In-depth comparative analysis of Tritrichomonas foetus transcriptomics reveals novel genes linked with adaptation to feline host.

Tritrichomonas foetus is a flagellated parasite able to infect cattle, cats, and pigs. Despite its prevalence, feline tritrichomonosis has received markedly less attention than venereal infection, and little information about the molecular mechanisms that participate in feline host infection is available. Through a bioinformatics approach, we integrated public transcriptomic data for three T. foetus isolates and explored the differences at transcript level with a focus on pathogenesis and adaptation processes, particularly for the feline isolate. Our analysis revealed higher abundance levels of predicted virulence factors, such as proteases and surface antigens. Additionally, by a comparative and expression analysis of T. foetus genes, we proposed putative virulence factors that could be involved in feline infection. Finally, we identified a great proportion of predicted transcription factors of the MYB protein family and, by a promoter analysis, we revealed that MYB-related proteins could participate in the regulation of gene transcription in T. foetus. In conclusion, this integrated approach is a valuable resource for future studies of host-pathogen interactions and identifying new gene targets for improved feline tritrichomonosis diagnosis and treatment.

Sex-specific effects of parasites on telomere dynamics in a short-lived passerine-the blue tit.

Parasitic infections potentially drive host's life-histories since they can have detrimental effects on host's fitness. Telomere dynamics is a candidate mechanism to underlie life-history trade-offs and as such may correlate with observed fitness reduction in infected animals. We examined the relationship of chronic infection with two genera of haemosporidians causing avian malaria and malaria-like disease with host's telomere length (TL) in a longitudinal study of free-ranging blue tits. The observed overall infection prevalence was 80% and increased with age, constituting a potentially serious selective pressure in our population. We found longer telomeres in individuals infected with a parasite causing lesser blood pathologies i.e. Haemoproteus compared to Plasmodium genus, but this only held true among males. Female TL was independent of the infection type. Our results indicate that parasitic infections could bring about other types of costs to females than to males with respect to TL. Additionally, we detected linear telomere loss with age, however a random regression analysis did not confirm significant heterogeneity in TL of first breeders and telomere shortening rates in further life.

Expression of infection-related immune response in European sea bass (Dicentrarchus labrax) during a natural outbreak from a unique dinoflagellate Amyloodinium ocellatum.

In the Mediterranean area, amyloodiniosis represents a major hindrance for marine aquaculture, causing high mortalities in lagoon-type based rearing sites during warm seasons. Amyloodinium ocellatum (AO) is the most common and important dinoflagellate parasitizing fish, and is one of the few fish parasites that can infest several fish species living within its ecological range. In the present study, A. ocellatum was recorded and collected from infected European sea bass (Dicentrarchus labrax) during a summer 2017 outbreak in north east Italy. Histological observation of infected ESB gill samples emphasized the presence of round or pear-shaped trophonts anchored to the oro-pharingeal cavity. Molecular analysis for small subunit (SSU) rDNA of A. ocellatum from gill genomic DNA amplified consistently and yielded 248 bp specific amplicon of A. ocellatum, that was also confirmed using sequencing and NCBI Blast analysis. Histological sections of ESB gill samples were addressed to immunohistochemical procedure for the labelling of ESB igm, inos, tlr2, tlr4, pcna and cytokeratin. Infected gills resulted positive for igm, inos, pcna and cytokeratin but negative to tlr-2 and tlr-4. Furthermore, ESB immune related gene response (innate immunity, adaptive immunity, and stress) in the course of A. ocellatum infection using quantitative polymerase chain reaction (qpcr) for infected gills and head kidney was analysed. Among the twenty three immune related gene molecules tested, cc1, il-8, il-10, hep, cox-2, cla, cat, casp9, and igt were significantly expressed in diseased fish. Altogether, these data on parasite identification and expression of host immune-related genes will allow for a better understanding of immune response in European sea bass against A. ocellatum and could promote the development of effective control measures.

Differential expression of microRNAs in Portunus trituberculatus in response to Hematodinium parasites.

Latest studies have indicated that microRNAs (miRNAs) play important roles in defending against bacterial and viral infections in marine crustacean, whereas little is known regarding the immunological roles of crustacean miRNAs in response to parasitic infection. To further reveal the host-parasite interactions between the parasitic dinoflagellate Hematodinium and its crustacean hosts, we applied the high-throughput sequencing technology to identify and characterize miRNAs in the Chinese swimming crab Portunus trituberculatus challenged with the Hematodinium parasite at a timescale of 16 days (d). A total of 168 miRNAs were identified and 51 miRNAs were differentially expressed in the hepatopancreas tissues of affected hosts. Eleven of the differentially expressed miRNAs were selected and verified by the quantitative real-time RT-PCR (qRT-PCR), manifesting the consistency between the high throughout sequencing and qRT-PCR assays. Further analysis of the putative target genes indicated that various immune-related pathways (e.g. endocytosis, Fc gamma R-mediated phagocytosis, lysosome, ECM-receptor interaction, complement and coagulation cascades, antigen processing and presentation, focal adhesion, etc.) and signal transduction pathways (e.g. JAK-STAT signaling pathway, MAPK signaling pathway, p53 signaling pathway, etc.) were mediated by the differentially expressed miRNAs. The results presented fundamental knowledge on the immunological roles of crustacean miRNAs and contributed to the better understanding of hosts' miRNAs-mediated immunity against the parasitic infection.

The secreted Theileria annulata Ta9 protein contributes to activation of the AP-1 transcription factor.

Theileria annulata is an obligate intracellular protozoan parasite of the phylum Apicomplexa. Theileria sporozoites invade bovine leukocytes and develop into a multinucleate syncytial macroschizont that causes uncontrolled proliferation and dissemination of infected and transformed leukocytes. Activator protein 1 (AP-1) is a transcription factor driving expression of genes involved in proliferation and dissemination and is therefore a key player in Theileria-induced leukocytes transformation. Ta9 possesses a signal peptide allowing it to be secreted into the infected leukocyte cytosol and be presented to CD8 T cells in the context of MHC class I. First, we confirmed that Ta9 is secreted into the infected leukocyte cytosol, and then we generated truncated versions of GFP-tagged Ta9 and tested their ability to activate AP-1 in non-infected HEK293T human kidney embryo cells. The ability to activate AP-1-driven transcription was found to reside in the C-terminal 100 amino acids of Ta9 distant to the N-terminally located epitopes recognised by CD8+ T cells. Secreted Ta9 has therefore, not only the ability to stimulate CD8+ T cells, but also the potential to activate AP-1-driven transcription and contribute to T. annulata-induced leukocyte transformation.

Perkinsus infection is associated with alterations in the level of global DNA methylation of gills and gastrointestinal tract of the oyster Crassostrea gasar.

Bivalves are filter feeders that obtain food from seawater that may contain infectious agents, such as the protozoan parasites Perkinsus marinus and P. olseni that are associated with massive mortalities responsible for losses in the aquaculture industry. Despite all physical and chemical barriers, microorganisms cross epithelia and infect host tissues to cause pathologies. Epigenetics mechanisms play important roles in a variety of human processes, from embryonic development to cell differentiation and growth. It is currently emerging as crucial mechanism involved in modulation of host-parasite interactions and pathogenesis, promoting discovery of targets for drug treatment. In bivalves, little is known about epigenetic mechanism in host parasite interactions. The objective of the present study was to evaluate the effect of Perkinsus sp. infections on DNA methylation levels in tissues of Crassostrea gasar oysters. Samples were collected in 2015 and 2016 in the Mamanguape River estuary (PB). Oyster gills were removed and used for Perkinsus sp. DIAGNOSIS: Gills (G) and gastrointestinal tract (GT), as well as cultured P. marinus trophozoites were preserved in 95% ethanol for DNA extractions. DNA methylation levels were estimated from G and GT tissues of uninfected (n=60) and infected oysters (n=60), and from P. marinus trophozoites, by ELISA assays. Results showed that the mean prevalence of Perkinsus sp. infections was high (87.3%) in 2015 and moderate (59.6%) in 2016. DNA methylation levels of G and GT tissues were significantly lower in infected oyster than in uninfected oysters, suggesting that infections are associated with hypomethylation. Methylation level was significantly higher in G than in GT tissues, indicating a likely tissue-specific mechanism. P. marinus trophozoites showed 33% methylation. This was the first study that confirms alterations of DNA methylation in two tissues of C. gasar oysters in association with Perkinsus sp. infections.

Pale and dark morphs of tawny owls show different patterns of telomere dynamics in relation to disease status.

Parasites are expected to exert long-term costs on host fecundity and longevity. Understanding the consequences of heritable polymorphic variation in disease defence in wild populations is essential in order to predict evolutionary responses to changes in disease risk. Telomeres have been found to shorten faster in malaria-diseased individuals compared with healthy ones with negative effects on longevity and thereby fitness. Here, we study the impact of haemosporidian blood parasites on telomere dynamics in tawny owls, which display a highly heritable plumage colour polymorphism. Previously, it has been shown that blood parasites have morph-specific impact on body mass maintenance. Here, we show that telomeres shortened faster in individuals with shorter breeding lifespan. Telomere length was negatively associated with the degree of pheomelanic brown coloration and shorter in infected than uninfected individuals. The rate of telomere shortening between breeding seasons was faster in darker pheomelanic individuals and suppression of parasite intensity between seasons was associated with faster telomere shortening in the paler individuals but not in darker ones. We propose that morph-specific physiological profiles cause differential telomere shortening and that this is likely to be a mechanism involved in previously documented environment-driven survival selection against the pheomelanic morph in this population.

MYD88 and functionally related genes are associated with multiple infections in a model population of Kenyan village dogs.

The purpose of this study was to seek associations between immunity-related molecular markers and endemic infections in a model population of African village dogs from Northern Kenya with no veterinary care and no selective breeding. A population of village dogs from Northern Kenya composed of three sub-populations from three different areas (84, 50 and 55 dogs) was studied. Canine distemper virus (CDV), Hepatozoon canis, Microfilariae (Acantocheilonema dracunculoides, Acantocheilonema reconditum) and Neospora caninum were the pathogens studied. The presence of antibodies (CDV, Neospora), light microscopy (Hepatozoon) and diagnostic PCR (Microfilariae) were the methods used for diagnosing infection. Genes involved in innate immune mechanisms, NOS3, IL6, TLR1, TLR2, TLR4, TLR7, TLR9, LY96, MYD88, and three major histocompatibility genes class II genes were selected as candidates. Single nucleotide polymorphism (SNP) markers were detected by Sanger sequencing, next generation sequencing and PCR-RFLP. The Fisher´s exact test for additive and non-additive models was used for association analyses. Three SNPs within the MYD88 gene and one TLR4 SNP marker were associated with more than one infection. Combined genotypes and further markers identified by next generation sequencing confirmed associations observed for individual genes. The genes associated with infection and their combinations in specific genotypes match well our knowledge on their biological role and on the role of the relevant biological pathways, respectively. Associations with multiple infections observed between the MYD88 and TLR4 genes suggest their involvement in the mechanisms of anti-infectious defenses in dogs.

Amyloodinum ocellatum in Dicentrarchus labrax: Study of infection in salt water and freshwater aquaponics.

This investigation is aimed to improve the knowledge on the physiological alterations occurring at morphological and molecular level in European sea bass naturally infected by A. ocellatum and reared at different salinities. European sea bass juveniles (Dicentrarchus labrax) weighing 20 ± 0.5 g were divided in three aquaponics systems: CTRL, reared at 20 ppt salinity; AFI, reared in freshwater (0 ppt) and infected with the dinoflagellate Amyloodinium ocellatum; ASI, reared at 20 ppt salinity and infected with A. ocellatum. Beta vulgaris plants were introduced in each of the aquaponic systems. Temperature was increased 1 °C every second day from 18 to 25 °C during the experiment. At the end of the trial, liver, brain, intestine and gills were sampled for molecular and histological analyses. A. ocellatum affected D. labrax growth (insulin-like growth factor I, IGF-I) and appetite (Neuropeptide Y, NPY) signals in ASI. Immune system was activated in ASI by the presence of parasites by producing higher levels of Interleukin-1 (IL-1) and Tumor Necrosis Factor α (TNFα). Peroxisome proliferator-activated receptor α (PPAR α), codifying for a protein involved in lipid metabolism, was upregulated in ASI because of the necessity to produce energy to maintain homeostasis. On the contrary, A. ocellatum did not cause signs of infection in AFI as confirmed by gene expression and histological analysis, that were similar to CTRL. However, in freshwater reared fish, a modification of lipid metabolism was observed through a reduction in PPARα gene expression and hepatic lipid content.

Extreme heterogeneity in parasitism despite low population genetic structure among monarch butterflies inhabiting the Hawaiian Islands.

Host movement and spatial structure can strongly influence the ecology and evolution of infectious diseases, with limited host movement potentially leading to high spatial heterogeneity in infection. Monarch butterflies (Danaus plexippus) are best known for undertaking a spectacular long-distance migration in eastern North America; however, they also form non-migratory populations that breed year-round in milder climates such as Hawaii and other tropical locations. Prior work showed an inverse relationship between monarch migratory propensity and the prevalence of the protozoan parasite, Ophryocystis elektroscirrha. Here, we sampled monarchs from replicate sites within each of four Hawaiian Islands to ask whether these populations show consistently high prevalence of the protozoan parasite as seen for monarchs from several other non-migratory populations. Counter to our predictions, we observed striking spatial heterogeneity in parasite prevalence, with infection rates per site ranging from 4-85%. We next used microsatellite markers to ask whether the observed variation in infection might be explained by limited host movement and spatial sub-structuring among sites. Our results showed that monarchs across the Hawaiian Islands form one admixed population, supporting high gene flow among sites. Moreover, measures of individual-level genetic diversity did not predict host infection status, as might be expected if more inbred hosts harbored higher parasite loads. These results suggest that other factors such as landscape-level environmental variation or colonization-extinction processes might instead cause the extreme heterogeneity in monarch butterfly infection observed here.

Differential haemoparasite intensity between black sparrowhawk (Accipiter melanoleucus) morphs suggests an adaptive function for polymorphism.

Recent research suggests that genes coding for melanin based colouration may have pleiotropic properties, in particular conveying raised immune function. Thus adaptive function of polymorphism may be associated with parasite resistance. The black sparrowhawk Accipiter melanoleucus is a polymorphic raptor with two morphs. Over most of its range the light morph is commonest, however within the recently colonised Western Cape of South Africa the dark morph predominates. The species breeds in winter throughout South Africa, however unlike in the rest of the species' South African range, the Western Cape experiences a winter rainfall regime, where arthropod vectors which transmit haematozoan parasites may be more abundant. We hypothesise that the higher frequency of dark morph birds in this region may be due to their improved parasite resistance, which enables them to cope with higher parasite pressure. If so, we predict that dark morph black sparrowhawks would have lower parasite burdens than light morph birds. Within our population the prevalence of the two most common haematozoan parasites was high, with 72% of adults infected with Haemoproteus nisi and 59% of adults infected with Leucocytozoon toddi. We found no difference in prevalence for either parasite between adult morphs, or between chicks of different parental morphs. However, within adults infected with H. nisi, infection intensity was significantly higher in light morphs than dark morphs. This suggests that dark morphs have lower parasite loads than light morphs due to resistance rather than morph-specific habitat exploitation. Greater resistance to Haemoproteus parasites may therefore be one of the mechanisms through which dark morph black sparrowhawks have a selective advantage in this region and may explain why they are most common in our study area. In other regions, the cost to benefit ratio may be in favour of the light morph, where parasites are less abundant or virulent.

Comparison of hatchery and field performance between a whirling-disease-resistant strain and the Ten Sleep strain of rainbow trout.

A whirling-disease-resistant strain of rainbow trout Oncorhynchus mykiss (GRHL strain) derived from a backcross of an F1 hybrid of two strains (German strain x Harrison Lake strain) with German strain females, was compared with the Ten Sleep (TS) strain of rainbow trout. The GRHL strain had consistently superior growth and feed conversion in two consecutive hatchery trials. Hatching and mortality rates were similar between strains. Both strains were stocked into two Utah reservoirs (Hyrum, Porcupine), and a third, Causey Reservoir, was monitored as a control for seasonal variation in prevalence of Myxobolus cerebralis. A total of 1,323 salmonids captured by gill net in spring and fall sampling between 2006 and 2008 were tested for M. cerebralis via pepsin-trypsin digest methods. Only eight of these (< 1% per species) had clinical signs consistent with whirling disease. In both reservoirs, GRHL survived better than the TS and had higher growth rates. The prevalence of M. cerebralis was significantly lower for GRHL (18.1%) than TS (50.0%) in Porcupine Reservoir. In Hyrum Reservoir the trend was similar, but prevalence was lower and did not significantly differ between GRHL (9.6%) and TS (23.1%). For infected fish, no significant differences were observed between strains in myxospore counts in either Hyrum (GRHL = 911-28,244 spores/fish [spf], TS = 1,822-155,800 spf) or Porcupine (GRHL = 333-426,667spf, TS = 333-230,511 spf) reservoirs. Unmarked rainbow trout in both reservoirs had significantly higher myxospore counts than stocked fish of either strain. There were significant differences in M. cerebralis prevalence and myxospore loads among other naturally reproducing salmonids in the reservoirs. The trend in susceptibility was cutthroat trout Oncorhynchus clarkii > kokanee Oncorhynchus nerka > brown trout Salmo trutta. The GRHL performed well in both hatchery and field settings and is recommended for stocking programs.

Bonamia exitiosa (Haplosporidia) observed infecting the European flat oyster Ostrea edulis cultured on the Spanish Mediterranean coast.

Bonamia exitiosa and Bonamia ostreae are parasites that reproduce within the haemocytes of several oyster species. In Europe, the host species is the flat oyster Ostrea edulis. The parasite B. ostreae has been responsible for mortalities since the late 1970s throughout the European Atlantic coast. B. exitiosa was first detected, in 2007, on this continent in flat oysters cultured in Galicia (NW Spain). Since then, the parasite has also been detected in France, Italy and the United Kingdom. The bays of the Ebro Delta in the south of Catalonia represent the main bivalve culture area in the Mediterranean coast of Spain. Previous information from the area includes reports of several flat oyster pathogens, including the notifiable parasite Marteilia refringens. However, the status with regard to Bonamia parasites was uncertain. In the present study, a Bonamia parasite was observed in flat oysters cultured in the Alfacs Bay of the Ebro Delta by histology and real-time PCR. PCR-RFLP and sequencing suggested the presence of B. exitiosa. Finally, phylogenetic analyses of the studied Bonamia isolates corroborated B. exitiosa infection. M. refringens was also observed in the same oyster batch, and co-infection with both parasites was also detected. This is the first detection of B. exitiosa, in Catalonia and the Spanish Mediterranean coast. The impact of the parasite on the Mediterranean flat oyster activity needs to be urgently addressed.

Diversity of avian haemosporidians in arid zones of northern Venezuela.

Arid zones of northern Venezuela are represented by isolated areas, important from an ornithological and ecological perspective due to the occurrence of restricted-range species of birds. We analysed the prevalence and molecular diversity of haemosporidian parasites of wild birds in this region by screening 527 individuals (11 families and 20 species) for parasite mitochondrial DNA. The overall prevalence of parasites was 41%, representing 17 mitochondrial lineages: 7 of Plasmodium and 10 of Haemoproteus. Two parasite lineages occurred in both the eastern and western regions infecting a single host species, Mimus gilvus. These lineages are also present throughout northern and central Venezuela in a variety of arid and mesic habitats. Some lineages found in this study in northern Venezuela have also been observed in different localities in the Americas, including the West Indies. In spite of the widespread distributions of some of the parasite lineages found in northern Venezuela, several, including some that are relatively common (e.g. Ven05 and Ven06), have not been reported from elsewhere. Additional studies are needed to characterize the host and geographical distribution of avian malaria parasite lineages, which will provide a better understanding of the influence of landscape, vector abundance and diversity, and host identity on haemosporidian parasite diversity and prevalence.

Redescription and molecular analysis of Myxobolus shantungensis Hu, 1965 (Myxozoa: Myxosporea) infecting common carp Cyprinus carpio haematopterus.

Myxobolus shantungensis Hu, 1965 infects gill arches of common carp Cyprinus carpio haematopterus (Temminck and Schlegel), causing serious pathological effects on host fish. An inadequate original description and absence of molecular data make accurate early diagnosis challenging. To augment the original description, M. shantungensis is redescribed here using morphological and molecular biological methods. Mature spores of M. shantungensis were ellipsoidal or apple shaped in frontal view and lemon shaped in lateral view, averaging 8.2 ± 0.3 µm (8.0-9.0 µm) × 10.1 ± 0.5 µm (9.2-11.1 µm) × 6.9 ± 0.3 µm (6.0-7.4 µm). Some spores had three to four "V"-shaped valve edge markings on the posterior of the spore. The two equal polar capsules were oval, measuring 4.3 ± 0.3 µm (3.8-5.0 µm) × 3.2 ± 0.2 µm (2.8-3.5 µm), situated at the anterior extremity of the spore. Polar filaments coiled with six to seven turns. Scanning electron microscopy revealed that the surface of mature spores of M. shantungensis was generally pitted with a number of irregular ridges in shape. M. shantungensis is also characterized on the molecular level using the sequence of the small subunit ribosomal RNA gene. A BLAST search revealed that this sequence did not match any available sequences in GenBank.

A fresh look at the origin of Plasmodium falciparum, the most malignant malaria agent.

From which host did the most malignant human malaria come: birds, primates, or rodents? When did the transfer occur? Over the last half century, these have been some of the questions up for debate about the origin of Plasmodium falciparum, the most common and deadliest human malaria parasite, which is responsible for at least one million deaths every year. Recent findings bring elements in favor of a transfer from great apes, but are these evidences really solid? What are the grey areas that remain to be clarified? Here, we examine in depth these new elements and discuss how they modify our perception of the origin and evolution of P. falciparum. We also discuss the perspectives these new discoveries open.

Molecular responses of Ostrea edulis haemocytes to an in vitro infection with Bonamia ostreae.

Bonamiosis due to the parasite Bonamia ostreae is a disease affecting the flat oyster Ostrea edulis. B. ostreae is a protozoan, affiliated to the order of haplosporidia and to the cercozoan phylum. This parasite is mainly intracellular, infecting haemocytes, cells notably involved in oyster defence mechanisms. Suppression subtractive hybridisation (SSH) was carried out in order to identify oyster genes differentially expressed during an infection of haemocytes with B. ostreae. Forward and reverse banks allowed obtaining 1104 and 1344 clones respectively, among which 391 and 480 clones showed a differential expression between both tested conditions (haemocytes alone versus haemocytes in contact with parasites). ESTs of interest including genes involved in cytoskeleton, respiratory chain, detoxification membrane receptors, and immune system were identified. The open reading frames of two selected genes (galectin and IRF-like) were completely sequenced and characterized. Real time PCR assays were developed to study the relative expression of candidate ESTs during an in vitro infection of haemocytes by live and dead parasites. Haemocyte infection with B. ostreae induced an increased expression of omega glutathione S-transferase (OGST), superoxide dismutase (SOD), tissue inhibitor of metalloproteinase (TIMP), galectin, interferon regulatory factor (IRF-like) and filamin genes.

Observation of a Bonamia sp. infecting the oyster Ostrea stentina in Tunisia, and a consideration of its phylogenetic affinities.

The small non-commercial oyster Ostrea stentina co-occurs with commercially important Ostrea edulis in the Mediterranean Sea, yet its disposition with respect to the destructive pathogens Bonamia ostreae and Marteilia refringens is unknown. We began an evaluation of the Bonamia spp. infection status of O. stentina from Hammamet, Tunisia, in June 2007 using polymerase chain reaction diagnostics followed by histology and in situ hybridization. Of 85 O. stentina sampled, nine were PCR-positive for a Bonamia sp. using a Bonamia genus-specific assay; of these nine, one displayed the uninucleate microcells associated with oyster hemocytes characteristic of Bonamia spp. There was no associated pathology. DNA sequencing of the parasite from this one infected individual revealed it to be of a member of the Bonamia exitiosa/Bonamia roughleyi clade, an identification supported by positive in situ hybridization results with probes specific for members of this clade, and by the morphology of the parasite cells: nuclei were central, as in B. exitiosa, not eccentric, as in B. ostreae. There is no basis for identifying the Tunisian parasite as either B. exitiosa or B. roughleyi, however, as these species are genetically indistinguishable. Likewise, there is no basis for identifying any of the other Bonamia spp. with affinities to the B. exitiosa/B. roughleyi clade, from Argentina, Australia, Spain, and the eastern USA, as one or the other of these named species. Though they are clearly distinct from Bonamia perspora and B. ostreae, justification for drawing species boundaries among the primarily austral microcells with affinities to B. exitiosa and B. roughleyi remains elusive.

Molecular epizootiology of canine hepatozoonosis in Croatia.

An epizootiological survey was conducted to investigate the prevalence of hepatozoonosis in a population of 924 apparently asymptomatic dogs from different regions of Croatia. DNA was isolated from canine blood and screening PCR on the 666 bp fragment of 18S rRNA revealed that 108 (11.8%) of dogs were infected. Positive samples were confirmed by partial sequencing of the 18S rRNA gene. The consensus sequences, derived from various sequence data sets, were compared with sequences of 18S ssrRNA of Hepatozoon spp. available in GenBank. The alignments revealed 106 Hepatozoon canis and two Hepatozoon sp. sequences. Among H. canis isolates, we found a certain amount of heterogeneity, while both Hepatozoon sp. isolates were identical to the Spanish isolate (Accession No. AY600625) from Clethrionomys glareolus. On the basis of eight commonly mutated nucleotide positions in the partial 18S rRNA gene sequence, we divided the H. canis isolates into five groups. The results obtained indicate a higher prevalence and significance of hepatozoonosis in Croatia than previously believed and demonstrate that the organisms belonging to H. canis that infect European dogs are genetically very heterogeneous.

Phenotypic differences on the outcome of the host-parasite relationship: behavior of mice of the CBi stock in natural and experimental infections.

Investigation of defined animal models may help to elucidate the role of the host genetic background in the development and establishment of a parasitic infection. Four lines of mice obtained by disruptive selection for body conformation (CBi+, CBi-, CBi/C and CBi/L) and the unselected control line CBi were examined in their response to different parasites to assess whether these distinct genotypes showed differences in their resistance to natural and experimental parasitosis. Protozoans (Trichomonas muris and Spironucleus muris) and nemathelminths (Syphacia obvelata and Aspiculurus tetraptera) were found naturally parasitizing the mice's intestines. CBi/C and CBi were the only genotypes in which T. muris was found. CBi- was least resistant to S. muris. The helminth parasitic burden showed differences between sexes within genotypes (males had a higher burden than females) and among genotypes (CBi/L males had the lowest burden). CBi/L animals were also most resistant to experimental challenge with Heligmosomoides polygyrus and Trypanosoma cruzi. Since all the animals examined shared a common habitat throughout the study and were equally exposed to infection, the phenotypic differences in the natural enteroparasitism herein described evince genetic differences among lines in the host-parasite relationship. This interpretation is further supported by the differences in the response to the experimental challenge to H. polygyrus and T. cruzi.