Molecular epidemiology and clinical manifestations of human cryptosporidiosis in Sweden.

This study describes the epidemiology and symptoms in 271 cryptosporidiosis patients in Stockholm County, Sweden. Species/genotypes were determined by polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) of the Cryptosporidium oocyst wall protein (COWP) and 18S rRNA genes. Species were C. parvum (n=111), C. hominis (n=65), C. meleagridis (n=11), C. felis (n=2), Cryptosporidium chipmunk genotype 1 (n=2), and a recently described species, C. viatorum (n=2). Analysis of the Gp60 gene revealed five C. hominis allele families (Ia, Ib, Id, Ie, If), and four C. parvum allele families (IIa, IIc, IId, IIe). Most C. parvum cases (51%) were infected in Sweden, as opposed to C. hominis cases (26%). Clinical manifestations differed slightly by species. Diarrhoea lasted longer in C. parvum cases compared to C. hominis and C. meleagridis cases. At follow-up 25-36 months after disease onset, 15% of the patients still reported intermittent diarrhoea. In four outbreaks and 13 family clusters, a single subtype was identified, indicating a common infection source, which emphasizes the value of genotyping for epidemiological investigations.

Genetic differences in internal transcribed spacer 1 between Dermanyssus gallinae from wild birds and domestic chickens.

We investigated the presence of the poultry red mite or the chicken mite, Dermanyssus gallinae De Geer, Acari: Dermanyssidae, in wild bird populations in four different geographical regions of Sweden. The mites identified as D. gallinae were compared genetically with D. gallinae from egg-producing poultry farms in the same regions. The small subunit (SSU) gene, the 5.8S ribosomal RNA (rRNA) gene and the two internal transcribed spacers (ITS) of the rRNA genes were used in the genetic analysis. All D. gallinae mites had identical SSU rRNA, 5.8S rRNA and ITS2 sequences independent of their origin. By contrast, we identified significant differences in the ITS1 sequences. Based on the differences in the ITS1 sequences, the mites could be divided into two genotypes, of wild and domesticated origin, with no variation within the groups. These results imply that wild bird populations are of low importance, if any, as natural reservoirs of D. gallinae in these four geographical regions of Sweden.

Isospora orlovi infection in suckling dromedary camel calves (Camelus dromedarius) in Kenya.

Outbreaks of isosporosis in young suckling dromedary camel calves (Camelus dromedarius) in Dubai, UAE and in Kenya were recently described. In the former outbreak the pathogen was shown to be Isospora orlovi by morphological features and was later characterized molecularly. In the present study, we have made a longitudinal investigation of 159 suckling dromedary calves < or =12 weeks of age belonging to 8 ranched camel herds (M1) in Northern Kenya. The study was carried out during 18 months. In three of the herds frequent samples were taken irregularly every 1-6 weeks. All calves < or =12 weeks of age present in the respective herds were sampled during the visits. In addition, 91 calves of the same age group but belonging to 42 pastoral herds (M2) in Northern Kenya were point sampled at convenience. Faecal samples from each calf were taken and the faeces were investigated for coccidia. Samples found with coccidian oocysts were suspended in a 2% potassium dichromate solution. Isospora sp. was identified and samples with relatively high numbers of Isospora sp. were analysed molecularly. The SSU rRNA gene and internal transcribed spacer 1 (ITS1) were amplified with primers complementary to conserved regions of the SSU rRNA gene in eukaryotes as well as a conserved part of the 5.8S rRNA gene of Eimeria. A relatively high number of the calves exhibited diarrhoea, 30.2% and 41.8% in the M1 and M2 herds, respectively. Isospora sp. was only found in diarrhoeic calves or in calves convalescent from recent scouring periods. No calf >8 weeks of age was found to be excreting Isospora sp. The parasite was only found in calves < or =4 weeks of age in the M1 herds and in the M2 herds in calves <8 weeks of age. Of the M1 and M2 calves exhibiting diarrhoea, 20.8% and 26.3% excreted Isospora sp., respectively. Morphologically the Isospora sp. was similar to I. orlovi and sequence analysis of the SSU rRNA gene from four Kenyan isolates (unfortunately only from the pastoral herds, M2) and ITS 1 segments from three of the isolates from Kenya and one from Dubai, confirmed that the Isospora isolates belonged to the species I. orlovi, and that the sequences were similar to the Dubai isolates.

Quantitative analysis of parasite DNA in the blood of immunized and naïve mice after infection with Neospora caninum.

Real-time PCR was used to study the duration and level of parasitaemia in mice immunized with immune-stimulating complexes (iscoms) containing recombinant NcSRS2, one of the immunodominant surface antigens of Neospora caninum. After challenge infection, blood was collected daily for 9 days. During this period the amounts of parasite DNA detected in immunized mice were significantly lower (P<0.001), and the duration of parasitaemia appeared to be shorter, than in non-immunized controls. Furthermore, the degree of parasitaemia seemed to correlate well with the amount of N. caninum DNA in the brain 3 weeks post-inoculation and with disease severity measured as changes in body weight. These results indicate that the protective immunity induced by the NcSRS2-iscoms was sufficient to reduce the level of parasitaemia, which probably reduced the number of parasites reaching the brain, and could be the reason for the reduction in brain parasite load and clinical symptoms. Furthermore, real-time PCR was found to be a sensitive means for rapid assessment of N. caninum in blood.

Molecular typing of Cryptosporidium parvum associated with a diarrhoea outbreak identifies two sources of exposure.

An outbreak of cryptosporidiosis associated with exposure to outdoor swimming-pool water affected an estimated 800-1000 individuals. PCR products were obtained from faecal specimens from 30 individuals who tested positive for Cryptosporidium oocysts. RFLP and sequencing analyses showed that all individuals were infected with Cryptosporidium parvum. Among the infected individuals, five had just swum in an adjacent indoor pool during the same period, and had no identified contact with individuals linked to the outdoor pool. With the use of subgenotyping based on analysis of three mini- and microsatellite loci, MS1, TP14, and GP15, we could identify two sources of exposure. One subtype was associated with the outdoor pool and another with the indoor pool. These data demonstrate that the use of mini- and microsatellite loci as markers for molecular fingerprinting of C. parvum isolates are valuable in the epidemiological investigation of outbreaks.

Effect of acaricides on the activity of glutathione transferases from the parasitic mite Sarcoptes scabiei.

Glutathione transferases (GSTs) are a family of multifunctional enzymes with fundamental roles in cellular detoxication. Here we report the molecular characterization of 3 recombinant GSTs belonging to the mu- and delta-class from the parasitic mite Sarcoptes scabiei. Kinetic constants were determined, and the effect of acaricides, including organothiophosphates, pyrethroid esters, a formamidine, a macrocyclic lactone, an organochlorine as well as a bridged diphenyl acaricide, on the activity of the GSTs were tested using 1-chloro-2,4-dinitrobenzene (CDNB) as model substrate. Our results showed that enzymes from the same class and with high amino acid sequence identity have significantly different kinetic properties. For instance, one mu-class GST lost more than 50% of its activity in the presence of one of the organothiophosphates while the activity of the second mu-class GST was only slightly reduced under identical conditions. Tertiary structure modulations indicated that structural differences were the crucial factor for the different kinetic patterns observed. Genome analysis showed that the two mu-class GSTs are organized in tandem in the S. scabiei genome. Taken together these results show that GSTs might be involved in the metabolism of acaricides in S. scabiei.

Neospora caninum IgG avidity tests: an interlaboratory comparison.

Avidity tests can be used to discriminate between cattle that are acutely and chronically infected with the intracellular parasite Neospora caninum. The aim of this study was to compare the IgG avidity ELISA tests being used in four European laboratories. A coded panel of 200 bovine sera from well documented naturally and experimentally N. caninum infected animals were analysed at the participating laboratories by their respective assay systems and laboratory protocols. Comparing the numeric test results, the concordance correlation coefficients were between 0.479 and 0.776. The laboratories categorize the avidity results into the classes "low" and "high" which are considered indicative of recent and chronic infection, respectively. Three laboratories also use an "intermediate" class. When the categorized data were analysed by Kappa statistics there was moderate to substantial agreements between the laboratories. There was an overall better agreement for dichotomized results than when an intermediate class was also used. Taken together, this first ring test for N. caninum IgG avidity assays showed a moderate agreement between the assays used by the different laboratories to estimate the IgG avidity. Our experience suggests that avidity tests are sometimes less robust than conventional ELISAs. Therefore, it is essential that they are carefully standardised and their performance continuously evaluated.

Population genetics of the bovine/cattle lungworm (Dictyocaulus viviparus) based on mtDNA and AFLP marker techniques.

Mitochondrial DNA (mtDNA) sequence data and amplified fragment length polymorphism (AFLP) patterns were compared for the lungworm Dictyocaulus viviparus, a nematode parasite of cattle. Eight individual D. viviparus samples from each of 8 herds in Sweden and 1 laboratory isolate were analysed, with the aim of describing the diversity and genetic structure in populations using different genetic markers on exactly the same DNA samples. There was qualitative agreement between the whole-genome AFLP data and the mtDNA sequence data, both indicating relatively strong genetic differentiation among the Swedish farms. However, the AFLP data detected much more genetic variation than did the mtDNA data, even after allowing for the different inheritance patterns of the markers, and indicated that there was much less differentiation among the populations. The mtDNA data therefore seemed to be more informative about the most recent history of the parasite populations, as the general patterns were less obscured by detailed inter-relationships among individual worms. The 4 mtDNA genes sequenced (1542 bp) showed consistent patterns, although there was more genetic variation in the protein-coding genes than in the structural RNA genes. Furthermore, there appeared to be at least 3 distinct genetic groups of D. viviparus infecting Swedish cattle, 1 of which was predominant and showed considerable differentiation between farms, but not necessarily within farms. Second, the 2 smaller genetic groups occurred on farms where the predominant group also occurred, suggesting that these farms have had multiple introductions of D. viviparus.

Characterization of an atypical antigen from Sarcoptes scabiei containing an MADF domain.

We have cloned a cDNA encoding a novel antigen from a Sarcoptes scabiei (Acari) cDNA library by immunoscreening with sera from S. scabiei-infected dogs. The antigen is encoded by a 2,157 bp mRNA with a predicted open reading frame of 719 amino acids (molecular weight 79 kDa). Our sequence analysis identified the presence of a MADF domain in the N-terminus, and downstream of this domain there was a region of low sequence complexity. This latter region contained several blocks of triplets and quadruplets of polar amino acids (Asn, Gln and Ser), and these 3 amino acids represented 39.7% of all amino acids. The antigen was named Atypical Sarcoptes Antigen 1 (ASA1) since the MADF domain normally is found in proteins involved in transcriptional regulation. In addition, 15 out of 62 S. scabiei-infected dogs reacted with a purified recombinant version of ASA1 in Western blot analysis. With immunohistochemistry we could show that ASA1 is expressed throughout the parasite, and that IgG specific for ASA1 binds to the inside wall of the mite's burrow. To our knowledge, this is the first description of an antigen containing an MADF domain.

An interlaboratory comparison of immunohistochemistry and PCR methods for detection of Neospora caninum in bovine foetal tissues.

Seven European laboratories contributed to a multi-centre evaluation of detection techniques for Neospora caninum in bovine foetuses. Six laboratories participated in immunohistochemistry (IHC) testing. All seven laboratories participated in PCR testing, but the results from one laboratory were not included in the analysis, because of contamination problems in the preparation of the samples. A coded panel of tissue sections from 36 infected and non-infected foetuses was used to evaluate the IHC detection of parasites. A coded panel consisting of 44 homogenized foetal brain samples from natural bovine abortion cases and 32 spiked samples were used to evaluate the PCR methods. Inclusion of a duplicate dilution series of spiked samples was used to evaluate detection limits and repeatability. IHC methods had a relatively low sensitivity, but a high specificity. There was considerable variation in IHC results between participating laboratories, which may be partly explained by examination practices that depended on the experience of the operator. In addition, the use of different antibody reagents, different antibody dilutions, and different enzymatic treatments of tissues may have contributed to the observed variation. PCR methods generally had a higher sensitivity than IHC methods and also a high specificity. The agreement between the majority scores of IHC and PCR methods was low. False positive PCR results indicated contamination problems in some instances. Agreement between the PCR results of the various laboratories was better, compared with the IHC results. There appeared to be no clear relationship between the PCR format (i.e. single or nested) and diagnostic sensitivity. Consequently, an improvement of diagnostic performance of PCR might possibly be achieved by optimizing DNA extraction methods.

Isolation of Neospora caninum from a calf in Malaysia.

In order to attempt isolate the protozoan parasite Neospora caninum, an N. caninum seropositive pregnant Sahiwal Friesian cross heifer from a large-scale dairy farm in Malaysia was kept for observation until parturition at the Veterinary Research Institute, Ipoh. The heifer gave birth to a female calf that was weak, underweight and unable to rise. Precolostral serum from the calf had an N. caninum indirect fluorescent antibody test titre of 1:3200. It died 12 h after birth and necropsy was performed. Brain homogenate from the calf was inoculated into 10 BALB/c mice that were kept for 3 months after which brain tissue from the mice was inoculated onto 24 h fresh monolayer Vero cell lines. The cell cultures were examined daily until growth of intracellular protozoa was observed. DNA of the organisms from the cell cultures was analyzed by PCR and DNA sequencing. DNA fragments of the expected size were amplified from the isolate using N. caninum-specific primers, and sequence analysis of ITS1 clearly identified the isolate as N. caninum. This is the first successful isolation of N. caninum from a bovine in Malaysia, and the isolate is designated Nc-MalB1.

Genetic diversity assessed by amplified fragment length polymorphism analysis of the parasitic nematode Dictyocaulus viviparus the lungworm of cattle.

We have examined the population genetic structure in a collection of nine isolates of the parasitic lungworm Dictyocaulus viviparus. Eight of the isolates were sampled from cattle in geographically separated farms throughout south-central Sweden, and one isolate was a laboratory strain that has been maintained in experimentally infected calves for almost four decades. A total of 72 worms were examined, with eight individual worms from the same individual host representing each isolate. The genetic variation as revealed by amplified fragment length polymorphism analysis using four selective primer combinations was high. Depending on the primer combination a total of 66-79 restriction fragments were amplified, with 26-44 peaks of similar complexity from each of the isolates. The heterozygosity within populations was relatively small, as were the population mutation and immigration rates, which seemed to be in neutral equilibrium. The genetic diversity was therefore reasonably well structured in the field; and the laboratory isolate was quite distinct from the field samples. There was no relationship between the patterns of genetic diversity and the geographical proximity of the farms. The estimates of heterozygosity were much larger and more consistent than those previously estimated for this nematode species using mitochondrial sequencing, and the genetic structuring was thus much less pronounced and the gene flow greater. We attribute these differences in estimation to the broader sampling of loci available using amplified fragment length polymorphism markers, which may therefore constitute a superior technique for the study of patterns of lungworm diversity. Furthermore, the data estimating gene flow for D. viviparus was less than previously reported for closely related species in North America. This might be related to different rates of movements of infected hosts. It seems likely that lungworm infections are rather persistent on different farms, and the sudden outbreaks of disease that can be observed with host movements are most likely to be related to the introduction of susceptible stock.

Expressed sequence tag analysis of Sarcoptes scabiei.

Sarcoptes scabiei is an important parasitic mite in both man and animals. Little is known about the molecular interactions between this pathogen and its host. This is in part explained by the paucity of mite-derived material, including antigens. To extend the knowledge of the molecular repertoire in S. scabiei, we have performed a gene survey by an expressed sequence tag (EST) analysis. A total of 1020 ESTs were generated from an S. scabiei cDNA library. The average sequence read was 510 bp after editing and the overall sequencing success was 89%. Clustering of the sequences resulted in 76 clusters, comprising 36% of the ESTs. Sequence similarity searches showed that almost half of the S. scabiei ESTs could be assigned a putative identity. Many of these transcripts shared similarity with genes involved in basic metabolism and cellular organization. In the data set we also identified several proteases and other types of potential allergens implicated in various disease mechanisms. A relatively large fraction of the ESTs coded for different proteins carrying protease inhibitor-like domains. The clones with no similarity to previously identified genes constituted 11% of our transcripts. The EST data generated in this study will be a valuable resource in further studies of the biology of S. scabiei and in the identification of genes that can serve as potential targets in the control of the parasite.

Phylogeny of Dictyocaulus (lungworms) from eight species of ruminants based on analyses of ribosomal RNA data.

In this study, we conducted phylogenetic analyses of nematode parasites within the genus Dictyocaulus (superfamily Trichostrongyloidea). Lungworms from cattle (Bos taurus), domestic sheep (Ovis aries), European fallow deer (Dama dama), moose (Alces alces), musk ox (Ovibos moschatus), red deer (Cervus elaphus), reindeer (Rangifer tarandus) and roe deer (Capreolus capreolus) were obtained and their small subunit ribosomal RNA (SSU) and internal transcribed spacer 2 (ITS2) sequences analysed. In the hosts examined we identified D. capreolus, D. eckerti, D. filaria and D. viviparus. However, in fallow deer we detected a taxon with unique SSU and ITS2 sequences. The phylogenetic position of this taxon based on the SSU sequences shows that it is a separate evolutionary lineage from the other recognized species of Dictyocaulus. Furthermore, the analysis of the ITS2 sequence data indicates that it is as genetically distinct as are the named species of Dictyocaulus. Therefore, either this taxon needs to be recognized as a new species, or D. capreolus, D. eckerti and D. viviparus need to be combined into a single species. Traditionally, the genus Dictyocaulus has been placed as a separate family within the superfamily Trichostrongyloidea. The present molecular phylogenetic analyses support the placement as a separate family, but the current data do not support the placement of the Dictyocaulidae within the Trichostrongyloidea without a reassessment of the placement of the superfamily Strongyloidea. While D. eckerti has been regarded as the one and only lungworm species of cervids, this study showed that 4 host species including 3 members of Cervidae (moose, reindeer, red deer) and 1 Bovidae (musk ox) were infected with this parasite. Host ranges of D. viviparus (cattle), D. filaria (sheep) and D. capreolus (moose and roe deer) were more restricted. No clear pattern of co-evolution between the dictyocaulid taxa and their bovid and cervid hosts could be determined.

Redescription of Neospora caninum and its differentiation from related coccidia.

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums.

Pyrosequencing as a method for grouping of Listeria monocytogenes strains on the basis of single-nucleotide polymorphisms in the inlB gene.

By using pyrosequencing (i.e., sequencing by synthesis) 106 strains of different serovars of Listeria monocytogenes were rapidly grouped into four categories based on nucleotide variations at positions 1575 and 1578 of the inlB gene. Strains of serovars 1/2a and 1/2c constituted one group, and strains of serovars 1/2b and 3b constituted another group, whereas serovar 4b strains were separated into two groups.

Paramyosin from the parasitic mite Sarcoptes scabiei: cDNA cloning and heterologous expression.

The burrowing mite Sarcoptes scabiei is the causative agent of the highly contagious disease sarcoptic mange or scabies. So far, there is no in vitro propagation system for S. scabiei available, and mites used for various purposes must be isolated from infected hosts. Lack of parasite-derived material has limited the possibilities to study several aspects of scabies, including pathogenesis and immunity. It has also hampered the development of high performance serological assays. We have now constructed an S. scabiei cDNA expression library with mRNA purified from mites isolated from red foxes. Immunoscreening of the library enabled us to clone a full-length cDNA coding for a 102.5 kDa protein. Sequence similarity searches identified the protein as a paramyosin. Recombinant S. scabiei paramyosin expressed in Escherichia coli was recognized by sera from dogs and swine infected with S. scabiei. We also designed a small paramyosin construct of about 17 kDa that included the N-terminal part, an evolutionary variable part of the helical core, and the C-terminal part of the molecule. The miniaturized protein was efficiently expressed in E. coli and was recognized by sera from immunized rabbits. These data demonstrate that the cDNA library can assist in the isolation of important S. scabiei antigens and that recombinant proteins can be useful for the study of scabies.

Identification of Dictyocaulus spp. in ruminants by morphological and molecular analyses.

Lungworms of the genus Dictyocaulus from cattle, roe deer, and moose in Sweden were subjected to morphological and molecular analyses. The objectives of the study were to investigate whether mixed or monospecific Dictyocaulus infections occur in Swedish cattle and whether wild cervids may act as reservoirs. The morphological characters examined were thickness and shape of the buccal capsule wall (BCW) and total spicular length (TSL). Morphometry was also done on the total body length, and BCW thickness and length. In the molecular identification, we used a PCR-linked hybridization assay to probe worm DNA with species-specific oligonucleotide probes to the second internal transcribed spacer (1TS2). The results showed that the BCW shape was the most reliable morphological character for identification. Significant differences were observed in this character, but an overlap occurred between lungworms from each of the host species. With the hybridization assay, all lungworms from cattle were identified as D. viviparus, whereas those from roe deer represented a novel Dictyocaulus species demonstrating that each host had a monospecific lungworm infection. In moose, 61 (78.2%) worms belonged to the new species and 17 (21.8%) were D. eckerti. This study shows the usefulness of hybridization assay as an epidemiological tool for the specific identification of lungworms of cattle and wild cervids.

MPS1: a small, evolutionarily conserved zinc finger protein from the protozoan Toxoplasma gondii.

Within the expressed sequence tag (EST) dataset of Toxoplasma gondii we have identified several ESTs encoding a protein similar to the small zinc finger protein MPS1. In human it is suggested that MPS1 plays a role as a transcriptional mediator in response to various growth factors and it is used as a tumour marker in sera from cancer patients. However, in rat a cDNA sequence homologous to MPS1 encodes ribosomal protein S27. To further characterise MPS1 in T. gondii we transformed tachyzoites with a c-Myc-tagged version of the Toxoplasma MPS1 cDNA, flanked by SAG1 sequences. Western blot analysis showed that the Myc-MPS1 was only poorly expressed in the stable transformants. In contrast, Northern blot analysis demonstrated that the Myc-MPS1 mRNA was abundantly transcribed and that the endogenous level of MPS1 mRNA was not affected.

ITS2 sequences of Dictyocaulus species from cattle, roe deer and moose in Sweden: molecular evidence for a new species.

Total DNA was isolated from adult lungworms of the genus Dictyocaulus, collected from cattle, moose (Alces alces) and roe deer (Capreolus capreolus) in Sweden. The second ribosomal internal transcribed spacer was amplified with PCR, and DNA sequences were determined from nine individual worms that all came from different hosts in order to avoid analysis of siblings. The sequence data obtained were aligned and compared with similar data derived from German lungworm isolates from cattle and fallow deer (Cervus dama). These analyses clearly showed that specimens of the cattle lungworm, Dictyocaulus viviparus, were almost identical irrespective of their geographical origin. However, when the second internal transcribed spacer sequence of D. viviparus was compared with that of lungworms from moose and roe deer, major differences were noticed. Although lungworms collected from these cervids had identical second internal transcribed spacer sequences, they proved to be genetically different from Dictyocaulus eckerti of German fallow deer, displaying a 66.5% similarity. In an evolutionary tree, inferred by maximum likelihood analysis, the Dictyocaulus species from cattle and wild cervids clustered as compared with Dictyocaulus filaria from sheep. The study has thus demonstrated that A. alces and C. capreolus in Sweden are parasitised with a Dictyocaulus species that is different from D. viviparus and D. eckerti, indicating that we are dealing with a new species in moose and roe deer.