Piroplasm Species Research Articles

Molecular-phylogenetic analyses of Babesia and Theileria species from small mammals and their ticks in northern China suggest new reservoirs of bovine and equine piroplasms

Babesia and Theileria species (Apicomplexa: Piroplasmida) are tick-borne protozoan parasites that can cause mild to severe infection in humans, wildlife, livestock and companion animals. To date, reports on the molecular study of piroplasms from wild living small mammals and their ticks are still limited, especially in Asia. This study encompassed an extensive survey involving 907 liver samples and 145 ixodid ticks from 16 different species of small mammals (Rodentia, Lagomorpha, Eulipotyphla). These were collected in 13 cities and counties in northern China. DNA extracts from these samples were screened for the presence of piroplasm 18S rRNA gene. Samples that tested positive were further evaluated for other genetic markers of piroplasms, including the cox1 gene and the ITS1-5.8S rDNA-ITS2 region. Several piroplasm species were identified, including Babesia sp. tavsan2, Babesia occultans, Theileria sp. Xinjiang, Theileria equi, and Theileria sp. Kalecik. Among these, Theileria sp. Xinjiang was shown to be the most prevalent. Importantly, Babesia sp. tavsan2 was identified in the tick Rhipicephalus sanguineus from the Yarkand hare and Theileria sp. Kalecik in Hyalomma asiaticum from the long-eared hedgehog, in line with the detection of these pathogens in tissue samples of the relevant hosts. This study further disclosed the presence of DNA from B. occultans and T. equi, typically found in cattle and horses respectively, with an additional discovery in small mammals. Moreover, Theileria sp. Kalecik, which was first detected in small-sized mammals, and Babesia sp. tavsan2, were both reported for the first time in China.

Tick-borne piroplasms and trypanosomes incidentally detected in eastern grey kangaroos (Macropus giganteus) during a mortality and morbidity event in southern New South Wales, Australia

Tick-borne haemoparasites, including piroplasms and trypanosomes, are almost ubiquitous in Australian wildlife, with some associated with health impacts to individual animals and declining wildlife populations. An array of ecologically distinct piroplasm and trypanosome species occur throughout Australia although many of these species and their sylvatic ecologies are poorly characterised. Between May 2022 and October 2023, an anecdotally reported localised eastern grey kangaroo (Macropus giganteus) morbidity/mortality event occurred in coastal southern New South Wales, Australia, characterised by animals presenting with blindness, emaciation, lethargy, ataxia, and astasia. Here we used molecular techniques to identify tick-borne piroplasms (Babesia and Theileria) and trypanosomes in affected animals. Blood (n = 89) and liver (n = 19) samples were collected after the humane euthanasia of wild animals due to welfare concerns, and brief notes on the animal's health were recorded. In total, 20 (22.5%) animals were infected with tick-borne haemoparasites, including a novel Theileria sp. nov. (14, 15.7%), Babesia macropus (2, 2.2%), Trypanosoma gilletti (5, 5.6%), and Trypanosoma vegrandis (1, 1.1%). Liver samples were also screened for Wallal and Warego viruses due to animals' blindness, but were negative. This is the first report of T. gilletti and T. vegrandis in eastern grey kangaroos, although they have been previously reported in high numbers in ticks which commonly parasites this host. The novel Theileria sp. was previously reported in questing Ixodes holocyclus and in ticks from an opportunistically collected eastern grey kangaroo and red-necked wallaby (Notamacropus rufogriseus). However, we show for the first time this Theileria sp. can occur widely in eastern grey kangaroos. Ultimately, this small study did not intend, and is not able to draw inference regarding the pathogenicity of these haemoparasites to eastern grey kangaroos and it is likely that other factors, such as chronic Phalaris grass toxicity, had a role in this localised mortality/morbidity event.

Comparative genome-wide identification and characterization of SET domain-containing and JmjC domain-containing proteins in piroplasms

BackgroundSET domain-containing histone lysine methyltransferases (HKMTs) and JmjC domain-containing histone demethylases (JHDMs) are essential for maintaining dynamic changes in histone methylation across parasite development and infection. However, information on the HKMTs and JHDMs in human pathogenic piroplasms, such as Babesia duncani and Babesia microti, and in veterinary important pathogens, including Babesia bigemina, Babesia bovis, Theileria annulata and Theileria parva, is limited.ResultsA total of 38 putative KMTs and eight JHDMs were identified using a comparative genomics approach. Phylogenetic analysis revealed that the putative KMTs can be divided into eight subgroups, while the JHDMs belong to the JARID subfamily, except for BdJmjC1 (BdWA1_000016) and TpJmjC1 (Tp Muguga_02g00471) which cluster with JmjC domain only subfamily members. The motifs of SET and JmjC domains are highly conserved among piroplasm species. Interspecies collinearity analysis provided insight into the evolutionary duplication events of some SET domain and JmjC domain gene families. Moreover, relative gene expression analysis by RT‒qPCR demonstrated that the putative KMT and JHDM gene families were differentially expressed in different intraerythrocytic developmental stages of B. duncani, suggesting their role in Apicomplexa parasite development.ConclusionsOur study provides a theoretical foundation and guidance for understanding the basic characteristics of several important piroplasm KMT and JHDM families and their biological roles in parasite differentiation.

Diversity of Babesia spp. in skunks from selected states in the United States of America.

Babesia species are intraerythrocytic protozoan parasites that infect a variety of hosts. The goal of this study was to evaluate the piroplasm species present in skunks in various states in the United States and determine whether there was any geographic variation. Spleen, whole blood, or blood on filter paper were received from Pennsylvania, Kentucky, North Carolina, South Carolina, Georgia, Missouri, Louisiana, Texas, Kansas, and California, and were tested for Babesia sp. We tested four species of skunks including striped skunk (Mephitis mephitis, n=72), eastern spotted skunk (Spilogale putorius, n=28), western spotted skunk (Spilogale gracilis, n=15), and hog-nosed skunk (Conepatus leuconotus, n=11). A PCR assay targeting the 18S rRNA region and cox1 region were used to determine if skunks were infected with piroplasms and for phylogenetic analyses. A total of 48.4% (61/126) of skunks tested positive for a Babesia species. Both the 18S and cox1 analysis supported a skunk-specific Babesia microti-like sp. of carnivores as well as a species in the B. microti complex that is phylogenetically unique from both B. microti of humans and the B. microti-like sp. of carnivores. In the 18S analysis, there was a third species of Babesia in hog-nosed skunks in the western piroplasm group. This study shows that at least three species of piroplasms occur in skunk species in the United States and further highlights the importance of phylogenetic analyses and the use of multiple gene targets when studying piroplasms.

Babesia ovis secreted antigen-1 is a diagnostic marker during the active Babesia ovis infections in sheep.

Ovine babesiosis caused by Babesia ovis is an economically significant disease. Recently, a few B. ovis-specific proteins, including recombinant B. ovis secreted antigen-1 (rBoSA1), have been identified. Immunological analyses revealed that rBoSA1 resides within the cytoplasm of infected erythrocytes and exhibits robust antigenic properties for detecting anti-B. ovis antibodies. This protein is released into the bloodstream during the parasite's development. It would be possible to diagnose active infections by detecting this secretory protein. For this purpose, a rBoSA1-specific polyclonal antibody-based sandwich ELISA was optimized in this study. Blood samples taken from the naturally (n: 100) and experimentally (n: 15) infected sheep were analyzed for the presence of native BoSA1. The results showed that native BoSA1 was detectable in 98% of naturally infected animals. There was a positive correlation between parasitemia level in microscopy and protein density in sandwich ELISA. Experimentally infected animals showed positive reactions from the first or second day of inoculations. However, experimental infections carried out by Rhipicephalus bursa ticks revealed the native BoSA1 was detectable from the 7th day of tick attachment when the parasite began to be seen microscopically. Sandwich ELISA was sensitive enough to detect rBoSA1 protein at a 1.52 ng/ml concentration. Additionally, no serological cross-reactivity was observed between animals infected with various piroplasm species, including Babesia bovis, B. bigemina, B. caballi, B. canis, B. gibsoni, Theileria equi, and T. annulata. Taken collectively, the findings show that the rBoSA1-specific polyclonal antibody-based sandwich ELISA can be successfully used to diagnose clinical B. ovis infections in sheep at the early stage.

First Molecular Evidence of Babesia vogeli, Babesia vulpes, and Theileria ovis in Dogs from Kyrgyzstan.

Tick-borne parasitic diseases cause mild to severe infections among vertebrate hosts, including dogs. Species in the genus Babesia are important tick-borne pathogens and have worldwide distributions. Although there are data on the prevalence and distribution of Babesia species among dogs around the world, there is no information available in Kyrgyzstan, according to a literature review. In this study, 337 dogs were screened by nested PCR for the presence of the 18S small subunit ribosomal RNA (18S SSU rRNA) gene of piroplasm species. Overall prevalence was 6.23% (21/337) for Babesia/Theileria spp. DNA sequencing of positively tested samples revealed that eighteen samples were infected with Babesia vogeli (B. vogeli) (5.34%), two samples with B. vulpes (0.59%), and one sample with Theileria ovis (T. ovis) (0.29%). The phylogenetic analyses and nucleotide sequences in contrast with those present in GenBank revealed that two nucleotide substitutions (594th and 627th) were found between B. vogeli isolates, including ours, indicating that the mutation is relatively rare. The sequences of other pathogens obtained in this study confirmed 100% nucleotide identity with B. vulpes and T. ovis sequences in GenBank. To the best of our knowledge, B. vogeli, B. vulpes, and T. ovis were detected for the first time in dogs from Kyrgyzstan, and it is thought that results will contribute to the understanding of the epidemiology of canine tick-borne pathogens in the country.

Small Ruminant Piroplasmosis: High Prevalence of Babesia aktasi n. sp. in Goats in Türkiye.

Small ruminant piroplasmosis is the hemoparasitic infection of sheep and goats caused by Babesia and Theileria species responsible for clinical infections with high mortality outcomes. The disease is transmitted by ixodid ticks and prevalent in the tropical and subtropical regions of the world, including Türkiye. A prevalence survey, using molecular methods, is conducted in this study to determine the frequency of newly defined Babesia aktasi n. sp. and other tick-borne piroplasm species in small ruminants in Turkiye. A total of 640 blood samples from sheep (n = 137) and goats (n = 503) were analyzed by nested PCR-based reverse line blot (RLB) hybridization. The results show that 32.3% (207/640) of apparently healthy, small ruminants are infected with three Theileria and two Babesia species. Babesia aktasi n. sp. was the most prevalent species in goats, with 22.5% of samples being positive, followed by B. ovis (4%), T. ovis (2.8%), T. annulata (2.6%), and Theileria sp. (0.6%). None of the sheep samples were positive for Babesia aktasi n. sp.; however, 51.8% were infected with T. ovis. In conclusion, the findings reveal that B. aktasi n. sp. is highly prevalent in goats, but absent in sheep. In future studies, experimental infections will determine whether B. aktasi n. sp. is infectious to sheep, as well as its pathogenicity in small ruminants.

A survey of piroplasms in white-tailed deer (Odocoileus virginianus) in the southeastern United States to determine their possible role as Theileria orientalis hosts.

In 2017, clinical disease and mortality in cattle associated with Theileria orientalis Ikeda was reported in Virginia, U.S. The exotic tick, Haemaphysalis longicornis, is a competent vector for this species. White-tailed deer (Odocoileus virginianus) are commonly infested with H. longicornis in the eastern U.S. and are also infected with several genotypes of piroplasms such as a Theileria sp. (often called Theileria cervi-like), Babesia odocoilei, and Babesia sp. H10. However, it is currently unknown if deer are susceptible to T. orientalis and can act as potential hosts. In this study, we tested 552 white-tailed deer samples from the southeastern U.S. to determine the presence of T. orientalis. We used a PCR-RFLP to test 293 (53%) of these samples to distinguish between piroplasm genera. A total of 189 white-tailed deer were positive with Theileria, 47 were positive with Babesia, and 57 did not amplify. Because this assay does not determine species, we sequenced 30 random samples targeting a fragment of the 18S rRNA gene. Although a high diversity of Theileria and Babesia spp. were detected, none were T. orientalis. All 552 samples were then screened with a T. orientalis specific real-time PCR protocol, but none were positive for T. orientalis. Our data suggests that white-tailed deer are commonly infected with piroplasm species but not T. orientalis.

Tick distribution and detection of Babesia and Theileria species in Eastern and Southern Kazakhstan

Piroplasmosis is an economically important tick-borne disease worldwide. However, little is known about the presence of Babesia spp. and Theileria spp. in ticks in Eastern and Southern Kazakhstan (ESK). During 2016 – 2019, adult ticks (at 26 sampling sites in 16 districts of 5 oblasts in ESK) were collected. Tick species were identified according to morphological and molecular characteristics. Two fragments (487 bp and 438 bp) of 18S ribosomal RNA (18S rRNA) were used to determine piroplasm species in representative 698 ticks. The genotype characteristics of Babesia caballi and Theileria equi were further analyzed by longer 18S rRNA gene fragments. A total of 6107 adult ticks (4558 parasitizing ticks and 1549 off-host ticks), including 4665 hard ticks and 1442 soft ticks, were collected from their natural hosts (cattle, horses, sheep, camels, shepherd dogs and hedgehogs) and the surrounding environment, respectively. Among the hard tick species, Dermacentor marginatus (62.59%, 2920/4665) was the most abundant, followed by Hyalomma asiaticum (19.36%, 903/4665) and Hyalomma detritum (9.95%, 464/4665). All soft ticks were identified as Argas persicus. 16S ribosomal DNA (16S rDNA) phylogenic analysis showed that several tick species in Kazakhstan, as exemplified by Haemaphysalis erinacei and D. marginatus, clustered together with conspecific ticks reported from China. Five species of piroplasms, i.e. Babesia occultans, Babesia caballi, Theileria ovis, Theileria annulata and Theileria equi, were detected in 698 representative ticks. Genotype E of T. equi in Almaty, and genotype A of B. caballi in Almaty and South Kazakhstan were identified.

First report of Theileria buffeli/Theileria orientalis group and identification of piroplasms via Nested PCR-based RLB Hybridization assay in zebu cattle in the Western Highlands of Cameroon

Piroplasms infections are tick-borne diseases caused by haemoparasite of the genus Theileria or Babesia. They have a great impact on livestock production, especially cattle in sub-Saharan countries. However, data on the prevalence of bovine piroplasms and their genetic diversity are scanty in Cameroon. This study was aimed at highlight the species composition and determine the prevalence of piroplasms infecting cattle in the Western Highlands of Cameroon. To achieve this aim, blood samples from a total of 162 cattle were collected and examined using Reverse Line Blot hybridization (RLB) assay. The amplified hypervariable V4 region of the 18S rRNA gene of bovine piroplasms species, including Theileria parva, T. annulata, T mutans, T. velifera, T. buffeli/T. orientalis, T. taurotragi, Theileria sp (buffalo), Babesia bovis, B. bigemina, B. divergens, B. major and B. occultans was hybridized against species-specific probes. RLB hybridization assay revealed the presence of four piroplasms species with the overall prevalence of infection of 82.1%. Theileria velifera (71.6%) was the most prevalent species followed by Theileria mutans (43.21%), Theileria buffeli/T. orientalis (5.55%) and Babesia bigemina (3.7%). However, the study provided the first molecular evidence for the presence of T. buffeli/T. orientalis group species in cattle in Cameroon. Higher overall prevalence of infection of tick-borne pathogens was observed in this study area as well as the increase in prevalence and widespread of T. velifera and the observance of a new species of piroplasms. These results are an indication that special attention should be given to epizootiological investigations alongside well-adopted control programs.

Eco-epidemiology of equine piroplasmosis and its associated tick vectors in Europe: A systematic literature review and a meta-analysis of prevalence.

When studying a vector-borne disease, an eco-epidemiological approach is vital for a comprehensive understanding of how the pathogen circulates amongst populations. Equine piroplasmosis (EP), a tick-borne disease caused by the protozoans Babesia caballi and Theileria equi, is endemic in the Mediterranean basin of Europe and causes both animal health and economic issues for the equine sector. With no vaccine available, defining the episystem of the disease can help to identify which components of the host-pathogen-vector-environment system to target to improve preventive measures. In this systematic literature review, we collected relevant data on the eco-epidemiology of EP in Europe. The 62 studies remaining after the selection procedure explored potential vectors, indicators of parasite circulation and putative risk factors of EP. Eight hard tick species were identified as potential vectors of one or both piroplasm species. Meta-analyses were then conducted on prevalence and seroprevalence data in equids in European countries, demonstrating an estimated seroprevalence of 30% and 8% and prevalence of 25% and 2% for T. equi and B. caballi, respectively. Finally, herd management practices and environmental risk factors analysed in studies showed no real consensus between studies, but revealed a general trend highlighting age and exposure to ticks as risk factors, and vaccination as a protective factor. Through this study, we point out that only a few studies have focused on disease management practices and even fewer have studied the effect of environmental parameters on equid infections. Further investigation in these areas is required to better characterize the eco-epidemiology of EP and risk factors associated with this disease.

SPECIFIC MOLECULAR DETECTION OF PIROPLASMS AND CHARACTERIZATION OF β-TUBULIN FOR A NOVEL BABESIA SPECIES IN SIKA DEER (CERVUS NIPPON YESOENSIS).

Piroplasms, which include Babesia spp. and Theileria spp., are protozoan parasites carried by ticks and commonly cause disease in animals and humans. Those caused by Babesia spp. manifest as fever, anemia, and hemoglobinuria, while Theileria spp. can lead to high fever, diarrhea, and lymphadenopathy. Recently, Theileria capreoli and an undescribed Babesia sp. were detected for the first time in sika deer (Cervus nippon yesoensis) from Hokkaido; however, there is limited information available on their epidemiology in Japan. Here, a touchdown polymerase chain reaction and reverse line blot hybridization were used to perform an epidemiological survey of T. capreoli and Babesia sp. using blood samples from 82 sika deer in Hokkaido, Japan. This was followed by partial sequencing and phylogenetic analysis of the 18S rRNA and β-tubulin genes to characterize both piroplasm species. A total of 43 (52.4%) and 3 (3.7%) of the sika deer were positive for T. capreoli and Babesia sp., respectively. The β-tubulin gene partial sequences for Babesia sp. were distinct from those of Babesia spp. in GenBank. Phylogenetic analysis showed that the unknown Babesia sp. is more closely related to B. bigemina and B. ovata than other Babesia spp. based on the β-tubulin gene. Further studies are required to understand the ecology of these tick-borne pathogens in Japan.

Targeted Next-Generation Sequencing and Informatics as an Effective Tool to Establish the Composition of Bovine Piroplasm Populations in Endemic Regions

Protists of the genera Babesia and Theileria (piroplasms) cause some of the most prevalent and debilitating diseases for bovines worldwide. In this study, we established and used a next-generation sequencing-informatic approach to explore the composition of Babesia and Theileria populations in cattle and water buffalo in a country (Pakistan) endemic for these pathogens. We collected individual blood samples from cattle (n = 212) and water buffalo (n = 154), extracted genomic DNAs, PCR-amplified the V4 hypervariable region of 18S small subunit rRNA gene from piroplasms, sequenced amplicons using Illumina technology, and then analysed data using bioinformatic platforms. The results revealed piroplasms in 68.9% (252/366) samples, with overall occurrence being markedly higher in cattle (85.8%) than in water buffaloes (45.5%). Babesia (B.) occultans and Theileria (T.) lestoquardi-like species were recorded for the first time in Pakistan, and, overall, T. annulata was most commonly detected (65.8%) followed by B. bovis (7.1%), B. bigemina (4.4%), and T. orientalis (0.5%), with the genetic variability within B. bovis being pronounced. The occurrence and composition of piroplasm species varied markedly across different agro-ecological zones. The high detection of T. annulata in asymptomatic animals suggested a relatively high level of endemic stability of tropical theileriosis in the bovine population.

Utilizing attached hard ticks as pointers to the risk of infection by Babesia and Theileria species in sika deer (Cervus nippon yesoensis), in Japan.

Ticks are hematophagous ectoparasites that have a significant impact on their animal hosts. Along with mosquitoes, they are the main arthropod vectors of disease agents in domestic animals, wildlife and humans. To investigate the occurrence and prevalence of piroplasmids in ticks, DNA was extracted from 519 hard ticks collected from 116 hunted Hokkaido sika deer (Cervus nippon yesoensis). The success of the DNA extraction was confirmed by touchdown PCR targeting the mitochondrial 16S rDNA gene of ticks. Touchdown PCR and reverse line blot (RLB) hybridization targeting the 18S rRNA gene were used to detect 14 piroplasm species. All hard ticks parasitizing Hokkaido sika deer were identified as belonging to the genera Ixodes and Haemaphysalis. In total 163 samples (31.4%) were positive for Babesia and Theileria spp. among tick species according to RLB hybridization. Tick DNA hybridized to the oligonucleotide probes of Theileria sp. Thrivae (27.0% of ticks; 140/519), Theileria capreoli (10.6%; 55/519), Babesia divergens-like (1.7%; 9/519), Babesia sp. (Bab-SD) (0.6%; 3/519), Babesia microti U.S. (0.4%; 2/519), and B. microti Hobetsu (0.4%; 2/519). The partial sequencing and phylogenetic analyses of the 18S rRNA gene confirmed the RLB hybridization results. Further investigations are needed to reveal the epidemiology and respective vectors of these pathogens.

Molecular detection and identification of piroplasms (Babesia spp. and Theileria spp.) and Anaplasma phagocytophilum in questing ticks from northwest Spain.

Anaplasma phagocytophilum and some piroplasm species are pathogens mainly transmitted by Ixodes ricinus. Considering that this tick species is predominant in north-western Spain, individual specimens (652 nymphs, 202 females and 202 males) and 23 larval pools were processed to determine the prevalence of these pathogens in questing I. ricinus from that region. Additionally, Dermacentor marginatus, Dermacentor reticulatus, Ixodes frontalis and Ixodes acuminatus were individually analysed. The groESL operon as well as the 16S rRNA and msp2 genes of Anaplasma were analysed. Similarly, piroplasms were identified at the 18S rRNA gene and the ITS1 of Babesia spp. and Theileria spp. Babesia venatorum (1.5%), A. phagocytophilum (0.7%), Babesia microti (0.3%) and Theileria sp. OT3 (0.2%) were detected in I. ricinus. A single I. frontalis (8.3%) tested positive to A. phagocytophilum. Although a low percentage of I. ricinus were infected with A. phagocytophilum and piroplasms, a potentially human pathogenic variant of A. phagocytophilum was detected, and both Babesia species found were zoonotic. Since the vector of Theileria sp. OT3 remains unknown, further investigations are needed to unravel the role of I. ricinus in the transmission of this piroplasm.

An outbreak of bovine babesiosis in February, 2019, triggered by above average winter temperatures in southern England and co-infection with Babesia divergens and Anaplasma phagocytophilum

BackgroundBovine babesiosis, commonly known as redwater fever, is a sporadic tick-borne disease in the United Kingdom. Outbreaks occur during the spring, summer and autumn months when ticks are active. This study reports the findings of an investigation of an outbreak of bovine babesiosis during the winter month of February, 2019.MethodsDNA from blood, organ and tick samples taken from affected cattle were tested for the presence of piroplasm and Anaplasma phagocytophilum DNA using PCRs directed to the 18S rRNA gene and msp2 gene respectively. The species of piroplasm was confirmed by sequencing.ResultsBabesia divergens DNA was detected in the blood of five cattle displaying clinical signs of babesiosis within a herd of twenty. This parasite was also detected in three of ten ticks removed from one of the affected cattle. In addition, A. phagocytophilum was detected in three cattle tested and two of ten of the ticks.ConclusionsAn outbreak of bovine babesiosis during February is unusual as the tick vector, Ixodes ricinus, does not generally become active until temperatures rise later in the year. February of 2019 was unusual as average temperatures during the first week of the month reached over 10 °C, well above historical averages that are typically below 5 °C, and a temperature at which ticks can become active. This unusual weather event is likely to have triggered tick questing, that combined with a co-infection with two tick-borne pathogens caused the severe outbreak of disease.

Detection of novel piroplasmid species and Babesia microti and Theileria orientalis genotypes in hard ticks from Tengchong County, Southwest China

To reveal the genetic diversity of Babesia microti and Theileria orientalis in Southwest China, we conducted a molecular survey of piroplasms in hard ticks in a China-Myanmar border county. Host infesting and questing ticks were collected from Tengchong County in 2013 and 2014. Piroplasm infection in ticks was detected by PCR, and then, phylogenetic analysis was conducted to study the genetic diversity of the pathogens identified in ticks. All in all, six piroplasm species comprising of B. microti; B. orientalis; a novel Babesia species designated Babesia sp. Tengchong, China; T. orientalis; T. luwenshuni; and an as yet undescribed piroplasmid species referred to as Piroplasmid sp. Tengchong, China, have been identified after screening goat- and cattle-attached ticks. In addition, B. bigemina has been identified by screening questing ticks. Phylogenetic analysis based on the 18S rRNA and partial β-tubulin gene revealed two novel potentially zoonotic genotypes designated B. microti Tengchong-Type A and B. The T. orientalis genotypes identified in the present study represent the seven known genotypes 1-5, 7, and N3 as revealed by phylogenetic analysis of 18S rRNA and MPSP genes. Importantly, an additional genotype designated N4 has also been identified in this study, which brings the number of recognized T. orientalis genotypes to a total of twelve. Thus, besides the two novel species, Babesia sp. Tengchong, China, closely related to Babesia species isolated from yak and Piroplasmid sp. Tengchong, China, our study demonstrates that additional novel B. microti and T. orientalis genotypes exist in Southwest China.

Molecular evaluation of piroplasms and hematological changes in canine blood stored in a clinical laboratory in Niterói, Rio de Janeiro

Piroplasm species were analyzed by molecular tools in total 31 blood samples from positive dogs, previously checked by stained slides, stored until DNA extraction between 2016 to 2018 in the laboratory Clinical Analyzes in Niterói, Rio de Janeiro. The piroplasms were identified by PCR, targeting the 18S rRNA gene and sequencing. From the total number of samples only 24 (77.4%) were positive and show adequate nucleotide sequences for interpretation with identity between 93%-100% with Babesia vogeli in compared to the sequences isolated of infected dogs from other states in Brazil deposited on GenBank. Most of dogs infected with B. vogeli had anemia (62.5%) and thrombocytopenia (95.8%). The findings of this study are compatible with previous reports in the literature and highlight B. vogeli as the most incriminated species in canine piroplasmosis in Brazil, and thrombocytopenia the hematological alteration most frequently identified in this infection. It is important to note that this is the first study involving the molecular characterization of piroplasms in the metropolitan region of Rio de Janeiro, based on PCR followed by sequencing.

Sequence analyses at mitochondrial and nuclear loci reveal a novel Theileria sp. and aid in the phylogenetic resolution of piroplasms from Australian marsupials and ticks.

The order Piroplasmida encompasses two main families: Babesiidae and Theileriidae, containing tick-borne pathogens of veterinary and medical importance worldwide. While only three genera (Babesia, Cytauxzoon and Theileria) comprising piroplasm parasites are currently recognised, phylogenetic studies at the 18S rRNA (18S) gene suggest that these organisms represent at least ten lineages, one of which comprises the relatively unique and highly diverse Theileria spp. from Australian marsupials and ticks. As an alternative to analysing 18S sequences alone, sequencing of mitochondrial genes has proven to be useful for the elucidation of evolutionary relationships amongst some groups of piroplasms. This research aimed to characterise piroplasms from Australian native mammals and ticks using multiple genetic markers (18S, cytochrome c, oxidase subunit III (cox3) and cytochrome B (cytB)) and microscopy. For this, nearly complete piroplasm-18S sequences were obtained from 32 animals belonging to six marsupial species: eastern bettong (Bettongia gaimardi), eastern quoll (Dasyurus viverrinus), eastern grey kangaroo (Macropus giganteus), swamp wallaby (Wallabia bicolor), quokka (Setonix brachyurus) and Gilbert’s potoroo (Potorous gilbertii). The organisms detected represented eight novel Theileria genotypes, which formed five sub-clades within the main marsupial clade containing previously reported Australian marsupial and tick-derived Theileria spp. A selection of both novel and previously described Australian piroplasms at the 18S were also successfully characterised, for the first time, at the cox3 and cytB loci, and corroborated the position of Australian native theilerias in a separate, well-supported clade. Analyses of the cox3 and cytB genes also aided in the taxonomic resolution within the clade of Australian Piroplasmida. Importantly, microscopy and molecular analysis at multiple loci led to the discovery of a unique piroplasm species that clustered with the Australian marsupial theilerias, for which we propose the name Theileria lupei n. sp.

Development of a deep amplicon sequencing method to determine the species composition of piroplasm haemoprotozoa

Piroplasmosis is caused by tick-borne haemoprotozoa of the genera Theileria and Babesia. These parasitic infections can seriously impact on the health of livestock and production. Piroplasms of multiple species can be present in a single host, but reliable molecular diagnostic tools are needed in order to understand the composition of these complex parasite communities. Theileria and Babesia vary in their epidemiology, drug sensitivity, pathogenicity and interaction with co-infecting species, but are similar in that infected animals become persistent carriers after recovery from primary infection, acting as reservoir hosts. Here, we describe for the first time the use of a deep amplicon sequencing platform to identify proportions of piroplasm species in co-infecting communities and develop the concept of a “haemoprotobiome”. First, four phenotypically-verified species of Theileria and Babesia were used to prepare mock DNA pools with random numbers of the parasites amplified by four different numbers of PCR cycles to assess sequence representation for each species. Second, we evaluated the detection threshold of the deep amplicon sequencing assay for each of the four species and to assess the accuracy of proportional quantification of all four species. Finally, we applied the assay to the field samples to afford insight of the species composition of piroplasm communities in small and large ruminants in the Punjab province of Pakistan. The “haemoprotobiome” concept has several potential applications in veterinary and human research, including understanding of responses to drug treatment; parasite epidemiology and ecology; species interactions during mixed infections; and parasite control strategies.