A rare Cryptosporidium parvum genotype associated with infection of lambs and zoonotic transmission in Italy.

An outbreak of cryptosporidiosis occurred in a mixed sheep/cattle farm of Central Italy in October 2011. A total of 450 ovines (250 sheep and 200 lambs) and 140 bovines (130 cows and 10 calves) were housed in two separated units, at the time of the outbreak. About half of the lambs had diarrhea due to Cryptosporidium sp. with a mortality rate of 80%; calves were not infected. Genomic DNA was extracted from an archived slide and from fecal specimens, and the parasite was identified as Cryptosporidium parvum by PCR and sequence analysis at the CpA135 gene. Genotyping at the GP60 gene showed the presence of a very rare genotype, IIaA20G2R1. Shortly after the outbreak was identified, the son of the farm's owner, aged 18 months, experienced an acute gastroenteritis and was hospitalized due to recurrent episodes of diarrhea, fever, vomiting and lack of appetite. The feces tested negative for bacteria and viruses, whereas cryptosporidiosis was diagnosed by microscopy and an immunochromatographic test. Molecular typing identified the C. parvum genotype IIaA20G2R1 in the feces of the child. This is the first case of transmission of cryptosporidiosis in Italy involving lambs as source of oocysts infectious to humans.

Introduction

Cryptosporidiosis is a common enteric infection of mammals, including domestic and wild animals, and humans (Tzipori and Widmer, 2008). The infection is transmitted through the fecal-oral route, and the extreme robustness of the oocysts, the infective stage of Cryptosporidium parasites, accounts for the importance of indirect transmission through ingestion of contaminated water and food (Smith et al., 2006). In humans, cryptosporidiosis is mainly due to Cryptosporidium hominis, a species with a predominant anthroponotic cycle, and to Cryptosporidium parvum, a zoonotic species with a significant impact on young ruminants (Cacciò et al., 2005, Xiao, 2010). C. hominis is the species responsible for the majority of human cases in the United States, Sub-Saharan Africa, and Asia, while C. parvum accounts for more human cases in Europe and particularly in the United Kingdom (Chako et al., 2010).

Among livestock, many studies have focused on the bovine host, particularly dairy calves, and the zoonotic role played by these animals is well established (Xiao, 2010). In comparison, less is known about cryptosporidiosis in goats and sheep and discordant opinions exist regarding the zoonotic potential of Cryptosporidium sp. oocysts shed by these animals (Robertson, 2009). Epidemiologic investigations have demonstrated the role of sheep in human cryptosporidiosis more than 20 years ago (Casemore, 1989), and this has been further supported by molecular studies, particularly in the UK (reviewed by Chalmers and Giles, 2010).

It is well established that sheep can be infected with different Cryptosporidium species, including C. parvum, Cryptosporidium bovis, Cryptosporidium xiaoi, and Cryptosporidium ubiquitum, whereas C. hominis, Cryptosporidium suis, Cryptosporidium andersoni, Cryptosporidium fayeri and the pig genotype II have been reported in a very low number of animals (Robertson, 2009, Xiao, 2010). Of these species, only C. parvum is of major concern from the perspective of public health, whereas C. ubiquitum has been associated only with sporadic cases in defined geographic areas (Fayer et al., 2010). The prevalence of C. parvum in sheep is very high in flocks in the UK (Smith et al., 2010), but it is reported to be very low in surveys conducted in Belgium, Norway, Australia, Brazil, China and the USA, where C. ubiquitum and C. xiaoi predominate (Ryan et al., 2005, Santín et al., 2007, Geurden et al., 2008, Robertson et al., 2010, Wang et al., 2010, Fiuza et al., 2011).

In this report, we have used molecular techniques to investigate a case of cryptosporidiosis in a child and to show that oocysts shed by lambs were the source of infection.

Materials and methods

Source of isolates

On November 5, 2011, a slide was prepared from a fecal sample taken directly from the rectum of a lamb brought to the Istituto Zooprofilattico Sperimentale (IZS) of Grosseto (Tuscany region, Central Italy) for post-mortem examination. The presence of Cryptosporidium spp. was investigated microscopically after staining with Kynioun (TCS Biosciences Ltd.), and by an ELISA test (Cryptosporidium parvum Elisa Kit, EuroClone).

On November 29, 2011, a fecal sample from the son of the farm's owner, aged 18 months, was collected at the “Ospedale della Misericordia di Grosseto” in Tuscany. On December 2, 2011, fecal samples were collected on farm from 21 adult sheep, 6 lambs and 6 calves. Fat and debris were removed from fecal samples by extraction with ether, followed by centrifugation and washing of the sediment with phosphate buffered saline (PBS). The presence of Cryptosporidium sp. oocysts on the concentrated fecal samples was assessed by immunofluorescence using a commercial kit (Merifluor, Meridian Biosciences).

DNA isolation and molecular characterization

DNA was extracted directly from the concentrated animal and human feces using a commercial kit (QiAmp Stool DNA mini kit, Qiagen). DNA was also extracted from the slide by scraping the surface and washing it with TE buffer (Tris 10 mM, EDTA 1 mM pH = 8.0). Three cycles of freezing in liquid nitrogen and thawing at 80 °C in a thermal block were performed to disrupt the oocysts recovered from the slide. DNA was then extracted using a commercial kit (QiAmp DNA mini kit, Qiagen, Milan, Italy).

PCR amplification of fragments of the CpA135 and of the GP60 genes was performed as described previously (Tosini et al., 2010, Strong et al., 2000) on a Veriti 96 thermocycler (Applied Biosystems). PCR products were purified using spin columns and sequenced on both strands. Sequences were assembled using SeqMan version 7.1 (DNAStar). BLAST search against the GenBank database was used to identify C. parvum GP60 genotypes. The novel GP60 sequence determined in this work is available in the GenBank database under the accession number JX110617.

Results and discussion

In October 2011, an outbreak of cryptosporidiosis occurred in a farm located in the province of Grosseto (Tuscany, Central Italy), that housed 450 ovines (250 sheep and 200 lambs) and 140 bovines (130 cows and 10 calves). The infection occurred only in lambs, and about 50% of the animals were affected, with a mortality rate close to 80%. The lambs presented with severe, yellowish liquid diarrhea, loss of weight, and depression.

On November 5, 2011, a lamb was brought to the IZS to carry out post-mortem examination and identify a potential cause of death. The animal presented pulmonary edema, liver congestion and catarrhal enteritis. Samples from liver, gut, lung, kidney and an intracardiac cloth were tested for pathogenic bacteria; Clostridium perfringens was isolated from the liver and the kidney, whereas Escherichia coli was isolated from the gut. The possible eziopathogenic role of these bacteria was investigated but further analysis revealed that enteropathogenic (ETEC), enterotoxic (EPEC) or enterohemorrhagic (EHEC) E. coli, as well as C. perfringens toxins were absent. The gut also tested negative for Coronavirus and Rotavirus. On the other hand, Cryptosporidium sp. oocysts were detected microscopically after Kynioun staining, and cryptosporidiosis was further confirmed by an ELISA test. On December 2, 2011, fecal samples were collected from 21 adult sheep, 6 lambs and 6 calves and tested for the presence of Cryptosporidium sp. oocysts. Only one sample from a lamb tested positive by immunofluorescence.

The son of the farm's owner, a child aged 18 months, experienced an acute enteritis in concomitance with the outbreak of cryptosporidiosis in farmed lambs. The main symptom was non-bloody diarrhea, without fever and vomiting, that lasted for at least four weeks. Laboratory tests ruled out common bacterial species (Salmonella, Campylobacter and Shigella) as the cause of diarrhea, and the child was prescribed with a rehydration therapy and probiotics. However, his conditions worsened and he was hospitalized on November 10, 2011, because of episodes of diarrhea, vomiting, dehydration and lack of appetite. Laboratory tests ruled out Rotavirus and Adenovirus as a cause of diarrhea, whereas both microscopy and immunochromatography revealed the presence of Cryptosporidium sp. The child received a parenteral rehydration therapy for 18 h and during the hospitalization he had no further episodes of vomiting or diarrhea, and no fever. He started to eat and drink normally and was discharged on November 13, 2011. Notably, a fecal sample collected from the child during a control visit on November 29, 2011, tested positive for Cryptosporidium by immunofluorescence, indicating an incomplete clearance of the infection.

The child had no direct contact with the infected animals; therefore the exposure must have been indirect, likely by an accidental ingestion of oocysts carried on his father's working clothes and/or footwear. Indeed, the father used to pick up his child from a childcare centre directly from the farm.

To identify the species of Cryptosporidium associated with the infection of lambs and of the child, a fragment of the CpA135 gene was amplified by PCR and sequenced. The analysis revealed 100% homology with C. parvum (GenBank acc. no. AJ006593) in 4 lamb samples, including 2 microscopically negative samples collected on December 2, 2011, and in the sample from the child. The identification of C. parvum is consistent with the notion that clinical cryptosporidiosis in sheep is caused only by this species and presents as a mild to severe yellowish liquid diarrhea with a strong odor, loss of weight, depression, and death usually involving animals up to one month of age (Smith et al., 2005). Samples from 6 calves and 21 adult sheep tested negative at this assay.

To identify C. parvum subtypes, the 5′ region of the GP60 gene, which contains polymorphic trinucleotide repeats, was amplified from the 5 samples that were positive at the CpA135 assay. The IIaA20G2R1subtype, named according to the nomenclature introduced by Sulaiman et al. (2005), was identified in the sample from the lamb collected on November 5, 2011, as well as in the sample from the child collected on November 29, 2011. Thus, the same and rare C. parvum GP60 genotype was identified in the feces from the lamb and the child. The IIaA20G2R1subtype was also identified in 2 samples from lambs collected on December 2, 2011, whereas the IIaA15G2R1 subtype was detected in another lamb also collected on December 2, 2011.

Molecular subtyping of C. parvum is an important tool for investigating the zoonotic potential of oocysts shed by livestock and the most commonly employed technique is sequencing of the highly polymorphic GP60 gene (Xiao, 2010). To date, only a modest number of specimens from sheep have been subgenotyped using this approach, and this has shown the presence of two families, known as IIa and IId (Díaz et al., 2010). The IIa family is commonly found in livestock, and the subtypes identified in sheep are also found in calves (Quílez et al., 2008, Díaz et al., 2010, Smith et al., 2010), and some have been associated with infection in humans (Table 1 ). On the contrary, the family IId is strongly associated with lambs, at least in Spain (Quílez et al., 2008), and with goat kids in Europe (Geurden et al., 2008, Quílez et al., 2008, Drumo et al., 2012).

Table 1

Subtypes in the GP60 family IIa of Cryptosporidium parvum identified in lambs, their distribution in other hosts and link to human cryptosporidiosis.

GP60 subtype	Host range	Geographic distribution	Link to human cryptosporidiosis	Setting
IIaA15G2R1	Sheep, calf, goat, human	Worldwide	Yes	Visit to open farm, bottle feeding lambs
IIaA16G3R1	Sheep, calf, human	Worldwide	Unknown
IIaA17G1R1	Sheep, calf, human	Worldwide	Yes	Visit to open farm, animal contact, one lamb scouring
IIaA18G2R1	Sheep, calf, human	Worldwide	Unknown
IIaA18G3R1	Sheep, calf, human	Worldwide	Unknown
IIaA20G2R1	Sheep, calf, human	Italy, Northern Ireland	Yes	Outbreak in lambs, indirect transmission to a child

In Italy, very little is known about cryptosporidiosis in sheep, but the presence of C. parvum has been reported by sequence analysis of the oocyst wall protein gene in 26 isolates from Central Italy (Paoletti et al., 2009). In a recent study that focused on the population structure of C. parvum in livestock (Drumo et al., 2012), 21 isolates from lambs were genotyped at seven loci, including GP60. The GP60 subtypes IIaA15G2R1, IIaA16G3R1 and IIaA18G2R1 were identified, in agreement with the result obtained in other studies.

In this work, a rare subtype, IIaA20G2R1, has been found associated with infection of lambs and with zoonotic transmission of cryptosporidiosis to a young child. The infection in lambs was particularly severe, with a mortality rate close to 80%, and the parasite was the only pathogen identified, as tests for both bacteria and viruses were negative. The analysis of fecal samples collected from lambs after the outbreak indicated that two genotypes, IIaA20G2R1 and IIaA15G2R1, were present in the farm. It should be noted that the IIaA20G2R1 subtype has been previously identified only in neonatal calves in Northern Ireland (Thompson et al., 2007), but not in humans or in other animals. This findings, together with the timing in the onset of symptoms, i.e., first in animals and then in the child, strongly suggest that the child acquired the infection from oocysts shed by the infected lambs. However, transmission must have been indirect, as the children had no contact with the infected animals, and could have resulted from an accidental exposure to oocysts carried by his father on working clothes. Interestingly, the child was excreting oocysts even after the regression of symptoms, albeit in much lower number, but no transmission occurred among other family members.

In summary, we described a case of zoonotic transmission of cryptosporidiosis to a young child in Italy, and demonstrated the involvement of a rare C. parvum genotype that was also associated with an outbreak in lambs.

Conflict of interest statement

The authors declare no conflicts of interest.