he protozoan parasite genus Cryptosporidium has been linked to humans and to more than 240 species of animals worldwide and is considered one of the most important causative agents of diarrhea. Infections with this parasite have significant impacts on public health and animal husbandry, and Cryptosporidium spp. have been considered potential biowarfare agents in addition to causing severe infections in immunocompromised individuals. To date, at least 20 Cryptosporidium spp. have been confirmed, including C. parvum, C. hominis, C. meleagridis, C. felis, C. canis, C. suis, C. muris, C. andersoni, C. ubiquitum, with more than 60 genotypes of an undefined species[1-4], including horse, rabbit, pig genotype II and the skunk and chipmunk I genotypes that have been identified in humans, and therefore considered zoonotic pathogens[5-10]. This broad spectrum of Cryptosporidium species with the potential of affecting humans suggests that a better understanding of the reservoir species involved in zoonoses will be needed for the development of effective public health preventive strategies. Furthermore, since effective drug treatments or vaccines against cryptosporidiosis have not been developed, a clear understanding of the epidemiology of this parasite will be essential to the development of control and prevention strategies.
Most Cryptosporidium studies have focused on assessing water contamination and disease outbreaks affecting humans and cattle but little is known about the role of ovines in the context of cryptosporidiosis in China. The present study was designed to estimate the prevalence of this parasite among pre-weaned ovines from Aba Tibetan and Qiang Autonomous Prefecture.
MATERIALS AND METHODS
The study was carried out in the Aba Tibetan and Qiang Autonomous Prefecture in the Sichuan Province, China. Fecal samples were collected from 213 pre-weaned lambs selected randomly from eight farms between July 29 to August 4, 2009. Animal demographic data including sex, age, and sampling sites were recorded at the time of collection.
Approximately 20 g of each stool specimen was collected, 1 to 2 drops of each specimen was smeared onto a glass slide and stained using the modified acid-fast staining technique prior to microscopic examination. The remaining sample was stored in a 2.5% aqueous potassium dichromate solution at 4 °C until use.
Oocyst-positive samples were washed three times with deionized water and centrifuged at1 800× g for 10 min. Genomic DNA was isolated using the QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions with one minor adjustment where the fecal suspension was heated for 10 min at 95 °C. DNA was stored at -20 °C before it was used in polymerase chain reaction (PCR) amplifications.
PCR Amplification and Analysis
Nested PCR was used to amplify an approximately 840 base pair (bp) long fragment of the 18S rRNA gene locus using two sets of oligonucleotide primers: 5’-TTCTAGAGCTAATACATGCG-3’ and 5’-CCCATTTCCTTCGAAACAGGA-3’ for primary PCR and 5’-GGAAGGGTTGTATTTATTAGATAAAG-3’ and 5’-CTCATAAGGTGCTGAAGGAGTA-3’ for secondary PCR[11-13]. Amplification reactions were carried out in 25 μL volumes consisting of 12.5 μL Taq Green Master Mix (2X), 1 μL of each primer (10X), 9.5 μL nuclease-free water and 1 μL DNA. Reaction conditions were comprised of a hot start at 94 °C for 1 min followed by 35 cycles at 94 °C for 10 s, 55 °C for 30 s, and 72 °C for 1 min, and a final extension at 72 °C for 10 min. Amplified regions were separated using 2% agarose gel electrophoresis and vizualized following ethidium bromide staining.
Amplified secondary PCR products were subjected to bi-directional sequencing with secondary primers performed by Invitrogen (Shanghai, China).
Sequences were compared by homology (basic local alignment search tool; BLAST) against sequences present in the National Center for Biotechnology Information (NCBI) database (http://blast.ncbi.nlm.nih.gov/) and multiple alignments were performed using ClustalX (version 1.83). Phylogenetic relationship analyses were performed using MEGA software (version 4.1; Biodesign Institute, Tempe, AZ, USA).
Study Animals and Sample Analysis
Samples collected from pre-weaned ovines from eight farms (A-H) are described in Table 1. All animals presented in good health despite collected fecal samples being loose. Following light microscopic examination, 31 oocyst-positive samples (14.6% incidence from eight farms) were identified using a modified acid fast staining technique (Figure 1).
C. parvum, C. hominis, C. meleagridis, C. felis, C. canis, C. suis, C. muris, C. andersoni, C. ubiquitum, Cryptosporidium horse, rabbit, skunk, and chipmunk I genotypes have been recognized as zoonotic pathogens, especially cervine genotype infections in sheep and goats, suggesting that these animals may play larger roles as reservoir species than that previously believed, posing a significant public health threat. Causape et al. previously used a modified Ziehl-Neelsen technique to examine the incidence of C. parvum infection by screening fecal samples from 583 lambs (aged 1 day to 3 months) and 205 ewes (>one year of age) in northeastern Spain. The Spain study identified that 59% of lambs and 7.8% of ewes were Cryptosporidium-positive and statistical analysis demonstrated that the prevalence of this parasite in lambs was significantly associated with younger age and the form of diarrhea. Further, Bomfim et al. examined 105 stool samples from dairy goats in Rio de Janeiro, Brazil, using centrifuge- flotation techniques and safranine-methylene blue staining, and found a Cryptosporidium incidence of 4.8%.
Karanis et al. found 15 Cryptosporidium- positive goats in 42 farms in the Qinghai province of China using immunofluorescence; one sample was identified as a C. bovis-like genotype and a second isolate was novel. Geurden et al. launched a cross-sectional epidemiological study to define the prevalence of Cryptosporidium in lambs and goat kids in Belgium and, using quantitative immunofluorescence, identified 18/137 positive lambs and 14/148 positive goat kids. Furthermore, molecular characterization demonstrated that the cervine genotype was predominant in lambs but only C. parvum in goat kids. Wang et al. microscopically examined stool samples concentrated using Sheather’s sugar flotation technique and stained using the modified acid-fast stain sheep samples from the Henan Province (China). They demonstrated that a 4.8% Cryptosporidium oocyst incidence and that the cervine genotype was the major Cryptosporidium genotype identified, which was similar to that reported in this present study. The Henan Province data combined with the data presented here suggested that the cervine genotype may be the main Cryptosporidium genotype in China.
To date, most Cryptosporidium prevalence studies in sheep and goats focused on microscopic examination diagnostic techniques and lacked much more sensitive assays. Except for the above-mentioned districts in China, other countries in different regions of the world, for example, Turkey[20-23], Mexico[24-25] and the west-central region of Poland also used these techniques, suggesting that more sensitive PCR-based assays are need for assessing Cryptosporidium infection rates.
Paoletti et al. combined ELISA and PCR assays to detect Cryptosporidium in lambs in Italy and reported that C. parvum was the major species identified (17.45% prevalence), suggesting that surveillance of sheep populations for Cryptosporidium infections would be an important public health prevention strategy. Fayer and Santin isolated a new species, C. xiaoi, with a prevalence of 6.96% (5/72) from sheep following a multi-locus analysis of SSU-rDNA, HSP-70 and actin gene sequences. In Western Australia, Yang et al. screened 477 stool samples using PCR to amplify the 18S rRNA locus and successfully identified 10 Cryptosporidium cervine genotype isolates, suggesting that the C. parvum/C. hominis qPCR assay was more sensitive than the nested 18S PCR analysis. Ryan et al. genotyped Cryptosporidium collected from sheep also at the 18S rRNA locus and identified three Cryptosporidium species and five different genotypes, including 33 Cryptosporidium cervine isolates. In Maryland, Santin et al. utilized PCR to identify Cryptosporidium in the feces of pregnant ewes after parturition and from each of their lambs at three different times after birth and demonstrated that Cryptosporidium was present in 25% in the ewes and 77.4% in lambs. In addition, they also identified the cervine genotype and lambs with mixed Cryptosporidium spp. infections.
Several limitations in this study included: (1) insufficient stool for molecular characterization; (2) if the oocyst counts were below the microscopic detection thresholds, those samples would not have been screened by PCR; and (3) due to lack of sheep younger <6 months of age (and limited number of samples), additional research will be needed to establish a correlation between prevalence and age in addition to the effect of climate on infection status.
The prevalence of Cryptosporidium in pre-weaned ovine populations from Aba Tibetan and Qiang Autonomous Prefecture was 14.6% using a modified acid-fast staining technique. All Cryptosporidium isolates identified in the present study belonged to the C. ubiquitum, similar to a Henan Province study which identified the Cryptosporidium cervinve genotype (74/82), C. andersoni (4/82), and C. xiaoi (4/82) using an 18S rRNA-based PCR assay. Taken together, these data suggested that sheep are an important reservoir for the C. ubiquitum and other Cryptosporidium spp, suggesting that surveillance of these animal populations for the presence of Cryptosporidium is important to public health.
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