Zoonotic Cryptosporidium parvum infections are mainly caused by IIa and IId subtypes. As most biological characterizations have been performed on IIa subtypes, the biological and genetic characteristics of IId subtypes in China are not clear. We evaluated the infection and genetic characteristics of IId isolates in interferon-γ-knockout mice using qPCR to quantify oocyst shedding, histological examination to monitor pathological changes and comparative genomic analyses to identify infectivity and virulence-associated differences. Compared with the reference IIa isolate, mice infected with the IId isolates had significantly higher and longer oocyst shedding and lower body weight gain. In addition, the four IId isolates examined differed significantly in infectivity (as indicated by the half infective dose), oocyst shedding duration, and pathogenicity. Comparative genomic analysis indicated that the IId isolates had three more subtelomeric genes than the reference IIa isolate and 5385–5548 nucleotide substitutions, with the hypervariable genes mostly in two blocks on chromosome 1. In contrast, the four IId isolates differed from each other by 77–1,452 nucleotides, with virulence-associated sequence differences mainly in nine genes within a 28-kb block on chromosome 6. These data indicate the newly emerged C. parvum IId subtypes in China have high animal infectivity and unique genomic characteristics.
Cryptosporidium spp. are important causes of diarrhea in humans, ruminants, and other mammals. Comparative genomic analysis indicated that genetically related and host-adapted Cryptosporidium species have different numbers of subtelomeric genes encoding the Cryptosporidium-specific MEDLE family of secreted proteins, which could contribute to differences in host specificity. In this study, a Cryptosporidium parvum-specific member of the protein family MEDLE-2 encoded by cgd5_4590 was cloned and expressed in Escherichia coli. Immunofluorescent staining with antibodies generated from the recombinant protein showed the expression of the protein in sporozoites and development stages. In vitro neutralization assay with the antibodies partially blocked the invasion of sporozoites. These results support the potential involvement of MEDLE-2 in the invasion of host cells.
The small Cryptosporidium genome (∼9 Mb) has over 20 copies of genes encoding insulinase-like proteases (INS), suggesting that these enzymes may have important biological functions in the pathogen and could be developmentally regulated. In this study, INS-5, a unique member of the INS family in Cryptosporidium parvum, was cloned and expressed in Escherichia coli BL21 (DE3). In addition to the predicted INS-5 of ∼78 kDa, smaller fragments of ∼70, ∼55, and ∼30 kDa were simultaneously generated. After purification through a nickel-nitrilotriacetic acid affinity column, the full recombinant protein obtained was used to prepare polyclonal antibodies. Antibodies raised against INS-5 recognized the recombinant protein and native protein in sporozoite extracts. Further characterization of INS-5 included qRT-PCR assessment of gene expression; immunofluorescence localization of the protein expression in sporozoites, merozoites, and other developmental stages; and neutralization of invasion of C. parvum in vitro. The results obtained indicated that although INS-5 was expressed in sporozoites and merozoites, the high gene expression was from 36 to 48 h of the in vitro culture after invasion. Anti-INS-5 antibodies partially neutralized the invasion (inhibition rate = 38.5%). Results of this study suggest that INS-5 plays some role in the invasion and growth of C. parvum.
To grow and survive within host cells, Toxoplasma must scavenge necessary nutrients from hosts to support its parasitism. Transporters located in the plasma membrane of the parasites play critical roles in nutrient acquisition. Toxoplasma encodes a large number of transporters, but so far, only a few have been characterized.
Nonhuman primates (NHPs) are considered an important source of parasitic zoonoses. A study in 2010 revealed high prevalence of Cryptosporidium spp. in free-ranging rhesus monkeys (Macaca mulatta) in a public park in Guiyang, southwestern China, which called for the control of disease in animals and long-term epidemiological tracking of Cryptosporidium spp. After the initiation of a series of public health interventions, we collected 2,402 fecal samples from monkeys and 123 water samples from lakes in the park on six occasions during 2013-2019. They were analyzed and genotyped for Cryptosporidium spp. using PCR and sequence analyses of the small subunit rRNA gene. The C. hominis and C. parvum identified were further subtyped by sequence analysis of the 60 kDa glycoprotein gene. Compared with the high prevalence of Cryptosporidium spp. in fecal samples (10.9% or 45/411) and water samples (47.8% or 11/23) in 2010, only 18 (0.7%) fecal samples and 3 (2.4%) water samples collected in the present study were positive for Cryptosporidium spp., including C. hominis (n = 9) and C. parvum (n = 12). The former belonged to the NHP-adapted IfA17G2R3 subtype, while the latter mostly belonged to rodent-adapted IIpA9. Therefore, the detection rate and genetic diversity of Cryptosporidium spp. during this study period were much lower than those before the public health interventions, and there was a switch from common occurrence of anthroponotic C. hominis subtypes to sporadic occurrence of NHP-adapted C. hominis and rodent-adapted C. parvum subtypes.
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