Occurrence and molecular typing of cryptosporidium species in neonatal calves with diarrhoea

Introduction

Neonatal calf diarrhoea costs the European dairy industry an estimated €500 million annually through mortality, treatment expenses, and reduced growth performance, and Cryptosporidium infection is implicated in roughly one third of these cases [1] . Unlike bacterial or viral causes of scours, cryptosporidiosis has no licensed drug treatment for calves in most jurisdictions, leaving producers reliant on supportive therapy and hygiene measures to limit its impact [2] . The parasite's thick-walled oocysts resist common disinfectants, persist in the farm environment for months, and require an infectious dose as low as 10-50 oocysts to establish infection in a neonatal calf features that make control exceptionally difficult once the organism enters a herd [3] . Molecular advances over the past two decades have revealed that what was once considered a single "Cryptosporidium of cattle" is actually a complex of at least four species with distinct host preferences and zoonotic potential [4] . C. parvum infects both calves and humans and is the species of greatest public health concern; outbreaks linked to contaminated water or direct animal contact are reported regularly across Europe [5] . C. bovis and C. ryanae appear to be cattle-adapted and are not associated with human disease, while C. andersoni infects the abomasum of older cattle and rarely causes clinical diarrhoea [6] . Distinguishing among these species is impossible by microscopy alone, since oocyst morphology overlaps extensively, and requires PCR-based methods targeting the 18S ribosomal RNA gene followed by RFLP or sequencing [7] . Within C. parvum, subtyping at the gp60 locus has identified more than 20 subtype families globally [8] . In Western Europe, the IIa family predominates in cattle and is the most frequently implicated in zoonotic transmission, whereas the IId family, more common in sheep and goats, has been increasingly reported in Irish and British calves [9] . Mapping the subtype landscape within a region matters because different subtypes may vary in virulence, environmental persistence, and potential for waterborne spread [10] . Ireland's dairy sector is overwhelmingly pasture-based, with a strongly seasonal calving pattern concentrated in January-April. This creates a burst of susceptible neonates in a short window, often housed at high density in calf sheds, conditions that favour rapid Cryptosporidium cycling [11] . Despite the importance of the disease, the most recent molecular survey of Cryptosporidium in Irish calves was published over a decade ago and covered only a single county [12] . An updated, multi-region assessment using current subtyping methodology is overdue. This research aimed to determine the occurrence and species composition of Cryptosporidium in diarrhoeic neonatal calves across six counties in southern Ireland, and to characterise C. parvum isolates at the gp60 subtype level in order to assess the prevailing zoonotic risk profile.

Material and Methods

Study area description

The research was conducted across six counties in the Munster province of southern Ireland: Cork (south and north divisions), Kerry, Limerick, Tipperary, and Waterford. These counties collectively account for approximately 35% of the national dairy cow population. Sampling farms were selected from the records of local veterinary practitioners who reported neonatal calf diarrhoea outbreaks during the 2023 spring calving season (January-May). A total of 42 commercial dairy farms (6-8 per region) participated, with herd sizes ranging from 80 to 340 milking cows.

Material

Fresh faecal samples were collected rectally from 387 neonatal calves (aged 1-35 days) presenting with clinical diarrhoea, defined as the passage of liquid or semi-liquid faeces for at least 24 hours. Samples were transported on ice to the National Institute of Veterinary Parasitology, Cork, within six hours of collection and processed immediately or stored at 4°C for no longer than 48 hours before analysis [13] . Calf age, sex, breed, and farm management details (housing type, colostrum protocol, herd cryptosporidiosis history) were recorded on a standardised data sheet.

Methods

Microscopic detection of Cryptosporidium oocysts was performed by mZN staining of thin faecal smears. Samples containing acid-fast oocysts (4-6 µm diameter) were classified as positive [7] . DNA was extracted from oocystpositive samples using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol, with an additional bead-beating step (FastPrep-24, MP Biomedicals) to disrupt oocyst walls. Species identification was achieved by nested PCR amplification of an approximately 830-bp fragment of the 18S rRNA gene, using outer primers (5′-TTCTAGAGCTAATACATGCG-3′ / 5′-CCCATTTCCTTCGAAACAGGA-3′) and inner primers (5′-GGAAGGGTTGTATTTATTAGATAAAG-3′ / 5′-AAGGAGTAAGGAACAACCTCCA-3′), following established cycling conditions [14] . PCR products were digested with SspI and VspI restriction endonucleases, and fragments were resolved on 2% agarose gels to identify species-specific banding patterns. For gp60 subtyping, a subset of 78 C. parvum-positive isolates (selected to represent each region proportionally) underwent nested PCR amplification of the gp60 gene, followed by bidirectional Sanger sequencing (Eurofins Genomics, Ebersberg, Germany). Sequences were aligned against GenBank reference sequences using MEGA v.11 and assigned to subtype families according to established nomenclature [8,15] . Data were analysed using SPSS v.28. Prevalence was expressed with 95% confidence intervals (CI). Associations between Cryptosporidium positivity and calf age, season, and region were tested by chi-squared or Fisher's exact test. Significance was set at P<0.05.

Ethical considerations

Sample collection was carried out as part of routine diagnostic veterinary investigation of diarrhoeic calves and did not require separate animal ethics approval under Irish legislation (Health Products Regulatory Authority, Statutory Instrument No. 543 of 2012). Written informed consent was obtained from all participating farmers prior to sample collection. No additional invasive procedures were performed beyond rectal faecal sampling, which is a standard diagnostic practice.

Results

Of the 387 faecal samples screened by mZN staining, 214 (55.3%; 95% CI: 50.2-60.3%) were positive for Cryptosporidium oocysts. Among the 214 positive samples subjected to molecular analysis, nested PCR successfully amplified the 18S rRNA gene in 196 (91.6%). RFLP analysis identified four Cryptosporidium species: C. parvum in 119 samples (60.7%), C. bovis in 37 (18.7%), C. ryanae in 28 (14.5%), and C. andersoni in 12 (6.1%). Mixed infections involving two species were detected in 16 calves (8.4% of positives), most commonly C. parvum plus C. bovis (n = 11). Strong sporadic cases The IIa family accounted for 82.1% of subtyped isolates. IIaA15G2R1 was the single most common subtype, a finding consistent with its status as the dominant zoonotic subtype in Western Europe [8,9] .

Comprehensive interpretation

The data paint a clear epidemiological picture: C. parvum is the overwhelmingly dominant Cryptosporidium species in diarrhoeic neonatal calves across southern Ireland, peaking in the second week of life, and the prevailing subtypes belong to the highly zoonotic IIa family. The concurrent presence of non-pathogenic species (C. bovis, C. ryanae) underscores the necessity of molecular typing microscopy alone would overestimate the proportion of clinically and zoonotically significant infections. These findings have direct implications for both calf-health management and occupational safety on dairy farms.

Seasonal variations

When monthly prevalence was compared, a clear seasonal pattern emerged. January samples (early calving season) had the lowest positivity rate (41.2%), rising sharply to a peak of 63.8% in March before declining to 48.4% in May as the calving season wound down. This trajectory mirrors the buildup of environmental oocyst contamination in calf sheds that occurs as successive batches of calves are housed in the same space without adequate cleaning between groups [3,11] . The March peak also coincides with the period of highest calf density, when many farms house 30-50 neonates simultaneously. C. parvum accounted for a greater share of infections in February-March (68-71%) than in April-May (52-55%), suggesting that pathogenic species may benefit disproportionately from crowding-related transmission pressure.

Discussion

The overall Cryptosporidium prevalence of 55.3% in diarrhoeic calves agrees closely with findings from other temperate European dairy regions. A recent Dutch survey reported 52.7% positivity in pre-weaned calves [1] , while a Scottish molecular survey found 58.4% [12] . The dominance of C. parvum (60.7% of typed isolates) is likewise consistent with the European consensus that this species drives the great majority of clinical neonatal cryptosporidiosis [4,5] . However, the combined proportion of C. bovis and C. ryanae (33.2%) is higher than in most comparable reports, where these cattleadapted species typically account for 15-25% of isolates [6] . One possible explanation is the broad age range sampled here (1-35 days): C. bovis prevalence tends to increase from the third week of life onward as C. parvum wanes, and including calves up to 35 days may have captured more C. bovis infections than research restricted to the first two weeks [4,7] . The GP60 subtype profile dominated by IIaA15G2R1 (46.2%) aligns with molecular epidemiological data from the United Kingdom and continental Europe, where IIaA15G2R1 is the most commonly reported zoonotic subtype in both calves and human patients [8,9] . The detection of IIdA20G1 in 17.9% of isolates is notable. This subtype, originally linked to small ruminants, has been appearing with increasing frequency in calves on mixed-species farms across the British Isles, raising questions about cross-species transmission on holdings where cattle share pastures or housing with sheep and goats [10] . The strong age-dependent pattern with peak prevalence in the second week of life has practical implications for control timing. Interventions such as enhanced calf-pen hygiene, reduced stocking density, and strategic use of halofuginone lactate (the only licensed anti-Cryptosporidium product for calves in the EU) should be concentrated in the first 14 days after birth to intercept the window of highest susceptibility and shedding [2,3] . Delaying action until clinical diarrhoea appears is likely too late, as oocyst excretion typically begins two to three days before the onset of loose faeces [14] .

Limitations

Some limitations of this research deserve acknowledgement.

The convenience sampling approach relying on veterinary practitioner referrals rather than random farm selection may overestimate prevalence by targeting farms with known diarrhoea problems. Only diarrhoeic calves were sampled, so the true herd-level prevalence, including subclinical shedders, was not captured. GP60 subtyping was performed on a proportional subset (78 of 119 C. parvum isolates) rather than the full set, which may have missed rare subtypes present at low frequency. The research covered a single calving season; year-to-year variation in species and subtype composition cannot be assessed from a cross-sectional design. Finally, environmental sampling of calf-shed surfaces and water sources was not included and would have strengthened the epidemiological interpretation.

Conclusion

The For the field of veterinary parasitology and public health, these findings carry significance in two directions. On the animal health side, they underscore the need for early, proactive hygiene interventions concentrated in the first two weeks of calf life, when C. parvum transmission is at its peak.

On the public health side, the high prevalence of IIa subtypes means that dairy farm workers, their families, and anyone in contact with calf faeces or contaminated water are at meaningful risk of zoonotic cryptosporidiosis a message that warrants inclusion in occupational health guidance for the agricultural sector. Several open questions emerge. Does longitudinal sampling across multiple calving seasons reveal year-to-year shifts in subtype composition, or is the IIaA15G2R1 dominance a stable feature of the Irish cattle population? What is the contribution of subclinically infected calves to environmental oocyst loads information that requires sampling of apparently healthy animals alongside diarrhoeic ones? Can wholegenome sequencing of C. parvum isolates resolve transmission chains between calves and human cases in the same catchment, moving beyond the limited discriminatory power of gp60 subtyping alone? And does the rising frequency of the IIdA20G1 subtype on mixed-species farms signal an emerging cross-species transmission pathway that could complicate control efforts? Answering these questions will require coordinated, longitudinal surveillance programmes integrating veterinary and public health data an investment justified by the scale of Cryptosporidium's economic and zoonotic impact.

Acknowledgement

Acknowledgements Institutional supportThe authors thank the National Institute of Veterinary Parasitology, Cork, for providing laboratory facilities, molecular diagnostic equipment, and cold-storage infrastructure essential to this research.

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