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.

Visceral Haemoproteus minutus Infection in a Major Mitchell's Cockatoo (Lophochroa leadbeateri).

A 1-year-old major Mitchell's cockatoo (Lophochroa leadbeateri) was presented for evaluation of weakness, diarrhea with undigested seeds in the droppings, and weight loss. Leukocytosis with severe heterophilia, monocytosis, and lymphocytosis was noted on the complete blood count. Altered plasma biochemical parameters included a slight increase in creatine kinase and mild hypoproteinemia. Two blood smears before and after 2 days of treatment revealed mild polychromasia and anisocytosis but no blood parasites. Radiographic and computed tomographic imaging of the cockatoo were helpful in identifying airsacculitis, pneumonia, and gastrointestinal motility disorders. The patient died 5 days after treatment for the presenting clinical problems. On the gross postmortem examination, dark red foci in the ventricular muscle layers and 1-3-mm white foci in the myocardium, opaque air sacs, and dark lungs were identified. Histopathologic examination of submitted tissue samples found severe granulomatous ventriculitis and myocarditis with intralesional Haemoproteus species megalomeronts. Qualitative polymerase chain reaction testing for the cytochrome b (cyt b) gene performed on pooled heart, liver, kidney, and intestinal tissues identified 99.5% homology to Haemoproteus minutus. This case report demonstrates the expansion of the geographic range of H minutus to France and potentially to Belgium, which may compromise breeding and conservation of Australian parrots living outdoors. Challenging diagnosis, rapid disease progression, and the absence of validated treatment protocols for psittacine patients suggest that the use of preventive measures to reduce the presence of insect vectors such as hippoboscid flies and biting midges (Culicoides) should be considered. Haemoproteus minutus should be considered and potentially screened by polymerase chain reaction testing on blood samples, especially in the case of highly susceptible avian species (eg, Australian parrots in Europe) that present with sudden weakness, heterophilic leukocytosis, and monocytosis associated with mild anemia.

Proteomic analysis of proteins released by Tritrichomonas foetus: Identification of potential targets for the development of new diagnostic methods.

Bovine trichomonosis (BT), a disease of the bovine urogenital tract, is caused by the protozoan Tritrichomonas foetus (Tf). Tf causes endometritis, infertility, and premature death of the embryo, which generates considerable economic losses. The proteins released can mediate fundamental interactions between the pathogen and the host, triggering factors associated with the symptomatology, immune evasion and pathogenesis characteristic of the species. However, little is known about the profile of the proteins released by Tf. In order to contribute to their knowledge, we performed an isolation protocol and a proteomic profiling of the supernatant (SN) content of six Tf isolates. A total of 662 proteins present in the SN of Tf were detected, out of which 121 were shared by the six isolates, while the remaining 541 were found in at least one of the isolates studied. The comparative analyses using the databases of Tf strain genome K revealed 32.9% of uncharacterized proteins. The bioinformatic analyses showed that the main molecular functions predicted were binding (47.9%) and catalytic activity (38.2%). Additionally, we performed immunodetection assays to evidence the antigenic potential of SN proteins. Interestingly, we observed great ability to detect SN proteins from all six isolates using serum from immunized mice and infected bulls. A complementary mass spectrometry assay allowed us to determine that the proteins that showed the strongest signal intensity in the immunoassays were Grp78 (A0A1J4IZS3) and Ap65 (A0A1J4JSR1). This work represents the first proteomic characterization of Tf SN proteins and their antigenic potential, which might be interesting for the future design of new diagnosis and treatment methods for BT.

Detection of Tritrichomonas foetus by RT-rtPCR in pooled bovine preputial washings.

Trichomonosis is a venereal disease of cattle caused by the protozoan Tritrichomonas foetus. T. foetus infection in cattle herds can be economically costly for cattle producers; therefore, testing is important for detection of the agent. Given that bulls are considered to be subclinical carriers of T. foetus, it is important to detect T. foetus infection prior to movement and/or breeding season. We have described previously the development of an updated set of PCR primers and probes that offer increased sensitivity of T. foetus detection in preputial washings collected in PBS by utilizing reverse-transcription real-time PCR (RT-rtPCR) that targets the 5.8S ribosomal RNA of the T. foetus organism. Here, we report improvements in the updated RT-rtPCR reagents as well as the evaluation of testing of pooled preputial washings. We found that up to 5 preputial washings can be pooled, similar to routine testing practices (InPouch culture), without reducing the sensitivity of detection of T. foetus.

Serum biochemical changes in cats with naturally acquired feline cytauxzoonosis.

OBJECTIVE: The geographical distribution of feline cytauxzoonosis is expanding in the US. Clinical signs of feline cytauxzoonosis, including lethargy, anorexia, and icterus, are similar to hepatic lipidosis and cholangiohepatitis. Hematologic and serum biochemical abnormality patterns may assist practitioners in prioritizing feline cytauxzoonosis as a differential diagnosis over hepatic lipidosis and cholangiohepatitis. SAMPLE: Hematology and serum biochemical profiles of cats with naturally acquired feline cytauxzoonosis, hepatic lipidosis, or cholangiohepatitis. PROCEDURES: Retrospective search and analysis of the Kansas State Veterinary Diagnostic Laboratory or Kansas State University Veterinary Health Center records between January 2007 and June 2018 for cats with cytauxzoonosis, hepatic lipidosis, or cholangiohepatitis. RESULTS: Patients with acute feline cytauxzoonosis presented with frequent nonregenerative anemia (20/28 [71%]), leukopenia (23/28 [82%]), thrombocytopenia (23/23 [100%]), hyperbilirubinemia (27/28 [97%]), hypoalbuminemia (26/28 [93%]), reduced (18/28 [64%]) or low normal (10/28 [36%]) serum ALP activity, and hyponatremia (23/28 [82%]). Reduced ALP activity was unique to cats with feline cytauxzoonosis relative to hepatic lipidosis and cholangiohepatitis. No correlation between the severity of anemia and the magnitude of hyperbilirubinemia was identified in feline cytauxzoonosis patients. CLINICAL RELEVANCE: The combination of nonregenerative anemia, leukopenia, thrombocytopenia, hyperbilirubinemia, and reduced serum ALP activity in icteric cats may increase the clinical suspicion, but is not pathognomonic, for acute feline cytauxzoonosis. Hematologic and serum biochemical abnormalities of naturally acquired feline cytauxzoonosis are like those reported with feline bacterial sepsis. Blood smear evaluation for intraerythrocytic Cytauxzoon felis piroplasms, tissue aspirates for schizont-laden macrophages, and/or molecular testing are required to diagnose feline cytauxzoonosis.

A novel one-step multiplex PCR protocol to detect avian haemosporidian parasites in the subgenus Haemoproteus (Kruse, 1890) used to quantify parasite prevalence in domestic pigeons (Columba livia) in Turkey.

Infections of avian haemosporidian parasites are regularly identified by molecular methods including multiplex PCR, which allows researchers to distinguish mixed infections of parasites from multiple genera. Here we extend the utility of a previously designed multiplex PCR by designing a primer set specific to parasites of the subgenus Haemoproteus (genus: Haemoproteus). The updated one-step multiplex PCR protocol we describe here allows for the detection of the genera Plasmodium and Leucocytozoon and the two subgenera (Haemoproteus and Parahaemoproteus) of the genus Haemoproteus. A sensitivity analysis showed that the multiplex PCR could amplify DNA of parasites in the subgenus Haemoproteus at very low levels of infection. We used this multiplex PCR to identify haemosporidian infections in 250 adult domestic pigeons (Columba livia) in Turkey. All samples were also screened by microscopy and a widely used nested PCR to compare with the results of multiplex PCR, to detect low levels of parasitemia, and to identify possible abortive infections. In total, 71 pigeons (28.4%) were found to be infected by all three methods. The multiplex PCR protocol successfully detected and discriminated both subgenera Haemoproteus and Parahaemoproteus infections. We compared our results with previous host species records to assess the host specificity of the parasite lineages we found. Our findings provide novel data on the prevalence of avian haemosporidians in domestic pigeons and demonstrate the utility of the new one-step multiplex PCR protocol for the determination of mixed avian haemosporidian infections. We expect that this protocol will contribute to a better understanding of the distribution, epizootiology, and ecology of avian haemosporidians.

Cytauxzoonosis.

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.

Untangling the actual infection status: detection of avian haemosporidian parasites of three Malagasy bird species using microscopy, multiplex PCR, and nested PCR methods.

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.

Molecular detection of Tritrichomonas foetus in bovine samples: a novel real-time polymerase chain reaction (PCR) assay targeting EF1-alpha-Tf1 and a comparative study of published PCR techniques.

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.

Simultaneous occurrence of inflammatory bowel disease and trichomonosis in a Maine coon cat.

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.

Apparition simultanée d'une maladie inflammatoire de l'intestin et d'une trichomonose chez un chat Maine coon. Une chatte Maine coon de 2 ans, pesant 4,2 kg, stérilisée, a été référée à l'hôpital vétérinaire pour une évaluation d'hyporexie, de croissance lente et de diarrhée chronique, intermittente, mucoïde, sanglante, volumineuse et fétide. La diarrhée avait été observée depuis que le chat avait été acquis en chatterie à l'âge de 4 mois; avec une aggravation aiguë dans les 5 jours avant la présentation. La palpation abdominale a révélé une douleur modérée. L'examen échographique a montré un épaississement de la paroi jéjunale et des anses iléales, une augmentation de l'échogénicité de la muqueuse jéjunale et une hypertrophie des ganglions lymphatiques jéjunaux et iléocoliques. L'histopathologie des biopsies intestinales de pleine épaisseur a montré une entérite modérée, diffuse, lymphoplasmocytaire, érosive avec hémorragie et oedème. Une colite érosive diffuse, lymphoplasmocytaire avec fibrose interstitielle légère et hémorragie a également été notée. La biopsie ganglionnaire iléo-caecale montrait une lymphadénite à éosinophiles. Sur la base de l'évaluation immunohistochimique d'échantillons intestinaux avec des anticorps CD3 et CD79a, un diagnostic de lymphome a été écarté. Le test de réaction en chaîne par la polymérase sur les matières fécales était positif pour Tritrichomonas foetus. Sur la base de ces résultats, une maladie inflammatoire de l'intestin et une trichomonose ont été diagnostiquées. Le traitement du chat comprenait un régime hypoallergénique et un supplément oral d'acides gras oméga-3, en association avec de la prednisolone, pour gérer la maladie inflammatoire de l'intestin. Le ronidazole a été administré pour cibler Tritrichomonas foetus. Le chat était cliniquement normal lors d'un examen de suivi après 6 mois de traitement.(Traduit par Dr Serge Messier).

Molecular survey of Leishmania spp. in skin samples of capybaras (Hydrochoerus hydrochaeris) from different areas of Brazil / Investigação molecular de Leishmania spp. em amostras de pele de capivaras (Hydrochoerus hydrochaeris) de diferentes regiões do Brasil

Leishmaniases comprise a spectrum of diseases caused by protozoan parasites of the genus Leishmania, with some species of rodents being incriminated as reservoirs. The capybara is the largest extant rodent species in the world and is widely distributed in South America. The occurrence of infection by Leishmania spp. was investigated in capybaras captured in Brazil during 2015­2019 from established populations in five highly anthropic areas of the state of São Paulo and two natural areas of the states of Mato Grosso and Mato Grosso do Sul. A total of 186 individuals were captured and subjected to abdominal skin biopsy. All skin samples were Leishmania kDNA-negative, suggesting that capybaras have no role in the transmission cycles of Leishmania species in the studied areas despite the well-known role of other rodents in the life cycle of Leishmania spp.(AU)

As leishmanioses compreendem um espectro de doenças causadas por protozoários do gênero Leishmania e algumas espécies de roedores são incriminadas como reservatórios de Leishmania spp. As capivaras compreendem a maior espécie de roedores existentes e são amplamente distribuídas na América do Sul. Para investigar a ocorrência de infecção por Leishmania spp. em capivaras, durante os anos de 2015-2019 capivaras foram capturadas em cinco áreas antrópicas do estado de São Paulo e em duas áreas naturais dos estados do Mato Grosso e do Mato Grosso do Sul, todos esses ambientes com populações de capivaras estabelecidas. Um total de 186 indivíduos foram capturados e submetidos à biópsia de pele abdominal. Todas as amostras de pele foram negativas para o alvo kDNA, assim, os dados sugerem que nas áreas estudadas as capivaras não têm papel no ciclo de transmissão de espécies de Leishmania spp., apesar do papel bem conhecido de outros roedores no ciclo de vida de Leishmania spp.(AU)

Evaluation of various sample sources for the cytologic diagnosis of Cytauxzoon felis.

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 = <.001). Novice observers were more likely to agree with experts when identifying schizont-laden macrophages in splenic aspirates (sensitivity = 77.1%, specificity = 94.4%) versus lymph node aspirates (sensitivity = 52.8%, specificity = 96.4%) or blood films (sensitivity = 41.7%, specificity = 96.9%). CONCLUSIONS AND CLINICAL IMPORTANCE: Schizont-laden macrophages are most frequently identified in spleen, even by novice observers. If the diagnosis of acute cytauxzoonosis cannot be confirmed via blood film, then splenic, followed by peripheral lymph node aspirates can be considered.

CYTAUXZOON FELIS DNA DETECTION IN HEALTHY CATS FROM RIO DE JANEIRO, BRAZIL.

Feline cytauxzoonosis is a disease caused by Cytauxzoon felis, a protozoan that infects the red blood cells and macrophages. It is responsible for an acute and often fatal disease in domestic cats. The purpose of this study was to investigate the occurrence of C. felis infections in healthy cats. Piroplasm forms were seen in the erythrocytes of 2 cats, and C. felis DNA was identified by polymerase chain reaction (PCR) in one of them. The results demonstrate that erythrocytic piroplasmids associated with tick-borne parasitic protozoa may be found circulating in the blood of healthy cats in Rio de Janeiro. These can be differentiated from the morphologically similar forms of species such as Babesia by analysis of DNA, thereby demonstrating the potential for further studies of feline populations in Brazil.

Taguchi method for the optimization of three loop mediated isothermal amplification procedures for Tritrichomonas foetus detection.

Taguchi method was used to optimize loop mediated isothermal amplification tests aimed to amplify segments of the elongation factor 1a1 (tf-ef1a1), the 5,8 ribosomal gene (tf-5,8 r) or the beta tubulin 2 (tf-btub2) from the protozoan parasite Tritrichomonas foetus. L9 orthogonal array and quadratic loss functions that penalize deviations from prediction values revealed the effect of amplification reaction components. Analysis of variance (ANOVA) decomposed the contribution of individual factors to a small Ct. Confirmation experiments established that optimum conditions were predictable, verifiable and reproducible. Primers concentration conditioned the non-specific amplification of tf-ef1a1 while betaine and magnesium concentration contributed to accelerate the time to reach a positive threshold in tf-ef1a1, tf-5,8 r and tf-btub2. The general strategy of simple and robust experimental design holds potential as a general optimization protocol for LAMP tests in every diagnostic laboratory.

Account and Utilization of Blood Lipid Profiles: Lipid Levels Predicted Hemosporidian Infection in Migrating Northern Saw-Whet Owls of Eastern North America.

Noting lipidomic changes following the parasitism of migrating birds, the metabolic needs of which are primarily fueled by lipids, can deepen our understanding of host-parasite interactions. We identified lipids of migrating Northern saw-whet owls (Aegolius acadicus) using collision-induced dissociation mass spectrometry, compared the lipidomic signatures of hemoparasite-infected and noninfected individuals, and performed cross-validation analyses to reveal associations between parasite infection and lipid levels. We found significantly lower levels of lipid classes phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), and sphingomyelin (SM) in infected Northern saw-whet owls than in the noninfected individuals. Conversely, we found higher levels for certain lysoPS and lysoPE species, and variable lipid level changes for free fatty acid (FFA) species. Reporting lipidomic changes observed between hemosporidian-infected and noninfected Northern saw-whet owls can strengthen our understanding of the mechanisms governing parasite proliferation in this species. Furthermore, our analysis indicated that lipidomic signatures are better predictors of parasite infection than the log-adjusted mass/wing chord body index, a metric commonly used to assess the influence of hemosporidia infection on the health of birds. Establishing a lipidomic profile for Northern saw-whet owls that provides baseline lipid levels during fall migration may assist future studies assessing causes of reductions in breeding brought about from subtle differences in behaviors such as delayed migration.

A case of "misplaced" Tritrichomonas foetus infection in a dog in Northern Italy.

An 8-year-old, spayed female Rottweiler dog, under immunosuppressant treatment for protein-losing enteropathy (PLE) and intestinal lymphangiectasia, was presented for anorexia, poor general conditions and episodes of diarrhea. A subcutaneous mass between the caudal abdominal mammary glands was found. A fine-needle aspiration cytology was performed and revealed the presence of inflammatory cells mixed with pear- or round-shaped microorganisms with cytomorphological features of flagellated protozoan trophozoites, belonging to Trichomonadida order. The final diagnosis of Tritrichomonas foetus infection was achieved with molecular analysis (Real-Time PCR) and sequencing. T. foetus has often been reported as a pathogenic agent in cattle and cats, and rarely reported in dogs, where its pathogenetic role is poorly understood. To the author's knowledge, this is the first case of T. foetus infection described in a location different from the gastrointestinal tract in a dog.

Use of molecular tools for the diagnosis of rangeliosis by Rangelia vitalii in Argentina: A case report.

Vector-borne pathogens are responsible for serious emerging diseases and Rangelia vitalii, the etiologic agent of canine rangeliosis, is one of the most pathogenic tick-borne pathogens for dogs in South America. This protozoan is transmitted by the Amblyomma aureolatum tick bite and the clinical features associated to the disease are fever, hemolytic anemia, jaundice, hepatosplenomegaly and bleeding from natural orifices, mainly from the ear egde. The reports of canine rangeliosis in Argentina are scarce. In the present study we report the detection of Rangelia vitalii in a naturally infected dog from Gualeguay, Entre Ríos, Argentina with history of tick infestation and clinical signs compatible with rangeliosis. An initial blood sample was positive to piroplasmids by blood smear examination and the molecular amplification of a fragment of the 18SrRNA gene. Sequencing of the fragment confirmed the pathogen identity. After treatment with imidocarb dipropionate, the clinical signs remitted and the blood smear tested negative.

First molecular detection of Haemoproteus spp. and Plasmodium spp. in eared doves (Zenaida auriculata) in Brazil.

The aim of this study was to verify the presence and identify the species of haemosporidian parasites in eared doves (Zenaida auriculata) in Brazil. Two hundred and eleven male and female eared doves were trap-captured in four different regions of Londrina city, in southern Brazil. Whole blood was collected in EDTA tubes through heart puncture after euthanasia in a CO2 chamber. A nested PCR targeting the mitochondrial cytochrome b gene (cyt b) of Haemoproteus spp./Plasmodium spp. was performed, followed by an enzymatic digestion to identify the genus. Phylogenetic trees were constructed to determine the closely related species. Out of 211 eared doves, 209 (99.05%) were positive for Haemoproteus spp. and/or Plasmodium spp. RFLP analysis showed that 72.72% (152/209) of eared doves were positive only for Haemoproteus spp., 6.22% (13/209) were positive only for Plasmodium spp., and 21.05% (44/209) of eared doves had mixed infections. Genetic analysis found four samples that were homologous with Haemoproteus multipigmentatus and one that was homologous with Plasmodium sp. This is the first molecular study of hemoparasites from eared doves in Brazil, and it is also the first description of H. multipigmentatus and Plasmodium spp. infection in eared doves in Brazil.

Using a multistate occupancy approach to determine molecular diagnostic accuracy and factors affecting avian haemosporidian infections.

The use of a sensitive and accurate parasite detection methodology is crucial in studies exploring prevalence of parasites in host populations or communities, and uncertainty in identifying parasite genera and/or lineages may limit the understanding of host-parasite interactions. Here, we used a multistate occupancy approach that accounts for imperfect detection to assess whether sex and breeding season influenced the prevalence of a specific Haemoproteus lineage (TARUF02) in a white-lined tanager population. Likewise, we explored whether the probability of detecting the target parasite in an infected bird using PCR and sequencing analyses may be influenced by season and host sex. We found little evidence that sex influenced the probability of an individual host being infected by a haemosporidian parasite. Conversely, we found that the probability of infection by Haemoproteus TARUF02 was ~30% higher during the breeding season, reflecting a higher prevalence of this parasite in this season. The probability that PCR detects DNA of haemosporidian parasite was higher for female birds, suggesting that they are more prone to be parasitized with parasitemia levels that are more successfully detected by molecular analysis. Sequencing successfully determined the Haemoproteus TARUF02 lineage in 60% of samples collected during the breeding season and 84% of samples collected during the non-breeding season. Understanding the ecology of hosts and aspects of their physiology that may influence the parasite infection is essential to better understanding of hemoparasite infections and how parasites influence their native hosts, through decreasing reproductive success, lifespan, and/or survival.