INTRODUCTION
Cryptosporidium and Giardia are two common intestinal protozoan parasites with worldwide prevalence in humans and many other animals (Xiao and Fayer, Reference Xiao and Fayer2008). The sources of infection for cryptosporidiosis and giardiosis are human and animal feces, with the pathogens being transmitted by the fecal–oral route via direct contact or ingestion of contaminated food or water (Xiao et al. Reference Xiao, Lal and Jiang2004; Smith and Nichols, Reference Smith and Nichols2006). Both pathogens are responsible for diarrhoea; however, the clinical signs of infection vary depending on the age and health of the infected hosts and the genetic background and infective dose of the parasites (Xiao and Fayer, Reference Xiao and Fayer2008). Life threatening diarrhoea has been reported in human cryptosporidiosis patients infected with HIV (Nahrevanian and Assmar, Reference Nahrevanian and Assmar2008; Sodqi et al. Reference Sodqi, Marih, Lahsen, Bensghir, Chakib, Himmich and El Filali2012).
Molecular epidemiological data have revealed that each parasite genera comprises different species and genotypes, some of which are specific to humans while others are zoonotic (Xiao and Fayer, Reference Xiao and Fayer2008). Of the six recognized Giardia species, Giardia duodenalis is the only one that infects humans. To date, eight assemblages (A to H) of G. duodenalis have been identified, and the two major assemblages (A and B) responsible for 99·2% of human giardiasis cases (Sprong et al. Reference Sprong, Cacciò and van der Giessen2009) have broad host specificity and are transmitted zoonotically (Feng and Xiao, Reference Feng and Xiao2011). Within the genus Cryptosporidium, to date, 27 species and more than 70 genotypes have been identified, with new genotypes still being found (Elwin et al. Reference Elwin, Hadfield, Robinson, Crouch and Chalmers2012; Kváč et al. Reference Kváč, Hofmannová, Hlásková, Květoňová, Vítovec, McEvoy and Sak2014). Additionally, at least 13 Cryptosporidium species and three genotypes have been isolated from humans (Xiao, Reference Xiao2010; Helmy et al. Reference Helmy, Krücken, Nöckler, von Samson-Himmelstjerna and Zessin2013). Among them, Cryptosporidium parvum, one of the two most common species causing human cryptosporidiosis, is generally considered to be zoonotically transmitted (Xiao, Reference Xiao2010).
Thus, molecular epidemiological investigations of animal cryptosporidiosis and giardiasis are important for public health. In fact, epidemiological studies of Cryptosporidium and Giardia have been conducted in many countries and areas of the world. However, these studies mainly involve economically important farm animals or pets that live in close contact with humans.
Rodents are highly successful in adapting to a variety of environments throughout the world, making them extremely abundant. These animals often live in close association with anthropogenic environments and endanger public health by destruction and contamination of food and by spreading various diseases. It has been reported that rodents can carry more than 200 pathogens; hence, they can act as vectors for various pathogens, with 57 of them, including Cryptosporidium and Giardia, being zoonotic pathogens (Zhao et al. Reference Zhao, Cheng, Gong and Hou2010). To date, three Giardia species (Giardia microti, Giardia muris and G. duodenalis) have been isolated from naturally infected rodents (Lebbad et al. Reference Lebbad, Mattsson, Christensson, Ljungström, Backhans, Andersson and Svärd2010; Backhans et al. Reference Backhans, Jacobson, Hansson, Lebbad, Lambertz, Gammelgard, Saager, Akande and Fellström2013), and within G. duodenalis, assemblages A, B, C, E, F and G have been identified (Feng and Xiao, Reference Feng and Xiao2011). Within Cryptosporidium, eight species and more than ten genotypes have been found in rodents, including C. parvum, Cryptosporidium muris, Cryptosporidium ubiquitum, Cryptosporidium meleagridis, Cryptosporidium scrofarum, Cryptosporidium wrairi, Cryptosporidium tyzzeri (mouse genotype I) and Cryptosporidium andersoni, as well as rat genotypes (I to IV), mouse genotypes (II, III and the Naruko genotype), a ferret genotype, chipmunk genotype I and a hamster genotype (Kimura et al. Reference Kimura, Edagawa, Okada, Takimoto, Yonesho and Karanis2007; Kvác et al. Reference Kvác, Hofmannová, Bertolino, Wauters, Tosi and Modrý2008; Lv et al. Reference Lv, Zhang, Wang, Jian, Zhang, Ning, Wang, Feng, Wang, Ren, Qi and Xiao2009a , Reference Lv, Feng, Qi, Yang, Jian, Ning and Zhang b ; Paparini et al. Reference Paparini, Jackson, Ward, Young and Ryan2012; Ren et al. Reference Ren, Zhao, Zhang, Ning, Jian, Wang, Lv, Wang, Arrowood and Xiao2012; Backhans et al. Reference Backhans, Jacobson, Hansson, Lebbad, Lambertz, Gammelgard, Saager, Akande and Fellström2013; Ng-Hublin et al. Reference Ng-Hublin, Singleton and Ryan2013; Silva et al. Reference Silva, Richtzenhain, Barros, Gomes, Silva, Kozerski, de Araújo Ceranto, Keid and Soares2013).
Currently, in China, only one genotyping and subtyping study on Cryptosporidium isolates from wild and laboratory animals and pet rodents has been conducted (Lv et al. Reference Lv, Zhang, Wang, Jian, Zhang, Ning, Wang, Feng, Wang, Ren, Qi and Xiao2009a ), and no published studies are available on the molecular identification and genetic characterization of rodent-derived Giardia isolates. Commensal rodents are known to live in close quarters with humans and where human activities occur, and they are commonly found in premises where farm animals are kept (Backhans et al. Reference Backhans, Jacobson, Hansson, Lebbad, Lambertz, Gammelgard, Saager, Akande and Fellström2013). The aims of this study, therefore, were to determine the prevalence of Cryptosporidium and Giardia in commensal rodents captured from animal farms and in the neighbourhoods near these farms, and to characterize the parasite isolates collected from these areas at the genotype and subtype levels. We also assessed the zoonotic potential of the Cryptosporidium and Giardia isolates collected herein by aligning the sequences obtained with published sequences of parasite isolates from GenBank.
MATERIALS AND METHODS
Study sites and rodent collections
In a study from June 2010 to December 2011, a total of 232 rodents were captured from four animal farms and other locations close to such farms using traps. Thirty-three Asian house rats (Rattus tanezumi) were captured from a pig farm and in the neighbourhood near the farm in Longyan City, Fujian Province. One-hundred and sixty-eight brown rats (Rattus norvegicus) and 31 house mice (Mus musculus) were captured on cattle and sheep farms or in the neighbourhoods near these two farms in Zhengzhou City, Henan Province. All the animals were transported to our laboratory in a cooler with ice packs within 48 h of being euthanized by CO2 inhalation.
Ethical and regulatory guidelines
All the animals were handled and cared for according to the Chinese Laboratory Animal Administration Act of 1998. The research of protocol was reviewed and approved by the Research Ethics Committee and the Animal Ethical Committee of Henan Agricultural University and Harbin Medical University.
Fecal sample collection and examination
Fresh fecal material (approximately 300–500 mg) was collected directly from the intestinal section of each rodent. Lugol's iodine staining method and Sheather's sugar flotation technique and were used to screen for the presence of Giardia cysts and Cryptosporidium oocysts, respectively. Wet smears were examined using a bright-field microscope with 10× and 40× magnification. Samples positive for Giardia cysts and/or Cryptosporidium oocysts were stored in a solution of 2·5% potassium dichromate at 4 °C prior to DNA extraction.
DNA extraction
Potassium dichromate was removed from the fecal specimens that were positive for either or both of the two parasite genera with distilled water by centrifugation at 1500 g at room temperature, three times. Genomic DNA was extracted directly from approximately 200 mg of each specimen using a QIAamp DNA Mini Stool Kit (Qiagen, Hilden, Germany) according to the manufacturer's procedures. DNA was eluted in 200 mL of AE elution buffer (provided in the kit) and the DNA preparations were stored at −20 °C prior to PCR analysis.
Giardia and Cryptosporidium genotyping and subtyping
The identity of genotypes and subtypes of Giardia cysts in the fecal samples was confirmed by DNA sequence analysis of the tpi, gdh and bg genes of this parasite using three distinct nested PCR protocols. tpi, gdh and bg PCR products of approximately 530, 530 and 510 bp, respectively, were amplified corresponding to the partial sequences of these genes (Cacciò et al. Reference Cacciò, De Giacomo and Pozio2002, Reference Cacciò, Beck, Lalle, Marinculic and Pozio2008; Sulaiman et al. Reference Sulaiman, Fayer, Bern, Gilman, Trout, Schantz, Das, Lal and Xiao2003; Lalle et al. Reference Lalle, Pozio, Capelli, Bruschi, Crotti and Cacciò2005). When DNA preparations were positive for Giardia at one or two, but not all three gene loci, they were subjected to two more repeated PCR amplifications at the locus or loci that were originally PCR-negative for Giardia.
Cryptosporidium oocysts in the fecal samples were identified at the species and genotype levels using nested PCR amplification of a SSU rRNA gene fragment of ~830 bp (Xiao et al. Reference Xiao, Morgan, Limor, Escalante, Arrowood, Shulaw, Thompson, Fayer and Lal1999). Subtyping of C. parvum isolates was conducted by nested PCR amplification of a gp60 gene fragment of ~800–850 bp (Alves et al. Reference Alves, Xiao, Sulaiman, Lal, Matos and Antunes2003).
DNA sequencing and analysis
All secondary PCR products were sequenced using the same PCR primers used for the secondary PCRs on an ABI PRISMTM 3730 DNA Analyser (Applied Biosystems, Carlsbad, CA, USA), using a BigDye Terminator v3·1 Cycle Sequencing kit (Applied Biosystems). The accuracy of the sequencing data was confirmed by sequencing the PCR products in both directions, and further PCR products were sequenced if necessary. The genotype and subtype identities of the Giardia and Cryptosporidium isolates were established by comparing the nucleotide sequences obtained with each other and with published GenBank sequences using the Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and ClustalX 1·83 (http://www.clustal.org/).
RESULTS
Prevalence and distribution of Giardia and Cryptosporidium
Giardia and Cryptosporidium were detected in all three rodent species we examined. In general, Cryptosporidium was more prevalent than Giardia in the rodents, based on the fact that 8·2% (19/232) and 6·0% (14/232) of the fecal samples were positive for Cryptosporidium oocysts and Giardia cysts, respectively. However, there was no statistically significant difference in the prevalence of the two parasites (P > 0·05). The infection rates for Giardia and Cryptosporidium differed in the three types of commensal rodents we examined. Giardia cysts and Cryptosporidium oocysts were detected in 18·2% (6/33) and 6·1% (2/33) of Asian house rats (R. tanezumi), respectively, while Giardia cysts and Cryptosporidium oocysts were detected in 7·1% (12/168) and 6·6% (11/168) of brown rats (R. norvegicus), respectively. Giardia and Cryptosporidium were both detected in one fecal specimen from a house mouse (M. musculus), with an infection rate of 3·2% (1/31) observed (Table 1).
Prevalence and molecular identification of Giardia and Cryptosporidium in three commensal rodent species
Note: The bars denote negative results at the locus.
a The results were all based on the following microscopy methods: Sheather's sugar flotation technique for Cryptosporidium oocysts and Lugol's iodine stain method for Giardia cysts.
Genotyping and subtyping of Giardia
In total, 12, 14 and 10 Giardia isolates were successfully amplified and sequenced at the tpi, bg and gdh loci, respectively. The sequence analysis revealed that all the Giardia isolates were assemblage G of G. duodenalis. The nucleotide sequences were identical to each other at all three loci, with the tpi, bg and gdh gene sequences sharing 100% sequence similarity with those of G. duodenalis assemblage G from a brown rat (R. norvegicus) in Sweden (GenBank: EU781013), another brown rat (R. norvegicus) in Sweden (GenBank: EU769221), and from a mouse in Australia (GenBank: AY178748), respectively.
Genotyping and subtyping of Cryptosporidium
In total, 19 fecal samples positive for Cryptosporidium oocysts were successfully amplified and sequenced at the SSU rRNA gene loci, revealing the presence of C. parvum (n = 12) and C. muris (n = 7). All the C. parvum SSU rRNA sequences were identical to each other and shared 100% sequence identity with those isolated from humans (GenBank: AB089290 from Japan and EU331237 from the Czech Republic), a sheep (GenBank: JN247404 from Brazil), a yellow-bellied marmot (GenBank: KF626381) and cattle (GenBank: HQ009805 from China, AF108864 from Australia, KC476546 from Poland, AB441687 from Iran and AB513881 from Egypt). All seven C. muris isolates had the same SSU rRNA sequence and shared 100% sequence identity with those from humans (GenBank: AJ307669 from Kenya), rodents (GenBank: JN172969 from Sweden, AB089284 from Japan, KF419208 and GQ121018 from China, EU245045 from America), monkeys (GenBank: GU319781 and GU319782 from China), bactrian camels (GenBank: EU245044 from the Czech Republic), ostriches (GenBank: GQ227706 from China), pythons (GenBank: EU553588, EU553592 from Spain), and giraffes (GenBank: FJ883577 from the Czech Republic).
All 12 C. parvum isolates were subtyped by sequence analysis of the gp60 gene. However, only nine of them produced the expected PCR products, which were identified as IIdA15G1. The same genotype (sequence) has been found in humans (GenBank: FJ897784, JF268647, JF268648 and HQ241928 from India; EF576977 from the Netherlands), cattle (GenBank: AB560740 from Iran), a goat (GenBank: HM627529 from India), a lamb (GenBank: EU549717 from Spain) and a hamster (GenBank: GQ121027 from China) by BLAST searching GenBank using the gp60 gene sequences obtained herein.
DISCUSSION
The epidemiological roles that rodents play in many zoonotic cycles are a cause for concern because these animals can transmit disease-causing agents to domestic animals and humans (Backhans et al. Reference Backhans, Jacobson, Hansson, Lebbad, Lambertz, Gammelgard, Saager, Akande and Fellström2013). In the present study, Giardia cysts and Cryptosporidium oocysts were detected in all three rodent species we collected in China. The overall prevalence of Cryptosporidium, as judged by microscopy, was higher than that of Giardia with 8·2% (19/232) for Cryptosporidium and 6·0% (14/232) for Giardia.
The average infection rate (6·0%) for Giardia was lower in commensal rodents than the rates reported previously for other rodent species (22·2–100%) (Fernández-Álvarez et al. Reference Fernández-Álvarez, Martín-Alonso, Abreu-Acosta, Feliu, Hugot, Valladares and Foronda2014). In Flanders (Belgium), of the 80 pet chinchillas (Chinchilla lanigera) that were screened, 53 (66·3%) excreted Giardia cysts (Levecke et al. Reference Levecke, Meulemans, Dalemans, Casaert, Claerebout and Geurden2011). Giardia infection was found in all of the common voles (Microtus arvalis) and bank voles (Clethrionomys glareolus) screened in Poland (Bednarska et al. Reference Bednarska, Bajer, Sinski, Girouard, Tamang and Graczyk2007), while Giardia infection rates of 65·9% in muskrats (Ondatra zibethicus) in America (Bitto and Aldras, Reference Bitto and Aldras2009) and 22·2% in pet hamsters (Mesocricetus auratus, Phodopus sungorus, Phodopus campbelli and Phodopus roborovskii) in China (Lv et al. Reference Lv, Feng, Qi, Yang, Jian, Ning and Zhang2009b ) were reported. In fact, detection method choice is a major factor influencing infection rate calculations in epidemiological studies. The highest and lowest infection rates (100 and 22·2%) mentioned above were based on the use of combined fluorescent in situ hybridization and direct fluorescent antibody techniques, or microscopic examination after iodine wet mount staining, respectively (Bednarska et al. Reference Bednarska, Bajer, Sinski, Girouard, Tamang and Graczyk2007; Lv et al. Reference Lv, Feng, Qi, Yang, Jian, Ning and Zhang2009b ). In the present study, the low infection rate we observed might be partially attributable to the type of morphological examination method used.
All of the 14 isolates that were positive for Giardia cysts were identified as G. duodenalis assemblage G in the present study (based on sequence analysis of the tpi, bg and gdh genes), with one from a house mouse, two from Asian house rats and the remaining 11 from brown rats. Additionally, we noted that PCR amplified 100% (14/14) of samples at the bg locus, followed by 85·7% (12/14) at the tpi locus and 71·4% (10/14) at the gdh locus. The factors that can influence the efficiency of PCR amplification of a target sequence are numerous. Because the same quality and quantity of DNA templates (from the same commercial DNA extraction kit) were used for the PCRs in our study, along with the same DNA polymerase, the degree with which the primers matched the DNA templates is likely to be the main reason for the amplification differences seen with the different sets of primers. In fact, intra- and inter-assemblage recombination and meiotic sex have been seen in assemblage A to G isolates from humans and other animals (Lasek-Nesselquist et al. Reference Lasek-Nesselquist, Welch, Thompson, Steuart and Sogin2009). The results suggest, therefore, that the primer sequences best-matched the binding region of the bg locus.
Sequence analysis revealed that for all three loci, all the G. duodenalis assemblage G isolate sequences were identical to each other, suggesting the conservative nature of the tpi, gdh and bg genes of assemblage G. The same tpi, bg and gdh gene sequences were found in a brown rat in Sweden (Lebbad et al. Reference Lebbad, Mattsson, Christensson, Ljungström, Backhans, Andersson and Svärd2010), and a mouse in Australia (unpublished), respectively. Indeed, to date, assemblage G has only been reported in rodents (Lebbad et al. Reference Lebbad, Mattsson, Christensson, Ljungström, Backhans, Andersson and Svärd2010; Feng and Xiao, Reference Feng and Xiao2011; Backhans et al. Reference Backhans, Jacobson, Hansson, Lebbad, Lambertz, Gammelgard, Saager, Akande and Fellström2013; Fernández-Álvarez et al. Reference Fernández-Álvarez, Martín-Alonso, Abreu-Acosta, Feliu, Hugot, Valladares and Foronda2014). Previous molecular epidemiological studies reported the presence of three Giardia species in rodents: G. duodenalis, G. muris and G. microti (Lebbad et al. Reference Lebbad, Mattsson, Christensson, Ljungström, Backhans, Andersson and Svärd2010). Within G. duodenalis, assemblages A, B, C, E, F and G have been identified in these animals (Feng and Xiao, Reference Feng and Xiao2011). Assemblage B was predominant in chinchillas (C. lanigera) in some geographical areas, accounting for the fact that 93·6% (29/31) of the G. duodenalis isolates belonged to assemblage B in Italy (Veronesi et al. Reference Veronesi, Piergili Fioretti, Morganti, Bietta, Moretta, Moretti and Traversa2012). In Belgium, all the Giardia isolates sequenced successfully were identified as assemblage B based on sequence analysis of the bg gene, while 85·7% of Giardia isolates were confirmed to be assemblage B of G. duodenalis based on the assemblage-specific PCR targeting the tpi gene (Levecke et al. Reference Levecke, Meulemans, Dalemans, Casaert, Claerebout and Geurden2011). Likewise, all the Giardia isolates from beavers and 62·5% muskrats captured in Maryland, USA were identified as G. duodenalis assemblage B (Sulaiman et al. Reference Sulaiman, Fayer, Bern, Gilman, Trout, Schantz, Das, Lal and Xiao2003). Of the 62 trap-captured beavers in Massachusetts, USA, four were confirmed to be infected with assemblage B (Fayer et al. Reference Fayer, Santín, Trout, DeStefano, Koenen and Kaur2006). Of course, in the present study, we could not rule out the presence of other G. duodenalis assemblages and other Giardia species in our samples because genotype/assemblage-specific PCR was not used. It has been established that genus-specific primers preferentially amplify the predominant species or genotypes/assemblages during PCR. Hence, the present data suggest a small possibility for zoonotic transmission of giardiasis assemblages A and B in the areas investigated. It is difficult for cysts in a low intensity in the fecal specimens to infect hosts successfully even if there were assemblages A and B isolate.
Cryptosporidium oocysts were detected by microscopy in 8·2% of 232 fecal specimens from the three species of commensal rodents caught in the present study. Previous epidemiological studies confirmed the presence of Cryptosporidium in nearly 40 rodent species, with Cryptosporidium infection rates ranging from 5·0 to 39·2% (Lv et al. Reference Lv, Zhang, Wang, Jian, Zhang, Ning, Wang, Feng, Wang, Ren, Qi and Xiao2009a ). To date, eight Cryptosporidium species and more than ten genotypes have been identified in a variety of rodents (Kimura et al. Reference Kimura, Edagawa, Okada, Takimoto, Yonesho and Karanis2007; Kvác et al. Reference Kvác, Hofmannová, Bertolino, Wauters, Tosi and Modrý2008; Lv et al. Reference Lv, Zhang, Wang, Jian, Zhang, Ning, Wang, Feng, Wang, Ren, Qi and Xiao2009a , Reference Lv, Feng, Qi, Yang, Jian, Ning and Zhang b ; Paparini et al. Reference Paparini, Jackson, Ward, Young and Ryan2012; Ren et al. Reference Ren, Zhao, Zhang, Ning, Jian, Wang, Lv, Wang, Arrowood and Xiao2012; Backhans et al. Reference Backhans, Jacobson, Hansson, Lebbad, Lambertz, Gammelgard, Saager, Akande and Fellström2013; Ng-Hublin et al. Reference Ng-Hublin, Singleton and Ryan2013; Silva et al. Reference Silva, Richtzenhain, Barros, Gomes, Silva, Kozerski, de Araújo Ceranto, Keid and Soares2013). Cryptosporidium parvum and C. muris were identified here in all 19 isolates that were positive for Cryptosporidium oocysts based on the SSU rRNA gene sequence analysis.
Cryptosporidium parvum is one of the two most common species of Cryptosporidium in humans (Xiao and Fayer, Reference Xiao and Fayer2008). It has also been found in ruminants, pigs, horses, alpacas, carnivores and some rodent species, with cattle being regarded as the major reservoirs for this parasite (Lv et al. Reference Lv, Zhang, Wang, Jian, Zhang, Ning, Wang, Feng, Wang, Ren, Qi and Xiao2009a ; Xiao, Reference Xiao2010). In the present study, gp60 gene sequence analysis showed that all nine of the C. parvum isolates amplified and sequenced successfully belonged to the subtype IIdA15G1. This subtype was identified in brown rats for the first time in the present study. Currently, at least 14 subtype families (IIa to Iii and IIk to IIo) of C. parvum have been identified based on sequence analysis of the gp60 gene, and IIa is the predominant subtype family in humans and other animals worldwide, whereas IId is another major zoonotic subtype reported in Europe, Asia, Egypt, and Australia (Xiao, Reference Xiao2010; Wang et al. Reference Wang, Zhang, Axén, Bjorkman, Jian, Amer, Liu, Feng, Li, Lv, Zhao, Qi, Dong, Wang, Sun, Ning and Xiao2014). The IIdA15G1 subtype has been found in human cases of cryptosporidiosis in the Netherlands and India (Wielinga et al. Reference Wielinga, de Vries, van der Goot, Mank, Mars, Kortbeek and van der Giessen2008; Ajjampur et al. Reference Ajjampur, Liakath, Kannan, Rajendran, Sarkar, Moses, Simon, Agarwal, Mathew, O'Connor, Ward and Kang2010). This subtype was also identified in C. parvum isolates from cattle in Iran (Nazemalhosseini-Mojarad et al. Reference Nazemalhosseini-Mojarad, Haghighi, Taghipour, Keshavarz, Mohebi, Zali and Xiao2011), goats in India (Rajendran et al. Reference Rajendran, Ajjampur, Chidambaram, Kattula, Rajan, Ward and Kang2011), lambs in Spain (Quílez et al. Reference Quílez, Torres, Chalmers, Hadfield, Del Cacho and Sánchez-Acedo2008), Siberian chipmunks and hamsters in China (Lv et al. Reference Lv, Zhang, Wang, Jian, Zhang, Ning, Wang, Feng, Wang, Ren, Qi and Xiao2009a ). The above findings that the same gp60 gene sequences of the C. parvum isolates have been found in humans and animals, suggest that the brown rats infected with C. parvum in the areas we investigated might pose a threat to local people and farm animals by shedding human-infective C. parvum oocysts in their feces, thereby contaminating the environment and food sources. In China, only a small number of C. parvum parasites have been obtained. Besides the subtype IIdA15G in the Siberian chipmunks and hamsters mentioned above, in recent years, another seven C. parvum subtypes have been identified; these include IIaA15G2R1, IIaA16G2R1, IIaA14G1R1, IIaA14G2R1 and IIaA16G3R1 from yaks (Mi et al. Reference Mi, Wang, Li, Huang, Zhou, Li, Lei, Cai and Chen2013), IIcA5G3a from monkeys (Ye et al. Reference Ye, Xiao, Ma, Guo, Liu and Feng2012), IIdA19G1 from cattle (Wang et al. Reference Wang, Wang, Sun, Zhang, Jian, Qi, Ning and Xiao2011; Zhang et al. Reference Zhang, Yang, Liu, Wang, Zhang, Shen, Cao and Ling2013), HIV patients (Wang et al. Reference Wang, Zhang, Zhao, Zhang, Zhang, Guo, Liu, Feng and Xiao2013) and wastewater (Li et al. Reference Li, Xiao, Wang, Zhao, Zhao, Duan, Guo, Liu and Feng2012) in China.
Cryptosporidium muris was identified in all three species of commensal rodents (Asian house rats, brown rats and house mice) in the present study. Cryptosporidium muris is a Cryptosporidium species that has been found in a variety of rodents, some mammals and one species of bird (tawny frogmouth) (Lv et al. Reference Lv, Zhang, Wang, Jian, Zhang, Ning, Wang, Feng, Wang, Ren, Qi and Xiao2009a ). Cryptosporidium muris has also been identified in human cryptosporidiosis cases in many countries, including Thailand (Tiangtip and Jongwutiwes, Reference Tiangtip and Jongwutiwes2002), Iran (Azami et al. Reference Azami, Moghaddam, Salehi and Salehi2007), India (Muthusamy et al. Reference Muthusamy, Rao, Ramani, Monica, Banerjee, Abraham, Mathai, Primrose, Muliyil, Wanke, Ward and Kang2006), Indonesia (Katsumata et al. Reference Katsumata, Hosea, Ranuh, Uga, Yanagi and Kohno2000), Saudi Arabia (Brikan et al. Reference Brikan, Salem, Beeching and Hilal2008), Kenya (Gatei et al. Reference Gatei, Ashford, Beeching, Kamwati, Greensill and Hart2002, Reference Gatei, Greensill, Ashford, Cuevas, Parry, Cunliffe, Beeching and Hart2003, Reference Gatei, Wamae, Mbae, Waruru, Mulinge, Waithera, Gatika, Kamwati, Revathi and Hart2006), Peru (Palmer et al. Reference Palmer, Xiao, Terashima, Guerra, Gotuzzo, Saldías, Bonilla, Zhou, Lindquist and Upton2003) and France (Guyot et al. Reference Guyot, Follet-Dumoulin, Lelievre, Sarfati, Rabodonirina, Nevez, Cailliez, Camus and Dei-Cas2001). Currently, there have been no human cryptosporidiosis cases caused by C. muris in China. The above findings indicate that rodents infected with C. muris pose a threat to local inhabitants and are of public health importance.
ACKNOWLEDGEMENTS
The authors would like to thank Xianggen Wang (Director of the Center of Henan Province Dairy Cattle Breeding) for his help with sample collection, and Dr Changshen Ning (Henan Agricultural University) and Dr Fuchun Jian (Henan Agricultural University) for their scientific support.
FINANCIAL SUPPORT
This study was supported in part by the State Key Program of the National Natural Science Foundation of China (31330079) and the National Natural Science Foundation of China (U1204328), as well as the Innovation Scientists and Technicians Troop construction Projects of Henan Province (134200510012), and the Program for Science and Technology Innovative Research Team in the University of Henan Province (012IRTSTHN005).
