Cytauxzoonosis in Indiana, USA: a case series of cats infected with Cytauxzoon felis (2018-2022).

CASE SERIES SUMMARY: This case series describes six cases involving seven cats naturally infected with Cytauxzoon felis in Indiana, USA. Medical records were retrospectively reviewed and all available information on signalment, history, clinical and diagnostic findings, treatment, outcome and pathology was reported. Cats infected with C felis were domestic shorthairs, were aged between 2 and 9 years and all but one of the cats were male. The seven infected cats originated from five counties in southwestern Indiana. Six of seven cats were found to have acute cytauxzoonosis based on clinical signs, gross pathologic lesions, observation of C felis in tissues and/or detection of C felis DNA. One cat was identified as a subclinical survivor cat with no known clinical history of cytauxzoonosis. RELEVANCE AND NOVEL INFORMATION: The reported cases are the first confirmed reports of acute and chronic cytauxzoonosis in cats from Indiana and document an expansion in the range of C felis. Veterinary practitioners in Indiana should consider infection with C felis as a differential diagnosis for cats that present with fever, inappetence, lethargy, depression, dehydration, dyspnea, hemolytic crisis, anorexia or icterus. Administration of approved acaricides to cats currently offers the best protection and control against C felis infection.

Unresolved haemosporidia of the Australian skink, Egernia stokesii.

The Australian skink Egernia stokesii had been recognised as a host of two species of Plasmodium, Plasmodium mackerrasae and P. circularis; nevertheless, molecular data are available for only a single haemosporidian species of this host. Its sequences are labelled as "Plasmodium sp." or "Plasmodium mackerrasae", but morphological characteristics of this isolate are unavailable. Phylogenetic analyses of these sequences placed them into the clade of the genus Haemocystidium. In this study, blood samples of six E. stokesii were analysed by both, molecular and microscopic methods to clarify the haemosporidia of this lizard. Application of these approaches offered discordant results. Whereas sequence analysis clustered our isolates with lizard species of Haemocystidium, morphology of blood stages is more akin to Plasmodium than Haemocystidium. However, limited sampling, indistinguishable nuclei/merozoites and risk of possible hidden presence of mixed infection prevent reliable species identification of detected parasites or their description as new species of Haemocystidium.

Concurrent Histomonas meleagridis and Hemorrhagic Enteritis Virus Infection in a Turkey Flock with Recurrent History of Blackhead Disease.

Intestinal health is one of the key factors required for the growth and production of turkeys. Histomoniasis (blackhead disease), caused by a protozoan parasite, Histomonas meleagridis, is a reemerging threat to the turkey industry. Increased incidences of histomoniasis have been reported in recent years due to withdrawal of antihistomonas treatments. H. meleagridis affects ceca and causes cecal inflammation and necrosis. H. meleagridis migrates from ceca to the liver and causes liver necrosis, resulting in high mortalities. Ironically, field outbreaks of histomoniasis are not always associated with high mortalities, while low mortalities have also been documented. There are several exacerbating factors associated with high mortality rates in histomoniasis outbreaks, with concurrent infection being one of them. Recurrent histomoniasis outbreaks in a newly constructed barn were documented, and concurrent infection of H. meleagridis and hemorrhagic enteritis virus was confirmed. Currently, neither commercial vaccines nor prophylactic or therapeutic solutions are available to combat histomoniasis. However, there are treatments, vaccines, and solutions to minimize or prevent concurrent infections in turkeys. In addition to implementing biosecurity measures, measures to prevent concurrent infections are critical steps that the turkey industry can follow to reduce mortality rates and minimize the production and economic losses associated with histomoniasis outbreaks.

Infección simultánea por Histomonas meleagridis y el virus de la enteritis hemorrágica en una parvada de pavos con antecedentes recurrentes de enfermedad de la cabeza negra. La salud intestinal es uno de los factores clave necesarios para el crecimiento y producción de los pavos. La histomoniasis (enfermedad de la cabeza negra), causada por un parásito protozoario, Histomonas meleagridis, es una amenaza reemergente para la industria del pavo. En los últimos años se ha informado de un aumento de la incidencia de histomoniasis debido al retiro de los tratamientos con antihistomonas. Histomonas meleagridis afecta los ciegos y causa inflamación y necrosis cecal. Histomonas meleagridis migra desde los ciegos al hígado y causa necrosis hepática, lo que resulta en una alta mortalidad. Irónicamente, los brotes de histomoniasis en el campo no siempre se asocian con una mortalidad elevada, aunque también se han documentado mortalidades bajas. Hay varios factores exacerbantes asociados con altas tasas de mortalidad en los brotes de histomoniasis, siendo la infección concurrente uno de ellos. Se documentaron brotes recurrentes de histomoniasis en un alojamiento avícola recién construido y se confirmó la infección concurrente de H. meleagridis y el virus de la enteritis hemorrágica. Actualmente no se dis-pone de vacunas comerciales ni soluciones profilácticas o terapéuticas para combatir la histomoniasis. Sin embargo, existen tratamientos, vacunas y soluciones para minimizar o prevenir infecciones concurrentes en los pavos. Además de implementar medidas de bioseguridad, las medidas para prevenir infecciones concurrentes son pasos críticos que la industria del pavo puede seguir para reducir las tasas de mortalidad y minimizar las pérdidas económicas y de producción asociadas con los brotes de histomoniasis.

High prevalence of haemosporidian parasites in Eurasian jays.

Avian haemosporidians are vector-borne parasites, infecting a great variety of birds. The order Passeriformes has the highest average infection probability; nevertheless, some common species of Passeriformes have been rather poorly studied. We investigated haemosporidians in one such species, the Eurasian jay Garrulus glandarius (Corvidae), from a forest population in Hesse, Central Germany. All individuals were infected with at least one haemosporidian genus (overall prevalence: 100%). The most common infection pattern was a mixed Haemoproteus and Leucocytozoon infection, whereas no Plasmodium infection was detected. Results on lineage diversity indicate a rather pronounced host-specificity of Haemoproteus and Leucocytozoon lineages infecting birds of the family Corvidae.

Molecular characterization of Haemoproteus enucleator with emphasis on the host and geographic distribution.

Haemoproteus species (Haemosporida, Haemoproteidae) are cosmopolitan and highly diverse blood parasites of birds that have been neglected in avian medicine. However, recent discoveries based on molecular diagnostic markers show that these pathogens often cause marked damage to various internal organs due to exo-erythrocytic development, sometimes resulting in severe and even lethal avian haemoproteosis, including cerebral pathologies. Molecular markers are essential for haemoproteosis diagnostics, but the data is limited, particularly for parasites transmitted in tropical ecosystems. This study combined microscopic and molecular approaches to characterize Haemoproteus enucleator morphologically and molecularly. Blood samples were collected from the African pygmy kingfisher Ispidina picta in Cameroon, and the parasite was identified using morphological characters of gametocytes. The analysis of partial cytochrome b sequences (cytb) identified a new Haemoproteus lineage (hISPIC03), which was linked to the morphospecies H. enucleator. Illustrations of blood stages were provided and the phylogenetic analysis showed that the new lineage clustered with five other closely related lineages belonging to the same morphospecies (hALCLEU01, hALCLEU02, hALCLEU03, hISPIC01, and hALCQUA01), with a maximum genetic distance between these lineages of 1.5 % (7 bp difference) in the 478 bp cytb sequences. DNA haplotype network was developed and identified geographic and host distribution of all lineages belonging to H. enucleator group. These lineages were almost exclusively detected in African kingfishers from Gabon, Cameroon, South Africa, and Botswana. This study developed the molecular characterization of H. enucleator and provides opportunities for diagnostics of this pathogen at all stages of its life cycle, which remains undescribed in all its closely related lineages.

In vitro efficacy testing of ivermectin against Tritrichomonas foetus trophozoites.

There are no recommended drugs to treat cattle infected with the protozoan Tritrichomonas foetus (TF). Ivermectin, widely used in the treatment of intestinal parasites, was found effective against some protozoa growing in vitro. Here, its effectiveness against a TF line was investigated. Trophozoites were incubated in media with increasing concentrations of ivermectin and mortality was determined after 24 h. Ivermectin killed cells with a mean maximum effective concentration (EC50) of 2.47 µg mL-1. The effective concentration of ivermectin was rather high for a formulation suitable for systemic treatment. However, topical treatment of animals against TF could still be considered and tested.

Novel phylogenetic clade of avian Haemoproteus parasites (Haemosporida, Haemoproteidae) from Accipitridae raptors, with description of a new Haemoproteus species.

Avian haemosporidian parasites (order Haemosporida, phylum Apicomplexa) are blood and tissue parasites transmitted by blood-sucking dipteran insects. Three genera (Plasmodium, Haemoproteus and Leucocytozoon) have been most often found in birds, with over 270 species described and named in avian hosts based mainly on the morphological characters of blood stages. A broad diversity of Haemoproteus parasites remains to be identified and characterized morphologically and molecularly, especially those infecting birds of prey, an underrepresented bird group in haemosporidian parasite studies. The aim of this study was to investigate and identify Haemoproteus parasites from a large sample comprising accipitriform raptors of 16 species combining morphological and new molecular protocols targeting the cytb genes of this parasite group. This study provides morphological descriptions and molecular characterizations of two Haemoproteus species, H. multivacuolatus n. sp. and H. nisi Peirce and Marquiss, 1983. Haemoproteus parasites of this group were so far found in accipitriform raptors only and might be classified into a separate subgenus or even genus. Cytb sequences of these parasites diverge by more than 15% from those of all others known avian haemosporidian genera and form a unique phylogenetic clade. This study underlines the importance of developing new diagnostic tools to detect molecularly highly divergent parasites that might be undetectable by commonly used conventional tools.

Title: Nouveau clade phylogénétique de parasites de rapaces Accipitridae du genre Haemoproteus (Haemosporida, Haemoproteidae), avec description d'une nouvelle espèce d'Haemoproteus. Abstract: Les parasites hémosporidies aviaires (ordre Haemosporida, phylum Apicomplexa) sont des parasites sanguins et tissulaires transmis par des insectes diptères hématophages. Trois genres (Plasmodium, Haemoproteus et Leucocytozoon) ont été le plus souvent trouvés chez les oiseaux, avec plus de 270 espèces décrites et nommées chez les hôtes aviaires en fonction principalement des caractères morphologiques des stades sanguins. Une grande diversité des Haemoproteus reste à identifier et à caractériser morphologiquement et génétiquement, en particulier ceux qui infectent les oiseaux de proie, un groupe d'oiseaux sous-représenté dans les études sur les hémosporidies. Le but de cette étude était d'étudier et d'identifier les Haemoproteus à partir d'un large échantillon comprenant des rapaces accipitriformes de 16 espèces, en combinant des protocoles morphologiques et de nouveaux protocoles moléculaires ciblant les gènes cytb de ce groupe de parasites. Cette étude fournit des descriptions morphologiques et des caractérisations moléculaires de deux espèces d'Haemoproteus, H. multivacuolatus n. sp. et H. nisi Peirce and Marquiss, 1983. Les Haemoproteus de ce groupe n'ont jusqu'à présent été trouvés que chez les rapaces accipitriformes et pourraient être classés dans un sous-genre ou même un genre distinct. Les séquences cytb de ces parasites divergent de plus de 15 % de celles de tous les autres genres d'hémosporidies aviaires connus et forment un clade phylogénétique unique. Cette étude souligne l'importance de développer de nouveaux outils de diagnostic pour détecter des parasites moléculairement très divergents qui pourraient être indétectables par les outils conventionnels couramment utilisés.

Molecular Survey of Haemosporidian Parasites in Procellariiformes Sampled in Southern Brazil, 2013-22.

The order Procellariiformes includes several species of seabirds that perform long-distance migrations crossing all the oceans. These movements may contribute to the dispersal and exchange of hemoparasites, such as haemosporidians. There is a lack of studies regarding the order Haemosporida in Procellariiformes, and, to date, only the genus Plasmodium has been reported. This survey investigated the occurrence of the three genera of haemosporidians, Plasmodium, Haemoproteus, and Leucocytozoon, in samples collected between 2013 and 2022 from 95 individuals of 14 species of Procellariiformes from southern Brazil, including live animals in rehabilitation centers, individuals caught as incidental bycatch, and carcasses found along the coast. A total of 171 samples of blood and fragments of liver and spleen were analyzed, with extracted DNA being subjected to a nested PCR followed by phylogeny analysis. All animals were negative for Plasmodium spp. and Leucocytozoon spp., but one Black-browed Albatross (Thalassarche melanophris) and one Manx Shearwater (Puffinus puffinus) specimen were positive for Haemoproteus spp. The sequences obtained from positive seabirds did not show 100% similarity with other known lineages available in the MalAvi database and thus were probably novel lineages. However, one sequence clustered together with Haemoproteus noctuae, a parasite from Strigiformes, while the other was grouped with Haemoproteus columbae, which is classically related to Columbiformes. These results suggest that both positive animals may have become infected when beached or in rehabilitation centers by a spillover of vectors from local birds. This highlights the importance of surveillance of the health of Procellariiformes regarding the possibility of dissemination of new pathogens in different bird populations.

Co-infecting Haemoproteus species (Haemosporida, Apicomplexa) show different host tissue tropism during exo-erythrocytic development in Fringilla coelebs (Fringillidae).

Avian haemosporidians of the genera Plasmodium, Haemoproteus, and Leucocytozoon are common blood parasites in wild birds all over the world. Despite their importance as pathogens potentially compromising host fitness and health, little is known about the exo-erythrocytic development of these parasites, particularly during co-infections which predominate in wildlife. This study aimed to address this issue using Haemoproteus parasites of Fringilla coelebs, a common bird species of the Western Palearctic and host to a variety of haemosporidian parasite lineages. Blood and tissue samples of 20 F. coelebs, positive for haemosporidians by blood film microscopy, were analysed by PCR and sequencing to determine cytochrome b lineages of the parasites. Tissue sections were examined for exo-erythrocytic stages by histology and in situ hybridization applying genus-, species-, and lineage-specific probes which target the 18S rRNA of the parasites. In addition, laser microdissection of tissue stages was performed to identify parasite lineages. Combined molecular results of PCR, laser microdissection, and in situ hybridization showed a high rate of co-infections, with Haemoproteus lineages dominating. Exo-erythrocytic meronts of five Haemoproteus spp. were described for the first known time, including Haemoproteus magnus hCCF6, Haemoproteus fringillae hCCF3, Haemoproteus majoris hCCF5, Haemoproteus sp. hROFI1, and Haemoproteus sp. hCCF2. Merogonic stages were observed in the vascular system, presenting a formerly unknown mode of exo-erythrocytic development in Haemoproteus parasites. Meronts and megalomeronts of these species were distinct regarding their morphology and organ distribution, indicating species-specific patterns of merogony and different host tissue tropism. New pathological aspects of haemoproteosis were reported. Furthermore, phylogenetic analysis of Haemoproteus spp. with regard to their exo-erythrocytic stages points towards separation of non-megalomeront-forming species from megalomeront-forming species, calling for further studies on exo-erythrocytic development of haemosporidian parasites to explore the phylogenetic character of this trait.

Seasonal Variation in Detection of Haemosporidia in a Bird Community: A Comparison of Nested PCR and Microscopy.

In a 2-yr study on prevalence of Haemosporidia in an avian community in Ithaca, New York, USA, we tested the hypothesis that apparent seasonal variation in prevalence is influenced by the detection protocol. We confirmed a higher detection of Haemosporidia using a molecular diagnosis technique (PCR) than by microscopy; this further increased when the PCR test was triplicated. Microscopic examination and PCR techniques have different specificity and sensitivity and therefore different probabilities of detecting hemoparasites. Birds with chronic infections or sampled during winter often have very low parasitemia, and such infections may be missed by microscopy but detected by PCR. Haemosporidian prevalence was higher during the breeding season than during the nonbreeding season regardless of the method used. Detection of Leucocytozoon spp. infection from blood smears using microscopy was challenging.

Bovine neutrophils kill the sexually-transmitted parasite Tritrichomonas foetus using trogocytosis.

The protozoan parasite Tritrichomonas foetus (T. foetus) is the causative organism of bovine trichomonosis (also referred to as trichomoniasis), a sexually-transmitted infection that reduces fertility in cattle. Efforts to control trichomonosis on cattle farms are hindered by the discouragement of antibiotic use in agriculture, and the incomplete, short-lived protection conferred by the current vaccines. A more complete mechanistic understanding of what effective immunity to T. foetus entails could enable the development of more robust infection control strategies. While neutrophils, the primary responders to infection, are present in infected tissues and have been shown to kill the parasite in vitro, the mechanism they use for parasite killing has not been established. Here, we show that primary bovine neutrophils isolated from peripheral blood rapidly kill T. foetus in vitro in a dose-dependent manner, and that optimal parasite killing is reduced by inhibitors of trogocytosis. We also use imaging to show that bovine neutrophils surround T. foetus and trogocytose its membrane. These findings are consistent with killing via trogocytosis, a recently described novel neutrophil antimicrobial mechanism.

Haemosporidian parasite infections of Malagasy Philepittidae and Nectariniidae are driven by phylogeny rather than ecology.

The nectarivorous common sunbird asity (Neodrepanis coruscans) is phylogenetically closely related to the frugivorous velvet asity (Philepitta castanea), yet it shares similar habitat and foraging behaviour as the Malagasy sunbirds (Cinnyris spp.). As ecological factors have been shown to influence blood parasite prevalence, it should be tested whether parasite abundance, prevalence and diversity of N. coruscans are more similar to the sunbirds than to its relative. Therefore, blood samples (n = 156) and smears (n = 60) were tested for different blood parasites (Haemosporida, trypanosomes, filarioid nematodes) using molecular and microscopic methods. High prevalence of haemosporidian parasites was observed in all bird taxa, with rates ranging from 23% in N. coruscans to 84.6% in C. notatus. The Malagasy Cinnyris spp. exhibited a high occurrence of mixed haemosporidian infections (>76%) with various specialized lineages. Within the Philepittidae family, no Haemoproteus infection was detected and just a few cases of mixed infections. Nectariniidae species predominantly had specialized haemosporidian lineages, while Philepittidae had infections mainly caused by generalist lineages. These findings emphasize the diverse range of blood parasites in Nectariniidae, while additionally highlighting the high diversity of trypanosomes and filarioid nematodes in Philepittidae. Additionally, several newly discovered haemosporidian lineages, Trypanosoma isolates and filarioid nematode isolates were identified. Notably, Philepittidae exhibited a lower prevalence of avian haemosporidian parasites compared to Nectariniidae, possibly due to potential resistance mechanisms. Despite N. coruscans sharing similar habitat and behavioural ecology with both Cinnyris spp., it closely resembles its relative, P. castanea, in all aspects of haemosporidian parasitism.

Contribution to ecological environmental factors and the occurrence of haemosporidians in birds in Zhongar Alatau National Park, Kazakhstan.

In addition to the presence of a suitable host and vector, the prevalence of haemosporidians is influenced by several important factors, including the environmental conditions of the habitat, which depend on broader geographic characteristics. The aim of this study is to perform a preliminarily assessment of the distribution of blood parasites in birds from the mountainous area of Zhongar Alatau NP and to find potential new sites for research on their ecology in Kazakhstan. The results of this research constitute the first report on the occurrence of blood parasites from this area. A total of 58 birds, from the order Passeriformes and one individual from the order Caprimulgiformes, were examined during the study. The overall prevalence of infections caused by haemosporidian parasites (Haemoproteus, Leucocytozoon) was 18.6%. Neither the genus Plasmodium nor the presence of trypanosomes and microfilariae was detected in the birds examined. Three birds (5.1% prevalence) were infected with parasites of the genus Haemoproteus, in all eleven positive birds the analyses showed the presence of parasites of the genus Leucocytozoon (18.6% prevalence). The presence of parasites genus Haemoproteus was detected only in birds that were also infected with Leucocytozoon parasites. More infections with parasites of the genus Leucocytozoon are predicted due to the higher altitude and ecological factors at the capture sites, which are more favourable for the development of vectors of this genus. The species Haemoproteus majoris was detected in the host Emberiza cioides and species Haemoproteus minutus in host Turdus merula. Other species of this genus in the hosts Cyanistes cyanus and Turdus atrogularis were not determined. The species Leucocytozoon fringilinarum was detected in the hosts Cyanistes cyanus and Parus major, Leucocytozoon dubreuili was detected in Turdus atrogularis and Turdus merula. In the other host species Aegithalos caudatus, Emberiza cioides and Periparus aterus, it was not possible to dermine the species of the genus Leucocytozoon.

Very low prevalence of haemosporidian parasites in two species of marsh terns.

Vector-transmitted haemosporidians are among the most common parasites in birds, but our knowledge of the inter-specific patterns of infection rates and the parasite community composition is far from complete because of the unequal distribution of the screening effort across bird families and genera. To assess infection rates and the diversity of haemosporidians from the genera Plasmodium, Haemoproteus, and Leucocytozoon in marsh terns, which represent poorly explored in this regard genus of the family gulls, terns, and skimmers (Laridae), we screened two species: the Whiskered Tern (Chlidonias hybrida) and the Black Tern (Chlidonias niger). We sampled these long-distance migratory birds on breeding grounds: the Whiskered Tern in south-central Poland and north-central Ukraine, and the Black Tern-in north-central Ukraine. We found that birds from both species were infected only sporadically, with prevalence at the population level not exceeding 3.4%. Only parasites from the genera Plasmodium and Leucocytozoon were detected. There was neither an inter-specific difference nor a difference between populations of the Whiskered Tern in infection rates. In total, we registered three lineages-one Plasmodium and two Leucocytozoon-that were previously recorded in other bird species, and two unidentified Plasmodium infections. One of the lineages (Leucocytozoon LARCAC02) represents a specialist parasite with the host range restricted to larids and geographic range restricted to Poland, and two others (Plasmodium SGS1 and Leucocytozoon CIAE02) represent generalist parasites with very broad host and geographic ranges. This study reinforces the existing evidence that terns host parasites from genera Haemoproteus, Plasmodium, and Leucocytozoon only sporadically.

Prevalence of Leucocytozoon infection in domestic birds in Ghana.

Leucocytozoon is a haemosporidian parasite known to cause leucocytozoonosis in domestic and wild birds in most parts of the world. It is an important pathogen, as some species can be pathogenic, especially in domestic birds. One of the factors affecting poultry health management worldwide is parasitism. However, the study of haemosporidian parasites in Ghana is still lacking. This study sought to assess the prevalence and diversity of Leucocytozoon parasites in domestic birds in Ghana. Blood samples were collected from domestic birds in Ghana's Bono and Eastern regions to screen for Leucocytozoon parasites. Thin blood smears were prepared for microscopy and DNA was extracted from whole blood kept in ethylenediaminetetraacetic acid (EDTA) tubes for PCR. Due to the large number of samples, real-time PCR was performed to amplify the conserved rDNA gene. Two different nested PCR protocols were performed on the positive samples obtained from real-time PCR results, to amplify a partial region of the mitochondrial cytochrome b gene and the amplicons were sequenced. Sequencing revealed six new lineages of Leucocytozoon sp. recovered in 976 individual domestic birds and these sequences were deposited in the National Center for Biotechnology Information (NCBI) GenBank. An overall Leucocytozoon prevalence of 11.6% was reported in all birds sampled. The most prevalent lineage LGHA146 (GenBank accession no. OM643346) (93.8%) was found infecting 3 bird species, Gallus gallus, Meleagris gallopavo, and Anas platyrhynchos. Phylogenetic analysis revealed that the new lineages (GenBank accession nos. OM643342, OM643343, OM643344, OM643345, OM643346, and OM643347), reported in this study were closely related to Leucocytozoon schoutedeni. We suggest that further studies be conducted to evaluate the effect of these parasite species on the general well-being of poultry in Ghana.

Mitochondrial genome amplification of avian haemosporidian parasites from single-infected wildlife samples using a novel nested PCR approach.

Haemosporidian parasites that infect birds (Apicomplexa: Haemosporida) are blood parasites that require an invertebrate host (vector) and a vertebrate host for their lifecycle and cause malaria-like diseases. This group of parasites has provided valuable insights into host specificity, virulence, and parasite dispersal. Additionally, they have played a significant role in reshaping our understanding of the evolutionary history of apicomplexans. In order to accurately identify species and to address phylogenetic questions such as the timing of the haemosporidian radiation, the use of a sufficiently large genetic data set is crucial. However, acquiring this genetic data poses significant challenges. In this research, a sensitive nested PCR assay was developed. This assay allows for the easy amplification of complete mitochondrial genomes of haemosporidian parasites in birds, even during the chronic stage of infection. The effectiveness of this new nested PCR assay was evaluated using blood and tissue samples of birds with verified single parasite infections from previous studies. The approach involves amplifying four overlapping fragments of the mitochondrial genome and requires DNA extracts from single-infected samples. This method successfully amplified the complete mitochondrial genomes of 24 distinct haemosporidian parasite lineages found in various bird species. This data is invaluable for conducting phylogenetic analyses and accurately defining species. Furthermore, this study proposes the existence of at least 15 new haemosporidian parasite species based on the genetic information obtained. Data regarding pGRW04, previously categorized as Plasmodium relictum like pSGS1 and pGRW11, indicates that the pGRW04 lineage is actually a separate, hidden Plasmodium species.

Prolonged survival of venereal Tritrichomonas foetus parasite in the gastrointestinal tract, bovine fecal extract, and water.

IMPORTANCE: Nowadays, the routine herd diagnosis is usually performed exclusively on bulls, as they remain permanently infected, and prevention and control of Tritrichomonas foetus transmission are based on identifying infected animals and culling practices. The existence of other forms of transmission and the possible role of pseudocysts or cyst-like structures as resistant forms requires rethinking the current management and control of this parasitic disease in the future in some livestock regions of the world.

Leucocytozoon cariamae n. sp. and Haemoproteus pulcher coinfection in Cariama cristata (Aves: Cariamiformes): first mitochondrial genome analysis and morphological description of a leucocytozoid in Brazil.

The distribution of avian haemosporidians of the genus Leucocytozoon in the Neotropics remains poorly understood. Recent studies confirmed their presence in the region using molecular techniques alone, but evidence for gametocytes and data on putative competent hosts for Leucocytozoon are still lacking outside highland areas. We combined morphological and molecular data to characterize a new Leucocytozoon species infecting a non-migratory red-legged seriema (Cariama cristata), the first report of a competent host for Leucocytozoon in Brazil. Leucocytozoon cariamae n. sp. is distinguished from the Leucocytozoon fringillinarum group by its microgametocytes that are not strongly appressed to the host cell nucleus. The bird studied was coinfected with Haemoproteus pulcher, and we present a Bayesian phylogenetic analysis based on nearly complete mitochondrial genomes of these 2 parasites. Leucocytozoon cariamae n. sp. morphology is consistent with our phylogenetic analysis indicating that it does not share a recent common ancestor with the L. fringillinarum group. Haemoproteus pulcher and Haemoproteus catharti form a monophyletic group with Haemocystidium parasites of Reptilia, supporting the polyphyly of the genus Haemoproteus. We also discussed the hypothesis that H. pulcher and H. catharti may be avian Haemocystidium, highlighting the need to study non-passerine parasites to untangle the systematics of Haemosporida.

Avian Haemosporidians Infecting Short- and Long-Distance Migratory Old World Flycatcher Species and the Variation in Parasitaemia After Endurance Flights.

PURPOSE: Avian haemosporidians are widespread parasites, occurring in many bird families and causing pathologies ranging from rather benign infections to highly virulent diseases. The state of knowledge about lineage-specific intensities of haemosporidian infection (i.e., parasitaemia) is mainly based on infection experiments conducted under laboratory conditions. The levels and range of parasitaemia in natural host-parasite associations as well as their influencing factor remain largely unexplored. METHODS: Thus, we explored the parasitaemia of four songbird species (i.e., European Robins, Black and Common Redstarts and Whinchats) during migration by screening individuals upon landing on an insular passage site after extensive endurance flights to (1) describe their natural host-parasite associations, (2) quantify parasitaemia and (3) explore potential host- and parasite-related factors influencing parasitaemia. RESULTS: We found 68% of Whinchats to be infected with haemosporidians, which is more frequent than any other of the studied host species (30-34%). Furthermore, we confirmed that parasitaemia of Haemoproteus infections was higher than average Plasmodium infections. Median parasitaemia levels were rather low (parasite cells in 0.01% of hosts' red blood cells) and varied largely among the different parasite lineages. However, we found four individuals hosting infections with parasitaemia higher than typical chronic infections. CONCLUSIONS: Based on the known transmission areas of the respective lineages, we argue that these higher intensity infections might be relapses of consisting infections rather than acute phases of recent primary infections.

The 18S rRNA genes of Haemoproteus (Haemosporida, Apicomplexa) parasites from European songbirds with remarks on improved parasite diagnostics.

BACKGROUND: The nuclear ribosomal RNA genes of Plasmodium parasites are assumed to evolve according to a birth-and-death model with new variants originating by duplication and others becoming deleted. For some Plasmodium species, it has been shown that distinct variants of the 18S rRNA genes are expressed differentially in vertebrate hosts and mosquito vectors. The central aim was to evaluate whether avian haemosporidian parasites of the genus Haemoproteus also have substantially distinct 18S variants, focusing on lineages belonging to the Haemoproteus majoris and Haemoproteus belopolskyi species groups. METHODS: The almost complete 18S rRNA genes of 19 Haemoproteus lineages of the subgenus Parahaemoproteus, which are common in passeriform birds from the Palaearctic, were sequenced. The PCR products of 20 blood and tissue samples containing 19 parasite lineages were subjected to molecular cloning, and ten clones in mean were sequenced each. The sequence features were analysed and phylogenetic trees were calculated, including sequence data published previously from eight additional Parahaemoproteus lineages. The geographic and host distribution of all 27 lineages was visualised as CytB haplotype networks and pie charts. Based on the 18S sequence data, species-specific oligonucleotide probes were designed to target the parasites in host tissue by in situ hybridization assays. RESULTS: Most Haemoproteus lineages had two or more variants of the 18S gene like many Plasmodium species, but the maximum distances between variants were generally lower. Moreover, unlike in most mammalian and avian Plasmodium species, the 18S sequences of all but one parasite lineage clustered into reciprocally monophyletic clades. Considerably distinct 18S clusters were only found in Haemoproteus tartakovskyi hSISKIN1 and Haemoproteus sp. hROFI1. The presence of chimeric 18S variants in some Haemoproteus lineages indicates that their ribosomal units rather evolve in a semi-concerted fashion than according to a strict model of birth-and-death evolution. CONCLUSIONS: Parasites of the subgenus Parahaemoproteus contain distinct 18S variants, but the intraspecific variability is lower than in most mammalian and avian Plasmodium species. The new 18S data provides a basis for more thorough investigations on the development of Haemoproteus parasites in host tissue using in situ hybridization techniques targeting specific parasite lineages.