Development and evaluation of a TaqMan® real-time PCR assay for species-specific detection of Ehrlichia canis.

The aim of this work was to develop a real-time PCR assay with a TaqMan® probe that detects a species-specific part of the 16S rDNA gene of Ehrlichia canis. Canine blood samples (n = 207), collected and tested by a conventional PCR assay within a study conducted by De Salvo et al., were simultaneously analyzed with the novel designed real-time PCR, and the results of both assays were compared. The agreement between the two methods was 97.6 % with a kappa value of 0.92186. Hereby, the standard error was 0.034416 and the 95 % confidence interval from 0.8544 to 0.98931. While the conventional PCR assay showed false negative results (2.42 %; 5/207), the real-time PCR assays showed a specificity of 100 %. The results of the current study showed that the developed assay presents sensitivity and specificity for the detection of E. canis in blood samples, adding a new tool for the diagnosis of this pathogen.

Description of bovine babesiosis and anaplasmosis outbreaks in northern Uruguay between 2016 and 2018.

Bovine babesiosis and anaplasmosis cause important economic losses in livestock production. In Uruguay, the main aetiological agents of bovine babesiosis and anaplasmosis are Babesia bovis, B. bigemina and Anaplasma marginale. The aim of this work was to describe the outbreaks of bovine babesiosis and anaplasmosis in northern Uruguay between 2016 and 2018. Convenience sampling was carried out. We worked with blood and organ samples from bovines with clinical signs and autopsy findings compatible with babesiosis and anaplasmosis. A total of 140 presumptive outbreaks were studied. Epidemiological information such as place, date of occurrence, age, number of sick and dead animals, clinical signs, autopsy findings, the presence of ticks and health management that involved injectables were registered. The diagnoses were carried out by blood and organ smears stained with Giemsa and confirmed by multiplex PCR. There were 83 (59.2%) positive outbreaks, comprising 35 (42.2%) A. marginale, 19 (22.9%) B. bigemina, 18 (21.7%) B. bovis and 11 (13.2%) mixed infections (Babesia spp. + A. marginale). Cows were the most commonly affected category. The clinical signs and autopsy findings with a significant association (p ≤ 0.05) were anaemia, pale mucous membranes, fever, jaundice, ataxia and aggressiveness, splenomegaly, and orange discolouration of the liver. Babesiosis had a seasonal occurrence, mainly in autumn, while anaplasmosis cases were recorded throughout the year. The use of injectable agents was associated with A. marginale transmission. This work contributes updated information about epidemiological and clinical patterns of bovine babesiosis and anaplasmosis in northern Uruguay, which is important for implementing preventive measures and control.

Molecular detection and phylogenetic relationship of Haemosporida parasites in free-ranging wild raptors from Brazil.

The order Haemosporida is widely distributed parasitizing members of the Aves class. In birds of prey, infection with Plasmodium spp. parasites varies from an apathogenic form to a clinical syndrome. However, studies on Haemosporida in raptors from the neotropical region are scarce. The aim of this study was to investigate natural infection by Plasmodium spp., Haemoproteus spp. and Leucocytozoon spp. in free-ranging wild raptors from southern Brazil. For this, we sampled 206 individuals of 21 species: 94 live-trapped Southern Caracaras (Caracara plancus) and 112 raptors from other species that were brought to rehabilitation centers. The presence of infection was investigated using a nested-PCR for Haemosporida performed on blood samples. Overall, 56 out of 206 birds were positive for Plasmodium spp./Haemoproteus spp. Twenty-two percent (21/94) of the C. plancus samples were positive. Of the 112 wild raptors rescued, 31% (35/112) of those belonging to 15 other species tested positive. No sample was positive for Leucocytozoon spp. Herein, we demonstrated nine lineages of Haemosporidian parasites (eight Plasmodium sp. and one Haemoproteus sp.) in free-living species of Brazilian birds of prey, being six of them potential novel lineages. It suggests that information currently available on South-American haemosporidian from these birds greatly underestimate the potential lineage diversity in this region.

Vector-Borne Blood Parasites of the Great-Tailed Grackle (Quiscalus mexicanus) in East-Central Texas, USA.

Great-tailed grackles (Quiscalus mexicanus) have dramatically expanded into North America over the past century. However, little is known about the blood that parasites they support. Here, for the first time, we document an assemblage of trypanosome, haemosporida, and filarial nematodes co-circulating in invasive great-tailed grackles. Between February and July, 2015, 61 individuals were captured in an urban environment of College Station, Texas. Field microscopy and molecular diagnostics indicate that 52% (24/46) were visually infected with filarioid nematodes, 24% (11/46) with avian trypanosomes, and 73% (n = 44/60) with haemosporida parasites, such as Haemoproteus (Parahaemoproteus) and Plasmodium cathemerium. Overall, 87% of great-tailed grackles were infected with blood parasites. Although 50% of individuals hosted parasites from multiple phylum, no patterns of parasite assembly were observed. Results indicate that great-tailed grackles can support a relatively high level of blood parasitism. However, the consequences for avian health remain to be determined.

Eficacia de la asociación oxitetraciclina - isometamidium en el control de anaplasmosis y tripanosomosis bovina / Efficacy of the association oxitetracicline - isometamidium on the control of bovine anaplasmosis and tripanosomosis / Eficácia da associacao oxitetraciclina-isometamidium no controle da anaplasmose e tripanosomose bovina

Resumen Objetivo: Evaluar la eficacia de la asociación cloruro de isometamidium+clorhidrato de oxitetraciclina (Isopan® Laboratorio Reveex de Venezuela) en el control de infecciones por Tripanosoma vivax y Anaplasma marginale en bovinos. Materiales y métodos: Doce bovinos de aproximadamente un año de edad fueron asignados al azar, a dos grupos de seis animales cada uno. Un grupo fue inoculado por vía venosa con 10 mL de sangre con una parasitemia de 3,5% de Ana- plasma marginale en extendido sanguíneo coloreado; el segundo grupo se inoculó, por la misma vía, con cinco mL de sangre de una cepa de campo de Trypanosoma vivax con una parasitemia de tres flagelados por campo en un frotis sanguíneo delgado coloreado. En todas las terneras, la temperatura rectal, el hematocrito y la parasitemia fueron registrados con frecuencia diaria o inter-diaria hasta los días 49 posinoculación y 27 postratamiento en el caso de A. marginale y hasta los días 11 posinoculación y 21 postratamiento en el caso de T. vivax. Resultados: La asociación oxitetraciclina-isometamidium fue 100% eficaz contra Trypanosoma vivax desde un día después de su aplicación y por el término de 29 días experimentales. La asociación tuvo una eficacia promedio contra Anaplasma marginale de 75,59% aunque cuatro de las terneras requirieron un segundo tratamiento contra Anaplasma entre los días 10 y 27 después del tratamiento. Conclusiones: La asociación oxitetraciclina - isometamidium mostró una eficacia de 100% contra Trypanosoma vivax y de 75,59% en promedio, contra Anaplasma marginale, aunque no previno un nuevo episodio de anaplasmosis entre los días 10 y 27 después del tratamiento.

Abstract Objective: To evaluate the efficacy of the association Isometamidium Chloride + Oxytetracycline Hydrochloride (Isopan® Reveex Laboratories, Venezuela) on the control of infections by Trypanosoma vivax and Anaplasma marginale in bovine. Materials and methods: Twelve one-year old heifers were randomly assigned to two groups of six animals each. Heifers on one group were each inoculated intravenously with 10 mL of blood showing a 3.5% parasitemia with Anaplasma marginale on stained blood smears. Heifers in the other group were inoculated intravenously with five mL of blood containing a field strain of T. vivax with a parasitemia of 3 flagellates per microscopic field in a stained blood smear. In all heifers, rectal temperature, haematocrit and parasitemia were monitored daily or every other day until days 49 post inoculation and 27 post treatment in the case of A. marginale and 11 post inoculation and 21 post treatment in the case of T. vivax. Results: The association oxitetracicline-isometamidium was 100% efficacious against T. vivax from day one after administration and until the 21th experimental day. The association showed an average efficacy of 75.59% against A. marginale, though four heifers required a second treatment against Anaplasma between days 10 and 27 after treatment. Conclusions: The association Oxitetracicline - isometamidium showed a 100% efficacy against T. vivax and an average of 75.59% efficacy against A. marginale but did not prevent a second anaplasmosis episode between days 10 and 27 following the first treatment.

Resumo Objetivo: Avaliar a eficácia da associação cloreto de isometamidio + cloridrato de oxitetraciclina (Isopan® Laboratorio Reveex de Venezuela) no controle de infecções por Trypanosoma vivax e Anaplasma marginale em bovinos. Materiais e métodos: Doze bovinos de aproximadamente 1 ano de idade foram distribuídos aleatoria- mente em dois grupos de 6 animais cada. Um grupo foi inoculado por via venosa com 10 mL de sangue com parasitemia de Anaplasma marginale de 3,5% em esfregaço de sangue corado; o segundo grupo foi inoculado, pela mesma via, com cinco mL de sangue de uma cepa de Trypanosoma vivax com parasitemia de três flagelados por campo em esfregaço de cor fina. Em todos os bezerros, temperatura retal, hematócrito e parasitemia foram registrados diariamente ou inter-diariamente até os dias 49 pós-inoculação e 27 pós-tratamento no caso de A. marginale e até os dias 11 pós-inoculação e 21 pós-tratamento no caso de A. de T. vivax. Resultados: A associação oxitetraciclina-isometamídio foi 100% eficaz contra o Trypanosoma vivax um dia após sua aplicação e pelo prazo de 29 dias experimentais. A associação teve uma eficácia média contra o Anaplasma marginale de 75,59%, embora quatro dos bezerros precisassem de um segundo tratamento contra o Anaplasma entre os dias 10 e 27 após o tratamento. Conclusões: A associação oxitetraciclina-isometamídio mostrou eficácia de 100% contra Trypanosoma vivax e 75,59% em média contra Anaplasma marginale, embora não tenha prevenido novo episódio de anaplasmose entre os dias 10 e 27 após o tratamento.

Haemoparasites in endemic and non-endemic passerine birds from central Mexico highlands.

Haemosporidian parasites of birds are found worldwide and include the genera Haemoproteus, Plasmodium and Leucocytozoon. Infection with haemosporidian parasites can affect host physical condition and reproductive success. The aim of this study was to identify the blood parasites and parasitaemia in endemic and non-endemic passerine birds from central Mexico highlands. This study included 157 passerines representing 29 species from 17 families. Overall, 30.6% (48/157) of the birds were infected with blood parasites. Of those, Haemoproteus spp. were found in 14.0% (n = 22), Leucocytozoon spp. 12.1% (n = 19) and microfilariae 0.6% (n = 1). Blood parasites were found in 71.4% (5/7) of endemic bird species and 45.4% (10/22) of non-endemic species. Medium to high parasitaemia (number of parasites/number erythrocytes) was observed in birds with infections of Haemoproteus spp. and Leucocytozoon spp. Co-infections 3.8% (n = 6) were observed in two species of endemic birds. This study contributes to the knowledge of haemoparasites in endemic and non-endemic passerine birds from central Mexico highlands. Additional investigation on the molecular identification of haemosporidian parasites, pathogenicity and health status of these birds is necessary.

Haemocystidium spp., a species complex infecting ancient aquatic turtles of the family Podocnemididae: First report of these parasites in Podocnemis vogli from the Orinoquia.

The genus Haemocystidium was described in 1904 by Castellani and Willey. However, several studies considered it a synonym of the genera Plasmodium or Haemoproteus. Recently, molecular evidence has shown the existence of a monophyletic group that corresponds to the genus Haemocystidium. Here, we further explore the clade Haemocystidium spp. by studying parasites from Testudines. A total of 193 individuals belonging to six families of Testudines were analyzed. The samples were collected in five localities in Colombia: Casanare, Vichada, Arauca, Antioquia, and Córdoba. From each individual, a blood sample was taken for molecular analysis, and peripheral blood smears were made, which were fixed and subsequently stained with Giemsa. The prevalence of Haemocystidium spp. was 1.55% (n = 3/193); all infected individuals belonged to Podocnemis vogli (Savanna Side-necked turtle) from the department of Vichada. This is the first report of Haemocystidium spp. in Colombia and in this turtle species. The phylogenetic analysis of a mitochondrial cytb fragment revealed Haemocystidium spp. as a monophyletic group and as a sister taxon of Haemoproteus catharti and the genus Plasmodium. Haemocystidium spp. are difficult to identify by morphology only. As a result, it is possible that some of the taxa, such as Haemocystidium (Simondia) pacayae, represent a species complex. The parasite found in our study is morphologically indistinguishable from Haemocystidium (Simondia) pacayae reported in Peru. However, the new lineage found in P. vogli shows a genetic distance of 0.02 with Hae. pacayae and 0.04 with Hae. peltocephali. It is proposed that this divergent lineage might be a new species. Nevertheless, additional molecular markers and ecological features could support this hypothesis in the future.

Rangelia vitalii and Hepatozoon canis coinfection in pampas fox Lycalopex gymnocercus from Santa Catarina State, Brazil / Infecção por Rangelia vitalii e Hepatozoon canis em Lycalopex gymnocercus proveniente do Estado de Santa Catarina, Brasil

Rangelia vitalii is a haemoparasite that infects erythrocytes, white blood cells and the cytoplasm of endothelial cells of blood capillaries of canids in South America, and has been detected in both domestic dogs and sylvatic canids. Hepatozoon canis is a parasite that infects neutrophils and monocytes of many mammalian hosts. This study reports the infection of Lycalopex gymnocercus from Santa Catarina, Brazil, with R. vitalii and H. canis. The piroplasm was observed on both blood smears and molecular tests. Many large piroplasms were detected inside the erythrocytes, with round, oval, or teardrop-shaped organism, that occurred singly or in pairs. They had an abundant, pale blue cytoplasm and decentral dark red small nucleus. The animal was also infected with H. canis that was detected only by molecular tests. The majority of haematological and biochemistry parameters were within the reference values for domestic dog and wild canids.(AU)

Rangelia vitalii é um hemoparasita que infecta eritrócitos, macrófagos e células endoteliais de canídeos na América do Sul, e vem sendo detectado tanto em cães domésticos quanto em canídeos silvestres. Hepatozoon canis é um parasita que infecta monócitos e neutrófilos de mamíferos. No presente estudo, é descrita a infecção de Lycalopex gymnocercus, proveniente de Santa Catarina, Brasil, por R. vitalii e H. canis. O piroplasma foi diagnosticado nos esfregaços sanguíneos e por técnicas moleculares. Nos eritrócitos foram observados vários merozoítos grandes, ovais, arredondados ou em forma de gota, ocorrendo isoladamente ou em pares. Estes piroplasmas apresentavam citoplasma abundante, corado em azul claro, com núcleo pequeno, avermelhado e descentralizado. O animal apresentou coinfecção com H. canis, que foi diagnosticado somente pelos testes moleculares. A maior parte dos parâmetros hematológicos e bioquímicos do animal estava dentro dos valores de referência para cães domésticos e canídeos silvestres.(AU)

Rangelia vitalii and Hepatozoon canis coinfection in pampas fox Lycalopex gymnocercus from Santa Catarina State, Brazil / Infecção por Rangelia vitalii e Hepatozoon canis em Lycalopex gymnocercus proveniente do Estado de Santa Catarina, Brasil

Abstract Rangelia vitalii is a haemoparasite that infects erythrocytes, white blood cells and the cytoplasm of endothelial cells of blood capillaries of canids in South America, and has been detected in both domestic dogs and sylvatic canids. Hepatozoon canis is a parasite that infects neutrophils and monocytes of many mammalian hosts. This study reports the infection of Lycalopex gymnocercus from Santa Catarina, Brazil, with R. vitalii and H. canis. The piroplasm was observed on both blood smears and molecular tests. Many large piroplasms were detected inside the erythrocytes, with round, oval, or teardrop-shaped organism, that occurred singly or in pairs. They had an abundant, pale blue cytoplasm and decentral dark red small nucleus. The animal was also infected with H. canis that was detected only by molecular tests. The majority of haematological and biochemistry parameters were within the reference values for domestic dog and wild canids.

Resumo Rangelia vitalii é um hemoparasita que infecta eritrócitos, macrófagos e células endoteliais de canídeos na América do Sul, e vem sendo detectado tanto em cães domésticos quanto em canídeos silvestres. Hepatozoon canis é um parasita que infecta monócitos e neutrófilos de mamíferos. No presente estudo, é descrita a infecção de Lycalopex gymnocercus, proveniente de Santa Catarina, Brasil, por R. vitalii e H. canis. O piroplasma foi diagnosticado nos esfregaços sanguíneos e por técnicas moleculares. Nos eritrócitos foram observados vários merozoítos grandes, ovais, arredondados ou em forma de gota, ocorrendo isoladamente ou em pares. Estes piroplasmas apresentavam citoplasma abundante, corado em azul claro, com núcleo pequeno, avermelhado e descentralizado. O animal apresentou coinfecção com H. canis, que foi diagnosticado somente pelos testes moleculares. A maior parte dos parâmetros hematológicos e bioquímicos do animal estava dentro dos valores de referência para cães domésticos e canídeos silvestres.

First report and description of a Cyrilia sp. (Apicomplexa: Haemogregarinidae) from a freshwater Cururu Stingray Potamotrygon cf. histrix (Elasmobranchii: Potamotrygonidae), from the Amazon Region, Brazil.

A haemogregarine is described in 12 cururu stingray (Potamotrygon cf. histrix), from Mariuá Archipelago, Negro River, in the Brazilian Amazon Basin. All animals, both male and female, were parasitized by the haemogregarine and parasitaemia varied between 0.8% and 10% of erythrocytes. The stages observed included trophozoites or merozoites, suspected meronts, and gamonts presumed to be of two types, macrogamonts and microgamonts. Most stages were observed inside mature erythrocytes, while others were extracellular. The stages observed were most similar to those characteristics of the genus Cyrilia, than to any other fish haemogregarine and may represent a new Cyrilia species.

Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF) of T. b. brucei: (i) fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes) and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme) in glycosomes, (ii) enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes) and a GAPDH isoenzyme in the cytosol, (iii) malate dehydrogenase in cytosol and (iv) glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD+/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.