Bovine piroplasmosis-anaplasmosis and clinical signs of tropical theileriosis in the plains of Djurdjura (north Algeria).

The study was conducted during tick activity season over a period of 5 years in the Djurdjura Plains, Algeria. A total of 299 cattle (Holstein, Montbeliard, Fleckvieh and crossbred animals) with clinical signs were included in this study. A total of 171 animals were found positive for at least one pathogen by Giemsa-stained blood smears examination Theileria annulata (136/299, 45.5%), Babesia bovis (14/299, 4.7%), B. bigemina (3/299, 1.0%) and Anaplasma marginale (12/299, 4.0%) were identified. Six animals were co-infected by T. annulata and A. marginale. Although no ticks were collected from diseased animals, clinical signs in cattle were hyperthermia (120/136, 88.3%), gluttony followed by anorexia (113/136, 83.1%), lymph node enlargement (99/136, 72.8%), anaemia (82/136, 60.3%), icterus (58/136, 42.6%) and haemoglobinuria (36/136, 26.5%). Gluttony followed by anorexia was considered highly suggestive of an incubation of tropical theileriosis as shown by a higher receptivity index (IR = 0.89-1). This clinical sign is evident in young Montbeliard and young Holstein males with anaemia (IR = 1) and icterus (IR = 0.78-0.81) which is earlier than haemoglobinuria (IR = 0.51-0.54). The incidence of T. annulata was maximum in July (n = 57), as well as B. bovis (n = 6) and A. marginale (n = 13). These results highlight the preponderance of tropical theileriosis in north-central Algeria, where gluttony followed by anorexia is probably a prodromal symptom during the incubation period of the disease.

Survey of Anaplasma phagocytophilum and Anaplasma sp. 'Omatjenne' infection in cattle in Africa with special reference to Ethiopia.

BACKGROUND: As evidence of the infection of domestic animals by Anaplasma phagocytophilum and Anaplasma sp. 'Omatjenne' is presently becoming available, understanding the epidemiological and ecological significance of infection is important to quantify the clinical and socio-economic impact of the diseases they cause. METHODS: The first aim of this study was to analyse the occurrence of A. phagocytophilum and Anaplasma sp. 'Omatjenne' in cattle samples collected from selected African countries using a polymerase chain reaction and restriction enzyme fragment length polymorphism. Secondly, this study was aimed at the molecular identification of Ehrlichia spp. and Anaplasma spp. infection in ruminants raised under different production systems in selected sites in central Ethiopia. RESULTS: In total, 695 samples from cattle in six African countries were analysed. Overall, 45 positive results were obtained for Anaplasma sp. 'Omatjenne' (6.47%) and 19 for A. phagocytophilum (2.73%). Anaplasma sp. 'Omatjenne' was detected in all countries except Tanzania while A. phagocytophilum was detected only in samples from Ethiopia. The proportion of samples tested positive for Anaplasma sp. 'Omatjenne' ranged from 1.2% in Morocco to 16% in Rwanda. The occurrence of both agents is now confirmed in African cattle. For the survey in Ethiopia a semi-nested 16S rDNA polymerase chain reaction followed by restriction fragment length polymorphism was used for the identification of Ehrlichia spp. and Anaplasma spp. in blood samples. Randomly selected samples were also analysed by pCS20 polymerase chain reaction for the detection of E. ruminantium. Positive results were obtained for E. ruminantium and five species of Anaplasma including a zoonotic species. To our knowledge, this is the first report of infection of domestic ruminants with A. phagocytophilum, A. ovis and Anaplasma sp. 'Omatjenne' in Ethiopia. CONCLUSION: The present study showed widespread occurrence of Anaplasma sp. 'Omatijenne' in African cattle and five Anaplasma species in Ethiopia.

Prevalence of bovine theileriosis in North Central region of Algeria by real-time polymerase chain reaction with a note on its distribution.

To determine the presence and distribution of bovine theileriosis in the North Central region of Algeria, 358 DNA samples and 359 blood smears were analyzed from nine provinces. Theileria DNA extracted from cattle blood was amplified by fluorescence resonance energy transfer polymerase chain reaction (FRET-PCR). Blood smears were examined for Theileria piroplasms by microscopical examination (ME) of Giemsa-stained slides. While microscopical identification revealed only 42 animals being infected with Theileria piroplasms, PCR-positive amplification using Theileria genus-specific primers was obtained from 132 Theileria spp. (P < 0.0001). Among the 132 positives, 108 animals (81.8 %) were found positive of Theileria annulata, while 24 (18.2 %) were found positive for Theileria sp. (P < 0.0001). However, melting curve analysis of these latter samples revealed the presence of two different peaks, 51.5 ± 0.5 °C corresponding to Theileria sp1 and 52.5 ± 0.5 °C for Theileria sp2. Cloning and sequencing of Theileria sp1 and Theileria sp2 using the Cox primers indicated that these species are very closely related to Theileria buffeli. There is a highly significant difference in the distribution of theileriosis between different provinces (P < 0.0001). This disparity between provinces is probably due to differences in tick contact, influenced by the subhumid bioclimatic gradient and differences in agricultural land use.

Transfection of live, tick derived sporozoites of the protozoan Apicomplexan parasite Theileria parva.

Theileria parva is an important veterinary protozoan causing the tick-borne disease East Coast fever. Transfection of Theileria parasites will facilitate the investigation of many aspects of this apicomplexan infection and its unique host-parasite interaction. The pathogen has the extraordinary capacity of transforming B and T cells into clonally dividing cancerous cell lines in a reversible way. Sequence data of the entire T. parva genome are available and in vitro infected cell lines can easily be generated, thereby eliminating the use of animals in the evaluation of the evolution of the transfected sporozoites. Here we report, for the first time, on experiments towards successful transient transfection of T. parva sporozoites, making use of a new generation transfection device. Plasmid DNA containing the strong EF-1α promoter and an Azami Green reporter gene were integrated by nucleofection into freshly purified T. parva sporozoites. Expression of Azami Green was detected with a fluorescence microscope and confirmed by counter staining with a monoclonal directed against a sporozoite protein. Despite the fact that transfection efficiencies are still low, this is the first step towards a stable infection method of T. parva parasites. In the long run, transfected parasites might become an alternative way to induce immunity without clinical signs.

Towards the PCR-based identification of Palaearctic Culicoides biting midges (Diptera: Ceratopogonidae): results from an international ring trial targeting four species of the subgenus Avaritia.

BACKGROUND: Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are biological vectors of internationally important arboviruses. To understand the role of Culicoides in the transmission of these viruses, it is essential to correctly identify the species involved. Within the western Palaearctic region, the main suspected vector species, C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, have similar wing patterns, which makes it difficult to separate and identify them correctly. METHODS: In this study, designed as an inter-laboratory ring trial with twelve partners from Europe and North Africa, we assess four PCR-based assays which are used routinely to differentiate the four species of Culicoides listed above. The assays based on mitochondrial or ribosomal DNA or microarray hybridisation were tested using aliquots of Culicoides DNA (extracted using commercial kits), crude lysates of ground specimens and whole Culicoides (265 individuals), and non-Culicoides Ceratopogonidae (13 individuals) collected from across Europe. RESULTS: A total of 800 molecular assays were implemented. The in-house assays functioned effectively, although specificity and sensitivity varied according to the molecular marker and DNA extraction method used. The Obsoletus group specificity was overall high (95-99%) while the sensitivity varied greatly (59.6-100%). DNA extraction methods impacted the sensitivity of the assays as well as the type of sample used as template for the DNA extraction. CONCLUSIONS: The results are discussed in terms of current use of species diagnostic assays and the future development of molecular tools for the rapid differentiation of cryptic Culicoides species.

Outcome of acute East African trypanosomiasis in a Polish traveller treated with pentamidine.

BACKGROUND: African trypanosomiasis is a parasitic infection sporadically imported to Europe by tourists or immigrants returning from endemic areas. We present the first and an unusual case of East African trypanosomiasis imported to Poland by a patient returning from a tourist trip to Uganda and Rwanda, which was successfully treated with pentamidine. CASE PRESENTATION: A 61-year-old Polish man was admitted to the Department because of high-grade fever and multi-organ dysfunction after a tourist trip to East Africa. He experienced a single tsetse fly bite during a safari trip to the Queen Elizabeth National Park in Uganda. On admission, his clinical status was severe, with high fever of 41ºC, preceded by chills, bleeding from the gums and oral mucosa, haemorrhages at the sites of venipuncture, numerous ecchymoses, fine-spotted skin rash, tachycardia, hepatosplenomegaly, dehydration, jaundice, dyspnoea, hypoxaemia, generalised oedema and oliguria. There was a typical non-painful trypanosomal chancre with central necrosis and peripheral erythema on his left arm. Laboratory investigations showed leucopenia, thrombocytopenia, haemolytic anaemia, hyperbilirubinaemia, hypoglycaemia, elevated creatinine and urea, high activity of aminotransferases, elevated levels of inflammatory markers, hypoproteinaemia, proteinuria, abnormal clotting and bleeding times, low fibrinogen level, metabolic acidosis, and electrolyte disturbances. A peripheral blood smear showed numerous Trypanosoma brucei trypomastigotes with a massive parasitaemia of 100,000/µl. T. brucei rhodesiense subspecies was finally identified on the basis of the characteristic serum resistance-associated gene using a polymerase chain reaction, and a seroconversion of specific immunoglobulin M and G antibodies in the peripheral blood by enzyme-linked immunosorbent assay. Serological tests for T. brucei gambiense subspecies were negative. A severe clinical course of acute rhodesiense trypanosomiasis with renal failure, respiratory distress, disseminated intravascular coagulation syndrome, haemolysis, liver insufficiency and myocarditis was confirmed. Intensive anti-parasitic and symptomatic treatment was immediately instituted, including intravenous pentamidine, plasmaphereses, oxygen therapy, blood transfusion, catecholamine administration, and fluid infusions, as well as haemostatic, hepatoprotective, anti-inflammatory, antipyretic and diuretic drugs. The final outcome was a full recovery with no late sequelae. CONCLUSION: Sleeping sickness should always be considered in the differential diagnosis of fever in people returning from safari trips to the national parks or nature reserves of sub-Saharan Africa.

Evaluation of a real-time PCR test for the detection and discrimination of theileria species in the African buffalo (Syncerus caffer).

A quantitative real-time PCR (qPCR) assay based on the cox III gene was evaluated for the simultaneous detection and discrimination of Theileria species in buffalo and cattle blood samples from South Africa and Mozambique using melting curve analysis. The results obtained were compared to those of the reverse line blot (RLB) hybridization assay for the simultaneous detection and differentiation of Theileria spp. in mixed infections, and to the 18S rRNA qPCR assay results for the specific detection of Theileria parva. Theileria parva, Theileria sp. (buffalo), Theileria taurotragi, Theileria buffeli and Theileria mutans were detected by the cox III assay. Theileria velifera was not detected from any of the samples analysed. Seventeen percent of the samples had non-species specific melting peaks and 4.5% of the samples were negative or below the detection limit of the assay. The cox III assay identified more T. parva and Theileria sp. (buffalo) positive samples than the RLB assay, and also detected more T. parva infections than the 18S assay. However, only a small number of samples were positive for the benign Theileria spp. To our knowledge T. taurotragi has never been identified from the African buffalo, its identification in some samples by the qPCR assay was unexpected. Because of these discrepancies in the results, cox III qPCR products were cloned and sequenced. Sequence analysis indicated extensive inter- and intra-species variations in the probe target regions of the cox III gene sequences of the benign Theileria spp. and therefore explains their low detection. The cox III assay is specific for the detection of T. parva infections in cattle and buffalo. Sequence data generated from this study can be used for the development of a more inclusive assay for detection and differentiation of all variants of the mildly pathogenic and benign Theileria spp. of buffalo and cattle.

Whole-genome sequencing of Theileria parva strains provides insight into parasite migration and diversification in the African continent.

The disease caused by the apicomplexan protozoan parasite Theileria parva, known as East Coast fever or Corridor disease, is one of the most serious cattle diseases in Eastern, Central, and Southern Africa. We performed whole-genome sequencing of nine T. parva strains, including one of the vaccine strains (Kiambu 5), field isolates from Zambia, Uganda, Tanzania, or Rwanda, and two buffalo-derived strains. Comparison with the reference Muguga genome sequence revealed 34 814-121 545 single nucleotide polymorphisms (SNPs) that were more abundant in buffalo-derived strains. High-resolution phylogenetic trees were constructed with selected informative SNPs that allowed the investigation of possible complex recombination events among ancestors of the extant strains. We further analysed the dN/dS ratio (non-synonymous substitutions per non-synonymous site divided by synonymous substitutions per synonymous site) for 4011 coding genes to estimate potential selective pressure. Genes under possible positive selection were identified that may, in turn, assist in the identification of immunogenic proteins or vaccine candidates. This study elucidated the phylogeny of T. parva strains based on genome-wide SNPs analysis with prediction of possible past recombination events, providing insight into the migration, diversification, and evolution of this parasite species in the African continent.

A Theileria parva isolate of low virulence infects a subpopulation of lymphocytes.

Theileria parva is a tick-transmitted protozoan parasite that infects and transforms bovine lymphocytes. We have previously shown that Theileria parva Chitongo is an isolate with a lower virulence than that of T. parva Muguga. Lower virulence appeared to be correlated with a delayed onset of the logarithmic growth phase of T. parva Chitongo-transformed peripheral blood mononuclear cells after in vitro infection. In the current study, infection experiments with WC1(+) γδ T cells revealed that only T. parva Muguga could infect these cells and that no transformed cells could be obtained with T. parva Chitongo sporozoites. Subsequent analysis of the susceptibility of different cell lines and purified populations of lymphocytes to infection and transformation by both isolates showed that T. parva Muguga sporozoites could attach to and infect CD4(+), CD8(+), and WC1(+) T lymphocytes, but T. parva Chitongo sporozoites were observed to bind only to the CD8(+) T cell population. Flow cytometry analysis of established, transformed clones confirmed this bias in target cells. T. parva Muguga-transformed clones consisted of different cell surface phenotypes, suggesting that they were derived from either host CD4(+), CD8(+), or WC1(+) T cells. In contrast, all in vitro and in vivo T. parva Chitongo-transformed clones expressed CD8 but not CD4 or WC1, suggesting that the T. parva Chitongo-transformed target cells were exclusively infected CD8(+) lymphocytes. Thus, a role of cell tropism in virulence is likely. Since the adhesion molecule p67 is 100% identical between the two strains, a second, high-affinity adhesin that determines target cell specificity appears to exist.

Analyses of genes encoding Theileria parva p104 and polymorphic immunodominant molecule (PIM) reveal evidence of the presence of cattle-type alleles in the South African T. parva population.

Restriction fragment length polymorphism analysis of PCR products (PCR-RFLP) and sequencing of the variable region of the p104 and PIM genes was performed on samples obtained from South African T. parva parasites originating from cattle on farms with suspected theileriosis and from buffalo. p104 and PIM PCR-RFLP profiles similar to those of the T. parva Muguga stock, an isolate that causes ECF in Kenya, were obtained from three of seven cattle samples collected on a farm near Ladysmith in KwaZulu-Natal Province. Amino acid sequences of the p104 and PIM genes from two of these samples were almost identical to the T. parva Muguga p104 and PIM sequences. This result supports findings from a recent p67 study in which p67 alleles similar to those of the T. parva Muguga stock were identified from the same samples. While these results suggest the presence of a cattle-derived T. parva parasite, reports of cattle-to-cattle transmission could not be substantiated and ECF was not diagnosed on this farm. Although extensive diversity of p104 and PIM gene sequences from South African T. parva isolates was demonstrated, no sequences identical to known cattle-type p104 and PIM alleles were identified from any of the buffalo T. parva samples analyzed. 'Mixed' PIM alleles containing both cattle- and buffalo-type amino acid motifs were identified for the first time, and there appeared to be selection of cattle-type and 'mixed'-type PIM sequences in the cattle samples examined.

Old focus of cysticercosis in a senegalese village revisited after half a century.

The objective of this epidemiological study was to determine whether cysticercosis and especially neurocysticercosis is endemic in Soutou village about half a century after the 1962 outbreak. This study was carried out from September 2009 to February 2010. It involved a questionnaire administration, serology, treatment, coproscopy and neuro-imaging. Four hundred and three serum samples were collected from the village people, which covered 94% of the village population. By using a parallel combination of the antigen-detection ELISA and the enzyme-linked immunoelectrotransfer blot (EITB) a cysticercosis seroprevalence of 11.9% (95% CI: 8.9-15.4%) was found. Cerebral CT-scans showed that 23.3% (10/43) of the seropositives were affected by neurocysticercosis. Four out of these 43 (9.3%) were tapeworm carriers. Seropositivity was significantly associated to older age groups (41-60 years old; p=0.001 and 61-91 years old; p=0.028) and absence of a household toilet (p=0.001). It can be concluded that Soutou village is an active focus of Taenia solium cysticercosis about 50 years after the first reported epidemic outbreak.

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.

Development and validation of a PCR-RFLP test to identify African Rhipicephalus (Boophilus) ticks.

The cattle tick Rhipicephalus (Boophilus) microplus has recently invaded West Africa and caused anxiety amongst farmers in Ivory Coast, as livestock production was severely affected. The introduction of this tick species has remained unnoticed for several years, as all the members of this genus are very similar in appearance. To overcome the cumbersome morphological identification of the four closely related R. (Boophilus) spp. in the region, a PCR-RFLP test, based on a part of the second internal transcribed spacer ribosomal DNA (ITS2), was developed. The molecular tool was successfully validated with a large number of ticks recently collected from West Africa and that were identified both morphologically and genetically. The tool developed is simple, fast, reliable and reproducible; hence it can be routinely applied for species identification.

Folding properties of the hepatitis B core as a carrier protein for vaccination research.

The hepatitis B core (HBc) protein has been used successfully in numerous experiments as a carrier for heterologous peptides. Folding and capsid formation of the chimeric proteins is not always achieved easily. In silico analyses were performed to provide further comprehension of the feasibility for predicting successful capsid formation. In contrast to previous work, we show that common in silico predictions do not ensure assembly into particles. We included new considerations regarding capsid formation of HBc fusion proteins. Not only the primary sequence and the length of the inserts seem important, also the rigidity, the distance between the N and the C-terminus and the presence of cysteines, which could form disulphide bonds, could influence proper capsid formation. Furthermore, new conformational insights were formulated when linkers were added to create extra flexibility of the chimeric particles. Different hypotheses were suggested to clarify the obtained results. To this extent, the addition of glycine-rich linkers could lower high rigidity of the insert, removal of the strain of the core protein or ease interaction between the HBc and the insert. Finally, we observed specific changes in capsid formation properties when longer linkers were used. These findings have not been reported before in this and other virus-like particle carriers. In this study, we also propose a new high-yield purification protocol for fusion proteins to be used in vaccination experiments with the carrier protein or in comparative studies of particulate or non-particulate HBc fusion proteins.

Four p67 alleles identified in South African Theileria parva field samples.

Previous studies characterizing the Theileria parva p67 gene in East Africa revealed two alleles. Cattle-derived isolates associated with East Coast fever (ECF) have a 129bp deletion in the central region of the p67 gene (allele 1), compared to buffalo-derived isolates with no deletion (allele 2). In South Africa, Corridor disease outbreaks occur if there is contact between infected buffalo and susceptible cattle in the presence of vector ticks. Although ECF was introduced into South Africa in the early 20th century, it has been eradicated and it is thought that there has been no cattle to cattle transmission of T. parva since. The variable region of the p67 gene was amplified and the gene sequences analyzed to characterize South African T. parva parasites that occur in buffalo, in cattle from farms where Corridor disease outbreaks were diagnosed and in experimentally infected cattle. Four p67 alleles were identified, including alleles 1 and 2 previously detected in East African cattle and buffalo, respectively, as well as two novel alleles, one with a different 174bp deletion (allele 3), the other with a similar sequence to allele 3 but with no deletion (allele 4). Sequence variants of allele 1 were obtained from field samples originating from both cattle and buffalo. Allele 1 was also obtained from a bovine that tested T. parva positive from a farm near Ladysmith in the KwaZulu-Natal Province. East Coast fever was not diagnosed on this farm, but the p67 sequence was identical to that of T. parva Muguga, an isolate that causes ECF in Kenya. Variants of allele 2 were obtained from all T. parva samples from both buffalo and cattle, except Lad 10 and Zam 5. Phylogenetic analysis revealed that alleles 3 and 4 are monophyletic and diverged early from the other alleles. These novel alleles were not identified from South African field samples collected from cattle; however allele 3, with a p67 sequence identical to those obtained in South African field samples from buffalo, was obtained from a Zambian field isolate of a naturally infected bovine diagnosed with ECF. The p67 genetic profiles appear to be more complex than previously thought and cannot be used to distinguish between cattle- and buffalo-derived T. parva isolates in South Africa. The significance of the different p67 alleles, particularly the novel variants, in the epidemiology of theileriosis in South Africa still needs to be determined.

Specific detection and identification of African trypanosomes in bovine peripheral blood by means of a PCR-ELISA assay.

The aim of the present study was to develop a PCR-ELISA assay for the detection and differentiation of the main African pathogen trypanosomal species present in peripheral blood of cattle. The proposed methodology allows to specifically differentiate Trypanosoma congolense, Trypanosoma vivax and the subgenus Trypanozoon, by means of a sensitive universal PCR amplifying trypanosome DNA followed by an ELISA-based hybridization with three highly specific probes. The semi-nested PCR had a sensitivity of 15 fg, 15 fg, and 0.15 fg of DNA from T. vivax, T. congolense, and Trypanosoma brucei brucei, respectively that is sufficient to detect parasites in blood during the chronic phase of the disease. Biotinylated second round asymmetric PCR amplification products were used in an ELISA set up using three species-specific probes for the diagnosis of T. congolense (type Riverine, Kilifi or Savannah), T. vivax and T. brucei brucei. A factor O.D. of 0.082 was determined on blood samples from bovines (n=18) from a non-endemic area in Africa. In a pilot study of blood samples of naturally and experimentally Trypanosoma infected cattle previously characterized by PCR-RFLP (n=42), a high rate of concordance (93.3%) was found between PCR-RFLP and PCR-ELISA. There is a good ratio between positive and negative O.D. values (3.00 vs. 0.1) and the technique can also be used to distinguish mixed infections.

Development and evaluation of a real-time polymerase chain reaction test for the detection of Theileria parva infections in Cape buffalo (Syncerus caffer) and cattle.

Corridor disease, caused by the tick-borne protozoan parasite Theileria parva, is a controlled disease in South Africa. The Cape buffalo is the reservoir host and uninfected buffalo have become sought-after by the game industry in South Africa, particularly for introduction into Corridor disease-free areas. A real-time polymerase chain reaction (PCR) test for detection of T. parva DNA in buffalo and cattle was developed to improve the sensitivity and specificity of the official diagnostic test package in South Africa. Oligonucleotide primers and hybridization probes were designed based on the 18S ribosomal RNA (rRNA) gene. Amplification of control DNA using Theileria genus-specific primers resulted in detection of T. taurotragi and T. annulata, in addition to T. parva. A T. parva-specific forward primer was designed which eliminated amplification of all other Theileria species, except for Theileria sp. (buffalo); however only the T. parva product was detected by the T. parva-specific hybridization probe set. The real-time PCR assay requires less time to perform, is more sensitive than the other molecular assays previously used in T. parva diagnostics and can reliably detect the parasite in carrier animals with a piroplasm parasitaemia as low as 8.79 x 10(-4)%.