Sarcocystis spp. diversity in the roe deer (Capreolus capreolus) from Lithuania and Spain.

The roe deer (Capreolus capreolus) has been identified as an intermediate host for six known Sarcocystis species, S. capreolicanis, S. entzerothi, S. gracilis, S. linearis, S. oviformis, and S. silva. In this study, we identified Sarcocystis species in the diaphragm and tongue muscles from the Lithuanian and Spanish roe deer, respectively, on the basis of a microscopic examination and DNA analysis. A total of 43 and 27 sarcocysts were isolated and characterized from the Lithuanian and Spanish roe deer, respectively. Overall six Sarcocystis species were identified in roe deer from Lithuania, and only three of them, S. gracilis, S. linearis, and S. silva were found to have infecting animals from Spain. The current paper represents first molecular results of Sarcocystis species in the Spanish roe deer. Furthermore, transmission electron microscopy examination revealed specific wall structure of sarcocysts studied, S. linearis was characterized by ribbon-like villar protrusions (vp) (type 8a), and S. oviformis was distinguished by elongated vp resembling spades or mushroom-like structures (type 39). Based on 18S rDNA and cox1 sequences, Sarcocystis species from the roe deer showed considerable intraspecific genetic variability. However, similar values of intraspecific genetic variation were estimated at both genes analysed. The highest variability was observed for S. capreolicanis and S. linearis in both genes and for S. silva at cox1. Consequently, the level of genetic variability of Sarcocystis from the roe deer varied depending on species rather than on gene analysed or geographical area.

A comparative test of ixodid tick identification by a network of European researchers.

This study reports the results of a comparative test of identification of ticks occurring in Western Europe and Northern Africa. A total of 14 laboratories were voluntarily enrolled in the test. Each participant received between 22 and 25 specimens of adult and nymphal ticks of 11 species: Dermacentor marginatus, D. reticulatus, Haemaphysalis punctata, Hyalomma lusitanicum, Hy. marginatum, Ixodes ricinus, I. hexagonus, Rhipicephalus annulatus, R. bursa, R. rossicus, and/or R. sanguineus s.l. Ticks were morphologically identified by three of the co-authors and the identification confirmed by a fourth co-author who used molecular methods based on several genes. Then ticks were randomly selected and blindly distributed among participants, together with a questionnaire. Only specimens collected while questing and, if possible, in the same survey, were circulated. Because of the random nature of the test, a participant could receive several specimens of the same species. Species in the different genera had variable misidentification rates (MR) of 7% (Dermacentor), 14% (Ixodes), 19% (Haemaphysalis), 36% (Hyalomma), and 54% (Rhipicephalus). Within genera, the MR was also variable ranging from 5.4% for I. ricinus or 7.4% for D. marginatus or D. reticulatus to 100% for R. rossicus. The test provided a total misidentification rate of 29.6% of the species of ticks. There are no significant differences in MR according to the sex of the tick. Participants were requested to perform a second round of identifications on the same set of ticks, using only purposely prepared keys (without illustrations), circulated to the enrolled participants, including 2 species of the genus Dermacentor, 8 of Haemaphysalis, 10 of Hyalomma, 23 of Ixodes, and 6 of Rhipicephalus. The average MR in the second round was 28%: 0% (Dermacentor), 33% (Haemaphysalis), 30% (Hyalomma) 18% (Ixodes), and 50% (Rhipicephalus). Species which are not reported in the countries of a participating laboratory had always highest MR, i.e. purely Mediterranean species had highest MR by laboratories in Central and Northern Europe. Participants expressed their concerns about a correct identification for almost 50% of the ticks of the genera Hyalomma and Rhipicephalus. The results revealed less than total confidence in identifying the most prominent species of ticks in the Western Palearctic, and underpin the need for reference libraries for specialists involved in this task. Results also showed that a combination of certain genes may adequately identify the target species of ticks.

Characterization of attenuated Theileria annulata vaccines from Spain and the Sudan.

Theileriosis caused by Theileria annulata can be effectively prevented by vaccination with attenuated, cultured schizonts. Although these attenuated vaccines have been applied for a long time, not much is known about the fate of the vaccine strain in the field. Here, two experimental Spanish vaccine strains originating in Cádiz and Cáceres, and one Sudanese strain are studied to address the development of a carrier status and the infectivity for Hyalomma ticks. Moreover, the heterogeneity of the merozoite surface protein, Tams1, was analyzed in search for an attenuation marker. Using the sensitive reverse line blot (RLB) hybridization, the development of a low level carrier status was demonstrated in the Cáceres and Sudanese line vaccinated calves. Although no signal was detected in the Cádiz line vaccinated calves, seroconversion against the schizont stage was observed, as it was in all other calves. The experimental transmission of T. annulata by Hyalomma ticks to naïve calves was unsuccessful for all cell line inoculated calves. Tams1 heterogeneity indicated a clonal selection of parasites during the process of attenuation, but the Tams1 sequence itself has no connection with the attenuation status. In conclusion, a carrier status develops in attenuated schizont culture vaccinated calves, but is not infective for Hyalomma ticks. Based on these data, the risk for spread of the vaccine strains in the field may be very low.

Histopathological changes in sheep experimentally infected with Babesia ovis.

Histopathological study was made of 12 Merino sheep - five splenectomized and seven intact - experimentally infected with Babesia ovis. Non-purulent encephalitis; initially exudative and subsequently interstitial pneumonia; pericarditis, myocarditis and haemorrhagic endocarditis; centrilobular necrotic hepatitis; hyperplasia of the lymphoreticular system; necrosis and vascular changes in adrenal glands were observed. The kidney was the most severely affected organ, exhibiting acute tubular necrosis typical of kidney shock syndrome. The lesions observed were suggestive of hypovolemic shock culminating in haemorrhagic diathesis owing to consumptive coagulopathy. Additionally, the massive release of catabolites from lysis and necrosis apparently produced endotoxic shock.

Pathological changes in kidneys of dogs with natural Leishmania infection.

A study was made of the nephropathy in canine leishmaniasis produced in ten adult dogs naturally infected with Leishmania infantum. Renal function analyses were performed (uraemia, creatinaemia, plasma proteins, biochemistry and urinary sediment), the humoral immune response (fluorescent antibodies and levels of serum IgG, IgM and IgA) was assessed and histopathological studies were carried out. Correlation of the results showed acute renal insufficiency which was reversible in two animals (endotheliomesangial glomerulonephritis) and irreversible in four cases corresponding to glomerulonephritis in its Type I and Type II proliferative forms; extensive increase in the glomerular basal membrane, proliferation of mesangial cells and growth of the mesangial matrix were observed, as was a widespread incidence of immune complex deposits. Two animals showed chronic renal insufficiency. Lack of renal changes (minimal-changes glomerulonephritis) in two dogs was accounted for in one animal by an almost complete absence of symptoms and in the other by chronic viscerocutaneous symptoms; neither showed more than a slight immunoglobulin response.

Changes in the plasma concentrations of lipids and lipoprotein fractions in dogs infected with Leishmania infantum.

A study was made of serum concentrations of cholesterol, phospholipids, triglycerides, cholesterol bound to high-density lipoproteins (HDL), HDL1-cholesterol, HDL2-cholesterol and cholesterol bound to low-density lipoproteins (LDL) in 16 dogs naturally infected with Leishmania infantum (ZMON-1) taken from an endemic focus. Results were compared with those of a control group of ten healthy dogs. Statistically significant increases in cholesterol, triglyceride and LDL-cholesterol levels were observed. There was, however, a statistically significant decrease in HDL-cholesterol level, mainly at the expense of the HDL2-cholesterol subfraction. Cholesterol transport is therefore shown to undergo changes which may be attributed to the consumptive evolution of the disease, immunocomplex deposits in cells, hepatic disorders and interactions between the parasite and the normal cholesterol metabolism of the host.

A Case of Human Oral Myiasis by Lucilia sericata in a Hospitalized Patient in Extremadura, Spain.

Myiasis is the term used to describe infestations, both obligatory and accidental, in vertebrate animals and humans by dipteral larvae. The oral cavity is rarely affected by this infestation and the circumstances which can lead to oral myiasis include persistent mouth opening together with poor hygiene, or facial traumatism. We present a case of oral myiasis by larvae of Lucilia sericata, a species present in the Iberian Peninsula, in a hospitalized patient with surgical problems.

Theileria (Babesia) equi and Babesia caballi infections in horses in Galicia, Spain.

The control of equine piroplasmosis is becoming increasingly important to maintain the international market open to the horse industry. The purpose of this study was to demonstrate the occurrence of equine piroplasmosis (Theileria equi and Babesia caballi) in Galicia, north-west Spain, and to compare haematological and serum biochemistry parameters between non-parasitaemic horses and horses parasitaemic with T. equi and B. caballi. Sixty serum samples (control group) were taken from healthy horses pastured on two farms, and examined for evidence of equine T. equi and B. caballi infection by indirect fluorescent antibody test (IFAT). Of the 60 samples, 24 (40%) and 17 (28.3%) samples were positive for T. equi and B. caballi, respectively. Twelve (20%) samples were positive for both parasites. Haematology and serum biochemistry were compared between controls and a series of 36 horses clinically affected by T. equi (25) or B. caballi (11). Compared with the healthy group, there was a 43% and 37% decrease in the haematocrit for T. equi and B. caballi infection, respectively. Parasitaemic horses presented an intense anaemia and serum biochemistry signs of liver damage. The anaemia was more severe in T. equi-infected than in B. caballi-infected horses. Our results suggest that equine piroplasmosis is widespread in the region and is a cause for concern.

Detection of Theileria annulata in blood samples of carrier cattle by PCR.

We report the detection of Theileria annulata, the causative agent of tropical theileriosis, by PCR in blood samples obtained from carrier cattle. The assay employs primers specific for the gene encoding the 30-kDa major merozoite surface antigen of T. annulata. A 721-bp fragment was amplified from blood samples taken monthly from calves experimentally infected with one of four different stocks of T. annulata originating in either Mauritania, Portugal, Spain, or Turkey. At the end of the experiment, five animals carried the infection for 12 months and two animals remained infected for 15 months. DNAs from six other Theileria species, T. parva, T. mutans, T. sergenti, T. buffeli, T. velifera, and T. taurotragi, were not amplified. Moreover, DNAs from four other hemoparasites (Anaplasma centrale, Anaplasma marginale, Babesia bovis, and Babesia bigemina) were also not amplified. As a control, primers derived from the small subunit rRNA gene of Theileria spp. amplified a 1.1-kb DNA fragment from all Theileria species examined but not from the other four hemoparasites. As few as two to three parasites per microliter of infected blood in a 50-microliters sample volume were detected by Southern or microplate hybridization with a T. annulata-specific cDNA probe. In addition, 92 field samples obtained from cattle in Spain were tested; 22% were positive in blood smears, 40% were positive by immunofluorescent antibody test, and 75% were positive for T. annulata by PCR. The method provides a useful diagnostic tool for detecting T. annulata carrier cattle.