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.

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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BackgroundCurrent available malaria diagnostic methods each have some limitations to meet the need for real-time and large-scale screening of asymptomatic and low density malaria infection at community level. It was proposed that malaria parasite-specific low molecular-weight metabolites could be used as biomarkers for the development of a malaria diagnostic tool aimed to address this diagnostic challenge. In this study, high resolution metabolomics (HRM) was employed to identify malaria parasite-specific metabolites in Plasmodium falciparum in vitro culture samples.MethodsSupernatants were collected at 12 hours interval from 3% haematocrit in vitro 48-hour time-course asynchronized culture system of P. falciparum. Liquid chromatography coupled with high resolution mass spectrometry was applied to discover potential parasite-specific metabolites in the cell culture supernatant. A metabolome-wide association study was performed to extract metabolites using Manhattan plot with false discovery rate (FDR) and hierarchical cluster analysis. The significant metabolites based on FDR cutoff were annotated using Metlin database. Standard curves were created using corresponding chemical compounds to accurately quantify potential Plasmodium-specific metabolites in culture supernatants.ResultsThe number of significant metabolite features was 1025 in the supernatant of the Plasmodium infected culture based on Manhattan plot with FDR q=0.05. A two way hierarchical cluster analysis showed a clear segregation of the metabolic profile of parasite infected supernatant from non-infected supernatant at four time points during the 48 hour culture. Among the 1025 annotated metabolites, the intensities of four molecules were significantly increased with culture time suggesting a positive association between the quantity of these molecules and level of parasitaemia: i) 3-methylindole, a mosquito attractant, ii) succinylacetone, a haem biosynthesis inhibitor, iii) S-methyl-L-thiocitrulline, a nitric oxide synthase inhibitor, and iv) O-arachidonoyl glycidol, a fatty acid amide hydrolase inhibitor, The highest concentrations of 3-methylindole and succinylacetone were 178 ± 18.7 pmoles at 36 hours and 157±30.5 pmoles at 48 hours respectively in parasite infected supernatant.ConclusionHRM with bioinformatics identified four potential parasite-specific metabolite biomarkers using in vitro culture supernatants. Further study in malaria infected human is needed to determine presence of the molecules and its relationship with parasite densities.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0651-1) contains supplementary material, which is available to authorized users.