Concentrations,Viability, and Distribution of Cryptosporidium Genotypes in Lagoons of Swine Facilities in the Southern Piedmont and in Coastal Plain Watersheds of Georgia

Waste lagoons of swine operations are a source of Cryptosporidium oocysts. Few studies, however, have reported on oocyst concentrations in swine waste lagoons; none have reported on oocyst viability status, nor has there been a systematic assessment of species/genotype distributions across different types of swine facilities. Ten swine waste lagoons associated with farrowing, nursery, finishing, and gestation operations were each sampled once a month for a year. Oocysts were extracted from triplicate 900-ml effluent samples, enumerated by microscopy, and assessed for viability by dye exclusion/vital stain assay. DNA was extracted from processed samples, and 18S ribosomal DNA (rDNA) genes were amplified by PCR and sequenced for species and genotype identification. Oocysts were observed at each sampling time at each lagoon. Annual mean concentrations of total oocysts and viable oocysts ranged between 24 and 51 and between 0.6 and 12 oocysts ml⁻¹ effluent, respectively. The species and genotype distributions were dominated (95 to 100%) by Cryptosporidium suis and Cryptosporidium pig genotype II, the latter of which was found at eight of the lagoons. The lagoon at the gestation facility was dominated by Cryptosporidium muris (90%), and one farrowing facility showed a mix of pig genotypes, Cryptosporidium muris, and various genotypes of C. parvum. The zoonotic C. parvum bovine genotype was observed five times out of 407 18S rDNA sequences analyzed. Our results indicate that pigs can have mixed Cryptosporidium infections, but infection with C. suis is likely to be dominant.Over the last few decades, pork production in North America has undergone significant growth and centralization into large concentrated swine (Sus scrofa) operations with more animals on fewer farms (18). A consequence of the increase in numbers of swine per facility is a concomitant increased concentration of swine waste. Present housing facilities for swine are designed to collect feces and urine in wastewater lagoons, in which the waste undergoes anaerobic transformations. One of several public health concerns over swine lagoons is the potential presence of infectious bacteria, viruses, and protozoa (4). Because of the notoriety given to swine waste lagoon spills in the coastal flood plain of North Carolina that were associated with a series of hurricanes in 1998 and 1999 (21), large-scale swine operations have become a focus of environmental and public health concerns.The cause of the massive outbreak of cryptosporidiosis in Milwaukee, WI, in 1993 was afterwards determined to be Cryptosporidium hominis, the human genotype of C. parvum and an obligate parasite of humans (33, 44). At the time, however, it was thought to be caused by C. parvum (22). Because of this initial misidentification of the cryptosporidial source of the outbreak, the connection between C. parvum and large-scale confined livestock operations has become a focused area of research. Although manure-associated outbreaks of C. parvum have implicated bovine sources, a Canadian study found that the prevalence of Cryptosporidium in swine lagoons was greater than that in dairy liquid manure (9). Olson et al. (24) also reported the presence of Cryptosporidium oocysts of undetermined genotype at four of six hog operations in Canada. Atwill et al. (2) observed C. parvum oocysts in feces of feral pigs. Hutchison et al. (13) observed C. parvum oocysts of undetermined genotype in 5 and 13% of fresh and stored fecal samples, respectively, from pigs of undeclared age. Guselle et al. (10) followed the course of a naturally occurring C. parvum infection in 33 weaned pigs. Following the protocol of the genetic analysis of Morgan et al. (23), Guselle et al. (10) identified this C. parvum genotype as being adapted to pigs. At the time, the zoonotic potential of this C. parvum pig-adapted genotype was considered uncertain (23).Recently, two genotypes of Cryptosporidium have been recognized as host adapted to swine: Cryptosporidium suis (formerly Cryptosporidium pig genotype I) and Cryptosporidium pig genotype II (28, 29). Xiao et al. (37) reported on an immunocompromised person who was infected with a Cryptosporidium pig genotype and thus implicated Cryptosporidium from swine as potentially zoonotic and a public health concern. Before molecular methods were developed to differentiate pig genotypes of Cryptosporidium from other species, C. parvum was thought to infect 152 species of mammals and consist of several cryptic species (6). An extensive survey of swine effluent from swine finishing operations in Ireland indicated a prevalence of both C. suis and Cryptosporidium pig genotype II (39). Hamnes et al. (11) reported prevalence of both C. suis and Cryptosporidium pig genotype II in feces of suckling pigs across Norway and thus implicated farrowing operations as sources of this parasite.Other than the prevalence of Cryptosporidium in feces of young pigs and effluent lagoons of older pigs in finishing operations, little comprehensive data on oocyst concentrations, viability of oocysts, and distributions of Cryptosporidium species and genotypes have been reported. No systematic study of swine lagoon effluents from large-scale facilities has been reported for the four separate stages of swine development, (i) breeding and gestation, (ii) farrowing (parturition), (iii) nursery (in which weaned piglets are kept until 8 to 9 weeks of age), and (iv) finishing (in which 8- to 9-week-old pigs are kept to market weight). The objective of this investigation was to determine for 1 year the frequencies, concentrations, viability statuses, and distributions of Cryptosporidium species and genotypes in lagoons associated with the four types of swine operations in the Southern Piedmont and in coastal plain watersheds of Georgia.