Origin and history to date of the World Association for the Advancement of Veterinary Parasitology (WAAVP) African Foundation.

The origin of the World Association for the Advancement of Veterinary Parasitology (WAAVP) African Foundation is described. The 16th WAAVP Conference held in South Africa in 1997 generated a surplus of ZAR 430 460 (US$ 70 116). This was invested and a foundation established to manage the fund with the intention of using it to the mutual advantage of the WAAVP and African veterinary parasitologists. To date, more than 110 scholarship applications have been screened, and 51 full and partial scholarships awarded to young African veterinary parasitologists to attend subsequent biennial WAAVP Conferences. This investment has grown into a very successful endowment currently valued at US$ 206 553. This article is written in response to many queries across the globe about the origin of this fund and how it has been invested, managed, sustained and utilised.

Live vaccines against bovine babesiosis.

Bovine babesiosis is an important tick-borne disease caused by Babesia bovis, B. bigemina and B. divergens. The first steps taken in the development of an effective vaccination strategy against bovine babesiosis followed the observations that animals, recovered from natural infection with Babesia were strongly protected against subsequent challenge. Further investigation indicated that the use of donor blood from recovered animals to infect recipient animals did not produce the severe form of the disease. The past century has seen a refinement of this original carrier-donor system to one using attenuated less virulent strains with standardized doses of known parasite concentration to ensure reliability. With the implementation of good manufacturing practices further changes were necessary in the production of these vaccines, such as freezing for long-term storage to allow sufficient time for pre-release safety and effectivity testing. Regardless of these improvements the vaccines are not without problems and breakdowns and breakthroughs occur from time to time. Despite considerable research efforts into the development of alternative more consumer friendly vaccines, none is immediately forthcoming and the live attenuated babesiosis vaccines are still used in many countries.

Possible death of a buffalo calf (Syncercus caffer) due to suspected heartwater (Ehrlichia ruminantium).

Rickettsial organisms resembling Ehrlichia ruminantium (the causative organism of heartwater) were demonstrated in brain smears and formalin-fixed brain sections derived from a buffalo calf that died on a private game reserve in northern KwaZulu-Natal. The possibility that the tick-free environment of a quarantine boma may have affected the calf's immunity, is discussed. These findings suggest that monitoring heartwater in wild ruminants and making brain smears as a routine during post mortem evaluations of wild ruminants, should be encouraged.

A sero-epidemiological survey of blood parasites in cattle in the north-eastern Free State, South Africa.

A survey to determine the incidence of parasites in cattle (n = 386) was conducted in the north eastern Free State between August 1999 and July 2000. Giemsa-stained blood smears were negative for blood parasites. A total of 94% of the cattle were sero-positive for Babesia bigemina by indirect fluorescent antibody test while 87% were sero-positive for Anaplasma by enzyme-linked immunosorbent assay. The observation of negative blood smears but high incidence of positive serological results for Anaplasma and Babesia for the same group of cattle indicates that this area is endemic for these diseases but with a stable disease situation. All the animals were sero-negative for B. bovis and this is probably because the tick vector (Boophilus microplus) which transmits the disease is not present in the Free State Province. Two tick species belonging to the family ixodidae were found on cattle, namely Boophilus decoloratus and Rhipicephalus evertsi evertsi. In the present study significant differences in seasonal burdens of B. decoloratus occurred, with the highest infestations recorded from February to June. The presence of R. evertsi evertsi throughout the year without any or with small fluctuations in winter months was observed, with a peak from February to May.

The expression of RoTat 1.2 variable surface glycoprotein (VSG) in Trypanosoma evansi and T. equiperdum.

In order to define whether the variable antigenic type RoTat 1.2 is restricted to Trypansoma evansi and could be used as antigen in serological tests to differentiate T. evansi from Trypansoma equiperdum, the appearance of RoTat 1.2-specific antibodies in rabbits, experimentally infected with T. evansi and T. equiperdum, respectively, was analyzed. Ten strains of T. evansi and 11 strains of T. equiperdum originating from Asia, Europe, Africa and Latin America were tested. Rabbit pre-infection sera and sera of days 7, 14, 25, 35 post-infection (p.i.) were analyzed for the presence of antibodies reactive with RoTat 1.2 in immune trypanolysis, ELISA/T. evansi and CATT/T. evansi. Within the duration of the infection (maximum 35 days), all T. evansi as well as 9 out of 11 T. equiperdum infected rabbits became positive in all these tests. The rabbits infected with T. equiperdum OVI (South Africa) and BoTat 1.1 (Morocco) remained negative in the immune trypanolysis test although the latter rabbit became positive in the CATT/T. evansi and ELISA/T. evansi. On the contrary, both rabbits were positive in immune trypanolysis when tested against their respective infecting population. From these data, we conclude that most T. equiperdum strains express isoVATs of RoTat 1.2. This explains, in part, why antibody tests based on T. evansi RoTat 1.2 cannot reliably distinguish between infections caused by T. evansi and those caused by T. equiperdum unless it can be proven that most described T. equiperdum are actually misclassified T. evansi.

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.

Residual effect of antibabesial drugs on the live redwater blood vaccines.

It has been demonstrated that the attenuated organisms used in the unfrozen South African Babesia bovis and B. bigemina (redwater) vaccines are susceptible for longer periods to the residual effect of the anti-babesial drugs diminazene and imidocarb dipropionate than the virulent field strains. Reports of vaccine failures in some animals vaccinated with the frozen South African redwater vaccines after prophylactic treatment with imidocarb dipropionate have led us to reinvestigate the validity of the recommended prescribed waiting periods. Results indicated that waiting periods before administration of the frozen B. bovis and B. bigemina vaccines in animals that have been treated with diminazene at 3.5 mg/kg live weight, compare favorably with results initially obtained for the unfrozen vaccines at 4 and 8 weeks, respectively. However, the inhibitory effect of imidocarb dipropionate at 3.0 mg/kg live weight on the infectivity of both frozen B. bovis and B. bigemina vaccines is longer than previously anticipated and necessitated changing the minimum waiting periods before administration of these vaccines from 8 to 12 weeks and 16 to 24 weeks, respectively.

Expression of RoTat 1.2 cross-reactive variable antigen type in Trypanosoma evansi and T. equiperdum.

The variable antigen type (VAT) RoTat 1.2 has been cloned from a T. evansi strain, isolated in 1982 from a water buffalo in Indonesia. All T. evansi isolates hitherto tested express this VAT. In a study on the differential diagnosis of T. equiperdum and T. evansi in horses, we investigated serological evidence for the expression of RoTat 1.2 in 11 T. evansi and six T. equiperdum populations originating from Asia, Europe, Africa, and the Americas. Preinfection sera and sera of days 7, 14, 25, and 35 post-infection (p.i.) were analyzed for the presence of antibodies reactive with RoTat 1.2 in immune trypanolysis, ELISA/T. evansi and CATT/T. evansi. Within the duration of the experiment, all rabbits infected with T. evansi became positive in the three serological tests. Five out of six rabbits infected with T. equiperdum also became positive in the three tests. Only one T. equiperdum strain (the OVI strain from South Africa) did not induce the production of antibodies reactive with RoTat 1.2 and thus might not contain or express a VSG that shares epitopes similar to those on the RoTat 1.2 VSG. The data lead to the conclusion that T. equiperdum can express VSGs containing epitopes serologically similar to those in the T. evansi RoTat 1.2 VAT. This explains, in part, why the antibody detection tests based on Ro Tat 1.2 VSG cannot reliably distinguish between the infections caused by T. evansi and those caused by T. equiperdum. There are no data that contradict the possibility that the putative T. equiperdum strains, which express VSGs with epitopes similar to those on RoTat 1.2, are actually T. evansi.

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.

Continuous in vitro cultivation of Babesia caballi in serum-free medium.

Experiments were undertaken to develop a serum-free medium for the in vitro cultivation of Babesia caballi, a tick-borne hemoprotozoan parasite, one of the causative agents of equine piroplasmosis. A modified HL-1 medium supplemented with horse serum, L-glutamine, antibiotics, and hypoxanthine was used. B. caballi organisms were continuously cultivated at 37 degrees C in microaerophilous stationary-phase culture in a humidified atmosphere containing 5% CO2 in air before exposure to serum-free culture conditions. For serum-free propagation, lipid-rich bovine serum albumin (LR-BSA), alone or with chemically defined lipids (CDL), were added instead of serum. Media containing LR-BSA alone or LR-BSA and CDL in various amounts supported the in vitro propagation of B. caballi. Growth was maintained for more than 6 months. The growth rates obtained in serum-free media were similar to those previously obtained in traditional serum-containing medium.

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.

Monoclonal antibody against Babesia equi: characterization and potential application of antigen for serodiagnosis.

Monoclonal antibody (MAb) BEG3 was produced against Babesia equi parasites to define a species-specific antigen for diagnostic use. The MAb reacted with single, paired, and Maltese cross forms of B. equi, and no reaction was observed with this MAb on acetone-fixed Babesia caballi, Babesia ovata, or Babesia microti parasites in the indirect immunofluorescent antibody test. Confocal laser and immunoelectron microscopic studies showed that the antigen which was recognized by this MAb was located on the surface of B. equi parasites. This MAb recognized a 19-kDa protein of B. equi antigen and did not react with B. caballi antigen or normal horse erythrocytes in immunoblot analysis. This MAb also significantly inhibited the in vitro growth of the B. equi parasite. Preliminary studies using partially purified antigen, which was separated by high-pressure liquid chromatography and recognized by the MAb, suggested that it is a suitable antigen for enzyme-linked immunosorbent assay detection of anti-B. equi antibodies in naturally infected horse sera.

Prevalence of equine piroplasmosis in Central Mongolia.

Antigen for the indirect fluorescent antibody test (IFAT) was routinely prepared from infected erythrocytes from horses experimentally infected with Babesia equi and Babesia caballi. With the successful establishment of in vitro cultures of B. equi and B. caballi, it is now possible to employ culture-derived antigens in this test. In this study, in vitro-propagated B. equi- and B. caballi-infected erythrocytes were used as antigen in the IFAT. Various modifications to an established protocol had to be implemented to allow repeatable results. Cultures with 3-4% parasitized erythrocytes were found to be most suitable. As cross-reactions of control sera on heterologous antigen were observed at serum dilutions of up to 1/40, a reciprocal titre of 80 was considered to be positive. In positive samples, specific fluorescence of Babesia parasites and/or erythrocyte membranes was observed. Fifteen sera from Babesia-free horses from Japan all tested negative in the IFAT. One hundred and ten field-horse sera from Central Mongolia were investigated in this study. The results indicate that both B. equi and B. caballi are endemic in horses in Central Mongolia, with 88.2% and 84.5% of horses being seropositive to B. equi and B. caballi, respectively.

In vitro cultivation of Babesia equi: detection of carrier animals and isolation of parasites.

By means of an in vitro culture technique, 75 samples of horse blood were examined for Babesia equi, a causative agent of equine piroplasmosis. At the time of culture initiation, 15 samples were microscopically positive for B. equi, and this was subsequently confirmed by culture diagnosis. Sixty samples showed no parasites in Giemsa-stained thin blood smears. However, after the culturing process, parasites were found in blood smears of 36 of these samples. The sensitivity of the in vitro culture method was such that 2.5 microliters (1/40 of the usual volume used for the above-mentioned samples) of packed erythrocytes obtained from a carrier horse still yielded positive results after cultivation. Cultures were initiated from blood samples stored for up to 120 h at 8 degrees C in vacuum tubes containing EDTA as anticoagulant. These results show that the in vitro culture method is highly sensitive. It can be used to identify B. equi carrier horses, to evaluate the effects of chemotherapeutic intervention, and to isolate field strains of B. equi for further characterization.

Evaluation of a 3 ml heartwater (cowdriosis) infective blood vaccine dose.

Three milliliters of blood from the present commercially produced heartwater infective blood vaccine (Ball3 stock) was experimentally tested in sheep and cattle for infectivity and efficacy. Results obtained for this vaccine dose were statistically not different from results for the prescribed 5 ml vaccine dose.

A sero-epidemiological survey of equine piroplasmosis in the northern and eastern Cape Provinces of South Africa.

Serum samples from yearling Thoroughbred horses (n = 176) in the magisterial districts of Colesberg, Venterstad, and Wodehouse in the Northern and Eastern Cape Provinces were collected between September and November 1995 to determine the prevalence of antibodies to Babesia equi and Babesia caballi in these regions. Samples were examined for specific antibodies using the indirect fluorescent antibody test. The 95% confidence intervals for the prevalence of serum antibodies in the 3 districts combined varied from 47% to 61% for B. equi and from 26% to 40% for B. caballi. Antibody prevalence did not correlate with the known distributions of the tick vectors (Rhipicephalus evertsi evertsi and Hyalomma truncatum). Colts had a significantly higher prevalence of antibodies against B. caballi than fillies. No such difference could be determined for B. equi.

The possible role of two common three-host ticks, Rhipicephalus appendiculatus and Amblyomma hebraeum, in the transmission of bovine leukosis virus.

The possible role of Rhipicephalus appendiculatus and Amblyomma hebraeum in the mechanical and transstadial transmission of bovine leukosis virus (BLV) was investigated. BLV-free laboratory strains of R. appendiculatus and A. hebraeum nymphal ticks (n = 400) were fed on a BLV-infected and a negative control bovine. At various intervals after engorgement the ticks were homogenised and injected subcutaneously into BLV-negative sheep. Adult R. appendiculatus and A. hebraeum, which had fed as nymphs on the BLV-infected bovine, were then allowed to feed on BLV-negative sheep. A control sheep was also injected intravenously with blood from the infected bovine. Only the control sheep that received blood from the BLV-positive bovine seroconverted 9 months later. All the other surviving sheep remained serologically negative during the 13 months observation period. It is suggested that the nymphal stages of these ticks probably do not play a role in the transstadial transmission of BLV in southern Africa. The significance of these results is discussed.

Isolation of a South African vector-specific strain of Babesia canis.

A South African strain of Babesia canis parasites was isolated and shown to be vector-specific to only one of the two vectors in the region, Haemaphysalis leachi. This tick was found to transmit the parasite in its adult instar. When infected as larvae, the ticks would not transmit in the proceeding nymphal instar. The vector-specific strain was named the 'Thomas strain' after one of the dogs involved in isolating it. A survey revealed a prevalence of > 50% of this strain in four widely separated areas of the country. Rhipicephalus sanguineus, which transmits B. canis vogeli elsewhere, has not been shown to be a vector of the South African strain of B. canis.