Cryptosporidium parvum is one of the major causes of neonatal calf diarrhoea resulting in reduced farm productivity and compromised animal welfare worldwide. Livestock act as a major reservoir of this parasite, which can be transmitted to humans directly and/or indirectly, posing a public health risk. Research reports on the prevalence of Cryptosporidium in ruminants from east Mediterranean countries, including Cyprus, are limited. This study is the first to explore the occurrence of Cryptosporidium spp. in cattle up to 24 months old on the island of Cyprus. A total of 242 faecal samples were collected from 10 dairy cattle farms in Cyprus, all of which were screened for Cryptosporidium spp. using nested-PCR amplification targeting the small subunit of the ribosomal RNA (18S rRNA) gene. The 60 kDa glycoprotein (gp60) gene was also sequenced for the samples identified as Cryptosporidium parvum-positive to determine the subtypes present. The occurrence of Cryptosporidium was 43.8% (106/242) with at least one positive isolate in each farm sampled. Cryptosporidium bovis, Cryptosporidium ryanae and C. parvum were the only species identified, while the prevalence per farm ranged from 20–64%. Amongst these, the latter was the predominant species, representing 51.8% of all positive samples, followed by C. bovis (21.7%) and C. ryanae (31.1%). Five C. parvum subtypes were identified, four of which are zoonotic—IIaA14G1R1, IIaA15G1R1, IIaA15G2R1 and IIaA18G2R1. IIaA14G1R1 was the most abundant, representing 48.2% of all C. parvum positive samples, and was also the most widespread. This is the first report of zoonotic subtypes of C. parvum circulating in Cyprus. These results highlight the need for further research into the parasite focusing on its diversity, prevalence, host range and transmission dynamics on the island.

One hundred and seventy-seven (177) bulk tank milk samples were analyzed with a commercially available real-time polymerase chain reaction kit and 11 (6.21%), 41 (23.16%), and 58 (32.77%) tested positive for Mycoplasma bovis, Staphylococcus aureus, and Streptococcus agalactiae, respectively. Statistical analysis revealed a significant relationship between the presence of S. aureus and S. agalactiae. Enumeration of somatic cells was performed in the same samples by flow cytometry. The somatic cell counts were found higher in S. aureus and S. agalactiae positive samples. No association was found between M. bovis presence and somatic cells counts. Low internal assay control Ct values were found to be related with high somatic cell counts. Noticeably, this is the first report for the presence of M. bovis in Cyprus. Therefore, its presence was confirmed by bulk tank milk culture, conventional PCR, and next generation sequencing. Furthermore, M. bovis was typed with multilocus sequencing typing and was allocated to sequence type 29 (ST 29). Real-time PCR in bulk tank milk samples is a useful tool to detect mammary infections, especially for neglected pathogens such as M. bovis.

Cryptosporidium spp. are globally important enteric pathogens in livestock and a leading cause of neonatal calf diarrhoea, with zoonotic potential. This study presents the first nationwide molecular survey of Cryptosporidium spp. and C. parvum gp60 subtypes in dairy cattle in Cyprus. A total of 517 faecal samples were collected from dams (n = 256) and their 3–7-day-old calves (n = 261) across 18 farms in five districts. Samples were screened by nested PCR and sequencing of the SSU rRNA and gp60 genes. Overall, Cryptosporidium spp. prevalence was 28.2%, with higher rates in calves (39.5%) than in dams (16.8%). Cryptosporidium parvum was the dominant species (85.6%), followed by C. bovis (8.2%), C. andersoni (4.8%), and C. ryanae (0.7%). Eight C. parvum gp60 subtypes were identified, six from family IIa and two from IId, the latter reported for the first time in Cypriot cattle. Subtypes IIaA14G1R1 and IIdA16G1 were strongly associated with severe diarrhoea, while IIaA17G2R1 predominated in asymptomatic calves. Several identified subtypes have been implicated in human outbreaks globally. Cyprus’s confined livestock population and strategic location at the crossroads of Europe, Asia, and Africa provide a unique context for understanding Cryptosporidium transmission. This study establishes essential baseline data on the prevalence, molecular diversity, and zoonotic potential of Cryptosporidium in an island setting. The findings underscore the need for integrated surveillance and One Health strategies to control transmission, protect public health, and monitor the emergence of high-risk subtypes locally and beyond.

Cryptosporidium parvum is one of the major causes of neonatal calf diarrhoea resulting in reduced farm productivity and compromised animal welfare worldwide. Livestock act as a major reservoir of this parasite, which can be transmitted to humans directly and/or indirectly, posing a public health risk. Research reports on Cryptosporidium prevalence from the east Mediterranean region are scarce with even less originating from Cyprus. This study is the first to explore occurrence of Cryptosporidium spp. in young dairy calves on the island. A total of 242 faecal samples were collected from 10 dairy cattle farms in Cyprus, all of which were screened for Cryptosporidium spp. using nested-PCR amplification targeting the small subunit of the ribosomal RNA (18S rRNA) gene. The 60 kDa glycoprotein (gp60) gene was also sequenced for the samples identified as C. parvum positive to determine the subtypes present. Occurrence of Cryptosporidium was 43.8% (106/242) with at least one positive isolate in each farm sampled. Prevalence per farm ranged from 20¬‒64%, while C. bovis, C. ryanae and C. parvum were the only species identified. Amongst those, the latter was the predominant species, representing 51.8% of all positive samples. Four zoonotic subtypes were identified – IIaA14G1R1, IIaA15G1R1, IIaA15G2R1 and IIaA18G2R1. IIaA14G1R1 was the most abundant; representing 48.2% of all C. parvum positive samples and was also the most widespread. This is the first report of zoonotic subtypes of C. parvum circulating in Cyprus. These results highlight the need for further research into the parasite focusing on its diversity, prevalence, host-range and transmission dynamics on the island.