New Cryptosporidium parvum subtypes of IIa subfamily in dairy calves from Brazil.

Bovine cryptosporidiosis is mainly caused by four distinct species: Cryptosporidium parvum, C. bovis, C. ryanae and C. andersoni. The first, C. parvum, is a major concern in livestock causing economic losses, in addition to public health impact because of its zoonotic characteristics. The present study aimed to determine the occurrence of different species and subtypes of Cryptosporidium using molecular techniques. A total of 143 fecal samples were collected from calves from three dairy farms located in the state of Rio de Janeiro, Brazil. Saturated sugar centrifugal flotation method was used for the microscopic evaluation of the samples. Among these samples, 19.6% (28) were positive by microscopy, and 82.1% (23) of these 28 samples had their diagnosis confirmed by PCR using 18S as gene target. After sequencing, three species of Cryptosporidium were found to infect calves in different age groups. In pre-weaning phase (<2 months), 10% (3/30) of the calves were infected with C. parvum, whereas 14.2% (16/113) of post-weaning calves (≥2 months) were observed to be infected with C. andersoni and 1.8% (2/113) by C. ryanae with the latter diagnosed for the first time in the state of Rio de Janeiro. Those samples identified as C. parvum were further characterized at the GP60 locus, and PCR products were cloned. Eight different subtypes (IIaA20G2R1, IIaA20G2R2, IIaA19G2R1, IIaA19G2R2, IIaA18G1R1, IIaA18G2R2, IIaA16G3R2 and IIaA14G2R2) of C. parvum were identified, all belonging to the IIa family subtype, which is considered of high zoonotic potential. The subtypes mentioned above have not yet been detected in Brazilian cattle, and four of these subtypes (IIaA20G2R2, IIaA19G2R2, IIaA18G2R2 and IIaA14G2R2) had not been diagnosed elsewhere in calves until this study.

Phylogenetic Relationships Among Selected Heteroderoidea Based on 18S and ITS Ribosomal DNA.

In a study of relationships among selected cyst-forming and noncyst-forming species of Heteroderoidea, combined sequences comprised of DNA from part of the conserved 18S ribosomal RNA gene (rDNA) plus the complete ITS rDNA segment were more similar to analyses based on the ITS data alone than to analyses based on the 18S data alone. One of the two noncyst-forming species, Ekphymatodera thomasoni, grouped with cyst-forming species of Heteroderoidea. Bilobodera flexa, also a noncyst-forming species, was separated from all the other taxa by a long branch. Afenestrata koreana, with a weakly sclerotized cyst, grouped closely with H. bifenestra. These observations suggest that phylogenetic analyses using molecular data may aid in our understanding of the evolution of cyst formation in nematodes, including the possibility of secondary loss. The usefulness of molecular phylogenetic analyses in nematodes may depend more on the particular selection of taxa than on mere addition of data from additional genes.