Cryptosporidium species and subtypes identified in human domestic cases through the national microbiological surveillance programme in Sweden from 2018 to 2022.

BACKGROUND: The intestinal protozoan parasite Cryptosporidium is an important cause of diarrheal disease worldwide. A national microbiological surveillance programme was implemented in Sweden in 2018 in order to increase knowledge of the molecular epidemiology of human cryptosporidiosis to better understand transmission patterns and potential zoonotic sources. This article summarises the results of the first five years of the surveillance programme. METHODS: Cryptosporidium-positive faecal and DNA samples from domestically acquired infections were collected from clinical microbiological laboratories in Sweden. Species and subtype determination was performed using 60 kDa glycoprotein and/or small subunit ribosomal RNA gene analysis. RESULTS: Between 2018 and 2022, 1654 samples were analysed and 11 different species were identified: C. parvum (n = 1412), C. mortiferum (n = 59), C. hominis (n = 56), C. erinacei (n = 11), C. cuniculus (n = 5), C. meleagridis (n = 3), C. equi (n = 2), C. ubiquitum (n = 2), and one each of C. canis, C. ditrichi and C. felis. Subtyping revealed seven subtype families of C. parvum (new subtype families IIy and IIz) and 69 different subtypes (11 new subtypes). The most common C. parvum subtypes were IIdA22G1c, IIdA24G1, IIdA15G2R1 and IIaA16G1R1b. For C. hominis, four different subtype families and nine different subtypes (two new subtypes) were identified. For additional species, two new subtype families (IIIk and VId) and nine new subtypes were identified. All successfully subtyped C. mortiferum cases were subtype XIVaA20G2T1, confirming previous findings in Sweden. Several outbreaks were identified of which the majority were foodborne and a few were due to direct contact with infected animals. CONCLUSION: Infection with C. parvum is the leading cause of human cryptosporidiosis acquired in Sweden, where more than 90% of domestic cases are caused by this zoonotic species and only a small proportion of cases are due to infection with other species. The rodent-associated C. mortiferum is considered an emerging zoonotic species in Sweden and the number of domestically acquired human cases has surpassed that of infection with C. hominis. A high diversity of species and subtypes, as well as diversity within the same subtype, was detected. Also, cryptosporidiosis appears to affect adults to a great extent in Sweden.

Extensive testing of a multi-locus sequence typing scheme for Giardia duodenalis assemblage A confirms its good discriminatory power.

BACKGROUND: The flagellated parasite Giardia duodenalis is a major and global cause of diarrhoeal disease. Eight genetically very distinct groups, known as assemblages A to H, have been recognized in the G. duodenalis species complex, two of which (assemblages A and B) infect humans and other mammalian hosts. Informative typing schemes are essential to understand transmission pathways, characterize outbreaks and trace zoonotic transmission. In this study, we evaluated a published multi-locus sequence typing (MLST) scheme for G. duodenalis assemblage A, which is based on six polymorphic markers. METHODS: We genotyped 60 human-derived and 11 animal-derived G. duodenalis isolates collected in Europe and on other continents based on the published protocol. After retrieving previously published genotyping data and excluding isolates whose sequences showed allelic sequence heterozygosity, we analysed a dataset comprising 146 isolates. RESULTS: We identified novel variants at five of the six markers and identified 78 distinct MLST types in the overall dataset. Phylogenetic interpretation of typing data confirmed that sub-assemblage AII only comprises human-derived isolates, whereas sub-assemblage AI comprises all animal-derived isolates and a few human-derived isolates, suggesting limited zoonotic transmission. Within sub-assemblage AII, isolates from two outbreaks, which occurred in Sweden and Italy, respectively, had unique and distinct MLST types. Population genetic analysis showed a lack of clustering by geographical origin of the isolates. CONCLUSION: The MLST scheme evaluated provides sufficient discriminatory power for epidemiological studies of G. duodenalis assemblage A.

Water quality modelling and quantitative microbial risk assessment for uMsunduzi River in South Africa.

South African rivers generally receive waste from inadequate wastewater infrastructure, mines, and farming activities, among others. The uMsunduzi River in KwaZulu-Natal, South Africa, is among these recipients with recorded poor to very poor water quality. To identify parts of the uMsunduzi River that are polluted by Cryptosporidium and Escherichia coli (E. coli), this study mapped out pollutants emanating from point and non-point sources using the Soil and Water Assessment Tool (SWAT). Streamflow calibration in the upper and lower reaches of the catchment showed good performance with R2 of 0.64 and 0.58, respectively. SWAT water quality output data were combined with a Quantitative Microbial Risk Assessment (QMRA) to understand the microbial health implications for people using river water for drinking, recreational swimming, and non-competitive canoeing. QMRA results for Cryptosporidium and pathogenic E. coli showed that the probability of infection for most users exceeds the acceptable level for drinking and recreation as outlined in the South African water quality guidelines, and by the World Health Organization (WHO). The results of this study can be used as a baseline to assess the economic and health implications of different management plans, resulting in better-informed, cost-effective, and impactful decision-making.

Prevalence, risk factor and diversity of Cryptosporidium in cattle in Latvia.

The epidemiology of Cryptosporidium spp. in Latvia was investigated by testing fecal samples from 926 animals aged from one day to 24 years for the presence of Cryptosporidium spp. oocysts. The samples were collected from 87 cattle farms and from four slaughterhouses, and analyzed by conventional and fluorescent microscopy, followed by Cryptosporidium species and C. parvum subtype differentiation. Moreover, using a questionnaire, we surveyed factors that could be relevant as risk factors of Cryptosporidium spp. infection on the farms. Cryptosporidium spp. were shed by 33.8% of the investigated cattle and at least one shedding animal was found on 77.8% of the farms. In the present study, all four Cryptosporidium species reported to commonly infect cattle and two additional Cryptosporidium species (C. scrofarum and C. ubiquitum) were identified. In addition, mix infections of C. parvum/C. bovis, C. bovis/C. ryanae, C. parvum/C. ryanae, C. parvum/C. andersoni and C. bovis/C. andersoni were observed. C. parvum and C. bovis was mostly prevalent in young animals (0-3 months old) and in addition, diarrhea associated with C. parvum infection was observed only in very young animals. Cryptosporidium andersoni and C. ryanae in age group 0-3 months was observed in low prevalence, while a higher proportion of animals with diarrhea associated with C. andersoni infection was observed in very young animals and with C. ryanae in animals age group 4-24 months. Eight previously described C. parvum subtypes were observed. The majority of the subtypes were in the IIa subtype family, while one subtype was identified from the IId subtype family. The most common subtype was IIaA15G2R1, which was found in 34.2% of the C. parvum successfully subtyped samples. The probability of Cryptosporidium spp. associated diarrhea in cattle decreased significantly with the age of the animals and a prolonged period during which calves were fed with milk.

TIDE Analysis of Cryptosporidium Infections by gp60 Typing Reveals Obscured Mixed Infections.

BACKGROUND: Cryptosporidiosis is a parasitic disease associated with potentially fatal diarrhea. The most used method in Cryptosporidium subtyping is based on the glycoprotein gene gp60. Each infection can represent a parasite population, and it is important to investigate the influence on transmission and virulence, as well as any impact on public health investigations. However, an easy-to-use method for detection is lacking. METHODS: Here we report on the use of the bioinformatic program TIDE for deconvolution of gp60 chromatograms. A combination of single oocyst analysis and cloning successfully confirmed the within-sample parasite population diversity. Retrospective sample analysis was conducted on archived chromatograms. RESULTS: For Cryptosporidium parvum, 8.6% multistrain infections (13 of 152) obscured by currently used consensus base calling were detected. Importantly, we show that single oocysts can harbor a mixed population of sporozoites. We also identified a striking dominance of unappreciated polymerase stutter artefacts in all 218 chromatograms analyzed, challenging the uncritical use of gp60 typing. CONCLUSIONS: We demonstrate the value of a new, easy-to-use analytical procedure for critical characterization of C. parvum and Cryptosporidium hominis in epidemiological investigations, also applicable retrospectively. Our findings illuminate the hidden parasite diversity with important implications for tracing zoonotic and person-to-person transmissions.

Dual RNA-seq transcriptome analysis of caecal tissue during primary Eimeria tenella infection in chickens.

BACKGROUND: Coccidiosis is an infectious disease with large negative impact on the poultry industry worldwide. It is an enteric infection caused by unicellular Apicomplexan parasites of the genus Eimeria. The present study aimed to gain more knowledge about interactions between parasites and the host immune system during the early asexual replication phase of E. tenella in chicken caeca. For this purpose, chickens were experimentally infected with E. tenella oocysts, sacrificed on days 1-4 and 10 after infection and mRNA from caecal tissues was extracted and sequenced. RESULTS: Dual RNA-seq analysis revealed time-dependent changes in both host and parasite gene expression during the course of the infection. Chicken immune activation was detected from day 3 and onwards with the highest number of differentially expressed immune genes recorded on day 10. Among early (days 3-4) responses up-regulation of genes for matrix metalloproteinases, several chemokines, interferon (IFN)-γ along with IFN-stimulated genes GBP, IRF1 and RSAD2 were noted. Increased expression of genes with immune suppressive/regulatory effects, e.g. IL10, SOCS1, SOCS3, was also observed among early responses. For E. tenella a general up-regulation of genes involved in protein expression and energy metabolism as well as a general down-regulation genes for DNA and RNA processing were observed during the infection. Specific E. tenella genes with altered expression during the experiment include those for proteins in rhoptry and microneme organelles. CONCLUSIONS: The present study provides novel information on both the transcriptional activity of E. tenella during schizogony in ceacal tissue and of the local host responses to parasite invasion during this phase of infection. Results indicate a role for IFN-γ and IFN-stimulated genes in the innate defence against Eimeria replication.

Benzylpenicillin-producing Trichophyton erinacei and methicillin resistant Staphylococcus aureus carrying the mecC gene on European hedgehogs - A pilot-study.

BACKGROUND: A high carriage rate of methicillin-resistant Staphylococcus aureus with the mecC gene (mecC-MRSA) has been described among Wild European hedgehogs (Europeaus erineaus). Due to this frequent occurrence, it has been suggested that hedgehogs could be a natural reservoir for mecC-MRSA. However, the reason why hedgehogs carry mecC-MRSA remains unknown, but it has been hypothesized that mecC-MRSA could have evolved on the skin of hedgehogs due to the co-occurrence with antibiotic producing dermatophytes. The aim of this pilot-study was therefore to investigate if hedgehogs in Sweden carry Trichophyton spp. and to provide evidence that these dermatophytes are able to produce penicillin or similar substances. In addition, the study aimed to identify if dermatophytes co-occurred with mecC-MRSA. METHODS: Samples were collected from hedgehogs (Europeaus erineaus) that were euthanized or died of natural causes. All samples were screened for dermatophytes and mecC-MRSA using selective cultivation methods. Suspected isolates were characterized using PCR-based methods, genome sequencing and bioinformatic analyses. Identification of penicillin was performed by ultra-high-performance liquid chromatography-tandem mass spectrometry. RESULTS: In total 23 hedgehogs were investigated, and it was shown that two carried Trichophyton erinacei producing benzyl-penicillin, and that these hedgehogs also carried mecC-MRSA. The study also showed that 60% of the hedgehogs carried mecC-MRSA. CONCLUSION: The pilot-study demonstrated that Trichophyton erinacei, isolated from Swedish hedgehogs, can produce benzylpenicillin and that these benzylpenicillin-producing T. erinacei co-occurred with mecC-MRSA. The study also reconfirmed the high occurrence of mecC-MRSA among hedgehogs.

Cryptosporidium chipmunk genotype I - An emerging cause of human cryptosporidiosis in Sweden.

Most cases of cryptosporidiosis in humans are caused by Cryptosporidium parvum or Cryptosporidium hominis. However, more uncommon species are increasingly being recognised to cause infection in humans. Here we report that Cryptosporidium chipmunk genotype I, which has various rodents as its natural host, is the third most common source of human cryptosporidiosis in Sweden. We also describe the first small outbreak of cryptosporidiosis caused by Cryptosporidium chipmunk genotype I and report the first case of zoonotic transmission of Cryptosporidium chipmunk genotype I from a red squirrel to a human. Cryptosporidium chipmunk genotype I was identified in 20 human cases, including 16 sporadic cases, three outbreak-related cases, and one zoonotic case, as well as in two squirrel samples. Gp60 subtyping which was successful for 19 human cases and two squirrel samples showed that all samples harboured the same subtype, XIVaA20G2T1. The work presented here suggests that red squirrel is a natural host of Cryptosporidium chipmunk genotype I and that infection with Cryptosporidium chipmunk genotype I is an emerging cause of domestic cryptosporidiosis in Sweden and a potential source of outbreaks.

A novel fragmented mitochondrial genome in the protist pathogen Toxoplasma gondii and related tissue coccidia.

Mitochondrial genome content and structure vary widely across the eukaryotic tree of life, with protists displaying extreme examples. Apicomplexan and dinoflagellate protists have evolved highly reduced mitochondrial genome sequences, mtDNA, consisting of only three cytochrome genes and fragmented rRNA genes. Here, we report the independent evolution of fragmented cytochrome genes in Toxoplasma and related tissue coccidia and evolution of a novel genome architecture consisting minimally of 21 sequence blocks (SBs) totaling 5.9 kb that exist as nonrandom concatemers. Single-molecule Nanopore reads consisting entirely of SBs ranging from 0.1 to 23.6 kb reveal both whole and fragmented cytochrome genes. Full-length cytochrome transcripts including a divergent coxIII are detected. The topology of the mitochondrial genome remains an enigma. Analysis of a cob point mutation reveals that homoplasmy of SBs is maintained. Tissue coccidia are important pathogens of man and animals, and the mitochondrion represents an important therapeutic target. The mtDNA sequence has been elucidated, but a definitive genome architecture remains elusive.

CryptoGenotyper: A new bioinformatics tool for rapid Cryptosporidium identification.

Cryptosporidium is a protozoan parasite that is transmitted to both humans and animals through zoonotic or anthroponotic means. When a host is infected with this parasite, it causes a gastrointestinal disease known as cryptosporidiosis. To understand the transmission dynamics of Cryptosporidium, the small subunit (SSU or 18S) rRNA and gp60 genes are commonly studied through PCR analysis and conventional Sanger sequencing. However, analyzing sequence chromatograms manually is both time consuming and prone to human error, especially in the presence of poorly resolved, heterozygous peaks and the absence of a validated database. For this study, we developed a Cryptosporidium genotyping tool, called CryptoGenotyper, which has the capability to read raw Sanger sequencing data for the two common Cryptosporidium gene targets (SSU rRNA and gp60) and classify the sequence data into standard nomenclature. The CryptoGenotyper has the capacity to perform quality control and properly classify sequences using a high quality, manually curated reference database, saving users' time and removing bias during data analysis. The incorporated heterozygous base calling algorithms for the SSU rRNA gene target resolves double peaks, therefore recovering data previously classified as inconclusive. The CryptoGenotyper successfully genotyped 99.3% (428/431) and 95.1% (154/162) of SSU rRNA chromatograms containing single and mixed sequences, respectively, and correctly subtyped 95.6% (947/991) of gp60 chromatograms without manual intervention. This new, user-friendly tool can provide both fast and reproducible analyses of Sanger sequencing data for the two most common Cryptosporidium gene targets.

Surveillance of foodborne parasitic diseases in Europe in a One Health approach.

In 2012, WHO/FAO ranked 24 foodborne parasites (FBP) using multicriteria decision analysis (MCDA) to provide risk assessors with a basis for prioritising control of highly ranked FBP on the global level. One conclusion was that ranking may differ substantially per region. In Europe, the same methodology was used to rank FBP of relevance for Europe. Of the 24 FBP, the top-five prioritised FBP were identified for Europe as Echinococcus multilocularis, Toxoplasma gondii, Trichinella spiralis, E. granulosus, and Cryptosporidium spp., all of which are zoonotic. The objective of the present study was to provide an overview of surveillance and reporting systems in Europe for these top five prioritised FBP in the human and animal populations, to identify gaps, and give recommendations for improvement. Information on the surveillance systems was collected from 35 European countries and analysed according to the five different regions. For most FBP, human surveillance is passive in most countries and regions in Europe and notification differs between countries and regions. Adequate surveillance programmes for these FBP are lacking, except for T. spiralis, which is notifiable in 34 countries with active surveillance in susceptible animals under EU directive. Although human and animal surveillance data are available for the five prioritised FBP, we identified a lack of consistency in surveillance and reporting requirements between national experts and European bodies. Recommendations for improved surveillance systems are discussed.

Dual RNA-Seq transcriptome analysis of chicken macrophage-like cells (HD11) infected in vitro with Eimeria tenella.

The study aimed to monitor parasite and host gene expression during the early stages of Eimeria tenella infection of chicken cells using dual RNA-Seq analysis. For this, we used chicken macrophage-like cell line HD11 cultures infected in vitro with purified E. tenella sporozoites. Cultures were harvested between 2 and 72 h post-infection and mRNA was extracted and sequenced. Dual RNA-Seq analysis showed clear patterns of altered expression for both parasite and host genes during infection. For example, genes in the chicken immune system showed upregulation early (2­4 h), a strong downregulation of genes across the immune system at 24 h and a repetition of early patterns at 72 h, indicating that invasion by a second generation of parasites was occurring. The observed downregulation may be due to immune self-regulation or to immune evasive mechanisms exerted by E. tenella. Results also suggested pathogen recognition receptors involved in E. tenella innate recognition, MRC2, TLR15 and NLRC5 and showed distinct chemokine and cytokine induction patterns. Moreover, the expression of several functional categories of Eimeria genes, such as rhoptry kinase genes and microneme genes, were also examined, showing distinctive differences which were expressed in sporozoites and merozoites.

Sarcoptic mange in the wild boar, Sus scrofa, in Sweden.

Sarcoptic mange caused by Sarcoptes scabiei has been present in the Swedish red fox (Vulpes vulpes) population since the 1970s. The disease has been described in other Swedish wildlife species, but not in the wild boar, Sus scrofa, until 2009. Single cases of sarcoptic mange have been diagnosed the last years in the expanding population of wild boar. This study aims to describe the histopathological lesions found on mangy wild boar and compare, by molecular methods, mites from wild boar cases with mites from mangy red foxes, raccoon dogs, and domestic pigs. Mangy wild boar with focal alopecia and clinical signs of pruritis were reported or submitted from various areas in southern Sweden to the National Veterinary Institute, Uppsala. The examined skin samples of wild boar infected with S. scabiei showed limited gross skin lesions, except for cases with severe exudative dermatitis. Histopathology of the affected wild boar skin samples showed an eosinophilic dermatitis with a variable hyperkeratosis and often low number of mites present. To study the relationship of S. scabiei mites isolated from different host species, a population genetics investigation was performed based on microsatellite markers. In total, 225 individual mites from eight individuals of four different host species; red fox (48 mites), wild boar (80 mites), domestic pig (48 mites) and raccoon dog (43 mites), were included in the study. In the phylogenetic analysis, all mites isolated from wild boar clustered together even though they originate from different geographical regions in Sweden. Mites from each individual host showed high similarity. The results indicate that wild boar mites differ from mites both from the red fox, raccoon dog, and domestic pig.

Subtype distribution of zoonotic pathogen Cryptosporidium felis in humans and animals in several countries.

Cryptosporidium felis is the major etiologic agent of cryptosporidiosis in felines and has been reported in numerous human cryptosporidiosis cases. Sequence analysis of the 60-kDa glycoprotein (gp60) gene has been developed for subtyping C. felis recently. In this study, 66 C. felis isolates from the United States, Jamaica, Peru, Portugal, Slovakia, Nigeria, Ethiopia, Kenya, China, India and Australia were subtyped using the newly established tool. Forty-four specimens yielded gp60 sequences, generating 23 subtypes clustered in 4 subtype families (XIXa, XIXc, XIXd and XIXe) with high bootstrap support in a phylogenetic analysis of sequence data. Among them, XIXa showed high genetic diversity at the nucleotide level, with the formation of 18 subtypes from both cats and humans with different geographic distribution. In contrast, all 11 XIXd isolates derived from humans from various countries had identical sequences. Results of this study improve our understanding of the genetic diversity, host specificity and transmission dynamics of C. felis.

Detection and molecular characterisation of Cryptosporidium spp. in Swedish pigs.

BACKGROUND: Cryptosporidium is a genus of apicomplexan parasites that cause enteric disease in vertebrates. In pigs, infections are most often asymptomatic, but may result in diarrhoea and poor growth. The most common species detected in pigs are C. suis and C. scrofarum with low zoonotic potential. C. parvum, with higher zoonotic potential, may also be found. As previous knowledge on the occurrence of Cryptosporidium in Swedish pigs is scarce, this was investigated in our study. Faecal samples from 13 pig herds were collected and a total of 222 pooled pen samples, from suckling piglets (n = 48), growers, aged 6-12 weeks (n = 57), fatteners, aged 13-24 weeks (n = 67) and adult animals (n = 50) were included. Samples were analysed using microscopy and positive samples were further analysed using polymerase chain reaction and sequencing of the 18S rRNA gene and the 28S rRNA gene to determine species. RESULTS: Cryptosporidium spp. were detected in all sampled herds and in 25% (56/222) of the individual pen samples. Infections were most common in growers and fatteners with 51% (29/57) and 35% (20/67) positive samples in each group, respectively. The piglets had 8% (4/48) positive samples and adults had 6% (3/50). Species determination showed C. suis and C. scrofarum in piglets and growers, C. scrofarum in the fatteners, and C. suis and C. parvum in the adults. Although no mixed infections could be confirmed we saw signs of double peaks in the 28S rRNA gene chromatograms, possibly indicating more than one species present per sample. CONCLUSION: Cryptosporidium spp. were detected on every sampled farm and in 25% of the individual pen samples in our study. We therefore conclude that Cryptosporidium spp. are present and likely common in Swedish pig herds, where pigs are loose and reared on solid floors. However, none of the farms reported any problems with poor weight gain, diarrhoea, or reduced appetite in their pig herds. The pig adapted C. suis and C. scrofarum were the predominant species identified. Two samples were positive for the more zoonotic C. parvum, and pigs should hence not be disregarded as a possible source of zoonotic cryptosporidiosis.

A single-cohort study of Cryptosporidium bovis and Cryptosporidium ryanae in dairy cattle from birth to calving.

In order to investigate the infection dynamics of Cryptosporidium bovis and C. ryanae, a two-year prospective cohort study was performed on a dairy farm known to be free of C. parvum. Sixteen calves were recruited when newborn. Faecal samples were collected weekly until calves were nine weeks old, then monthly until calving or culling. Samples (n = 455) were examined by fluorescence microscopy, and when positive the species were determined by DNA sequencing. In calves up to nine weeks, C. bovis was found in 58.5% of the samples, C. ryanae in 9.2%, and both C. bovis and C. ryanae in 3.1%. The prevalence of shedding calves peaked at 87.5% in week five, which is earlier than many international studies have shown for C. bovis. The cumulative incidence of C. bovis reached 100% when the calves were five weeks old. In four calves, the species detected changed from C. bovis to C. ryanae or the other way around, and two samples were a combination of both species. Several individuals shed oocysts sporadically up to 16 months of age. The highest oocysts per gram faeces count was seen in week three (3.6 × 106 OPG). Diarrhoea was not associated with oocyst shedding.

Why do we need training? - A "Training school on molecular methods used for foodborne parasite diagnostics in different matrices" is a example of knowledge transfer to foster research quality in EU.

Foodborne parasites with zoonotic potential are of particular concern for human health, being responsible for serious and potentially life threatening diseases. In the last decades, the development of molecular biology techniques have been successfully implemented for clinical diagnosis of FBPs in animal or human samples providing cheaper, less labor intensive, reliable and more sensitive tests. It is apparent from recent publications that unsubstantiated molecular methods for parasite detection that have undergone scant evaluation for sensitivity and specificity are becoming increasingly common. The aim of the organized Training Schools was to transfer knowledge on application, optimization and troubleshooting for methods used to extract, amplify, and sequence nucleic acids from contaminated matrices and isolated FBPs. The organized Training Schools fulfilled the trainees' expectations, whom acquired useful knowledge for their research activities.

Development of a gp60-subtyping method for Cryptosporidium felis.

BACKGROUND: Feline cryptosporidiosis is an increasing problem, especially in catteries. In humans, close contact with cats could be a potential source of infection although the risk of contracting cryptosporidiosis caused by Cryptosporidium felis is considered to be relatively low. Sequencing of the 60-kDa glycoprotein gene is a commonly used tool for investigation of the genetic diversity and transmission dynamics of Cryptosporidium species. However, until now the sequence of gp60 from C. felis has not been available and genotyping has been limited to less discriminatory markers, such as 18S rRNA, COWP and HSP70. METHODS: We have identified the gp60 orthologue within the genome sequence of C. felis, and used the sequence to design a nested PCR for subtyping purposes. A total of 128 clinical isolates of both feline and human origin, were used to evaluate the marker. RESULTS: Sequence analysis revealed large variations between the different samples. The C. felis gp60 lack the characteristic serine-tract found in many other cryptosporidian orthologues, instead it has an insertion of variable length (361-742 nt). Also, two cases of suspected zoonotic transmission of C. felis between cats and humans were successfully confirmed. CONCLUSIONS: We have identified the gp60 gene in C. felis and show how this highly variable marker can be used in epidemiological investigations.

Infection dynamics of Cryptosporidium bovis and Cryptosporidium ryanae in a Swedish dairy herd.

In order to investigate the infection dynamics of Cryptosporidium bovis and Cryptosporidium ryanae, a Swedish dairy farm known to be free of C. parvum was recruited for a one-year study. Fecal samples were collected every four weeks (a total of 13 occasions) from 20 randomly selected calves younger than 65 days. A total of 238 feces samples were collected directly from the rectum and then examined with immunofluorescence microscopy; those positive for Cryptosporidium spp. oocysts were further processed by molecular species determination. Oocysts were found in 92 samples, and in 72 of these the species was successfully determined: 87.5% C. bovis, 9.7% C. ryanae and 2.8% a mix of both species. No C. parvum was found. The prevalence of shedding calves was highest at ages 4 and 5 weeks; however, the feces with the highest oocyst per gram (OPG) were registered at 2-4 weeks of age. This is earlier than what has been found in most other studies, indicating that the infection dynamics of C. bovis/C. ryanae may be affected by the presence of C. parvum. The OPG for the positive calves ranged from 200 to 1.1 × 106 (geometric mean 6.88 × 103). The youngest calf in which C. bovis was identified was 5 days old, and the youngest calf in which C. ryanae was identified was 15 days old. Calves' housing type and seasonality were not associated with differences in the shedding of oocysts. Furthermore, there was no association between the presence of diarrhea and oocyst shedding.

Infection dynamics of Cryptosporidium bovis and Cryptosporidium ryanae in a Swedish dairy herd.

In order to investigate the infection dynamics of Cryptosporidium bovis and Cryptosporidium ryanae, a Swedish dairy farm known to be free of C. parvum was recruited for a one-year study. Fecal samples were collected every four weeks (a total of 13 occasions) from 20 randomly selected calves younger than 65 days. A total of 238 feces samples were collected directly from the rectum and then examined with immunofluorescence microscopy; those positive for Cryptosporidium spp. oocysts were further processed by molecular species determination. Oocysts were found in 92 samples, and in 72 of these the species was successfully determined: 87.5% C. bovis, 9.7% C. ryanae and 2.8% a mix of both species. No C. parvum was found. The prevalence of shedding calves was highest at ages 4 and 5 weeks; however, the feces with the highest oocyst per gram (OPG) were registered at 2-4 weeks of age. This is earlier than what has been found in most other studies, indicating that the infection dynamics of C. bovis/C. ryanae may be affected by the presence of C. parvum. The OPG for the positive calves ranged from 200 to 1.1 × 106 (geometric mean 6.88 × 103). The youngest calf in which C. bovis was identified was 5 days old, and the youngest calf in which C. ryanae was identified was 15 days old. Calves' housing type and seasonality were not associated with differences in the shedding of oocysts. Furthermore, there was no association between the presence of diarrhea and oocyst shedding.