Detection of Cryptosporidium spp. and Giardia duodenalis in small wild mammals in northeastern Brazil

This study investigated the occurrence of Giardia duodenalis and Cryptosporidium spp. in rodents and marsupials from the Atlantic Forest in southern Bahia, northeastern Brazil. Two hundred and four fecal samples were collected from different forest areas in the municipalities of Ilhéus, Una, Belmonte, and Mascote. Identifications were performed using PCR and nested PCR followed by sequencing of the gdh and tpi genes for G. duodenalis, and the gp60 and Hsp-70 genes for Cryptosporidium. The total frequency of positive PCR samples for both G. duodenalis and Cryptosporidium spp. was 5.4% (11/204). Giardia duodenalis occurred in 2.94% (4/136) of rodents and 2.94% (2/68) of marsupials. The prevalence of Cryptosporidium in rodents and marsupials was 1.47% (2/136) and 4.41% (3/68), respectively. In the areas sampled, the frequency of parasitism was 50% (7/14), while the Mascote region alone had no parasitized animals. The G. duodenalis subgenotype AI was identified in the rodent species Hylaeamys laticeps, Oecomys catherinae, Oligoryzomys nigripes and Akodon cursor, and in the marsupials Gracilinanus agilis and Monodelphis americana. In the rodents Rhipidomys mastacalis, H. laticeps and in the marsupial Marmosa murina the protozoa Cryptosporidium fayeri, Cryptosporidium parvum and Cryptosporidium ubiquitum with subtypes IIa and IVg by the gp60 gene were found. In conclusion, this study provides the genetic characterization of Giardia and Cryptosporidium species and genotypes in rodents and marsupials. And, these findings reinforce that the rodent and marsupial species mentioned above play a role as new hosts for Giardia and Cryptosporidium.

Subtyping Cryptosporidium xiaoi, a Common Pathogen in Sheep and Goats

Cryptosporidiosis is a significant cause of diarrhea in sheep and goats. Among the over 40 established species of Cryptosporidium, Cryptosporidium xiaoi is one of the dominant species infecting ovine and caprine animals. The lack of subtyping tools makes it impossible to examine the transmission of this pathogen. In the present study, we identified and characterized the 60-kDa glycoprotein (gp60) gene by sequencing the genome of C. xiaoi. The GP60 protein of C. xiaoi had a signal peptide, a furin cleavage site of RSRR, a glycosylphosphatidylinositol anchor, and over 100 O-glycosylation sites. Based on the gp60 sequence, a subtyping tool was developed and used in characterizing C. xiaoi in 355 positive samples from sheep and goats in China. A high sequence heterogeneity was observed in the gp60 gene, with 94 sequence types in 12 subtype families, namely XXIIIa to XXIIIl. Co-infections with multiple subtypes were common in these animals, suggesting that genetic recombination might be responsible for the high diversity within C. xiaoi. This was supported by the mosaic sequence patterns among the subtype families. In addition, a potential host adaptation was identified within this species, reflected by the exclusive occurrence of XXIIIa, XXIIIc, XXIIIg, and XXIIIj in goats. This subtyping tool should be useful in studies of the genetic diversity and transmission dynamics of C. xiaoi.

Subtype Characterization and Zoonotic Potential of Cryptosporidium felis in Cats in Guangdong and Shanghai, China

Cryptosporidiumfelis is an important cause of feline and human cryptosporidiosis. However, the transmission of this pathogen between humans and cats remains controversial, partially due to a lack of genetic characterization of isolates from cats. The present study was conducted to examine the genetic diversity of C. felis in cats in China and to assess their potential zoonotic transmission. A newly developed subtyping tool based on a sequence analysis of the 60-kDa glycoprotein (gp60) gene was employed to identify the subtypes of 30 cat-derived C. felis isolates from Guangdong and Shanghai. Altogether, 20 C. felis isolates were successfully subtyped. The results of the sequence alignment showed a high genetic diversity, with 13 novel subtypes and 2 known subtypes of the XIXa subtype family being identified. The known subtypes were previously detected in humans, while some of the subtypes formed well-supported subclusters with human-derived subtypes from other countries in a phylogenetic analysis of the gp60 sequences. The results of this study confirmed the high genetic diversity of the XIXa subtype family of C. felis. The common occurrence of this subtype family in both humans and cats suggests that there could be cross-species transmission of C. felis.

Molecular survey of Cryptosporidium spp. in calves from the state of Mato Grosso, Brazil

Cryptosporidium spp. is a protozoan that infects a wide range of vertebrate hosts; it has been reported to be the cause of severe illness or death in livestock worldwide, which leads to decreased performance and production losses, especially in young animals. This study investigated the presence of Cryptosporidium in calves from beef farms in the state of Mato Grosso, midwestern Brazil. For this purpose, fecal samples from 237 animals aged ? 45 days, raised in 20 rural properties were subjected to DNA extraction and nested polymerase chain reaction (nPCR) targeting 18S ribosomal RNA (18S rRNA) gene followed by sequencing. Additionally, positive samples, previously identified as Cryptosporidium parvum by sequencing and phylogenetic analyses based on 18S rRNA gene, were subsequently analyzed focusing the amplification and sequencing using nPCR of a fragment of the 60 kDa glycoprotein (gp60) gene. Of the 237 fecal samples analyzed by PCR (18S rRNA), 50 (21.1%) fecal samples were positive for Cryptosporidium spp., while 14 (70%) of the 20 properties had at least one positive animal. The following Cryptosporidium species were detected: C. bovis, C. parvum, and C. ryanae. Thereafter, two potentially zoonotic subtypes (IIaA15G2R1 and IIaA16G3R1) of C. parvum were identified based on gp60 gene sequences. This study resulted in the detection of subtype IIaA16G3R1 for the first time in South America and showed a wide distribution of the protozoan in beef farms in the studied area of the State.

Molecular detection of genotypes and subtypes of Cryptosporidium infection in diarrheic calves, lambs, and goat kids from Turkey

Background: Cryptosporidium spp. are enteric protozoan parasites that infect a wide range of animals as well as humans. The studies on Cryptosporidium infections of animals in Turkey are mostly rely on microscopic observation. Few data are available regarding the distribution of Cryptosporidium genotypes and subtypes infection. The aim of this study is to analyse the zoonotic potential of Cryptosporidium oocysts shed from young ruminant livestock. Methods: A total of 415 diarrheic fecal specimens from 333 calves, 67 lambs, and 15 goat kids were examined for the presence of Cryptosporidium oocysts by microscopy. Microscopic positive specimens were then analyzed for Cryptosporidium genotypes and subtypes detection by use of nested PCR of the small subunit ribosomal RNA (SSU rRNA) gene and the highly polymorphic 60 kDa glycoprotein (gp60) gene followed by sequence analyses. Results: The results of this study revealed that 25.6% (106 of 415) of the specimens were positive for Cryptosporidium spp. infection by microscopic examination and molecular analysis. We identified 27.4% (91/333), 19.4% (13/67), and 13.4% (2/15) of positivity in calves, lambs and goat kids, respectively. Genotyping of the SSU rRNA indicated that almost all positive specimens were of C . parvum , except for one calf which was of C. bovis . Sequence analysis of the gp60 gene revealed the most common zoonotic subtypes (IIa and IId) of C . parvum. We detected 11 subtypes (IIaA11G2R1, IIaA11G3R1, IIaA12G3R1, IIaA13G2R1, IIaA13G4R1, IIaA14G1R1, IIaA14G3R1, IIaA15G2R1, IIdA16G1, IIdA18G1, IIdA22G1); three of them (IIaA12G3R1, IIaA11G3R1 and IIaA13G4R1) was novel subtypes found in calves and lambs. Additionally, three subtypes (IIaA11G2R1, IIaA14G3R1, and IIdA16G1) were detected in calves, lambs, and goat kid for the first time in Turkey. Conclusions: The findings illustrate the high occurrence of Cryptosporidium infection in Turkey and suggest that calves, lambs, and goat kids are likely a major reservoir of C. parvum and a potential source of zoonotic transmission, which may have public health implications. Keywords: Calves, C. bovis, C. parvum , Cryptosporidium , Diarrhea, Goat kids, Lambs, Subtypes, Turkey.

Genetic diversity of Cryptosporidium parvum in neonatal calves in Xinjiang, China

Background The Cryptosporidium causes infection in wide spectrum of vertebrate hosts and is well known for leading to diarrhea and other gastrointestinal illness. To assess Cryptosporidium genetic diversity in neonatal calves and probe the cause for diarrhea of them, a total of 232 fecal samples from neonatal calves in 12 farms in Xinjiang were characterized for the presence of Cryptosporidium .Results The prevalence of Cryptosporidium was 38.4% (89/232), and three species were detected with SSU rRNA gene, including C. parvum (n = 88), C. ryanae (n = 9), and C. bovis (n = 1). Prevalence of C . parvum neonatal calves with diarrhea (52.6%, 51/97) was significantly higher than calves without diarrhea (28.1%, 38/135). All the C . parvum -positive samples were analyzed based on gp60 gene, IIdA15G1 (n = 35), IIdA20G1 (n = 21), IIdA14G1 (n = 17), and IIdA19G1 (n = 13) were successfully subtyped in this study.Conclusions These data indicated that C . parvum was a major contributor in diarrheal disease in neonatal calves, and C . parvum subtypes from neonatal calves in Xinjiang were high genetic diversity. Additionally, our findings implicating neonatal calves could be a potential source of human Cryptosporidium infection and provide further evidence for the uniqueness of C . parvum IId subtypes in cattle in China.

Evaluation of recombinant Cryptosporidium hominis GP60 protein and anti-GP60 chicken polyclonal IgY for research and diagnostic purposes

In this study, a method for expressing Cryptosporidium hominis GP60 glycoprotein in Escherichia coli for production of polyclonal anti-GP60 IgY in chickens was developed aiming future studies concerning the diagnosis, prevention and treatment of cryptosporidiosis. The full-length nucleotide sequence of the C. hominis gp60 gene was codon-optimized for expression in E. coli and was synthesized in pET28-a vector. Subcloning was performed on several different strains of BL21 E. coli. Temperature, time and inducer IPTG concentration assays were also performed and analyzed using SDS-PAGE. The optimal conditions were observed at a temperature of 37 °C, with overnight incubation and 1 mM of IPTG. Purification was performed by means of affinity chromatography using the AKTA Pure chromatography system and the Hi-Trap™ HP column (GE Healthcare). The recombinant protein GP60 (rGP60) thus generated was used to immunize laying hens owing the production of polyclonal IgY. Western blot and indirect immunofluorescence showed that the polyclonal antibody was capable of binding to rGP60 and to Cryptosporidium parvum sporozoites, respectively. The rGP60 and the IgY anti-rGP60 generated in this study may be used as templates for research and for the development of diagnostic methods for cryptosporidiosis.

Development and Application of a gp60-Based Typing Assay for Cryptosporidium viatorum

The apicomplexan intestinal parasites of the genusCryptosporidiumtake a major toll on human and animal health and are frequent causes of waterborne outbreaks. Several species and genotypes can infect humans, includingCryptosporidium viatorum, which, to date, has only been found in humans. Molecular characterization ofCryptosporidiumspp., critical to epidemiological analyses, is commonly based on gp60 gene analysis, which appears to require bespoke species- or group-specific PCR primers due to extensive genetic diversity across the genus. In this study, we amplified, sequenced, and characterized the gp60 gene ofC. viatorumfor the first time. Moreover, we developed and validated a gp60 typing assay for this species and applied it to 27 isolates originating from Asia, Africa, and Central America. A single subtype family, XVa, was identified containing multiple alleles.

Genotyping and subtyping ofGiardiaandCryptosporidiumisolates from commensal rodents in China

SUMMARYCryptosporidiumandGiardiaare two important zoonotic intestinal parasites responsible for diarrhoea in humans and other animals worldwide. Rodents, as reservoirs or carriers ofCryptosporidiumandGiardia, are abundant and globally widespread. In the present study, we collected 232 fecal specimens from commensal rodents captured in animal farms and farm neighbourhoods in China. We collected 33 Asian house rats, 168 brown rats and 31 house mice. 6·0% (14/232) and 8·2% (19/232) of these rodents were microscopy-positive forGiardiacysts andCryptosporidiumoocysts, respectively. All 14Giardiaisolates were identified asGiardia duodenalisassemblage G at a minimum of one or maximum of three gene loci (tpi, gdhandbg). By small subunit rRNA (SSU rRNA) gene sequencing,Cryptosporidium parvum(n= 12) andCryptosporidium muris(n= 7) were identified. The gp60 gene encoding the 60-kDa glycoprotein was successfully amplified and sequenced in nineC. parvumisolates, all of which belonged to the IIdA15G1 subtype. Observation of the same IIdA15G1 subtype in humans (previously) and in rodents (here) suggests that rodents infected withCryptosporidiumhave the potential to transmit cryptosporidiosis to humans.