Abstract
BackgroundCryptosporidium is an opportunistic pathogen that infects a wide variety of vertebrates. The aim of the present study was to characterize Cryptosporidium spp. isolates from Bactrian camels and to foster further understanding of the biological characteristics of the pathogen.MethodsFecal specimens were collected from two 4-year-old Bactrian camels resident at the Kaifeng City Zoo in China and examined for Cryptosporidium. Fecal specimens were screened using the floatation method, and then genomic DNA was extracted from the oocysts and identified by nested-PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene, the actin gene and the Cryptosporidium oocyst wall-protein (COWP) gene. Subtype analysis was performed based on four minisatellite (MS) loci (MS1, MS2, MS3 and MS16) that were aligned and phylogenetically analyzed to determine the species and subtype of Cryptosporidium. We then established a BALB/c mice infection model and further verified the results through clinical status, pattern of oocyst excretion and histological examination.ResultsCryptosporidium oocyst isolates from the two Bactrian camels had an average (± standard deviation) size of 7.49 ± 0.13 × 5.70 ± 0.10 μm (n = 50). The sequencing and phylogenetic analysis confirmed the species as C. muris. Multilocus sequence typing analysis indicated that the subtypes were M13, M4, M1 and M5. Following the inoculation of BALB/c mice, we found that the prepatent period and number of oocysts per gram increased with increasing infective dose. Oocysts were first detected in the feces of BALB/c mice at 7–8 days post-infection (dpi), with levels peaking twice thereafter, at 15–16 dpi and 19–20 dpi. Histology and scanning electron microscopy studies showed that the stomach contained gastric pits filled with Cryptosporidium that adhered to the surface of gastric mucosa gland epithelial cells, causing the latter to deform, swell and become disordered.ConclusionsThe findings of this study indicated that oocysts isolated from Bactrian camels were from C. muris. This is the first report of C. muris isolated from camels in China. More epidemiological data are needed to understand the prevalence and transmission of C. muris in camels in different geographic areas.Graphical abstract
Highlights
Cryptosporidium is an opportunistic pathogen that infects a wide variety of vertebrates
DNA sequencing indicated that the small ribosomal subunit (SSU) Small-subunit ribosomal RNA (rRNA) nucleotide sequences had 96% similarity with those of C. muris (GenBank accession number EU245044 in the Czech Republic and AJ307669 in Kenya)
The isolates formed a cluster with C. muris (CM), while the Cryptosporidium oocyst wall-protein (COWP) and actin nucleotide sequences had 100% similarity with those of C. muris (GenBank accession numbers KF419210 and KJ746834 in China, respectively) (Fig. 2a–c)
Summary
Cryptosporidium is an opportunistic pathogen that infects a wide variety of vertebrates. One of the drivers of this strategy is that different Cryptosporidium species and genotypes have a certain degree of morphological similarity, making identification by conventional microscopic examination difficult This difficulty has resulted in the development of a nested-PCR molecular diagnostic tool based on polymorphism of the small ribosomal subunit (SSU) gene. Due to the strong similarity between these two species in terms of their genetics and biology, researchers have developed a multiocus sequence typing (MLST) tool using polymorphic microsatellites and microsatellite repeat markers to distinguish between them [5]. This method has been used effectively for characterizing the population genetics of C. muris and C. andersoni [6]
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