Unusual cryptosporidiosis cases in Swedish patients: extended molecular characterization of Cryptosporidium viatorum and Cryptosporidium chipmunk genotype I.

Most human cases of cryptosporidiosis are caused by Cryptosporidium parvum or Cryptosporidium hominis, but the use of molecular diagnostic methods has revealed that several other less common species or genotypes can also be involved. Here, we describe two unusual causes of cryptosporidiosis, one being the recently described species Cryptosporidium viatorum and the other Cryptosporidium chipmunk genotype I. Two Swedish patients who were infected with C. viatorum had travelled to Kenya and Guatemala, respectively, and two others had been infected with Cryptosporidium chipmunk genotype I in Sweden. None of these four patients were immunocompromised, and all four showed classical symptoms of cryptosporidiosis. We performed extensive molecular characterization, including analysis of four loci. The two C. viatorum isolates were found to differ slightly at the 70-kDa heat shock protein locus, which may indicate a local geographical variation in this species that has previously been described exclusively on the Indian subcontinent.

Zoonotic transmission of Cryptosporidium meleagridis on an organic Swedish farm.

We believe that we present the first evidence of zoonotic transmission of the bird parasite, Cryptosporidium meleagridis. Despite being the third most common cause of human cryptosporidiosis, an identified zoonotic source has not been reported to date. We found Cryptosporidium oocysts in pigs, sheep/goats, hens and broiler chickens on a farm with suspected zoonotic transmission. By DNA analysis we identified C. meleagridis in samples from one human, three chickens and one hen. Sequencing of the ssrRNA and 70kDa Heat Shock Protein (HSP) genes showed identical C. meleagridis sequences in the human and chicken samples, which is evidence of zoonotic transmission. The HSP70 sequence was unique.

Potential application of serological tests on fluids from carcasses: detection of antibodies against Toxoplasma gondii and Sarcoptes scabiei in red foxes (Vulpes vulpes).

BACKGROUND: Serological surveys for disease investigation of wild animal populations require obtaining blood samples for analysis, which has logistic, ethic and economic difficulties. Applying serological test to fluids collected from dead animals is an alternative. The aim of this study was to assess if antibodies could be detected in two types of fluids collected from 56 carcasses of red foxes (Vulpes vulpes): pleural fluid and lung extract. FINDINGS: In 22 (39%) foxes antibodies against Sarcoptes scabiei were detected in both fluid types by ELISA and Western blot. In 46 (82%) foxes, antibodies against Toxoplasma gondii were detected in pleural fluid and in 41 (73%) in lung extract applying a Toxo-screen test (DAT). Antibodies were still detectable in the same fluids kept at room temperature for 28 days, although in fewer foxes (16 and 14 foxes tested for T. gondii in lung extract and pleural fluid respectively; and 1 and 4 tested for S. scabiei in lung extract and pleural fluid respectively. CONCLUSIONS: These results indicate the potential utility of using fluids from carcasses for antibody screening of wild animals at the population level.

Global teaching and training initiatives for emerging cohort studies.

A striking disparity exists across the globe, with essentially no large-scale longitudinal studies ongoing in regions that will be significantly affected by the oncoming non-communicable disease epidemic. The successful implementation of cohort studies in most low-resource research environments presents unique challenges that may be aided by coordinated training programs. Leaders of emerging cohort studies attending the First World Cohort Integration Workshop were surveyed about training priorities, unmet needs and potential cross-cohort solutions to these barriers through an electronic pre-workshop questionnaire and focus groups. Cohort studies representing India, Mexico, Nigeria, South Africa, Sweden, Tanzania and Uganda described similar training needs, including on-the-job training, data analysis software instruction, and database and bio-bank management. A lack of funding and protected time for training activities were commonly identified constraints. Proposed solutions include a collaborative cross-cohort teaching platform with web-based content and interactive teaching methods for a range of research personnel. An international network for research mentorship and idea exchange, and modifying the graduate thesis structure were also identified as key initiatives. Cross-cohort integrated educational initiatives will efficiently meet shared needs, catalyze the development of emerging cohorts, speed closure of the global disparity in cohort research, and may fortify scientific capacity development in low-resource settings.

Cryptosporidium infection in herds with and without calf diarrhoeal problems.

A case-control study was designed to investigate the role of different Cryptosporidium spp. in Swedish dairy herds with and without calf diarrhoeal problems. Faecal samples were collected from preweaned calves, young stock and cows. Cryptosporidium oocysts were detected by sodium chloride flotation and epifluorescence microscopy. Molecular diagnostics were used to identify Cryptosporidium species. Samples containing C. parvum were further analysed to determine subtypes. Calf faecal samples were also analysed for rotavirus, coronavirus and Escherichia coli F5+. Total protein was assessed in 1- to 8-day-old calves. A questionnaire was used to identify differences in management routines. Cryptosporidium infection was diagnosed in all herds, with equal prevalence in case and control herds in all three age groups. Cryptosporidium parvum, Cryptosporidium bovis, Cryptosporidium ryanae and Cryptosporidium andersoni were all identified, as were rotavirus, coronavirus and E. coli F5+. C. ryanae and C. andersoni were only detected in non-diarrhoeal samples, whereas the other pathogens were detected in both diarrhoeal and non-diarrhoeal samples. Diarrhoea was more common in case herd calves. Disinfection of single pens was more common in case herds and several other management routines seemed to differ although results were not significant.

Molecular characterisation of Cryptosporidium isolates from Swedish dairy cattle in relation to age, diarrhoea and region.

Cryptosporidium positive samples from 176 preweaned calves, young stock and cows of 48 herds were subjected to molecular characterisation of the 18S rRNA gene to determine which species are present in Swedish dairy cattle. In addition, samples characterised as Cryptosporidium parvum were further analysed at the GP60 gene to investigate distribution and zoonotic potential of subtypes. The 18S rRNA gene was successfully sequenced in 110 samples, with Cryptosporidium bovis in 83, C. parvum in 15, Cryptosporidium ryanae in 10, and Cryptosporidium andersoni in two samples. C. bovis was the most common species, being identified in 74% of calf samples, in 77% of young stock samples and in 100% of cow samples. The youngest calves infected with C. bovis were 7 days old, showing that the prepatent period is shorter than the previously stated 10 days. C. parvum was detected in 15 calves from nine farms, and samples were clustered in the southern parts of Sweden. Diarrhoeic calf samples contained C. parvum, C. bovis or C. ryanae. Sequencing of the GP60 gene was successful in 13 of the C. parvum samples. Eight subtypes, including three novel ones, were detected. Four of the subtypes have previously been identified in humans. This indicates that there is a zoonotic potential in C. parvum infected Swedish dairy calves.

From mouse to moose: multilocus genotyping of Giardia isolates from various animal species.

Giardia intestinalis is a protozoan parasite that consists of seven genetically distinct assemblages (A to G). Assemblage A and B parasites have been detected in a wide range of animals including humans, while the other assemblages (C to G) appear to have a narrower host range. However, the knowledge about zoonotic transmission of G. intestinalis is limited. To address this question, 114 Giardia isolates from various animals in Sweden including pets, livestock, wildlife and captive non-human primates were investigated by a sequence-based analysis of three genes (beta-giardin, glutamate dehydrogenase and triose phosphate isomerase). Assemblage A infections were detected in nine ruminants, five cats and one dog, while three sheep were infected with both assemblages A and E. Multilocus genotypes (MLGs) were defined for assemblage A, and three of these MLGs have previously been detected in Giardia isolates from humans. The newly described sub-assemblage AIII, until now reported mainly in wild hoofed animals, was found in one cat isolate. Assemblage B occurred in three monkeys, one guinea pig and one rabbit. The rabbit isolate exhibited sequences at all three loci previously detected in human isolates. The non-zoonotic assemblages C, D, E, F or G were found in the remaining 83 G. intestinalis isolates, which were successfully amplified and genotyped, generating a wide variety of both novel and known sub-genotypes. Double peaks in chromatograms were seen in assemblage B, C, D and E isolates but were never observed in assemblage A, F and G isolates, which can reflect differences in allelic sequence divergence. No evidence of genetic exchange between assemblages was detected. The study shows that multilocus genotyping of G. intestinalis is a highly discriminatory and useful tool in the determination of zoonotic sub-groups within assemblage A, but less valuable for subtyping assemblages B, C, D and E due to the high frequency of double peaks in the chromatograms. The obtained data also suggest that zoonotic transmission of assemblages A and B might occur to a limited extent in Sweden.

Limited sequence variation in the major sperm protein 1 (MSP) gene within populations and species of the genus Dictyocaulus (Nematoda).

Populations of the bovine lungworm, Dictyocaulus viviparus, are genetically structured based on variation in mtDNA and AFLP data. Our aim was to investigate if this genetic variability also is reflected in a protein recognized by the host immune system. We focused on the major sperm protein (MSP), a small and abundant protein used in diagnostic immunoassays, which has been shown to be variable in some nematodes but not others. MSP was sequenced using worm DNA from eight adult worms from each of nine populations whose genetic structure previously had been quantified. For comparison, we also analyzed MSP sequences of the closely related Dictyocaulus eckerti and Dictyocaulus capreolus and from nematodes with sequences deposited in GenBank. In contrast to previous results, this study shows that the MSP ofD. viviparus is similar to that of other nematodes. Almost no sequence variation, and thus no antigenic diversity, was detected in MSP between worms from different sub-populations or in the other Dictyocaulus species investigated. A functional test of a recombinant variant of the MSP showed that the expressed protein was recognized by antibodies in sera from infected cattle. This has practical implications for the development of species-specific markers, recombinant vaccines, and immunodiagnostics.

Coccidial infections in commercial broilers: epidemiological aspects and comparison of Eimeria species identification by morphometric and polymerase chain reaction techniques.

The objective of this study was to add to existing knowledge of the epidemiology and the aetiology of coccidial infections in commercial broiler flocks. Polymerase chain reaction (PCR) and morphometric identification of the Eimeria species were compared as means of differentiation in the field samples of faeces and litter. For morphometry, the Eimeria species were categorized into three groups based on lengths of the oocysts. Two random samples of commercial broilers were studied, one during 2000/01 and the other during 2003/04. The prophylactic regime (in-feed narasin), husbandry and methods applied were broadly the same for both subpopulations. Coccidial infection prevalence increased from approximately 45% to approximately 75% during this period, but infection levels (oocysts per gram of faeces) did not significantly change. There were substantial geographical differences in both prevalence and infection levels. A change in Eimeria species profile occurred during the study period. Five Eimeria species were identified at slaughter, by PCR targeting the ITS-1 region of the genome; Eimeria acervulina (100%), Eimeria tenella (77%), Eimeria maxima (25%), Eimeria praecox (10%) and Eimeria necatrix (2%). PCR and morphometric tentative identification were in complete agreement in only 49% of the cases.

A simplified protocol for molecular identification of Eimeria species in field samples.

This study aimed to find a fast, sensitive and efficient protocol for molecular identification of chicken Eimeria spp. in field samples. Various methods for each of the three steps of the protocol were evaluated: oocyst wall rupturing methods, DNA extraction methods, and identification of species-specific DNA sequences by PCR. We then compared and evaluated five complete protocols. Three series of oocyst suspensions of known number of oocysts from Eimeria mitis, Eimeria praecox, Eimeria maxima and Eimeria tenella were prepared and ground using glass beads or mini-pestle. DNA was extracted from ruptured oocysts using commercial systems (GeneReleaser, Qiagen Stoolkit and Prepman) or phenol-chloroform DNA extraction, followed by identification of species-specific ITS-1 sequences by optimised single species PCR assays. The Stoolkit and Prepman protocols showed insufficient repeatability, and the former was also expensive and relatively time-consuming. In contrast, both the GeneReleaser protocol and phenol-chloroform protocols were robust and sensitive, detecting less than 0.4 oocysts of each species per PCR. Finally, we evaluated our new protocol on 68 coccidia positive field samples. Our data suggests that rupturing the oocysts by mini-pestle grinding, preparing the DNA with GeneReleaser, followed by optimised single species PCR assays, makes a robust and sensitive procedure for identifying chicken Eimeria species in field samples. Importantly, it also provides minimal hands-on-time in the pre-PCR process, lower contamination risk and no handling of toxic chemicals.

Immunogenicity and protective effect against murine cerebral neosporosis of recombinant NcSRS2 in different iscom formulations.

Recombinant NcSRS2, a major immunodominant surface antigen of the intracellular protozoan parasite Neospora caninum, was used as a model antigen to compare the immunogenicity of iscoms prepared according to three different methods. Two NcSRS2 fusion proteins were used, one that was biotinylated upon expression in Escherichia coli and linked to Ni2+-loaded iscom matrix (iscom without any protein) via a hexahistidyl (His6)-tagged streptavidin fusion protein, and another that contained both a His6-tag and streptavidin (His6-SA-SRS2') and was coupled to either Ni2+-loaded or biotinylated matrix. While all three iscom preparations induced N. caninum specific antibodies at similar levels, His6-SA-SRS2' coupled to biotinylated matrix generated the strongest cellular responses measured as in vitro proliferation and production of interferon-gamma and interleukin-4 after antigen stimulation of spleen cells. However, the relationship between the levels of these cytokines as well as between IgG1 and IgG2a titres in serum induced by the three iscom preparations were similar, indicating that the balance between Th1 and Th2 responses did not differ. After challenge infection, mice immunised with His6-SA-SRS2' coupled to biotinylated matrix had significantly lower amounts of parasite DNA in their brains compared to the other immunised groups. Possible reasons for the performance of the different iscom formulations are discussed.

Prevalence of antibodies against Toxoplasma gondii and Neospora caninum in Hungarian red foxes (Vulpes vulpes).

In the present study we have investigated the seroprevalence to the protozoan parasites Toxoplasma gondii and Neospora caninum in 337 red foxes (Vulpes vulpes) from 16 out of 19 counties in Hungary. The foxes were originally collected within a National vaccination program against rabies. Antibodies to T. gondii were detected in as many as 228 (68%) of the foxes using a commercial direct agglutination test (DAT). In an indirect iscom ELISA, five foxes (1.5%) were positive for antibodies against N. caninum. The high prevalence of foxes positive for T. gondii might be explained by the widespread occurrence of the parasite in the diet of foxes. As a contrast, latent infections of N. caninum among red foxes in Hungary are much less common.

Neospora caninum emerges from the shadow of Toxoplasma gondii.

It is sometimes easy to make the mistake of assuming that everything that holds true for Toxoplasma gondii is also true for its relative Neospora caninum. However, a recurring theme in the recent review by Hemphill et al. is not the similarities but the striking differences between the two parasites.

Global patterns reveal strong population structure in Haemonchus contortus, a nematode parasite of domesticated ruminants.

We have examined the global population genetic structure of Haemonchus contortus. The genetic variability was studied using both amplified fragment length polymorphism (AFLP) and nad4 sequences of the mitochondrial genome. To examine the performance and information content of the two different marker systems, comparative assessment of population genetic diversity was undertaken in 19 isolates of H. contortus, a parasitic nematode of small ruminants. A total of 150 individual adult worms representing 14 countries from all inhabited continents were analysed. Altogether 1,429 informative AFLP markers were generated using four different primer combinations. Also, the genetic variation was high, which agrees with results from previous AFLP studies of nematode parasites of livestock. The genetic structure was high, indicating limited gene flow between the different isolates and populations from each continent mostly formed monophyletic groups in the phylogenetic analysis. However, for isolates representing Australia, Greece and one laboratory strain that originated from South Africa (WRS), there was no clear genetic relationship between the isolates and the distance between their geographical origins. Basically the same pattern was observed for the mitochondrial marker, although the phylogenetic analysis was less resolved than for AFLP. In contrast with previous findings on the population genetic structure of H. contortus, the calculation of population structure gave high values (Nst=0.59). The strong structure was present also for the four Swedish isolates (Nst=0.16) representing a small geographical area.

Persistent infection in a dairy herd with an unusual genotype of bovine Cryptosporidium parvum.

On two occasions, we identified Cryptosporidium parvum with an unusual banding pattern after restriction enzyme digestion of Cryptosporidium oocyst wall protein PCR fragments. The samples originated from a single farm with calf diarrhoea. The Cryptosporidium oocyst wall protein PCR fragment sequence revealed a point mutation in one of the recognition sites for RsaI. Our subgenotyping showed that samples from both samplings shared the same MS1 and GP15 alleles. Analysis of the TP14 microsatellite was less sensitive, and results were only obtained from two samples. Both had the same allele. This is, to our knowledge, the first description of a C. parvum with this Cryptosporidium oocyst wall protein genotype. More importantly, the genotype seems to be stable over time since we could detect it again among newborn animals almost 1 year after it was first identified.

Immunisation of mice against neosporosis with recombinant NcSRS2 iscoms.

The coccidian parasite Neospora caninum is an intracellular protozoan, causing abortion in cattle in many countries around the world. In this study, the protective potential of the major N. caninum surface antigen NcSRS2, expressed in Escherichia coli and formulated into immunostimulating complexes (iscoms), was investigated in an experimental mouse model. The recombinant protein was specially designed for binding to iscoms via biotin-streptavidin interaction. Two groups of 10 BALB/c mice were immunised twice, on days 0 and 28 with iscoms containing either the recombinant NcSRS2 (NcSRS2 iscoms) or similar iscoms with NcSRS2 substituted by an unrelated recombinant malaria peptide (M5) as a control (M5 iscoms). A third group of 10 age-matched BALB/c mice served as an uninfected control group. Immunisation with recombinant NcSRS2 iscoms resulted in production of substantial antibody titres against N. caninum antigen, while the mice immunised with M5 iscoms produced only very low levels of antibodies reacting with N. caninum antigen. After challenge infection with N. caninum tachyzoites on day 69, mice immunised with NcSRS2 iscoms showed only mild and transient symptoms, whereas the group immunised with M5 iscoms showed clinical symptoms until the end of the experiment at 31 days post inoculation. A competitive PCR assay detecting Nc5-repeats was applied to evaluate the level of parasite DNA in the brain. The amount of Nc5-repeats in the group vaccinated with NcSRS2 iscoms was significantly lower than in the control group given M5 iscoms. In conclusion, it was found that the recombinant NcSRS2 iscoms induced specific antibodies to native NcSRS2 and immunity sufficient to reduce the proliferation of N. caninum in the brains of immunised mice.

Functional analysis and localisation of a delta-class glutathione S-transferase from Sarcoptes scabiei.

The mite Sarcoptes scabiei causes sarcoptic mange, or scabies, a disease that affects both animals and humans worldwide. Our interest in S. scabiei led us to further characterise a glutathione S-transferase. This multifunctional enzyme is a target for vaccine and drug development in several parasitic diseases. The S. scabiei glutathione S-transferase open reading frame reported here is 684 nucleotides long and yields a protein with a predicted molecular mass of 26 kDa. Through phylogenetic analysis the enzyme was classified as a delta-class glutathione S-transferase, and our paper is the first to report that delta-class glutathione S-transferases occur in organisms other than insects. The recombinant S. scabiei glutathione S-transferase was expressed in Escherichia coli via three different constructs and purified for biochemical analysis. The S. scabiei glutathione S-transferase was active towards the substrate 1-chloro-2,4-dinitrobenzene, though the positioning of fusion partners influenced the kinetic activity of the enzyme. Polyclonal antibodies raised against S. scabiei glutathione S-transferase specifically localised the enzyme to the integument of the epidermis and cavities surrounding internal organs in adult parasites. However, some minor staining of parasite intestines was observed. No staining was seen in host tissues, nor could we detect any antibody response against S. scabiei glutathione S-transferase in sera from naturally S. scabiei infected dogs or pigs. Additionally, the polyclonal sera raised against recombinant S. scabiei glutathione S-transferase readily detected a protein from mites, corresponding to the predicted size of native glutathione S-transferase.

The current status of the small subunit rRNA phylogeny of the coccidia (Sporozoa).

There is no current comprehensive assessment of the molecular phylogeny of the coccidia, as all recently published papers either deal with subsets of the taxa or sequence data, or provide non-robust analyses. Here, we present a comprehensive and consistent phylogenetic analysis of the available data for the small-subunit ribosomal RNA gene sequence, including a number of taxa not previously studied, based on a Bayesian tree-building analysis and the covariotide model of evolution. The assumptions of the analysis have been rigorously tested, and the benefits and limitations highlighted. Our results provide support for a number of prior conclusions, including the monophyly of the families Sarcocystidae (cyst-forming coccidia) and Eimeriidae (oocyst-forming coccidia), but with bird-host Isospora species in the Eimeriidae and mammal-host species in the Sarcocystidae. However, it is clear that a number of previously reported relationships are dependent on the evolutionary model chosen, such as the placements of Goussia janae, Lankesterella minimia and Caryospora bigenetica. Our results also confirm the monophyly of the subfamilies Toxoplasmatinae and Sarcocystinae, but only some of the previously reported groups within these subfamilies are supported by our analysis. Similarly, only some of the previously reported groups within the Eimeriidae are supported by our analysis, and the genus Eimeria is clearly paraphyletic. There are unambiguous patterns of host-parasite relationship within the coccidia, as most of the well-supported groups have a consistent and restricted range of hosts, with the exception of the Toxoplasmatinae. Furthermore, the previously reported groups for which we found no support all have a diverse range of unrelated hosts, confirming that these are unlikely to be natural groups. The most interesting unaddressed questions may relate to Isospora, which has the fewest available sequences and host-parasite relationships apparently not as straightforward as elsewhere within the suborder.

The origin of Sarcoptes scabiei in wombats.

In 2002, Skerratt et al. phylogenetically analysed sequence data for several haplotypes of the parasitic mite Sarcoptes scabiei from wombat, human and dog hosts in Australia, to test scenarios concerning the origin and diversification of the scabies infections in wombats. Here we note that their substantive conclusions can be called into question by the choice of model used in their phylogenetic analysis, the lack of a root for their phylogenetic trees, and their interpretation of the evolutionary scenario.

Pyrosequencing analysis identifies discrete populations of Haemonchus contortus from small ruminants.

The genus Haemonchus consists of blood-sucking parasitic nematodes in the abomasum of ruminants. Members of this genus are responsible for extensive production losses, particularly of small ruminants in the tropics but are also found in temperate regions. In this study, we examined the internal transcribed spacers-1 and -2 of rRNA in Haemonchus spp. The rRNA region spanning the internal transcribed spacers-1, -2 and the 5.8S rRNA gene was amplified by PCR from each of 10 worms from Swedish sheep, a Swedish goat and Kenyan sheep. The fragments were sequenced and examined with respect to genetic differences fixed among the three isolates. These and additional worms were further analysed with Pyrosequencing technology. This technique allowed us to rapidly analyse 110 individuals in three putative polymorphic nucleotide positions that were initially identified with dideoxy sequencing. The geographical isolates could to a large extent be genetically distinguished, but none of the polymorphic positions were consistent among all individuals within each isolate. Furthermore an alignment of our sequences and a consensus sequence published for Haemonchus contortus revealed two differences in positions 123 and 196 in internal transcribed spacers-2. Although these positions were previously reported as heterogenic, no polymorphism was detected among the 30 worms sequenced in the present study. Modelling of the internal transcribed spacers-2 secondary structure based on our data also identified a new putative long-range interaction. The isolates are best described as populations. In conclusion, consistent differences were not identified and the studied isolates are therefore best described as discrete populations. This study also reveals for the first time the potential of Pyrosequencing technology as a tool in the analysis of nematode population genetics.