To cool or not to cool? Intestinal coccidians disrupt the behavioral hypothermia of lizards in response to tick infestation.

It is generally accepted that parasites exert negative effects on their hosts and that natural selection favors specific host responses that mitigate this impact. It is also known that some components of the host immune system often co-evolve with parasite antigens resulting in a host-parasite arms race. In addition to immunological components of the anti-parasitic response, host behavioral responses are also important in this arms race and natural selection may favor avoidance strategies that preclude contact with parasites, or shifts in the host's thermoregulatory strategy to combat active infections (e.g., behavioral fever). Ticks are widespread parasites with direct and indirect costs on their vertebrate hosts. Their saliva provokes hemolysis in the blood of their hosts and can transmit a plethora of tick-borne pathogens. We enquired whether tick infestation by Ixodes pacificus can provoke a thermoregulatory response in Sceloporus occidentalis. For this, we compared the thermoregulatory behavior of tick-infested lizards against tick-infested lizards co-infected with two different species of coccidians (Lankesterella occidentalis and Acroeimeria sceloporis). After this, lizards were kept in individual terraria with a basking spot and fed ad libitum. We found that tick-infested lizards sought cooler temperatures in proportion to their tick load, and this response was independent of the co-infection status by L. occidentalis. This was consistent in April and June (when tick loads were significantly lower) and suggests a conservative strategy to save energy which might have been selected to overcome tick infestations during phenological peaks of this parasite. However, this behavior was not observed in lizards co-infected with A. sceloporis, suggesting that co-infection with this intestinal parasite prompt lizards to be active. Cost of tick infestation was confirmed because housed lizards lost weight at a constant ratio to initial tick load, independently of other infections. The broader implications of these findings are discussed in the context of climate change.

Strategies to discover the structural components of cyst and oocyst walls.

Cysts of Giardia lamblia and Entamoeba histolytica and oocysts of Toxoplasma gondii and Cryptosporidium parvum are the infectious and sometimes diagnostic forms of these parasites. To discover the structural components of cyst and oocyst walls, we have developed strategies based upon a few simple assumptions. Briefly, the most abundant wall proteins are identified by monoclonal antibodies or mass spectrometry. Structural components include a sugar polysaccharide (chitin for Entamoeba, ß-1,3-linked glucose for Toxoplasma, and ß-1,3-linked GalNAc for Giardia) and/or acid-fast lipids (Toxoplasma and Cryptosporidium). Because Entamoeba cysts and Toxoplasma oocysts are difficult to obtain, studies of walls of nonhuman pathogens (E. invadens and Eimeria, respectively) accelerate discovery. Biochemical methods to dissect fungal walls work well for cyst and oocyst walls, although the results are often unexpected. For example, echinocandins, which inhibit glucan synthases and kill fungi, arrest the development of oocyst walls and block their release into the intestinal lumen. Candida walls are coated with mannans, while Entamoeba cysts are coated in a dextran-like glucose polymer. Models for cyst and oocyst walls derive from their structural components and organization within the wall. Cyst walls are composed of chitin fibrils and lectins that bind chitin (Entamoeba) or fibrils of the ß-1,3-GalNAc polymer and lectins that bind the polymer (Giardia). Oocyst walls of Toxoplasma have two distinct layers that resemble those of fungi (ß-1,3-glucan in the inner layer) or mycobacteria (acid-fast lipids in the outer layer). Oocyst walls of Cryptosporidium have a rigid bilayer of acid-fast lipids and inner layer of oocyst wall proteins.

Water-related parasitic diseases in China.

Water-related parasitic diseases are directly dependent on water bodies for their spread or as a habitat for indispensable intermediate or final hosts. Along with socioeconomic development and improvement of sanitation, overall prevalence is declining in the China. However, the heterogeneity in economic development and the inequity of access to public services result in considerable burden due to parasitic diseases in certain areas and populations across the country. In this review, we demonstrated three aspects of ten major water-related parasitic diseases, i.e., the biology and pathogenicity, epidemiology and recent advances in research in China. General measures for diseases control and special control strategies are summarized.

Survey and genetic characterization of wastewater in Tunisia for Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, Cyclospora cayetanensis and Eimeria spp.

The microbial diversity of wastewater used for irrigation and fertilization was assessed using specific polymerase chain reaction (PCR) assays to detect and genotype several pathogenic protists including Cryptosporidium spp., Giardia duodenalis, Cyclospora spp., Eimeria spp. and Enterocytozoon bieneusi. A total of 220 wastewater samples (110 raw, 110 treated) and 12 sludge samples were collected from 2005 to 2008 from 18 treatment plants located throughout Tunisia. Except for Cyclospora, which was detected only once, E. bieneusi (61%), G. duodenalis (28%), Cryptosporidium spp. (27%) and Eimeria spp. (45%) were frequently observed in wastewater and sludge. Sequencing of PCR products showed that C. hominis, C. andersoni, G. duodenalis sub-assemblage A-II and E. bieneusi genotypes D and IV were the most prevalent. An analysis of the distribution of 209 internal transcribed spacer sequences of E. bieneusi originating from wastewater at the 18 treatment plants showed a similar genetic diversity, regardless of the geographical location. The identification of these parasite species and genotypes and of host-specific Eimeria species indicates that the microbial quality of wastewater was impacted by humans, livestock and rodents. Given the public health risks that some of these parasites represent, guidelines on wastewater usage are needed to minimize human exposure to these pathogens.

[Development of a new hydrocarbon extract from the medicinal raw material of Circassian walnut (Juglans regia) and study of its antiparasitic activity].

The authors developed a technology for preparing a hydrocarbon extract from the medicinal raw material of Circassian walnut (Juglans regia), including its green fruits, green leaves, and fresh roots. To prepare the preparation, they obtained for the first time a new extragent called petroleum Russia that was found to contain more than hundred chemical compounds by chromatography mass spectrometry. The new agent was named irillen. Experiments on albino mice and albino rats established that the new agent was low toxic. The lethal doses of irillen were calculated: LD50 was 16377 +/- 457.5 mg/kg; LD16 = 12986.4 mg/kg; LD84 was 18976.6 mg/kg for albino mice; LD50 was 16998.0 +/- 535.4 mg/kg; LD16 = 12875.3 mg/ kg; LD84 = 18583.4 mg/kg for albino rats. The irillen prepared by the authors should be referred to as a low toxic and practically nontoxic agent (Toxicity Class IV and V). Irillen has a broad spectrum of antiparasitic activity. It is effective in treating toxocariasis in dogs, larval alveolar echinococcosis, ascaridiasis, and eimeriasis in chickens, and siphachiasis.

Assessment of the quality of multi-institutional data for population-based epidemiological studies: a case study of atoxoplasmosis in Bali mynahs (Leucopsar rothschildi).

To evaluate the potential to conduct population-based epidemiologic studies using multi-institutional data, the quality of data extracted from the MedARKS system was evaluated with regard to atoxoplasmosis in Bali mynahs (Leucopsar rothschildi). Data extracted for 338 Bali mynahs from 34 institutions were analyzed for completeness, correctness, and consistency and subjectively analyzed for accessibility. Data completeness was ascertained by analysis of missing records, tests, and blank fields. Two hundred and sixty four records had animal enclosure information missing, nine records were missing from the parasitology module, and 85 records did not include tests or results. Data correctness was assessed by evaluation of unclear and inaccurate results. From 2,432 parasitology records, 81 tests (3.3%) were not definitive because of uninterpretable entries. Data consistency was assessed by comparing the problem list to positive tests in the parasitology module and listing of buffy coat smears in the clinical pathology module. Overall, six different terms with 28 values were used to denote "positive for Atoxoplasma sp.," and a substantial number of discrepancies were found between problem lists, parasitology modules, and test findings among these data records. In general, this study showed that the data contained in a computer patient record (CPR) system for zoos has great promise for population-level studies if specific areas are addressed, including 1) reduced reliance on free-text data entry, 2) universal use of a standardized vocabulary, 3) use of methods to identify and track individual animals accurately and easily, 4) integration of data checks and maintenance methods, and 5) concerted use of "centralized" animal medical record information.

Morphologic, immunohistochemical, and molecular characterization of a novel Lankesterella protozoan in two White's tree frogs (Litoria caerulea).

Two White's tree frogs (Litoria caerulea) housed at a zoological park died after a short period of lethargy, weight loss, and edema. Detailed postmortem examinations were performed on both frogs, including bacterial cultures and complete histologic examinations. Intracytoplasmatic as well as free protozoan parasites were identified in multiple organs from both frogs. The parasites were identified within erythrocytes, leukocytes, endothelial cells, and hepatocytes. Immunohistochemistry demonstrated a cross-reaction with Toxoplasma gondii antisera. Parasite ultrastructural analysis was performed by transmission electron microscopy. The parasites demonstrated an apical complex containing a conoid, rhoptries, and micronemes, demonstrating it was a member of the phylum Apicomplexa. In addition, the parasites had bipolar paranuclear bodies, organelles that are typical of coccidian sporozoites. The organisms were tentatively identified as members of the genus Lankesterella on the basis of histologic and ultrastructural morphology. A portion of the 18s ribosomal RNA (rRNA) gene was amplified via a polymerase chain reaction, sequenced, and used in a Basic Local Alignment Search Tool search of the GenBank database. The 18s rRNA gene sequence was found to be most similar to gene sequences isolated from Lankesterella organisms (88%). In aggregate, these data support the classification of these protozoa as a novel species of Lankesterella. A causal relationship between frog morbidity and protozoal parasitism was not determined. This is the first report of Lankesterella sp. in White's tree frogs.

Detection of some intestinal protozoa in commercial fresh juices.

Fresh fruit juices are popular, but not always safe. For assessing the likelihood of infection with newly emerging intestinal protozoa, commercial fresh orange, lemon, sugar cane, strawberry, and mango juices were screened by wet mounts, Weber's modified trichrome and modified Ziehl-Neelsen stains. Protozoa viability was done by fluorescein-diacetate/propidium-iodide staining, and infectivity was performed in Swiss albino mice. Results showed that 35.43% were contaminated with one or more of Cryptosporidia, Microsporidia, and Cyclospora, as well as Giardia spp. Strawberry was the most contaminated juice (54.28%), while orange was the slightest (22.86%). Cryptosporidia was the highest contaminant (61.29%), and Cyclospora was the least (14.52%). Microsporidia spp. was the most robust contaminant which retained its viability and infectivity in juices in which it was detected. Moderately acidic strawberry and mango juices and alkaline sugar cane juice pose a possible threat, due to harboring the highest viable and infectious protozoa. Regarding highly acidic juices, viability and infectivity decreased in lemon, yet was not still risk free. Orange juice was comparatively safe, as viability dramatically declined, while infectivity was completely abolished. Hence consumers, especially high risk group, are placed at hazard of contracting intestinal protozoa infections, especially through moderately acidic and alkaline juices.

Gastrointestinal parasites of Howler monkeys (Alouatta palliata) inhabiting the fragmented landscape of the Santa Marta mountain range, Veracruz, Mexico.

In recent years populations of howler monkeys (Alouatta palliata) in southeastern Mexico have decreased substantially due to the transformation and loss of natural habitats. This is especially evident in the Santa Marta mountain range, Veracruz, Mexico where several studies have evaluated the impact of fragmentation on howler monkey populations in order to propose management programs for their conservation. The conditions generated by fragmentation likely change the rates of parasitic infection and could decrease howler survival. In this study, gastrointestinal parasite species richness, prevalence, and egg density of infection were determined in howler groups inhabiting five forest fragments at the Santa Marta mountain range. Two hundred and seventy-eight fresh fecal samples were collected between October 2002 and April 2003. Three parasite species were found during the dry and the wet season in all forest fragments sampled: one unidentified species of Eimeriidae; Trypanoxyuris minutus (Oxyuridae); and Controrchis biliophilus (Dicrocoeliidae). Both the prevalence of T. minutus and infection density for all parasites differed between seasons and fragments (the largest fragment consistently differed from other fragments). Host density, distance to the nearest town, fragment size, fragment shape, and total basal area of food trees explained parasite prevalence, but each species had a different pattern. Although parasite richness was lower, prevalence and density were higher than values reported for howlers in conserved forests. These results suggest that the establishment of biological corridors and animal translocation programs must take into account the parasite ecology of each fragment to avoid higher infection rates and preclude potential consequent mortality.

[Coccidia (Eimeriida) from fishes of the continental waters of Russia].

Data on the Coccidia parasitizing fishes of 12 orders (Acipenseriformes, Clupeiformes, Salmoniformes, Anguilliformes, Siluriformes, Atheriniformes, Gadiformes, Gasterosteiformes, Syngnatiformes, Mugiliformes, Scorpaeniformes, and Pleuronectiformes) are given. Authors and year of description, synonymy, morphofunctional organization of exogenic stages, and list of hosts are presented for each coccidian species.

Dynamics of transcription of immunomodulatory genes in endothelial cells infected with different coccidian parasites.

Sporozoites of Eimeria bovis and tachyzoites of Neospora caninum and Toxoplasma gondii are able to invade and to replicate in endothelial cells. Here we report on responses of bovine umbilical vein endothelial cells (BUVEC) in vitro to these coccidial infections by determining mRNA levels of the CXC chemokines GRO-alpha, IL-8 and IP-10, the CC chemokines MCP-1 and RANTES and of GM-CSF, COX-2 and iNOS relative to the level of housekeeping gene (GAPDH) transcription. T. gondii and N. caninum tachyzoites caused profound transcriptional upregulation of all genes in question. In general, upregulation started 2-4 h p.i. and maximum transcript levels were observed 4 h p.i. GRO-alpha and IL-8 gene transcription had decreased to almost control levels by 12 h p.i.; in the case of the other chemokines enhanced transcript levels persisted longer or showed a biphasic time-course. A similar time-course to CC chemokines was observed for GM-CSF mRNA, whilst COX-2 gene transcript peaks were detected at 2-4 h p.i. and 48-72 h p.i. iNOS mRNA levels increased from 4 to 48 h p.i. In contrast, E. bovis sporozoites failed to induce the transcription of CXC chemokine genes and of COX-2, and only caused moderate transcription upregulation of the other genes considered. In conclusion, infections of BUVEC with these coccidian parasites result in host cell activation associated with enhanced transcription of genes encoding for proinflammatory and immunomodulatory molecules, which are important for innate immune reactions and the transition to adaptive immunity. Differences between E. bovis versus T. gondii and N. caninum may illustrate a particular evasion strategy of E. bovis sporozoites, which is related to their need to persist in the host cell for a long period of time and to the avoidance of inflammatory process-induction.

Anticoccidial kinase inhibitors: identification of protein kinase targets secondary to cGMP-dependent protein kinase.

Trisubstituted pyrrole inhibitors of the essential coccidian parasite cGMP dependent protein kinase (PKG) block parasite invasion and show in vivo efficacy against Eimeria in chickens and Toxoplasma in mice. An imidazopyridine inhibitor of PKG activity with greater potency in both parasite invasion assays and in vivo activity has recently been identified. Susceptibility experiments with a Toxoplasma knock-out strain expressing a complementing compound-refractory PKG allele ('T761Q-KO'), suggest a role for additional secondary protein kinase targets. Using extracts from this engineered T. gondii strain and a radiolabeled imidazopyridine ligand, a single peak of binding activity associated with calmodulin-like domain protein kinase (CDPK1) has been identified. Like PKG, CDPK1 has been implicated in host cell invasion and exhibits sub-nanomolar sensitivity to the compound. Amino acid sequence comparisons of coccidian CDPKs and a mutational analysis reveal that the binding of the ligand to PKG and CDPK1 (but not other CDPK isoforms) is mediated by similar contacts in a catalytic site hydrophobic binding pocket, and can be blocked by analogous amino acid substitutions. Transgenic strains over-expressing a biochemically active but compound-refractory CDPK1 mutant ('G128Q') fail to show reduced susceptibility to the compound in vivo, suggesting that selective inhibition of this enzyme is not responsible for the enhanced anti-parasitic potency of the imidazopyridine analog. An alternative secondary target candidate, the alpha-isoform of casein kinase 1 (CK1alpha), shows sensitivity to the compound in the low nanomolar range. These results provide an example of the utility of the Toxoplasma model system for investigating the mechanism of action of novel anticoccidial agents.

Studies on serological cross-reaction of Neospora caninum with Toxoplasma gondii and Hammondia heydorni.

The enzyme-linked immunosorbent assay (ELISA) was used to examine cross-reactivity of Neospora caninum with Toxoplasma gondii and Hammondia heydorni. Anti-T. gondii mouse and cat sera cross-reacted with N. caninum soluble antigen (NLA), but not with the recombinant surface antigen (NcSRS2). Anti-H. heydorni dog sera showed no cross-reactivity with either the NLA antigen or the NcSRS2. Lack of cross-reactivity between anti-H. heydorni sera and N. caninum antigens, and the cross-reactivity of anti-T. gondii sera with the NLA suggest that N. caninum has common antigens to T. gondii except for NcSRS2 based on serology. In light of several studies suggesting a closer relationship between N. caninum and H. heydorni than with T gondii, examination of serological cross-reactivity with N. caninum may be necessary to further classify the parasites in addition to molecular and morphological studies and clarification of the life cycle.

Neospora caninum and Hammondia heydorni are separate species/organisms.

Neospora caninum and Hammondia heydorni are two coccidian parasites with morphologically similar oocysts in canine feces. It was recently proposed that they are one species. In this paper, we review the biology and morphology of these parasites and present evidence that N. caninum and H. heydorni are separate species.

Further remarks on Hammondia hammondi and the taxonomic importance of obligate heteroxeny.

In this opinion-paper reasons are given why Hammondia hammondi cannot be considered as a separate species, but should be kept as a species of the genus Toxoplasma, if not a strain of Toxoplasma gondii.

Invasion of different cell types by sporozoites of Eimeria species and effects of monoclonal antibody 1209-C2 on invasion of cells by sporozoites of several apicomplexan parasites.

Sporozoites of avian Eimeria species differed markedly in their ability to invade cells in vitro. Invasion by E. tenella and E. adenoeides was significantly greater in baby hamster kidney (BHK) and chicken cecal cell (CC) cultures than in primary chicken (PCK) or turkey kidney (PTK) cell cultures. Moreover, invasion of BHK cell cultures by E. adenoeides was significantly greater than that of other Eimeria species, and invasion by E. acervulina sporozoites was significantly lower. Monoclonal antibody 1209-C2 (MAb 1209-C2) reacted by immunofluorescent labeling (IFA) with refractile bodies of sporozoites of 5 species of Eimeria and Caryospora bigenetica, but not with sporozoites of Toxoplasma gondii, Hammondia hammondi, or Cryptosporidium parvum, which have no refractile bodies. The MAb also cross-reacted with formalin-fixed BHK, CC, turkey cecal (TC) cells, and PTK. Pretreatment of BHK cells with MAb 1209-C2 significantly reduced invasion of the cells by sporozoites of E. tenella, E. acervulina, E. meleagrimitis, and C. bigenetica, but did not alter invasion by T. gondii, C. parvum, or H. hammondia. Apparently, reactivity of MAB 1209-C2 with the sporozoites was required for inhibition of invasion despite the fact that the inhibition resulted from pre-treatment of the host cell. Conversely, although MAb 1209-C2 also reacted moderately with PTK and TC cells, pre-treatment of these cell cultures with the MAb did not inhibit invasion by either MAB 1209-C2-reactive or -nonreactive parasites. Collectively, the data indicated that refractile body antigens of sporozoites of Eimeria and Caryospora, which are recognized by MAb 1209-C2, may function in cellular invasion, but also suggest that cellular invasion is probably not mediated by interactions between the conserved epitopes in sporozoites and cultured host cells that are recognized by the MAb.

The taxonomic importance of obligate heteroxeny: distinction of Hammondia hammondi from Toxoplasma gondii--another opinion.

We enumerate identical and divergent findings concerning the obligate heteroxenous Hammondia hammondi and the facultatively homoxenous or heteroxenous Toxoplasma gondii. Differences exist in life-cycles, transmission, and host range, especially transmissibility to birds and mammals other than rodents, in ultrastructural morphology, immunity and serology in cats and to lesser degree in rodents, in DNA sequences and in isoenzymes. Because the recognition of obligate heteroxeny is essential to study these organisms and to recognize them as taxa, it is advantageous to give heteroxeny a generic rather than a specific value. Characterization of organisms with the life-cycle patterns of Hammondia, Sarcocystis, Frenkelia, and Toxoplasma is best achieved by means of the genera presently used.

Neospora caninum: its oocysts and its identity: an opinion.

A recently published opinion that Hammondia heydorni, Neospora caninum, and N. hughesi are a single species is not supported by genetic comparisons. Furthermore, the suggestion that oocysts shed by dogs after experimental infection with N. caninum might have been of another organism is not consistent with the finding that these oocysts induced N. caninum infections in mice.

Immunohistochemical study of the cyst of Besnoitia besnoiti.

The present study has been undertaken in order to provide information on the molecular structure of the cysts of Besnoitia besnoiti. To that end, immunohistochemical techniques have been used to investigate the expression of several enzymes and proteins implicated in the cellular membrane permeability of bradyzoites. Paraffin and frozen sections, which were obtained from subcutaneous tissue samples taken from naturally infected cattle (coming from northeast Spain), were treated with a panel of antibodies. These were specific for Na(+), K(+)-ATPase, alkaline phosphatase, calmodulin, S100 protein, heat shock proteins, hsp60, and hsp70. Positive-cysts for the said antibodies were found in 23.3% of the cows studied. Bradyzoites showed a positive immunoreaction in every positive cyst with respect to all these antibodies. In addition to the low percentage of positive animals, it is worth noting that positive and unstained cysts were observed in the same tissue section. These results suggest that bradyzoites may pass through both active and dormant metabolic phases.