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.     

Simultaneous detection of piroplasma infections in field Haemaphysalis qinghaiensis ticks by reverse line blotting

Piroplasmosis is caused by tick-borne haemoprotozoan parasites in the genera of Theileria and Babesia, in which numbers of agents are highly pathogenic for cattle, sheep and goats. We developed a reverse line blot (RLB) assay for detection and differentiation of four different parasites, in which 18S ribosomal RNA gene sequence was amplified with a set of universal primers specific for all members in the genera of Theileria and Babesia; and the probes were designed on the basis of hypervariable region 4 (V4 region) of 18S rRNA gene. Three Theileria and one Babesia can be detected simultaneously on this system and it was sensitive to detect a parasitemia level between 10⁻⁵ and 10⁻⁸%. A total of 149 Haemaphysalis qinghaiensis ticks collected from Lintan County of Gannan Tibetan Autonomous Region was tested by RLB. Among these, 136 tick samples were also tested by a nested PCR assay developed previously. After comparison of these results, it showed that more T. luwenshuni was detected in RLB assay, while more T. uilenbergi was detected in H. qinghaiensis ticks by nested PCR. The RLB has shown capability for simultaneous detection of four species of piroplasm in H. qinghaiensis ticks, indicating its usefulness for epidemiological studies of piroplasmosis.     

Simultaneous Detection and Identification of Common Cell Culture Contaminant and Pathogenic Mollicutes Strains by Reverse Line Blot Hybridization

We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with “universal” primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.     

Identification,genetic variation,and structural analysis of 18S rRNA of Theileria orientalis and Theileria velifera-like isolates from Myanmar

Ribosomal RNA genes have been widely used for the identification and phylogenetic analysis of various organisms, including parasitic protozoa. Here, we report nine near full-length Theileria orientalis 18S rRNA gene sequences from cattle from different areas of Myanmar. Phylogenetic analysis of the 18S rRNA genes revealed a considerably close genetic relationship among T. orientalis isolates from Australia, China, Japan, Korea, Myanmar, and Pakistan. We also obtained four Theileria velifera-like (Theileria cf. velifera) 18S rRNA gene sequences from two cattle and two water buffaloes from the northernmost area of Myanmar. The phylogenetic analysis of T. cf. velifera isolates from Myanmar along with T. velifera and T. cf. velifera isolates from African countries suggested an evolutionary lineage of greater complexity in T. velifera-related parasites. DNA alignment analysis indicated the presence of 51 and 55 nucleotide variation positions within the 18S rRNA genes from 15 T. orientalis and 11 T. velifera-related isolates, respectively. Alignment entropy analysis of the 18S rRNA sequences indicated that both T. orientalis and T. velifera-related isolates had three hyper variable regions, corresponding to V2, V4, and V7 regions in eukaryotes. The degree of variation was prominent in the V2 in T. orientalis and V4 in T. velifera-related isolates. The secondary structure analysis of the 18S rRNA predicted using minimum free energy algorism revealed that the structure of V4 region differed most significantly between T. orientalis and T. velifera. These results provide novel insights into common structures, variations and functions of small subunit rRNA in Theileria species.     

Molecular analysis of tick-borne protozoan and rickettsial pathogens in small ruminants from two South African provinces

Tick-borne protozoan and rickettsial diseases are a major threat to livestock in tropical and sub-tropical regions of Africa. In this study we investigated the presence and distribution of Theileria spp., Babesia ovis, Anaplasma ovis, Anaplasma phagocytophilum, Ehrlichia ruminantium and SFG Rickettsia in sheep and goats from Free State and KwaZulu-Natal provinces. A total of 91 blood samples were screened in this study, 61 from goats and 30 from sheep. PCR assay was conducted using primers based on Theileria spp. 18S rRNA, Babesia ovis (BoSSU rRNA), Anaplasma ovis (AoMSP4), Anaplasma phagocytophilum epank1, Ehrlichia ruminantium pCS20 and SFG Rickettsia OmpA. Overall infection rates of Theileria spp., Anaplasma ovis and Ehrlichia ruminantium were 18 (19.8%), 33 (36.3%) and 13 (14.3%), respectively. The co-infection of two pathogens were detected in 17/91 (18.7%) of all samples, goats having higher rates of co-infection compared to sheep. Phylogenetic tree analysis sequence of pCS20 gene of E. ruminantium of this study was found to be in the same clade with Kumm2 and Riverside strains both from South Africa. The phylogram of SSU rRNA of Theileria ovis had longer branch length compared to all other sequences most of which were from Asia and Middle East. This study provides important data for understanding the tick-borne diseases occurrence in the study area and it is expected to improve the approach for the diagnosis and control of these diseases.     

Genetic characterization of Strongyloides spp. from captive, semi-captive and wild Bornean orangutans (Pongo pygmaeus) in Central and East Kalimantan, Borneo, Indonesia

Orangutans (Pongo spp.), Asia's only great apes, are threatened in their survival due to habitat loss, hunting and infections. Nematodes of the genus Strongyloides may represent a severe cause of death in wild and captive individuals. In order to better understand which Strongyloides species/subspecies infect orangutans under different conditions, larvae were isolated from fecal material collected in Indonesia from 9 captive, 2 semi-captive and 9 wild individuals, 18 captive groups of Bornean orangutans and from 1 human working with wild orangutans. Genotyping was done at the genomic rDNA locus (part of the 18S rRNA gene and internal transcribed spacer 1, ITS1) by sequencing amplicons. Thirty isolates, including the one from the human, could be identified as S. fuelleborni fuelleborni with 18S rRNA gene identities of 98·5-100%, with a corresponding published sequence. The ITS1 sequences could be determined for 17 of these isolates revealing a huge variability and 2 main clusters without obvious pattern with regard to attributes of the hosts. The ITS1 amplicons of 2 isolates were cloned and sequenced, revealing considerable variability indicative of mixed infections. One isolate from a captive individual was identified as S. stercoralis (18S rRNA) and showed 99% identity (ITS1) with S. stercoralis sequences from geographically distinct locations and host species. The findings are significant with regard to the zoonotic nature of these parasites and might contribute to the conservation of remaining orangutan populations.     

Molecular detection of tick-borne protozoan parasites in sika deer (Cervus nippon) from western regions of Japan

The sika deer (Cervus nippon) is one of the most common species of wildlife in Japan. This study aimed to reveal the prevalence of tick-borne protozoan parasites in wild sika deer living in western Japan. We used nested polymerase chain reaction (PCR) to detect the 18S rRNA gene of tick-borne apicomplexan parasites (Babesia, Theileria, and Hepatozoon spp.) from 276 blood and liver samples from sika deer captured in the Yamaguchi, Oita, Kagoshima, Okayama, Ehime, Kochi, and Tokushima Prefectures. In total, 259 samples (259/276; 93.8%) tested positive in the nested PCR screening. Gene sequencing revealed that 99.6% (258/259) of positive samples contained Theileria sp. (sika 1), while Theileria sp. (sika 2), another Theileria species, was detected in only 3 samples. We also found that one sample from a sika deer captured in Kagoshima contained the gene of an unidentified Babesia sp. related to Babesia sp. Kh-Hj42, which was previously collected from tick in western Siberia. In conclusion, we found a high prevalence of piroplasms in sika deer from western Japan, and DNA analysis revealed that Theileria sp. (sika 1) had the highest infection rate.     

Development and evaluation of a novel loop-mediated isothermal amplification (LAMP) method targeting Theileria parasites infecting Yezo sika deer

The increasing Yezo sika deer (Cervus nippon yesoensis) population is creating a large problem. Yezo sika deer are an important blood meal source, and these deer contribute to the maintenance of tick populations. Theileria spp. infections in Yezo sika deer and T. orientalis infections in cows occur at high frequencies, and the same tick species infests both deer and cows. Therefore, a specific detection method to identify deer Theileria spp. is important. In this study, we establish a novel molecular detection method for identifying Theileria spp. from deer and tick samples using loop-mediated isothermal amplification (LAMP). This method targets a metalloprotease/cell division cycle protein gene homologue. Our LAMP protocol was able to detect deer Theileria and did not show cross reactivity with other closely related protozoan parasites, including T. orientalis. The LAMP method showed sensitivity and specificity equivalent to those of nested PCR performed on the same field samples from deer and ticks. These results demonstrate the applicability of LAMP to field surveys in which the detection of deer Theileria spp. is required. In conclusion, due to its simplicity, specificity, and reliability, we suggest our LAMP protocol as an appropriate method for routine surveys to detect Yezo sika deer and ticks infected with deer Theileria spp. parasites. Additionally, this LAMP method offers great promise as a useful tool to distinguish Yezo sika deer Theileria from related Theileria parasites present in livestock.     

Simultaneous detection and identification of common cell culture contaminant and pathogenic mollicutes strains by reverse line blot hybridization

We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with "universal" primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.     

Mitochondrial DNA variability in Giraffa camelopardalis: consequences for taxonomy, phylogeography and conservation of giraffes in West and central Africa

The giraffe (Giraffa camelopardalis) still survives in four countries of West and central Africa. The populations of Niger and Cameroon are generally assigned to the subspecies peralta, but those of Chad and the Central African Republic are taxonomically problematic, as they are referred to as either peralta, or antiquorum, or congoensis. In this study, a mitochondrial fragment of 1765 nucleotide sites, covering the complete cytochrome b gene, three transfer RNAs and a large part of the control region, was sequenced to assess the relationships between several populations of giraffe. The phylogenetic analyses performed on the 12 identified haplotypes indicate that northern giraffes constitute a natural group, distinct from that of southern giraffes. Surprisingly, the giraffes of Niger are found to be more closely related to the giraffes of East Africa (subspecies rothschildi and reticulata) than to those of central Africa. We conclude therefore that the subspecies peralta contains only the Niger giraffes, whereas the subspecies antiquorum includes all populations living in Cameroon, Chad, the Central African Republic, and southwestern Sudan. We suggest that the ancestor of the Nigerian giraffe dispersed from East to North Africa during the Quaternary period and thereafter migrated to its current Sahelian distribution in West Africa, in response to the development of the Sahara desert. This hypothesis implies that Lake Mega-Chad acted as a strong geographical barrier during the Holocene, preventing any contact between the subspecies peralta and antiquorum. Our study has direct implications for conservation management, as we show that no subspecies peralta is represented in any European zoos, only in Niger, with a small population of less than 200 individuals.     

Phylogenetic Analysis of Ruminant Theileria spp. from China Based on 28S Ribosomal RNA Gene

Species identification using DNA sequences is the basis for DNA taxonomy. In this study, we sequenced the ribosomal large-subunit RNA gene sequences (3,037-3,061 bp) in length of 13 Chinese Theileria stocks that were infective to cattle and sheep. The complete 28S rRNA gene is relatively difficult to amplify and its conserved region is not important for phylogenetic study. Therefore, we selected the D2-D3 region from the complete 28S rRNA sequences for phylogenetic analysis. Our analyses of 28S rRNA gene sequences showed that the 28S rRNA was useful as a phylogenetic marker for analyzing the relationships among Theileria spp. in ruminants. In addition, the D2-D3 region was a short segment that could be used instead of the whole 28S rRNA sequence during the phylogenetic analysis of Theileria, and it may be an ideal DNA barcode.     

The scent of the reticulated giraffe (Giraffa camelopardalis reticulata)

The giraffe (Giraffa camelopardalis) emits a scent that can be detected by humans over considerable distances. Dichloromethane extracts of hair samples from adult male and female reticulated giraffes (G. c. reticulata) were analysed by gas chromatography–mass spectrometry. Two highly odoriferous compounds, indole and 3-methylindole, identified in these extracts appear to be primarily responsible for the giraffe’s strong scent. Other major compounds identified were octane, benzaldehyde, heptanal, octanal, nonanal, p-cresol, tetradecanoic acid, hexadecanoic acid, and 3,5-androstadien-17-one; the last compound has not previously been identified from a natural source. These compounds may deter microorganisms or ectoparasitic arthropods. Most of these compounds are known to possess bacteriostatic or fungistatic properties against mammalian skin pathogens or other microorganisms. The levels of p-cresol in giraffe hair are sufficient to repel some ticks.     

Spatial ecology and habitat use of giraffe (Giraffa camelopardalis) in South Africa

The lack of long-term studies remains a limiting factor in understanding the home range, spatial ecology and movement of giraffes. We equipped eight giraffes with GPS satellite units and VHF capacity, which were built in to the collars for the remote collection of data on their movements and home ranges over two years on Khamab Kalahari Nature Reserve (KKNR) within the Kalahari region of South Africa. Giraffe numbers in KKNR dropped from 135 individuals to 111 in just five years, revealing the lack of knowledge about their required habitat needs, space use and diet. With over 1000 km² available for roaming within the reserve, habitat selection, principle and preferred food species played a significant role in home range size and overlap between individuals. These giraffes used an average annual home range of 206 km² (20 602 ha) as calculated by a 95% minimum convex polygon (MCP) with a standard deviation core home range calculated by a 50% MCP of 10.1 km² to satisfy their annual needs for survival and reproduction in their preferred vegetation. In the wet, hot season (summer: December–February) when food was abundant, giraffes frequented smaller areas (average 177 km²), while in the dry, cool season (winter: June to August) the mean home range size increased to approximately 245 km². Rainfall influenced spatial distribution since it determined vegetation productivity and leaf phenology. The different seasons influenced giraffe movements, while different vegetation types and season influenced their home range size. Season and food availability also influenced home range overlap between different giraffe herds. Home range overlap occurred when giraffes were forced to roam in overlapping areas during the dryer months when the winter deciduous nature of the majority of the tree species resulted in lower food availability. In winter, the overlap was approximately 31% and in autumn approximately 23%. During the wet and warmer months, overlapping was 15% in summer and 19% in spring, respectively. The percentage of time spent in different vegetation type areas was influenced by the abundance of the principal food species of that plant community. It is thus concluded that the movements of giraffes were primarily influenced by a combination of environmental factors such as season, rainfall and vegetation density.     

Milky hemolymph syndrome (MHS) in spiny lobsters, penaeid shrimp and crabs

Black tiger shrimp Penaeus monodon, European shore crab Carcinus maenas and spiny lobster Panulirus spp. can be affected by milky hemolymph syndrome (MHS). Four rickettsia-like bacteria (RLB) isolates of MHS originating from 5 geographical areas have been identified to date. The histopathology of the disease was characterized and a multiplex PCR assay was developed for detection of the 4 bacterial isolates. The 16S rRNA gene and 16-23S rRNA intergenic spacer region (ISR) were used to examine the phylogeny of the MHS isolates. Although the pathology of this disease appears similar in the various different hosts, sequencing and examination of the phylogenetic relationships reveal 4 distinct RLB involved in the infection process.     

Development and evaluation of real-time PCR assay for the detection of Babesia orientalis in water buffalo (Bubalus bubalis, Linnaeus, 1758)

Babesia orientalis is the causative agent of babesiosis in water buffalo (Bubalus babalis, Linnaeus, 1758). In this study, a TaqMan real-time PCR assay was developed for quantitative detection of B. orientalis in water buffalo. Hybridization probe and oligonucleotide primers were designed based on the v4 region of 18S rRNA gene. Detection limit was determined at 2 parasites. Blood samples were collected from experimentally infected water buffalo, as well as from 180 field samples, which were collected from 4 different geographical locations to the north and south of the Yangtse River. The parasite was detected by real-time PCR on day 2 until day 39 post-infection, while reverse line blot (RLB) was on day 6 until day 36 in experimentally infected water buffalo. For the results of 180 field samples, statistical analysis showed no significant difference in relative effectiveness of real-time PCR and RLB. The analysis also indicated that there was no difference in the prevalence of B. orientalis between the regions of south and north of the Yangtse River by both the real-time PCR assay and RLB detection. These results indicated that the parasite infection has spread to the north of the Yangtse River.     

Outcome of experimental porcine circovirus type 1 infections in mid-gestational porcine foetuses

Background

Lyme disease caused by Borrelia burgdorferi sensu lato complex is an important endemic zoonosis whose distribution is closely related to the main ixodid tick vectors. In China, isolated cases of Lyme disease infection of humans have been reported in 29 provinces. Ticks, especially ixodid ticks are abundant and a wide arrange of Borrelia natural reservoirs are present. In this study, we developed a reverse line blot (RLB) to identify Borrelia spp. in ticks collected from sheep and cattle in 7 Provinces covering the main extensive livestock regions in China.

Results

Four species-specific RLB oligonucleotide probes were deduced from the spacer region between the 5S-23S rRNA gene, along with an oligonucleotide probe which was common to all. The species specific probes were shown to discriminate between four genomic groups of B. burgdorferi sensu lato i.e. B. burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana, and to bind only to their respective target sequences, with no cross reaction to non target DNA. Furthermore, the RLB could detect between 0.1 pg and 1 pg of Borrelia DNA. A total of 723 tick samples (Haemaphysalis, Boophilus, Rhipicephalus and Dermacentor) from sheep and cattle were examined with RLB, and a subset of 667 corresponding samples were examined with PCR as a comparison. The overall infection rate detected with RLB was higher than that of the PCR test. The infection rate of B. burgdoreri sensu stricto was 40% in south areas; while the B. garinii infection rate was 40% in north areas. The highest detection rates of B. afzelii and B. valaisiana were 28% and 22%, respectively. Mixed infections were also found in 7% of the ticks analyzed, mainly in the North. The proportion of B. garinii genotype in ticks was overall highest at 34% in the whole investigation area.

Conclusion

In this study, the RLB assay was used to detect B. burgdorferi sensu lato in ticks collected from sheep and cattle in China. The results showed that B. burdorferi senso stricto and B. afzelii were mainly distributed in the South; while B. garinii and B. valaisiana were dominant in the North. Borrelia spirochaetes were detected in Rhipicephalus spp for the first time. It is suggested that the Rhipicephalus spps might play a role in transmitting Borrelia spirochaetes.     

RPS8—a New Informative DNA Marker for Phylogeny of Babesia and Theileria Parasites in China

Piroplasmosis is a serious debilitating and sometimes fatal disease. Phylogenetic relationships within piroplasmida are complex and remain unclear. We compared the intron–exon structure and DNA sequences of the RPS8 gene from Babesia and Theileria spp. isolates in China. Similar to 18S rDNA, the 40S ribosomal protein S8 gene, RPS8, including both coding and non-coding regions is a useful and novel genetic marker for defining species boundaries and for inferring phylogenies because it tends to have little intra-specific variation but considerable inter-specific difference. However, more samples are needed to verify the usefulness of the RPS8 (coding and non-coding regions) gene as a marker for the phylogenetic position and detection of most Babesia and Theileria species, particularly for some closely related species.     

Screening for potential <Emphasis Type="Italic">Striga hermonthica</Emphasis> fungal and bacterial biocontrol agents from suppressive soils in Western Kenya

Striga hermonthica is a hemiparasitic weed that causes huge grain yield losses to small-scale farmers in Africa. Effective biocontrol agents against S. hermonthica can sustainably mitigate these losses. This study characterized the biocontrol potential of culturable fungal and bacterial isolates from S. hermonthica suppressive soils of western Kenya. These isolates were screened for their ability to produce antibiotic compounds and extra cellular enzymes and also their ability to cause S. hermonthica seed decay. Genomic DNA of the selected bacterial and fungal isolates was extracted and partial characterization of 16S rRNA and 18S rRNA genes performed respectively. Analysis show that antibiosis and enzymatic properties of potential biocontrol isolates correlated positively. Isolate KY041696 recorded high antibiosis, enzymatic and seed decay values. This study also revealed that bioactive bacterial isolates belonged to Bacillus, Streptomyces and Rhizobium genera. In this study, no fungal isolate caused S. hermonthica seed decay. This study therefore provides baseline information on the potential biocontrol microbes against S. hermonthica in Western Kenya that could be exploited further in the management of the weed.     

Detection of Babesia caballi in Amblyomma variegatum ticks (Acari: Ixodidae) collected from cattle in the Republic of Guinea

A reverse line blot hybridisation (RLB) assay was applied to screen Amblyomma variegatum adult ticks (n = 504) collected from N'Dama cattle in the Republic of Guinea. In a PCR, the V1 hypervariable region of the 16S ribosomal RNA (rRNA) gene was amplified with a set of primers unique for species of the genera Anaplasma and Ehrlichia, and the V4 hypervariable region of the 18S rRNA gene was amplified with primers specific for members of the genera Theileria and Babesia. Amplified PCR products from A. variegatum ticks were hybridised onto a membrane, to which oligonucleotide probes species-specific for Ehrlichia/Anaplasma and Theileria/Babesia parasites were covalently linked. No pathogens belonging to Ehrlichia/Anaplasma species were found, while 10 DNA samples resulted positive for Babesia caballi and 5 samples for Theileria velifera. This is the first report of B. caballi in A. variegatum ticks. One of the B. caballi positive samples was sequenced. This new strain (BcabGuinea) showed a 97% similarity to the Z15104 B. caballi GenBank sequence.     

Molecular detection and characterization of Theileria sp. from hedgehogs (Paraechinus aethiopicus) in Saudi Arabia

In this study, we conducted molecular detection and characterization of piroplasms that infect the Ethiopian or desert hedgehogs (Paraechinus aethiopicus) in Saudi Arabia. Blood samples from 112 (68 males and 44 females) desert hedgehogs from Unaizah, Central Saudi Arabia were screened for Theileria/Babesia DNA using the polymerase chain reaction (PCR) employing specific primers amplifying the partial 18S small subunit rRNA gene. Theileria DNA was detected in 51 samples (45·5%), giving a prevalence of 45·5%. Theileria DNA was found in 33 (48·5%) males and 18 (40·9%) females, and there was no significant difference (P > 0·05) in the prevalence between males and females. Similarly, there was no significant difference (P > 0·05) in the prevalence between juveniles (40%) and adults (46·7%). There was a significant difference in the prevalence of Theileria in hedgehogs collected from May to September and the period from October to April (P = 0·003). Four haplotypes of Theileria sp. in hedgehogs were detected and designated as H1–H4. H1 was the predominant haplotype and found in 80·8% of the positive individuals. Partial sequences of the 18S rRNA of Theileria sp. from hedgehogs grouped with Theileria spp. that are benign. This study is the first report of the occurrence of Theileria spp. in Saudi Arabian desert hedgehogs.