Cryptosporidium infections in asymptomatic calves up to 4 months in Poland: a cross-sectional population study.

Cattle cryptosporidiosis is noted worldwide with varied frequency of infection prevalence depending on geographical, environmental and husbandry factors. In this study, the prevalence of Cryptosporidium infections in cattle was determined on the basis of molecular results obtained by testing 1601 faecal samples collected from calves up to 4 months of age housed in all Polish provinces from 2014 to 2018. Detection and identification of Cryptosporidium species was performed at the 18 small subunit ribosomal RNA (18S rRNA) locus by conducting PCR-RFLP analysis of the amplified DNA fragments. The prevalence of Cryptosporidium infections in the cattle population was 45.3% (CI 95%: 42.8-47.7; 725/1601). The infected animals were housed on 233/267 (87.3%) of monitored farms with regional prevalence ranging from 27.8 to 62%. The restriction pattern of 18S rRNA amplicons for positive samples was characteristic of C. parvum, C. bovis, C. ryanae, C. andersoni, and unexpectedly also of C. baileyi and C. suis. Infections of C. bovis and C. ryanae prevailed in the studied cattle population relegating C. parvum to third in prevalence. Likewise, mixed infections caused by C. bovis and C. ryanae as well as C. parvum and C. bovis were observed. A relationship between the infecting parasite species and animal breed was found. For instance, C. parvum prevailed in Black and White lowland breed, C. ryanae in Limousine cattle and C. andersoni in dairy animals of mixed dairy breeds. Furthermore, differences in prevalence of particular parasite species between cattle breeds were also shown.

Population genetics of Cryptosporidium parvum subtypes in cattle in Poland: the geographical change of strain prevalence and circulation over time.

BACKGROUND: Cryptosporidium parvum (C. parvum) is a cosmopolitan parasite that infects various livestock animals including cattle. Microsatellite typing tools for identification of C. parvum subtypes are currently employed to better understand the species-specific epidemiology of cattle cryptosporidiosis. The aim of this study was to analyse the population genetics of C. parvum strains infecting cattle and recognise geographical distribution and time-span correlations in subtype prevalence in Poland. In total, 1601 faecal samples were collected from 2014 to 2018 from healthy cattle from dairy, meat and mixed breeds at the age of 1 week to 4 months. The 267 farms visited were randomly selected and represented all Polish provinces. PCR-RFLP based identification of C. parvum at the 18 small subunit ribosomal RNA (SSU rRNA) locus was performed, followed by strain subtyping by GP60-PCR.  RESULTS: The overall prevalence of C. parvum in Polish cattle was estimated at 6.2% (100/1601). Animals below the age of 1 month were the major host for this parasite. Excluding one breed, that of dairy-meat mixed, there were no significant differences observed between breed and presence of C. parvum infections (95% TPIAll breeds: 1.67-73.53%; POPR = 0.05-0.95). Infected animals were detected in 15 out of 16 Polish provinces, with significant regional prevalence diffrences (Kruskal-Wallis rank sum test, Kruskal-Wallis χ2 = 13.46, p < 0.001). When the population genetics of C. parvum strains were analysed, 11 parasite subtypes from the IIa and IId genetic families were identified. Compared to other parasite strains, IIaA17G1R1 and IIaA17G2R1 appeared at statistically significantly higher frequency (F-test, F = 3.39; p = 0.0003). The prevalence of C. parvum subtypes in cattle was breed-related (Chi-squared test, χ2 = 143.6; p < 0.001). CONCLUSIONS: The analysis of the population genetics of C. parvum subtypes showed that strains from the IIa subtype family predominated in the tested cattle population. However, relations in changes of subtype prevalence and circulation over time were observed. They were associated with the disappearance of some strains and emergence of new variants from the same genetic family in different geographical locations.

Detection of Cryptosporidium parvum in a Red-Eared Slider Turtle (Trachemys scripta elegans), a Noted Invasive Alien Species, Captured in a Rural Aquatic Ecosystem in Eastern Poland.

PURPOSE: Little is known about cryptosporidiosis in turtles of invasive alien species (IAS) inhabiting European bodies of fresh water. In this article, we report an occurrence of Cryptosporidium parvum in a red-eared slider turtle (Trachemys scripta elegans) captured in a rural aquatic ecosystem in eastern Poland. METHODS: A pair of samples consisting of feces and scrapings of intestinal mucosa (taken during necropsy) were collected from 104 animals representing the four IAS turtle species red-eared slider, yellow-bellied slider, false map and Cumberland slider. The animals were trapped in running and standing freshwater ecosystems across the Lublin province. Parasite genomic DNA was extracted from samples using a modified alkali wash and a heat-lysis method and identification of the Cryptosporidium species was performed at the 18SSU rRNA and COWP loci. RESULTS: The presence of Cryptosporidium DNA was only detected in one sample of intestinal scraping collected from a red-eared slider. A phylogenetic analysis of a 18SSU rRNA gene fragment showed 100% sequence identity between the C. parvum strain isolated from the turtle and other C. parvum strains previously detected in cattle from the Lublin province. CONCLUSIONS: There was no clinical evidence that the red-eared slider turtle was truly infected rather than being merely a mechanical parasite carrier. Sporadic detection of this protozoan parasite in the studied population of IAS turtles could be associated with low natural occurrence of Cryptosporidium infections in this animal species. The results provide evidence for possible transmission of zoonotic Cryptosporidium species by IAS turtles.

Comparison of Cryptosporidium oocyst recovery methods for their applicability for monitoring of consumer-ready fresh shellfish.

Growing demand for fresh, unprocessed food favours the emergence of Cryptosporidium infections in humans. Mainly it is food of plant origin or unpasteurized milk which have been involved in food-borne outbreaks of cryptosporidiosis. So far consumption of shellfish contaminated with Cryptosporidium were not associated with human infections although such as possibility exists. In this study an attempt was undertaken to evaluate the analytical performance of three commonly used methods for recovery of Cryptosporidium oocysts from shellfish: i) pepsin digestion of shellfish in conjunction with immunomagnetic separation (IMS) of oocysts (method A), ii) pepsin-HCl treatment of shellfish homogenate without IMS (method B), and iii) a strainer method with direct oocyst extraction and separation from shellfish tissue using IMS (method C). Each method's performance was assessed according to the ISO standard requirements by testing shellfish homogenates seeded with different numbers of C. parvum oocysts. Two groups of parameters were compared, encompassing precision (coefficient of variation (CV)) and accuracy of measurements. These were described by linear regression models allowing calculation of the methods' limits of detection (LOD) and quantification (LOQ). In addition, oocyst recovery efficiencies from shellfish were calculated for each method. All three compared methods allowed for at least 66% recovery of Cryptosporidium oocysts from the tested samples. The best recovery (83.3-100%) in the whole range of tested suspensions was obtained for method C. The accuracy of method B was better (linearity of r2 = 0.9996 in the full measurement range) than that of method A (r2 = 0.968). Method C showed the best accuracy (r2 = 1) and precision (CV 0.2-14.1). Compared to other methods it was also characterised by the best LOD and LOQ, attaining ≅4 and ≅12 oocysts per 3 g of tested shellfish sample respectively. Despite a lack of the ability of method A to give the proportional results in oocysts recovery (non-linearity of the method) compared to the reference values, it achieved the highest LOD and LOQ values among the tested methods. As demonstrated here, the most efficient method for extraction of Cryptosporidium oocysts from shellfish tissues was method C employing sample homogenisation and separation of oocysts from tissue debris using IMS. Used alone this method does not in fact allow for identification of Cryptosporidium species but delivers quantitative results concerning the level of food contamination by parasites.

Diversity of Cryptosporidium species occurring in sheep and goat breeds reared in Poland.

The aim of this study was molecular identification of Cryptosporidium species and assessment of their prevalence in different breeds of sheep and goat reared in Poland. In addition, the relationship between animal age, breed type, and the frequency of Cryptosporidium infections was determined. Fecal samples from 234 lambs and 105 goat kids aged up to 9 weeks, representing 24 breeds and their cross-breeds were collected from 71 small ruminant farms across Poland. The identification of Cryptosporidium species was performed at the 18 SSU ribosomal RNA (rRNA) and COWP loci followed by subtyping of C. parvum and C. hominis strains at GP60 gene locus. The presence of Cryptosporidium DNA at the 18 SSU rRNA locus was detected in 45/234 (19.2%) lamb feces samples and in 39/105 (37.1%) taken from goats. The following Cryptosporidium species: C. xiaoi, C. bovis, C. ubiquitum, C. parvum, and C. hominis were detected in small ruminants. Infections caused by C. xiaoi were predominant without favoring any tested animal species. Subsequent GP60 subtyping revealed the presence of C. parvum IIaA17G1R1 subtype in sheep and IIdA23G1 subtype in goats. IIdA23G1 subtype was detected in a goat host for the first time. There were no significant differences found in frequency of infections between the age groups (<3 and 3-9 weeks) of lambs (P = 0.14, α > 0.05) or goat kids (P = 0.06, α > 0.05). In addition, there was no correlation observed between the frequency in occurrence of particular parasite species and breed type in relation to native sheep breeds (F = 0.11; P = 0.990 > 0.05). In the case of goats, more breed-related differences in parasite occurrence were found. The results of this study improve our knowledge on the breed-related occurrence of Cryptosporidium infections in the population of small ruminants reared in Poland.

Emergence of novel subtypes of Cryptosporidium parvum in calves in Poland.

The aim of the study was to identify the Cryptosporidium parvum subtypes circulating in Polish cattle and their distribution in relation to the age and health status of tested animals. In total, 779 fecal samples were obtained from young cattle originating from 237 farms. C. parvum strains were identified at the 18 small-subunit ribosomal RNA (SSU rRNA), COWP, and LIB13 loci and were subsequently analyzed by sequencing at the 60-kDa glycoprotein (GP60) locus for subtype determination. The presence of 71 C. parvum strains belonging to IIa, IId, or IIl subtype families was shown. The strains from the IIa allele family prevailed with IIaA17G1R1, IIaA17G2R1, and IIaA15G2R1 subtypes occurring frequently. Two novel subtypes IIaA10G1R1 and IIlA19R3 were detected for the first time in a bovine host. The highest C. parvum prevalence (22.5 %, confidence interval (CI) = 2.5 %) was observed among the youngest animals up to 2 weeks of age, followed by the prevalence among those aged 2 to 4 weeks (6.6 %, CI = 2.6 %) and then among older cattle (4.9 %, CI = 2.1). The occurrence of diarrhea in animals was associated with the presence of the IIaA16G1R1b subtype, while infections caused by IIaA15G2R1 strains were more likely to be asymptomatic. The geographical distribution of subtypes revealed that strains from the IIa subtype family were detected all over the country frequently compared to the IId and IIl subtypes, the sporadic appearances of which confirmed their endemic occurrence. Subtype analysis revealed the presence of zoonotic strains indicating cattle as a reservoir for human cryptosporidiosis.

Tracing enteric viruses in the European berry fruit supply chain.

In recent years, numerous foodborne outbreaks due to consumption of berry fruit contaminated by human enteric viruses have been reported. This European multinational study investigated possible contamination routes by monitoring the entire food chain for a panel of human and animal enteric viruses. A total of 785 samples were collected throughout the food production chain of four European countries (Czech Republic, Finland, Poland and Serbia) during two growing seasons. Samples were taken during the production phase, the processing phase, and at point-of-sale. Samples included irrigation water, animal faeces, food handlers' hand swabs, swabs from toilets on farms, from conveyor belts at processing plants, and of raspberries or strawberries at points-of-sale; all were subjected to virus analysis. The samples were analysed by real-time (reverse transcription, RT)-PCR, primarily for human adenoviruses (hAdV) to demonstrate that a route of contamination existed from infected persons to the food supply chain. The analyses also included testing for the presence of selected human (norovirus, NoV GI, NoV GII and hepatitis A virus, HAV), animal (porcine adenovirus, pAdV and bovine polyomavirus, bPyV) and zoonotic (hepatitis E virus, HEV) viruses. At berry production, hAdV was found in 9.5%, 5.8% and 9.1% of samples of irrigation water, food handlers' hands and toilets, respectively. At the processing plants, hAdV was detected in one (2.0%) swab from a food handler's hand. At point-of-sale, the prevalence of hAdV in fresh raspberries, frozen raspberries and fresh strawberries, was 0.7%, 3.2% and 2.0%, respectively. Of the human pathogenic viruses, NoV GII was detected in two (3.6%) water samples at berry production, but no HAV was detected in any of the samples. HEV-contaminated frozen raspberries were found once (2.6%). Animal faecal contamination was evidenced by positive pAdV and bPyV assay results. At berry production, one water sample contained both viruses, and at point-of-sale 5.7% and 1.3% of fresh and frozen berries tested positive for pAdV. At berry production hAdV was found both in irrigation water and on food handler's hands, which indicated that these may be important vehicles by which human pathogenic viruses enter the berry fruit chain. Moreover, both zoonotic and animal enteric viruses could be detected on the end products. This study gives insight into viral sources and transmission routes and emphasizes the necessity for thorough compliance with good agricultural and hygienic practice at the farms to help protect the public from viral infections.

Occurrence and molecular identification of Cryptosporidium species isolated from cattle in Poland.

Cryptosporidiosis is a disease reported in both humans and animals and is caused by a protozoan parasite Cryptosporidium. In most cases calves are infected by different Cryptosporidium species adapted to this animal host. Most infections are subclinical; however in some cases the watery diarrhea appears. The rapid development of new molecular diagnostic tools has provided an opportunity for better recognition of Cryptosporidium species and genotypes isolated from infected hosts. The aim of this study was an assessment of the prevalence of Cryptosporidium species in cattle herds in Poland. In total, 700 cattle fecal samples were tested. The examined cattle were at the age of 1 day to 6 years old. Overall 194 farms were monitored for parasite presence. Cryptosporidium detection in animal feces was performed using only molecular methods. Species identity of oocyst-positive samples were defined on the basis of PCR-RFLP and nucleotide sequence analysis of the amplified 18 SSU rRNA and COWP gene fragments. Cryptosporidium oocysts were detected in 119 (17%) of cattle feces. Most of the positive feces 55 (19.7%) were derived from young animals at the age of 1-4 weeks. The tested samples were positive for Cryptosporidium bovis, Cryptosporidium parvum, Cryptosporidium andersoni, and Cryptosporidium ryanae. C. parvum was not the most frequently detected parasite species, but calves below the age of 1 month were the major host for this parasite. The overall prevalence of C. parvum in Polish cattle herds was estimated at 5.1%. C. andersoni was the only species occurring in adult calves. The infected animals were housed in 67 (34.5%) of the monitored homesteads. Although C. bovis and C. ryanae were previously detected in cattle populations in many countries, this is the first report describing their presence in Polish cattle. Moreover, this is the first report on the prevalence of Cryptosporidium species in Polish cattle herds.