Paul Tshepo Matjila

Molecular detection of tick-borne protozoal and ehrlichial infections in domestic dogs in South Africa.

A total of 1,138 blood specimens were collected over a 6-year period (2000-2006) from domestic dogs in South Africa. Specimens from domestic dogs were obtained from the Onderstepoort Veterinary Academic Hospital (OVAH) in Pretoria, the Society for the Prevention of Cruelty to Animals (Johannesburg, Durban, East London and Bloemfontein) and private practices from four provinces (Gauteng, Mpumalanga, KwaZulu-Natal and Western Cape). All specimens were screened for Babesia, Theileria, Hepatozoon and Ehrlichia/Anaplasma species using PCR and Reverse Line Blot (RLB) assays. On RLB, 560/1,137 domestic dog-specimens were positive for one or more parasites. Of the positive domestic dog-specimens, 420 (75%) were infected with Babesia rossi; 82 (15%) dogs were infected with Theileria sp. (dog); 18 (3%) dogs were infected with Babesia vogeli; 14 (3%) specimens were infected with Ehrlichia canis. Mixed infections were also found: B. rossi and E. canis were detected in 12 (2%) specimens; B. vogeli and E. canis occurred in 7 (1%) specimens; Theileria sp. (dog) and E. canis in 3 (0.5%) specimens; B. rossi and B. vogeli in one specimen. B. rossi, B. vogeli and E. canis occurred simultaneously in one dog. There was also one incidental finding of a dog positive for Trypanosoma congolense. The results indicate that a wide range of tick-borne pathogens are circulating in the canine populations in South Africa.

Detection of a Theileria species in dogs in South Africa.

A Theileria species was detected by PCR in blood samples collected from dogs in the Pietermaritzburg area and was also found in dogs presented at the Outpatients Clinic of the Onderstepoort Veterinary Academic Hospital (OVAH), in the Pretoria area, South Africa. In the Pietermaritzburg area, 79 of the 192 samples were positive, while 3 out of 1137 of the Onderstepoort samples were positive. Three positive samples from Pietermaritzburg were co-infected with Ehrlichia canis. PCR positive samples were further analysed by the Reverse Line Blot (RLB) and sequence analysis. Phylogenetic analysis of the 18S rRNA full-length gene sequences of one sample (VT12) from Pietermaritzburg and two samples from OVAH (BC281 and BC295) revealed a close relationship with sequences of Theileria species (sable). Clinical signs of the dogs that were examined at Pietermaritzburg and OVAH included an immune-mediated condition with severe thrombocytopenia. These findings identify a Theileria sp. in dogs for the first time in South Africa and add yet another microorganism to the growing list of haemoprotozoan parasites infecting dogs worldwide. The clinical significance of this infection in dogs is poorly resolved.

Occurrence and diversity of bovine coccidia at three localities in South Africa

Information on the occurrence and diversity of coccidia infecting cattle in different farming systems in South Africa is lacking. The objective of the study was to determine the levels of infection of coccidia in cattle at three localities and to determine the Eimeria species occurring at each locality. Faecal samples (n=1936) were collected over a 13-month period from a dairy farm (Mallesons), a Bonsmara breeding farm (Kaalplaas) and a Nguni stud farm (Pienaars River). Samples were collected from adults >24 months old and calves <12 months old. The modified McMaster technique was used to determine OPG (oocysts per gram) of faeces. Samples with OPGs > or =2000 were allowed to sporulate in potassium dichromate solution at room temperature. Species identification was done by measurement and morphological characteristic of each species. At Mallesons, 29% of samples were positive, at Kaalplaas 50% and at Pienaars River 52%. The most prevalent species were Eimeria zuernii and E. ellipsoidalis at Mallesons, E. zuernii and E. bovis at Kaalplaas and E. bovis and E. zuernii at Pienaars River, respectively. Adults had very low OPGs. There was a pattern at the three localities in that high OPGs were only recorded in calves, which were also shedding the most pathogenic species.

Molecular detection of Babesia rossi and Hepatozoon sp. in African wild dogs (Lycaon pictus) in South Africa.

Blood specimens from wild dogs (n=301) were obtained from De Wildt Cheetah and Wildlife Centre (Pretoria) and five game reserves (4 in the North-West Province and 1 in Limpopo Province), South Africa. Specimens were screened for Babesia, Theileria, Hepatozoon and Ehrlichia/Anaplasma species using PCR and Reverse Line Blot (RLB) assays. Positive results were obtained in 18 (6%) wild dogs. Sixteen specimens were found positive for Babesia rossi and two dogs were Hepatozoon sp. positive. It appears that these tick-borne pathogens are not widely distributed in wild dog populations.

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

BackgroundCanine 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 (BrEMA 1) 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 BrEMA 1 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.MethodsBlood 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 BrEMA1 gene using an RT-PCR. In addition, ticks were collected from dogs found to be infested during sampling.ResultsOn 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.ConclusionsUp 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.

Sequence heterogeneity in the equi merozoite antigen gene (ema-1) of Theileria equi and development of an ema-1-specific TaqMan MGB assay for the detection of T. equi.

Although a quantitative real-time PCR assay (qPCR) assay for the detection of Theileria equi has been developed and evaluated, it is possible that additional, as yet undetected 18S rRNA gene sequence variants may exist. A qPCR assay targeting a different gene, used in conjunction with the T. equi 18S rRNA qPCR assay, could assist in the detection of all T. equi genotypes in field samples. A T. equi ema-1-specific qPCR (Ueti et al., 2003) was tested on 107 South African field samples, 90 of which tested positive for T. equi antibody using the immuno-fluorescent antibody test (IFAT). The qPCR assay performed poorly, as T. equi was detected in only 67 of the 90 IFAT-positive field samples at quantification cycle (C(q)) values ranging from 27 to 39.95. Furthermore, a high C(q) value of 36.18 was obtained from DNA extracted from a South African in vitro-cultured T. equi WL isolate [1.38% parasitized erythrocytes (PE)] when a low C(q) value (indicative of a high T. equi DNA concentration) was expected. Approximately 600 bp of the ema-1 gene from 38 South African samples were sequenced and BLASTN analysis confirmed all sequences to be merozoite surface protein genes, with an identity of 87.1-100% to previously published T. equi ema-1 gene sequences. Alignment of the sequences revealed extensive sequence variations in the target regions of the primers and probes (Ueti et al., 2003), explaining the poor performance of the qPCR assay. Based on these observations, we developed a new TaqMan minor-groove binder (MGB) probe-based qPCR assay, targeting a more conserved region of the ema-1 gene. This assay was shown to be efficient and specific, and the detection limit, defined as the concentration at which 95% of T. equi-positive samples are detected, was determined to be 1.4 x 10(-4)% PE. The two ema-1 assays were compared by testing 41 South African field samples in parallel. The results suggested that the new assay was more sensitive than the original assay, as T. equi was detected in more samples and at lower C(q) values when the new assay was used. Phylogenetic analyses of the 18S rRNA gene sequences and ema-1 amino acid sequences from the same samples showed inconsistencies between the clades, indicating that the T. equi 18S rRNA genetic groups previously identified in South Africa may not represent distinct T. equi lineages. It is possible that the different T. equi ema-1 genotypes could be related to antigenic variability and pathogenicity and may be associated with clinical differences in equine piroplasmosis cases, but this remains to be elucidated.

Preliminary Evaluation of the BrEMA1 Gene as a Tool for Associating Babesia rossi Genotypes and Clinical Manifestation of Canine Babesiosis

ABSTRACT Babesia rossi, an intraerythrocytic protozoan, causes a severe, often life-threatening disease of domestic dogs. Dogs treated early for B. rossi infection usually recover from the disease, but dogs left untreated or treated at a later stage of infection seldom survive. Dogs infected with B. rossi have varied clinical manifestations that can be categorized as uncomplicated (with a good prognosis) or complicated (with a poor prognosis). One hundred twenty-one blood samples were collected from dogs presented to the Onderstepoort Veterinary Academic Hospital and diagnosed with babesiosis by the use of a thin blood smear. An additional 20 samples were obtained from Babesia-infected dogs from private clinics around the Onderstepoort, Johannesburg, Durban, White River, and Cape Town areas. The samples were screened by PCR targeting the Babesia rossi erythrocyte membrane antigen gene (BrEMA1) and by sequencing of the polymorphic region (i.e., region with a variable number of hexapeptide repeats). Analysis of PCR products revealed 11 different gene profiles, visualized by gel electrophoresis. Twelve distinct BrEMA1 genotypes were identified by sequencing, but the numbers of hexapeptide repeats varied from 6 to 31 (classified as genotype6 to genotype31). The genotypes were retrospectively compared to the clinical case data. The most frequently encountered B. rossi parasites were those attributed to genotype19 (36.2%), genotype28 and genotype29 (20.6% each), and genotype11 (12.7%). These genotypes were also the ones associated with the poorest prognosis. This preliminary finding suggests clinically important differences between the various B. rossi genotypes identified.

In vitro culture and structural differences in the major immunoreactive protein gp36 of geographically distant Ehrlichia canis isolates

Ehrlichia canis, the etiologic agent of canine ehrlichiosis, is an obligate intracytoplasmic Gram-negative tick-borne bacterium belonging to the Anaplasmataceae family. E. canis is distributed worldwide and can cause serious and fatal infections in dogs. Among strains of E. canis, the 16S rRNA gene DNA sequences are highly conserved. Using this gene to genetically differentiate isolates is therefore difficult. As an alternative, the gene gp36, which encodes for a major immunoreactive protein in E. canis, has been successfully used to characterize the genetic diversity of this pathogen. The present study describes the isolation and continuous propagation of a Spanish and 2 South African isolates of E. canis in IDE8 tick cells. Subsequently, canine DH82 cell cultures were infected using initial bodies obtained from infected IDE8 cultures. It was possible to mimic the life cycle of E. canis in vitro by transferring infection from tick cells to canine cells and back again. To characterize these E. canis strains at the molecular level, the 16S rRNA and gp36 genes were amplified by PCR, sequenced, and aligned with corresponding sequences available in GenBank. All 16S rRNA sequences amplified in this study were identical to previously reported E. canis strains. Maximum likelihood analysis based on the gp36 amino acid sequences showed that the South African and Spanish strains fall into 2 well-defined phylogenetic clusters amongst other E. canis strains. The members of these 2 phylogenetic clusters shared 2 unique molecular properties in the gp36 amino acid sequences: (i) deletion of glycine 117 and (ii) the presence of an additional putative N-linked glycosylation site. We further show correlation between the putative secondary structure and the theoretical isoelectric point (pI) of the gp36 amino acid sequences. A putative role of gp36 as an adhesin in E. canis is discussed. Overall, we report the successful in vitro culture of 3 new E. canis strains which present different molecular properties in their gp36 sequences.