D.T. De Waal

Equine piroplasmosis: A review

Abstract This review focuses on equine piroplasmosis with specific reference to its distribution, diagnosis and clinical and pathological signs. The more common used drugs are discussed both with reference to treatment and chemosterilization. Areas requiring further research are also briefly mentioned.

Phylogeny and evolution of the piroplasms

Small subunit ribosomal RNA (srRNA) genes of three Theileria species, one Cytauxzoon and four Babesia species were amplified using the polymerase chain reaction (PCR), cloned and sequenced. Our sequences were aligned with srRNA sequences previously published for eight species of Apicomplexa, one ciliate and one dinoflagellate, the last two being included as free-living outgroup species. Phylogenetic relationships between the organisms were inferred by four independent methods of phylogenetic tree construction using the ciliate Oxytricha nova to root the trees. Our trees fail to show a consensus branching order. They do, however, clearly indicate that the theilerias form a monophyletic taxon derived from a paraphyletic group which includes the species B. equi, C. felis and B. rodhaini. The distance trees indicate that the babesias sensu stricto (B. canis, B. caballi, B. bigemina and B. bovis) form another monophyletic taxon which diverged before the theilerias separated from the above-mentioned paraphyletic group. The parsimony and maximum likelihood trees suggest that the babesias and theilerias are sister taxa, both of which were derived from the paraphyletic group.

Risk factors to tick infestations and their occurrence on horses in the state of São Paulo, Brazil

From December 1998 to March 1999, 40 stud farms were studied in the state of São Paulo, Brazil. During visits to farms, horses reared under grazing conditions were examined for the presence of ticks. On each farm visit, horse pastures were closely inspected and a questionnaire was given to the farm supervisor with the purpose of gaining information about ecological and management variables (independent variables) that could be associated with the presence and infestation levels of ticks on the farm (dependent variables). Three tick species were found during the study. Anocentor nitens, Amblyomma cajennense and Boophilus microplus were present on horses from 38 (95%), 20 (50%) and four (10%) farms, respectively. All farms that had A. cajennense or B. microplus infestations also had A. nitens infestations. Only one of the four farms with B. microplus infestations on the horses also had A. cajennense infestations. Two farms had all horses free of ticks. There was a strong association between the presence of infestation by B. microplus on horses and the simultaneous use of a grazing area by cattle and horses (P = 0.000). There was no statistical association between any of the independent variables and the presence or infestation level of A. nitens on the horses (P > 0.20). The presence of A. cajennense was statistically associated with the presence of at least one mixed overgrowth pasture in the farm (P = 0.001). A mixed overgrowth pasture means the presence of undesired plants such as bushes and shrubs in the pasture. The presence of high levels of A. cajennense on horses was also associated with the presence of at least one mixed overgrowth pasture in the farm (P = 0.026). The regular use of acaricides was statistically associated with the presence of ticks on the horses (P < 0.05), making this procedure a result of the inefficacy of controlling ticks on the farms. The occurrence of human infestation by ticks was statistically associated with the presence of A. cajennense on the horses (P=0.000). The presence of at least one mixed overgrowth pasture on the farm was associated (P = 0.000) to either higher horse densities and to farms that did not mow all the pastures once a year, indicating that mowing all the pastures at least once a year can be considered a protective factor against the presence of mixed overgrowth pastures on the farm, and consequently, against the presence of A. cajennense on the horses.

Besnoitia besnoiti: Studies on the definitive host and experimental infections in cattle

Domestic cats, 11 other species of carnivorous mammals, 6 species of snakes, and whitebacked vultures were tested for their possible role as definitive hosts ofBenoitia besnoiti by feeding with cystic material from chronically infected bovines. None of the species tested is a definitive host; hence, the life cycle of this parasite remains obscure. In attempts to produce clinical cases of besnoitiosis by experimental infection, bovines were inoculated IV, SC, and IP with cystozoites or tachyzoites. Immunosuppression of the animals was essential for the development of severe cases and skin lesions; cystozoites proved to be more pathogenic than tachyzoites.

Anaplasmosis Control and Diagnosis in South Africa

Abstract: Anaplasmosis is widespread in South Africa with more than 99% of the total cattle population at risk. Five tick species have been experimentally shown to be capable of transmitting Anaplasma in South Africa. Mechanical transmission through blood contaminated instruments and biting flies also occurs. Vaccination against Anaplasma marginale by administration of an Anaplasma centrale live‐blood vaccine has been practiced in this country since 1912. Although generally a mild pathogen, Anaplasma centrale can cause severe clinical reactions following vaccination and also does not afford complete protection against all A. marginale isolates. Anaplasmosis vaccine is routinely available in a deep‐frozen form and approximately 220,000 doses of vaccine are sold per annum. Microscopic examination of stained thin blood smears is still the most reliable and cost effective method of confirming a clinical diagnosis of anaplasmosis. Several diagnostic tests, such as the complement fixation tests, card agglutination test, and enzyme‐linked immunosorbent assay (ELISA) have been developed to identify carrier cattle. A competitive inhibition ELISA test, based on antibody binding to a recombinant MSP‐5 protein conserved among Anaplasma species, is routinely used at this laboratory.

Continuous in vitro cultivation of erythrocytic stages ofBabesia equi

The protozoan parasiteBabesia equi, a causative agent of equine piroplasmosis, was continuously cultivated in horse erythrocytes. The parasites were isolated from a carrier horse at a time when no parasite was detected in a thin blood smear. The culture medium consisted of modified medium 199 supplemented with 40% non-heat-inactivated horse serum in a humidified atmosphere containing 5% CO2, 2% O2, and 93% N2 at 37° C. Parasites were detected after 2 days in culture. When the percentage of parasitized erytrrocytes (PPE) reached 1%, the cultures were transferred into a humidified atmosphere of 5% CO2 in air. After 7 days the cultures were split at a ratio of 1∶2, and after another 5 days they were split at a ratio of 1∶4. From them on, cultures were split at a ratio of 1∶4 routinely at 2-day intervals. The PPE ranged between 10% and 25%. Supplementation with hypoxanthine was essential for the initiation and propagation of cultures. In established cultures, hypoxanthine could be replaced by equimolar concentrations of adenosine or guanosine. Parasites from cultures could be cryopreserved and resuscitated. Cultures were maintained for more than 300 days.

How does Trypanosoma equiperdum fit into the Trypanozoon group? A cluster analysis by RAPD and multiplex-endonuclease genotyping approach

The pathogenic trypanosomes Trypanosoma equiperdum, T. evansi as well as T. brucei are morphologically identical. In horses, these parasites are considered to cause respectively dourine, surra and nagana. Previous molecular attempts to differentiate these species were not successful for T. evansi and T. equiperdum; only T. b. brucei could be differentiated to a certain extent. In this study we analysed 10 T. equiperdum, 8 T. evansi and 4 T. b. brucei using Random Amplified Polymorphic DNA (RAPD) and multiplex-endonuclease fingerprinting, a modified AFLP technique. The results obtained confirm the homogeneity of the T. evansi group tested. The T. b. brucei clustered out in a heterogenous group. For T. equiperdum the situation is more complex: 8 out of 10 T. equiperdum clustered together with the T. evansi group, while 2 T. equiperdum strains were more related to T. b. brucei. Hence, 2 hypotheses can be formulated: (1) only 2 T. equiperdum strains are genuine T. equiperdum causing dourine; all other T. equiperdum strains actually are T. evansi causing surra or (2) T. equiperdum does not exist at all. In that case, the different clinical outcome of horse infections with T. evansi or T. b. brucei is primarily related to the host immune response.

A possible new piroplasm in lions from the Republic of South Africa.

A small piroplasm was detected in blood smears from lions (Panthera leo) in the Kruger National Park (KNP; Republic of South Africa) during 1991/1992. The parasite was identified provisionally as Babesia felis, but sera from these lions tested negative to B. felis antigen in the indirect immunofluorescent antibody test (IFAT). Blood from an infected lion was subsequently subinoculated into a domestic cat and two leopards in an attempt to identify the parasite. A lion also was infected with B. felis (from a cat). Serum samples collected from these animals were tested against B. felis, the KNP small piroplasm, and Cytauxzoon felis antigen in the IFAT. The serological results indicate that the KNP small piroplasm isolated from the lion is probably a distinct species from B. felis and C. felis.

Detection of Babesia equi in infected horses and carrier animals using a DNA probe

The ability of the Babesia equi repetitive probes, pSE2 and pSB20, to detect parasites in blood from experimentally infected, naturally infected and carrier animals was tested using a spot hybridization assay. The clinical course of the experimentally infected horses was monitored using microscopy, indirect fluorescent antibody tests, packed cell volume, temperature and the probe assay. The probes sensitively monitored the parasite level during the development of the disease and correlated well with the other parameters tested. The sensitivity of the probe assay was superior to that of light microscopy, and a parasitaemia equivalent to less than 0.0025% could be detected. Detection of B. equi DNA was possible in all natural cases tested and 20 of the 119 randomly selected horses were identified as carriers of B. equi parasites. Microscopy could identify parasites in only 8 of these carrier animals. These results show that the probes can detect B. equi parasites in carrier animals and that they are suitable for use in a laboratory-based assay for B. equi.

Anthelmintic-resistant nematodes in Irish commercial sheep flocks- the state of play

Anthelmintic resistance has been reported in most sheep producing countries. Prior to the mid 1990s, reports of anthelmintic resistance in Ireland were sparse and focused on benzimidazole, one of the three classes of anthelmintic available during this period. This evidence for efficacy issues on Irish farms combined with awareness that anthelmintic resistance was increasingly being reported in other countries prompted the need for more comprehensive investigations on Irish farms. Faecal egg count reduction and micro-agar larval development tests were employed to investigate resistance to benzimidazole, levamisole and macrocyclic lactone. There is compelling evidence for resistance to both benzimidazole (>88% of flocks) and levamisole (>39% of flocks). Resistance of nematode populations to macrocyclic lactone was suspected on a small number of farms (11%) but needs to be confirmed. The recent introduction of two new classes of anthelmintics, after over a 25 year interval, together with the evidence that anthelmintic resistance is reported within a relatively short time following the introduction of a new anthelmintic compound means that the challenge to the industry is immediate. Actions are urgently required to manage anthelmintic resistance so as to prolong the lifespan of anthelmintics.