Large-Scale International Validation of an Indirect ELISA Based on Recombinant Nucleocapsid Protein of Rift Valley Fever Virus for the Detection of IgG Antibody in Domestic Ruminants.

Diagnostic performance of an indirect enzyme-linked immunosorbent assay (I-ELISA) based on a recombinant nucleocapsid protein (rNP) of the Rift Valley fever virus (RVFV) was validated for the detection of the IgG antibody in sheep (n = 3367), goat (n = 2632), and cattle (n = 3819) sera. Validation data sets were dichotomized according to the results of a virus neutralization test in sera obtained from RVF-endemic (Burkina Faso, Democratic Republic of Congo, Mozambique, Senegal, Uganda, and Yemen) and RVF-free countries (France, Poland, and the USA). Cut-off values were defined using the two-graph receiver operating characteristic analysis. Estimates of the diagnostic specificity of the RVFV rNP I-ELISA in animals from RVF-endemic countries ranged from 98.6% (cattle) to 99.5% (sheep) while in those originating from RVF-free countries, they ranged from 97.7% (sheep) to 98.1% (goats). Estimates of the diagnostic sensitivity in ruminants from RVF-endemic countries ranged from 90.7% (cattle) to 100% (goats). The results of this large-scale international validation study demonstrate the high diagnostic accuracy of the RVFV rNP I-ELISA. Standard incubation and inactivation procedures evaluated did not have an adverse effect on the detectable levels of the anti-RVFV IgG in ruminant sera and thus, together with recombinant antigen-based I-ELISA, provide a simple, safe, and robust diagnostic platform that can be automated and carried out outside expensive bio-containment facilities. These advantages are particularly important for less-resourced countries where there is a need to accelerate and improve RVF surveillance and research on epidemiology as well as to advance disease control measures.

High prevalence of spotted fever group rickettsiae in Amblyomma variegatum from Uganda and their identification using sizes of intergenic spacers.

The spotted fever group (SFG) rickettsiae are obligate intracellular bacteria transmitted by ticks that cause several tick-borne rickettsioses in humans worldwide. This study was intended to determine the prevalence of SFG rickettsiae in Amblyomma variegatum from 7 districts across Uganda. In addition to sequencing of gltA and ompA genes, identification of Rickettsia species based on the sizes of highly variable intergenic spacers, namely, dksA-xerC, mppA-purC, and rpmE-tRNA(fMet) was carried out. Application of multiplex PCR for simultaneous amplification of 3 spacers combined with capillary electrophoresis separation allowed simple, accurate, and high-throughput fragment sizing with considerable time and cost savings. Rickettsia genus-specific real-time PCR detected 136 positives out of 140 samples, giving an overall prevalence of 97.1%. Most samples (n=113) had a size combination of 225, 195, and 341 bp for dksA-xerC, mppA-purC, and rpmE-tRNA(fMet), respectively, which was identical to that of R. africae, a causative agent of African tick bite fever. In addition, several samples had size variants in either dksA-xerC or rpmE-tRNA(fMet). Nonetheless, the partial sequences of gltA and ompA genes of samples of all size combinations showed the greatest similarity to R. africae (99.3-100% for gltA and 98.1-100% for ompA). Given these results, it is highly possible that the tested ticks were infected with R. africae or closely related species. This is a first report on molecular genetic detection of R. africae and its high endemicity in Uganda. Clinicians in this country should be aware of this pathogen as a cause of non-malarial febrile illness. This study provided a starting point for the development of Rickettsia species identification based on the sizes of intergenic spacers. The procedure is simple, rapid, and cost-effective to perform; hence it might be particularly well suited for preliminary species identification in epidemiological investigations. The results may be more detailed and reliable when simultaneous sequencing analysis is performed.

Diagnosis of cattle diseases endemic to sub-Saharan Africa: evaluating a low cost decision support tool in use by veterinary personnel.

BACKGROUND: Diagnosis is key to control and prevention of livestock diseases. In areas of sub-Saharan Africa where private practitioners rarely replace Government veterinary services reduced in effectiveness by structural adjustment programmes, those who remain lack resources for diagnosis and might benefit from decision support. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated whether a low-cost diagnostic decision support tool would lead to changes in clinical diagnostic practice by fifteen veterinary and animal health officers undertaking primary animal healthcare in Uganda. The eight diseases covered by the tool included 98% of all bovine diagnoses made before or after its introduction. It may therefore inform proportional morbidity in the area; breed, age and geographic location effects were consistent with current epidemiological understanding. Trypanosomosis, theileriosis, anaplasmosis, and parasitic gastroenteritis were the most common conditions among 713 bovine clinical cases diagnosed prior to introduction of the tool. Thereafter, in 747 bovine clinical cases estimated proportional morbidity of fasciolosis doubled, while theileriosis and parasitic gastroenteritis were diagnosed less commonly and the average number of clinical signs increased from 3.5 to 4.9 per case, with 28% of cases reporting six or more signs compared to 3% beforehand. Anaemia/pallor, weakness and staring coat contributed most to this increase, approximately doubling in number and were recorded in over half of all cases. Finally, although lack of a gold standard hindered objective assessment of whether the tool improved the reliability of diagnosis, informative concordance and misclassification matrices yielded useful insights into its role in the diagnostic process. CONCLUSIONS/SIGNIFICANCE: The diagnostic decision support tool covered the majority of diagnoses made before or after its introduction, leading to a significant increase in the number of clinical signs recorded, suggesting this as a key beneficial consequence of its use. It may also inform approximate proportional morbidity and represent a useful epidemiological tool in poorly resourced areas.

Development of multiple-locus variable-number tandem-repeat analysis for rapid genotyping of Ehrlichia ruminantium and its application to infected Amblyomma variegatum collected in heartwater endemic areas in Uganda.

The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a serious tick-borne disease in ruminants. The genetic diversity of organisms in the field will have implications for cross-protective capacities of any vaccine developed, and for an effective vaccine design strategy proper genotyping and understanding of existing genetic diversity in the field is necessary. We searched for variable-number tandem-repeat (VNTR) loci for use in a multi-locus VNTR analysis (MLVA). Sequencing analysis of 30 potential VNTRs using a panel of 17 reference strains from geographically diverse origins identified 12 VNTRs with allelic profiles differing between strains. Application of MLVA to 38 E. ruminantium-infected Amblyomma variegatum collected from indigenous cattle in 6 different districts of Uganda identified 21 MLVA types. The discriminatory power of MLVA was greater than that of map1 PCR-restriction fragment length polymorphism analysis, with which only 6 genotypes were obtained. The high discriminatory power as well as cost- effective performance of MLVA provide the potential for this technique to be applied in the future with respect to optimizing vaccine trials by identifying local strain diversity, and also raise the possibility of exploring the association between E. ruminantium genotypes and phenotypes such as pathological outcome in the ruminant host.

Multi-locus sequence typing of Ehrlichia ruminantium strains from geographically diverse origins and collected in Amblyomma variegatum from Uganda.

BACKGROUND: The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater in ruminants. A better understanding of the population genetics of its different strains is, however, needed for the development of novel diagnostic tools, therapeutics and prevention strategies. Specifically, the development of effective vaccination policies relies on the proper genotyping and characterisation of field isolates. Although multi-locus sequence typing (MLST) has been recently developed, only strains from geographically restricted collections have been analysed so far. The expansion of the MLST database to include global strains with different geographic origins is therefore essential. In this study, we used a panel of reference strains from geographically diverse origins and field samples of E. ruminantium detected from its vector, Amblyomma variegatum, in heartwater-endemic areas in Uganda. RESULTS: A total of 31 novel alleles (six, four, six, three, two, five, three, and two for gltA, groEL, lepA, lipA, lipB, secY, sodB, and sucA loci, respectively) and 19 novel sequence types (STs) were identified. Both neighbour-joining and minimum spanning tree analyses indicated a high degree of genetic heterogeneity among these strains. No association was observed between genotypes and geographic origins, except for four STs from West African countries. When we performed six different tests for recombination (GeneConv, Bootscan, MaxChi, Chimaera, SiScan, and 3Seq) on concatenated sequences, four possible recombination events were identified in six different STs. All the recombination breakpoints were located near gene borders, indicating the occurrence of intergenic recombination. All four STs that localized to a distinct group in clustering analysis showed evidence of identical recombination events, suggesting that recombination may play a significant role in the diversification of E. ruminantium. CONCLUSIONS: The compilation of MLST data set across the African continent will be particularly valuable for the understanding of the existing genetic diversity of field isolates in African countries. Comprehensive information on the degree of cross-protection between strains and further understanding of possible relationships between genotypes and phenotypes such as vaccine efficacy are expected to lead to the development of region-specific vaccination strategies.

Response of Nkedi Zebu and Ankole cattle to tick infestation and natural tick-borne, helminth and trypanosome infections in Uganda.

A cross-sectional study was conducted in Soroti district of Uganda to establish important traits of Nkedi Zebu and Ankole cattle regarding their production performance responses to natural infections of trypanosomes, gastrointestinal nematodes, Theileria parva, Babesia bigemina, Anaplasma marginale and tick infestations. Over four visits between October 2006 to August 2007, tick counts were performed and blood, faecal samples and sera were collected from the Nkedi Zebu (295) and Ankole (165) cattle from 86 herds in six locations per visit. Low parasitological prevalence of trypanosome infection (<6%) and high prevalence of gastrointestinal nematode infections (>30%) with low faecal egg counts (110-300 eggs per gramme (EPG)) were observed in the Nkedi Zebu and Ankole cattle. Both breeds had high, moderate and low mean counts of Rhipicephalus appendiculatus (18.0-24.0), Rhipecephalus (Boophilus) decoloratus (3.6-10.3) and Amblyomma variegatum ticks (1.7-4.3), respectively. In addition, both breeds had similar mean packed cell volumes (26.4-31.2) and a similar percentage of animals were anaemic (14.5-36.6%). The Nkedi Zebu cattle further had higher mean optical density (OD) values for antibodies against T. parva (1.093-1.445) and A. marginale infections (0.573-0.583), and significantly (P < 0.001) higher mean OD values of antibodies against B. bigemina infections (1.07-2.175) than the Ankole cattle: T. parva (1.030-1.302); A. marginale (0.442-0.603) and B. bigemina infections (0.863-2.154). The Ankole cows produced significantly more (P < 0.001) milk per day (2.68 L) than the Nkedi Zebu cows (1.98 L), and the Ankole oxen had significantly higher (P < 0.05) draught power output (2.57 days/acre) than the Nkedi Zebu oxen (2.93 days/acre). Liveweights of calves aged 0-12 months of both breeds were comparable, suggesting that the Nkedi Zebu and Ankole cattle under similar disease challenge exhibited similar growth rates. In conclusion, the Nkedi Zebu cattle seem to possess a higher degree of disease resistance against endemic parasitic diseases, while the Ankole cattle seem to possess a moderate degree of disease resistance coupled with a moderate production potential.

Development of loop-mediated isothermal amplification (LAMP) assays for rapid detection of Ehrlichia ruminantium.

BACKGROUND: The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a potential zoonotic disease of ruminants transmitted by ticks of the genus Amblyomma. The disease is distributed in nearly all of sub-Saharan Africa and some islands of the Caribbean, from where it threatens the American mainland. This report describes the development of two different loop-mediated isothermal amplification (LAMP) assays for sensitive and specific detection of E. ruminantium. RESULTS: Two sets of LAMP primers were designed from the pCS20 and sodB genes. The detection limits for each assay were 10 copies for pCS20 and 5 copies for sodB, which is at least 10 times higher than that of the conventional pCS20 PCR assay. DNA amplification was completed within 60 min. The assays detected 16 different isolates of E. ruminantium from geographically distinct countries as well as two attenuated vaccine isolates. No cross-reaction was observed with genetically related Rickettsiales, including zoonotic Ehrlichia species from the USA. LAMP detected more positive samples than conventional PCR but less than real-time PCR, when tested with field samples collected in sub-Saharan countries. CONCLUSIONS: Due to its simplicity and specificity, LAMP has the potential for use in resource-poor settings and also for active screening of E. ruminantium in both heartwater-endemic areas and regions that are at risk of contracting the disease.

A field study to estimate the prevalence of bovine African Trypanosomosis in Butaleja District, Uganda.

Prevalence of bovine trypanosomosis was determined from a total of 203 blood samples collected from Butaleja district, eastern Uganda. All samples were examined by microhematocrit centrifuge test (MHC), PCR and ELISA. ELISA was performed in accordance with the OIE standard procedures using Trypanosoma brucei gambiense procyclic form crude antigens. PCR were utilized to identify the species and the subspecies of trypanosome. The overall prevalence of bovine African trypanosomosis was 8.9% by MHC, and 45.3% by the ELISA. Since substantial number (12 out of 18) of MHC positive samples were negative in the PCR tests, we could not conclude the most epidemic trypanosome species in the studied area. Nevertheless, the PCR results suggests that the most prevalent trypanosome was T. b. brucei (31/203), followed by T. congolense (6/203). In addition, only a few (3/203) mixed infections of T. b. brucei and T. congolense was detected by the PCR. Results obtained from this study indicates that bovine trypanosomosis is endemic in Butaleja district, Uganda.