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Background Cytauxzoon felis is a life‐threatening protozoan disease of cats. Identification of schizont‐laden macrophages is a point‐of‐care diagnostic test for acute cytauxzoonosis. Hypothesis/Objectives The primary objective determined cytologic agreement between sample types to diagnose acute cytauxzoonosis. The secondary objective evaluated novices' ability to identify cytauxzoon organisms in blood films and tissue aspirates. Animals Thirty‐eight cats with suspected acute cytauxzoonosis and 5 controls examined postmortem. Methods Cases were prospectively submitted and collected. Blood film, lymph node, and splenic aspirates were blindly reviewed for sample quality, presence of schizont‐laden macrophages, and agreement between sample types. A subset of cases and controls were evaluated by 12 blinded novice observers to determine sensitivity and specificity for identifying organisms in various sample types. Results Acute cytauxzoonosis diagnosis was made on at least 1 sample type in 28/38 cats. Schizont‐laden macrophages were seen on 33% (10/30) of blood films, 56% (19/34) lymph node aspirates, 77% (26/34) splenic aspirates. Schizont‐laden macrophages were more likely seen on splenic than lymph node aspirates (McNemar's, P = .03) or blood film (McNemar's, P =

Theileria orientalis is an emerging apicomplexan pathogen of cattle occurring in areas populated by the principal vector tick, Haemaphysalis longicornis. Unlike transforming Theileria spp. that induce cancer-like proliferation of lymphocytes via their schizont stage, T. orientalis destroys host erythrocytes during its piroplasm phase resulting in anaemia. The underlying pathogenic processes of T. orientalis infection are poorly understood; consequently, there are no vaccines for prevention of T. orientalis infection and chemotherapeutic options are limited. To identify antigens expressed during the piroplasm phase of T. orientalis, including those which may be useful targets for future therapeutic development, we examined the proteome across three common genotypes of the parasite (Ikeda, Chitose and Buffeli) using preparations of piroplasms purified from bovine blood. A combination of Triton X-114 extraction, one-dimensional electrophoresis and LC-MS/MS identified a total of 1113 proteins across all genotypes, with less than 3% of these representing host-derived proteins. Just over three quarters of T. orientalis proteins (78%) identified were from the aqueous phase of the TX-114 extraction representing cytosolic proteins, with the remaining 22% from the detergent phase, representing membrane-associated proteins. All enzymes involved in glycolysis were expressed, suggesting that this is the major metabolic pathway used during the T. orientalis piroplasm phase. Proteins involved in binding and breakdown of haemoglobin were also identified, suggesting that T. orientalis uses haemoglobin as a source of amino acids. A number of proteins involved in host cell interaction were also identified which may be suitable targets for the development of chemotherapeutics or vaccines.

Piroplasmosis is a tick-borne haemolytic disease caused by different species of the Babesia and Theileria genera. Data on the prevalence of bovine piroplasms and their genetic diversity are scanty in Nigeria. Hence, this study reported the detection of some piroplasms in the blood of cattle in Abeokuta, Nigeria by the polymerase chain reaction (PCR). Blood samples were collected from 252 cattle and subjected to DNA extraction followed by PCR amplification of the partial region of 18S rRNA of the haemoprotozoans. Selected positive amplicons were unidirectionally sequenced and compared to the reference sequences from the Genbank. A total of 220 (87.3 %) cattle were positive for Theileria velifera and/or Babesia bigemina. The T. velifera was detected only in 163 (64.7 %) cattle, while 7 (2.8 %) cattle had a single infection with B. bigemina. Fifty cattle (19.8 %) had mixed infections with both parasites. There were no significant differences in piroplasm infections between the ages of cattle for both parasites. There were no significant differences in infection rates between the sexes for T. velifera, while the males had a significantly higher (P < 0.05) rate of infection for B. bigemina than the female cattle. The molecular detection of Babesia and Theileria species of cattle are reported for the first time in cattle in Abeokuta, Nigeria. This study, which confirmed the endemic nature of the parasites in cattle in the study area, stresses their importance in livestock health and production in Nigeria.

Increasing amount of data attest that (in the context of vector-borne infections) birds are not only important as hosts of blood-sucking arthropod vectors, but also as reservoirs of vector-borne pathogens. From 2015 to 2019 cadavers of 100 birds (from 45 species, nine orders) were collected in Hungary, and their organs were screened for DNA from a broad range of vector-borne bacteria with PCR and sequencing. Molecular analyses revealed the presence of Anaplasmataceae, and sequencing identified bacteria closely related to Neorickettsia helminthoeca and Ehrlichia chaffeensis in a Eurasian teal (Anas crecca) and a song thrush (Turdus philomelos), respectively. All samples were PCR negative for rickettsiae, borreliae, Francisella and Coxiella spp., as well as for piroplasms. To our knowledge, this is the first report of a Neorickettsia and an Ehrlichia sp., which belong to the phylogenetic groups of N. helminthoeca and E. chaffeensis, respectively, from Europe. The potential presence of these two vector-borne bacteria needs to be taken into account during future studies on the eco-epidemiology of Anaplasmataceae in Europe.

An HIV-positive man from Zimbabwe living in South Africa sought treatment for multiple clinical signs, including fever, weight loss, anemia, and splenomegaly. We identified in his blood an African rodent piroplasm, Anthemosoma garnhami, related to Babesia species. This finding extends the known geographic and host range of A. garnhami.

Over the last two decades, Cytauxzoon spp. has been conquering Eurasia, although this fact has only been brought to light through recent more intensive research after the discovery of C. manul in Pallas’ cat. In Europe, Cytauxzoon was detected mainly in southern countries and later in central Europe. This pathogen has now been found in Russia for the first time (50 km from Moscow), this being the most northern sighting in Eurasia. A captive serval (Leptailurus serval) was found to be infected. Hematological analysis showed a crucial decrease in the number of leukocytes and erythrocytes, as well as in hemoglobin concentration. Genetic analysis confirmed the presence of Cytauxzoon spp. in serval blood at the beginning of the disease period. The identical pathogen was found in one bobcat at the same breeding center. Two other haplotypes of Cytauxzoon spp. were obtained from domestic cats at the same location, identical to the samples from Italy. One new haplotype, which was sequenced for the first time, was found in 7/7 investigated Amur wildcats (100%). The high occurrence and diversity of these pathogens suggest that they are present in free-ranging domestic cats and wild felids in Russia, and may be considered a potential threat to the endangered species. Current research shows that the genetic diversity of this pathogen may be even higher than it was suggested previously. Further genetic research is necessary to describe the diversity and phylogeny of this pathogen in Eurasia.

This study, conducted in a nature reserve in southern Portugal, investigated the frequency and diversity of tick-borne piroplasms in six species of adult ixodid ticks removed from 71 fallow deer (Dama dama) and 12 red deer (Cervus elaphus), collected over the period 2012-2019. The majority of 520 ticks were Ixodes ricinus (78.5%), followed by Rhipicephalus sanguineus sensu lato, Hyalomma lusitanicum, Haemaphysalis punctata, Dermacentor marginatus, and Ixodes hexagonus. The R. sanguineus ticks collected from the deer were clearly exophilic, in contrast to the endophilic species usually associated with dogs. Four tick-borne piroplasms, including Theileria spp., and the zoonotic species, Babesia divergens and Babesia microti, were detected. B. divergens 18S rDNA, identical to that of the bovine reference strain U16370 and to certain strains from red deer, was detected in I. ricinus ticks removed from fallow deer. The sporadic detection of infections in ticks removed from the same individual hosts suggests that the piroplasms were present in the ticks rather than the hosts. Theileria sp. OT3 was found in I. ricinus and, along with T. capreoli, was also detected in some of the other tick species. The natural vector and pathogenic significance of this piroplasm are unknown. Funding was provided by a grant from the Health Institute Carlos III (PI20CIII/00037 to E.M. and L.G.M.), Spain and a grant from Alfonso X el Sabio Foundation (1.010.911 to N.F.), Spain. Sí

Dogs are known to host several tick-borne pathogens with zoonotic potential; however, scant information is available on the epidemiology of these pathogens in low-income tropical countries and in particular in sub-Saharan Africa. With the aim of investigating a wide range of tick-borne pathogens (i.e., Rickettsia spp., Anaplasma spp., Erhlichia spp., Borrelia spp., Hepatozoon spp. and Babesia spp.), 273 blood samples were collected from dogs in selected districts of Ethiopia and analyzed by real-time and/or end-point PCR. The results of the study showed that Hepatozoon canis was the most prevalent pathogen (53.8%), followed by Anaplasma phagocythophilum (7.0%), Babesia canis rossi (3.3%), Ehrlichia canis (2.6%) and Anaplasma platys (2.2%). Furthermore, five samples tested positive for Borrelia spp., identified as Borrelia afzelii (n = 3) and Borrelia burgdorferi (n = 2), and two samples for Rickettsia spp., identified as Rickettsia conorii (n = 1) and Rickettsia monacensis (n = 1). The finding of Anaplasma phagocythophilum and different species of the genera Borrelia and Rickettsia with zoonotic potential was unexpected and alarming, and calls for further investigation on the roles of dogs and on the tick, species acting as vector in this specific context. Other pathogens (Hepatozoon canis, Babaesia canis rossi, Anaplasma platys, Ehrlichia canis) are already known to have an important impact on the dogs’ health but have minor zoonotic potential as they were rarely or never reported in humans. Dogs from rural areas were found to be at higher risk for different pathogens, probably due to the presence of other wild canids in the same environment. The findings of the present study contribute to a better knowledge of the epidemiology of tick-borne pathogens, which is relevant to human and animal health.

Background Babesia spp. are protozoan parasites of great medical and veterinary importance, especially in the northern Hemisphere. Ticks are known vectors of Babesia spp., although some Babesia-tick interactions have not been fully elucidated. Methods The present review was performed to investigate the specificity of Babesia-tick species interactions that have been identified using molecular techniques in studies conducted in the last 20 years under field conditions. We aimed to indicate the main vectors of important Babesia species based on published research papers (n = 129) and molecular data derived from the GenBank database. Results Repeated observations of certain Babesia species in specific species and genera of ticks in numerous independent studies, carried out in different areas and years, have been considered epidemiological evidence of established Babesia-tick interactions. The best studied species of ticks are Ixodes ricinus, Dermacentor reticulatus and Ixodes scapularis (103 reports, i.e. 80% of total reports). Eco-epidemiological studies have confirmed a specific relationship between Babesia microti and Ixodes ricinus, Ixodes persulcatus, and Ixodes scapularis and also between Babesia canis and D. reticulatus. Additionally, four Babesia species (and one genotype), which have different deer species as reservoir hosts, displayed specificity to the I. ricinus complex. Eco-epidemiological studies do not support interactions between a high number of Babesia spp. and I. ricinus or D. reticulatus. Interestingly, pioneering studies on other species and genera of ticks have revealed the existence of likely new Babesia species, which need more scientific attention. Finally, we discuss the detection of Babesia spp. in feeding ticks and critically evaluate the data on the role of the latter as vectors. Conclusions Epidemiological data have confirmed the specificity of certain Babesia-tick vector interactions. The massive amount of data that has been thus far collected for the most common tick species needs to be complemented by more intensive studies on Babesia infections in underrepresented tick species. Graphical abstract

Healthcare professionals must be aware that babesiosis caused by reinfection might occur., Babesiosis developed in a 62-year-old immunocompetent physician, who had an uneventful recovery after receiving atovaquone and azithromycin. Three years later, babesiosis developed again, and he was again successfully given treatment. Clinical and laboratory evidence were highly supportive of Babesia reinfection. Healthcare professionals should be aware that reinfection might occur in babesiosis.

BackgroundEmerging long reads sequencing technology has greatly changed the landscape of whole-genome sequencing, enabling scientists to contribute to decoding the genetic information of non-model species. The sequences generated by PacBio or Oxford Nanopore Technology (ONT) be assembled de novo before further analyses. Some genome de novo assemblers have been developed to assemble long reads generated by ONT. The performance of these assemblers has not been completely investigated. However, genome assembly is still a challenging task.Methods and ResultsWe systematically evaluated the performance of nine de novo assemblers for ONT on different coverage depth datasets. Several metrics were measured to determine the performance of these tools, including N50 length, sequence coverage, runtime, easy operation, accuracy of genome and genomic completeness in varying depths of coverage. Based on the results of our assessments, the performances of these tools are summarized as follows: 1) Coverage depth has a significant effect on genome quality; 2) The level of contiguity of the assembled genome varies dramatically among different de novo tools; 3) The correctness of an assembled genome is closely related to the completeness of the genome. More than 30× nanopore data can be assembled into a relatively complete genome, the quality of which is highly dependent on the polishing using next generation sequencing data.ConclusionConsidering the results of our investigation, the advantage and disadvantage of each tool are summarized and guidelines of selecting assembly tools are provided under specific conditions.

Background Emerging long reads sequencing technology has greatly changed the landscape of whole-genome sequencing, enabling scientists to contribute to decoding the genetic information of non-model species. The sequences generated by PacBio or Oxford Nanopore Technology (ONT) be assembled de novo before further analyses. Some genome de novo assemblers have been developed to assemble long reads generated by ONT. The performance of these assemblers has not been completely investigated. However, genome assembly is still a challenging task. Methods and Results We systematically evaluated the performance of nine de novo assemblers for ONT on different coverage depth datasets. Several metrics were measured to determine the performance of these tools, including N50 length, sequence coverage, runtime, easy operation, accuracy of genome and genomic completeness in varying depths of coverage. Based on the results of our assessments, the performances of these tools are summarized as follows: 1) Coverage depth has a significant effect on genome quality; 2) The level of contiguity of the assembled genome varies dramatically among different de novo tools; 3) The correctness of an assembled genome is closely related to the completeness of the genome. More than 30× nanopore data can be assembled into a relatively complete genome, the quality of which is highly dependent on the polishing using next generation sequencing data. Conclusion Considering the results of our investigation, the advantage and disadvantage of each tool are summarized and guidelines of selecting assembly tools are provided under specific conditions.

Human babesiosis is a life-threatening infectious disease that causes societal and economic impact worldwide. Several species of Babesia cause babesiosis in terrestrial vertebrates, including humans. A one-day clinic was held in Ontario, Canada, to see if a red blood cell parasite, which is present in blacklegged ticks, Ixodes scapularis, is present in humans. Based on PCR testing and DNA sequencing of the 18S rRNA gene, we unveiled B. odocoilei in two of 19 participants. DNA amplicons from these two patients are almost identical matches with the type strains of B. odocoilei in GenBank. In addition, the same two human subjects had the hallmark symptoms of human babesiosis, including night sweats, chills, fevers, and profound fatigue. Based on symptoms and molecular identification, we provide substantive evidence that B. odocoilei is pathogenic to humans. Dataset reveals that B. odocoilei serologically cross-reacts with Babesia duncani. Clinicians must realize that there are more than two Babesia spp. in North America that cause human babesiosis. This discovery signifies the first report of B. odocoilei causing human babesiosis.

Parasitos da ordem Piroplasmorida causam doenças em animais silvestres e de companhia. Devido a isso, nessa tese estão incluídos três artigos científicos acerca do tema, com destaque para a espécie Rangelia vitalii e para espécies do gênero Babesia. O primeiro trabalho teve por objetivo relatar detecção molecular de parasitismo por R. vitalii em uma população uruguaia de Cerdocyon thous. Amostras de sangue e/ou baço de C. thous e Lycalopex gymnocercus encontrados atropelados nas principais vias do Uruguai foram submetidos à Reação em Cadeir de Polimerase (PCR) amplificando um gragmento de 551 pb do gene rRNA 18S para R. vitalii. Foram analisados 62 canídeos, sendo 38 C. thous e 24 L. gymnocercus. Cinco C. thous (13,16%) foram positivos para R. vitalii, com 99,5% a 100% de similaridade entre cada sequência, e nenhum dos L. gymnocercus amostrados foram positivos. Quando comparadas às amostras de R.vitalii disponíveis no GenBank, uma similaridade de 98,9% a 100% foi revelada. Os resultados da análise molecular sugerem que R. vitalii está circulando na população de raposas comedoras de caranguejo no Uruguai; no entanto, sua relevância veterinária para essas raposas permanece desconhecida. O segundo estudo objetivou detectar e quantificar a carga parasitária de R. vitalii em diferentes orgãos de canídeos domésticos e silvestres a fim de elucidar as distintas apresentações da infecção nestas espécies. Fragmentos de 22 órgãos coletados de cães domésticos (n=7) e silvestres (n=8) foram utilizados para quantificação histológica e molecular, através de PCR em tempo real do gene hsp70. Na histologia os vacúolos parasitóforos foram detectados nos tecidos de todos os cães que morreram com rangeliose, e em somente dois C. thous. A carga parasitária foi significativamente maior em cães domésticos nos tecidos do sistema digestivo, cardiorrespiratório, endócrino, genitourinário e músculo esquelético. No sistema hematopoiético, C. thous apresentou carga parasitária significativamente menor em linfonodos e tonsilas do que cães, enquanto no baço, medula óssea e sangue as detecções foram similares. No sistema nervoso central a detecção foi semelhante. Tanto em cães como em C. thous, o agente possivelmente se mantém de forma assexuada (merogonia). No entanto, C. thous provavelmente desenvolva uma fase esquizogônica limitada e/ou de curta duração, o que conferiria ao mesmo o possível caráter de reservatório do agente, em contrapartida com o cão, um provável hospedeiro acidental. O terceiro artigo é sobre espécies do gênero Babesia em procionídeos, e tem como objetivo de detectar e caracterizar filogeneticamente o parasitismo por piroplasmídeos em P. cancrivorus de vida-livre do Sul do Brasil. Amostras de baço e/ou pool de órgãos de quatro P. cancrivorus atropelados em estradas do sul do Brasil (#1 a #4), além de uma amostra de sangue de P. cancrivorus de vida livre (#5), foram submetidas a PCR para os genes 18S rRNA, hsp70 e cox1. Paralelamente à coleta de sangue, foi realizado esfregaço sanguíneo de ponta de orelha e cauda para análise citológica em busca de hemoparasitos. Duas amostras de P. cancrivorus foram positivas para o gene 18S rRNA, e uma dessas amostras foi positiva, também, para os genes hsp70 e cox1. À análise filogenética, uma dessas apresentou alta similariadade (99,75%) com Babesia sp. obtida em P. cancrivorus do Uruguai, pertencente ao clado das Babesia sensu stricto, enquanto a outra amostra se enquadrou no clado I, do grupo de Babesia microti-like, agrupando-se monofileticamente com as sequências de B. microti isoladas de Procyon lotor. Pelo conhecimento dos autores, essa é a primeira detecção de parasitismo por Babesia sp. em P. cancrivorus no Brasil; no entanto, análises filogenética mais amplas são necessárias em relação a B.microti- like a fim de elucidado se a espécie pertence a um genótipo zoonótico. Piroplasmida parasites cause a wide range of diseases in wild and domestic animals. The thesis presented herein includes three scientific papers regarding this subject, with an emphasis on Rangelia vitalii and Babesia sp. The first manuscripts aimed to detect R. vitalii parasitism in the Uruguayan wild fox population. DNA extracted from the blood and/or spleen samples of road-killed C. thous and L. gymnocercus found in northern Uruguay were subjected to polymerase chain reaction (PCR) to amplify a 551-bp fragment of the Rangelia 18S rRNA gene. A total of 62 wild canids, including 38 C. thous and 24 L. gymnocercus, were analyzed. Five crab-eating fox samples (13.2%) were positive for R. vitalii, with 99.5–100% identity between the sequences. All samples from pampas fox tested negative for R. vitalii. When compared with the R. vitalii sequences available in GenBank, a similarity of 98.9– 100% was revealed. Molecular analysis results suggest that R. vitalii is circulating in the crabeating fox population in Uruguay; however, its veterinary relevance for these foxes remains unknown.The second study aimed to detect and quantify the parasitic load of R. vitalii in different organs of domestic and wild canids in order to elucidate differences in clinical and pathological presentations of rangeliosis in these species. Fragments of 22 organs were collected from domestic (n = 7) and wild (n = 8) canids and later used for histological and molecular quantification, which was performed through real-time hsp70 PCR. Histologically, parasitophorous vacuoles were detected in tissues of all dogs that died due to rangeliosis, and only in two Cerdocyon thous. The parasitic load was significantly higher in domestic dogs in tissues of the digestive, cardiorespiratory, endocrine, genitourinary systems, and skeletal muscle. Regarding the hematopoietic system, C. thous had a significantly lower parasitic load in the lymph nodes and tonsils compared to dogs, while in the spleen, bone marrow, and blood the parasitic load was similar, as in the central nervous system. In domestic and wild canids (C. thous), the agent possibly maintains a prolonged asexual phase (merogony), but it probably develops a limited and/or short schizogonic phase in the C. thous, which would characterize the host as a reservoir, in contrast to the dog, a probable accidental host. In the third investigation, aimed to detect and phylogenetically characterize the parasitism by piroplasmids in free-living P. cancrivorus from Southern Brazil. Samples of spleen and/or organ pool of four P. cancrivorus killed by motor vehicle collision on highways of Rio Grande do Sul state, Southern Brazil (# 1 to # 4), in addition to a blood sample of free-living P. cancrivorus (# 5), were subjected to PCR for the 18S rRNA, hsp70 and cox1 genes. During clinical evaluation of P. cancrivorus #5, a sample of peripheral blood (tip of the ear and tail) was collected and a blood smear was made for cytological analysis in search of hemoparasites. Two samples of P. cancrivorus were positive for the 18S rRNA gene, and one of these samples was also positive for the hsp70 and cox1 genes. On phylogenetic analysis, one of these showed high similarity (99.75%) with Babesia sp. obtained previously from P. cancrivorus in Uruguay, belonging to the Babesia sensu stricto clade, while the other sample fell into clade I, Babesia microti –like group, grouping monophyletically with the B. microti sequences isolated from Procyon lotor. To the knowledge of the authors, this is the first detection of parasitism by Babesia sp. in P. cancrivorus in Brazil; however, more wide phylogenetic analyses are necessary for B. microti-like, in order to elucidate whether the species belongs to a zoonotic genotype.

Cytauxzoon felis, a tick-borne hemoprotozoal pathogen of felids, causes an acute, often-fatal disease in domestic cats. While public awareness of the disease has increased, few studies have evaluated the incidence of acute cytauxzoonosis cases and their associated risk factors. The objective of this study was to retrospectively review records of cats diagnosed with acute cytauxzoonosis in eastern Kansas from 2006–2019 using clinic records and determine: (i) feline cytauxzoonosis risk factors; and (ii) if cytauxzoonosis case incidence is increasing. Although inter-annual variation of acute cytauxzoonosis diagnosis was observed in the eastern Kansas domestic cat population, the overall incidence trend remained largely unchanged over the 14-year case review period. In comparison to ill (C. felis-unrelated) control cases, more acute cytauxzoonosis cases were diagnosed in spring and summer, suggesting a seasonal fluctuation of infection, with samples most commonly submitted from ≥1 year old, owned, male cats. Although cytauxzoonosis case submissions remained consistent over the broad study period, increasing tick vector and domestic cat reservoir populations may contribute to additional cytauxzoonosis case expansion in endemic areas. Investigating the incidence of acute cytauxzoonosis, patient risk factors, and ecological variables that influence disease transmission are important steps towards developing and communicating the need for effective cytauxzoonosis control strategies for high-risk cat populations.

Songbirds widely disperse ticks that carry a diversity of pathogens, some of which are pathogenic to humans. Among ticks commonly removed from songbirds, the blacklegged tick, Ixodes scapularis, can harbor any combination of nine zoonotic pathogens, including Babesia species. From May through September 2019, a total 157 ticks were collected from 93 songbirds of 29 species in the Canadian provinces of Ontario and Qu&eacute, bec. PCR testing for the 18S gene of Babesia species detected Babesia odocoilei in 12.63% of I. scapularis nymphs parasitizing songbirds in Ontario and Qu&eacute, bec, none of the relatively small numbers of Ixodes muris, Ixodes brunneus, or Haemaphysalis leporispalustris were PCR-positive. For ticks at each site, the prevalence of B. odocoilei was 16.67% in Ontario and 8.89% and 5.26% in Qu&eacute, bec. Of 31 live, engorged I. scapularis larvae and nymphs held to molt, 25 ticks completed the molt, five of these molted ticks were positive for B. odocoilei. PCR-positive ticks were collected from six bird species&mdash, namely, Common Yellowthroat, Swainson&rsquo, s Thrush, Veery, House Wren, Baltimore Oriole, and American Robin. Phylogenetic analysis documented the close relationship of B. odocoilei to Babesia canis canis and Babesia divergens, the latter a known pathogen to humans. For the first time in Canada, we confirm the transstadial passage of B. odocoilei in I. scapularis molting from larvae to nymphs. A novel host record reveals I. scapularis on a Palm Warbler. Our findings show that B. odocoilei is present in all mobile life stages of I. scapularis, and it is widely dispersed by songbirds in Ontario and Qu&eacute, bec.

Tick-borne pathogens cause economically significant diseases in cattle. Theileria spp. are parasitic protozoa and the causative agent of bovine theileriosis. Here we report the distribution and risk factors of bovine Theileria using blood samples taken between 2018 and 2019. Of 737 tested cattle, nine animals (1.2%) were positive for Theileria orientalis infection by 18S rRNA gene amplification. Further analysis of the infected samples using the T. orientalis major piroplasm surface protein (MPSP) gene revealed five different genotypes circulating in the population: Types 1, 2, 3, 7, and N3. To the best of our knowledge, this is the first research to describe the existence of the T. orientalis MPSP genotype N3 in South Korea. Although the prevalence of bovine T. orientalis was low, our study offers data on the geographical distribution and prevalence of bovine Theileria spp. in South Korea. Further studies are warranted to determine the correlation of clinical symptoms with parasite MPSP genotypes. Our data provide epidemiological information to help control bovine theileriosis in South Korea.

Theileria parva is a protozoan parasite that causes East Coast fever (ECF), an economically important disease of cattle in Africa. It is transmitted mainly by the tick Rhipicephalus appendiculatus. Research efforts to develop a subunit vaccine based on parasite neutralizing antibodies and cytotoxic T-lymphocytes have met with limited success. The molecular mechanisms underlying T. parva life cycle stages in the tick vector and bovine host are poorly understood, thus limiting progress toward an effective and efficient control of ECF. Transcriptomics has been used to identify candidate vaccine antigens or markers associated with virulence and disease pathology. Therefore, understanding how genes are expressed and where their products act in the pathogenic and infective forms of T. parva should shed more light on host-pathogen interactions in ECF and identify genes underlying difference in parasite stages as well as novel therapeutic targets. Recently, the first gene expression profiling of T. parva was conducted for the sporoblast, sporozoite and schizont stages. Sporozoite is infective to cattle whereas schizont is the major pathogenic form of the parasite. The schizont can differentiate into piroplasm which is infective to the tick vector. The present study was designed to extend the T. parva gene expression profiling to the piroplasm stage with reference to the schizont. Pairwise comparison revealed that 2,740 of a possible 4,084 protein coding genes were differentially expressed, with 1,407 (51%) genes up-regulated and 1,333 (49%) downregulated in the schizont relative to the piroplasm. In addition, over 160 genes were stage-specifically expressed. In general, there were more molecular functions, biological processes, subcellular localizations and pathways significantly enriched in the piroplasm than in the schizont. Using known antigens as benchmarks, we identified several new potential vaccine antigens, including TP04_0076 and TP04_0640, which were highly immunogenic in naturally T. parva-infected cattle. All the candidate vaccine antigens identified are yet to be investigated for their capacity to induce protective immune response against ECF.

Background Successful Cytauxzoon felis transmission studies have occurred using Amblyomma americanum adults acquisition-fed as nymphs on an experimentally infected domestic cat or Dermacentor variabilis adults fed as nymphs on a splenectomized bobcat. Here, we evaluated A. americanum and D. variabilis nymphs acquisition-fed as larvae on a C. felis-infected carrier domestic cat for competence to transmit the protozoan parasite as nymphs to naïve, healthy domestic cats. Methods Amblyomma americanum and D. variabilis larvae were applied to a chronically infected, parasitemic C. felis donor cat (Felis catus) and allowed to feed to repletion. Engorged larvae were collected and held through ecdysis. Three cats were each infested with 66 A. americanum or 66 D. variabilis emerged nymphs. Cytauxzoon felis infections in principal cats were determined by clinical signs and detection of circulating parasite by blood smear and PCR evaluation. Results Clinical signs of cytauxzoonosis were observed in cats infested with A. americanum nymphs beginning 12–15 days post-infestation (dpi). The same cats were PCR positive on 12–14 dpi; piroplasms were evident in blood smears at 16 dpi, and macrophage schizonts were observed in stained spleen impression smears in two animals at necropsy. Cats infested with acquisition-fed D. variabilis nymphs remained clinically normal and did not develop detectable parasitemia over the course of the study as determined by blood smear and PCR. Conclusions Cytauxzoon felis was successfully transmitted to domestic cats by A. americanum nymphs acquisition-fed as larvae on the donor cat. However, we were not able to transmit C. felis to healthy domestic cats with D. variabilis nymphs that were simultaneously acquisition-fed on the same donor cat. Results from this study suggest that larval and nymphal A. americanum likely play important roles in natural transmission cycles of C. felis.

Case summary A castrated male domestic shorthair cat from a wooded area in Missouri had recovered from typical severe cytauxzoonosis at 4 years of age, after intensive in-hospital supportive care and administration of atovaquone and azithromycin. At 11 years of age, the same cat again experienced an acute febrile illness compatible with cytauxzoonosis. Intraerythrocytic piroplasms typical of Cytauxzoon felis were identified by cytology. The owners opted for euthanasia but allowed collection of splenic and hepatic tissue for histopathologic examination. Schizont-laden macrophages were identified in both tissue specimens, confirming active cytauxzoonosis at the time of the cat’s death. Relevance and novel information Although cats that have recovered from cytauxzoonosis can harbor red blood cell piroplasms for many years without apparent clinical illness, repeat illness owing to either disease recrudescence or repeat infection has never been documented. In fact, recovered cats have been thought to be resistant to reinfection and subsequent illness. This report describes a cat that had recovered from documented cytauxzoonosis 7 years previously and then developed a subsequent clinical illness typical of cytauxzoonosis, which was accompanied not only by intraerythrocytic piroplasms, but also by schizont-laden tissue macrophages pathognomonic of clinical cytauxzoonosis.

Natural landscape alterations as a consequence of urbanisation are one of the main drivers in the movements of wildlife into metropolitan and peri-urban areas. Worldwide, these wildlife species are highly adaptable and may be responsible for the transmission of tick-borne pathogens including piroplasms (Babesia, Theileria and Cytauxzoon spp.) that cause piroplasmosis in animals and occasionally in humans. Little is known about piroplasms in the ticks of urban wildlife in Australia. Ticks from long-nosed bandicoots (Perameles nasuta; n = 71), eastern-barred bandicoots (Perameles gunnii; n = 41), northern-brown bandicoots (Isoodon macrourus; n = 19), southern-brown bandicoots (Isoodon obesulus; n = 4), bandicoot sp. (n = 2), flying foxes (Pteropus sp.; n = 3), black rats (Rattus rattus; n = 7), bush rats (Rattus fuscipes; n = 4), brushtail possums (Trichosurus vulpecula; n = 19), ringtail possums (Pseudocheirus peregrinus; n = 12), short-eared possums (Trichosurus caninus; n = 6), possum sp. (Trichosurus sp.; n = 8), and red foxes (Vulpes vulpes; n = 12) were analysed using piroplasm-specific 18S primers and Sanger sequencing. Seven Ixodes tasmani ticks from long-nosed bandicoots and bandicoots sp., three I. tasmani ticks and one Ixodes holocyclus tick from brushtail possums, and one Haemaphysalis longicornis tick from a red fox were positive for piroplasms. New genotypes, with sequences sharing 98% nucleotide similarities with Theileria sp. K1 detected in a burrowing bettong (Bettongia lesueur), were identified from bandicoot ticks. New genotypes were detected in ticks from brushtail possums, which shared 98% similarity with a Babesia sp. (JQ682877) previously identified in marsupials. Theileria orientalis was identified in the H. longicornis tick from the red fox. Babesia and Theileria spp. in the ticks parasitizing bandicoots and brushtail possums clustered closely with respective Babesia and Theileria clades derived from Australian marsupials. This represents the first detection of piroplasms in ticks parasitizing brushtail possums and a red fox in Australia., Graphical abstract Image 1, Highlights • First characterisation of novel Babesia sp. in ticks from brushtail possums in Australia. • First detection of Theileria orientalis in tick on a red fox in Australia. • Theileria sp. derived from Australian marsupials form a unique clade.

Rangelia vitalii, a tick-borne piroplasm that infects dogs, has been recently molecularly characterized in Brazil, Uruguay and Argentina. Studies on molecular characterization of these piroplasms in different Brazilian regions are scarce. The aim of this study was to evaluate clinical and hematological changes in dogs caused by R. vitalii in the mountainous region of the state of Rio de Janeiro. Blood samples from 36 dogs were evaluated for piroplasms and hematological disorders using light microscopy and molecular analysis. Blood samples from all the animals included in this study were confirmed to be positive for R. vitalii through genetic sequencing. Clinical signspresented by 24 of the 36 dogs of the study were evaluated during appointments or hospitalization within private practice. The most frequent clinical disorders in these dogs that were naturally infected with R. vitalii were fever, spontaneous cutaneous bleeding and diarrhea. Normochromic non-regenerative anemia and thrombocytopenia were the most common hematological disorders in these R. vitalii-positive dogs and therefore should be considered in hematological evaluations on suspected cases. Resumo Rangelia vitalii, um piroplasma transmitido por carrapatos que infecta cães, foi sendo recentemente caracterizado molecularmente no Brasil, Uruguai e Argentina. Nas diferentes regiões brasileiras são escassos os estudos acerca da caracterização molecular destes piroplasmídeos. O objetivo deste estudo foi avaliar as alterações clínicas e hematológicas em cães causadas por R. vitalii na região serrana do Rio de Janeiro. Amostras de sangue total de 36 cães foram examinadas quanto à presença de piroplasmas pela microscopia de luz, alterações hematológicas e análise molecular. Todos os cães do presente estudo foram positivos para R. vitalii através do sequenciamento genético. Dos 36 animais positivos para R. vitalii, 24 foram avaliados clinicamente. As alterações mais frequentemente observadas foram febre, sangramento cutâneo espontâneo e diarréia. Anemia normocítica normocrômica arregenerativa e trombocitopenia foram as alterações hematológicas mais observadas em cães positivos para R. vitalii, devendo ser consideradas na avaliação hematológica de cães suspeitos.

Human babesiosis is a life-threatening infectious disease that causes societal and economic impact worldwide. Several species of Babesia cause babesiosis in terrestrial vertebrates, including humans. A one-day clinic was held in Ontario, Canada, to see if a red blood cell parasite, which is present in blacklegged ticks, Ixodes scapularis, is present in humans. Based on PCR testing and DNA sequencing of the 18S rRNA gene, we unveiled B. odocoilei in two of 19 participants. DNA amplicons from these two patients are almost identical matches with the type strains of B. odocoilei in GenBank. In addition, the same two human subjects had the hallmark symptoms of human babesiosis, including night sweats, chills, fevers, and profound fatigue. Based on symptoms and molecular identification, we provide substantive evidence that B. odocoilei is pathogenic to humans. Dataset reveals that B. odocoilei serologically cross-reacts with Babesia duncani. Clinicians must realize that there are more than two Babesia spp. in North America that cause human babesiosis. This discovery signifies the first report of B. odocoilei causing human babesiosis.

Tick-borne zoonotic diseases have an economic and societal impact on the well-being of people worldwide. In the present study, a high frequency of Babesia odocoilei, a red blood cell parasite, was observed in the Huronia area of Ontario, Canada. Notably, 71% (15/21) blacklegged ticks, Ixodes scapularis, collected from canine and feline hosts were infected with B. odocoilei. Consistent with U.S. studies, 12.5% (4/32) of questing I. scapularis adults collected by flagging in various parts of southwestern Ontario were positive for B. odocoilei. Our data show that all B. odocoilei strains in the present study have consistent genetic identity, and match type strains in the GenBank database. The high incidence of B. odocoilei in the Huronia area indicates that this babesial infection is established, and is cycling enzootically in the natural environment. Our data confirm that B. odocoilei has wide distribution in southern Ontario.

Background Understanding how fauna translocation and antiparasitic drug treatment impact parasite community structure within a host is vital for optimising translocation outcomes. Trypanosoma spp. and piroplasms (Babesia and Theileria spp.) are known to infect Australian marsupials, including the woylie (Bettongia penicillata). However relatively little is known about these haemoparasites, or how they respond to management practices such as translocation. We monitored haemoparasites infecting woylies for up to 12 months during two fauna translocations to supplement existing woylie populations in three different sites (Dryandra, Walcott and Warrup East) within south-western Australia between 2014 and 2016, with the aim of investigating (i) how haemoparasite prevalence, Trypanosoma spp. richness and Trypanosoma spp. community composition varied over time and between different sites following translocation; and (ii) whether ivermectin treatment indirectly impacts haemoparasite prevalence. Using molecular methods, 1211 blood samples were screened for the presence of trypanosomes, and a subset of these samples (n = 264) were also tested for piroplasms. Results Trypanosomes and piroplasms were identified in 55% and 94% of blood samples, respectively. We identified five Trypanosoma species, two Theileria species, a single species of Babesia and a novel Bodo species. Trypanosoma spp. richness and the prevalence of haemoparasite co-infection increased after translocation. Prior to translocation, Trypanosoma spp. community composition differed significantly between translocated and resident woylies within Walcott and Warrup East, but not Dryandra. Six months later, there was a significant difference between translocated and resident woylies within Dryandra, but not Walcott or Warrup East. The response of haemoparasites to translocation was highly site-specific, with predominant changes to the haemoparasite community in translocated woylies occurring within the first few months following translocation. Ivermectin treatment had no significant effect on haemoparasite prevalence. Conclusions This study contributes to our understanding of haemoparasite dynamics in woylies following translocation. The highly site-specific and rapid response of haemoparasites to translocation highlights the need to better understand what drives these effects. Given that haemoparasite prevalence and composition of translocated and resident animals changed significantly following translocation, we propose that parasite monitoring should form an essential component of translocation protocols, and such protocols should endeavour to monitor translocated hosts and cohabiting species. Electronic supplementary material The online version of this article (10.1186/s13071-019-3370-6) contains supplementary material, which is available to authorized users.

Rangelia vitalii is a haemoparasite that infects erythrocytes, white blood cells and the cytoplasm of endothelial cells of blood capillaries of canids in South America, and has been detected in both domestic dogs and sylvatic canids. Hepatozoon canis is a parasite that infects neutrophils and monocytes of many mammalian hosts. This study reports the infection of Lycalopex gymnocercus from Santa Catarina, Brazil, with R. vitalii and H. canis. The piroplasm was observed on both blood smears and molecular tests. Many large piroplasms were detected inside the erythrocytes, with round, oval, or teardrop-shaped organism, that occurred singly or in pairs. They had an abundant, pale blue cytoplasm and decentral dark red small nucleus. The animal was also infected with H. canis that was detected only by molecular tests. The majority of haematological and biochemistry parameters were within the reference values for domestic dog and wild canids. Resumo Rangelia vitalii é um hemoparasita que infecta eritrócitos, macrófagos e células endoteliais de canídeos na América do Sul, e vem sendo detectado tanto em cães domésticos quanto em canídeos silvestres. Hepatozoon canis é um parasita que infecta monócitos e neutrófilos de mamíferos. No presente estudo, é descrita a infecção de Lycalopex gymnocercus, proveniente de Santa Catarina, Brasil, por R. vitalii e H. canis. O piroplasma foi diagnosticado nos esfregaços sanguíneos e por técnicas moleculares. Nos eritrócitos foram observados vários merozoítos grandes, ovais, arredondados ou em forma de gota, ocorrendo isoladamente ou em pares. Estes piroplasmas apresentavam citoplasma abundante, corado em azul claro, com núcleo pequeno, avermelhado e descentralizado. O animal apresentou coinfecção com H. canis, que foi diagnosticado somente pelos testes moleculares. A maior parte dos parâmetros hematológicos e bioquímicos do animal estava dentro dos valores de referência para cães domésticos e canídeos silvestres.

Background Cytauxzoonosis is an emerging tick-borne disease of domestic and wild felids. Cytauxzoon felis induces severe and often fatal disease in domestic cats. In Europe, clinical and subclinical infections caused by Cytauxzoon sp. are described. We report the first cases of Cytauxzoon sp. infection in domestic cats in Switzerland. Methods Clinical and laboratory data and results of PCR analyses were collected from Cytauxzoon sp. PCR-positive cats and the cats followed for up to 851 days. Results The cases were three two-month old kittens from the same litter (Cases 1–3) and two adult domestic shorthair cats (Cases 4 and 5). The cats originated from the north-west and west of Switzerland. Cases 1–3 presented with moderate to severe regenerative anaemia and intraerythrocytic inclusions. Cytauxzoon sp. was confirmed by PCR and sequencing. The kittens made a clinical and haematological recovery after blood transfusion and/or treatment with azithromycin and atovaquone, but erythroparasitaemia persisted. Case 4 presented with severe non-regenerative anaemia. Case 5 was healthy and used as a blood donor for Case 4. Following blood transfusion, Case 4 showed intraerythrocytic inclusions, and Cytauxzoon sp. was confirmed in both Cases 4 and 5 using PCR and sequencing. Case 4 achieved clinical and haematological remission after treatment with azithromycin, atovaquone and immunosuppressive drugs. Eight months later, Case 4 was presented again with anaemia but tested Cytauxzoon sp. PCR-negative. Sequencing of 1637 bp of the 18S rRNA gene of Cytauxzoon sp. revealed 100% nucleotide sequence identity among isolates of Cases 1–3 and between isolates of Cases 4 and 5, and 99% sequence identity between isolates of all cases. Phylogenetic analysis revealed the closest relationship of the Swiss isolates to Cytauxzoon sp. isolates from domestic cats and wild felids from France, Spain and Romania and to Cytauxzoon manul from a Pallas’s cat. Conclusions This is the first report of Cytauxzoon sp. infection in domestic cats in Switzerland. It is also the first report of infection in very young kittens and transmission of Cytauxzoon sp. to an adult cat by transfusion of blood from an asymptomatic cat. The cats recovered but some developed chronic asymptomatic erythroparasitaemia for up to 28 months. Domestic cats may act as reservoirs for Cytauxzoon sp. in Europe and blood donor cats should be screened for this agent by PCR.

Highlights • Detailed description of novel Babesia infection causing mortality in macropods. • First report of this infection in agile wallabies. • Information on the geographical incidence of this disease in the eastern states of Australia. • Comprehensive review of the clinical signs and pathology of the disease., Graphical Abstract, This is a retrospective study of 38 cases of infection by Babesia macropus, associated with a syndrome of anaemia and debility in hand-reared or free-ranging juvenile eastern grey kangaroos (Macropus giganteus) from coastal New South Wales and south-eastern Queensland between 1995 and 2013. Infection with B. macropus is recorded for the first time in agile wallabies (Macropus agilis) from far north Queensland. Animals in which B. macropus infection was considered to be the primary cause of morbidity had marked anaemia, lethargy and neurological signs, and often died. In these cases, parasitised erythrocytes were few or undetectable in peripheral blood samples but were sequestered in large numbers within small vessels of visceral organs, particularly in the kidney and brain, associated with distinctive clusters of extraerythrocytic organisms. Initial identification of this piroplasm in peripheral blood smears and in tissue impression smears and histological sections was confirmed using transmission electron microscopy and molecular analysis. Samples of kidney, brain or blood were tested using PCR and DNA sequencing of the 18S ribosomal RNA and heat shock protein 70 gene using primers specific for piroplasms. The piroplasm detected in these samples had 100% sequence identity in the 18S rRNA region with the recently described Babesia macropus in two eastern grey kangaroos from New South Wales and Queensland, and a high degree of similarity to an unnamed Babesia sp. recently detected in three woylies (Bettongia penicillata ogilbyi) in Western Australia.

Made available in DSpace on 2019-10-06T15:20:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-07-01. Added 1 bitstream(s) on 2019-10-09T18:34:45Z : No. of bitstreams: 1 S1984-29612018000300377.pdf: 1261854 bytes, checksum: f9b1dea231a876356b12e84c02a2b0f5 (MD5) Rangelia vitalii is a haemoparasite that infects erythrocytes, white blood cells and the cytoplasm of endothelial cells of blood capillaries of canids in South America, and has been detected in both domestic dogs and sylvatic canids. Hepatozoon canis is a parasite that infects neutrophils and monocytes of many mammalian hosts. This study reports the infection of Lycalopex gymnocercus from Santa Catarina, Brazil, with R. vitalii and H. canis. The piroplasm was observed on both blood smears and molecular tests. Many large piroplasms were detected inside the erythrocytes, with round, oval, or teardrop-shaped organism, that occurred singly or in pairs. They had an abundant, pale blue cytoplasm and decentral dark red small nucleus. The animal was also infected with H. canis that was detected only by molecular tests. The majority of haematological and biochemistry parameters were within the reference values for domestic dog and wild canids. Departamento de Parasitologia Instituto de Biociências Universidade Estadual Paulista – UNESP Departamento de Clínica e Cirurgia Veterinárias Escola de Veterinária Universidade Federal de Minas Gerais – UFMG Departamento de Medicina Veterinária Centro de Ciências Agroveterinárias – CAV Universidade do Estado de Santa Catarina – UDESC Centro de Ciências Exatas e Naturais Universidade Estadual do Maranhão – UEMA Departamento de Parasitologia Instituto de Biociências Universidade Estadual Paulista – UNESP

Made available in DSpace on 2018-12-11T17:09:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-06-01 Earthwatch Institute This study investigated the occurrence of Cytauxzoon felis and Babesia spp. in free-ranging jaguars (Panthera onca), domestic dogs (Canis lupus familiaris) and domestic cats (Felis catus) from the Cerrado, Amazon and Pantanal biomes of Brazil. Blood samples were collected from 30 jaguars, 129 dogs and 22 cats for detection of the 18S rRNA genes of piroplasmids. All of the jaguars from the Pantanal (n = 22) and Cerrado (n = 4) and three of four jaguars from the Amazon were positive for C. felis, but no dogs or cats were positive for the agent. All of the jaguars and domestic cats were negative for Babesia spp., while dogs from the Cerrado (7.9%; 5/63) and Amazon (10.6%; 5/47) biomes tested positive for the hemoparasite. Cytauxzoon nucleotide sequences detected were closely related to C. felis; and Babesia nucleotide sequences showed 100% of identity with Babesia vogeli. Although the pathogenicity of Cytauxzoon spp. genotypes that circulate in Brazil is still unknown, free-ranging jaguars probably play an important role in the maintenance of C. felis in nature. In addition, even though there is no evidence of the circulation of Babesia spp. between jaguars and dogs, the presence of this hemoparasite should be monitored in jaguar populations. Jaguar Conservation Fund/Instituto Onça-Pintada, Caixa Postal 193 Departamento de Medicina Veterinária Preventiva e Saúde Animal Faculdade de Medicina Veterinária e Zootecnia Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87 Instituto de Biociências de Botucatu UNESP—Universidade Estadual Paulista Campus de Botucatu Departamento de Parasitologia, Distrito de Rubião Júnior Instituto de Biologia Instituto de Ciências Biomédicas Universidade Federal de Uberlândia, Rua Ceará, s/n Department of Wildlife Fish and Conservation Biology University of California Davis, 1088 Academic Surge, One Shields Ave Instituto de Biociências de Botucatu UNESP—Universidade Estadual Paulista Campus de Botucatu Departamento de Parasitologia, Distrito de Rubião Júnior

Babesiosis, the hemolytic disease caused by Babesia, which is a tick-transmitted obligate intraerythrocytic protozoan parasite. This disease is responsible for significant mortality and morbidity rates and enormous economic losses to the livestock industry in tropical and subtropical regions worldwide. In this study, blood samples were collected from 141 pet dogs from Gansu, China, and analyzed for Babesia or Theileria spp. infection using specific PCR and sequencing based on 18S rRNA gene fragments. The results indicated that 18S rRNA gene sequences from 11 samples were similar to the 18S rRNA gene sequences in Babesia canis vogeli (2) and Theileria sinensis (9). The total infected rates of B. canis vogeli and T. sinensis were 1.4% (2/141) and 6.4% (9/141), respectively. This represents the first molecular report of T. sinensis in dogs worldwide and of B. canis vogeli in dogs from Gansu province of China. Furthermore, the finding of T. sinensis in dogs may represent the common infection of this parasite occurring in Gansu.

Canine rangeliosis, caused by the piroplasmid protozoon Rangelia vitalii, is currently recognized as a reemerging disease that affects domestic dogs in Brazil. In the present study, piroplasmid infection was searched in wild canids (20 Cerdocyon thous and 4 Lycalopex gymnocercus) in Brazil. Molecular analysis, based on PCR and DNA sequencing of a portion of the 18S rRNA gene, revealed that 30% (6/20) C. thous were infected by R. vitalii. Blood and bone marrow samples from one of the R. vitalii-infected C. thous were inoculated into a domestic dog, which developed clinical rangeliosis that was confirmed by molecular tests. However, the C. thous donor showed no clinical, hematological or biochemical alterations, even though its R. vitalii infection status was confirmed for at least 80 days. These observations suggest that R. vitalii is not as highly pathogenic for C. thous as it is for domestic dogs. Phylogenetic analysis inferred by the 18S rRNA gene placed R. vitalii embedded in the clade ‘Babesia sensu stricto’, consisting of a number of species that represent truly the genus Babesia. It is proposed that the species R. vitalii should be transferred to the genus Babesia. The present study expands our knowledge on the natural history of R. vitalii, suggesting that it might have a natural cycle involving the wild canid C. thous. Further studies are needed to confirm that C. thous is a natural reservoir of R. vitalii in Brazil.

Such surveillance can provide an early warning for emergence of this disease and measure disease underreporting., Human babesiosis is an emerging tick-borne disease caused by the intraerythrocytic protozoan Babesia microti. Its geographic distribution is more limited than that of Lyme disease, despite sharing the same tick vector and reservoir hosts. The geographic range of babesiosis is expanding, but knowledge of its range is incomplete and relies exclusively on reports of human cases. We evaluated the utility of tick-based surveillance for monitoring disease expansion by comparing the ratios of the 2 infections in humans and ticks in areas with varying B. microti endemicity. We found a close association between human disease and tick infection ratios in long-established babesiosis-endemic areas but a lower than expected incidence of human babesiosis on the basis of tick infection rates in new disease-endemic areas. This finding suggests that babesiosis at emerging sites is underreported. Vector-based surveillance can provide an early warning system for the emergence of human babesiosis.

Background Cytauxzoonosis is an emerging and life-threatening tick-borne feline disease caused by haemoprotozoan parasites of the genus Cytauxzoon. Information regarding epidemiological and clinical presentation of infections by species other than Cytauxzoon felis is scant. A case of Cytauxzoon sp. infection is described in a 2-year-old mixed breed male domestic cat from Portugal, presenting a history of acute lethargy, anorexia and pyrexia. Results Complete blood count revealed a severe anaemia, leucocytosis and thrombocytopenia. A pleural effusion was noticed on thoracic radiograph, and marked splenomegaly and free abdominal fluid were visualized by ultrasound. A molecular screening for the detection of causative agents of infectious anaemia was performed, and a positive result for Piroplasmorida was obtained. DNA sequencing of a 743 bp amplicon of the 18S rRNA gene (GenBank accession no. KU710344) revealed 99.9 % identity with Cytauxzoon manul. Conclusions This is the first report of Cytauxzoon sp. (clustering together with C. manul) in a felid from Portugal. Clinical manifestations along with molecular analysis suggest the hypothesis that domestic cats might be infected with and serve as a reservoir host for C. manul.

Equine piroplasmosis (EP) has been frequently described in donkeys in subtropical and tropical regions, but published data reflecting large scale surveys are very limited in Europe. The seroprevalence of Babesia caballi and Theileria equi was determined in a donkey population from Campania Region in Southern Italy using a commercial indirect fluorescent antibody test (IFAT), and the risk factors associated with the occurrence of the infection were assessed. Of 203 samples, the overall seroprevalence for EP was 57.1% (116/203), with 35.5% (72/203) for B. caballi and 44.3% (90/203) for T. equi. Co-infection was detected in 46 donkeys (22.6%). The distribution of IFAT antibody titres to B. caballi was: 1:80 (n = 67), 1:160 (n = 2), 1:320 (n = 3); while the distribution of IFAT antibody titres to T. equi was: 1:80 (n = 25), 1:160 (n = 42), 1:320 (n = 12), 1:640 (n = 8), 1:1280 (n = 3). All examined donkeys were asymptomatic, except one adult male (with a titre of 1:640 against T. equi) that showed clinical signs corresponding to the acute stage of EP, reported for the first time in Italy. The unique risk factor associated with a higher B. caballi seroprevalence was the presence of horses in the farms, while risk factors associated with a higher T. equi seroprevalence were poor body condition, presence of ruminants in the farms and milk production. The results indicate a high level of exposure in donkeys living in Southern Italy and suggest that donkeys may be an important reservoir of EP.

International audience; Single dilution ELISAs were standardised for the determination of antibody titres against Theileria annulata using three antigens namely soluble piroplasm, cellular schizont or soluble schizont antigens. Antibody titres of 20 cattle serum samples of known identity were determined by multi-dilution ELISA using the three antigens. The ratio of the optical density (OD) of known positive and known negative sera at different serum dilutions were calculated and termed as positive/negative (P/N) ratios. Coefficients of correlation (r) were calculated between the P/N ratios at different dilutions of known sera and their log$_{10}$ antibody titres by multi-dilution ELISA. The value of "r"was the highest at the dilution of 1:400. From the log$_{10}$ antibody titres of known sera and their P/N ratios at the dilution of 1:400, regression equations (Y = a $+$ bX, where Y = predicted log$_{10}$ titre, X = the P/N ratio at 1:400 dilution) were calculated separately for the three antigens. Thus, the equations Y = 1.63 $+$ 1.35X for soluble piroplasm, Y = 2.67 $+$ 0.547X for cellular schizont and Y = 1.817 $+$ 0.663X for soluble schizont antigens were derived. Test sera were diluted to 1:400 and their OD were read in duplicate wells and converted to P/N ratios. The antibody titres were predicted from the P/N ratios using the above mentioned regression equations. Twenty randomly selected sera tested by single and multi-dilution ELISAs showed non-significant differences ($P < 0.01$) between antibody titres. Antibody titres of 90 unknown field sera of cattle were determined by single dilution ELISA. The piroplasm antigen detected higher antibody titres followed by cellular schizont and soluble schizont antigens. The study revealed that a single dilution ELISA could be successfully used for field epidemiological studies of tropical theileriosis.; ELISA à dilution unique utilisant l'antigène de piroplasme soluble, de schizonte cellulaire ou de schizonte soluble pour la détection d'anticorps contre Theileria annulata. Un ELISA à dilution unique a été standardisé pour la détermination des titres en anticorps contre Theileria annulata, en utilisant trois antigènes : l'antigène de piroplasme soluble, de schizonte cellulaire, ou de schizonte soluble. Les titres en anticorps de 20 échantillons de sérums d'identité connue ont été déterminés par un ELISA à dilutions multiples, avec les trois antigènes. Le rapport de la densité optique (DO) de sérums positifs connus sur la DO de sérums négatifs connus, à différentes dilutions, a été calculé et a été appelé le rapport positif/négatif (P/N). Le coefficient de corrélation (r) entre les rapports P/N de sérums connus et leur titre en anticorps (log$_{10}$) déterminé par ELISA à dilutions multiples, a été calculé pour différentes dilutions des sérums. La plus forte valeur de "r "correspondait à une dilution de 1/400. A partir des titres (log10) en anticorps de sérums connus et leur rapport P/N à une dilution 1/400, des équations de régression ont été calculées séparément pour chaque antigène (Y = a $+$ bX, où Y = titre prédit en log$_{10}$ ; X = rapport P/N à la dilution 1/400). Les équations suivantes ont ainsi été dérivées : Y = 1,63 $+$ 1,35X pour le piroplasme soluble ; Y = 2,67 $+$ 0,547X pour le schizonte cellulaire, et Y = 1,817 $+$ 0,663X pour le schizonte soluble. Les sérums testés ont été dilués à 1/400 et leurs DO ont été lues chacune dans deux puits puis converties en rapport P/N. Les titres en anticorps ont été prédits à partir des rapports P/N, en utilisant les équations de régression correspondantes. Vingt sérums sélectionnés au hasard ont été testés à la fois par ELISA à dilutions multiples et par ELISA à dilution unique ; il n'y a pas eu de différence significative entre les titres en anticorps obtenus par les deux méthodes ($P < 0,01$). Les titres en anticorps de 90 sérums prélevés sur le terrain ont été déterminés par ELISA à dilution unique. Dans ces échantillons, l'antigène de piroplasme détectait les anticorps aux titres les plus élevés, suivi du schizonte cellulaire, puis du schizonte soluble. L'étude a révélé que l'ELISA à dilution unique peut être utilisée avec succès dans des études épidémiologiques de terrain sur la theilériose tropicale.

A 12-year old mixed breed neutered bitch from Misiones, Argentina, was presented with a history of fever and epistaxis. Blood, bone marrow, and lymph node samples were collected for hematology and cytology. Mild regenerative anemia was recorded and large, round, poorly stained piroplasms (> 2.5 μm) were found within erythrocytes in blood and lymph node smears. Nested PCR-RFLP on blood and bone marrow samples was positive for piroplasm DNA. The 18S rRNA gene of piroplasms was targeted. A restriction pattern of a previously unreported piroplasm was observed. The PCR product was sequenced, and the sequence obtained had 99% identity with the Rangelia vitalii sequences from Brazil when compared by BLAST analysis. Further characterization of the detected piroplasm consisted of nearly full-length sequencing (1668 bp) of the 18S rRNA gene of this organism. Those sequences were deposited in GenBank. A phylogenetic analysis indicated that they clustered together with R. vitalii from Brazil but separately from large Babesia species of dogs such as Babesia canis, and from species of Theileria of dogs as well. This is the first report of R. vitalii infection in Argentina, and the first case of canine rangeliosis diagnosed outside Brazil. Fil: Eiras, Diego Fernando. Laboratorio Diagnóstico en Animales Pequeños. Banfield; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina Fil: Craviotto, Maria Belen. Laboratorio Diagnóstico en Animales Pequeños. Banfield; Argentina Fil: Baneth, Gad. The Hebrew University Of Jerusalem; Israel Fil: Moré, Gastón Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina

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A new species of haematozoa, Babesia ugwidiensis sp. nov. from a cormorant is described. This is the first species of piroplasm to be recorded from the Phalacrocoracidae and the relationship of this parasite to other Babesia spp. from marine hosts is discussed.

The Iberian lynx (Lynx pardinus) is the most endangered felid in the world. Only about 160 individuals remain in 2 separate metapopulations in Southern Spain (Sierra Morena and Don˜ ana). We obtained blood samples of 20 lynxes captured from 2004 to 2006, and determined the prevalence of infection and genetic diversity of Cytauxzoon spp. using 18S rRNA PCR and sequence analysis. Prevalence of infection was 15 % (3 of 20). Cytauxzoon sp. was only detected in Sierra Morena. For phylogenetic analysis, we used the sequences reported in the present study and those characterized in different domestic and wild felids and ticks from North and South America, Asia and Europe. Three different Cytauxzoon sp. sequences were obtained. They were closely related to that obtained from a Spanish cat, but diverged in up to 1.0 % with respect to the only previously reported sequence from an Iberian lynx. Conversely, the latter sequence clustered together with C. manul sequences obtained from Pallas cats (Otocolobus manul) in Mongolia. Our analysis yields a separate cluster of C. felis sequences from cats, wild felids and ticks in the United States and Brazil. These results suggest that at least 2 different Cytauxzoon spp. may be present in Iberian lynx. The apparent absence in one of the areas, together with the possibility of fatal cytauxzoonosis in lynxes makes necessary disease risks to be taken into account in management conservation strategies, such as translocations and re-introductions.