Genetic diversity in Babesia bovis from southern Africa and estimation of B. bovis infection levels in cattle using an optimised quantitative PCR assay.

Babesia bovis is a causal agent of bovine babesiosis, a disease which leads to mortality and morbidity and impacts the cattle industry worldwide. We amplified, cloned and sequenced the B. bovis merozoite surface antigen-2b (msa-2b) gene (∼940 bp) and the near full-length 18S rRNA gene (∼1600 bp) from cattle samples from South Africa and Mozambique to determine sequence variation between B. bovis parasites in the region. A TaqMan quantitative real-time PCR (qPCR) assay (18S rRNA gene) was optimised for the detection of B. bovis and estimation of parasitaemia in field samples from cattle from southern Africa. Phylogenetic analysis grouped the Msa-2b sequences in six clades and these were 59.7 to 99.6% identical to reference sequences. Sequence variation amongst B. bovis 18S rRNA sequences was found at 2 to 36 positions, and the sequences were 97 to 99% identical to published sequences. Mismatches between the B. bovis 18S rRNA sequences and a previously published qPCR forward primer (BoF) were observed; therefore, we developed a new forward primer (BoF2), and optimised the qPCR assay. Six 10-fold dilution series of B. bovis infected erythrocytes (2 × 108 to 2 × 103 infected red blood cells [iRBC]/ml) were analysed in triplicate in each of six separate qPCR runs, to determine the efficiency of the assay. The qPCR assay amplified the B. bovis 18S rRNA gene with 92.0 to 94.9% efficiency. The detection limit of the qPCR assay was approximately 6 iRBCs/µl. The performance of the optimised assay to diagnose B. bovis in field samples was assessed by testing DNA from 222 field samples of cattle from South Africa and Mozambique using three methods: the optimised qPCR assay, the reverse line blot (RLB) hybridisation assay, and the previously published qPCR assay. The detection rate of B. bovis using the optimised qPCR assay (31.1%, 69/222) was significantly higher (p<0.001) than both that using RLB (20.7%, 46/222) and the previously published qPCR assay (5.4%; 12/222). The B. bovis parasitaemia in samples from infected cattle ranged from 6 iRBCs/µl to 101,852 iRBCs/µl of blood. Our study revealed marked sequence variation between B. bovis parasites from southern Africa. The optimised qPCR assay will be useful in epidemiological studies and clinical diagnosis of B. bovis in southern Africa, and can be used to determine parasitaemia and potential carrier status in cattle populations, which is essential in the control of babesiosis.

Molecular detection and characterisation of protozoan and rickettsial pathogens in ticks from cattle in the pastoral area of Karamoja, Uganda.

Ticks and tick-borne diseases (TBDs) significantly affect cattle production and the livelihoods of communities in pastoralist areas. Data on protozoan and rickettsial pathogens in ticks infesting cattle in Uganda is scanty; while it is an indicator of the likelihood of disease transmission and occurrence. A cross-sectional study was conducted amongst cattle in the Karamoja Region, northeastern Uganda, from July through September 2017, to determine the tick species diversity, identify protozoan and rickettsial pathogens in the ticks, and characterise pathogenic species by sequence and phylogenetic analyses. About 50 % of the ticks detected from each predilection site on each animal were collected from 100 purposively-selected cattle from 20 randomly-selected herds. Twelve tick species belonging to the genera Amblyomma, Rhipicephalus and Hyalomma were identified, the most abundant being Amblyomma lepidum (93.9 %), followed by Amblyomma variegatum (2.0 %) and Rhipicephalus evertsi evertsi (1.0 %). Tick species that have not been reported in recent studies amongst cattle in Uganda were found, namely Rhipicephalus pravus, Rhipicephalus praetextatus and Rhipicephalus turanicus. The ticks were grouped into 40 pools, by species and location, and the reverse line blot (RLB) hybridisation assay was used to detect pathogens from the ticks. The most frequently detected tick-borne parasites were Theileria mutans, Theileria velifera and Theileria parva, each observed in 25 % (10/40) of the tick pools. Tick-borne pathogens, namely Babesia rossi, Babesia microti and Theileria sp. (sable) that are not common to, or not known to infect, cattle were identified from ticks. The gene encoding Ehrlichia ruminantium pCS20 region, the Ehrlichia and Anaplasma 16S rRNA gene, and T. parva p67 sporozoite antigen gene were amplified, cloned and sequenced. Seven novel E. ruminantium pCS20 variants were identified, and these grouped into two separate clusters with sequences from other parts of Africa and Asia. The T. parva p67 sequences were of the allele type 1, and parasites possessing this allele type are commonly associated with East Coast fever in eastern Africa. Analysis of the Ehrlichia and Anaplasma 16S rRNA gene sequences showed that they were closely related to Rickettsia africae and to a new Ehrlichia species variant recently found in China. Our R. africae 16S rRNA sequences grouped with R. africae isolates from Nigeria, Egypt and Benin. The information on tick species diversity and pathogens in the various tick species provides an indicator of potential transmission amongst cattle populations, and to humans, and can be useful to estimate disease risk and in control strategies.

Babesia bicornis, Theileria bicornis and Theileria equi in metapopulations of two black rhinoceros (Diceros bicornis) subspecies in South Africa and their potential impact on conservation.

The two black rhinoceros subspecies (Diceros bicornis bicornis and D. b. minor) in South African conservation areas are managed as separate metapopulations. Since infection with Babesia bicornis can be fatal in black rhinoceroses, occurrence of this and other piroplasms in the two metapopulations was determined to assess possible risk. Blood specimens were collected from 156 black rhinoceroses: 80 from D. b. bicornis and 76 from D. b. minor. DNA was extracted; the V4 hypervariable region of the parasite 18S rRNA gene was amplified and subjected to the Reverse Line Blot (RLB) hybridization assay. There was a significant difference in occurrence of piroplasms: 18/80 (23%) in D. b. bicornis and 39/76 (51%) in D. b. minor. Theileria bicornis occurred in significantly more of the D. b. minor population (36/76; 47%) than the D. b. bicornis population (1/80; 1%); with B. bicornis the difference was not significant: D. b. bicornis 5/80 (6%) and D. b. minor 9/76 (11%). Three individuals were infected with Theileria equi. Results were confirmed using molecular characterization of the near full-length parasite 18S rRNA gene of 13 selected specimens. We identified four (Tb1, Tb2, Tb3 and Tb4) 18S rDNA sequence types for T. bicornis, two for B. bicornis (Bb1 and Bb2) and one for T. equi (Teq1). We furthermore identified T. bicornis haplotypes H1, H3 and H4 in 10 rhinoceroses; H3 was the most common haplotype identified. Rhinoceroses inhabiting more arid areas are apparently free of T. bicornis and B. bicornis, probably due to the absence or scarcity of vectors. When individuals are relocated for metapopulation management purposes, appropriate prophylactic action should be taken to minimise the risk of babesiosis, which could be fatal.

Improved detection of Babesia bigemina from various geographical areas in Africa using quantitative PCR and reverse line blot hybridisation.

Babesia bigemina is one of the aetiological agents of bovine babesiosis, which causes economic losses through mortality, loss of production and control costs. Effective means of detecting and quantifying B. bigemina in cattle populations is therefore important to inform control approaches. In order to examine the parasite genetic diversity in African countries, B. bigemina 18S rRNA genes from cattle from South Africa, Uganda and Angola were sequenced. The 25 distinct B. bigemina 18S rRNA gene sequences obtained in this study showed 99 to 100% identity with previously published sequences of strains from African and other continents. The sequences of the previously published B. bigemina 18S rRNA gene-specific quantitative PCR (qPCR) primers and probe, developed based on American and Asian strains, were conserved in the African B. bigemina sequences. The qPCR assay was evaluated using 10-fold and 2-fold serial dilutions of B. bigemina-infected erythrocytes to determine the efficiency and analytical sensitivity. The qPCR assay had an efficiency of 98.14 ± 1.71%, and the limit of detection was approximately 1.5 infected red blood cells (iRBCs) per microlitre (µl) of blood. The detection rate of B. bigemina from duplicates of field-collected blood samples from cattle from South Africa, Mozambique and Angola was 37% (30/81), 12% (6/49) and 50% (38/76), respectively. Reverse line blot hybridisation (RLB) results obtained from the same samples in previous studies, using a previously published B. bigemina-specific probe, detected the parasite DNA in only 1.5% (3/206) of the samples. A new B. bigemina-specific RLB oligonucleotide probe was designed in the hypervariable V4 region of the 18S rRNA gene. Screening of field blood samples from cattle showed that the new probe was specific, and its frequency of detection of B. bigemina was three times higher than the previously published probe. The qPCR assay and the newly developed B. bigemina-specific RLB probe provide good tools for epidemiological studies, which are essential in the control of bovine babesiosis.

A Comparison Between Manual Count, Flow Cytometry and Quantitative Real-Time Polymerase Chain Reaction as a Means of Determining Babesia rossi Parasitaemia in Naturally Infected Dogs.

PURPOSE: Light microscopic manual count is the current gold standard for parasite quantification. The ability to determine parasite density in whole blood is crucial to understanding disease pathogenesis and finding a suitable automated method of Babesia rossi parasite quantification would facilitate higher throughput and provide results that are more objective. This study investigated both peripheral capillary and central venous whole blood to estimate the correlations between light microscopy, flow cytometry and quantitative real-time polymerase chain reaction (qPCR). METHODS: Peripheral capillary and central venous blood were sampled from 40 naturally B. rossi-infected dogs and 10 healthy control dogs. Samples were analysed by reverse line blot hybridization assay to confirm a mono-B. rossi infection. Capillary blood parasite density was detected using light microscopic manual counting and venous blood parasitaemia detected by manual counts, flow cytometry and qPCR. RESULTS: A significant correlation was found between the venous manual counts and flow cytometry (rs = 0.465; P < 0.001), as well as qPCR (rs = - 0.500; P < 0.001). A significant correlation was also observed between the capillary manual counts compared to venous manual counts (rs = 0.793; P < 0.001), flow cytometry (rs = 0.399; P = 0.004), and qPCR (rs = - 0.526; P < 0.001). CONCLUSIONS: The study results suggest that qPCR is of value as an alternative to the gold standard manual count for detecting B. rossi parasitaemia in canine whole blood and that flow cytometry may be useful with further refinement of issues such as background fluorescence and the influence of reticulocytes.

Development and validation of a multiplex, real-time PCR assay for Babesia rossi and Babesia vogeli.

Canine babesiosis is caused by tick-transmitted intraerythrocytic protozoan parasites occurring worldwide. In southern Africa, babesiosis is caused by Babesia rossi and B. vogeli and is one of the most common and important infectious diseases affecting dogs. There is no reliable, rapid and sensitive method for the detection of these parasites, especially when parasitaemia is low. The aim of this study was to develop a sensitive and specific multiplex TaqMan® MGB PCR assay for the diagnosis of canine babesiosis infections occurring in southern Africa, and to discriminate between Babesia rossi and B. vogeli. The fitness of purpose of the assay was to confirm diagnosis of suspect or clinical cases, and estimate prevalence of infection for research purposes. A total of 648 published sequences were used to design the assay. A set of group-specific canine Babesia spp. primers were designed to amplify a 117 nucleotide region of the 18S rRNA gene of all canine Babesia spp. Species-specific TaqMan® MGB probes were developed for B. rossi, B. vogeli, B. canis and B. gibsoni, but analytical validation was only performed for B. rossi and B. vogeli as a multiplex assay. The assay had a broad dynamic range and amplified B. rossi and B. vogeli efficiently (98.6% and 94.7% respectively). The assay was sensitive, with a 95% LOD of 10-2.67% parasitized erythrocytes (PE) for B. rossi and 10-2.03% PE for B. vogeli, and specific, with no cross reaction between B. rossi and B. vogeli and no detection of other haemoparasites that infect dogs, such as Ehrlichia canis and Anaplasma platys. Consistent repeatability within and between PCR runs was shown. This assay will be able to accurately and rapidly confirm babesiosis in canines and allow for treatment to be administered in the early stages of the disease, speeding up the recovery time in affected dogs.

Occurrence of tick-borne haemoparasites in cattle in the Mungwi District, Northern Province, Zambia.

Little is known about the occurrence of haemoparasites in cattle in communal grazing areas of Mungwi District of Northern Province, Zambia. Clinical signs and post mortem lesions are pathognomonic of mixed tick-borne infections especially babesiosis, anaplasmosis and East Coast fever. The main objective of this study was to screen selected communal herds of cattle for tick-borne haemoparasites, and identify the tick vectors associated with the high cattle mortalities due to suspected tick-borne diseases in the local breeds of cattle grazing along the banks of the Chambeshi River in Mungwi District, Northern Province, Zambia. A total of 299 cattle blood samples were collected from July to September 2010 from Kapamba (n = 50), Chifulo (n = 102), Chisanga (n = 38), Kowa (n = 95) and Mungwi central (n = 14) in the Mungwi District. A total of 5288 ticks were also collected from the sampled cattle from April to July 2011. DNA was extracted from the cattle blood and the hypervariable region of the parasite small subunit rRNA gene was amplified and subjected to the reverse line blot (RLB) hybridization assay. The results of the RLB assay revealed the presence of tick-borne haemoparasites in 259 (86.6%) cattle blood samples occurring either as single (11.0%) or mixed (75.6%) infections. The most prevalent species present were the benign Theileria mutans (54.5%) and T. velifera (51.5%). Anaplasma marginale (25.7%), Babesia bovis (7.7%) and B. bigemina (3.3%) DNA were also detected in the samples. Only one sample (from Kapamba) tested positive for the presence of T. parva. This was an unexpected finding; also because the tick vector, Rhipicephalus appendiculatus, was identified on animals from Kowa (14.0%), Chisanga (8.5%), Chifulo (6.0%) and Kapamba (1.4%). One sample (from Kapamba) tested positive for the presence of Ehrlichia ruminantium even though Amblyomma variegatum ticks were identified from 52.9% of the sampled animals from all study areas. There was significant positive association between T. mutans and T. velifera (p < 0.001) infections, and between A. marginale and B. bovis (p = 0.005). The presence of R. microplus tick vectors on cattle was significantly associated with B. bovis (odds ratio, OR = 28.4, p < 0.001) and A. marginale (OR = 42.0, p < 0.001) infections, while A. variegatum presence was significantly associated with T. mutans (OR = 213.0, p < 0.001) and T. velifera (OR = 459.0, p < 0.001) infections. Rhipicephalus decoloratus was significantly associated with B. bigemina (OR = 21.6, p = 0.004) and A. marginale (OR = 28.5, p < 0.001). Multivariable analysis showed a significant association between location and tick-borne pathogen status for A. marginale (p < 0.001), T. mutans (p = 0.004), T. velifera (p = 0.003) and T. taurotragi (p = 0.005). The results of our study suggest that the cause of cattle mortalities in Mungwi during the winter outbreaks is mainly due to A. marginale, B. bovis and B. bigemina infections. This was confirmed by the clinical manifestation of the disease in the affected cattle and the tick species identified on the animals. The relatively low prevalence of T. parva, B. bigemina, B. bovis and E. ruminantium could indicate the existence of endemic instability with a pool of susceptible cattle and the occurrence of disease outbreaks.

Within guild co-infections influence parasite community membership: a longitudinal study in African Buffalo.

Experimental studies in laboratory settings have demonstrated a critical role of parasite interactions in shaping parasite communities. The sum of these interactions can produce diverse effects on individual hosts as well as influence disease emergence and persistence at the population level. A predictive framework for the effects of parasite interactions in the wild remains elusive, largely because of limited longitudinal or experimental data on parasite communities of free-ranging hosts. This 4-year study followed a community of haemoparasites in free-ranging African buffalo (Syncerus caffer). We detected infection by 11 haemoparasite species using PCR-based diagnostic techniques, and analyzed drivers of infection patterns using generalized linear mixed models to understand the role of host characteristics and season on infection likelihood. We tested for (i) effects of co-infection by other haemoparasites (within guild) and (ii) effects of parasites infecting different tissue types (across guild). We found that within guild co-infections were the strongest predictors of haemoparasite infections in the buffalo; but that seasonal and host characteristics also had important effects. In contrast, the evidence for across-guild effects of parasites utilizing different tissue on haemoparasite infection was weak. These results provide a nuanced view of the role of co-infections in determining haemoparasite infection patterns in free living mammalian hosts. Our findings suggest a role for interactions among parasites infecting a single tissue type in determining infection patterns.

The epidemiology of tick-borne haemoparasites as determined by the reverse line blot hybridization assay in an intensively studied cohort of calves in western Kenya.

The development of sensitive surveillance technologies using PCR-based detection of microbial DNA, such as the reverse line blot assay, can facilitate the gathering of epidemiological information on tick-borne diseases, which continue to hamper the productivity of livestock in many parts of Africa and elsewhere. We have employed a reverse line blot assay to detect the prevalence of tick-borne parasites in an intensively studied cohort of indigenous calves in western Kenya. The calves were recruited close to birth and monitored for the presence of infectious disease for up to 51 weeks. The final visit samples from 453 calves which survived for the study period were analyzed by RLB. The results indicated high prevalences of Theileria mutans (71.6%), T. velifera (62.8%), Anaplasma sp. Omatjenne (42.7%), A. bovis (39.9%), Theileria sp. (sable) (32.7%), T. parva (12.9%) and T. taurotragi (8.5%), with minor occurrences of eight other haemoparasites. The unexpectedly low prevalence of the pathogenic species Ehrlichia ruminantium was confirmed by a species-specific PCR targeting the pCS20 gene region. Coinfection analyses of the seven most prevalent haemoparasites indicated that they were present as coinfections in over 90% of the cases. The analyses revealed significant associations between several of the Theileria parasites, in particular T. velifera with Theileria sp. sable and T. mutans, and T. parva with T. taurotragi. There was very little coinfection of the two most common Anaplasma species, although they were commonly detected as coinfections with the Theileria parasites. The comparison of reverse line blot and serological results for four haemoparasites (T. parva, T. mutans, A. marginale and B. bigemina) indicated that, except for the mostly benign T. mutans, indigenous cattle seem capable of clearing infections of the three other, pathogenic parasites to below detectable levels. Although the study site was located across four agroecological zones, there was little restriction of the parasites to particular zones.

Tick-borne haemoparasites in African buffalo (Syncerus caffer) from two wildlife areas in Northern Botswana.

BACKGROUND: The African buffalo (Syncerus caffer) is a host for many pathogens known to cause economically important diseases and is often considered an important reservoir for livestock diseases. Theileriosis, heartwater, babesiosis and anaplasmosis are considered the most important tick-borne diseases of livestock in sub-Saharan Africa, resulting in extensive economic losses to livestock farmers in endemic areas. Information on the distribution of tick-borne diseases and ticks is scarce in Northern Botswana. Nevertheless, this data is necessary for targeting surveillance and control measures in livestock production at national level. METHODS: In order to address this gap, we analyzed 120 blood samples from buffalo herds for the presence of common tick-borne haemoparasites causing disease in livestock, collected in two of the main wildlife areas of Northern Botswana: the Chobe National Park (CNP, n=64) and the Okavango Delta (OD, n=56). RESULTS: Analysis of the reverse line blot (RLB) hybridization assay results revealed the presence of Theileria, Babesia, Anaplasma and Ehrlichia species, either as single or mixed infections. Among the Theileria spp. present, T. parva (60%) and T. mutans (37%) were the most prevalent. Other species of interest were Anaplasma marginale subsp. centrale (30%), A. marginale (20%), Babesia occultans (23%) and Ehrlichia ruminantium (6%). The indirect fluorescent antibody test (IFAT) indicated 74% of samples to be positive for the presence of T. parva antibodies. Quantitative real-time PCR (qPCR) detected the highest level of animals infected with T. parva (81% of the samples). The level of agreement between the tests for detection of T. parva positive animals was higher between qPCR and IFAT (kappa=0.56), than between qPCR and RLB (kappa=0.26) or the latter and IFAT (kappa=0.15). CONCLUSIONS: This is the first report of tick-borne haemoparasites in African buffalo from northern Botswana, where animals from the CNP showed higher levels of infection than those from OD. Considering the absence of fences separating wildlife and livestock in the CNP and the higher levels of some parasite species in buffalo from that area, surveillance of tick-borne diseases in livestock at the interface in the CNP should be prioritized.

Occurrence of tick-transmitted pathogens in dogs in Jos, Plateau State, Nigeria.

BACKGROUND: Canine babesiosis caused by Babesia rossi, transmitted by Haemaphysalis elliptica in South Africa, has also been reported from Nigeria. Although H. leachi (sensu lato) is widespread in sub-Saharan Africa, published literature on the occurrence of canine babesiosis is meagre. It has been postulated that the genotype of Babesia rossi Erythrocyte Membrane Antigen 1 (BrEMA1) may be linked to virulence of the specific isolate. The primary objective of this study was to detect and characterise tick-borne pathogens in dogs presented to a veterinary hospital using molecular techniques. In B. rossi-positive specimens, we aimed to determine whether the BrEMA1 gene occurred and to compare genotypes with those found in other isolates. Lastly, we wished to identify the tick species that were recovered from the sampled dogs. METHODS: Blood specimens (n = 100) were collected during January to March 2010 from domestic dogs presented at an animal hospital in Jos, Plateau State, Nigeria. They were screened for the presence of Babesia/Theileria and Ehrlichia/Anaplasma genomic DNA using PCR and Reverse Line Blot (RLB) assays. Positive B. rossi specimens were tested for the presence of the BrEMA1gene using an RT-PCR. In addition, ticks were collected from dogs found to be infested during sampling. RESULTS: On RLB, 72 (72%) of the specimens were positive for one or more haemoparasites. Of the positive specimens, 38 (53%) were infected with B. rossi; 9 (13%) with Theileria sp. (sable); 5 (7%) with either Ehrlichia canis or Anaplasma sp. Omatjenne, respectively; 3 (4%) with Theileria equi; and 1 (1%) with B. vogeli and E. ruminantium, respectively. Co-infections were detected in 13 (18%) of the specimens. Results of RT-PCR screening for the BrEMA1 gene were negative. A total of 146 ticks belonging to 8 species were collected and identified: Rhipicephalus sanguineus 107 (73%), Haemaphysalis leachi (sensu stricto) 27 (18%), R. turanicus 3 (2%), and Amblyomma variegatum, H. elliptica, R. lunulatus, R. muhsamae and R. senegalensis 1 (1%), respectively. CONCLUSIONS: Up to 8 tick-borne pathogens possibly occur in the dog population at Jos, with B. rossi being the most prevalent. The absence of the BrEMA1 gene suggests that B. rossi occurring in that area may be less virulent than South African isolates.

Theileriosis in six dogs in South Africa and its potential clinical significance.

Theileriosis is a tick-borne disease caused by a piroplasma of the genus Theileria that can causeanaemia and thrombocytopenia. Its clinical importance for dogs' remains poorly understood,as only some develop clinical signs. In this study, physical and laboratory findings, treatment and outcomes of six client-owned diseased dogs presented at the Onderstepoort Veterinary Academic Hospital are described retrospectively. In the dogs, Theileria species (n = 4) and Theileria equi (n = 2) were detected by a polymerase chain reaction (PCR)-reverse blothybridisation assay in blood samples, whilst PCR for Babesia, Anaplasma and Ehrlichia were negative. The most common physical findings were pale mucous membranes (five out of six dogs), bleeding tendencies (five out of six dogs) and lethargy (three out of six dogs). All dogs were thrombocytopenic [median 59.5 x 10(9)/L (range 13-199)] and five out of six dogs were anaemic [median haematocrit 18% (range 5-32)]. Bone marrow core biopsies performed in two dogs showed myelofibrosis. Theileriosis was treated with imidocarb dipropionate and the suspected secondary immune-mediated haematological disorders with prednisolone and azathioprine. Five dogs achieved clinical cure and post-treatment PCR performed in three out of five dogs confirmed absence of circulating parasitaemia. An immune-mediated response to Theileria species is thought to result in anaemia and/or thrombocytopenia in diseased dogs with theileriosis. A bleeding tendency, most likely secondary to thrombocytopenia and/or thrombocytopathy, was the most significant clinical finding in these cases. The link between thrombocytopenia, anaemia and myelofibrosis in theileriosis requires further investigation and theileriosis should be considered a differential diagnosis for dogs presenting with anaemia and/or thrombocytopenia in endemic tick-borne disease areas.

Evidence of vertical transmission of lumpy skin disease virus in Rhipicephalus decoloratus ticks.

Lumpy skin disease (LSD) is an economically important acute or sub-acute disease of cattle that occurs across Africa and in the Middle East. The aim of this study was to assess whether Rhipicephalus decoloratus ticks were able to transmit lumpy skin disease virus (LSDV) transovarially. Uninfected, laboratory-bred R. decoloratus larvae were placed to feed on experimentally infected "donor" cattle. After completion of the life cycle on donor animals, fully engorged adult female ticks were harvested and allowed to lay eggs. Larvae that hatched from these eggs were then transferred to feed on uninfected "recipient" cattle. The latter became viraemic and showed mild clinical disease with characteristic skin lesions and markedly enlarged precrural and subscapular lymph nodes. This is the first report of transovarial transmission of poxviruses by R. decoloratus ticks, and the importance of this mode of transmission in the spread of LSDV in endemic settings requires further investigation.

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.

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.

Identification of novel Babesia and Theileria species in South African giraffe (Giraffa camelopardalis, Linnaeus, 1758) and roan antelope (Hippotragus equinus, Desmarest 1804).

Blood specimens were received from five cases in which young adult giraffe, from different geographic origins in South Africa, showed sudden onset of disease and subsequently died. Additional specimens from two translocated giraffe, as well as one specimen from a roan antelope, were also included in the study. Blood slides from some of these animals showed the presence of piroplasms. DNA was extracted; the V4 hypervariable region of the 18S rRNA gene amplified and analyzed using the Reverse Line Blot (RLB) hybridization assay. PCR products failed to hybridize with any of the Babesia or Theileria species-specific probes, and only hybridized with the Babesia/Theileria genus-specific probe suggesting the presence of a novel species or variant of a species. Full-length 18S rDNA was amplified, cloned and the recombinants were sequenced. 18S rRNA gene sequence similarity analysis revealed the presence of novel piroplasm species in both healthy giraffe and a roan antelope and clinically sick or dead giraffe. Phylogenetic analysis grouped five of these organisms in the Babesia sensu stricto clade and three in the Theileria sensu stricto clade. Although parasites were observed in blood smears, there is no direct evidence that piroplasmosis caused the death of five giraffe, although it certainly seems to be likely.

Functional CD1d and/or NKT cell invariant chain transcript in horse, pig, African elephant and guinea pig, but not in ruminants.

CD1d-restricted invariant natural killer T cells (NKT cells) have been well characterized in humans and mice, but it is unknown whether they are present in other species. Here we describe the invariant TCR alpha chain and the full length CD1d transcript of pig and horse. Molecular modeling predicts that porcine (po) invariant TCR alpha chain/poCD1d/alpha-GalCer and equine (eq) invariant TCR alpha chain/eqCD1d/alpha-GalCer form complexes that are highly homologous to the human complex. Since a prerequisite for the presence of NKT cells is the expression of CD1d protein, we performed searches for CD1D genes and CD1d transcripts in multiple species. Previously, cattle and guinea pig have been suggested to lack CD1D genes. The CD1D genes of European taurine cattle (Bos taurus) are known to be pseudogenes because of disrupting mutations in the start codon and in the donor splice site of the first intron. Here we show that the same mutations are found in six other ruminants: African buffalo, sheep, bushbuck, bongo, N'Dama cattle, and roe deer. In contrast, intact CD1d transcripts were found in guinea pig, African elephant, horse, rabbit, and pig. Despite the discovery of a highly homologous NKT/CD1d system in pig and horse, our data suggest that functional CD1D and CD1d-restricted NKT cells are not universally present in mammals.

Identification of a novel Babesia sp. from a sable antelope (Hippotragus niger Harris, 1838).

Babesiosis in a sable antelope (Hippotragus niger Harris, 1838) was first reported in 1930; the parasite was named Babesia irvinesmithi. Recently, specimens from an adult sable that presented with a sudden onset of disease and that subsequently died during immobilization were submitted for molecular characterization. Microscopic examination of thin blood smears revealed the presence of small piroplasms. DNA was extracted from blood samples; the V4 variable region of the 18S rRNA gene was amplified and analyzed using the reverse line blot (RLB) assay. Amplicons did not hybridize with any of the Babesia or Theileria species-specific probes present on the blot and hybridized only with a Babesia or Theileria genus-specific probe, suggesting the presence of a novel species. The full-length 18S rRNA gene sequence was obtained and aligned with published sequences of related genera, and phylogenetic trees were constructed. Sequence similarity analyses indicated that a Babesia species, designated Babesia sp. (sable), was present. The sequence showed its highest similarity to B. orientalis and to an unnamed Babesia species previously detected in bovine samples. The latter was later established to be Babesia occultans. A Babesia sp. (sable)-specific RLB oligonucleotide probe was designed and used to screen 200 South African sable samples, but so far, no other sample has been found to be positive for the presence of Babesia sp. (sable) DNA. In summary, we identified a novel piroplasm parasite from a sable antelope that died from an unknown illness. While the parasite was observed in blood smears, there is no direct evidence that it was the cause of death.