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.

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.

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.

Characterization of the Borrelia burgdorferi RNase P RNA gene reveals a novel tertiary interaction.

Characterization of the RNase P RNA gene derived from Borrelia burgdorferi reveals covariation of the conserved nucleotides at positions corresponding to nucleotides 128 and 230 in Escherichia coli RNase P RNA (M1 RNA). Single base substitutions at either of these positions in M1 RNA resulted in a lack of complementation of the temperature-sensitive phenotype associated with rnpA49 in vivo whereas complementation was observed for the double mutant M1 RNA or wild-type M1 RNA. Our in vitro data showed that M1 RNA harbouring a substitution at 128 or 230 cleaved a tRNA precursor both in the absence and presence of C5 with reduced efficiency compared to the wild-type and the double mutant M1 RNA. We conclude that the nucleotides at positions 128 and 230 establish a long-range tertiary interaction in RNase P RNA. Our data also suggest that this interaction together with the identity of the nucleotide at position 230 is important for Pb2+ induced cleavage at specific positions in M1 RNA.

RNase P RNA structure and cleavage reflect the primary structure of tRNA genes.

The function of RNase P RNA depends on its folding in space. A majority of RNase P RNAs from various bacteria show a similar secondary structure to that of Escherichia coli (M1 RNA). However, there are exceptions as exemplified by the RNase P RNA derived from the low GC-content Gram-positive bacteria Bacillus subtilis and Mycoplasma hyopneumoniae (Hyo P RNA). Previous studies using M1 RNA and Hyo P RNA suggest differences both with respect to the kinetics of cleavage as well as to cleavage site recognition. Here we have studied cleavage by these two structurally different RNase P RNAs as a function of changes in the 5' leader and the 3'-terminal CCA motif in the substrate. Our data suggest that the nucleotide at the -2 position in the 5' leader plays a role both for cleavage site recognition and for the rate of cleavage. However, depending on the identity of the -2 residue differences in the cleavage pattern comparing these two types of RNase P RNAs were observed. The results also suggest that the identity of the -1/+73 base-pair in the substrate influences the cleavage site recognition process. These findings will be related to differences in structure comparing these types of RNase P RNAs and the "RCCA-RNase P RNA" interaction. In addition, our findings will be discussed with respect to the primary structure of the tRNA genes in different bacteria.

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.

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.

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.

Detection and identification of Mobiluncus species by direct filter hybridization with an oligonucleotide probe complementary to rRNA.

A hybridization assay for direct detection and identification of Mobiluncus species has been developed and tested. A [32P]-labelled synthetic oligonucleotide probe, complementary to a nucleotide sequence in the variable region V8 of Mobiluncus 16S ribosomal RNA, was utilized. One of the advantages of using rRNA as target molecule for the hybridization assays is the copy number of rRNA, which can be as high as 10(4), and that additionally three to six sites on the minus strand of the DNA gene can be utilized. This probe was found to be sensitive and to react with 62 of 68 tested typical or atypical Mobiluncus isolates. It was also specific, and was shown not to react with 96 tested unrelated bacterial species and isolates, including taxonomically closely related species like Actinomyces or Bifidobacterium spp., or with bacteria isolated from the vagina of both healthy persons with an undisturbed flora, as well as from patients suffering from the bacterial vaginosis syndrome (BV).

Oligonucleotide probes complementary to 16S rRNA for rapid detection of mycoplasma contamination in cell cultures.

Mycoplasma contamination of cell cultures is a menace to diagnostic and research procedures. Rapid and reliable detection methods are, therefore, sorely needed. After comparing 16S rRNA sequences from those mycoplasmas that contaminate cell cultures, three different oligonucleotide probes were constructed. Two of these probes were designed to be group-specific and one to be species-specific. The three oligonucleotide probes were designed to cover all mycoplasmas commonly isolated from cell cultures. Contaminated cell lines could easily be detected by a direct filter hybridization assay in which the probes were incubated jointly. The assay proved to be rapid and sensitive with the possibility to perform and evaluate the mycoplasma testing within one working day.

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.

The phylogeny of Mycoplasma bovis as determined by sequence analysis of the 16S rRNA gene.

The nucleotide sequence of the 16S rRNA gene of Mycoplasma bovis has been determined. Comparisons with other 16S rRNA sequences of mycoplasmas showed that Mycoplasma agalactiae is phylogenetically the closest relative. In total, only eight nucleotides differed between the M. bovis and M. agalactiae 16S rRNA sequences. The phylogenetic position of M. bovis with respect to other mycoplasmas was determined by sequence comparisons and from features in the secondary structure of 16S rRNA.

The phylogeny of Neospora caninum and Toxoplasma gondii based on ribosomal RNA sequences.

Neospora caninum is a newly described cyst-forming coccidium which is the cause of severe neurological disease in dogs. The parasite is morphologically similar to Toxoplasma gondii, but the two species can be differentiated serologically. In order to define the phylogenetic position of N. caninum, we have determined 16S-like rRNA sequences from three members of the family of Sarcocystidae: N. caninum, T. gondii, and Sarcocystis fusiformis. The 16S-like rRNA genes from the three parasites were amplified by polymerase chain reaction and the sequences were determined by direct solid-phase sequencing. The sequences derived were computer aligned with several other 16S-like rRNA sequences from protozoan parasites to construct phylogenetic trees. The study confirmed that N. caninum should be classified as a member of the family Sarcocystidae. However, because of the close relationship to T. gondii it seems questionable that N. caninum should be placed in a new genus.

In vitro amplification of the 16S rRNA genes from Mycoplasma bovis and Mycoplasma agalactiae by PCR.

Mycoplasma bovis and Mycoplasma agalactiae are two very closely related species which cause mastitis in cows and goats, respectively. M. bovis can also cause arthritis and respiratory disease in cattle. It has recently been shown that the 16S rRNA sequences differ only in 8 nucleotide positions between the two species [J.G. Mattsson, B. Guss and K.-E. Johansson (1994) FEMS Microbiol. Lett., 115: 325-328]. These nucleotide differences are distributed over the molecule in such a way that it is difficult to design specific identification systems, based on PCR only, for M. bovis and M. agalactiae. Two different PCR systems based on 16S rRNA sequence data have, however, been designed for these two species. The forward primers were identical in the two systems and complementary to a segment of the evolutionarily variable region V2. The reverse primers were complementary to the variable region V6, in which there are two nucleotide differences between M. bovis and M. agalactiae. The size of the PCR products, generated with these primers, was 360 bp. Cross-amplification was obtained with the two species in the heterologous PCR systems, but with approximately a 100-fold lower efficiency. Cross-amplification was not obtained with any other bovine or caprine mycoplasma except for Mycoplasma sp. strain A1343 of the caprine group 7. The detection limit of the PCR system for M. bovis with a reference culture was 4 x 10(2) CFU/ml and of the PCR system for M. agalactiae 2 x 10(2) CFU/ml. The M. bovis-PCR system was used to analyze nasal samples of calves from a herd where an outbreak of pneumonia had occured and it proved possible to detect M. bovis in these samples.

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.

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.

Species-specific oligonucleotide probes complementary to 16S rRNA of Mycoplasma gallisepticum and Mycoplasma synoviae.

Mycoplasma gallisepticum and M synoviae are important avian pathogens causing respiratory diseases which result in great economic losses in poultry farming. Two oligonucleotide probes, complementary to the variable region V8 of 16S rRNA from the avian mycoplasmas M gallisepticum and M synoviae have, therefore, been designed and used in direct filter hybridisation experiments. Both probes gave strong hybridisation signals with their homologous targets, whereas no cross-hybridisations were obtained with any of the other avian mycoplasmas tested. It was possible to detect 2-3 x 10(4) mycoplasma organisms by direct filter hybridisation experiments with radiolabelled probes. The probes were also used to analyse several laboratory strains and field isolates of M gallisepticum and M synoviae with complete agreement between the probe technique and the other methods used for species determination. Atypical M gallisepticum strains also gave strong hybridisation signals with the M gallisepticum specific probe.

Specificity of oligonucleotide probes complementary to evolutionarily variable regions of 16S rRNA from Mycoplasma hyopneumoniae and Mycoplasma hyorhinis.

Mycoplasma is the common name for the smallest free-living microorganisms, the Mollicutes. Mycoplasma hyopneumoniae is of great importance in veterinary medicine, causing enzootic pneumonia in pigs. M hyorhinis can cause polyserositis and may cause pneumonia in piglets. Oligonucleotides complementary to variable regions of 16S rRNA from these mycoplasmas were designed and used as probes for detection and identification of these mycoplasmas. The probe complementary to 16S rRNA of M hyorhinis gave a very weak cross-hybridisation with M hyosynoviae in filter hybridisation experiments, but not with any of the other porcine mycoplasmas tested. Three oligonucleotide probes complementary to M hyopneumoniae 16S rRNA were tested. One of the probes (Mhp6/30) was found to be specific to M hyopneumoniae, but the other two gave cross-hybridisation with M flocculare. Using the Mhp6/30 probe in direct filter hybridisation experiments, it proved possible to detect M hyopneumoniae in lung biopsies from experimentally infected pigs.

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.

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.