Molecular identification and epidemiological comparison of Cryptosporidium spp. among different pig breeds in Tibet and Henan, China

Background Cryptosporidium spp. are important zoonotic pathogens infecting a wide range of vertebrate hosts, and causing moderate to severe diarrhea in humans. Cryptosporidium infections are frequently reported in humans and animals worldwide, but little research has been done on local pig breeds such as Tibetan pigs and Yunan Black pigs and imported pig breeds such as Landrace pigs in China. Therefore, a total of 1089 pig fecal samples from four intensive farms in four areas of China, including Tibetan pigs from Gongbujiangda County (n = 180) and Mainling County (n = 434), Tibet, Yunan Black pigs from Sanmenxia, Henan Province (n = 246), and Landrace pigs from Kaifeng, Henan Province (n = 229), and were screened for the presence of Cryptosporidium with microscopy and nested PCR amplification of the small subunit rRNA gene. Results The total infection rate of Cryptosporidium in 1089 fecal samples of three different pig breeds was 2.11% (23/1089), and the infection rates of Tibetan pigs, Yunan Black pigs, and Landrace pigs were 0.49% (3/614), 0.41% (1/246), and 8.30% (19/229), respectively. The prevalence of Cryptosporidium infection was significantly higher in weaned piglets (1–2 months) (4.36%, 21/482) than in younger and older age groups (p < 0.01). Sequence analysis of positive samples revealed that there was no mixed infection in our study population, which included 12 cases of C. suis mono-infections (52.17%, 12/23) and 11 cases of C. scrofarum mono-infections (47.83%, 11/23). C. suis was identified in one pre-weaned piglet (< 1 month) and 11 weaned piglets (1–2 months), while C. scrofarum was only detected in 10 weaned piglets (1–2 months) and one finished pig (> 2 months). Conclusions This is the first report on the identification of Cryptosporidium spp. in Tibetan pigs, and our findings also elucidate the occurrence and distribution of Cryptosporidium in three different pig breeds in Tibet and Henan, China. More molecular epidemiological studies are required to better clarify the prevalence and public health significance of Cryptosporidium in different pigs.


Background
Cryptosporidium is an important parasitic pathogen, generally causing self-limiting diarrhea in livestock, wild animals, and humans. However, it may cause severe debilitating disease in immunocompromised patients, especially those with acquired immune deficiency syndrome. The pathogen is transmitted via the fecal-oral route in both humans and animals, usually through the ingestion of contaminated water or food.
There is an extensive genetic variation within the genus Cryptosporidium, with 37 valid Cryptosporidium species recognized to date [1][2][3][4]. Of these, C. suis, C. scrofarum (formerly Cryptosporidium pig genotype II), C. parvum, C. muris, C. tyzzeri (formerly Cryptosporidium mouse genotype I), and C. andersoni have been identified from pigs [5,6]. There are also reports that both C. hominis and C. meleagridis can infect pigs [7][8][9]. Interestingly, cases of C. suis and C. scrofarum infection have been reported in humans in recent years, suggesting that these two pig-adapted Cryptosporidium species are potentially zoonotic [10][11][12]. C. suis was first identified in a 24-year old HIV patient in Peru in 2002 [13], and was then identified in HIV patients in Peru and China in 2007 and 2013, respectively [14,15]. C. suis has also been found in patients with digestive system ailments in the United Kingdom and Madagascar [12,16]. At present, there has only been one reported case of C. scrofarum infection in humans, which occurred in the Czech Republic in 2009 [17].
Landrace pigs are very famous in the world, mainly because of its good reproductive performance and fast growth, but have poor stress resistance [18]. Some indigenous Chinese pig breeds have lower growth rates and lean meat content, compared with conventional western pig breeds, but they are better able to survive in harsh environments. Yunan Black pigs, whose mother is one of some excellent local pig breeds in Henan, were bred by introducing Duroc pigs lineages in a large proportion (62.5%). Its main characteristics are slow growth but good meat quality. Tibetan pigs, also known as ginseng pigs, are mainly found in the eastern region of the Qinghai-Tibet Plateau, the northwest of Yunnan Province, and in the southwest of Gansu Province, all of which have an average elevation of 3000-6000 m. Tibetan pigs are strongly adapted to the natural environment of the plateaus, including tolerance to cold and hypoxia [19]. As a result of economic development, the breeding mode has been transformed from free-grazing to large-scale feed-lots. Despite the large amounts of data on Cryptosporidium spp. in pigs worldwide, there have been very few studies on the occurrence and distribution of Cryptosporidium spp. in pigs in China. At present, there is no information on the prevalence of Cryptosporidium spp. in Tibetan pigs in Tibet, China. Therefore, the aim of this study was to examine the prevalence, identity, and molecular characteristics of Cryptosporidium spp. in three different pig breeds in Tibet and Henan, China, and to estimate their zoonotic potential.

Sample collection
From March to June 2016, a total of 1089 fresh faecal samples, each of~20 g, were collected from four intensive farms in four areas of China, and each farm was visited only once ( Fig. 1; Table 1). The samples were numbered and the sample details were recorded, including sampling time, geographic information and growth stage. During specimen collection, only the inner portion of each faecal sample was collected to ensure no environmental contamination. Each fresh faecal sample was separately collected into sterile gloves before adding 2.5% potassium dichromate and then placed into containers filled with ice packs and immediately transported to the laboratory. Upon arrival, each specimen was first treated by Sheather's sugar flotation technique, and then examined by microscopy to detect Cryptosporidium oocysts at a bright-field microscope with 100 × and 400 × magnification. All fecal specimens were stored at 4°C prior to DNA extraction.

DNA extraction and nested polymerase chain reaction amplification
Prior to DNA extraction, fecal samples were washed with distilled water to remove the potassium dichromate. Subsequently, genomic DNA was extracted from approximately 200 mg of semi-purified product using an E.Z.N.A.R Stool DNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA) as per the manufacturer's instructions. The extracted DNA was stored in 200-μl volume of Solution Buffer (supplied with the kit) at − 20°C until use.

Sequence analysis
All of the secondary amplification products were sequenced in both directions on an ABI PRISM 3730 XL DNA Analyzer using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). To ensure sequence accuracy, two-directional sequencing was used. To identify Cryptosporidium species, the resulting sequences were subjected to BLAST analysis against sequences in the GenBank database (http:// blast.ncbi.nlm.nih.gov), and aligned using Clustal X 2.1 software (http://www.clustal.org/).

Statistical analysis
The Cryptosporidium infection rates were evaluated using Regression Analysis in SPSS 22.0 software for  Windows with 95% confidence intervals (CI), and probability leval (P) of < 0.05 were considered as statistically significant.

Species distribution
Sequence analysis of the SSU rRNA gene fragment revealed the presence of two Cryptosporidium species: C. suis (n = 12) and C. scrofarum (n = 11), and also showed 100% nucleotide identity to a pig-derived sequence (GenBank accession number: GU254174) and pig-derived sequences (GenBank accession numbers: KU668895 and KP704557) respectively. All sequence data has been deposited in the GenBank database under accession numbers MH174659, MH174663, MH178034, and MH178036.
In Henan, two Cryptosporidium species were indentified, including 12 C. suis-positive samples and 8 C. scrofarum-positive samples, while only 3 C. scrofarum-positive samples were found in Tibet. There were many Cryptosporidium -positive samples in imported Landrace pigs, including 11 C. suis-positive samples and 8 C. scrofarum-positive samples, but fewer Cryptosporidium-positive samples in local pig breeds, including 3 C. scrofarum-positive samples in Tibetan pigs and one C. suis-positive sample in Yunan Black pigs. In addition, C. suis was only indentified in pigs aged < 1 month old (n = 1) and 1-2 months old (n = 11), and C. scrofarum was only indentified in pigs aged 1-2 months old (n = 10) and > 2 months old (n = 1).

Discussion
Among the 23 Cryptosporidium-positive samples identified by PCR, only 5 were detected by microscopy, which may be caused by the low concentration of Cryptosporidium oocyst in fecal specimens. In this study, the Cryptosporidium prevalence in Henan was 4.21%, which is lower than that in many reported areas of China, such as Guangdong (8. [21][22][23][24][25][26][27]. Surprisingly, the infection rate of Cryptosporidium in Tibet is lower than in any reported areas ( Table 2). The present study also indicated that the Cryptosporidium infection rates in both local pig breeds were lower than that in imported pig breed. The observed Cryptosporidium prevalence rates of local pig breeds, including 0.49% in Tibetan pigs and 0.41% in Yunan Black pigs, were also apparently lower than that reported in some foreign pig breeds in North America (17.85-55.74%), Europe (5.36-74.67%), and Asia (59.68-71.43%), and in wild boar in the Czech Republic (16.68%) and Spain (16.75%) ( Table 3) [6,[28][29][30][31][32][33][34][35][36][37][38][39]. Many factors, including management system, specimen size and diagnostic technique, may be also responsible for the differences in the prevalence of Cryptosporidium among different pig breeds and different geographic areas.
Cryptosporidium infections are common in pigs and have been found in all age groups worldwide. However, many studies have shown that the highest rates of Cryptosporidium infection occur in weaned piglets (1-2 months), with the infection rates ranging from 20.63 to 83.72% indentified by PCR [6,23,26,27]. In this study, the prevalence of Cryptosporidium in weaned piglets (1-2 months) was at a lower level, but still consistent with the studies of others that weaned piglets (1-2 months) was higher than that in other two age groups. Therefore, although the age distribution of Cryptosporidium infection rates in pigs has not been clearly concluded up to now, we can generally assume that the rate of Cryptosporidium infection in pigs aged 1-2 months is greater than that of other age groups. It is difficult to explain why previous studies have found that conventionally-reared piglets < 1 month of age are reliably infected under the conditions examined. The age distribution of Cryptosporidium infection may also be affected by a number of other factors, including changes in the intestinal environment of the animals caused by diet or age.
Many studies have shown that C. suis and C. scrofarum appear to be the main pig-adapted species, and some other Cryptosporidium species were also identified in pigs, sometimes with mixed infections. Oddly, there was no mixed infection in our study. In this study, C. suis and C. scrofarum were both found in Henan, which was the same as that in Shaanxi and other regions that have been reported [22-24, 26, 27] (Table 2), while C. scrofarum was only identified in Tibet, which was the same as that in Guangdong and other regions that have been reported [21,25] (Table 2). So far, mixed infections of C. suis and C. scrofarum were only reported in Heilongjiang and Shanghai, China [23,26] (Table 2). C. suis and C. scrofarum were detected in local Yunan Black pigs and Tibetan pigs in this study, respectively. However, C. suis and C. scrofarum were fairly common, and mixed infections of C. suis and C. scrofarum were reported in some foreign breeds in Japan, Austria, The Czech Republic, and Poland [6,30,32,33,35] (Tables 3). Interestingly, C. parvum, C. muris, and mouse genotype strains have also been reported in pigs in several countries [32,34,36,37,39] (Tables 3). In addition, our results also showed that 12 samples from one pre-weaned piglet (< 1 month) and 11 weaned piglets (1-2 months) contained C. suis, while 11 samples from 10 weaned piglets (1-2 months) and one finished pig (> 2 months) contained C. scrofarum. These findings are consistent with a previous report showing that the different Cryptosporidium species are age-specific in pigs, and piglets are more susceptible to C. suis infection while older pigs are more susceptible to C. scrofarum [24]. There are many factors leading to differences in the identification of single or mixed infection, with insufficient testing accuracy being a major cause. A comparative study found that one Cryptosporidium-positive sample was typed as C. muris on the basis of Sanger sequencing but was identified as a C. muris and C. tyzzer mixed infection by highthroughput sequencing, which has a superior depth of coverage, especially for mixed infections in clinical samples [40]. Another study found that C. suis and C. scrofarum in mixed infections were successfully identified using Illumina sequencing technology [41]. However, to improve identification rates of Cryptosporidium species or genotypes in mixed-infection samples, more sensitive tools with greater resolution are required.

Conclusions
In conclusion, although the levels of Cryptosporidium infection were lower in three different pig breeds in Tibet and Henan, especially in Tibetan pigs and Yunan Black pigs, the two indentified Cryptosporidium species, including C. suis and C. scrofarum are both zoonotic. Importantly, this is the first epidemiological investigation of the prevalence and risk factors of Cryptosporidium in Tibetan pigs from Tibet. At present, there is no effective drug treatment or vaccine for porcine cryptosporidiose. Therefore, measures such as strengthening the

Availability of data and materials
The data supporting the findings are included in the manuscript. Representative nucleotide sequences generated in this study were submitted to the GenBank database under the accession numbers MH174659 and MH178034 for C. suis, MH178036 and MH174663 for C. scrofarum. Additional data and materials are available upon request from the corresponding author.
Authors' contributions LZ conceived and designed the experiments; SZ, DL and CZ performed the experiments; SZ, LZ, YW and Y-K C analyzed the data; S-M Z, CN, and GZ contributed reagents/materials/Únalysis tools, SZ, Y-C C and JH wrote the paper. All authors critically read and contributed to the manuscript and approved the final version.

Ethics approval
This study was performed in accordance with the Chinese Laboratory Animal Administration Act of 1988. Before the experiments, the research protocol was reviewed and approved by the Research Ethics Committee of Henan Agricultural University. Permission was obtained from all managers of the study pig farms before the fecal samples were collected.

Consent for publication
Not applicable.