Molecular characterization of Cryptosporidium strains from foals: preliminary results

Cryptosporidium spp., a common enteric pathogen of animals and humans, has been involved as a cause of diarrhea in foals both immunocompetent and immunocompromised (Sgorbini et al, 2003, Ippologia, 4: 5-9; Grinberg et al, 2009, New Zeal Vet J, 57: 284-289; Perrucci et al, 2011, Vet Paras 182: 333-336). Several aspect of this infection in horse, such as prevalence, clinical onset, risk factors, economic relevance and the species status are not completely defined (Veronesi et al, 2009 Zoon Pub Health, 57: 510-517). So far, the literature on equine cryptosporidiosis, describe the presence of Cryptosporidium parvum “cattle” genotype and Cryptosporidium “horse” genotype (Grinberg et al, 2003 Vet Rec, 153: 628-631; Grinberg et al, 2008 J Clin Microbiol, 46: 2396-2398; Chalmers et al, 2005, Vet Rec, 156: 49-50; Chalmers et al, 2009, Eurosurveillance, 14: 1-9; Xiao et al, 2009 J Clin Microbiol, 47: 3017-3020). AIM: During the last horse breeding seasons at the Neonatal Intensive Care Unit of Department of Veterinary Medical Science Alma Mater Studiorum-University of Bologna during past several cases of cryptosporidiosis have been observed in foals and mare. With the aim of undertake a study of epidemiology of these outbreak and in order to set up adequate control measures, a molecular study on Cryptosporidium spp. isolates from foals hosted in this structure in 2007 and from diarrheic goat housed in 2006 in a separate experimental stable have been carried out. Furthermore a human isolate from a technician with gastro-intestinal symptoms working on goat during the outbreak was also analyzed. MATERIALS AND METHODS. The following fecal samples, tested positive by Ziehl-Neelsen stain and stored frozen, were examined: 4 from foals with diarrheic syndrome, 1 from goat and 1 from man. All the samples were subjected to DNA extraction with QIAamp DNA Stool Mini Kit (Qiagen) and to nested PCR, amplifying the 18S rRNA (Miller et al, 2006, J Microbiol Meth, 65: 367-379) and the gene encoding the Cryptosporidium oocyst wall protein (COWP) (Traversa et al, 2008, Mol Cell Probes, 22: 122-128). The PCR products were sequenced in both direction by ABI 3730 DNA Analyzer at StarSEQ GmbH (Mainz, Germany). RESULTS. Sequence analysis of both genes revealed that all the isolates (foal, goat and human) were identical to each other and showed 100% identity with the Cryptosporidium parvum “cattle” genotype. CONCLUSIONS. The presence of C. parvum “cattle” genotype has already been recorded all around the world in a wide host range comprising human (Chalmers et al, 2005, Vet Rec, 156: 49-50; Grinberg et al, 2003, Vet Rec, 153: 628-630; Grinberg et al, 2008, J Clin Microbiol, 46: 2396-2398; Imhasly et al, 2009, Arch Tierheilkd, 151: 21-26; Veronesi et al, 2009, Zoon Pubbl Health, 57: 510-517; Traversa et al., 2010, Parassitologia, 52: 214; Perrucci et al, 2011, Vet Parasitol, 182: 333-336; Grinberg et al., 2012, Vet Rec, 153: 628-631) while only few researches reported the presence of Cryptosporidium “horse genotype” in foals (Ryan et al, 2003, Appl Environ Microbiol, 69: 4302-4307; Xiao and Fayer, 2008, Int J Parasitol, 38: 1239-1255; Xiao and Feng, 2008, FEMS Immunol Med Microbiol, 53: 309-323) with a single case of human infection (Xiao et al, 2009, J Clin Microbiol, 47: 3017-3020). Our results confirm the data obtained by Grinberg et al (2012) on the evidence that C. parvum “cattle” genotype could be a co-factor of foals diarrhea. The isolation of the same genotype also from goat and humans, suggest the possible cross-transmission between animals and man, pointing out the zoonotic professional risk linked to the presence of C. parvum “cattle” genotype as already described in previous studies reporting outbreak of cryptosporidiosis in vet students (Pohjola et al, 1986, Scand J Infect Dis, 18: 173-178; Levine et al, 1988, J Am Vet Med Ass, 193: 1413-1414; Reif et al, 1989, Am J Public Health, 79: 1528-1530; Preiser et al, 2003, J Am Coll Health, 51: 213-215; Gait et al, 2008, Vet Rec, 162: 843-845). Further investigations are necessary in order to verify the presence of sub-genotypes by studying other more suitable genes such as the GP60 and the Hsp70 in order to link the epidemiologically related isolates