Haemoprotozoa in wild short-beaked echidnas (Tachyglossus aculeatus).

The occurrence and clinical significance of the protozoal parasite reported as Hepatozoon tachyglossi in wild short-beaked echidnas (Tachyglossis aculeatus) have long been uncertain, as has its potential as a prognostic indicator. This retrospective survey of free-ranging short-beaked echidnas admitted to a wildlife hospital used morphological evidence to identify a H. tachyglossi prevalence of 56%, with parasitaemias affecting 0%-36% of monocytes. There was no statistical association between H. tachyglossi intensity and clinical status (P-value = 0.12; 95% confidence interval = 0.1 to 1.3), nor between the presence of H. tachyglossi and age, reason for admission, outcome, season or location. Piroplasms, presumed to be Theileria tachyglossi, were concurrently identified in the erythrocytes of 88% of short-beaked echidnas with no association between age, outcome, season or location, but a statistical association with the location where the animal was found (either on a road, airport runway, exposed urban area, or entangled). Given the current results, intracellular parasitism due to H. tachyglossi may be considered as an incidental finding on haematologic examination of short-beaked echidnas and is likely not an effective prognostic indicator. Further research using molecular tools is required to resolve the uncertain identity of H. tachyglossi which has been based on morphologic characteristics alone.

A community approach of pathogens and their arthropod vectors (ticks and fleas) in dogs of African Sub-Sahara.

BACKGROUND: Arthropod-borne pathogens and their vectors are present throughout Africa. They have been well-studied in livestock of sub-Saharan Africa, but poorly in companion animals. Given the socio-economic importance of companion animals, the African Small Companion Animal Network (AFSCAN), as part of the WSAVA Foundation, initiated a standardized multi-country surveillance study. METHODS: Macro-geographic variation in ectoparasite (ticks and fleas) and pathogen communities in dogs was assessed through molecular screening of approximately 100 infested dogs in each of six countries (Ghana, Kenya, Nigeria, Tanzania, Uganda and Namibia), both in rural and urban settings. The most important intrinsic and extrinsic risk factors within the subpopulation of infested dogs were evaluated. RESULTS: Despite the large macro-geographic variation in the dogs screened, there was no consistent difference between East and West Africa in terms of the diversity and numbers of ticks. The highest and lowest numbers of ticks were found in Nigeria and Namibia, respectively. Most often, there was a higher diversity of ticks in rural habitats than in urban habitats, although the highest diversity was observed in an urban Uganda setting. With the exception of Namibia, more fleas were collected in rural areas. We identified tick species (including Haemaphysalis spinulosa) as well as zoonotic pathogens (Coxiella burnetti, Trypanosoma spp.) that are not classically associated with companion animals. Rhipicephalus sanguineus was the most abundant tick, with a preference for urban areas. Exophilic ticks, such as Haemaphysalis spp., were more often found in rural areas. Several multi-host ticks occurred in urban areas. For R. sanguineus, housing conditions and additional pets were relevant factors in terms of infestation, while for a rural tick species (Haemaphysalis elliptica), free-roaming dogs were more often infested. Tick occurrence was associated to the use of endoparasiticide, but not to the use of ectoparasiticide. The most prevalent tick-borne pathogen was Hepatozoon canis followed by Ehrlichia canis. High levels of co-parasitism were observed in all countries and habitats. CONCLUSIONS: As dogs share a common environment with people, they have the potential to extend the network of pathogen transmission to humans. Our study will help epidemiologists to provide recommendations for surveillance and prevention of pathogens in dogs and humans.

Phylogeography and population differentiation in Hepatozoon canis (Apicomplexa: Hepatozoidae) reveal expansion and gene flow in world populations.

BACKGROUND: Hepatozoon canis is a protozoan transmitted to dogs and other wild carnivores by the ingestion of ticks containing mature oocysts and is considered the principal cause of canine hepatozoonosis in the world. Here, we examined ribosomal RNA 18S gene sequence variation to determine the genetic differences and phylogeographic diversity of H. canis from various geographical areas around the world. METHODS: We used 550 publicly available sequences of H. canis from 46 countries to assess haplotype relationships, geographical structure, genetic diversity indices, and relationships among populations. We performed neutrality tests and pairwise comparisons of fixation index (FST) values between groups and pairwise comparisons of FST values between populations. To determine whether populations are structured, analyses of molecular variance (AMOVAs) and spatial analysis of molecular variance (SAMOVA) were performed. RESULTS: The dataset of H. canis yielded 76 haplotypes. Differentiation among populations indicated that there is no phylogeographical structure (GST = 0.302 ± 0.0475). Moreover, when samples were grouped by continents a significant FST was obtained, meaning that populations were genetically differentiated. The AMOVA showed that 57.4% of the genetic variation was explained by differences within populations when all locations were treated as a single group and revealed that there is no population structure when populations are grouped into two, three, and four groups (FCT, p > 0.05), suggesting that dispersal between populations is high. SAMOVA revealed significant FCT values for groups K = 5. The Tajima's D and Fu's Fs show that populations have undergone recent expansion, and the mismatch distribution analysis showed population expansion (multimodal distribution). CONCLUSIONS: The current molecular data confirmed that H. canis does not show phylogeographic or population structure. The haplotypes exhibit low genetic differentiation, suggesting a recent expansion due to gene flow among populations. These results provide pivotal information required for future detailed population genetic analysis or to establish control strategies of this parasite.

Hepatozoon cevapii (Apicomplexa: Hepatozoidae) in the Thamnodynastes lanei snake (Colubridae, Tachymenini) from the Eastern Amazon, Brazil.

The aim of the present study was to verify the occurrence of hemogregarines in the colubrid snake Thamnodynastes lanei from the eastern Amazon region of Brazil. Intraerythrocytic gamonts with mean dimensions of 14.8 ± 1.8 × 4.0 ± 0.7 µm and encapsulated gamonts with mean dimensions of 15.3 ± 1.1 × 4.8 ± 0.5 µm were observed. Through morphological and molecular data based on the partial 18S rDNA gene, the parasite was identified as Hepatozoon cevapii, originally described in the viperid snake Crotalus durissus terrificus from the southeast region of Brazil. Thus, the findings of the present study extend the geographic range of H. cevapii and provide novel Hepatozoon-snake associations.

Hepatozoon in Eurasian red squirrels Sciurus vulgaris, its taxonomic identity, and phylogenetic placement.

Adeleorid apicomplexan parasites of the genus Hepatozoon Miller, 1908 are broadly distributed among the rodents. Broader molecular data on Hepatozoon from Palaearctic squirrels are necessary for evaluation of diversity and origin of Hepatozoon in Eurasian red squirrel Sciurus vulgaris populations, considering ongoing invasion by Gray squirrel S. carolinensis. Our report brings a set of molecular data from a population of S. vulgaris in the Czech Republic, non-invaded by any invasive squirrel species. Cadavers of 41 Eurasian red squirrels were examined using nested PCR targeting 18S rRNA gene; 30 animals tested positive for the presence of Hepatozoon spp. DNA in at least one tissue. Phylogenetic analysis of obtained sequence types revealed relatedness to sequences of Hepatozoon sp. from S. vulgaris from Spain and the Netherlands, forming a sister clade to Hepatozoon isolates from other European rodents. The fact that all available 18S rRNA gene sequences form a monophyletic clade is interpreted as a presence of a single Hepatozoon species in S. vulgaris in continental Europe, most probably Hepatozoon sciuri. The presented molecular data on the Hepatozoon from European squirrels provides a basis for future studies on possible exchange of Hepatozoon species between Eurasian red and gray squirrels.

DESCRIPTION OF THE FIRST KLOSSIA SPECIES (APICOMPLEXA: EUCOCCIDIORIDA: ADELEORINA: ADELEIDAE) INFECTING A PULMONATE LAND SNAIL, TRIODOPSIS HOPETONENSIS (MOLLUSCA: POLYGYRIDAE), IN NORTH AMERICA.

Snails identified as Triodopsis hopetonensis were collected (n = 18) from the University of Arkansas-Fayetteville campus in December 2018. Additional snails were collected in April 2019 (n = 9) and in September 2019 (n = 9). Kidney tissues were examined using light microscopy, and polysporocystic oocysts were observed. Sporulated oocysts (n = 2) measured 78 ± 3.4 µm × 76 ± 2.9 µm with an irregular oocyst residuum and contained an estimated 44-55 tetrazoic sporocysts. The sporocysts (n = 10) measured 13 ± 0.5 µm × 11 ± 1.5 µm with an indistinct, irregularly placed, sporocyst residuum and usually contained 4 sporozoites, although an octozoic variant was observed. DNA was extracted from the snail kidney tissues and used as a template for polymerase chain reaction (PCR). PCR was used to determine the infection status of the snails; 13 of 36 (36%) specimens were found to be infected with a new Klossia species, and only 3 (8%) of these infections were detected using light microscopy. The complete nuclear 18S ribosomal DNA (1,800 bp) and mitochondrial genomes (6,775 bp) were generated, and they differentiated this parasite from the type species Klossia helicina and support the description of this new Klossia species, Klossia razorbacki n. sp. This is the first Klossia species to be described from any North American snail.

Parasites in a hotspot: diversity and specificity patterns of apicomplexans infecting reptiles from the Socotra Archipelago.

Although parasites represent a major component of biodiversity, they remain poorly assessed, especially in remote regions. In this study, we screened 461 reptiles from Socotra, the largest and most biologically diverse archipelago in Arabia. Using 18S rRNA primers, we detected various apicomplexan parasites, namely haemogregarines, sarcocystids and eimeriids. Haemogregarines were the most common and genetically diverse, followed by sarcocystids (genus Sarcocystis) and eimeriids (genera Isospora and Lankesterella). All were related to parasites of other reptiles, including species from Arabia, Northern Africa and Asia. Like their 29 endemic reptile hosts, almost all Socotran parasites presented high genetic divergence and ecological differences from those found elsewhere, and probably represent undescribed endemic species. Among hosts, skinks were the most parasitized, which contrasted with similar studies from other areas, probably due to their more generalist diet and habitat use. As expected due to its high species richness, geckos harboured the highest parasite diversity in the archipelago. Parasite diversity also seemed to be correlated to island size, as the largest island harboured most haplotypes. This study emphasizes the importance of screening parasites in wild hosts from remote regions and of considering host ecology to understand disease transmission across taxa.

First record of Hepatozoon spp. in domestic cats in Greece.

Feline hepatozoonosis is an emerging disease of domestic and wild felids though there is limited knowledge of this infection, e.g. regarding geographical distribution and parasite species involved. The present study evaluated microscopically and molecularly the occurrence of Hepatozoon spp. in domestic cats from insular (Crete, Mykonos and Skopelos) and continental (prefectures of Attica and Thessaloniki) Greece. Out of 282 cats examined, 72 (25.5 %) scored positive by PCR for Hepatozoon spp. and of them, 9 (12.5 %) showed gamonts on the blood smear microscopic examination. Sequences obtained from 35 of the amplicons proved the presence of two haplotypes of Hepatozoon felis. One, herein called H1 (34/35 amplicons) resulted 100 % identical with H. felis from Italy and isolates from other continents, and ∼98 % similar with a H. felis isolate causing severe clinical signs in Austria. The haplotype H2, found in a cat in Skopelos, had ∼94 % identity with H1, with H. felis isolates from Italy, Israel, Spain, a ∼92 % identity with the isolate from Austria, and ∼94-98 % with isolates from South Africa. These are the first records of H. felis in cat populations from Greece and indicate that the infection may be present at high prevalences in different regions of the country. Furthermore, the results of the molecular and phylogenetic analysis support a recent hypothesis indicating the existence of a species-complex classification for H. felis. Further studies aiming at elucidating the genetic make-up of Hepatozoon populations and possible variations in terms of geographic distribution and clinical relevance are necessary. The importance of a continuous epizootiological monitoring is crucial for the establishment of preventative and control measures protecting the health of cats living in or travelling to enzootic areas.

Molecular detection of vector-borne agents in ectoparasites and reptiles from Brazil.

Trombidiformes and Mesostigmata mites, as well as Ixodida ticks, infest ectothermic tetrapods worldwide, potentially acting as vectors of bacteria, viruses and protozoa. The relationship among ectoparasites, transmitted pathogenic agents (e.g., Borrelia spp., Coxiella spp., Hepatozoon spp., and Rickettsia spp.) and ectothermic hosts has been scarcely investigated. This research focuses on a large collection of Brazilian herpetofauna screened for the presence of arthropod ectoparasites and vector-borne microbial agents. Reptiles (n = 121) and amphibians (n = 49) from various locations were infested by ectoparasites. Following genomic extraction, microbial agents were detected in 81 % of the Acari (i.e. n = 113 mites and n = 26 ticks). None of the mites, ticks and tissues from amphibians yielded positive results for any of the screened agents. Blood was collected from reptiles and processed through blood cytology and molecular analyses (n = 48). Of those, six snakes (12.5 %) showed intraerythrocytic alterations compatible with Hepatozoon spp. gamonts and Iridovirus inclusions. Hepatozoon spp. similar to Hepatozoon ayorgbor and Hepatozoon musa were molecularly identified from seven hosts, two mite and two tick species. Rickettsia spp. (e.g., Rickettsia amblyommatis, Rickettsia bellii-like, Rickettsia sp.) were detected molecularly from four mite species and Amblyomma rotundatum ticks. Phylogenetic analyses confirmed the molecular identification of the above-mentioned microbial agents of mites and ticks related to snakes and lizards. Overall, our findings highlighted that the Brazilian herpetofauna and its ectoparasites harbour potentially pathogenic agents, particularly from the northern and south-eastern regions. The detection of several species of spotted fever group Rickettsia pointed out the potential role of ectothermic hosts and related arthropod ectoparasites in the epidemiological cycle of these bacteria in Brazil.

The red fox (Vulpes vulpes), a possible reservoir of Babesia vulpes, B. canis and Hepatozoon canis and its association with the tick Dermacentor reticulatus occurrence.

The red fox (Vulpes vulpes) is known to be a reservoir host of various vector-borne protozoan parasites. Yet, the impact of these parasitic infections on the health status of the red fox is unknown. In this study, we compared the prevalence of haemoprotozoa in juvenile and adult foxes from four regions in Poland. The abundance of questing adult Dermacentor reticulatus ticks was shown to differ between regions and include a tick-endemic and non-endemic region, as well as two zones in which D. reticulatus recently expanded: an eastern and a western zone. Spleen and kidney enlargement indices were compared between infected, co-infected and non-infected foxes to evaluate pathogenic output of parasitic infections. Blood samples were collected from 383 necropsied red foxes and 25 live-trapped red fox cubs. The weights of spleens, kidneys and bodies were recorded for each necropsied individual. Blood and spleen DNA samples were screened by PCR to detect 18S rRNA and cox1 gene fragments of Babesia spp., and 18S rRNA of Hepatozoon spp. Prevalence of infection and mean organ weight indices were compared in relation to fox age and sex, origin (zone) and infection status. Hepatozoon canis (174/381; 45.7%) and Babesia vulpes (117/381; 30.7%) were the dominant pathogens infecting adult foxes. Babesia canis DNA was detected in 9 (2.4%) individuals. Two fox cubs from Western Poland were infected with B. vulpes (2/14; 14%), while H. canis infection (16/25; 59%) was detected in cubs from all three regions where trapping was carried out. Infection with B. vulpes was associated with significantly higher weights of spleens and kidneys. Spleen weight/ body weight and mean kidney weight/ body weight indices were shown as relevant in evaluating changes occurring during infection with B. vulpes. Babesia and Hepatozoon spp. infections differed in red foxes from D. reticulatus-endemic, non-endemic and newly inhabited areas. The prevalence of B. canis in foxes from four regions of Poland reflects the prevalence recorded in questing adult D. reticulatus collected in these areas. This finding suggests a contribution of red foxes to the establishment of new foci of B. canis-infected D. reticulatus ticks in areas recently invaded by this tick species. Spleen and kidney enlargement was identified in foxes infected with B. vulpes, particularly in individuals co-infected with B. vulpes and H. canis.

Molecular detection and characterization of Hepatozoon canis in stray dogs from Cuba.

Canine hepatozoonosis caused by Hepatozoon canis is a worldwide distributed tick-borne disease of domestic and wild canids that is transmitted by ingestion of Rhipicephalus sanguineus sensu lato (s.l.) ticks. The present study was aimed to determine the prevalence of Hepatozoon infections in 80 stray dogs from Havana Province in Cuba, and to confirm the species identity and phylogenetic relationships of the causative agent. Samples were screened by microscopical examination of thin blood smears for the presence of Hepatozoon spp. gamonts and by genus-specific SYBR green-based real-time PCR assay targeting the 18S rRNA gene. Direct microscopy examination revealed Hepatozoon gamonts in the peripheral blood of 8 dogs (10.0%; 95% CI: 4.80-18.0%), while 38 animals (47.5%; 95% CI: 36.8-58.4%) were PCR-positive, including all microscopically positive dogs. Hence, the agreement between the two detection methods was 'poor' (κ = 0.20). Hematological parameters did not differ significantly between PCR-positive and PCR-negative dogs (p > 0.05). The DNA sequences of the 18S rRNA gene of the Hepatozoon spp. from Cuban dogs showed a nucleotide identity >99% with those of 18S rRNA sequences of Hepatozoon canis isolates from Czech Republic, Brazil and Spain. Phylogenetic analysis revealed that obtained sequences clustered within the Hepatozoon canis clade, different from the Hepatozoon felis or Hepatozoon americanum clades. The present study represents the first molecular characterization of Hepatozoon canis in stray dogs within Cuba.

Molecular detection and characterization of tick-borne hemoparasites and Anaplasmataceae in dogs in major cities of Malawi.

Tick-borne pathogens (TBPs) in dogs have attracted much attention over the last decade since some are now known to be zoonotic and pose a threat to both animal and human health sectors. Despite the increase in the number of studies on canine TBPs worldwide, only a few studies have been conducted in resource-limited countries where research priority is given to food animals than companion animals. In the present study, the occurrence of TBPs of the genera Babesia, Hepatozoon, Anaplasma, and Ehrlichia was investigated in 209 owned and stray dogs in three major cities in Malawi through molecular techniques. Among the examined dogs, 93 (44.5%) were infected with at least one TBP. The detection rates were 23.1% for Babesia rossi, 2.9% for B. vogeli, 19.1% for Hepatozoon canis, 2.4% for Anaplasma platys, and 3.8% for Ehrlichia canis. This is the first molecular study that has provided evidence that dogs in Malawi are infected with TBPs. Sensitization is required for veterinary practitioners, dog handlers, and pet owners as the detected pathogens affect the animals' wellbeing. Further studies focusing on rural areas with limited or no access to veterinary care are required to ascertain the extent of the TBP infection in dogs.

Morphological and molecular data of Hepatozoon ursi in two brown bears (Ursus arctos) in Turkey.

Species of Hepatozoon Miller, 1908 are vector-borne parasites that infect domestic and wild animals worldwide. Hepatozoon ursi Kubo, Uni, Agatsuma, Nagataki, Panciera et al., 2008 was reported from bears (Ursidae) in Japan and India. The present study represents the first report of infection with H. ursi in Turkish brown bears (Ursus arctos Linnaeus) by microscopic and molecular analysis. Two dead brown bears were found in Uzundere and Pasinler districts of Erzurum. Blood and visceral organ (spleen and liver) samples were delivered to laboratory by the Nature Conservation and National Parks officers. Detected gamonts were evaluated based on morphological features and confirmed as gamonts of H. ursi. The size of gamonts and parasitemia were 8.2 × 3.5 µm (6.9-8.7 × 3.0-3.9 µm; n = 12) and 0.6% (6/1000 leukocytes), respectively. The blood and visceral organ samples were positive for species of Hepatozoon by PCR targeting partial sequence of 18S rDNA. Sequence analysis of newly obtained sequences of H. ursi showed 98.8-100% identity with previously sequenced isolates of H. ursi. Sequences of H. ursi from Erzurum were identical to each other and showed 100% identity with isolates of H. ursi from ticks Ixodes ricinus (Linnaeus), Rhipicephalus turanicus Pomerantzev and Hyalomma marginatum Koch collected from two brown bears in Turkey (GenBank accession numbers MN463021, MN463022, MN905023). Analysis of partial sequences of the 18S rRNA gene of H. ursi showed that Turkish isolates differ in NT substitutions found at three different positions [72 (A→G), 537 (A→G) and 570 (A→T)]. This study provides morphological and molecular data of H. ursi infection in brown bears from two districts of Erzurum, Turkey. Further studies are needed to elucidate whether brown bears have any eco-epidemiologic importance in the life cycle of H. ursi in wildlife.

Detection of tick-borne pathogens in Rhipicephalus sanguineus sensu lato and dogs from different districts of Portugal.

Dogs are highly exposed to pathogens transmitted by ectoparasites. The Mediterranean climate of Southern Europe, together with the presence of stray and/or neglected pets in close proximity with humans, contribute for tick expansion and stand for increased risk to infections in humans due to the zoonotic potential of many of these agents. The aim of this study was to perform a molecular survey in dogs (suspected of tick-borne disease and/or infested with ticks), as well as in ticks collected from those animals, from 12 districts of Portugal to investigate the occurrence of Rickettsia spp. and other tick-borne pathogens (Babesia, Ehrlichia, Anaplasma and Hepatozoon). Additionally, a serological survey of spotted fever group Rickettsia in Portuguese dogs was performed using an in-house immunofluorescence assay (IFA). A total of 200 whole-blood samples and 221 Rhipicephalus sanguineus s. l. ticks were collected from dogs. A total of 14 (7 %) blood samples and 10 (4.5 %) ticks yielded presumptively positive 420-bp amplicons using the Rickettsia spp. partial ompB nested PCR. Screening of the ompB-positive samples using the gltA gene showed 8 positive ticks. All Rickettsia ompB and gltA sequences had the highest identity with R. massiliae. The Rickettsia-positive dogs were further tested for other tick-borne pathogens and were found to be infected with Babesia spp. (n = 5), but not with Ehrlichia, Anaplasma or Hepatozoon. Of the 149 dog serum specimens tested in the serological assay, 103 (69 %) were positive for IgG antibodies against spotted fever group Rickettsia. Antibodies were found in dogs from all the studied districts, in 55 (53 %) of the stray and in 48 (47 %) of the owned dogs. Our study detected and characterized for the first time R. massiliae in dogs from Portugal, broadening the geographical range of this canine pathogen and adding knowledge to the impact of this disease in dogs.

Development and application of multiplex PCR assay for the simultaneous detection of Babesia vogeli, Ehrlichia canis and Hepatozoon canis in dogs.

A multiplex PCR assay was standardized and evaluated to simultaneously detect the DNA of Babesia vogeli, Ehrlichia canis and Hepatozoon canis in dogs of selected districts of Punjab state, India. Amplicons of 602 bp, 380 bp and 306 bp corresponding to B. vogeli (18S rRNA gene), E. canis (VirB9 gene), and H. canis (18S rRNA gene) were obtained, without any non-specific amplification. The results of multiplex PCR assay were further compared with the corresponding singleplex PCR assay. The diagnostic sensitivity and specificity of multiplex PCR assay with respect to singleplex PCR assay in the detection of B. vogeli, E. canis and H. canis varied from 50% to 100% and 92.08% to 98.79%, respectively revealing "moderate" to "very good" agreement by kappa value statistics. Blood samples from 322 dogs collected from selected districts of Punjab state, India, when screened by microscopy revealed the prevalence of B. vogeli, E. canis and H. canis as 0.31%, 0.93% and 1.86%, respectively whereas with multiplex PCR assay the values were 0.93%, 10.24% and 4.65%, respectively, with concurrent infection of E. canis & H. canis (1.86%) and B. vogeli & E. canis (0.31%). The diagnostic sensitivity and specificity of multiplex PCR assay with respect to microscopy in the detection of B. vogeli, E. canis and H. canis varied from 69.15% to 100% and 85.11% to 92.33%, respectively revealing "fair" agreement by kappa value statistics and the data was statistically significant. The analytical sensitivity of multiplex PCR assay in the detection of B. vogeli, E. canis and H. canis was 100 pg, 10 pg and 0.1 pg, respectively, whereas the values for the singleplex counterpart were 0.1 pg, 0.01 pg and 0.01 pg. Furthermore, various risk factors viz. age, breed, sex, season and districts were non-significantly associated with the prevalence of these haemoparasites except for E. canis that revealed a significant association with districts by multiplex PCR assay. Therefore the multiplex PCR assay developed may be useful in identification of the aetiological agents of these diseases during their early phase, which may in turn be useful in development of better health care and appropriate treatment of suspected dogs, particularly in endemic regions.

Scarcity of Hepatozoon americanum in Gulf Coast tick vectors and potential for cultivating the protozoan.

American canine hepatozoonosis (ACH) is a debilitating tick-borne disease characterized by pyrexia, body wasting, myopathy, mucopurulent ocular discharge, and periosteal proliferation. The causative agent, Hepatozoon americanum, is an apicomplexan that utilizes the Gulf Coast tick, Amblyomma maculatum, as its definitive host and vector. Unlike most tick-borne disease agents, H. americanum is not transmitted via a tick bite, but is transmitted when canids ingest a tick vector that contains sporulated oocysts within the tick hemocoel or paratenic hosts with cystozoites. Our understanding of H. americanum prevalence is based on its detection in the intermediate host, wild or domestic canids, with domestic canids often showing clinical signs at the time of diagnosis. The frequency of H. americanum in A. maculatum, on the other hand, is unknown; this gap in our knowledge hinders our understanding of transmission risk. Furthermore, current diagnostic assays are limited in efficacy, and serologic assays are not widely available. To begin to address gaps in our knowledge, we developed a TaqMan® multiplex qPCR assay for H. americanum detection in A. maculatum tick extracts and evaluated infection rates in questing adult A. maculatum. Additionally, we used a co-culture system to expose H. americanum stages to host cells for in vitro development. Results from qPCR analysis of over 500 tick extracts revealed no positive samples; this suggests both low transmission risk by adult Gulf Coast tick ingestion in the sampled areas, and that surveillance should be focused in areas where ACH has been diagnosed at higher frequencies. Hepatozoon americanum was detectable by qPCR in co-culture of an infected canine buffy coat with ISE6 (Ixodes scapularis embryonic) tick cells, and microscopic examination of samples from those days revealed some structures that were suspicious for developing stages. These data are a starting point for future work to advance our understanding of H. americanum transmission and mechanisms of disease in canids with ACH.

Molecular detection of pathogens in ticks and fleas collected from companion dogs and cats in East and Southeast Asia.

BACKGROUND: Ticks and fleas are considered amongst the most important arthropod vectors of medical and veterinary concern due to their ability to transmit pathogens to a range of animal species including dogs, cats and humans. By sharing a common environment with humans, companion animal-associated parasitic arthropods may potentially transmit zoonotic vector-borne pathogens (VBPs). This study aimed to molecularly detect pathogens from ticks and fleas from companion dogs and cats in East and Southeast Asia. METHODS: A total of 392 ticks and 248 fleas were collected from 401 infested animals (i.e. 271 dogs and 130 cats) from China, Taiwan, Indonesia, Malaysia, Singapore, Thailand, the Philippines and Vietnam, and molecularly screened for the presence of pathogens. Ticks were tested for Rickettsia spp., Anaplasma spp., Ehrlichia spp., Babesia spp. and Hepatozoon spp. while fleas were screened for the presence of Rickettsia spp. and Bartonella spp. RESULT: Of the 392 ticks tested, 37 (9.4%) scored positive for at least one pathogen with Hepatozoon canis being the most prevalent (5.4%), followed by Ehrlichia canis (1.8%), Babesia vogeli (1%), Anaplasma platys (0.8%) and Rickettsia spp. (1%) [including Rickettsia sp. (0.5%), Rickettsia asembonensis (0.3%) and Rickettsia felis (0.3%)]. Out of 248 fleas tested, 106 (42.7%) were harboring at least one pathogen with R. felis being the most common (19.4%), followed by Bartonella spp. (16.5%), Rickettsia asembonensis (10.9%) and "Candidatus Rickettsia senegalensis" (0.4%). Furthermore, 35 Rhipicephalus sanguineus ticks were subjected to phylogenetic analysis, of which 34 ticks belonged to the tropical and only one belonged to the temperate lineage (Rh. sanguineus (sensu stricto)). CONCLUSION: Our data reveals the circulation of different VBPs in ticks and fleas of dogs and cats from Asia, including zoonotic agents, which may represent a potential risk to animal and human health.

Molecular detection of Hepatozoon canis in dogs and ticks in Shaanxi province, China.

Hepatozoon canis, transmitted by Rhipicephalus sanguineus, is a tick-borne pathogen and causes canine hepatozoonosis. Until now, only limited previous studies were conducted on the molecular detection and characterization of Hepatozoon sp. in dogs in China. Blood samples were collected from 93 sick dogs that were clinically diagnosed as babesiosis but tested negative for Babesia, and 103 apparently healthy dogs, as well as their infesting ticks in Xi'an and Hanzhong cities, Shaanxi province of China. PCR amplifying partial 18S rRNA gene was used to detect the DNA of Hepatozoon sp. Genetic and phylogenetic analysis were performed to determine the Hepatozoon species. Our results demonstrated that H. canis was identified from the sick dogs and the infested ticks in Hanzhong, with no significant differences of prevalence between both genders and ages. No positive blood or tick samples were found in Xi'an. Moreover, all the 18S rRNA gene sequences recovered from both dogs and the infested ticks showed a high genetic similarity with each other, and also presented a close relationship with other known sequences in and outside China. In conclusion, H. canis was identified in babesiosis-suspected dogs and ticks infesting them in Shaanxi, China, although the association between clinical signs and H. canis need further study.

Hepatozoon canis in hunting dogs from Southern Italy: distribution and risk factors.

Hepatozoon canis is a hemoprotozoan organism that infects domestic and wild carnivores throughout much of Europe. The parasite is mainly transmitted through the ingestion of infected ticks containing mature oocysts. The aims of the present survey were to determine the prevalence of H. canis in hunting dogs living in Southern Italy and to assess potential infection risk factors. DNA extracted from whole blood samples, collected from 1433 apparently healthy dogs living in the Napoli, Avellino, and Salerno provinces of Campania region (Southern Italy), was tested by a quantitative real-time polymerase chain reaction (qPCR) assay to amplify H. canis. Furthermore, the investigated dog population was also screened by qPCR for the presence of Ehrlichia canis, a major tick-borne pathogen in Southern Italy, in order to assess possible co-infections. Two hundred dogs were H. canis PCR-positive, resulting in an overall prevalence of 14.0% (CI 12.2-15.9). Breed category (P < 0.0001), hair coat length (P = 0.015), and province of residence (P < 0.0001) represented significant risk factors for H. canis infection. The presence of H. canis DNA was also significantly associated with E. canis PCR positivity (P < 0.0001). Hunting dogs in Campania region (Southern Italy) are frequently exposed to H. canis, and the infection is potentially associated with close contact with wildlife. Further studies are needed to assess the pathogenic potential of H. canis, as well as the epidemiological relationships between hunting dogs and wild animal populations sharing the same habitats in Southern Italy.

The genetic diversity of blood parasites within the freshwater turtles Mauremys leprosa and Emys orbicularis in Tunisia reveals coinfection with Haemogregarina spp.

Haemogregarina species are apicomplexan blood parasites infecting vertebrates such as fish, lizards, and turtles. Due to the high morphological similarity of the erythrocytic stages infecting host species, it has always been a challenge to identify the true diversity of these parasites. Therefore, taxonomic studies are presently based on the combination of morphological and molecular data. In Tunisia, two species of Haemogregarina have been reported within the freshwater turtle Mauremys leprosa (Geoemydidae) for more than 40 years. Since M. leprosa occurs in the same aquatic environments as Emys orbicularis (Emydidae) in Tunisia, our objectives were to assess parasite diversity and specificity on the basis of both morphological and molecular approaches. The turtles were surveyed and sampled across six aquatic areas of Tunisia. Among the 39 specimens of M. leprosa and seven of E. orbicularis that were trapped and investigated, the presence of haemogregarines was detected in the blood of turtles only at sites where leeches were observed. Three 18S variants were identified, which corresponded to three distinct Haemogregarina species, among which one was identified as Haemogregarina stepanowi. The two other species that were detected are likely new to science. Because we show the occurrence of more than one blood parasite species within a single host specimen, our study provides the first report of coinfection with molecularly distinct Haemogregarina spp.