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.

The role of parasitic diseases as causes of mortality in small ruminants in a high-potential farming area in central Kenya.

A 15-year retrospective study was performed to determine the role of parasitic diseases in causing mortalities in small ruminants. In total, 115 (32 %) sheep were diagnosed as having been killed by parasitic diseases out of 366 that died as a result of disease. The major cause of mortality was helminthosis (63 % of all parasitic cases). Most of the helminthosis cases were attributed to haemonchosis (40% of parasitic cases). Heartwater was the second most important parasitic disease (27% of all parasitic cases). Ninety-five (26%) goats were diagnosed to have been killed by parasitic diseases out of 365 cases presented at the post mortem facility. Helminthosis was the most frequent cause of mortality (55% of the total parasitic diseases). Twenty-six goats were killed by haemonchosis (27% of all parasitic diseases). Heartwater was the second most important parasitic disease, accounting for about 20% of all parasitic diseases. These findings indicate that viable helminth and tick control strategies should be devised in order to reduce mortality caused by helminthosis and heartwater and thereby achieve improved productivity.

A polarized Th1 type immune response to Cowdria ruminantium infection is detected in immune DBA/2 mice.

Immune responses to Cowdria ruminantium, an intracellular organism that causes heartwater in domestic ruminants, were characterized in a DBA/2 mouse model. Immunity induced by infection and treatment was adoptively transferable by splenocytes and could be abrogated by in vivo depletion of T cells but not by inhibition of nitric oxide synthase using NG-monomethyl-L-arginine. IgG2a and IgG2b C. ruminantium-specific responses were detected in immune mice. Culture supernatants of splenocytes from immune DBA/2 mice, which were stimulated with crude C. ruminantium antigens or recombinant major antigenic proteins 1 or 2, contained significant levels of interferon (IFN)-gamma and interleukin (IL)-6, but insignificant levels of IL-1alpha, IL-2, IL-4, IL-5, IL-10, IL-12, tumor necrosis factor-alpha (TNF), and nitric oxide. A similar response was detected during primary infection, although IFN-gamma levels decreased significantly during clinical illness and then increased following natural or antibiotic-aided recovery. These data support the conclusion that protective immunity to C. ruminantium in DBA/2 mice is mediated by T cells and is associated with a polarized T helper 1 type of immune response. This murine model could be utilized to screen for protective C. ruminantium antigens that provoke Th1 type immune responses and for evaluation of these antigens in recombinant vaccines against heartwater.

Effects of tick infestation and tick-borne disease infections (heartwater, anaplasmosis and babesiosis) on the lactation and weight gain of Mashona cattle in south-eastern Zimbabwe.

The effects of ticks and tick-borne disease infections on the lactation and weight gain of Mashona cattle were studied at Mbizi Quarantine Station in the south-eastern lowveld of Zimbabwe. Twenty-nine Mashona cows were allocated to 2 balanced groups and kept in separate paddocks at a stocking rate of one animal per 8 ha. One group received regular acaricide treatment to control bont (Amblyomma hebraeum) and other ticks. The other group was left untreated. The cows were artificially inseminated. The acaricide-treated cows and calves were essentially tick free throughout the experiment, while the untreated cows and calves were continuously tick infested. There was a drought-related decline in tick infestations in the second year of the experiment. Antibodies to Cowdria ruminantium, Babesia bigemina and Anaplasma marginale were detected in cows and calves from both groups, though the untreated group had significantly higher titres to C. ruminantium (P < 0.001). The total, measured amount of milk suckled by untreated calves was significantly more than treated calves (273 kg vs. 241 kg, P < or = 0.05). By interpolating between the twice weekly measurements, it was calculated that over the entire lactation untreated calves suckled an average of 935 kg/hd vs. 837 kg/hd for the treated group. There were no statistical differences in the weights of the 2 groups of calves at birth, weaning, 180 and 210 days post partum (P < 0.05). For cows, there were no statistically significant differences in gestation periods (288 vs. 279 days), reconception rates or weight patterns over time (P < 0.05). The results show that intensive acaricide treatment in areas of Zimbabwe where heartwater is enzootically stable is uneconomical. The maintenance of enzootic stability for tick-borne diseases through minimal tick control is clearly a more economic and practical control option.

Correlation between antibodies to Cowdria ruminantium (rickettsiales) in cattle and the distribution of Amblyomma vector ticks in Zimbabwe.

Cowdriosis, caused by Cowdria ruminantium, is transmitted by Amblyomma ticks, which are widely distributed in Zimbabwe. To assess the distribution of this disease in Zimbabwe, cattle either exposed to Amblyomma ticks or maintained in areas free from these ticks were tested for antibodies to Cowdria. A total of 324 sera were tested using competitive ELISA and the indirect fluorescent antibody test (IFAT). At diptanks in Amblyomma-infested areas 52% (n = 95) and 26% (n = 47) of sera were positive by cELISA and IFAT, respectively. At diptanks in Amblyomma-free areas 11% (n = 125) and 10% (n = 134) of sera were positive by cELISA and IFAT, respectively. The results were significantly different between Amblyomma-infested and tick-free areas (chi 2 = 24.73, P < or = 0.005 for IFAT and chi 2 = 57.53, P < or = 0.005 for cELISA). High background readings in field sera, possibly due to cross-reactive antibodies to Ehrlichia spp., complicated the determination of a realistic cut-off point, especially in cELISA. On the basis of the distribution of Amblyomma ticks, currently a large part of Zimbabwe can be considered endemic for the disease.

Adult Amblyomma hebraeum burdens and heartwater endemic stability in cattle.

Several conclusions of importance to studies on the epidemiology of heartwater were drawn from an investigation in which the numbers of adult Amblyomma hebraeum ticks, to which a closed herd of Hereford cattle were exposed over a period of 6 1/2 years, were manipulated. With a tick Cowdria ruminantium infection rate of 3-5%, an endemically stable situation was created by dipping the herd only when an average of 10 adult male and female A. hebraeum ticks were counted on 10 animals. When the average was increased to 15 during the calving period, 97% of calves acquired a tick-mediated immunity at the age of 6 months. Because only adult ticks confined to the hindquarters are counted, this procedure is recommended as a feasible and practical guideline to stock owners wishing to determine a dipping programme that would ensure endemic stability. The indirect fluorescent antibody test gave a true reflection of the infection rate through ticks in calves 3-6 months old, but not in older animals that had been re-infected more than once. This is because on one hand antibody may persist for 2 years after withdrawal from tick exposure and on the other the artificial re-infection of cattle with a tick acquired immunity is not always followed by a rise in antibody titres and may even result in seronegativity. Four cows infected and re-infected through ticks, remained immune to challenge for 2 years after withdrawal from tick exposure. Within the confines of one farm 3 isolates of C. ruminantium that differed in pathogenicity and immunogenicity were recovered from ticks. One of these isolates was almost non-pathogenic to cattle.

The ecology of the African vectors of heartwater, with particular reference to Amblyomma hebraeum and Amblyomma variegatum.

The hosts, sites of attachment, life cycle, habitat requirements and seasonal abundance of Amblyomma astrion, Amblyomma cohaerens, Amblyomma gemma, Amblyomma hebraeum, Amblyomma lepidum, Amblyomma marmoreum, Amblyomma pomposum, Amblyomma sparsum, Amblyomma tholloni and Amblyomma variegatum, the 10 potential vectors of heartwater in Africa, are listed. Factors influencing the distribution and abundance of the ticks as well as interactions with other species and the role of predators and pathogens are discussed.

Heartwater in the Caribbean: isolation of Cowdria ruminantium from Antigua.

Adult Ambylomma variegatum ticks were collected from 184 cattle, 13 sheep and one goat in Antigua, and ground in phosphate buffered saline. The resultant supernates were cryopreserved in liquid nitrogen. Five supernate pools, each derived from approximately 100 ticks collected from different herds, were thawed and each was inoculated intravenously into a separate experimental goat. One goat exhibited a febrile response with Cowdria ruminantium demonstrable in brain biopsies; after recovery, this animal showed no reaction to a lethal challenge with a Guadeloupe isolate of C ruminantium.