Prevalence and Identity of Cryptosporidium spp. in Pig Slurry

Abstract

Cryptosporidium spp. were detected in 25 of 56 pig slurry samples from 33 Irish farms by PCR and DNA sequencing. The organisms detected included C. suis, Cryptosporidium pig genotype II, and C. muris. We concluded that Cryptosporidium oocysts can persist in treated slurry and potentially contaminate surface water through improper discharge or uncontrolled runoff.

Cryptosporidium spp. are intestinal protozoans that occur in many animal species, including pigs (1, 3, 21). Two Cryptosporidium spp., C. suis and an unnamed species (Cryptosporidium pig genotype II), may cause enteritis in pigs, although this conclusion was based on a limited number of studies (9, 12, 13). Even though the pathogenicity of Cryptosporidium spp. in pigs is equivocal (2, 3, 21), the recent identification of C. suis in an asymptomatic human immunodeficiency virus-positive person indicates that Cryptosporidium spp. from pigs have the potential to be zoonotic pathogens (18).

Spreading of slurry onto pasture and tillage land is a common practice that is used for disposal of animal waste from intensive animal husbandry units (4). However, spreading of slurry during inappropriate weather conditions has resulted in pollution of water through direct runoff into streams and rivers and contamination of groundwater. Recently, the health impact of this practice has attracted attention, because many microbes found in animal feces, such as Salmonella spp., Shigella spp., Campylobacter spp., Escherichia coli O157, and Yersinia enterocolitica, can cause illness in humans (4).

Cryptosporidium oocysts are known to survive for extended periods in fecal material, much longer than most bacterial agents (6, 10, 17). In controlled studies, it has been found that spreading of animal slurry can lead to the release of large oocyst loads into the environment, especially after a heavy rainfall, which can result in deterioration of the quality of surface water in a watershed (15). In this pilot study, we examined the prevalence and identity of Cryptosporidium oocysts in pig slurry from commercial pig farms in Ireland.

Sample collection.

Pig slurry was obtained from fattening units on 33 commercial pig farms in Northern Ireland and the Republic of Ireland in the spring of 2002 and in the spring and autumn of 2004. Twenty-four samples were obtained from one farm in County Meath in 2002, and single samples were obtained from 32 other farms (Table 1).

TABLE 1.

Prevalence of Cryptosporidium spp. in pig slurry from 33 pig-fattening units in Ireland

Farm	Sample type	No. of positive samples/total no.	Cryptosporidium species found (detection rate)
County Meath farm	Nonseparated slurry	5/8	C. suis (50.0%), pig genotype II (25.0%)
	Liquid fraction	6/7	C. suis (85.7%), pig genotype II (14.3%), C. muris (14.3%)
	Solid fraction	1/9	C. suis (11.1%)
32 other farms	Nonseparated slurry	13/32	C. suis (15.6%), pig genotype II (25.0%)

For the majority of the farms (32 farms) approximately 50 g of nonseparated slurry was collected from the main storage tank in a sterile plastic container and transported at the ambient temperature to the laboratory for analysis. For the remaining farm in County Meath, three types of slurry samples were taken from the main storage tank, in which feces, urine, and wash water from the fattening operation had been collected for the previous 4 months. The sampling procedure involved initial collection of 2 kg of nonseparated slurry, from which a 200-μl aliquot was taken and tested for Cryptosporidium. The remainder of the sample or another 2 kg of nonseparated slurry was separated mechanically into solid and liquid fractions, using a rotascreen (Carier model RS1000; ApT Solutions, Ballyclare, United Kingdom) equipped with a 1.5-mm perforated drum. By pumping slurry between a set of compression rollers and a rotating perforated cylinder drum screen, the rotascreen unit separated whole slurry into liquid and solid material. A total of 24 samples (8 nonseparated slurry, 9 separated solid, and 7 separated liquid samples) were collected on this farm and analyzed for the presence of Cryptosporidium spp.

Cryptosporidium genotyping.

About 200 μl of each slurry sample was washed twice in a microcentrifuge tube with 1 ml of distilled water and centrifuged at 12,000 × g for 15 min prior to DNA extraction with a QIAamp DNA stool mini kit (QIAGEN Inc.) (23). Cryptosporidium species and genotypes were determined by a technique based on PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the small-subunit (SSU) rRNA gene (19, 23). In this technique, a 826- to 864-bp fragment of the SSU rRNA gene was amplified by nested PCR. For detection and differentiation of Cryptosporidium species and genotypes, 10 μl of the secondary PCR product was subjected to restriction digestion with SspI (New England BioLabs, Beverly, MA) and VspI (GIBCO BRL, Grand Island, NY). Differences in SspI and VspI banding patterns after 2% agarose electrophoresis were used to identify the Cryptosporidium species and genotypes (23). Extracted DNA from each slurry sample was analyzed at least twice by the PCR-RFLP method using 1.0 μl of DNA as the template. A positive control (C. serpentis DNA) and a negative control (no template DNA) were included in each PCR run. All PCR products were sequenced in both directions using an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA) in order to verify the results of the PCR-RFLP analysis. The sequences obtained were aligned with each other and with the sequences of known Cryptosporidium spp. using the software ClustalX (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/).

Prevalence and identities of Cryptosporidium spp. in pig slurry.

SSU rRNA PCR products that were of the expected size (∼830 bp) were obtained from 12 of the 24 (50%) slurry samples collected from 1 farm in County Meath and from 13 (41%) samples collected from the remaining 32 farms (Table 1). In a detailed examination of the localization of oocysts in the three components (liquid, solids, and nonseparated slurry) from the County Meath farm, we observed that six of seven liquid samples, one of nine solid samples, and five of eight nonseparated slurry samples were positive for Cryptosporidium. The higher rate of Cryptosporidium detection in the liquid fractions of the separated slurry was likely due to the fact that that a large portion of the slurry samples could be analyzed when liquid fractions rather than sediments were used for DNA extraction.

Restriction analyses with the SspI and VspI enzymes suggested that most of the PCR products belonged to two Cryptosporidium spp. previously reported to be present in pigs, C. suis and pig genotype II. C. suis was found in 11 samples from the County Meath farm and in five samples from five other farms, whereas Cryptosporidium pig genotype II was found in three samples from the County Meath farm and in eight samples from eight other farms (Fig. 1). One Cryptosporidium sp. was identified in the slurry from the majority of positive farms. However, three species were found on the farm in County Meath. In addition, two of the samples from this farm were positive for both C. suis and pig genotype II, and one sample was positive for C. suis and a C. muris/C. andersoni-like parasite (Table 1).

FIG. 1.

RFLP patterns of two common Cryptosporidium spp. in pig slurry. The upper panel shows SspI digestion products, and the lower panel shows VspI digestion products. Lanes 1 to 3, Cryptosporidium pig genotype II; lane 4, C. suis; lane 5, C. serpentis (positive control); lane 6, 100-bp size markers.

The results of DNA sequencing confirmed that all PCR products were products from three Cryptosporidium spp. Twenty PCR products (two PCR products for some samples) from the County Meath farm and 13 PCR products from 13 other farms were sequenced. Fifteen PCR products from the County Meath farm and five PCR products from five other farms yielded sequences identical to each other and to the C. suis sequence deposited in the GenBank database under accession no. AF115377, which was originally obtained from a pig in Australia (22). Four PCR products from the County Meath farm and eight PCR products from eight other farms generated a second type of sequences that were identical to each other and to a 474-bp SSU rRNA sequence of Cryptosporidium pig genotype II deposited in the GenBank database (accession no. AY271721), which was obtained from a pig in Australia (13). The PCR product of the C. muris/C. andersoni RFLP pattern from the sample from the County Meath farm generated an SSU rRNA sequence identical to the sequence (accession no. AF093498) reported for C. muris obtained from a rock hyrax in the United States (20). There was complete agreement between the RFLP and DNA sequence genotyping results.

Public health implications.

Nursing piglets and weanlings have been shown to be infected with two Cryptosporidium spp., C. suis and pig genotype II (13). The results reported here suggest that early infections in young pigs may persist in older pigs or that old pigs can be reinfected with the same Cryptosporidium spp. The only other Cryptosporidium sp. found in this study was C. muris, which could have originated from either infected pigs or rodents. Even though C. muris infection has never been reported in pigs, this species does have the widest host range of all Cryptosporidium spp., and this range includes humans (7). Recently, it has been shown that cattle are infected with four different Cryptosporidium spp. at different ages (14). This is unlikely for pigs, as fatteners in this study were also infected with the same two species found in piglets.

The viability of the oocysts was not studied; thus, the infectivity of Cryptosporidium oocysts after more than 4 months of storage was not known. Previously, it was shown that C. parvum can survive the bovine manure storage and slurry treatment process (4, 5). Even though the more recognized zoonotic pathogen C. parvum was not found in pig slurry in this study, two of the three Cryptosporidium spp. in pig slurry, C. muris and C. suis, have been identified in some human cases (8, 18). Oocysts of C. muris and pig genotype II were previously found in surface and source water in Europe and Australia (11, 16). Thus, spreading of pig slurry onto pasture or crops may lead to increases in the occurrence of these and other zoonotic enteric pathogens in water.