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Avian haemosporidian may affect the host from body damage to the extinction of a population. Knowledge of their status may help in future avifauna conservation plans. Hence, their status in two bird groups of India and their phylogenetic relationships with other known lineages of the world were examined.Cytochrome b gene sequences (479 bp) generated from India and available at MalAvi database were used to study the avian haemosporidian prevalence and phylogenetic analysis of lineages at local and world levels.One common (COLL2) and only once in the study (CYOPOL01, CHD01, CYORUB01, EUMTHA01, GEOCIT01) haemosporidian lineages were discovered. 5.88% prevalence of haemosporidian infection was found in 102 samples belonging to 6 host species. Haemoproteus prevalence was 4.90% across five host species (Phylloscopus trochiloides, Cyornis poliogenys, C. hainanus dialilaemus, C. rubeculoides, Eumiyas thalassinus) and Plasmodium prevalence was 0.98% in Geokichla citrina. Spatial phylogeny at the global level showed that COLL2 lineage, found in C. poliogenys in India, was genetically identical to H. pallidus lineages (COLL2) in parts of Africa, Europe, North America, Malaysia, and the Philippines. The Plasmodium lineage (GEOCIT01) was related to PADOM16 in Egypt, but the sequences were only 93.89% alike.Four new lineages of Haemoproteus and one of Plasmodium were reported. COLL2 similarity with other H. pallidus lineages may suggest their hosts as possible infection sources.

Espinilho savanna ("seasonal steppe savanna") is a unique vegetation formation of the Pampas biome that is found near the tri-border of Brazil, Uruguay, and Argentina. The Yellow Cardinal (Gubernatrix cristata) is a flagship species of this ecosystem, but it is classified as "critically endangered" in Brazil due to habitat loss and poaching for the illegal trade. Population supplementation through the release of individuals that were captive-bred or apprehended by authorities from the illegal trade has been considered as a conservation strategy for this species; however, the risk of pathogen introduction is a critical concern. We used microscopy and molecular methods to investigate the occurrence of blood parasites in wild passerines (n = 64, including three Yellow Cardinals) at Espinilho State Park, Rio Grande do Sul, Brazil, and in captive Yellow Cardinals (n = 30) at three facilities in Brazil. Haemosporidian parasites were detected in the blood smears of 10.9% of the wild passerines, comprising the morphospecies Haemoproteus erythrogravidus in Rufous-collared Sparrow (Zonotrichia capensis), H. quiscalus in Grayish Baywing (Agelaioides badius), and H. tyranni in Great Kiskadee (Pitangus sulphuratus); these are the southernmost records for these morphospecies and their first record for the Pampas biome. No haemosporidian parasites were detected in the blood smears of the Yellow Cardinals, wild or captive. Microfilariae were detected in the blood smears of 14.1% of the wild passerines, including all wild Yellow Cardinals, and in 43.3% of captive Yellow Cardinals. Trypanosoma sp. was detected in the blood smear of one captive Yellow Cardinal. Nested PCR and gene sequencing of the cyt-b gene of Haemoproteus/Plasmodium was used to test a subset of wild passerines and captive Yellow Cardinals, allowing for the molecular barcoding of H. quiscalus lineage AGEBAD04 and H. tyranni lineage PITSUL01; additionally, DNA identical to that of lineage PITSUL01 was detected in the blood of one captive Yellow Cardinal. This study provides valuable data to support the conservation management of the Yellow Cardinal and other threatened passerines from the Pampas and highlights the need for further studies on the epidemiology and pathology of filarioid worms and trypanosomes in passerines from this biome.

Histomonosis (syn. blackhead disease) is caused by the protozoan parasite Histomonas meleagridis and can result in high mortality in turkey flocks, a situation driven by the limitation of prophylactic and therapeutic interventions. Multi-locus sequence typing confirmed the existence of two genotypes, with the vast majority of reported histomonosis outbreaks being caused by genotype 1 in contrast to only a few detections of genotype 2. For the first time, genotype 2 of H. meleagridis was successfully isolated from an outbreak of histomonosis in a flock of 5-week-old turkeys and a clonal culture was established. Using this culture, an experimental infection was performed in naïve turkeys. The animal trial reflected the observations from the field outbreak and coincided with a previously reported case of histomonosis caused by genotype 2, albeit no mortality was observed in the infected birds whereas 17.1% mortality was noticed in the field outbreak from appearance of disease until slaughter. Post mortem investigations demonstrated that lesions were restricted to the caeca in the field outbreak and the experimental trial. In parallel with the experimental reproduction of pathological changes, an oral vaccination of day-old turkeys with a monoxenic genotype 1 vaccine was carried out to determine efficacy against a genotype 2 challenge. Successful vaccine uptake was characterized by the presence of the vaccine in the caeca determined by qPCR and immunohistochemistry (IHC). Excretion of the vaccine strain was confirmed prior challenge, with the majority of birds developing antibodies. The new monoxenic vaccine was able to minimize lesions in the caeca demonstrating heterologous protection. No parasites were detected in the liver by IHC in any of the vaccinated birds, compared to non-vaccinated animals. However, in 6 out of 17 birds of the vaccinated group a positive signal was obtained by real time PCR from liver samples with 2 positives being typeable by conventional PCR as genotype 2. Overall, H. meleagridis genotype 2 infection was successfully reproduced. Experimental vaccination with a genetically distantly related genotype 1 was able to reduce lesions, supporting protection by a recently developed vaccine candidate as an efficacious prophylactic strategy.

The parasite T. foetus causes trichomonosis in cattle but is generally asymptomatic in males. Thus, many bulls carrying the disease go unnoticed, making the detection of T. foetus in bulls an important aspect for its control. Due to drawbacks posed by its cultivation, PCR is a preferred option for diagnostic laboratories. Most published PCR protocols target the genomic region compring the 18S, 5.8S, and 28S rRNA genes and internal transcribed spacers 1 and 2 (rRNA-ITS region), homologous to that of other Tritrichomonas species. There is minimal information on alternative genetic targets and no comparative studies have been published. We compared a protocol based on the microsatellite TfRE (called H94) and five protocols based on the rRNA-ITS region (called M06, M15, G02, G05, and N02). We also designed and evaluated a novel PCR-based assay on the EF1-alpha-Tf1 gene (called V21). The analytical sensitivity and specificity assays for the PCR protocols were performed according to the World Organisation for Animal Health (OIE) directives and the comparative study was performed with a widely used PCR (M06) on clinical samples from 466 breeding bulls. V21 showed a high degree of agreement with our reference M06 (kappa = 0.967), as well as M15 (kappa = 0.958), G05 (kappa = 0.948), and H94 (kappa = 0.986). Protocols H94 and V21 appear to be good approaches for confirming clinical cases in preputial bull samples when genomic regions alternative to rRNA-ITS are required. By contrast, N02 gave false negatives and G02 false positives.

Background Cytauxzoon spp. infection is believed to be a newly emerging tick-borne disease in felids in Europe, with three species of the haemoparasite having recently been differentiated in wild felids. In Switzerland, rare infections have been documented in domestic cats in the west and northwest of the country, the first of which was in 2014. The aims of the present study were: (i) to characterize a Cytauxzoon spp. hotspot in domestic cats in central Switzerland; (ii) to elucidate the geographic distribution of Cytauxzoon spp. in domestic cats in Switzerland; (iii) to assess suspected high-risk populations, such as stray and anaemic cats; and (iv) to investigate the newly emerging nature of the infection. Cytauxzoon spp. were further differentiated using mitochondrial gene sequencing. Methods The overall study included samples from 13 cats from two households in central Switzerland (study A), 881 cats from all regions of Switzerland (study B), 91 stray cats from a hotspot region in the northwest of Switzerland and 501 anaemic cats from across Switzerland (study C), and 65 Swiss domestic cats sampled in 2003 and 34 European wildcats from eastern France sampled in the period 1995–1996 (study D). The samples were analysed for Cytauxzoon spp. using real-time TaqMan quantitative PCR, and positive samples were subjected to 18S rRNA, cytochrome b (CytB) and cytochrome c oxidase subunit I (COI) gene sequencing. Results In study A, six of 13 cats from two neighbouring households in central Switzerland tested postive for Cytauxzoon spp.; two of the six infected cats died from bacterial infections. In studies B and C, only one of the 881 cats (0.1%; 95% confidence interval [CI]: 0–0.3%) in the countrywide survey and one of the 501 anaemic cats (0.2%; 95% CI: 0–0.6%) tested postive for Cytauxzoon spp. while eight of the 91 stray cats in the northwest of Switzerland tested positive (8.8%; 95% CI: 3.0–14.6%). In study D, Cytauxzoon spp. was detected in one of the 65 domestic cat samples from 2003 (1.5%; 95% CI: 0–4.5%) and in ten of the 34 European wildcat samples from 1995 to 1996 (29%; 95% CI: 14.2–44.7%). The isolates showed ≥ 98.6% sequence identities among the 18S rRNA, CytB and COI genes, respectively, and fell in the subclade Cytauxzoon europaeus based on CytB and COI gene phylogenetic analyses. Conclusions The study challenges the newly emerging nature of Cytauxzoon spp. in central Europe and confirms that isolates from domestic cats in Switzerland and European wild felids belong to the same species. Graphical Abstract

Widespread surveys of avian haemosporidia (Plasmodium, Haemoproteus, and Leucocytozoon) in wild birds have substantially advanced information on the haemosporidian fauna of Japan. However, many areas and bird species remain insufficiently investigated. Bird carcasses collected for personal specimen collection seldom reach academic audience particularly in the veterinary field. The presence of avian haemosporidia was investigated in these personally collected bird carcasses, in order to better understand the avian haemosporidian fauna in Japan. Bird carcasses were donated through personal contact upon approval of the study. Tissue samples were collected from the birds and examined for haemosporidian parasites using nested-PCR targeting the cytochrome b gene. One hundred and forty-three birds of 85 species were donated, including 34 species and two subspecies that were molecularly or collectively investigated for the first time in Japan. Avian haemosporidian DNA was detected from 37 of the 134 tested birds (27.61%). In 8 bird species, avian haemosporidia was detected for the first time. Twenty-nine lineages were detected, including 8 novel and 9 known lineages detected in Japan for the first time. Furthermore, 16 lineages were detected from novel host species. While information that could be drawn was limited and risk management of zoonotic diseases needs re-consideration, these findings expanded information on the host range and distribution of several lineages. Collectively, this method of investigation using personally collected bird samples can provide important additions to more fully understand the avian haemosporidian fauna of Japan, as well as other areas with limited investigations.

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 =

The specialist versus generalist strategies of hemoparasites in relation to their avian host, as well as environmental factors, can influence their prevalence, diversity and distribution. In this paper we investigated the influence of avian host species, as well as the environmental and geographical factors, on the strategies of Haemoproteus and Plasmodium hemoparasites. We determined prevalence and diversity by targeting their cytochrome b (Cytb) in a total of 2,590 passerine samples from 138 localities of Central and South America, and analysed biogeographic patterns and host-parasite relationships. We found a total prevalence of 23.2%. Haemoproteus presented a higher prevalence (15.3%) than Plasmodium (4.3%), as well as a higher diversity and host specificity. We determined that Plasmodium and Haemoproteus prevalences correlated positively with host diversity (Shannon index) and were significantly influenced by bird diversity, demonstrating a possible "amplification effect". We found an effect of locality and the avian family for prevalences of Haemoproteus and Plasmodium. These results suggest that Haemoproteus is more specialist than Plasmodium and could be mostly influenced by its avian host and the Andes Mountains.

Bovine trichomonosis is caused by Tritrichomonas foetus. Thirty-three US states have state rules on this disease and render it reportable due to potential huge economic losses to cattle industry. The various rules of different states generally mandate testing and culling T. foetus-positive bulls as well as prohibiting import of T. foetus-positive animals. Wyoming has enforced these rules for over 20 year beginning in 2000. From 2017 to 2019, 3 years in a row, not even one T. foetus-positive bull has been detected throughout the entire state among over ten thousand bulls tested annually. Wyoming is the first US state to achieve total control and eradication of bovine trichomonosis by testing and culling T. foetus-positive bulls.

Background Haemosporidioses are common in birds and their manifestations range from subclinical infections to severe disease, depending on the involved parasite and bird species. Clinical haemosporidioses are often observed in non-adapted zoo or aviary birds, whereas in wild birds, particularly passerines, haemosporidian infections frequently seem to be asymptomatic. However, a recent study from Austria showed pathogenic haemosporidian infections in common blackbirds due to high parasite burdens of Plasmodium matutinum LINN1, a common parasite in this bird species, suggesting that virulent infections also occur in natural hosts. Based on these findings, the present study aimed to explore whether and to what extent other native bird species are possibly affected by pathogenic haemosporidian lineages, contributing to avian morbidity. Methods Carcasses of passerine birds and woodpeckers were collected during a citizen science-based survey for avian mortality in Austria, from June to October 2020. Tissue samples were taken and examined for haemosporidian parasites of the genera Plasmodium, Haemoproteus and Leucocytozoon by nested PCR and sequencing the mitochondrial cytb barcode region, histology, and chromogenic in situ hybridization applying genus-specific probes. Results From over 160 dead bird reportings, 83 carcasses of 25 avian species were submitted for investigation. Overall haemosporidian infection rate was 31%, with finches and tits prevailing species counts and infections. Sequence analyses revealed 17 different haplotypes (4 Plasmodium, 4 Haemoproteus, 9 Leucocytozoon), including 4 novel Leucocytozoon lineages. Most infected birds presented low parasite burdens in the peripheral blood and tissues, ruling out a significant contribution of haemosporidian infections to morbidity or death of the examined birds. However, two great tits showed signs of avian malaria, suggesting pathogenic effects of the detected species Plasmodium relictum SGS1 and Plasmodium elongatum GRW06. Further, exo-erythrocytic tissue stages of several haemosporidian lineages are reported. Conclusions While suggesting generally little contribution of haemosporidian infections to mortality of the investigated bird species, the findings indicate a possible role of certain haemosporidian lineages in overall clinical manifestation, either as main causes or as concurrent disease agents. Further, the study presents new data on exo-erythrocytic stages of previously reported lineages and shows how citizen science can be used in the field of haemosporidian research. Graphic abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12936-021-03949-y.

Avian malaria (Plasmodium) and related genera (Haemoproteus and Leucocytozoon) are diverse and widespread parasites. Despite the extent of knowledge on avian haemosporidian parasites, information about domestic and wild bird's blood parasites is overall insufficient in Iran. Prevalence of the haemosporidian parasites’ and phylogenetic relationship of lineages are studied by using molecular and morphological results of 152 examined hosts belonging to 17 species. Molecular analysis for haemosporidian detections demonstrated overall prevalence 22.36%. Inspected hosts mostly belonging to Common Pigeons (Columba livia) parasitized by Haemoproteus spp., and Hooded Crows (Corvus cornix) and Carrion Crow (C. corone) were identified as hosting Plasmodium spp. Detected lineages COLIV03, COQUI05, LINN01, ROFI04 and SGS01 are identified as new reports from Iran. We detected no evidence of Leucocytozoon lineages, while the high prevalence of H. columbae was found in Common Pigeons. Such investigation on avian blood parasites contributes to providing new information on the prevalence, epidemiology and geographical distribution of haemosporidian parasites circulating in domestic, pets and wild birds., Despite the extent of knowledge on avian haemosporidian parasite, information about domestic and wild bird's blood parasites is overall insufficient in Iran. Prevalence of the haemosporidian parasite' and phylogenetic relationship of lineages was estimated by molecular and morphological results of 152 examined hosts belonging to 17 species.

Cytauxzoon felis is a hematoprotozoan parasite with a complex life cycle involving a tick-vector and a mammalian host. The mammalian hosts are all felidae but in the bobcat reservoir host, the parasite typically causes only a brief, self-resolving illness followed by a prolonged subclinical infection. In domestic cats, however, infection often leads to an acute febrile illness characterized by severe morbidity and mortality. Diagnosis is based on microscopic identification of parasites or molecular testing. Treatment for ill cats is expensive, difficult, and often unsuccessful. Prevention is quite possible and depends on avoidance of feeding by vector ticks.

Background Pentatrichomonas hominis inhabits the digestive tracts of several vertebrates, such as humans, monkeys, pigs, dogs, cats and rats. This protozoan was originally considered a commensal of the digestive tract but has subsequently been identified as a potential zoonotic parasite and a causative agent of diarrhoea. Molecular techniques are considered more sensitive and specific to detect P. hominis. This study aimed to determine the presence and genetic diversity of P. hominis in animals in Thailand. A total of 403 faecal samples were collected from 119 cats, 55 dogs, 73 goats, 35 monkeys, 55 cattle and 66 pigs, and the presence of P. hominis was determined using the nested polymerase chain reaction method. Sequence analysis of small-subunit ribosomal RNA genes was used to determine the genotype of the organism. Results Twenty-six samples (26/403, 6.45%) were positive for P. hominis. The highest prevalence was found in cats (21/119; 17.65%), followed by cattle (3/55; 5.45%) and dogs (2/55; 3.64%). Seven out of 26 nucleotides demonstrated 100% sequence identity with existing sequences; additionally, 16 novel sequence patterns were identified. All nucleotide sequences of P. hominis-positive samples were shown in the same branch with the previously described P. hominis sequences found in humans, dogs and goat. Conclusion This is the first study on P. hominis infections in animals in Thailand. Our findings revealed that the prevalence of P. hominis was significantly higher in cats than in cattle and dogs. Cats were the main reservoir host; however, P. hominis can infect several kinds of animals. Therefore, the proper waste management of animals is necessary to reduce and prevent infection in the community.

Understanding the drivers of infection risk helps us to detect the most at-risk species in a community and identify species whose intrinsic characteristics could act as potential reservoirs of pathogens. This knowledge is crucial if we are to predict the emergence and evolution of infectious diseases. To date, most studies have only focused on infections caused by a single parasite, leaving out co-infections. Yet, co-infections are of paramount importance in understanding the ecology and evolution of host-parasite interactions due to the wide range of effects they can have on host fitness and on the evolutionary trajectories of parasites. Here, we used a multinomial Bayesian phylogenetic modelling framework to explore the extent to which bird ecology and phylogeny impact the probability of being infected by one genus (hereafter single infection) or by multiple genera (hereafter co-infection) of haemosporidian parasites. We show that while nesting and migration behaviours influenced both the probability of being single- and co-infected, species position along the slow-fast life-history continuum and geographic range size were only pertinent in explaining variation in co-infection risk. We also found evidence for a phylogenetic conservatism regarding both single- and co-infections, indicating that phylogenetically related bird species tend to have similar infection patterns. This phylogenetic signal was four times stronger for co-infections than for single infections, suggesting that co-infections may act as a stronger selective pressure than single infections. Overall, our study underscores the combined influence of hosts' evolutionary history and attributes in determining infection risk in avian host communities. These results also suggest that co-infection risk might be under stronger deterministic control than single infection risk, potentially paving the way toward a better understanding of the emergence and evolution of infectious diseases.

Bovine tritrichomonosis, a sexually transmitted disease caused by the protozoan Tritrichomonas foetus, is characterized by producing reproductive alterations in cattle. Carbohydrates on the surface of the uterine epithelium are involved in the process of adhesion and colonization of the protozoan. The murine model has proved to be an inexpensive, practical and representative alternative to study the lesions produced in the natural host. For this work, during the first stage, 6-8 week old female BALB/c mice were inoculated with 24 different T. foetus isolates in order to classify them according to their pathogenicity. Then, seven isolates were selected and processed with lectin histochemistry to determine if the differences in pathogenicity corresponded to the changes found in the uterine carbohydrate expression pattern. In this work, we demonstrate the differences in the expression of the carbohydrate pattern between infected and uninfected mice. In addition, within the group of infected mice, differences were found in the degree of pathogenicity of the isolates, thus evidencing their biological variability.

We determined the prevalence of diseases and pathogens associated with mortality in beef cattle in the State of Rio Grande do Sul, Brazil, based on pathology laboratory submissions. Postmortem examinations were conducted on 1,277 beef cattle that died between 2008 and 2018. Information regarding age, time of the year, breed, and regional location were analyzed statistically. Most cattle were from the surrounding region of Porto Alegre, and 78.7% of the analyzed cases had diagnostic value. The diagnostic category with most cases was infectious and/or parasitic diseases (60%), followed by toxic and toxicoinfectious (25%). Most cases occurred in the fall. Major disease conditions identified included hemoprotozoal infection (18.2%), rabies (8.2%), and plant intoxications by Senecio spp. (8.5%) and Pteridium arachnoideum (4.6%). Hemoprotozoal infection occurred at a higher frequency in young cattle, mainly in animals up to 1 y old. Intoxication by Senecio spp. was more frequent in cattle 2–3 y old.

Diseases can play a role in species decline. Among them, haemosporidian parasites, vector-transmitted protozoan parasites, are known to constitute a risk for different avian species. However, the magnitude of haemosporidian infection in wild columbiform birds, including strongly decreasing European turtle doves, is largely unknown. We examined the prevalence and diversity of haemosporidian parasites Plasmodium, Leucocytozoon and subgenera Haemoproteus and Parahaemoproteus in six species of the order Columbiformes during breeding season and migration by applying nested PCR, one-step multiplex PCR assay and microscopy. We detected infections in 109 of the 259 screened individuals (42%), including 15 distinct haemosporidian mitochondrial cytochrome b lineages, representing five H. (Haemoproteus), two H. (Parahaemoproteus), five Leucocytozoon and three Plasmodium lineages. Five of these lineages have never been described before. We discriminated between single and mixed infections and determined host species-specific prevalence for each parasite genus. Observed differences among sampled host species are discussed with reference to behavioural characteristics, including nesting and migration strategy. Our results support previous suggestions that migratory birds have a higher prevalence and diversity of blood parasites than resident or short-distance migratory species. A phylogenetic reconstruction provided evidence for H. (Haemoproteus) as well as H. (Parahaemoproteus) infections in columbiform birds. Based on microscopic examination, we quantified parasitemia, indicating the probability of negative effects on the host. This study provides a large-scale baseline description of haemosporidian infections of wild birds belonging to the order Columbiformes sampled in the northern hemisphere. The results enable the monitoring of future changes in parasite transmission areas, distribution and diversity associated with global change, posing a potential risk for declining avian species as the European turtle dove.

Background Culicoides biting midges (Diptera, Ceratopogonidae) are known vectors of avian Haemoproteus parasites. These parasites cause diseases, pathology and even mortality in birds. The diversity of biting midges in Europe is great, but only four Culicoides species are known to be vectors of avian Haemoproteus parasites. In general, our knowledge about the role of the particular Culicoides species in the transmission of Haemoproteus parasites remains insufficient. Information gaps hinder a better understanding of parasite biology and the epizootiology of parasite-caused diseases. The aim of this study was to determine new Culicoides species involved in the transmission of Haemoproteus parasites. Methods Biting midges were collected using a UV trap as well as sticky traps installed in bird nest boxes. Individual parous females were diagnosed for the presence of haemoproteids using both PCR-based and microscopic methods. Results We collected and dissected 232 parous Culicoides females from 9 species using a UV trap and 293 females from 11 species from bird nest boxes. Culicoides obsoletus was the dominant species collected using a UV trap, and Culicoides kibunensis dominated among midges collected in nest boxes. PCR-based screening showed that 5.2% of parous biting midges collected using a UV trap and 4.4% of midges collected from nest boxes were infected with avian haemosporidian parasites. Haemoproteid DNA was detected in C. kibunensis, Culicoides pictipennis, Culicoides punctatus, Culicoides segnis and Culicoides impunctatus females. The sporozoites of Haemoproteus minutus (genetic lineages hTURDUS2 and hTUPHI01) were detected in the salivary glands of two C. kibunensis females using microscopy, and this finding was confirmed by PCR. Conclusions Culicoides kibunensis was detected as a new natural vector of Haemoproteus minutus (hTURDUS2 and hTUPHI01). Haemoproteid DNA was detected in females from five Culicoides species. This study contributes to the epizootiology of avian Haemoproteus infections by specifying Culicoides species as vectors and species that are likely to be responsible for the transmission of haemoproteids in Europe. Graphical Abstract

Background: Swine species are an important source of meat production worldwide, except in Islamic countries where pig breeding and pork consumption are forbidden. Hence, they are often neglected in these regions. A considerable number of wild boars (Sus scrofa) inhabit Iranian territories, particularly in dense forests of north, west and southwest of the country, but our knowledge regarding their parasites is very limited. Objective: The lack of a comprehensive record in this connection encouraged us to review the whole works of literature in the country. Methods: The current review presents all the information about the parasitic diseases of wild boar in Iran extracted from articles available in both Persian and English databases until June 2017. Results: So far, 8 genera of protozoa (Toxoplasma, Balanthidium, Tritrichomonas, Blastocystis, Entamoeba, Iodamoeba, Chilomastix and Sarcocystis) and 20 helminth species, including four cestode species, two trematode species, thirteen nematode species as well as a single species of Acanthocephala have been described in Iranian wild boars. Conclusion: This review sheds light on the veterinary and public health aspects of the parasitic diseases of wild boars in the country and alerts authorities for future preventive measures.

Parasites of the haemosporidian genus Polychromophilus have exclusively been described in bats. These parasites belong to the diverse group of malaria parasites, and Polychromophilus presents the only haemosporidian taxon that infects mammalian hosts in tropical as well as in temperate climate zones. This study provides the first information of Polychromophilus parasites in the lesser Asiatic yellow bat (Scotophilus kuhlii) in Thailand, a common vespertilionid bat species distributed in South and Southeast Asia. The gametocyte blood stages of the parasites could not be assigned to a described morphospecies and molecular analysis revealed that these parasites might represent a distinct Polychromophilus species. In contrast to Plasmodium species, Polychromophilus parasites do not multiply in red blood cells and, thus, do not cause the clinical symptoms of malaria. Parasitological and molecular investigation of haemosporidian parasites of wildlife, such as the neglected genus Polychromophilus, will contribute to a better understanding of the evolution of malaria parasites.

Parasites can significantly influence the ecology, behaviour and physiology of their hosts sometimes with remarkable effects on their survivorship. However, endemic parasites or those not associated with obvious clinical disease have been partly neglected in the past decades comparatively to the most pathogenic ones. Apicomplexa are an important example of blood parasites that have been broadly investigated, although it can be difficult to determine the effects of infections at the population level, especially in widespread species. Such is the case of the common wall lizard (

Avian haemosporidian parasites have received considerable attention in ecology and evolution as a result of their wide distribution and ease of detection. However, conventional PCR-based detection methods may sometimes underestimate haemosporidian mixed infections, which are frequent in natural populations. This underestimation is due to differences in PCR sensitivity for detection of lineages within the mixed infections. Therefore, we designed new primers to amplify sequences that were not detected by the conventional primers and examined if our primers were useful for accurate detection of mixed infections. Blood samples were collected from 32 wild birds captured in Hokkaido, and 16 of these were positive for Leucocytozoon using the conventional primers, while 15 were positive using our primers. All positively amplified samples were sequenced, and we found that the conventional primers detected 16% (5/32) of multiple infections and none of them was a novel lineage, whereas our primers detected 44% (14/32) of multiple infections and ten of them were novel lineages. A phylogenetic analysis showed that the new primers can detect a wide range of Leucocytozoon lineages compared with that detected by the conventional primers. The results indicate that our primers are particularly suitable for revealing unique strains from multiple infections. Highly variable multiple infections in the same population of birds at the same location were found for the first time. We revealed a higher diversity of Leucocytozoon lineages in nature than expected, which would provide more information to better understand parasite diversity and host-vector interactions in wildlife.

A 2-year-old, 4.2 kg, spayed female, Maine coon cat was referred to the veterinary hospital for evaluation of hyporexia, slow growth, and chronic, intermittent, mucoid, bloody, voluminous, and fetid diarrhea. The diarrhea had been observed since the cat was acquired from a cattery at 4 months of age; with acute worsening in the 5 d before presentation. Abdominal palpation revealed moderate pain. Ultrasonographic examination showed thickening of the jejunal wall and ileal loops, increased echogenicity of the jejunal mucosa, and enlargement of the jejunal and ileocolic lymph nodes. Histopathology of full-thickness intestinal biopsies showed moderate, diffuse, lymphoplasmacytic, erosive enteritis with hemorrhage and edema. Diffuse, lymphoplasmacytic, erosive colitis with mild, interstitial fibrosis and hemorrhage was also noted. The ileocecal lymph node biopsy showed eosinophilic lymphadenitis. Based on the immunohistochemical evaluation of intestinal samples with CD3 and CD79a antibodies, a diagnosis of lymphoma was ruled out. Fecal polymerase chain reaction testing was positive for Tritrichomonas foetus. Based on these results, inflammatory bowel disease and trichomonosis were diagnosed. Treatment for the cat included a hypoallergenic diet and an oral omega-3 fatty acid supplement, in conjunction with prednisolone, to manage the inflammatory bowel disease. Ronidazole was administered to target the Tritrichomonas foetus. The cat was clinically normal during a follow-up examination after 6 months of treatment.

Previous studies found a relationship between blood parasite infection and bird gender, with higher prevalence in males. Some studies also found a relationship between host plumage color and parasitic infection, while others did not. Here, we investigated the blood parasite prevalence in correlation with sex and plumage color in free-range chickens (Gallus gallus domesticus) in China. We analyzed a total of 297 blood samples, out of which 234 chickens tested positive for haemosporidian parasites, with 78.5% parasite prevalence. Out of 139 males, 118 tested positive with 84.8% parasite prevalence while 116 of 158 female samples tested positive (73.4%). Leucocytotozoon was the most frequent genus isolated (193 infected individuals /234 birds), followed by Plasmodium (41 infected individuals/234 birds), with no Haemoproteus parasites being detected. There were no significant differences in the body parameters and chicken color plumages with regards to the infection status. Our study indicated that blood parasite infection was significantly different between male and female chickens, with infection prevalent in males.

Tritrichomonas foetus is a flagellated parasite able to infect cattle, cats, and pigs. Despite its prevalence, feline tritrichomonosis has received markedly less attention than venereal infection, and little information about the molecular mechanisms that participate in feline host infection is available. Through a bioinformatics approach, we integrated public transcriptomic data for three T. foetus isolates and explored the differences at transcript level with a focus on pathogenesis and adaptation processes, particularly for the feline isolate. Our analysis revealed higher abundance levels of predicted virulence factors, such as proteases and surface antigens. Additionally, by a comparative and expression analysis of T. foetus genes, we proposed putative virulence factors that could be involved in feline infection. Finally, we identified a great proportion of predicted transcription factors of the MYB protein family and, by a promoter analysis, we revealed that MYB-related proteins could participate in the regulation of gene transcription in T. foetus. In conclusion, this integrated approach is a valuable resource for future studies of host–pathogen interactions and identifying new gene targets for improved feline tritrichomonosis diagnosis and treatment.

Cytauxzoon felis is a tick-borne haemoprotozoan parasite that often causes fatal disease in domestic cats. Histological studies have described substantial pulmonary pathology due to cytauxzoonosis. Published reports were not found describing the thoracic radiographic signs associated with acute cytauxzoonosis in cats. The purpose of this retrospective descriptive study was to describe thoracic radiographic findings in a group of felines with confirmed acute cytauxzoonosis. A total of 37 cats with confirmed cytauxzoonosis and with available thoracic radiographs were included. A subset of 7 cats in this sample also had histopathologic evaluation of their lung parenchyma. Thoracic radiographs were retrieved and reviewed. A bronchial pulmonary pattern was identified as the most common finding (n = 27/37; 73%). Other radiographic findings included cardiomegaly (n = 19/37; 51%), interstitial pattern (n = 17/37; 46%), pleural effusion (n = 12/37; 32%), arterial vascular distension (n = 10/37; 27%), arterial and venous distension (n = 10/37; 27%), and venous distension (n = 1/37; 3%). The primary histological features present in 7 cats with additional histopathologic evaluation, similar to previously published studies, were vascular occlusion. Our study suggests that, despite severe histologic evidence of disease, there are no pathognomonic thoracic radiographic findings in cats with acute cytauxzoonosis.

Parasites of the genus Haemoproteus are vector-borne avian haemosporidia commonly found in bird species of the world. Haemoproteus infections are typically considered relatively benign in birds. However, some Haemoproteus species cause severe disease and mortality, especially for captive birds removed from their original habitat. In September 2018, a captive 15-year-old snowy owl (Bubo scandiacus), kept in a zoological garden of Japan, died subacutely after presenting leg dysfunction. This case showed significantly low PCV and elevated AST, ALT, CK, and LDH values. Many megalomeronts with prominent morphological characteristics of Haemoproteus were observed in the left leg muscles. Those megalomeronts exhibited multilocular structures and were internally filled with merozoites. A new lineage of Haemoproteus was detected by subsequent PCR for the cytochrome b (cytb) gene of avian haemosporidia from DNA extracted from several organ tissues. The detected lineage was classified in the subgenus Parahaemoproteus and was similar to those from the wild birds inhabiting the region including the study area, suggesting that this snowy owl likely acquired its infection from wild birds. This is the first report of a fatal case of a captive bird with a locally transmitted Haemoproteus infection in Japan. We considered the pathogenicity of this infection in conjunction with the clinical course and hematology results. We surmise that snowy owls may be particularly susceptible to infection with Haemoproteus parasites, and warming northern temperatures may exacerbate the overall health of these and other high latitude birds. Further research into the prevalence of Haemoproteus in wild birds near zoological gardens and potential biting midge vectors is necessary for the ex situ conservation of introduced birds.

Background Atovaquone and azithromycin (A&A) with supportive care improve survival rates in cats with cytauxzoonosis. Resistance to atovaquone via parasite cytochrome b gene (cytb) mutations occurs in other Apicomplexan protozoans but is not described in Cytauxzoon felis. Objective To serially characterize the C. felis cytb sequences from a cat that remained persistently infected after A&A treatment. Animal A cat with naturally occurring C. felis infection. Methods Case report of the anemic cat persistently infected with C. felis before, during and after A&A treatment. Cytauxzoon felis cytb genes were amplified and sequenced before, during and after A&A treatment. Results Cytauxzoon felis was detected before, during and after A&A treatment including samples collected 570 days after treatment. After A&A treatment, the cat's anemia improved slightly. Cytb sequencing revealed only wild-type cytb methionine (M128) in samples collected before treatment. In samples collected after treatment, the cytb coded for isoleucine (M128I) and valine (M128I) at 2- and 4-months after treatment. These M128I and M128V mutations persisted even after a repeat treatment course with a higher dose atovaquone combined with the standard dose of azithromycin. Conclusions and clinical importance This report documents C. felis atovaquone resistance associated with M128 cytb mutations. This study suggests parasites with mutations of cytb M128 can be selected and impart resistance to A&A treatment even with higher atovaquone dosing.

Background Piroplasms are vector-borne intracellular hemoprotozoan parasites that infect wildlife and livestock. Wildlife species are reservoir hosts to a diversity of piroplasms and play an important role in the circulation, maintenance and evolution of these parasites. The potential for likely spillover of both pathogenic and non-pathogenic piroplasm parasites from wildlife to livestock is underlined when a common ecological niche is shared in the presence of a competent vector. Method To investigate piroplasm diversity in wildlife and the cattle population of the greater Kafue ecosystem, we utilized PCR to amplify the 18S rRNA V4 hyper-variable region and meta-barcoding strategy using the Illumina MiSeq sequencing platform and amplicon sequence variant (ASV)-based bioinformatics pipeline to generate high-resolution data that discriminate sequences down to a single nucleotide difference. Results A parasite community of 45 ASVs corresponding to 23 species consisting of 4 genera of Babesia, Theileria, Hepatozoon and Colpodella, were identified in wildlife and the cattle population from the study area. Theileria species were detected in buffalo, impala, hartebeest, sable antelope, sitatunga, wild dog and cattle. In contrast, Babesia species were only observed in cattle and wild dog. Our results demonstrate possible spillover of these hemoprotozoan parasites from wildlife, especially buffalo, to the cattle population in the wildlife-livestock interface. Conclusion We demonstrated that the deep amplicon sequencing of the 18S rRNA V4 hyper-variable region for wildlife was informative. Our results illustrated the diversity of piroplasma and the specificity of their hosts. They led us to speculate a possible ecological cycle including transmission from wildlife to domestic animals in the greater Kafue ecosystem. Thus, this approach may contribute to the establishment of appropriate disease control strategies in wildlife-livestock interface areas. Graphical Abstract

Made available in DSpace on 2020-12-12T02:20:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Amphibians are among the most threatened vertebrate groups in the world, and the main causes include climate change, habitat destruction, and emerging diseases. Herein, we investigated the occurrence and characterized molecularly Apicomplexa in anurans from southeastern Brazil. Forty individuals from seven anuran species were sampled in São Paulo state. In the molecular analyses, one Leptodactylus latrans and one Rhinella diptycha were positive in PCR assays for species of Hepatozoon. Two L. latrans were also positive for coccidian infections (Lankesterella sp. and an unidentified coccidian species). Phylogenetic analysis based on 18S rDNA clustered the sequences detected in anurans from the present study with Hepatozoon spp. detected in reptiles and other anurans from Brazil, albeit they were separate from Hepatozoon haplotypes detected in frogs from Africa and North America. Our study showed, for the first time, the molecular detection of Lankesterella sp. and another coccidian in L. latrans. Additionally, co-infection by different species of Hepatozoon haplotypes and an unidentified coccidian in anurans from Brazil was documented. Department of Morphology and Animal Physiology of the Faculty of Agricultural and Veterinary Sciences UNESP-FCAV Laboratório de Imunoparasitologia Departamento de Patologia Veterinária Faculdade de Ciências Agrárias e Veterinárias Júlio de Mesquita Filho (UNESP), Campus de Jaboticabal, Via de Acesso Prof. Paulo Donato Castellane, s/n, Zona Rural Department of Morphology and Animal Physiology of the Faculty of Agricultural and Veterinary Sciences UNESP-FCAV Laboratório de Imunoparasitologia Departamento de Patologia Veterinária Faculdade de Ciências Agrárias e Veterinárias Júlio de Mesquita Filho (UNESP), Campus de Jaboticabal, Via de Acesso Prof. Paulo Donato Castellane, s/n, Zona Rural

The protozoan parasite Tritrichomonas foetus causes early embryonic death in cattle which results in severe economic loss. In the United States, there are no drugs are approved for treatment of this pathogen. In this study, we evaluated in vitro anti-protozoal effects of compounds from an open access chemical library against T. foetus trophozoites. An initial high-throughput screen identified 16 compounds of interest. Further investigation revealed 12 compounds that inhibited parasite growth and 4 compounds with lethal effects. For lethal compounds, dose-response curves were constructed and the LD50 was calculated for laboratory and field strains of T. foetus. Our experiments revealed chemical scaffolds that were parasiticidal in the micromolar range, and these scaffolds provide a starting point for drug discovery efforts. Further investigation is still needed to investigate suitability of these scaffolds and related compounds in food animals. Importantly, open access chemical libraries can be useful for identifying compounds with activity against protozoan pathogens of veterinary importance., Graphical abstract Image 1, Highlights • No legal treatments are available for bovine trichomoniasis in the United States. • The open access malaria box was screened for compounds with activity against T. foetus trophozoites. • Identification of several scaffolds meriting further investigation for suitability in food animals.

Equines are subject to infection with many parasites, which threaten their health. In the present study, we systematically reviewed existing literature on the prevalence of endo‐ and ectoparasites of equines in Iran. Major electronic databases, including PubMed, PubMed Central, Google Scholar, Science Direct and Scientific Information Database (SID), were searched (Last updated 11/05/2018) for relevant literature of parasites that have been identified from equines in Iran. Of the 1809 titles produced by bibliographic search, 38 were included in the review. Twenty‐seven of the studies were on horses, six on donkeys, three on both horses and donkeys, and one study was on both horses and mules. Furthermore, 24 of the studies reported infections caused by protozoa, thirteen by helminths, two by ectoparasites, and one by both protozoa and helminths. The overall pooled prevalence of parasitic infection was 28.8% (95%CI: 22.9–35.7, I 2 = 93.4%). Helminths were the most prevalent parasites 46.7% (95% CI: 24.1–70.7, I 2 = 96.0%). Furthermore, donkeys were the most affected equine, with a prevalence of 70.7% (95% CI: 53.2–83.7, I 2 = 92.5%). The protozoa frequently reported included nine species belonging to the genera: Neospora, Toxoplasma, Theileria, Babesia and Eimeria. Also, the helminths frequently reported included 21 species belonging to the genera: Strongylus, Dicrocoelium, Oxyuris, Habronema, Echinococcus, Dictyocaulus, Cyathostomum, Probstmayria, Anoplocephala, Setaria and Fasciola. Ticks were the only ectoparasites frequently reported. Parasitic fly species of the genera Gasterophilus were also reported. The study‐level risk of bias was likely to be high because of differences in study design. Parasitic infections of equines in Iran are frequent and caused by a diversity of parasites, which threatens the health and well‐being of these animals. Further research is needed in the area to identify the risk factors of infection for effective control of the parasites., In this study, we systematically reviewed existing literature on the prevalence of endo‐ and ecto‐parasites of equines in Iran.

Amebiasis is a worldwide parasitic zoonosis, with symptoms of abdominal discomfort, indigestion, diarrhea, and even death. However, limited information about the prevalence of Entamoeba spp. in experimental nonhuman primates (NHPs) in southwestern China is available. The objective of the current study was to investigate the frequency and species identity of Entamoeba to evaluate potential zoonotic risk factors for Entamoeba spp. infection in experimental NHPs. A total of 505 fecal samples were collected from NHPs (macaques) and analyzed by PCR analysis the small subunit rRNA (SSU rRNA) gene of Entamoeba spp. Forty-seven specimens were positive for Entamoeba spp., and the prevalence of Entamoeba spp. was 9.31% (47/505). Significant differences in the prevalence rates among the three breeds (P = 0.002

Infection patterns of avian haemosporidians result from the evolution of their associations with hosts, and can be shaped by multiple biotic factors. However, at the level of parasite species, few studies have investigated the details of the temporal dynamics of infection patterns in wild bird communities. We hereby studied a wild bird community in southern Sweden to investigate two generalist parasites (cyt b lineages PARUS1 and WW2) of the morphological species Haemoproteus majoris in their main host species (tits and warblers, respectively) to look for seasonal (spring to autumn) and age class related variation in infection patterns. For both lineages, we detected a similar temporal pattern in prevalence and infection intensity, with peak levels during the main nesting season in adults and a few weeks later in juveniles. Infections in juveniles were detected as soon as they started to be caught by mist nets, implying that they became infected when still in the nest or during the first weeks post-fledging. The initially high intensities in juveniles were followed by a significant decrease during the hatching year, emphasising the importance of studying haemosporidian infections in nestlings and fledglings. Both prevalence and infection intensity in adults increased from spring to early summer, either due to spring relapses or new infections. Both prevalence and infection intensity declined in adults at the time when independent juveniles of the respective species started to appear, suggesting that the rate of parasite withdrawal from blood exceeded the rates of new infections gained and relapses of previous infections. Prevalence in both juveniles and adults approached zero towards the end of the summer.

Rodents and other micromammals constitute important reservoirs of infectious diseases; their role in the life cycle of apicomplexan parasites such as Toxoplasma gondii, Neospora caninum, and Sarcocystis spp. still needs clarification. In the present study, we analyzed by PCR and Sanger sequencing methods the presence of specific parasite DNA within brain and heart tissues of 313 individuals of five synanthropic small mammal species (Apodemus sylvaticus, Mus spretus, M. musculus, Rattus rattus, and Crocidura russula) collected in Barcelona metropolitan area (NE Spain). In addition, PCR-RFLP and microsatellites were also used as tools for genotypic characterization of T. gondii and N. caninum, respectively. Specific DNA of T. gondii, N. caninum, and Sarcocystis spp. was detected in 0.3% (n = 1), 1.3% (n = 4), and 3.8% (n = 12) of the animals, respectively. No mixed infections were observed. Crocidura russula stood out as the main host for Sarcocystis spp. Toxoplasma gondii-specific DNA detected in a house rat was genetically characterized by PCR-RFLP, presenting type II and III alleles (SAG1 [II], SAG3 [II], GRA6 [II], c22-8 [III], Apico [III]). Also, unsuccessful DNA sequencing and microsatellite typing were attempted in N. caninum-positive samples, which suggested a lack of PCR specificity and open avenues to speculate the host competence of rodents for N. caninum. Likewise, Sarcocystis spp. identity was studied by alignment and phylogenetic analyses of cox1 and 28S rRNA sequences from the 14 positive samples. It resulted in at least three unknown organisms closely similar (95.7-100% cox1-sequence homology) to Sarcocystis pantherophisi from the Eastern rat snake (Pantherophis alleghaniensis) (KU891603), suggesting together with 28S rRNA sequences analyses, three Sarcocystis sp. with a life cycle conformed by rodents as intermediate host (IH) and snakes as definitive hosts (DH) infecting the periurban micromammals surveyed. Prevalence figures found in this first survey carried out in Spain agree with other international studies focused on periurban areas. Further surveys should be conducted in farms and their surroundings in order to unravel the role of wild micromammals in the epidemiology of such protozoan parasites affecting our livestock, and therefore human population.

The objectives of the current study were to assess the preliminary status of Cytauxzoon felis (C. felis) infection among outdoor cats in Mashhad, Iran and also to compare clinicopathological findings between C. felis infected and non-infected cats. Blood samples were collected from 100 outdoor domestic cats between April and September in 2019. Infection with C. felis was determined using microscopic observation of giemsa-stained blood smears and molecular analysis. The piroplasms was microscopically detected in 5 (5%) of the blood smears with low parasitemia. The presence of C. felis was confirmed in one positive microscopy sample by PCR. The molecular assay revealed that 19 cats (19%) were infected with C. felis. Hematological and some serum biochemical factors were evaluated in both of the infected and non-infected cats. There was no association between C. felis infection and age, gender, and laboratory findings except for hematocrit (Hct) and concentration of total protein and globulin. Clinical signs such as fever, dehydration, lethargy, and icterus were observed only in 15.78% (3/19) of the infected cats, while 84.22% (16/19) were asymptomatic. Laboratory findings such as non-regenerative anemia, thrombocytopenia, neutrophilic leukocytosis hyperproteinemia, hypoalbuminemia, and hyperbilirubinemia were detected in the clinically infected cats. This study revealed the relatively high frequency of C. felis infection in outdoor domestic cats in Mashahd, Iran. The predominance of asymptomatic infection likely indicates that these cats may be infected with low-virulent strains of C. felis.

Blastocystis is a zoonotic protozoan parasite frequently identified in the intestinal tract of humans and a vast variety of animals, worldwide. Here, we assessed the prevalence of Blastocystis and its subtypes in stool samples of raccoons. Stool samples from 30 raccoons were collected. Total DNA was extracted, and the barcoding region of the small subunit ribosomal rRNA (SSU rRNA) gene was amplified and sequenced. Specific fragment for Blastocystis was successfully amplified in five samples (16.66%). Sequencing analysis revealed ST1, ST2, and ST3 among 1, 2, and 2 Blastocystis-positive samples. Our results documented the presence of Blastocystis subtypes 1-3 in raccoons. Subtype 1 showed higher similarity to the human isolates of Blastocystis. However, it seems that raccoons may emerge as reservoirs for Blastocystis and may be linked to zoonotic transmission of the protist.

Pathogens pose significant threats to pollinator health and food security. Pollinators can transmit diseases during foraging, but the consequences of plant species composition for infection is unknown. In agroecosystems, flowering strips or hedgerows are often used to augment pollinator habitat. We used canola as a focal crop in tents and manipulated flowering strip composition using plant species we had previously shown to result in higher or lower bee infection in short-term trials. We also manipulated initial colony infection to assess impacts on foraging behavior. Flowering strips using high-infection plant species nearly doubled bumble bee colony infection intensity compared to low-infection plant species, with intermediate infection in canola-only tents. Both infection treatment and flowering strips reduced visits to canola, but we saw no evidence that infection treatment shifted foraging preferences. Although high-infection flowering strips increased colony infection intensity, colony reproduction was improved with any flowering strips compared to canola alone. Effects of flowering strips on colony reproduction were explained by nectar availability, but effects of flowering strips on infection intensity were not. Thus, flowering strips benefited colony reproduction by adding floral resources, but certain plant species also come with a risk of increased pathogen infection intensity.

Histomoniasis is currently a re-emerging disease of major significance for many commercial turkey and broiler breeder production companies because of the unavailability of drugs or vaccines. The protozoa Histomonas meleagridis (HM) requires the presence of enteric microflora to promote the disease. The objectives of this research note were to evaluate the effect of dietary administration of sodium chlorate (SC) and sodium nitrate (SN) in vitro and in vivo for HM prophylaxis in poults. A total of 128 day-of-hatch female poults obtained from a commercial hatchery were wing-tagged and randomly assigned into 1 of 4 experimental groups: negative control (NC), positive control, dietary inclusion of SC (3,200 ppm) and SN (500 ppm). Poults from groups SC and SN started on their respective diets on day 12. All groups, except the NC, were challenged with 2 × 105 HM on day 19. Controls were fed a basal diet, identical to the treatment diets but not supplemented with SC or SN. Body weight gain (BWG) was determined weekly, starting on day 1 until day 28, and postchallenge morbidity and mortality were recorded. On day 28 of age, all surviving poults were lesion scored for hepatic and cecal lesions. Ceca and distal ileum were collected on day 28 for bacterial recovery on selective media for total aerobic, lactic acid bacteria, or gram-negative bacteria. The addition of SC and SN in the in vitro growth of HM greatly reduced the growth of the protozoa after 20 h of incubation when compared with the control nontreated group (P

Background Fecal polymerase chain reaction (PCR) testing for Tritrichomonas foetus is considered the most sensitive means for diagnosis of infection but results could be influenced by fecal collection technique and prior use of antimicrobial drugs. Objectives To establish any association between fecal collection technique or treatment history and results of fecal PCR testing for T. foetus. Animals Fecal samples from 1717 cats submitted by veterinarians between January 2012 and December 2017. Methods This study used a retrospective analysis. T. foetus PCR test results from 1808 fecal samples submitted for diagnostic testing were examined for their association with method of fecal collection and prior antimicrobial treatments. Data were collected from sample submission form. Results Positive T. foetus PCR test results were obtained for 274 (16%) cats. Fecal samples collected via fecal loop had increased probability of positive PCR test results (odds ratio [OR] 2.04, 95% confidence interval [CI] 1.31-3.17, P = .002) compared to samples collected by colonic flush. There was no association between PCR test results and treatment history, treatment type, or prior treatment with ronidazole. After an initial positive PCR test, 4/19 (21%; 95% CI 2.7%-39.4%) cats treated with ronidazole had a second positive test result. Conclusions and clinical importance Results of this study support that fecal samples collected by loop might be better for PCR diagnosis of T. foetus infection. Lack of association of ronidazole with PCR test results and a 21% all-potential-causes failure rate of ronidazole in cats with preconfirmed infection are important limitations to use of this drug.

Background Several Tritrichomonas species have been found in mammalian hosts. Among these trichomonads T. foetus is often found in the urogenital tract of cattle and the gastrointestinal tract of the domestic cat, resulting in sexually transmitted bovine trichomonosis and fecal-orally transmitted feline trichomonosis, respectively. The aims of the current study were to molecularly characterize clinical isolates of T. foetus in cattle populations in Wyoming, South Dakota, and Montana of the United States of America and to phylogenetically analyze Tritrichomonas species of mammalian hosts. Results DNA sequencing of rRNA genes showed over 99% identity of the newly described isolates to other bovine isolates. Further, T. foetus isolates of various mammalian hosts originated in different geographic regions worldwide were clustered into two well-defined clades by phylogenetic analysis of rRNA and cysteine protease 2 genes. Clade I consisted of isolates originated from cattle, pig, and human whereas clade II contained isolates of cat and dog. Conclusion It is concluded that all mammalian Tritrichomonas spp. apparently belong to T. foetus. Analysis of more sequences is warranted to support this conclusion.

Histomoniasis, caused by the protozoan parasite Histomonas meleagridis, is a disease to which turkeys are especially susceptible. Currently, no chemoprophylaxis compounds are available to mitigate this disease. Boric acid (BA) exhibits antifungal, antiseptic, and antiviral properties and has been used in the treatment of yeast infections. Based on these characteristics, an experiment was conducted to evaluate whether BA might be an efficacious prophylaxis against challenge with wild-type H. meleagridis (WTH). On day-of-hatch, poults were randomly assigned to either a basal control diet or a BA diet (basal diet + 0.2% BA). Groups consisted of a nonchallenged control (NC; basal diet), 0.2% BA + challenge (BA; 0.2% BA diet), and a positive-challenged control (PC; basal diet). On day 21, challenged groups were intracloacally inoculated with 2 × 105 WTH cells/turkey, and lesions were evaluated on day 14 postchallenge. Individual body weights were recorded on day 0, 21, and 35 to calculate the prechallenge and postchallenge body weight gain (BWG). The BA group resulted in lower prechallenge day 0 to day 21 BWG (P = 0.0001) than the NC group. Postchallenge day 21 to day 35, BWG was also lower (P = 0.0503) in the BA group than the PC group. No differences between the BA and PC groups were detected for mortalities associated with histomoniasis. Moreover, liver and cecal lesions were not statistically different between the BA and PC groups. Taken together, these data suggest that BA was not efficacious in the prevention or reduction of histomoniasis disease severity when provided at 0.2% dietary concentration under these experimental conditions.

Background Histomonas meleagridis is an anaerobic, intercellular parasite, which infects gallinaceous birds such as turkeys and chickens. In recent years, the reemergence of Histomoniasis has caused serious economic losses as drugs to treat the disease have been banned. At present, H. meleagridis research focuses on virulence, gene expression analysis, and the innate immunity of the host. However, there are no studies on the differentially expressed miRNAs (DEMs) associated with the host inflammatory and immune responses induced by H. meleagridis. In this research, high-throughput sequencing was used to analyze the expression profile of cecum miRNA at 10 and 15 days post-infection (DPI) in chickens infected with Chinese JSYZ-F strain H. meleagridis. Results Compared with the controls, 94 and 127 DEMs were found in cecum of infected chickens at 10 DPI (CE vs CC) and 15 DPI (CEH vs CCH), respectively, of which 60 DEMs were shared at two-time points. Gene Ontology (GO) functional enrichment analysis of the target genes of DEMs indicated that 881 and 1027 GO terms were significantly enriched at 10 and 15 DPI, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG, www.kegg.jp/kegg/kegg1.html) pathway enrichment analysis of the target genes of DEMs demonstrated that 5 and 3 KEGG pathways were significantly enriched at 10 and 15 DPI, respectively. For previous uses, the Kanehisa laboratory have happily provided permission. The integrated analysis of miRNA–gene network revealed that the DEMs played important roles in the host inflammatory and immune responses to H. meleagridis infection by dynamically regulating expression levels of inflammation and immune-related cytokines. Conclusion This article not only suggested that host miRNA expression was dynamically altered by H. meleagridis and host but also revealed differences in the regulation of T cell involved in host responses to different times H. meleagridis infection.

Haemosporidian blood parasites are widely used in evolutionary ecological research when exploring the effects of parasites on different life-history traits of their bird hosts. However, their roles in bird migration are less studied. If these parasites deteriorate the body condition of the birds strongly, they might negatively affect the whole migration phenology and the survival of the birds as well. In our study, we tested the relationships between infection for parasite genera (Haemoproteus or Plasmodium), the three most frequent parasite lineages and body condition (body mass, fat deposit), and the timing of autumn migration in the European Robin (Erithacus rubecula). We found that mean body mass and fat scores did not differ between parasitized and non-parasitized individuals, but infected juveniles arrived later than their non-infected counterparts. The difference in the arrival time of parasitized and non-parasitized birds was greater in the case of Haemoproteus infections. However, when we analysed the effects of the distinct parasite lineages separately, we found that prevalence of parasite lineages correlated with the body mass, fat storage, and timing of autumn migration of the birds in a different direction. Our results therefore emphasize the importance of testing the impacts of the different parasites individually, because possible lineage-specific effects on bird condition during migration might exist.

The development of new molecular methods has significantly improved the detection and identification of avian haemosporidian parasites (Plasmodium, Haemoproteus and Leucocytozoon) compared to microscopic examination. Very large numbers of previously hidden Haemosporida species of a wide range of avian hosts have thus been discovered in the last two decades. However, test parameters of the various detection methods remain largely unevaluated. In this study, the merits of microscopy, multiplex PCR, and nested PCR were compared to identify the infection status of three Malagasy bird species. A total of 414 blood samples of Hypsipetes madagascariensis, Foudia omissa and F. madagascariensis, as well as 147 blood smears, were examined for haemosporidian infection. Thirty-four lineages of haemosporidian parasites could be identified, of which six have been detected for the first time. Microscopy, multiplex and nested PCR showed differences in detection rate, most likely due to low parasitemia of chronically infected birds. The combination of both PCR methods yielded the best results. In particular, detection of multiple infections could be greatly improved and will enable more precise prevalence estimates of individual haemosporidian species in wild birds in the future.

Background The order Accipitriformes comprises the largest group of birds of prey with 260 species in four families. So far, 21 haemosporidian parasite species have been described from or reported to occur in accipitriform birds. Only five of these parasite species have been characterized molecular genetically. The first part of this study involved molecular genetic screening of accipitriform raptors from Austria and Bosnia-Herzegovina and the first chromogenic in situ hybridization approach targeting parasites in this host group. The aim of the second part of this study was to summarize the CytB sequence data of haemosporidian parasites from accipitriform raptors and to visualize the geographic and host distribution of the lineages. Methods Blood and tissue samples of 183 accipitriform raptors from Austria and Bosnia-Herzegovina were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR, and tissue samples of 23 PCR-positive birds were subjected to chromogenic in situ hybridization using genus-specific probes targeting the parasites’ 18S rRNAs. All published CytB sequence data from accipitriform raptors were analysed, phylogenetic trees were calculated, and DNA haplotype network analyses were performed with sequences from clades featuring multiple lineages detected in this host group. Results Of the 183 raptors from Austria and Bosnia-Herzegovina screened by PCR and sequencing, 80 individuals (44%) were infected with haemosporidian parasites. Among the 39 CytB lineages detected, 18 were found for the first time in the present study. The chromogenic in situ hybridization revealed exo-erythrocytic tissue stages of Leucocytozoon parasites belonging to the Leucocytozoon toddi species group in the kidneys of 14 infected birds. The total number of CytB lineages recorded in accipitriform birds worldwide was 57 for Leucocytozoon, 25 for Plasmodium, and 21 for Haemoproteus. Conclusion The analysis of the DNA haplotype networks allowed identifying numerous distinct groups of lineages, which have not yet been linked to morphospecies, and many of them likely belong to yet undescribed parasite species. Tissue stages of Leucocytozoon parasites developing in accipitriform raptors were discovered and described. The majority of Leucocytozoon and Haemoproteus lineages are specific to this host group, but most Plasmodium lineages were found in birds of other orders. This might indicate local transmission from birds kept at the same facilities (raptor rescue centres and zoos), likely resulting in abortive infections. To clarify the taxonomic and systematic problems, combined morphological and molecular genetic analyses on a wider range of accipitriform host species are needed.

Bats (order, Chiroptera) account for more than one-fifth of all mammalian species in the world and are infected by various intra-erythrocytic parasites of the family Plasmodiidae (Apicomplexa: Haemosporida), including Polychromophilus Dionisi, 1899. Recent advance in the molecular characterization of haemosporidian isolates has enabled their accurate identification, particularly in the last decade. Studies are actively conducted in tropical regions, Europe, and Australia; however, data on haemosporidian infection in bats in Asian temperate areas, including Japan, remain limited. In this study, 75 bats of 4 species (Miniopterus fuliginosus, Myotis macrodactylus, Rhinolophus nippon, and Rhinolophus cornutus) were captured at three sites in western Japan (Yamaguchi Prefecture), and haemosporidian parasites were screened microscopically and molecularly via nested polymerase chain reaction (PCR) targeting the cytochrome b (cytb), cytochrome c oxidase subunit I (cox-1), apicoplast caseinolytic protease C (clpc), and nuclear elongation factor 2 (EF2) genes. The survey detected Polychromophilus melanipherus in 15 (40.5%) miniopterid bats (M. fuliginosus) and Polychromophilus murinus in 6 (46.2%) vespertilionid bats (M. macrodactylus), whereas none of the 25 rhinolophid bats (R. nippon and R. cornutus) was infected, indicating the robust host specificity for miniopterid (P. melanipherus) and vespertilionid (P. murinus) bats regardless of orthotopic nesting. The 15 Polychromophilus cytb sequences obtained from 11 miniopterid and 4 vespertilionid bats were classified into six cytb haplotypes (three for each species), showing no region-specific variation in a phylogenetic tree of Polychromophilus isolates in the Old World. Similarly, multiple haplotypes (seven for cox-1 and nine for clpc) and genotypes (three for EF2) were characterized for the Japanese isolates of Polychromophilus, and the results were consistent with those based on a haemosporidian cytb analysis. Bat flies (Nycteribia allotopa and another undetermined Nycteribia sp.) collected from the body surface of bats harbored Polychromophilus oocysts on the external surface of the midgut. This is the first study to report the isolation and molecular characterization of Polychromophilus spp. in miniopterid and vespertilionid bats in the temperate area of Asia (western Japan). Future studies should evaluate the global prevalence of haemosporidian infections in bats.