Seasonal variation in the prevalence and molecular epidemiology of Cryptosporidium infection in dairy cattle in the New York City Watershed

We conducted cross-sectional studies in the New York City Watershed to ensure a valid estimate of the risk associated with Cryptosporidium infection in dairy herds. Our aims were to obtain species-specific estimates of the prevalence of Cryptosporidium in dairy cattle and to investigate seasonal variations in prevalence. We validated our empirical estimates using a Bayesian approach. Samples were collected on 32 study farms, once in each of 3 different seasons using an age-stratified sampling design. The overall prevalence of Cryptosporidium parvum-like species and Cryptosporidium andersoni among the 1911 animals tested by the flotation method was 5% and 1%, respectively. Among preweaned calves (<65 days of age), the prevalence of C. parvum-like species was twice as high in the summer (26%) compared with the winter (11%). Herd prevalence showed the same seasonal trend. Preweaned calves were also shedding C. andersoni at an average intensity of 20 oocysts per gram of feces. We did not detect C. parvum-like oocysts in cattle older than 5 months. Sequencing of a portion of the 18s rRNA gene revealed that in the summer, 42% of the C. parvum-like oocysts shed by preweaned calves were zoonotic, compared with >74% during the rest of the year. Both empirical and stochastic methods revealed a summer peak in the prevalence of C. parvum-like oocysts in preweaned calves. Determining whether seasonal variation in the prevalence and proportion of Cryptosporidium species shed by preweaned calves is due to management practices or ecological factors will have important implications for effective control of this parasite.     

CP2 gene as a useful viability marker for Cryptosporidium parvum

The validity of the CP2 gene of Cryptosporidium parvum as a viability marker was evaluated using absolute quantitative real-time polymerase chain reaction (qPCR) assays. Total ribonucleic acid (RNA) was isolated from live and heat-killed C. parvum oocysts, and complementary deoxyribonucleic acid was synthesized and used as a template. The most accurate number of viable C. parvum oocysts was predicted when the CP2 gene was used as a target gene. The lower detection limit of the CP2 gene was ten oocysts, which was the most sensitive among examined target genes. With heat shock induction, only hsp70 messenger RNA (mRNA) was induced, and the predicted viable oocyst number was increased by heat shock for this marker. The CP2, hsp70, Cryptosporidium oocyst wall protein, and β-tubulin mRNAs were not detected in heat-killed oocysts, but the 18S ribosomal ribonucleic acid (rRNA) showed heat stability until 48 h after heat killing. Although the 18S rRNA demonstrated the fastest response in crossing point (CP) value among the examined primer sets in qPCR, overestimation of viable oocysts was noted in the analysis with this gene. In conclusion, the CP2 gene was identified as the most sensitive, reliable, and accurate candidate of a viability marker of C. parvum by qPCR evaluation.     

Molecular characterization of Cryptosporidium isolates from high-excreting young dairy calves in dairy cattle herds in Western France

Ninety-two Cryptosporidium sp.-positive fecal samples of dairy diarrheic or non-diarrheic calves from 30 cattle herds in Normandy (France) were selected. Here, the aim was to investigate the species of Cryptosporidium excreted as well as the subtypes of Cryptosporidium parvum found in 7–17-day-old dairy calves. Excretion levels were comprised between 2?×?10⁴ and 4?×?10⁷ oocysts per gram of feces. Here, a nested 18S SSU rRNA PCR associated with sequencing was performed for identification of Cryptosporidium species and revealed the presence of C. parvum in most cases (80/82), except for two animals which were infected with Cryptosporidium bovis. Then, C. parvum samples were submitted to gp60 PCR. For 39 samples from 24 different herds, a multilocus analysis based on four mini-microsatellites loci (MM19, MM5, MSF, and MS9-Mallon) were conducted. These results were combined with sequence analysis of the gp60 to obtain multilocus types (MLTs). Here, C. parvum gp60 genotyping identified three subtypes in the IIa zoonotic allele family: IIaA15G2R1 (88 %), IIaA16G3R1 (10 %), and IIaA19G2R1 (2 %), and we identified 12 MLTs. The MS9-Mallon locus was reported as the most polymorphic (five alleles). The most common MLT was MLT 1 with 15 samples in 10 farms: (MS9-M: 298, MSF: 165, MM5: 264, MM19: 462, and gp60 subtype: IIaA15G2R1). When comparing diarrheic and non-diarrheic fecal samples, no difference was seen for distribution of Cryptosporidium species, C. parvum gp60 subtypes, and MLTs. Here, in a range of oocyst excretion of 10⁴–10⁷ opg, both in diarrheic and non-diarrheic calves, infection was mainly due to C. parvum and to the zoonotic subtype: IIaA15G2R1.     

Cryptosporidium infection in livestock and first identification of Cryptosporidium parvum genotype in cattle feces in Taiwan

Fecal survey by modified Ziehl-Neelsen (MZN) method and immunofluorescence assay (IFA) of Cryptosporidium infection in cattle and goats in Taiwan showed a prevalence of 37.6% (173/460) and 35.8% (44/123), respectively. In addition to the calves, adult cattle were also found to be shedding Cryptosporidium oocyst. No significant difference was observed between diarrheic and non-diarrheic cattle feces with regard to the presence of Cryptosporidium oocyst. Two groups of oocysts with different diameter sizes, possibly indicating two different species, were observed in the cattle feces. By PCR analysis using primers directed against the 18S rRNA gene, followed by sequencing of the amplicon, we were able to confirm that one of the oocyst species belong to that of the bovine genotype of Cryptosporidium parvum. This is the first identification of a genotype of C. parvum oocyst in the feces of cattle in Taiwan.     

Improved immunofluorescence assay for detection of Giardia and Cryptosporidium from asymptomatic adult cervine animals

We tested an improved immunofluorescence assay (IFA) in detecting Giardia and Cryptosporidium from feces of asymptomatic adult cervine animals. Samples were concentrated by sucrose flotation before being stained by fluorescent monoclonal antibody and examined microscopically. The detection limit was determined as 500 G. intestinalis cysts or 200 C. parvum oocysts/g of sample. Among the 82 samples collected from adult fallow deer, Columbian black-tailed deer, and Tule elk in northern California, 3 (3.7%) contained G. intestinalis cysts, which were confirmed by a species-specific polymerase chain reaction (PCR) following immunomagnetic capture (IC) of cysts. C. parvum oocysts were detected in a total of 13 (15.9%) samples, and oocysts from 2 such samples were smaller than oocysts from the other 11 samples. C. parvum identification was also confirmed by specific IC-PCR and sequencing of the PCR product. In addition, a C. muris-like organism was detected in 2 (2.4%) samples. Findings obtained with the improved IFA confirmed that cysts/oocysts may pass unnoticed in adult cervine animals and that subclinically infected individuals could serve as potential carriers of infection for humans and other animals via contaminated feces or water. Received: 19 January 1999 / Accepted: 29 March 1999     

Multicentric Evaluation of a New Real-Time PCR Assay for Quantification of Cryptosporidium spp. and Identification of Cryptosporidium parvum and Cryptosporidium hominis

Cryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocompromised patients. Laboratory diagnosis of cryptosporidiosis relies on microscopy, antigen detection, and nucleic acid detection and analysis. Among the numerous molecular targets available, the 18S rRNA gene displays the best sensitivity and sequence variations between species and can be used for molecular typing assays. This paper presents a new real-time PCR assay for the detection and quantification of all Cryptosporidium species associated with the identification of Cryptosporidium hominis and Cryptosporidium parvum. The sensitivity and specificity of this new PCR assay were assessed on a multicentric basis, using well-characterized Cryptosporidium-positive and -negative human stool samples, and the efficiencies of nine extraction methods were comparatively assessed using Cryptosporidium-seeded stool samples and phosphate-buffered saline samples. A comparison of extraction yields showed that the most efficient extraction method was the Boom technique in association with mechanical grinding, and column extraction showed higher binding capacity than extraction methods based on magnetic silica. Our PCR assay was able to quantify at least 300 oocysts per gram of stool. Satisfactory reproducibility between laboratories was observed. The two main species causing human disease, Cryptosporidium hominis and Cryptosporidium parvum, were identified using a duplex real-time PCR assay with specific TaqMan minor-groove-binding ligand (MGB) probes for the same amplicon. To conclude, this one-step quantitative PCR is well suited to the routine diagnosis of cryptosporidiosis since practical conditions, including DNA extraction, quantification using well-defined standards, and identification of the two main species infecting humans, have been positively assessed.     

Description of fecal shedding of Cryptosporidium parvum oocysts in experimentally challenged dairy calves

The objective was to describe the probability of Cryptosporidium parvum fecal oocyst shedding at different magnitudes of exposure, the pattern of fecal shedding over time, and factors affecting fecal shedding in dairy calves. Within the first 24 h of life, 36 calves were experimentally challenged with C. parvum oocysts at one of four possible magnitudes of oral exposure (1?×?10³, 1?×?10⁴, 1?×?10⁵, and 1?×?10⁶ oocysts), and 7 control calves were sham dosed. Fecal shedding occurred in 33 (91.7 %) experimentally challenged calves and in none of the control calves. There was a difference in the log-total number of oocysts counted per gram of feces dry weight among the four exposure groups; calves with the lowest magnitude of exposure (1?×?10³ oocysts) shed less than the other three groups. At higher magnitudes of exposure, there was more variability in the range of fecal oocyst shedding. There was an inverse relationship between the log-total amount of oocysts counted per gram of feces dry weight and the number of days to the onset of fecal shedding per calf, i.e., the more time that elapsed to the onset of fecal shedding, the fewer oocysts that were shed. The pattern of fecal shedding over time for all calves shedding oocysts was curvilinear; the number of oocysts increased with time, reached a peak, and declined. Therefore, the dynamics of oocyst shedding can be influenced in part by limiting exposure among calves and delaying the onset of fecal oocyst shedding.     

Application of recombinant Cryptosporidium parvum P23 for isolation and prevention

Cryptosporidium parvum is a coccidian protozoan that causes diarrhea in immunocompromised humans and newborn animals. Billions of oocysts of C. parvum can be released from the infected calves and can contaminate the environment. The severity of the disease depends on the immunological status of the individual. Oocysts of Cryptosporidium are extremely resistant to many environmental stresses, and no effective disinfectant and curative agent against this organism is available. In our study, recombinant C. parvum P23 was prepared for application in the isolation and prevention of cryptosporidiosis. P23 is a glycoprotein that belongs to a family of protein of 23–27 kDa and is believed to be expressed in the different life stages of the parasite. Immunostaining analysis using the indirect fluorescent antibody test showed that P23 could be recognized on the surface of the oocysts. The antibody prepared in rabbit against P23 was bound to Sepharose 4B and used for the isolation of oocysts. The results showed that the prepared column was able to bind specifically only the oocysts. The effect of specific recombinant C. parvum IgY antibody against infection with C. parvum was examined in a mouse model. For this aim, purified egg yolk antibody prepared from immunized hens was used to analyze the protective effect of recombinant P23 specific antibody in immunosuppressed adult mice. The results showed more than 70 % reduction in oocyst shedding after challenge with 1?×?10⁴ oocysts. These results support previous studies of other investigators regarding the protective effect of P23 as an antigen against C. parvum infection and showed that it could be possible to design a passive immunization strategy against C. parvum based on the anti-P23 yolk antibody in animals and immunosuppressed humans.     

Assessment of cryptodiag for diagnosis of cryptosporidiosis and genotyping Cryptosporidium species

The performance of a new commercial PCR-enzyme-linked immunosorbent assay (ELISA) (Cryptodiag; Bio Advance, France) for the diagnosis of cryptosporidiosis and the identification of Cryptosporidium hominis and C. parvum from stool samples was examined. This test is based on PCR amplification of Cryptosporidium DNA extracted from stools, followed by an ELISA based on hybridization with Cryptosporidium sp.-, C. hominis-, or C. parvum-specific probes. In spiking experiments, approximately five oocysts were detected either in water or in stool suspensions while assessing for the efficient removal of stool PCR inhibitors. No cross-reactivity was observed in the detection of C. parvum and C. hominis using the respective specific probes. Thirty-three fecal samples from patients with microscopically proven cryptosporidiosis and 118 from patients with or without other digestive protozoan infections were tested by Cryptodiag, blinded to the results of microscopy. Compared to microscopy, the sensitivity of Cryptodiag was 97.0% (32/33) and 100% (33/33), including the gray zone, and specificity was 98.3% (116/118) and 96.6% (114/118), including the gray zone. Among 34 positive results, Cryptodiag identified 19 due to C. hominis, 8 due to C. parvum, and 7 due to Cryptosporidium spp. Genotyping by Cryptodiag agreed with reference typing methods in 85% of cases of C. parvum or C. hominis infections. Cryptodiag proved to be reliable and sensitive for the diagnosis of cryptosporidiosis. The use of specific probes allowed the identification of C. hominis and C. parvum, i.e., the two main species responsible for human cryptosporidiosis, and rapidly provided information on the possible source of infection.     

Cryptosporidium oocysts in Bent mussels (Ischadium recurvum) in the Chesapeake Bay

Filter-feeding molluscan shellfish can concentrate environmentally derived waterborne pathogens of humans, which can be utilized in the sanitary assessment of water quality. In the present study, oocysts of Cryptosporidium were detected in Bent mussels (Ischadium recurvum) at two Chesapeake Bay sites from which C. parvum-contaminated oysters had previously been collected. Spiking of Cryptosporidium-free blue mussel (Mytilus edulis) tissue with C. parvum oocysts showed a 51.1% recovery rate of oocysts, giving an oocyst detection limit of 19 oocysts/0.7 ml of mussel tissue homogenate. The results indicate that Bent mussels, which are common throughout the Chesapeake Bay region, may prove to be useful as biological indicators of water contamination with Cryptosporidium oocysts. Received: 24 November 1998 / Accepted: 20 January 1999     

The first detection of <Emphasis Type="Italic">Cryptosporidium</Emphasis> deer-like genotype in cattle in Japan

The general perception is that cattle are major reservoirs for Cryptosporidium parvum infections in humans and that C. parvum is a major cause of diarrhea and production loss in cattle. Adult cattle may play an important role as cryptic carrier of the infection. Cryptosporidium spp. in asymptomatic adult dairy cattle from some farms around Osaki area, Miyagi prefecture, Japan, was examined on a field visit during August, 2007, by polymerase chain reaction techniques for detection, genotyping, and subtyping. Cryptosporidium oocysts were detected in the feces of five out of 50 animals. Of the five Cryptosporidium-positive specimens available for molecular analysis, C. parvum was identified in three specimens, Cryptosporidium deer-like genotype in one, and Cryptosporidium andersoni in one specimen. Amplification of Cpgp60 from C. andersoni and Cryptosporidium deer-like genotype samples revealed that these samples have light concurrent C. parvum infection. Sequence analysis of the 60-kDa glycoprotein gene indicated that all C. parvum samples are IIa subtype. Detection of Cryptosporidium deer-like genotype is geographically unique in Japan. The genetic diversity of Cryptosporidium in dairy cattle in Japan may be much greater than that reported before.     

Detection of Cryptosporidium parvum in Horses: Thresholds of Acid-Fast Stain, Immunofluorescence Assay, and Flow Cytometry

Feces collected from three asymptomatic horses and seeded with Cryptosporidium parvum oocysts (10¹ to 10⁶/g of feces) were evaluated by acid-fast staining (AF), an immunofluorescent antibody (IFA) technique, and flow cytometry. The thresholds of detection were 5 × 10⁵ oocysts/g of feces for the IFA and AF techniques and 5 × 10⁴ oocysts/g for flow cytometry.     

Antibody Response of Healthy Adults to Recombinant Thrombospondin-Related Adhesive Protein of Cryptosporidium 1 after Experimental Exposure to Cryptosporidium Oocysts

Thrombospondin-related adhesive protein of Cryptosporidium 1 (TRAP-C1) belongs to a group of proteins that are also found in Toxoplasma gondii, Eimeria tenella, and Plasmodium species. TRAP-related proteins are needed for gliding motility, host-cell attachment, and invasion. The objective of this study was to characterize the antibody response to recombinant TRAP-C1 (rTRAP-C1) in healthy volunteers exposed to C. parvum and their association with clinical illness. A total of 31 healthy adult volunteers participated. Seven volunteers received the C. parvum TAMU isolate (inocula, 10 to 300 oocysts), and 24 volunteers received the C. parvum UCP isolate (500 to 10⁵ oocysts). The total antibody (immunoglobulin M [IgM], IgG, and IgA) response to rTRAP C-1 was measured by enzyme-linked immunosorbent assays prior to and after exposure to Cryptosporidium parvum (days 0 to 45). Results of this study showed that individuals who were uninfected demonstrated higher reactivity at baseline compared to those who became infected. After challenge, increases in antibody reactivity were seen on days 30 and 45 compared to the results seen on days 0 to 5. The increases in antibody reactivity were statistically significant in subjects with diarrhea and with or without detectable oocysts compared to the results seen with those who were uninfected and asymptomatic. These findings suggest that increases in antibody reactivity to rTRAP-C1 occur after recent exposure to C. parvum.     

Molecular characterization of Cryptosporidium spp. in dairy calves from the state of São Paulo, Brazil

Cryptosporidiosis is a common protozoan disease observed in a wide range of vertebrate hosts, including ruminants. Cattle can be a potential reservoir of Cryptosporidium spp., leading to environmental contamination with oocysts of zoonotic species. The molecular characterization of Cryptosporidium spp. isolated from cattle from the state of S?o Paulo, Brazil, was accomplished using nested polymerase chain reaction for amplification of fragments of the 18S rRNA gene and the glycoprotein GP60 gene, following sequencing of amplified fragments. Positivity for Cryptosporidium was found in 10.7% (21/196) of the samples. Four species of Cryptosporidium were identified: C. andersoni, C. bovis, C. parvum subtype IIaA15G2R1, and C. ryanae. To the best of our knowledge, this is the first report of infection by C. ryanae and C. parvum IIaA15G2R1 in cattle from Brazil.     

An evaluation of primers amplifying DNA targets for the detection of <Emphasis Type="Italic">Cryptosporidium</Emphasis> spp. using <Emphasis Type="Italic">C. parvum</Emphasis> HNJ-1 Japanese isolate in water samples

The performance of polymerase chain reaction (PCR) procedures for the detection of Cryptosporidium parvum HNJ-1 strain (genotype II) oocysts purified from mice using published protocols was evaluated. Oocysts were concentrated from fecal samples of infected severe combined immunodeficiency (SCID) mice by sucrose flotation and were then purified by immunomagnetic separation method. The genotype of C. parvum was established as type II by restriction fragment length polymorphism (RFLP) analysis. Water samples were spiked with different numbers of oocysts, determined by limiting dilution. Genomic DNA was extracted and used for PCR assays targeting various Cryptosporidium species genes (Beta-Tubulin, COWP, 70 kDa HSP, SSU rRNA, ITS1, TRAP-C1 and TRAP-C2 gene). DNA from oocyst numbers of more than 1 × 10⁴ was detected using each of the primers. However, when using lower oocyst numbers, the tools based on 9 of the 16 different primer assays gave sufficient results. Assays using the remaining seven primers gave less than satisfactory results. A new primer set, named VKSS-F1/2 and VKSS-R1/2, that target the 18 SSU rRNA gene of C. parvum was constructed and applied.The VKSS-F1/2 and VKSS-R1/2 assays amplified DNA isolated from spiked samples in 206 of 211 trials (97.6%). This illustrates the difficulty of detecting low numbers of Cryptosporidium spp. oocysts by molecular methods when working with environmental samples.     

Quantitative assessment of viable Cryptosporidium parvum load in commercial oysters (Crassostrea virginica) in the Chesapeake Bay

The epidemiological importance of increasing reports worldwide on Cryptosporidium contamination of oysters remains unknown in relation to foodborne cryptosporidiosis. Thirty market-size oysters (Crassostrea virginica), collected from each of 53 commercial harvesting sites in Chesapeake Bay, MD, were quantitatively tested in groups of six for Cryptosporidium sp. oocysts by immunofluorescent antibody (IFA). After IFA analysis, the samples were retrospectively retested for viable Cryptosporidium parvum oocysts by combined fluorescent in situ hybridization (FISH) and IFA. The mean cumulative numbers of Cryptosporidium sp. oocysts in six oysters (overall, 42.1±4.1) were significantly higher than in the numbers of viable C. parvum oocysts (overall, 28.0±2.9). Of 265 oyster groups, 221 (83.4%) contained viable C. parvum oocysts, and overall, from 10–32% (mean, 23%) of the total viable oocysts were identified in the hemolymph as distinct from gill washings. The amount of viable C. parvum oocysts was not related to oyster size or to the level of fecal coliforms at the sampling site. This study demonstrated that, although oysters are frequently contaminated with oocysts, the levels of viable oocysts may be too low to cause infection in healthy individuals. FISH assay for identification can be retrospectively applied to properly stored samples.     

Susceptibility and Serologic Response of Healthy Adults to Reinfection with Cryptosporidium parvum

Healthy adults are susceptible to infection with small numbers of Cryptosporidium parvum oocysts, resulting in self-limited infection. We investigated if infection of humans with C. parvum is protective 1 year after primary exposure. At 1 year after a primary challenge with 30 to 10⁶ oocysts, 19 healthy immunocompetent adults were rechallenged with 500 oocysts and monitored for the development of infection and/or illness. Oocyst excretion was quantitated by direct immunofluorescence with a C. parvum-specific monoclonal antibody, and anti-C. parvum antibodies in serum were detected by an enzyme-linked immunosorbent assay. Fewer subjects shed oocysts after the second exposure (3 of 19; 16%) than after the first exposure (12 of 19; 63%) (P < 0.005). Although the rates of diarrhea were comparable after each of the two exposures, the clinical severity as determined by the mean number of unformed stools passed was lower after reexposure (11.25 versus 8.62; P < 0.05). The number of anti-Cryptosporidium immunoglobulin G and A seroconversions increased after secondary exposure. However, the C. parvum serum antibody response did not correlate with the presence or absence of infection.     

Molecular characterisation of Cryptosporidium: an emerging parasite

Purpose: The aim of the present study was to determine the prevalence of Crytposporidium in local population and to understand its epidemiology by molecular methods. Methods: Faecal samples from 681 children and 804 adults, admitted to tertiary care hospitals in twin cities of Hyderabad and Secunderabad with complaints of diarrhoea; and six calves with diarrhoea, were screened for Cryptosporidium oocysts by microscopy and enzyme linked immunosorbent assay (ELISA). Polymerase chain reaction restriction fragment length polymorphism (PCR RFLP) based identification of Cryptosporidium species in positive specimens was done to elucidate epidemiology of Cryptosporidium. Results: Cryptosporidium was found in 52 (7.6%) children and 7(0.9%) adults and 1(16.6%) calf with diarrhoea. The prevalence of Cryptosporidium in children below five years of age was 8.2% and 14.3% in children in the age group of six months to one year. Of the 42 samples genotyped 29 (69%) were C. hominis and 8 (19%) were C. parvum and 5 (11.9%) were mixed infection with the two species. Conclusions: Children in the age group of six months to one year were found to be the most vulnerable. The occurrence of C. parvum, in nearly one third of cases in the present series indicates that the zoonotic transmission is of considerable significance in the epidemiology of Cryptosporidiosis in the study area.     

Identification and determination of the viability of Giardia lamblia cysts and Cryptosporidium parvum and Cryptosporidium hominis oocysts in human fecal and water supply samples by fluorescent in situ hybridization (FISH) and monoclonal antibodies

In the present study, fluorescent in situ hybridization (FISH) and monoclonal antibodies (MAbs) were evaluated for species-specific detection and viability determination of Giardia lamblia, Cryptosporidium parvum, and Cryptosporidium hominis in human fecal and water supply samples. A total of 50 fecal human samples positive for G. lamblia cysts, 38 positive for C. parvum, and 23 positive for C. hominis were studied. Also, 18 water supply samples positive for Giardia spp. and Cryptosporidium spp. by the United States Environmental Protection Agency (USEPA) Method 1623 were studied by FISH and fluorescein isothiocyanate (FITC)-conjugated MAbs. Eighteen percent of the fecal samples parasitologically positive for G. lamblia presented viable and nonviable cysts, and 5% of those positive for Cryptosporidium spp. presented viable and nonviable oocysts. Of the 18 water supply samples analyzed, 6 (33%) presented Giardia spp. viable and nonviable cysts and 2 (11%) presented viable and nonviable Cryptosporidium spp. oocysts. G. lamblia identification was confirmed by polymerase chain reaction (PCR) and sequencing of the β-giardin gene in the fecal and water samples found positive by FISH and FITC-conjugated MAbs. C. parvum and Cryptosporidium muris were identified, by PCR and sequencing of the small subunit of ribosomal RNA gene, in seven and one water samples, respectively. Our results confirm that this technique enables simultaneous visualization, species-specific identification, and viability determination of the organisms present in human fecal and water supply samples.     

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.