross-reactivity with the carrier molecules. All five MAbs were of the IgG1 isotype. Each of the five MAbs was assessed in a competitive ELISA for isometamidium; in each case, the minimum level of detection was approximately 10 ng/ml. Each MAb exhibited approximately 0.1% cross-reactivity with the anti-trypanosomal compound diminazene. However, based on their cross-reactivity with the anti-trypanosomal compound homidium, the MAbs could be divided into two groups; IL-A 1001, IL-A 1002, and IL-A 1003, produced using an isometamidium-HSA conjugate as an immunogen, exhibited low levels of cross-reactivity (approximately 0.1%). In contrast, 5F7.B7 and 5F7.C9, produced using an isometamidium-PTG conjugate as an immunogen, exhibited high levels of cross-reactivity.

Identification of Babesia bigemina infected erythrocyte surface antigens containing epitopes conserved among strains.

The presence of previously uncharacterized antigens (new antigens) on the surface of intact erythrocytes infected with three strains of Babesia bigemina from Kenya and one each from Puerto Rico, Mexico, St. Croix, and Texcoco-Mexico was demonstrated by indirect immunofluorescent antibody (IFA) reactions. These antigens were not strain specific because antibodies in bovine immune serum to either the Mexico or Kenya isolates reacted with all seven strains tested. Homologous and heterologous immune serum antibodies bound a maximum of 83% and 55%, respectively, of intact erythrocytes infected with the Kenya-Ngong strain but not uninfected erythrocytes. Both sera caused agglutination of only infected erythrocytes. Antibodies eluted from the surface of glutaraldehyde (0.25%) fixed infected erythrocytes had IFA reaction patterns among strains similar to those of immune sera before elution. Eluted antibodies were used to determine if these antigens were protein and encoded by B. bigemina. Eluted antibodies bound seven parasite-encoded proteins of 240, 220, 66, 62, 58, 52 and 38 kDa in an erythrocyte surface-specific immunoprecipitation reaction of 35S-methionine labelled proteins. It was concluded that the surface of B. bigemina infected erythrocytes had parasite-encoded proteins and that these proteins had surface exposed epitopes that were conserved among the seven strains examined which were from two continents.

Variation in sensitivity of Trypanosoma congolense to diminazene during the early phase of tsetse-transmitted infection in goats.

Twenty-five goats were randomly allocated to five groups of five animals each and infected with Trypanosoma congolense IL 3274 via the bites of infected Glossina morsitans centralis. At intervals of 1, 4, 8, 12 or 19 days following infection, each group of five animals was treated intramuscularly with diminazene aceturate at a dose of 7.0 mg kg-1 body weight (b.w.). While treatment on Day 1 eliminated infections in all five goats, treatment on Day 19 did not cure any of the animals; in groups treated 4, 8 or 12 days following infection, two of five goats in each group were cured. Since the alteration in apparent resistance of T. congolense IL 3274 between Day 1 and Day 19 could have been due to alteration in expression of drug resistance by trypanosomes as the population expanded, the experiment was repeated using trypanosomes that reappeared in the animals that had been treated with diminazene aceturate on Day 19. On Day 36, when all five animals were parasitaemic, five groups of teneral G. m. centralis, each containing 160 flies, were fed on one occasion on each of the five goats (one group of testse flies per goat). Thereafter, each group of tsetse flies was maintained on clean rabbits. When infective, five flies from each group were allowed to feed on two naive goats each (i.e. two goats per group of tsetse flies). One animal in each pair was treated 24 h after infection with diminazene aceturate at a dose of 7.0 mg kg-1 b.w., the other was treated on Day 19, when parasitaemic, with the same drug dosage. As before, treatment 24 h following infection eliminated infections in all animals, but when treatment was delayed until Day 19, trypanosomes in all animals were refractory to treatment. Thus, although tsetse flies were infected with trypanosomes that had arisen in infected goats following treatment with diminazene aceturate at a dose of 7.0 mg kg-1 b.w., when the same flies were allowed to feed on clean goats, the resultant infections were sensitive to treatment with the same drug dosage when administered 24 h following infection. These data therefore indicate that there is a significant alteration in diminazene sensitivity of IL 3274 between Day 1 and Day 19 and that this is associated with an alteration in the resistance phenotype of the trypanosomes.

New methods for the diagnosis of Babesia bigemina infection.

Accurate diagnosis of Babesia bigemina infection, an economically important tick-transmitted protozoan parasite of cattle, is essential in the management of disease control and in epidemiological studies. The currently used methods of diagnosis are blood smear examination and serological tests which include agglutination and immunofluorescence tests. These tests have been used in the fild but because they lack sensitivity and specificity, newer and improved methods of diagnosis are being developed. The quantitative buffy coat (QBC) method, using microhaematocrit tubes and acridine orange staining allows rapid and quicker diagnosis of B. bigemina and other blood parasites compared to light microscopic examination of stained smears. Parasite specific monoclonal antibodies have been used in antigen/antibody capture enzymelinked immunosorbent assays with greater sensitivity and specificity than previously described serological tests. Similarly, DNA probes, derived from a repetitive sequence of the B. bigemina genome, offer a method of detecting very small numbers of parasites which are undetectable by conventional microscopy. An extrachromosomal DNA element, present in all the tick-borne protozoan parasites so far tested, provides an accurate means of differentiating mixed parasite populations in infected animals. These improved methods will greatly facilitate epidemiological studies.

New methods for the diagnosis of Babesia bigemina infection

Accurate diagnosis of Babesia bigemina infection, an economically important tick-transmitted protozoan parasite of cattle, is essential in the management of disease control and in epidemiological studies. The currentlyused methods of diagnosis are blood smear examination and serological tests which include agglutination and immunofluorescence tests. These testes have been used the fild but because they lack sensitivity and specificity, never and improved methods of diagnosis are being developed. The quantitative buffy coat (OBC) method, using microhaematocrit tubes and acridine orange staining allows rapid and quicker diagnosis of B. bigemina and other blood parasites compared to light microscopic examination of stained smears. Parasite specific monoclonal antibodies have been used in antigen/antibody capture enzymelinked immunosorbent assays with grater sensitivity and specificity than previously described serological tests. Similary, DNA probes, derived from a repetitive sequence of the B. bigemina genome, offer a method of detecting very small numbers of parasites which are undetectable by conventional microscopy. An extrachromosomal DNA element, present in all the tick-borne protozoan parasites so far tested, provides an accurate means of diferentiating mixed parasite populations in infected animals. These improved methods will greatly facilitate epidemiological studies

Identification of a Theileria mutans-specific antigen for use in an antibody and antigen detection ELISA.

Purified piroplasms of Theileria mutans were used to immunize BALB/c mice to generate monoclonal antibodies (MoAbs). The MoAbs recognized an antigen of a relative molecular mass of 32 kDa in Western blots. This antigen was also recognized by sera from cattle which had recovered naturally from experimental tick-transmission or infections induced by the blood stages of T. mutans. The MoAbs did not react, in indirect immunofluorescence or enzyme-linked immunosorbent assays (ELISA), with the common haemoparasites of cattle, namely, T. parva, T. annulata, Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma congolense, T. vivax or T. brucei. An antigen capture ELISA was established with two of the MoAbs which recognized different epitopes on the 32 kDa molecule. Using this test it was possible to detect circulating antigens or immune complexes in sera collected from cattle during the acute or chronic phases of infection. When the purified 32 kDa protein was used as antigen in a micro-ELISA to detect circulating antibodies in both experimental and field cattle sera, it was found that the titres of antibodies ranged between 1:20 and 1:10,240. Results of this study indicate that the antigen and immune complex capture assays and the antibody detection ELISA can be complementary in the immunodiagnosis of acute and chronic T. mutans infections. Moreover, the tests are useful in the differential diagnosis of the disease and for epidemiological studies.

Long-acting oxytetracycline prophylaxis to protect susceptible cattle introduced into an area of Kenya with endemic East Coast fever.

Two field trials were carried out in successive years at the Ngong Veterinary Farm, Kenya, in which young cattle, previously unexposed to tick-borne diseases, were introduced into an area with endemic East Coast fever while protected by a series of injections of a long-acting oxytetracycline. In 1984, 12 animals which received injections of 20 mg/kg of the drug on days 0, 7, 14 and 21 after introduction, together with 12 untreated controls, were exposed without tick control until clinical disease occurred. All 12 control animals contracted East Coast fever by day 24 and 10 of them died. Five of the 12 injected animals had detectable parasites, and one of them required antitheilerial treatment. In 1985, four groups of 10 calves were introduced. One group received injections of 20 mg/kg of oxytetracycline on days 7 and 14, one group received injections on days 7, 14 and 21, and a third group received injections on days 7, 12 and 17; the fourth group (controls) had no treatment until clinical disease occurred. By day 35 all the control animals had contracted the disease and one had died despite antitheilerial treatment. Three injections of oxytetracycline suppressed the disease so that mild reactions occurred in only four animals in each group, but two injections failed to prevent severe reactions in two animals and mild reactions in four others.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of East Coast fever immunisation and different acaricidal treatments on the productivity of beef cattle.

A trial was performed on a farm in the Coast Province of Kenya to study the effects of East Coast fever immunisation and different acaricidal treatments on the productivity of immunised and unimmunised beef cattle. Eighty cattle were immunised against Theileria parva parva (Marikebuni) by the infection and treatment method and a similar group was left as an unimmunised control. Immunisation had no deleterious effect on the cattle. After immunisation, the immunised and control groups were each subdivided into four groups of 20 and each subgroup was managed under a different tick control regimen. The tick control regimen were, acaricidal spraying twice a week or once every three weeks, the application of acaricide-impregnated ear-tags, and no tick control. During a nine-month exposure period there were 18 cases of East Coast fever among the 80 immunised cattle, three which were severe and the others mild. Among the 80 unimmunised cattle there were 57 cases of East Coast fever, 50 of which were severe. The highest morbidity and mortality occurred in the groups under limited tick control or without tick control. Overall weight gain in the immunised cattle, irrespective of the tick control regimen, was better than the weight gain in the unimmunised groups. Within the immunised groups, the weight gain of the cattle sprayed twice weekly was comparable to the weight gain of the animals with acaricidal ear-tags and was significantly higher than the weight gains in the groups sprayed once every three weeks or with tick control. Preliminary cost/benefit analysis showed that it was uneconomical to maintain unimmunised cattle under limited or no tick control.(ABSTRACT TRUNCATED AT 250 WORDS)