To provide information on the transmission dynamics of cryptosporidial infections in domestic small ruminants and the potential role of sheep and goats as a source for human cryptosporidiosis, Cryptosporidiumpositive isolates from 137 diarrheic lambs and 17 goat kids younger than 21 days of age were examined by using genotyping and subtyping techniques. Fecal specimens were collected between 2004 and 2006 from 71 sheep and 7 goat farms distributed throughout Aragón (northeastern Spain). Cryptosporidium parvum was the only species identified by restriction analyses of PCR products from small-subunit rRNA genes from all 154 microscopy-positive isolates and the sequencing of a subset of 50 isolates. Sequence analyses of the glycoprotein (GP60) gene revealed extensive genetic diversity within the C. parvum strains in a limited geographical area, in which the isolates from lambs exhibited 11 subtypes in two subtype families (IId and IIa) and those from goat kids displayed four subtypes within the family IId. Most isolates (98%) belonged to the subtype family IId, whereas only three isolates belonged to the most widely distributed family, IIa. Three of the four most prevalent subtypes (IIdA17G1a, IIdA19G1, and IIdA18G1) were previously identified in humans, and five subtypes (IIdA14G1, IIdA15G1, IIdA24G1, IIdA25G1, and IIdA26G1) were novel subtypes. All IId subtypes were identical to each other in the nonrepeat region, except for subtypes IIdA17G1b and IIdA22G1, which differed by a single nucleotide polymorphism downstream of the trinucleotide repeats. These findings suggest that lambs and goat kids are an important reservoir of the zoonotic C. parvum subtype family IId for humans.

The abundance, seasonal activity patterns and development rates of the tick Ixodes ricinus (L.) (Acari: Ixodidae), as well as microclimate features of the site of study, are described for a 9-year-long study (1994-2002) in north-central Spain. According to drag captures, larvae had a unimodal activity pattern, with a maximum observed around July-August, whereas nymphs displayed a bimodal pattern (May-June and August-September) with strong dominance of spring activity. An inversion of this pattern, with larger autumn peak, was observed in years with humid summers. Adults showed a small spring peak and a large autumn one. In the later years of the study, a small increase in the adult spring peak of activity was noticed, correlated with mild winters. Over the entire period of study, a clear increase in the total tick abundance was detected. Statistically significant differences between years were observed for some climate variables (saturation deficit, winter temperatures and number of days with temperatures above 6 degrees C), but a consistent and constant pattern of change was not observed in any climate variable. Temperature requirements for developing stages showed a sharp decrease in weeks 35-51 (eggs) and 38-50 (larvae and nymphs), a feature attributed to the presence of the morphogenetic diapause, beginning around September. Development rates obtained under quasi-natural conditions were almost twice those reported for other sites, suggesting an adaptation of this local, largely isolated I. ricinus population. According to drag captures and field-obtained development rates, interchange of nymphs between the two cohorts is common in this site, and seems to be influenced by the winter temperature and the date of larval engorgement.

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Faecal samples from 554 bovines randomly selected at 30 farms in Aragón were examined to investigate the prevalence of Cryptosporidium and Giardia infections. C. parvum oocysts were identified by using the Ziehl-Neelsen modified technique in 109 (19.7%) bovines ranging from 3 days old to adults. Positive animals were found in 19 (63.3%) farms. As much as 44.4% of calves aged 3-4 days were infected, but infection rates peaked at 6-15 days of age (76.7%). Nevertheless, prevalence was also high in weanling calves aged 1.5-4 months (14%), fattening calves and heifers 4-24 months old (7.7%) and adults (17.8%). Diarrhoea was recorded in 78.6% of suckling and 29.4% of weanling calves infected by C. parvum, but it was only found to be statistically associated with infection in suckling calves (P < 0.01). All calves shedding moderate or many oocysts had diarrhoea, whereas asymptomatic infection was always correlated with few oocysts in faeces. Cryptosporidial infections were always asymptomatic in bovines older than 4 months. Giardia cysts were identified in 65 bovines (11.7%) from 16 (53.3%) of the farms surveyed. Infection rates were significantly higher in suckling (14.1%) and weanling calves (38%) than in bovines older than 4 months (2.2%) (P < 0.001). Diarrhoea was recorded in 45.5% of suckling and 10.9% of weanling calves infected by Giardia, but it was not found to be statistically associated with infection. In fact, infection rates were higher in non-diarrhoeic than in diarrhoeic calves.

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Faecal specimens from 287 diarrhoeic calves younger than 21 days, collected over a 2-year period (2006-2007) from 82 dairy cattle farms in 14 provinces across the north of Spain, were examined for the presence of Cryptosporidium oocysts. Overall, 63 farms (76.8%) and 166 calves (57.8%) tested positive by microscopy. In order to elucidate the genetic diversity, selected positive specimens from 149 calves originating from 61 farms in the 14 provinces were examined by genotyping and subtyping techniques. Cryptosporidium parvum was the only species identified by PCR-RFLP of SSU rDNA from all 149 isolates and sequencing of a subset of 50 isolates, except for 2 specimens that were identified as C. bovis. Sequence analyses of the glycoprotein (GP60) gene revealed that most C. parvum isolates (98%) belonged to the subtype family IIa and 2 isolates were identified as the novel subtype IIdA23G1. Subtype IIaA15G2R1 was the most common and widely distributed (80.3% of the 61 farms), followed by subtype IIaA16G3R1 (14.7%), whereas the remaining IIa subtypes (IIaA16G2R1, IIaA17G2R1, IIaA18G3R1, IIaA19G3R1) were restricted to 1-3 farms. All these C. parvum IIa subtypes have previously been described in human patients, indicating that most isolates from diarrhoeic calves in northern Spain have zoonotic potential.

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