Piroplasm Species Research Articles

Great diversity of Piroplasmida in Equidae in Africa and Europe, including potential new species.

Piroplasms are Apicomplexa tick-borne parasites distributed worldwide. They are responsible for piroplasmosis (theileriosis and babesiosis) in Vertebrata and are therefore of medical and economic importance. Herein, we developed a new real time PCR assay targeting the 5.8S rRNA gene and three standard PCR assays, targeting 18S rRNA, 28S rRNA, and cox1 genes, for the detection of piroplasmids. These assays were first optimized and screened for specificity and sensitivity. Then, they were used to study a total of 548 blood samples and 97 ticks collected from Equidae in four sub-Saharan countries (Senegal, Democratic Republic of the Congo, Chad, and Djibouti) and France (Marseille and Corsica). DNA of piroplasms was detected in 162 of 548 (29.5%) blood samples and in 9 of 97 (9.3%) ticks. The highest prevalence in blood samples was observed in Chad in 2016 with 72.9% positivity rate. Sequencing allowed the identification of four species of piroplasms, including two potentials new species. Theileria equi was mainly found. The highest prevalence was observed in Senegal (14 positive out of 23, 60.87%). Babesia caballi was detected in one horse in Senegal. Two new potential Theileria species were detected: Theileria sp. "Africa", observed in all areas excepted in Marseille and Theileria sp. "Europa", observed in Marseille and Corsica. In conclusion, sensitive and specific PCR assays were developed for epidemiological studies of Piroplasmida. The circulation of multiple species of piroplasms, including two potentials new species, observed among Equidae from sub-Saharan Africa and France.

First molecular survey of piroplasm species in cattle from Kyrgyzstan.

Bovine piroplasmosis is a tick-borne disease caused by apicomplexan hemoparasites of the genera Theileria and Babesia. This study was carried out to assess the presence and frequency of piroplasm parasites in apparently healthy cattle in Kyrgyzstan. A total of 454 blood samples were collected from animals of various ages in eight villages located in the Chu valley and around the Lake Issyk Kul. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified with a set of primers specific targeting members of the genera Theileria and Babesia. Amplified PCR products were hybridized onto a membrane to which generic and species-specific oligonucleotide probes were covalently linked. The results revealed the presence of three piroplasm species (Theileria orientalis, Babesia major, Theileria annulata). Theileria orientalis was the most prevalent species (32.8%; CI 28.5-37.3). Babesia major was the only species of Babesia found in any of the samples (1.3%; CI 0.5-2.8). The co-existence of Theileria annulata and T. orientalis was detected in nine animals (1.9%; CI 0.9-3.7). BLAST search revealed that the Theileria sequences shared 100% identity with the recently reported sequences for T. buffeli and T. annulata. The sequence of B. major was also 100% identical to an existing B. major sequence. This molecular survey provides important epidemiological data for control of bovine piroplasmosis caused by T. orientalis, B. major, and T. annulata in Kyrgyzstan.

Prevalence, distribution, and diversity of cryptic piroplasm infections in raccoons from selected areas of the United States and Canada.

The order Piroplasmida contains a diverse group of intracellular parasites, many of which can cause significant disease in humans, domestic animals, and wildlife. Two piroplasm species have been reported from raccoons (Procyon lotor), Babesia lotori (Babesia sensu stricto clade) and a species related to Babesia microti (called B. microti-like sp.). The goal of this study was to investigate prevalence, distribution, and diversity of Babesia in raccoons. We tested raccoons from selected regions in the United States and Canada for the presence of Babesia sensu stricto and Babesia microti-like sp. piroplasms. Infections of Babesia microti-like sp. were found in nearly all locations sampled, often with high prevalence, while Babesia sensu stricto infections had higher prevalence in the Southeastern United States (20–45% prevalence). Co-infections with both Babesia sp. were common. Sequencing of the partial 18S rRNA and cytochrome oxidase subunit 1 (cox1) genes led to the discovery of two new Babesia species, both found in several locations in the eastern and western United States. One novel Babesia sensu stricto sp. was most similar to Babesia gibsoni while the other Babesia species was present in the ‘western piroplasm’ group and was related to Babesia conradae. Phylogenetic analysis of the cox1 sequences indicated possible eastern and western genetic variants for the three Babesia sensu stricto species. Additional analyses are needed to characterize these novel species; however, this study indicates there are now at least four species of piroplasms infecting raccoons in the United States and Canada (Babesia microti-like sp., Babesia lotori, a novel Babesia sensu stricto sp., a novel western Babesia sp.) and a possible fifth species (Babesia sensu stricto) in raccoons in Japan.

The role of healthy dog carriers of Babesia microti-like piroplasms

BackgroundWhile in Europe Babesia canis has been traditionally held responsible for canine piroplasmosis, Babesia microti-like piroplasm (Bml) infection is being ever more observed in dogs, with the first clinical cases reported in northwestern Spain. This study examines the epidemiological role of healthy dogs living in endemic areas of Bml infection in Spain. The data obtained were used to describe the clinical status and map the geographical distribution of Bml infection in healthy dogs in northwestern Spain.ResultsBlood samples and ticks were taken from 756 healthy dogs representatively across the whole Galicia region (northwestern Spain): stray (n = 211), hunting dogs (n = 333) and pets (n = 212). Blood samples were tested by microscopy parasite observation, nested PCR-RFLP and sequencing. Piroplasm infection prevalences in healthy dogs from northwestern Spain were 17.1% (129/756) by PCR and 3.4% (26/756) by microscopy observation. The species found by PCR were: 2.2% (17/756) for B. canis and 15.1% (114/756) for Bml. Co-infection with B. canis and Bml was noted in 2 dogs. The higher prevalences detected were Bml in hunting dogs (25.5%; 85/333) and B. canis in stray dogs (6.6%; 14/211). In fox-hunting dogs from any area and dogs from the A Coruña Province, significantly higher prevalences of Bml infection were detected (P < 0.001). Upon physical examination, tick infestation was observed: 130 ticks in 18 hunting and three pet dogs. These were subsequently identified as Rhipicephalus sanguineus (s.l.) (49.2%), Ixodes hexagonus (38.5%), Ixodes ricinus (6.9%) and Dermacentor reticulatus (5.4%). Among the more prevalent ticks infesting healthy carrier dogs were I. hexagonus, followed by D. reticulatus and I. ricinus.ConclusionsBabesia canis and Bml were the only piroplasm species found infecting healthy dogs in Galicia, the prevalence of Bml being higher than of B. canis. Factors correlated with a higher Bml infection risk were being a hunting dog and living in the A Coruña Province. Healthy dogs travelling to other countries could act as carriers and probably contribute to the spread of Bml infection in dogs and wild carnivores throughout Europe.

Molecular detection of vector borne pathogens in anemic and thrombocytopenic dogs in southern Brazil

Arthropod-borne pathogens are medically important because of their ability to cause diseases in their hosts. The purpose of this study was to detect the occurrence of Ehrlichia spp., piroplasmids and Hepatozoon spp. in dogs with anemia and thrombocytopenia in southern Brazil. EDTA-whole blood was collected from 75 domestic dogs presenting anemia or/and thrombocytopenia from Guarapuava, state of Paraná, Brazil. DNA samples were subjected to conventional PCR assays for Ehrlichia spp. (dsb), piroplasmids (18S rRNA) and Hepatozoon spp. (18S rRNA), followed by sequencing and phylogenetic analyses. Among the 75 dogs, one (1.33%) was positive for Hepatozoon sp. and six (8%) were positive for piroplasmids in 18S rRNA cPCR assays. None of the dogs showed positive results in Ehrlichia spp.-cPCR targeting dsb gene. The phylogenetic analyses revealed that three piroplasm sequences were clustered with Rangellia vitalii, while one sequence was grouped with B. vogeli. The only sequence obtained from Hepatozoon spp.-PCR protocol was pooled with H. canis. Therefore, there is urgent need for differential molecular diagnosis of the two piroplasm species cited as etiological agents in clinical cases of canine hemoparasitic diseases, given the higher pathogenic potential of R. vitalii than of B. vogeli.

Theileria sp. in water buffaloes from Maranhão State, northeastern Brazil.

Anaplasma marginale and piroplasm species are widespread among Brazilian cattle herds. Both of these tick-borne pathogens hamper livestock production and cause a significant economic impact. Although buffaloes have demonstrated a high level of adaptability, data on tick-borne pathogens are scarcely reported in Brazil. Thus, the aim of this study was to screen water buffaloes from the state of Maranhão for piroplasm and A. marginale occurrence using PCR assays. All samples were negative for A. marginale. One of the 287 (0.35%) water buffaloes tested was positive for Theileria sp. Sequencing of the 18S rDNA fragment (356 bp) showed that the Theileria sp. identified was closely related to the T. buffeli /orientalis group. Future studies on the clinical signs of infection and the main vector in this country are needed.

Molecular survey of piroplasm species from selected areas of China and Pakistan

BackgroundPiroplasmosis is an important animal disease that is a major constraint to the development of the livestock industry, often resulting in significant economic losses. Therefore, there is an urgent need to further understand the etiology of this and other tick-borne infections.MethodsBlood samples were collected from tick-infested animals from the Chakwal, Jhang, and Faisalabad districts of Punjab, Pakistan and from peri-urban areas around Hohhot, Inner Mongolia, China to investigate the presence of Babesia and Theileria species. In total, 450 blood samples were collected with FTA cards from cattle of the study areas of Pakistan; the genomic (g)DNA of one hundred and twenty samples from cattle in Inner Mongolia were provided by the Lanzhou Veterinary Research Institute, China. Following the extraction of gDNA, the 18S rRNA gene (V4 hypervariable region) of piroplasms was amplified in all samples using semi-nested PCR. Positively identified samples were sequenced for the identification of Theileria and Babesia species. The partial full-length sequence of 18S rDNA was amplified for species confirmation of Theileria-positive samples, whereas the RAP-1c gene was amplified for Babesia bigemina-positive samples.ResultsSemi-nested PCR results revealed that 144 (25.26%) samples were positive for piroplasms. Theileria annulata was the most prevalent species (115/144; 20.17%), followed by Theileria orientalis (16/144; 2.80%). Among Babesia, the only species recorded was Babesia bigemina (13/144; 2.28%).ConclusionThe present study reveals new data on the prevalence of piroplasm species in bovine populations of selected areas of China and Pakistan and their phylogenetic relationships. It is also the first detailed report of T. orientalis from native animals in Pakistan.

Genetic diversity and prevalence of piroplasm species in equids from Turkey.

Equine piroplasmosis (EP) is a protozoon disease caused by Babesia caballi and Theileria equi transmitted by ticks from the Ixodidae family. This study investigated the genetic heterogeneity and diversity of piroplasm genotypes using the Reverse Line Blotting (RLB) technique for piroplasm species in equids in Turkey. A total of 233 blood samples from 142 horses and 91 donkeys were collected in Şanlıurfa, Tunceli, and Iğdır. The RLB assay was performed for simultaneous detection of piroplasm species. The prevalence of piroplasm infection was 33.5% (95% CI; 27.4-39.9). T. equi was the most common species (%20.2; 95% CI 15.2-25.9), whereas B. caballi infection was not detected. Nine samples hybridized to the Theileria/Babesia and Theileria catch-all probes but did not hybridize to any species-specific probe tested, suggesting the presence of unrecognized Theileria variants or genotypes. Nine samples that gave Theileria spp. signal and two samples that gave T. equi specific signal were sent for sequence analysis and the A and D genotypes of T. equi were found. In addition, T. annulata, B. ovis, and B. canis species were detected in the samples and verified by sequence analysis in Turkey. More research is needed to understand the clinical impact of atypical piroplasm infections in equids.

Simultaneous detection of Theileria annulata and Theileria orientalis infections using recombinase polymerase amplification

Theileriosis is a disease of domesticated animals in tropical and subtropical countries and causes significant reductions in livestock productivity. The arid region of Punjab in Pakistan is notorious for the presence of the vector tick (Acari: Ixodidae) and tick-borne diseases, such as theileriosis and babesiosis. The distribution of Theileria annulata and T. orientalis in the Chakwal district of Punjab was determined by developing a multiplex recombinase polymerase amplification (RPA) assay as a scientific basis for formulating control strategies for bovine theileriosis. Specificity was evaluated using DNA from related piroplasm species, while analytical sensitivity was calculated using a long fragment of the enolase gene. A total of 188 blood samples were collected on FTA cards (Whatman®) from tick-infested asymptomatic breeds of cattle (Bos indicus, Bos taurus, and Bos indicus × Bos taurus) in the study district. Finally, infections with of T. annulata and T. orientalis were detected using the multiplex RPA and compared with the conventional multiplex polymerase chain reaction (PCR). The multiplex RPA specifically amplified 282-bp and 229-bp fragments of the enolase gene from T. annulata and T. orientalis and had no cross-reaction with other piroplasm species. It was determined that 45 (23.9%) and 5 (2.6%) out of 188 blood samples were positive for T. annulata and T. orientalis, respectively, when examined using RPA. Multiplex PCR detection indicated that 32 (17.0%) and 3 (1.6%) blood samples were positive for T. annulata and T. orientalis, respectively. In the present study, a specific RPA method was developed for simultaneous differentiation and detection of T. annulata and T. orientalis infections and used for the first time for the detection of the two bovine Theileria infections.

Endemic, exotic and novel apicomplexan parasites detected during a national study of ticks from companion animals in Australia

BackgroundApicomplexan tick-borne pathogens that cause disease in companion animals include species of Babesia Starcovici, 1893, Cytauxzoon Neitz & Thomas, 1948, Hepatozoon Miller, 1908 and Theileria Bettencourt, Franca & Borges, 1907. The only apicomplexan tick-borne disease of companion animals that is known to occur in Australia is babesiosis, caused by Babesia canis vogeli Reichenow, 1937 and Babesia gibsoni Patton, 1910. However, no molecular investigations have widely investigated members of Apicomplexa Levine, 1980 in Australian ticks that parasitise dogs, cats or horses, until this present investigation.ResultsTicks (n = 711) removed from dogs (n = 498), cats (n = 139) and horses (n = 74) throughout Australia were screened for piroplasms and Hepatozoon spp. using conventional PCR and Sanger sequencing. The tick-borne pathogen B. vogeli was identified in two Rhipicephalus sanguineus Latreille ticks from dogs residing in the Northern Territory and Queensland (QLD). Theileria orientalis Yakimov & Sudachenkov, 1931 genotype Ikeda was detected in three Haemaphysalis longicornis Neumann ticks from dogs in New South Wales. Unexpectedly, the exotic tick-borne pathogen Hepatozoon canis James, 1905 was identified in an Ixodes holocyclus Neumann tick from a dog in QLD. Eight novel piroplasm and Hepatozoon species were identified and described in native ticks and named as follows: Babesia lohae n. sp., Babesia mackerrasorum n. sp., Hepatozoon banethi n. sp., Hepatozoon ewingi n. sp., Theileria apogeana n. sp., Theileria palmeri n. sp., Theileria paparinii n. sp. and Theileria worthingtonorum n. sp. Additionally, a novel cf. Sarcocystidae sp. sequence was obtained from Ixodes tasmani Neumann but could not be confidently identified at the genus level.ConclusionsNovel species of parasites in ticks represent an unknown threat to the health of companion animals that are bitten by these native tick species. The vector potential of Australian ticks for the newly discovered apicomplexans needs to be assessed, and further clinical and molecular investigations of these parasites, particularly in blood samples from dogs, cats and horses, is required to determine their potential for pathogenicity.

Piroplasmoses bovines dans les provinces de Skikda et d’Oum El Bouaghi (nord-est de l’Algérie) : étude épidémiologique et estimation des pertes de production laitière

Au cours d’une enquête menée dans 88 fermes de deux provinces du nord-est de l’Algérie – Oum El Bouaghi et Skikda –, 89 cas cliniques de piroplasmoses, soit un taux d’infection de 11,6 % des bovins examinés, ont été enregistrés entre mai et septembre 2011 dans 86,4 % des fermes visitées. Trois espèces de piroplasmes ont été identifiées dans les étalements sanguins colorés au Giemsa : Theileria annulata (chez 94,0 % des bovins malades), Babesia bovis (33,7 %) et Babesia bigemina (3,4 %), avec 31 % de co-infections. Il n’y avait pas de différence significative de prévalence entre les deux provinces ni entre les races bovines. La theilériose tropicale a provoqué une baisse significative de la production laitière, estimée à 319 litres par vache infectée pendant les deux mois suivant la maladie. Sept espèces de tiques ont été collectées dans les fermes pendant l’étude. Le pic d’infestation a été observé en juillet pour Hyalomma scupense (syn. H. detritum), H. lusitanicum, H. anatolicum, Rhipicephalus bursa et R. turanicus, et en août pour H. marginatum et H. excavatum. Une corrélation positive a été observée entre les cas cliniques de theilériose tropicale bovine et la charge parasitaire en H. scupense, d’une part, et entre les cas cliniques de babésioses et la charge parasitaire en R. bursa, d’autre part.

Development of an indirect ELISA based on the recombinant Spm2 protein for detection of tropical theileriosis

Tropical theileriosis, caused by Theileria annulata, is distributed worldwide and causes great economic losses in dairy. The reliable diagnostic method is critical for prevention and control of the disease. In this study, a sporozoite and macroschizont gene 2 (spm2) protein from T. annulata was used to develop an indirect ELISA for tropical theileriosis. Specificity test showed that there were no cross-reactions with antibodies raised against other bovine piroplasm species using ELISA or western blotting. The specificity and sensitivity were 98.4% and 98.7%, respectively, with a threshold of 35.5% of the specific mean antibody rate (AbR). Furthermore, a total of 196 field sera samples collected from Xinjiang and Gansu provinces were detected by the spm2 ELISA and IFA. The results obtained with the spm2 ELISA and IFA in this study had the moderate agreement. The average positive rates of T. annulata sera samples detected in the present study were close to the prevalence of previous reports in these endemic areas. This indicated that the Spm2 ELISA could be used as a reliable diagnostic tool for serological survey of T. annulata infection in areas where Theileria parva is not present.

Development and validation of a duplex real-time PCR assay for the diagnosis of equine piroplasmosis

BackgroundEquine piroplasmosis (EP) is an economically significant infection of horses and other equine species caused by the tick-borne protozoa Theileria equi and Babesia caballi. The long-term carrier state in infected animals makes importation of such subclinical cases a major risk factor for the introduction of EP into non-enzootic areas. Regulatory testing for EP relies on screening of equines by serological methods. The definitive diagnosis of EP infection in individual animals will benefit from the availability of sensitive direct detection methods, for example, when used as confirmatory assays for non-negative serological test results. The objectives of this study were to develop a real-time quantitative polymerase chain reaction (qPCR) assay for simultaneous detection of both agents of EP, perform comprehensive evaluation of its performance and assess the assay’s utility for regulatory testing.ResultsWe developed a duplex qPCR targeting the ema-1 gene of T. equi and the 18S rRNA gene of B. caballi and demonstrated that the assay has high analytical sensitivities for both piroplasm species. Validation of the duplex qPCR on samples from 362 competitive enzyme-linked immunosorbent assay (cELISA)-negative horses from Canada and the United States yielded no false-positive reactions. The assay’s performance was further evaluated using samples collected from 430 horses of unknown EP status from a highly endemic area in Brazil. This set of samples was also tested by a single-target 18S rRNA qPCR for T. equi developed at the OIE reference laboratory for EP in Japan, and a previously published single-target 18S rRNA qPCR for B. caballi whose oligonucleotides we adopted for use in the duplex qPCR. Matching serum samples were tested for antibodies to these parasites using cELISA. By the duplex qPCR, T. equi-specific 18S rRNA qPCR and cELISA, infections with T. equi were detected in 87.9% (95% confidence interval, CI: 84.5–90.7%), 90.5% (95% CI: 87.3–92.3%) and 87.4% (95% CI: 84.0–90.2%) of the horses, respectively. The B. caballi prevalence estimates were 9.3% (95% CI: 6.9–12.4%) by the duplex qPCR and 7.9% (95% CI: 5.7–10.9%) by the respective single-target qPCR assay. These values were markedly lower compared to the seroprevalence of 58.6% (95% CI: 53.9–63.2%) obtained by B. caballi-specific cELISA. The relative diagnostic sensitivity of the duplex qPCR for T. equi was 95.5%, as 359 of the 376 horses with exposure to T. equi confirmed by cELISA had parasitemia levels above the detection limit of the molecular assay. In contrast, only 39 (15.5%) of the 252 horses with detectable B. caballi-specific antibodies were positive for this piroplasm species by the duplex qPCR.ConclusionsThe duplex qPCR described here performed comparably to the existing single-target qPCR assays for T. equi and B. caballi and will be more cost-effective in terms of results turnaround time and reagent costs when both pathogens are being targeted for disease control and epidemiological investigations. These validation data also support the reliability of the ema-1 gene-specific oligonucleotides developed in this study for confirmatory testing of non-negative serological test results for T. equi by qPCR. However, the B. caballi-specific qPCR cannot be similarly recommended as a confirmatory assay for routine regulatory testing due to the low level of agreement with serological test results demonstrated in this study. Further studies are needed to determine the transmission risk posed by PCR-negative equines with detectable antibodies to B. caballi.

Molecular Detection of Theileria annulata in Cattle from Different Regions of Punjab, Pakistan, by Using Recombinase Polymerase Amplification and Polymerase Chain Reaction.

Piroplasmosis is one of the most important diseases of livestock, constraining optimal production and leading to economic loss. This study was carried out to detect Theileria annulata by using 2 different molecular techniques: recombinase polymerase amplification (RPA) and conventional polymerase chain reaction (PCR). Blood samples were collected from 274 ticks infesting asymptomatic cattle from several counties in the Chakwal, Faisalabad, and Jhang districts of Punjab Province in Pakistan by using FTA cards. After extraction of genomic DNA, each sample was subjected to RPA optimized to amplify a 281-bp fragment of the Enolase gene for T. annulata. The specificity of the test was confirmed using positive DNA samples of related piroplasm species, whereas the analytical sensitivity was calculated using different serial dilutions of a long fragment of the same gene. The RPA results were positive for 48 (17.51%) of 274 samples. All 274 samples were screened using conventional PCR, and 21 (7.66%) samples were positive for T. annulata. All the samples that were RPA positive but PCR negative were sequenced, which confirmed the results of RPA. The highest positive rate was found in Chakwal district, followed by Faisalabad and Jhang districts. This study demonstrates the application of highly sensitive and specific rapid diagnostic methods for T. annulata to a regional screening program. This is the first report of tick-borne disease from Pakistan by using RPA.

Deep-sequencing to resolve complex diversity of apicomplexan parasites in platypuses and echidnas: Proof of principle for wildlife disease investigation

The short-beaked echidna (Tachyglossus aculeatus) and the platypus (Ornithorhynchus anatinus) are iconic egg-laying monotremes (Mammalia: Monotremata) from Australasia. The aim of this study was to demonstrate the utility of diversity profiles in disease investigations of monotremes. Using small subunit (18S) rDNA amplicon deep-sequencing we demonstrated the presence of apicomplexan parasites and confirmed by direct and cloned amplicon gene sequencing Theileria ornithorhynchi, Theileria tachyglossi, Eimeria echidnae and Cryptosporidium fayeri. Using a combination of samples from healthy and diseased animals, we show a close evolutionary relationship between species of coccidia (Eimeria) and piroplasms (Theileria) from the echidna and platypus. The presence of E. echidnae was demonstrated in faeces and tissues affected by disseminated coccidiosis. Moreover, the presence of E. echidnae DNA in the blood of echidnas was associated with atoxoplasma-like stages in white blood cells, suggesting Hepatozoon tachyglossi blood stages are disseminated E. echidnae stages. These next-generation DNA sequencing technologies are suited to material and organisms that have not been previously characterised and for which the material is scarce. The deep sequencing approach supports traditional diagnostic methods, including microscopy, clinical pathology and histopathology, to better define the status quo. This approach is particularly suitable for wildlife disease investigation.

Molecular identification and characterization of piroplasm species in Hokkaido sika deer (Cervus nippon yesoensis), Japan

Babesia and Theileria species are tick-borne protozoan parasites that have a veterinary and zoonotic importance. In order to investigate the prevalence and genetic diversity of these parasites, a total of 269 sika deer blood DNA samples collected from Hokkaido, Japan, were examined for Babesia and Theileria species by touch-down PCR targeting the 18S rRNA gene. Reverse line blot (RLB) hybridization was then used to detect 12 piroplasm species. The results revealed that 95.5% (257/269), 94.1% (253/269), 14.1% (38/269), 87.7% (236/269) and 11.5% (31/269) of the examined PCR products hybridized with the probes which were designed to detect all Babesia and Theileria spp., all Theileria spp., all Babesia spp., Theileria sp. Thrivae and Babesia divergens-like, respectively. The 18S rRNA gene partial sequences were divided into Theileria sp. Thrivae, T. capreoli, B. divergens-like and an undescribed Babesia species. This study showed the first detection of the undescribed Babesia sp. from Japan. Therefore, more studies are required to understand the ecology of the newly detected tick-borne pathogens in Hokkaido.

A survey of canine haemoprotozoan parasites from Turkey, including molecular evidence of an unnamed Babesia.

Canine tick-borne apicomplexan parasites have emerged in recent years, showing a wider geographic distribution and increased global prevalence. A reverse line blot assay was performed on 219 blood samples collected from domestic dogs for simultaneous detection of all named canine piroplasm species as well as Hepatozoon canis. Ten samples hybridized to the Theileria/Babesia and Babesia catch all probes but did not hybridize to any species-specific probe tested, suggesting the presence of an unrecognized Babesia species or genotype. Sequencing results showed 91.5%, 91.9%, 92.4%, 92.4%, and 89.2% similarity to B. canis, B. vogeli, B. rossi, B. gibsoni, and B. conradae, respectively. The highest homology (98.1-98.5%) observed was with unnamed Babesia sp. isolates (Ludhiana and Malbazar) described in dogs, Babesia sp. of buffalo origin, Babesia sp. Kashi 2, and Babesia orientalis, along with Babesia occultans of cattle origin. The partial cox1 sequence indicated that this isolate was most similar to Babesia sp. 1 HG-2012, with an identity of 86.5%. The survey revealed high prevalence of haemoprotozoans in domestic dogs (57.5%, CI 50.7-64.2), with Hepatozoon canis the most prevalent (54.3%, CI 47.5-61.117%), followed by Babesia sp. (4.6%, CI 2.2-8.2), B. vogeli (1.4%; CI 0.3-3.9), and B. canis (0.4%, CI 0-2.5). Combined infection of Hepatozoon canis and Babesia sp. was detected in five (2.3%, CI 0.7-5.2) samples and of H. canis and B. vogeli in two (0.9%, CI 0.1-3.2) dogs. The study contributes insight into the distribution and phylogenetic diversity of canine piroplasms in Turkey.

Identification of 12 Piroplasms Infecting Ten Tick Species in China Using Reverse Line Blot Hybridization.

Piroplasmosis, a disease of domestic and wild animals, is caused by tick-borne protozoa in the genera of Theileria and Babesia. There is limited information available about the prevalence of piroplasmosis in ticks in China, and to assess the potential threat of piroplasmosis in China, we investigated the infections of ovine and bovine Babesia and Theileria species in ticks collected from cattle, yaks, sheep, horses, and camels in several regions of China where tick-borne diseases have been reported. In total, 652 ticks were collected from the animals in 6 provinces of China. Babesia spp. and Theileria spp. were detected with a PCR-RLB method and identified by sequencing. Overall, 157 ticks (24.1%) were infected with 5 Babesia and 4 Theileria species. Among tested tick samples, 134 (20.6%) were single infections with 1 of 7 piroplasm species, with Theileria annulata (118/652, 18.1%) being dominant. Only 23 (3.5%) tick samples were double or triple infected, Theileria luwenshuni and Theileria sinensis (18/652, 2.8%) were frequently observed in co-infections. Some piroplasm species were carried by ticks that were not previously reported to be vectors.

Redescription of Babesia ardeae Toumanoff, 1940, a parasite of Ardeidae, including molecular characterization.

Among the actual 16 identified and thought to be valid avian piroplasm species, certain parasites are only known from their original description with no subsequent report. Babesia ardeae Toumanoff, 1940 is one of them. It was described from a single sacrificed gray heron (Ardea cinerea) from Vietnam and had never been reported since this date despite inhabiting a very common avian host. The present study reports the accidental rediscovery of B. ardeae from an injured wild gray heron rescued in Singapore. This report confirms the existence of this parasite species in the gray heron from Southeast Asia, highlights the similarities with the original description, provides additional morphologic and morphometric data, and designates neotype material for B. ardeae. Additionally, the report also furnishes the first molecular data about B. ardeae with the amplification and sequencing of the near-full-length 18S rRNA gene sequence and its comparison with the other available sequences of avian piroplasms. Phylogenetic analysis based on this gene was performed to study the relationship of B. ardeae with the other piroplasms from mammals and birds and indicated that B. ardeae appears as a brother group of a clade formed by several avian piroplasm species isolated from seabirds, altogether clustering in a well-supported clade related to the "Babesia duncani group" and protothelerids. Scarcity of this parasite is discussed as well as its taxonomy in relation to the conundrum about the systematics of piroplasms.

Anaplasma phagocytophilum and Babesia spp. in roe deer (Capreolus capreolus), fallow deer (Dama dama) and mouflon (Ovis musimon) in Germany

Infections with the tick-borne pathogens Anaplasma phagocytophilum and Babesia spp. can cause febrile disease in several mammalian species, including humans. Wild ruminants in Europe are suggested to serve as reservoir hosts for particular strains or species of these pathogens. The aims of this study were to investigate the occurrence of A. phagocytophilum and Babesia spp. in roe deer (Capreolus capreolus), fallow deer (Dama dama) and mouflon (Ovis musimon orientalis) in Germany, and the diversity and host association of genetic variants of A. phagocytophilum and Babesia species. From 2009 to 2010, 364 spleen samples from 153 roe deer, 43 fallow deer and 168 mouflon from 13 locations in Germany were tested for DNA of A. phagocytophilum and Babesia spp. by real-time PCR or conventional PCR, respectively. Variants of A. phagocytophilum were investigated with a nested PCR targeting the partial 16S rRNA gene, and species of piroplasms were identified by sequencing. DNA of A. phagocytophilum was detected in 303 (83.2%) samples: roe deer, 96.1% (147/153); fallow deer, 72.1% (31/43); and mouflon, 74.4% (125/168). Sequence analysis of 16S rRNA-PCR products revealed the presence of nine different genetic variants. DNA of Babesia spp. was found in 113 (31.0%) samples: roe deer, 62.8% (96/153); fallow deer, 16.3% (6/43); and mouflon, 6.5% (11/168). Babesia capreoli, Babesia sp. EU1 (referred to also as B. venatorum), B. odocoilei-like and a Theileria species were identified. Co-infections with A. phagocytophilum and Babesia spp. were detected in 30.0% of the animals which were tested positive for A. phagocytophilum and/or Babesia spp. Roe deer had a significantly higher percentage of co-infections (60.8%), followed by fallow deer (14.0%) and mouflon (6.5%). Thus, the results suggest that roe deer plays a key role in the endemic cycles of the pathogens investigated.