Molecular epidemiology of parasitic protozoa and Ehrlichia canis in wildlife in Madrid (central Spain).

Wildlife species are involved in the transmission of diverse pathogens. This study aimed to monitor raccoons (Procyon lotor), American minks (Neovison vison), and red foxes (Vulpes vulpes) as potential reservoirs in central Spain. Specifically, 200 spleen and fecal samples (from 194 raccoons, 3 minks, and 3 foxes) were analyzed molecularly by PCR/qPCR and sequencing for the presence of piroplasmids, Hepatozoon spp., Toxoplasma gondii, and Ehrlichia canis infections in the Community of Madrid (Spain). Biological samples were obtained in the years 2014, 2015, and 2016. No pathogen DNA was found in fecal samples. In contrast, analysis of raccoon spleen samples revealed that Toxoplasma was the most prevalent pathogen (prevalence 3.6 ± 2.6%), followed by Hepatozoon canis and E. canis (each with a prevalence of 2.57 ± 2.2%). Hepatozoon canis was also diagnosed in all three of the analyzed foxes. Analysis of yearly prevalence showed that tick-borne pathogens were less frequent in raccoon in 2015, a dry and warm year compared both to 2014 and 2016. These data suggest that fecal PCR assays are unsuitable for detection of DNA of non-erythrocytic pathogens. Furthermore, they demonstrate that the raccoon (an invasive species often living in proximity to domestic areas) and the red fox are putative reservoirs for pathogenic organisms in the Community of Madrid.

Development and optimization of new culture media for Acanthamoeba spp. (Protozoa: Amoebozoa).

The isolation and growth in axenic liquid media of Acanthamoeba strains is necessary in order to carry out primary in vitro drug screening. Amoebic isolates which are hard to grow in the current liquid media have been reported. Such circumstances hampers the ability of conducting drug sensitivity tests. Therefore, finding suitable universal growth media for Acanthamoeba species is required. The present study was aimed at the development of liquid medium suitable for growing a fastidious (F) genotype T3 Acanthamoeba isolate, and eventually for other genotypes of this genus as well. Trophozoite growth was indirectly monitored by respiration analysis with oxygen-sensitive microplates (OSM) and further confirmed by manual counting. Media were empirically designed and tested first in a non-fastidious (NF) T3 isolate and then tested with 14 different strains, including the fastidious one. Combinations of nutritive components such as meat/vegetable broth, LB medium, malt and skimmed milk led to the design of new media suitable for culturing all the isolates tested, in conditions similar to those obtained in standard culture media such as PYG or CERVA.

Nest ecology of blood parasites in the European roller and its ectoparasitic carnid fly.

Haemosporidian parasites are considered the most important vector-borne parasites. However, vector identity and ecology is unknown for most such host-vector-parasite systems. In this study, we employ microscopic and molecular analyses to examine haemosporidian prevalence in a migratory, cavity-nesting bird, European roller Coracias garrulus, and its nidicolous blood-feeding ectoparasite Carnus hemapterus. This system is unique in that the ectoparasite is confined to a near-closed environment, in contrast to the free-wandering system of haematophagous dipterans such as mosquitoes. Blood film analysis confirms previous works in that Haemoproteus parasites are widely prevalent in adult rollers and belong to a single species, Haemoproteus coraciae. Leucocytozoon sp. and Trypanosoma sp. also are detected in adult rollers at low intensities with this technique. By means of molecular analysis, we report for the first time Plasmodium sp. presence in C. garrulus. Based on PCR results, Plasmodium parasites are relatively less prevalent than Haemoproteus parasites (20% vs. 31%) in rollers. In contrast, haemosporidian prevalences show the opposite trend for Carnus flies: Plasmodium sp. occurrence (62%) clearly predominates over that of Haemoproteus sp. (5%). A comparison between roller and Carnus samples reveals a significantly higher prevalence of Plasmodium sp. in Carnus samples. Insect survey and phylogenetic analysis suggest Culicoides flies as Haemoproteus sp. vectors, which appear to readily transmit the parasite in southern Spain. This study does not find support for Carnus flies to serve as biological or mechanical vectors of haemosporidians. In spite of this, nidicolous blood-feeding ectoparasites, such as carnid flies, appear as a suitable model for studies on the occurrence and temporal dynamics of avian haemosporidians such as Plasmodium sp. present at low intensities.

A molecular survey of Piroplasmida and Hepatozoon isolated from domestic and wild animals in Burgos (northern Spain).

This study reports a molecular survey of Hepatozoon species and of the order Piroplasmida in the Province of Burgos, northern Spain. The diagnostic techniques employed included PCR and the sequencing of the 18S rRNA gene. Eighty-nine blood samples from domestic animals plus 138 blood/coagulated blood samples from wild mammals were examined. Theilerid protozoa were found at relatively high frequencies in bovines (14.6%) and horses (36%). Theileria buffeli, T. sergenti and T. annulata were diagnosed in cows. T. equi was common in horses and T. annae was found in a donkey for the first time. A new piroplasmid was found in the European badger (20%). This appears to be distantly related to both T. annae and a piroplasmid isolated from Lontra canadensis. A moderate prevalence (14%) for T. annae was recorded in red foxes. A species of hepatozoon was found in one bank vole (17%), while 28% of the red foxes examined were found to be infected with H. canis. Twenty-five wild house mice were studied and found not to be carriers of piroplasmids or Hepatozoon species. Wild boars, roe deer, hares, Apodemus sp. and moles were also negative for haematozoan infection. The present study indicates that piroplasmid protozoa are present at a low to moderate frequency in some domestic herbivores in the Burgos area. They also infect certain wild mammalian species, which may act as zoonotic carriers.

Parasitological and molecular characterization of a venezuelan isolate of babesia canis / Caracterización parasitológica y molecular de un aislado venezolano de Babesia canis

Babesiosis is one of the most important tick-borne hemoprotozoan diseases that affects dogs worldwide. In the last years, there has been a sharp increase in positive cases of Babesia canis infection in the Falcon State, Venezuela. Although recent studies conducted in South America suggest that B. canis vogeli might be the species present in Venezuela, no data on the pathogenesis and molecular characteristics of Venezuelan piroplasms are available at this moment. By this reason the present work was undertaken to describe the evolution of an experimental infection induced in dogs, as well as the first molecular characterization of the 18S rRNA gene of the Venezuelan B. canis isolate used for inoculating the experimental animals. Experimental infection was carried out in four dogs, two of them splenectomized. The animals were inoculated with 1 x 10(6) B. canis infected-red blood cells. Parasitemia, packed cell volume and body temperature were measured daily. Prepatent period of the infection oscillated between 2-6 days. All of the inoculated dogs developed a mild-type disease. Symptoms of canine piroplasmosis were moderate, and main clinical findings consisted in low parasitemia, short febrile period and slight anaemia. Imidocarb dipropionate showed to be the treatment of choice, since it cleared parasites from blood, as demonstrated by PCR assays. The partial sequence of the ssu rRNA gene of the Venezuelan piroplasm showed 100% identity with a Brazilian isolate of Babesia canis vogeli, which is in full concordance with the clinical signs caused by this subspecies in the experimental animals, as well as those reported previously by other authors.

La babesiosis, causada por un hemoparásito trasmitido a través de las garrapatas, es una de las enfermedades más importantes que afectan a los caninos mundialmente. En los últimos años se ha observado un aumento abrupto de los casos positivos a Babesia canis en el estado Falcón, Venezuela. Aunque los estudios recientes realizados en Suramérica sugieren que Babesia canis vogeli pudiera ser la especie presente en Venezuela, no existen datos disponibles sobre la patogénesis y las características moleculares de los piroplasmas venezolanos. Por esta razón se realizó el presente estudio, a fin de describir la evolución de una infección experimental inducida en caninos, así como realizar la primera caracterización molecular del gen 18S ARNr del aislado venezolano de Babesia canis utilizado para inocular los animales experimentales. La infección experimental se llevó a cabo en cuatro caninos, dos de ellos esplenectomizados. Los animales fueron inoculados 1 x 10(6) eritrocitos infectados con Babesia canis. La parasitemia, el hematocrito y la temperatura corporal fueron evaluados diariamente. El período prepatente de la infección osciló entre 2 y 6 días. Todos los caninos inoculados desarrollaron una enfermedad leve. Los síntomas de la babesiosis fueron moderados y los principales hallazgos consistieron en baja parasitemia, período febril corto y anemia leve. Se demostró, a través de las pruebas de PCR, la efectividad del dipropionato de imidocarb como tratamiento de elección para la eliminación de los parásitos sanguíneos. La secuencia parcial de la ssu rARN del piroplasma venezolano mostró 100% de identidad con un aislado brasileño de Babesia canis vogeli, lo que coincide plenamente con los síntomas causados por esta subespecie en los animales experimentales, así como por lo reportado previamente por otros autores.

Molecular characterization of the 18S rDNA gene of an avian Hepatozoon reveals that it is closely related to Lankesterella.

As a part of intensive study of blood parasite infections in a population of the passerine bird blue tit (Cyanistes caeruleus, Paridae), we detected a parasite species that, based on its morphological similarity, was tentatively identified as Hepatozoon parus, the only species of this parasite genus described from birds of this family. However, morphological measurements show that H. parus is slightly larger than the parasite detected in our population. A molecular characterization of the parasite species was conducted by amplification of the 18S rDNA gene, using primers that were reported previously to amplify in Hepatozoon sp. of water pythons. Additional primers were developed based on the new sequence obtained. The 1,484-bp fragment amplified reveals that the parasite from our bird population is more closely related to Lankesterella minima than to Hepatozoon species from other vertebrates according to analysis using the BLAST comparison method (93% identity). In addition, phylogenetic analyses using parsimony and Kimura procedures unequivocally related the parasite species detected by PCR with L. minima. The bootstrap values obtained were 97% and 100%, respectively. These results imply that this parasite is a species of a lankesterellid instead of Hepatozoon.

A new pentastomid from the black vulture.

This article describes a new pentastomid species from the abdominal air sacs of a black vulture (Aegypius monachus Linnaeus, 1766) from central Spain. The parasite's morphological characteristics (as shown by light and scanning electron microscopy) suggest that it should be classified in the new genus. It is the third pentastomid species described in birds and the first for the Accipitridae. The mouth is almost terminal, there are 2 pairs of hooks behind the mouth, and the genital pore is immediately posterior to these structures, placing the new species within the Cephalobaenida. The anterior and posterior hooks are similar in size and are flanked by parapodial lob. Cuticular tubercles are absent, false annulations can be seen, and the parasite's eggs have 2 layers.