Blood parasites (Trypanosoma, Leucocytozoon, Haemoproteus) in the Eurasian sparrowhawk (Accipiter nisus): diversity, incidence and persistence of infection at the individual level.

BACKGROUND: A high prevalence of parasites may result from life-long persistence of infection or from high reinfection rates. We have studied blood parasites in a breeding population of the accipitrid raptor, Eurasian sparrowhawk (Accipiter nisus), to determine parasite diversity and turnover. METHODS: During this 7-year study, 210 adult Eurasian sparrowhawks breeding in the city of Prague were checked for parasites using several diagnostic methods. RESULTS: In both female and male raptors, parasites of the genus Leucocytozoon were the most prevalent (92% and 85%, respectively) followed in decreasing order of prevalence by those of genus Trypanosoma (74% and 68%, respectively) and genus Haemoproteus (46% and 16%, respectively). The prevalence of all parasites increased with age in both sexes, with the females at each respective age having the higher prevalence. There was a positive association between Haemoproteus and Leucocytozoon infections. Persistence at the individual level was higher than incidence for Trypanosoma and Haemoproteus. In the case of Leucocytozoon and Trypanosoma, most individuals probably become infected in their first year of life or even before dispersal from the nest. The detected parasites belonged to Trypanosoma avium sensu stricto, Leucocytozoon sp. (haplotypes ACNI1 and ACNI3) and Leucocytozoon mathisi (haplotype ACNI4) and two new lineages of the Haemoproteus elani complex (ACCNIS6 and ACCNIS7). Detailed analysis of parasite lineages in individuals that were repeatedly sampled revealed lineage turnover that would otherwise remain hidden. Phylogenetic analysis revealed that the detected Haemoproteus belongs to a phylogenetically distant group whose taxonomic position requires further analysis. CONCLUSIONS: All three genera of blood parasites persist in infected individuals, thus enabling sustainability of vector transmission cycles. Prevalence increases with age; however, there is a high turnover of Leucocytozoon lineages. No clear evidence of parasite-induced mortality was found, and most of the individuals were infected early in life, particularly in the case of Leucocytozoon.

Targeted Mass Spectrometry Analysis of Clostridium perfringens Toxins.

Targeted proteomics recently proved to be a technique for the detection and absolute quantification of proteins not easily accessible to classical bottom-up approaches. Due to this, it has been considered as a high fidelity tool to detect potential warfare agents in wide spread kinds of biological and environmental matrices. Clostridium perfringens toxins are considered to be potential biological weapons, especially the epsilon toxin which belongs to a group of the most powerful bacterial toxins. Here, the development of a target mass spectrometry method for the detection of C. perfringens protein toxins (alpha, beta, beta2, epsilon, iota) is described. A high-resolution mass spectrometer with a quadrupole-Orbitrap system operating in target acquisition mode (parallel reaction monitoring) was utilized. Because of the lack of commercial protein toxin standards recombinant toxins were prepared within Escherichia coli. The analysis was performed using proteotypic peptides as the target compounds together with their isotopically labeled synthetic analogues as internal standards. Calibration curves were calculated for each peptide in concentrations ranging from 0.635 to 1101 fmol/µL. Limits of detection and quantification were determined for each peptide in blank matrices.

Kinetics of antibody response in BALB/c and C57BL/6 mice bitten by Phlebotomus papatasi.

BACKGROUND: Phlebotomine sand flies are blood-sucking insects transmitting Leishmania parasites. In bitten hosts, sand fly saliva elicits specific immune response and the humoral immunity was shown to reflect the intensity of sand fly exposure. Thus, anti-saliva antibodies were suggested as the potential risk marker of Leishmania transmission. In this study, we examined the long-term kinetics and persistence of anti-Phlebotomus papatasi saliva antibody response in BALB/c and C57BL/6 mice. We also tested the reactivity of mice sera with P. papatasi salivary antigens and with the recombinant proteins. METHODOLOGY/PRINCIPAL FINDINGS: Sera of BALB/c and C57BL/6 mice experimentally bitten by Phlebotomus papatasi were tested by ELISA for the presence of anti-saliva IgE, IgG and its subclasses. We detected a significant increase of specific IgG and IgG1 in both mice strains and IgG2b in BALB/c mice that positively correlated with the number of blood-fed P. papatasi females. Using western blot and mass spectrometry we identified the major P. papatasi antigens as Yellow-related proteins, D7-related proteins, antigen 5-related proteins and SP-15-like proteins. We therefore tested the reactivity of mice sera with four P. papatasi recombinant proteins coding for most of these potential antigens (PpSP44, PpSP42, PpSP30, and PpSP28). Each mouse serum reacted with at least one of the recombinant protein tested, although none of the recombinant proteins were recognized by all sera. CONCLUSIONS: Our data confirmed the concept of using anti-sand fly saliva antibodies as a marker of sand fly exposure in Phlebotomus papatasi-mice model. As screening of specific antibodies is limited by the availability of salivary gland homogenate, utilization of recombinant proteins in such studies would be beneficial. Our present work demonstrates the feasibility of this implementation. A combination of recombinant salivary proteins is recommended for evaluation of intensity of sand fly exposure in endemic areas and for estimation of risk of Leishmania transmission.

Trypanosoma culicavium sp. nov., an avian trypanosome transmitted by Culex mosquitoes.

A novel avian trypanosome, Trypanosoma culicavium sp. nov., isolated from Culex mosquitoes, is described on the basis of naturally and experimentally infected vectors and bird hosts, localization in the vector, morphological characters and molecular data. This study provides the first comprehensive description of a trypanosome species transmitted by mosquitoes, in which parasites form plugs and rosettes on the stomodeal valve. Trypanosomes occurred as long epimastigotes and short trypomastigotes in vectors and culture and as long trypomastigotes in birds. Transmission of parasites to bird hosts was achieved exclusively by ingestion of experimentally infected Culex mosquito females by canaries (Serinus canaria), but not by Japanese quails (Coturnix japonica), nor by the bite of infected vectors, nor by ingestion of parasites from laboratory cultures. Transmission experiments and the identity of isolates from collared flycatchers (Ficedula albicollis) and Culex mosquitoes suggests that the natural hosts of T. culicavium are insectivorous songbirds (Passeriformes). Phylogenetic analyses of small-subunit rRNA and glycosomal glyceraldehyde-3-phosphate dehydrogenase gene sequences demonstrated that T. culicavium sp. nov. is more related to Trypanosoma corvi than to other avian trypanosomes (e.g. Trypanosoma avium and Trypanosoma bennetti).

Biodiversity of avian trypanosomes.

We have studied the biodiversity of trypanosomes from birds and bloodsucking Diptera on a large number of isolates. We used two molecular approaches, random amplification of polymorphic DNA (RAPD) method, and sequence analysis of the small subunit ribosomal RNA (SSU rRNA) gene. RAPD method divided the isolates into 11 separate lineages. Phylogenetic analysis of the SSU rRNA gene was congruent with the RAPD. Morphometric analysis of kinetoplast width and cell length was in agreement with molecular data. Avian trypanosomes appeared polyphyletic on SSU rDNA tree; thus, they do not represent a taxonomic group. We propose that all lineages recovered by SSU analysis probably represent distinct species of avian trypanosomes. We discuss possible transmission ways and geographical distribution of new avian trypanosome lineages. Finally, we recommend methods that should be used for species determination of avian trypanosomes.

Reproductive anatomy and fecundity estimation of the haematophagous ectoparasite Carnus hemapterus.

The dynamics of host-parasite interactions depends to a large extent on the effect of host responses on parasite fitness. An increased research effort is currently being invested in the study of host influence on parasite fitness both at a population and at an individual level even though basic information (e.g. the reproductive anatomy of parasites) is frequently missing. Here, we study for the first time the reproductive system of the diptera Carnus hemapterus, a 2-mm long, highly mobile haematophagous fly parasitizing nestlings of a broad variety of bird species. Although this species is poorly known, it is being increasingly used for the study of host-parasite interactions. We also assess the reliability of a method to estimate fecundity based on the number of laid eggs per gravid female and analyse the effect of body size on fecundity estimates. Our results show that carnid flies are synovigenic, so that both the number of laid eggs at a given moment and the egg load represent only a fraction of the true potential fecundity. Moreover, laid eggs are probably a fraction of the number of mature eggs ready to be laid since females withheld seemingly mature eggs at oviposition. A high proportion of pregnant females did not lay eggs, and the number of eggs laid per pregnant female varied remarkably. The latter is explained partly by a positive relationship with body size (thorax length and abdomen width). Caution is needed when using the number of laid eggs as a shortcut estimation of fecundity in C. hemapterus. We propose some improvements to the method for assessing Carnus fertility.

Targeted metabolomic analysis of plasma samples for the diagnosis of inherited metabolic disorders.

Metabolomics has become an important tool in clinical research and diagnosis of human diseases. In this work we focused on the diagnosis of inherited metabolic disorders (IMDs) in plasma samples using a targeted metabolomic approach. The plasma samples were analyzed with the flow injection analysis method. All the experiments were performed on a QTRAP 5500 tandem mass spectrometer (AB SCIEX, U.S.A.) with electrospray ionization. The compounds were measured in a multiple reaction monitoring mode. We analyzed 50 control samples and 34 samples with defects in amino acid metabolism (phenylketonuria, maple syrup urine disease, tyrosinemia I, argininemia, homocystinuria, carbamoyl phosphate synthetase deficiency, ornithine transcarbamylase deficiency, nonketotic hyperglycinemia), organic acidurias (methylmalonic aciduria, propionic aciduria, glutaric aciduria I, 3-hydroxy-3-methylglutaric aciduria, isovaleric aciduria), and mitochondrial defects (medium-chain acyl-coenzyme A dehydrogenase deficiency, carnitine palmitoyltransferase II deficiency). The controls were distinguished from the patient samples by principal component analysis and hierarchical clustering. Approximately 80% of patients were clearly detected by absolute metabolite concentrations, the sum of variance for first two principle components was in the range of 44-55%. Other patient samples were assigned due to the characteristic ratio of metabolites (the sum of variance for first two principle components 77 and 83%). This study has revealed that targeted metabolomic tools with automated and unsupervised processing can be applied for the diagnosis of various IMDs.

Herpetomonas trimorpha sp. nov. (Trypanosomatidae, Kinetoplastida), a parasite of the biting midge Culicoides truncorum (Ceratopogonidae, Diptera).

Monoxenous trypanosomatid Herpetomonas trimorpha sp. nov. was isolated from the digestive tract of the biting midge Culicoides truncorum (Ceratopogonidae, Diptera). This species forms three distinct morphotypes in culture: the microflagellate promastigote, the small promastigote and the long promastigote. The last form is unique for the newly described species. Phylogenetic analyses of SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase genes showed that H. trimorpha sp. nov. is the closest relative of Herpetomonas ztiplika, another monoxenous trypanosomatid isolated from biting midges. However, morphological and randomly amplified polymorphic DNA analyses confirmed that H. trimorpha sp. nov. is distinct from H. ztiplika.

Cutaneous leishmaniasis caused by Leishmania infantum transmitted by Phlebotomus tobbi.

Transmission of cutaneous leishmaniasis (CL) caused by Leishmania infantum was studied in South Anatolia, Turkey. Small, non-ulcerating lesions prevailed and patients were negative in rK39 tests for antibody detection for human visceral leishmaniasis (VL). The most abundant sand fly species, Phlebotomus tobbi, was found positive for Leishmania promastigotes with a prevalence of 1.4% (13 out of 898 dissected females). The isolated strains were identical with those obtained from patients with CL and were typed as L. infantum. Phylogenetic analysis revealed similarity to MON-188 and a clear difference from the MON-1 clade. Blood-meal identification showed that P. tobbi feeds preferentially on cattle and humans. This finding, the high number of CL patients and relative scarcity of dogs in the focus, suggests that the transmission cycle could be anthroponotic.

Sergeia podlipaevi gen. nov., sp. nov. (Trypanosomatidae, Kinetoplastida), a parasite of biting midges (Ceratopogonidae, Diptera).

Three strains of a trypanosomatid protozoan were isolated from the midguts of two naturally infected species of biting midges [Culicoides (Oecacta) festivipennis and Culicoides (Oecacta) truncorum] and characterized by light and electron microscopy and by molecular techniques. Morphological characteristics and sequences of the 18S rRNA, 5S rRNA, spliced leader RNA and glycosomal glyceraldehyde-3-phosphate dehydrogenase genes indicate that the studied flagellates represent a novel phylogenetic lineage within the Trypanosomatidae. Based on phylogenetic analyses, the novel endosymbiont-free, monoxenous trypanosomatid was classified as Sergeia podlipaevi gen. nov., sp. nov. Interestingly, it is closely related to another trypanosomatid species that parasitizes the sand fly Lutzomyia evansi, a blood-sucking dipteran from South America. The type strain of S. podlipaevi sp. nov., ICUL/CZ/2000/CER3, was obtained from Malpighian tubes. Of 2518 females of seven species of biting midges trapped in the Czech Republic, more than 1.5 % were infected by trypanosomatid parasites. An unrelated insect species, Culicoides (Monoculicoides) nubeculosus, was experimentally infected with S. podlipaevi, demonstrating that its host range extends to different subgenera of biting midges.

In vitro stimulation of penetration gland emptying by Trichobilharzia szidati and T. regenti (Schistosomatidae) cercariae. Quantitative collection and partial characterization of the products.

Induction of penetration gland emptying by cercariae of the bird schistosomes Trichobilharzia szidati and T. regenti employing linoleic acid, linolenic acid, praziquantel and calcium ionophore A23187 showed that both postacetabular and circumacetabular cells released their content at chosen stimulant concentrations. The gland secretions consisted of soluble and insoluble parts. The former one adhering to the ground seemed to have different saccharide composition from the glands of Schistosoma mansoni. It bound labelled saccharides, thus exhibiting lectin-like activity. Protein profiles of the latter one were identical after stimulation by all four stimulants in T. szidati. The soluble secretions contained several proteolytic enzymes; 31 kDa and 33 kDa cysteine proteases were identified in E/S products of T. szidati and T. regenti, respectively. The circumacetabular glands contained a significant amount of calcium. Immunohistochemistry revealed that the origin of E/S products after in vitro stimulation is in both penetration glands and tegumental structures. No crossreactivity was observed between the bird schistosomes and a serum raised against S. mansoni elastase.