Cryptosporidiosis caused by Cryptosporidium parvum subtype IIdA15G1 at a dairy farm in Northwestern China

Background Cryptosporidium spp. are zoonotic parasites responsible for diarrhoeal diseases in animals and humans worldwide. Cattle are the most common mammalian species in which Cryptosporidium is detected, with pre-weaned calves considered to be reservoirs for zoonotic C. parvum. In October 2013, severe diarrhoea was observed in 396 pre-weaned calves at a farm in the Ningxia Autonomous Region of Northwestern China. 356 of the infected calves died despite antibiotic therapy. Findings 252 faecal samples were collected from the investigated farm. The identity of Cryptosporidium species was determined by polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) analysis, and by DNA sequence analysis of the small subunit (SSU) rRNA gene. C. parvum was subtyped using sequence analysis of the 60 kDa glycoprotein (gp60) gene. The highest infection rate of 83.3% (40/48) was seen in 2–3-week-old calves with diarrhoea, corresponding to the age at which animals died. Three Cryptosporidium species were identified, including C. parvum (n = 51), C. bovis (n = 1), and C. ryanae (n = 1). All C. parvum isolates were further identified as subtype IIdA15G1. Conclusions Cryptosporidium parvum was likely to be most responsible for diarrhoea and death. This is the first report of a cryptosporidiosis outbreak caused by C. parvum IIdA15G1 in Chinese dairy cattle.


Background
Species of Cryptosporidium are important zoonotic parasites that infect a wide range of vertebrate hosts, including humans [1]. There are extensive genetic variations within the Cryptosporidium genus. In addition to 27 recognized species of Cryptosporidium, more than 70 Cryptosporidium genotypes with no designated species names have been described [2][3][4][5]. Cryptosporidium can be transmitted by the faecal-oral route, via either direct contact or ingestion of contaminated food or water [1].
Cryptosporidiosis, caused by Cryptosporidium infection, is of great concern because of associated economic losses and the public health significance in humans. Diarrhoea is a typical clinical sign of human and animal cryptosporidiosis; dehydration, fever, nausea and anorexia are sometimes symptoms in infected hosts. Over 200 water-borne, food-borne, person-to-person, and zoonotic cryptosporidiosis outbreaks have been recorded [6]. The most famous cryptosporidiosis outbreak occurred in Milwaukee (Wisconsin, USA), where 403,000 people were infected through contaminated drinking water [7]. In recent years, cryptosporidiosis outbreaks have been reported in several European and American countries [8][9][10][11]. Molecular techniques have shown that C. parvum is the predominant species in cryptosporidiosis outbreaks, accounting for 50.8% of cases among 325 water-borne outbreaks of parasitic protozoan diseases worldwide [6].
Cattle are the most common mammalian species in which Cryptosporidium is detected; pre-weaned calves are considered reservoirs for zoonotic C. parvum [12]. Of 14 C. parvum subtype families (IIa, IIb, IIc, IId, IIe,  IIf, IIg, IIh, IIi, IIk, IIl, IIm, IIn, and IIo), IIa and IId are the two major zoonotic subtype families in animals and humans, while IIc and IIe are anthroponotic subtype families [13]. Other subtype families of C. parvum are occasionally seen in humans and other animals [2,14].
In China, Cryptosporidium infections have been reported in domestic animals, wild animals, and humans [13][14][15][16][17][18]. A cryptosporidiosis outbreak was noticed in a paediatric hospital in Shanghai [19]. In the current study we have briefly described a cryptosporidiosis outbreak that occurred at a dairy farm in the Ningxia Autonomous Region (Northwestern China). The infection rate and species distribution of Cryptosporidium were also determined.

Cryptosporidiosis outbreak
The investigated farm, located in the Ningxia Autonomous Region of Northwestern China, was established in July 2012 with 7000 dairy cattle (2600 calves and 4400 adult cattle). The breeding cattle were introduced from Australia and Uruguay, while forage was produced in the United States. Previously, vaccination was not done against rotaviruses, coronaviruses, and E. coli. Before this outbreak, Cryptosporidium oocysts were not detected using conventional faecal examination by microscopy. In October 2013, severe diarrhoea was observed in 396 preweaned calves on this farm. 356 of the calves, in particular the 2-3-week-olds, died despite being treated with antibiotics. We collected 30 faecal samples from calves with diarrhoea and subjected them to testing for gastrointestinal pathogens [Rota, Corona, E. coli F5 (K99), Crypto] using the commercial Digestive Kit (Real Bio-technology, Qingdao, China). One month later, the symptom of diarrhoea gradually disappeared in calves.

Ethics statement
During specimen collection, all animal experiments were conducted in accordance with the Chinese Laboratory Animal Administration Act 1988.Prior to experiment, the protocol of the current study wasreviewed and approved by the Research Ethics Committee of Henan Agricultural University (License no. 2014-018).

Sampling and microscopy examination
To determine infection rate and species distribution of Cryptosporidium, we obtained 252 faecal samples: 158 were from pre-weaned calves, 55 from post-weaned calves, and 39 from heifers and adults. Fresh faecal samples for each animal were collected immediately after defecation on the ground. Cryptosporidium oocysts in faecal samples were concentrated using Sheather's sugar flotation technique, with samples from pre-weaned calves subjected to formalin-ethyl acetate sedimentation [14].
Cryptosporidium-positive samples were determined by microscopy and stored in 2.5% potassium dichromate at 4°C prior to DNA extraction.

Molecular identification
Genomic DNA was extracted from Cryptosporidiumpositive faecal samples using the E.Z.N.A. Stool DNA kit (Omega Biotek Inc., Norcross, GA, USA) according to the manufacturer's recommended procedures. Cryptosporidium species were determined by polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) analysis, and by DNA sequence analysis of the small subunit (SSU) rRNA gene [20]. Subtyping of C. parvum was conducted using a nested PCR technique targeting the gp60 gene, which encodes the 60 kDa glycoprotein [2,21]. Amplicons were sequenced on an ABI PRISMTM 3730 XL DNA Analyzer using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Sequence accuracy was confirmed by twodirectional sequencing and by sequencing a fresh PCR amplicon if necessary. Sequence alignment was carried out using ClustalX 1.83 (ftp://ftp-igbmc.u-strasbg.fr/pub/ ClustalX/). Representative nucleotide sequences were deposited in GenBank (Accession numbers KM215133-KM215134, and KM873712-KM873713).

Findings and discussion
Using the Digestive Kit, we found that nine samples were positive for Cryptosporidium and four were positive for rotavirus. None of the samples analyzed with the Digestive Kit were positive for coronavirus or E. coli F5 (K99).
Diarrhoea is common in calves worldwide, and is considered a major cause of productivity decline and economic losses in the cattle industry [22]. Previous studies have indicated that rotaviruses, coronaviruses, E. coli, and C. parvum are all contributing factors to diarrhoea in calves. However, C. parvum is believed to be the primary cause of calf diarrhoea, and is a potential zoonotic agent [23,24]. Results from the Digestive Kit in combination with high Cryptosporidium prevalence (83.3%) in 2-3-week-old calves with diarrhoea were strong indicators that C. parvum was likely most responsible for diarrhoea and death.
An overall infection rate of 21% on this farm was higher than that observed in Heilongjiang (15.0%), Henan (13.0%), and Shannxi (3.4%) provinces in China [14,[25][26][27][28]. The highest level of prevalence (83.3%) in 2-3-week-old calves was similar to that seen in previous studies [29], and was consistent with the morbidity and mortality of calves in this age group.
The predominance of C. parvum infection in preweaned calves seen in this study was highly similar to those seen in most studies conducted in other countries [2,14,27,28]. It is known that C. parvum is a major etiological agent for sporadic cases, or water-and food-borne disease outbreaks in humans worldwide [9,[30][31][32][33]. As a major reservoir of C. parvum, pre-weaned calves might play an important role in zoonotic infections [12].
Previously, the C. parvum subtype IIdA15G1 has been detected in cattle, sheep and goats in Iran, Malaysia, and Spain [34][35][36][37]. It has also been found in humans from the Netherlands, Australia, Iran, Malaysia, and India [37][38][39][40][41]. The IIdA15G1 subtype identified in this study was unlikely to have arrived via Uruguay or the United States because the C. parvum IId subtype family has never been found in the Americas [13]. It is also unlikely that IIdA15G1 was introduced from Australia because C. parvum has not been detected in Australian breeding cattle. Thus, the source of the cryptosporidiosis outbreak could be attributed to C. parvum contamination in the area surrounding the farm. The IIdA15G1 subtype has been found in rodents in China, with C. parvum isolates from animals all belonging to the IId subtype [14,18,27,28]. Generally, IId is a major zoonotic subtype family reported in Europe (Hungary, Germany, Portugal, Sweden, Ireland, Spain, Belgium, Romania, the United Kingdom, the Netherlands, Slovenia, and Serbia and Montenegro), Asia (Kuwait, Iran, Jordan, India, Malaysia, and China), Africa (Egypt and Ethiopia), and Australia [13,14,42,43].
In comparison with cryptosporidiosis outbreaks in humans, only a few outbreaks have been reported in animals, including goats in Brazil and Oman [44,45], foals in New Zealand [46], cockatiels in Japan [47], stone curlews in the United Arab Emirates [48], alpaca crias in the United States [49], chelonians in Australia [50], and rabbits in Poland [51]. The findings from our current study provide further information regarding cryptosporidiosis outbreaks among animal populations.
In conclusion, this is the first report of a cryptosporidiosis outbreak in Chinese dairy cattle. The C. parvum IIdA15G1 subtype was responsible for the reported outbreak as determined by genotyping and subtyping tools. Further studies are required to improve our understanding of the transmission and public health significance of Cryptosporidium species in China.

Conclusions
This is the first report of a cryptosporidiosis outbreak in Chinese dairy cattle. The C. parvum IIdA15G1 subtype was responsible for the reported outbreak as determined by genotyping and subtyping tools.

Competing interests
The authors declare that they have no competing interests.
Authors' contributions RJW, LXZ conceived and designed the experiments; ZHC, JYH, and JQL performed the experiments; HYW, JFZ, ZJZ, and NNL analyzed the data; ZHC, RJW, and LXZ wrote the manuscript. All the authors have read and approved the final version of the manuscript.