RESUMEN
Genetic variation in Cryptosporidium, a common protozoan gut parasite in humans, is often based on marker genes containing trinucleotide repeats, which differentiate subtypes and track outbreaks. However, repeat regions have high replication slippage rates, making it difficult to discern biological diversity from error. Here, we synthesized Cryptosporidium DNA in clonal plasmid vectors, amplified them in different mock community ratios, and sequenced them using next-generation sequencing to determine the rate of replication slippage with dada2. Our results indicate that slippage rates increase with the length of the repeat region and can contribute to error rates of up to 20%.
Asunto(s)
Cryptosporidium , Replicación del ADN , Cryptosporidium/genética , Cryptosporidium/clasificación , Humanos , ADN Protozoario/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Código de Barras del ADN Taxonómico/métodos , Criptosporidiosis/parasitología , Variación GenéticaRESUMEN
Cryptosporidium parvum is a zoonotic apicomplexan parasite and a common cause of diarrheal disease worldwide. The development of vaccines to prevent or limit infection remains an important goal for tackling cryptosporidiosis. At present, the only approved vaccine against any apicomplexan parasite targets a conserved adhesin possessing a thrombospondin repeat domain. C. parvum possesses 12 orthologous thrombospondin repeat domain-containing proteins known as CpTSP1-12, though little is known about these potentially important antigens. Here, we explore the architecture and conservation of the CpTSP protein family, as well as their abundance at the protein level within the sporozoite stage of the life cycle. We examine the glycosylation states of these proteins using a combination of glycopeptide enrichment techniques to demonstrate that these proteins are modified with C-, O-, and N-linked glycans. Using expansion microscopy, and an antibody against the C-linked mannose that is unique to the CpTSP protein family within C. parvum, we show that these proteins are found both on the cell surface and in structures that resemble the secretory pathway of C. parvum sporozoites. Finally, we generated a polyclonal antibody against CpTSP1 to show that it is found at the cell surface and within micronemes, in a pattern reminiscent of other apicomplexan motility-associated adhesins, and is present both in sporozoites and meronts. This work sheds new light on an understudied family of C. parvum proteins that are likely to be important to both parasite biology and the development of vaccines against cryptosporidiosis.
Asunto(s)
Criptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Animales , Humanos , Cryptosporidium parvum/metabolismo , Criptosporidiosis/parasitología , Criptosporidiosis/prevención & control , Glicosilación , Cryptosporidium/metabolismo , Proteínas Protozoarias/química , Esporozoítos , Trombospondinas/metabolismoRESUMEN
The North African hedgehog (Atelerix algirus) is an introduced species from Northwest Africa and is currently distributed in the Canary Islands. This species of hedgehog has been studied as a reservoir of enteropathogens, including Cryptosporidium spp. However, there are no data at species level. Therefore, the aim of the present study was to identify the Cryptosporidium species present in a population of hedgehogs (n = 36) in the Canary Islands. Molecular screening was performed using conventional polymerase chain reaction (PCR) targeting the small subunit ribosomal RNA (18S rRNA) gene of Cryptosporidium spp. Seven of the 36 fecal samples (19.45%) were positive and confirmed by nested PCR targeting the 18S rRNA gene and Sanger sequencing. Cryptosporidium parvum and Cryptosporidium muris were identified in 11.1% (4/36) and 5.6% (2/36) of the samples, respectively, while one sample could only be identified at the genus level. The zoonotic subtypes IIdA15G1 (n = 1), IIdA16G1b (n = 1), and IIdA22G1 (n = 1) of C. parvum were identified by nested PCR followed by analysis of the 60 kDa glycoprotein (gp60) gene sequence. This study is the first genetic characterization of Cryptosporidium spp. in A. algirus, identifying zoonotic species and subtypes of the parasite.
Asunto(s)
Criptosporidiosis , Cryptosporidium , Erizos , Filogenia , Animales , Criptosporidiosis/parasitología , Criptosporidiosis/epidemiología , Cryptosporidium/genética , Cryptosporidium/clasificación , Cryptosporidium/aislamiento & purificación , ADN Protozoario/genética , ADN Ribosómico/genética , ADN Ribosómico/química , Heces/parasitología , Genotipo , Erizos/parasitología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Ribosómico 18S/genética , Análisis de Secuencia de ADN , EspañaRESUMEN
Genetic recombination plays a critical role in the emergence of pathogens with phenotypes such as drug resistance, virulence, and host adaptation. Here, we tested the hypothesis that recombination between sympatric ancestral populations leads to the emergence of divergent variants of the zoonotic parasite Cryptosporidium parvum with modified host ranges. Comparative genomic analyses of 101 isolates have identified seven subpopulations isolated by distance. They appear to be descendants of two ancestral populations, IIa in northwestern Europe and IId from southwestern Asia. Sympatric recombination in areas with both ancestral subtypes and subsequent selective sweeps have led to the emergence of new subpopulations with mosaic genomes and modified host preference. Subtelomeric genes could be involved in the adaptive selection of subpopulations, while copy number variations of genes encoding invasion-associated proteins are potentially associated with modified host ranges. These observations reveal ancestral origins of zoonotic C. parvum and suggest that pathogen import through modern animal farming might promote the emergence of divergent subpopulations of C. parvum with modified host preference.
Asunto(s)
Criptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Animales , Criptosporidiosis/parasitología , Cryptosporidium/genética , Cryptosporidium parvum/genética , Variaciones en el Número de Copia de ADN , Recombinación GenéticaRESUMEN
Here, a machine learning tool (YOLOv5) enables the detection of Cryptosporidium microorganisms using optical and phase contrast microscope images. The two databases were processed using 520 images (optical microscopy) and 1200 images (phase contrast microscopy). It used Python libraries to label, standardize the size, and crop the images to generate the input tensors to the YOLOv5 network (s, m, and l). It implemented two experiments using randomly initialized weights in optical and phase contrast microscope images. The other two experiments used the parameters for the best training time obtained before and after retraining the models. Metrics used to assess model accuracy were mean average accuracy, confusion matrix, and the F1 scores. All three metrics confirmed that the optimal model used the best epoch of optical imaging training and retraining with phase contrast imaging. Experiments with randomly initialized weights with optical imaging showed the lowest precision for Cryptosporidium detection. The most stable model was YOLOv5m, with the best results in all categories. However, the differences between all models are lower than 2%, and YOLOv5s is the best option for Cryptosporidium detection considering the differences in computational costs of the models.
Asunto(s)
Criptosporidiosis , Cryptosporidium , Humanos , Criptosporidiosis/diagnóstico por imagen , Microscopía , Imagen ÓpticaRESUMEN
Cryptosporidium parvum is a globally distributed zoonotic pathogen and a major cause of diarrhoeal disease in humans and ruminants. The parasite's life cycle comprises an obligatory sexual phase, during which genetic exchanges can occur between previously isolated lineages. Here, we compare 32 whole genome sequences from human- and ruminant-derived parasite isolates collected across Europe, Egypt and China. We identify three strongly supported clusters that comprise a mix of isolates from different host species, geographic origins, and subtypes. We show that: (1) recombination occurs between ruminant isolates into human isolates; (2) these recombinant regions can be passed on to other human subtypes through gene flow and population admixture; (3) there have been multiple genetic exchanges, and most are probably recent; (4) putative virulence genes are significantly enriched within these genetic exchanges, and (5) this results in an increase in their nucleotide diversity. We carefully dissect the phylogenetic sequence of two genetic exchanges, illustrating the long-term evolutionary consequences of these events. Our results suggest that increased globalization and close human-animal contacts increase the opportunity for genetic exchanges between previously isolated parasite lineages, resulting in spillover and spillback events. We discuss how this can provide a novel substrate for natural selection at genes involved in host-parasite interactions, thereby potentially altering the dynamic coevolutionary equilibrium in the Red Queens arms race.
Asunto(s)
Criptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Animales , Humanos , Cryptosporidium parvum/genética , Criptosporidiosis/parasitología , Cryptosporidium/genética , Filogenia , RumiantesRESUMEN
Cryptosporidium spp. remain a major cause of waterborne diarrhea and illness in developing countries and represent a significant burden to farmers worldwide. Cryptosporidium parvum virus 1 (CSpV1), of the genus Cryspovirus, was first reported to be present in the cytoplasm of C. parvum in 1997. Full-length genome sequences have been obtained from C. parvum from Iowa (Iowa), Kansas (KSU) and China. We aimed at characterizing the genome of CSpV1 from France and used sequence analysis from Cryptosporidium isolates to explore whether CSpV1 genome diversity varies over time, with geographical sampling location, C. parvum genetic diversity, or ruminant host species. A total of 123 fecal samples of cattle, sheep and goats were collected from 17 different French departments (57 diseased animal fecal samples and 66 healthy animal fecal samples). Subtyping analysis of the C. parvum isolates revealed the presence of two zoonotic subtype families IIa and IId. Sequence analysis of CSpV1 revealed that all CSpV1 from France, regardless of the subtype of C. parvum (IIaA15G2R1, IIaA17G2R1 and IIdA18G1R1) are more closely related to CSpV1 from Turkey, and cluster on a distinct branch from CSpV1 collected from C. parvum subtype IIaA15G2R1 from Asia and North America. We also found that samples collected on a given year or successive years in a given location are more likely to host the same subtype of C. parvum and the same CSpV1 strain. Yet, there is no distinct clustering of CSpV1 per French department or ruminants, probably due to trade, and transmission of C. parvum among host species. Our results point towards (i) a close association between CSpV1 movement and C. parvum movement, (ii) recent migrations of C. parvum among distantly located departments and (iii) incidental transmission of C. parvum between ruminants. All together, these results provide insightful information regarding CSpV1 evolution and suggest the virus might be used as an epidemiological tracer for C. parvum. Future studies need to investigate CSpV1's role in C. parvum virulence and on subtype ability to infect different species.
Asunto(s)
Enfermedades de los Bovinos , Criptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Enfermedades de las Cabras , Enfermedades de las Ovejas , Ovinos , Animales , Bovinos , Cabras , Cryptosporidium parvum/genética , Criptosporidiosis/epidemiología , Francia/epidemiología , Enfermedades de los Bovinos/epidemiología , Enfermedades de las Cabras/epidemiología , Enfermedades de las Ovejas/epidemiologíaRESUMEN
BACKGROUND: Cryptosporidium parvum is a protozoan parasite of medical and veterinary importance that causes neonatal diarrhea in many vertebrate hosts. In this study, we evaluated the efficacy of an affinity-purified antigen as a C. parvum vaccine candidate using ileal and liver tissues of experimentally infected neonatal mice by immunohistochemical profiling and immune scoring of CD4+, CD8+, Caspase-3, and nuclear factor kappa B (NF-κB). This vaccine was prepared from the C. parvum oocysts antigen using immune affinity chromatography with cyanogen bromide-activated Sepharose-4B beads. METHODS: Thirty neonatal mice were divided into three groups (10 mice/group): (1) non-immunized non-infected, (2) non-immunized infected (using gastric tubes with a single dose of 1 × 105 of C. parvum oocysts in 250 µl PBS solution 1 h before a meal) and (3) immunized (twice with 40 µg/kg of purified C. parvum antigen at 2-week intervals and then infected with 1 × 105 C. parvum oocysts simultaneously with the second group). After euthanizing the animals on the 10th day, post-infection, their ileal and liver tissues were collected and prepared for immunohistochemistry (IHC) staining to detect CD4+, CD8+, Caspase-3, and NF-κB levels, which are indicators for T helper cells, cytotoxic T cells, apoptosis, and inflammation, respectively. RESULTS: The IHC results showed that CD4+, CD8+, Caspase-3, and NF-κB expression varied significantly (P < 0.001) in both organs in all the groups. We also recorded high CD4+ levels and low CD8+ expression in the non-immunized non-infected mice tissues, while the opposite was observed in the non-immunized infected mice tissues. In the immunized infected mice, the CD4+ level was higher than CD8 + in both organs. While the Caspase-3 levels were higher in the ileal tissue of non-immunized infected than immunized infected mice ileal tissues, the reverse was seen in the liver tissues of both groups. Furthermore, NF-κB expression was higher in the liver tissues of non-immunized infected mice than in immunized infected mice tissues. Therefore, the IHC results and immune-scoring program revealed a significant difference (P < 0.001) in the CD4+, CD8+, Caspase-3, and NF-κB expression levels in both ileal and liver tissues of all mice groups, which might be necessary for immunomodulation in these tissues. CONCLUSIONS: The improvement observed in the immunized infected mice suggests that this vaccine candidate might protect against cryptosporidiosis.
Asunto(s)
Antígenos CD4 , Antígenos CD8 , Caspasa 3 , Criptosporidiosis , FN-kappa B , Vacunas Antiprotozoos , Animales , Ratones , Caspasa 3/biosíntesis , Caspasa 3/inmunología , Antígenos CD4/biosíntesis , Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/inmunología , Antígenos CD8/biosíntesis , Antígenos CD8/inmunología , Linfocitos T CD8-positivos/inmunología , Criptosporidiosis/prevención & control , Criptosporidiosis/parasitología , Cryptosporidium , Cryptosporidium parvum/inmunología , Inmunohistoquímica , FN-kappa B/biosíntesis , FN-kappa B/inmunología , Vacunas Antiprotozoos/uso terapéutico , VacunasRESUMEN
Cryptosporidium parvum is an important apicomplexan parasite causing severe diarrhea in both humans and animals. Calmodulin (CaM), a multifunctional and universal calcium-binding protein, contributes to the growth and development of apicomplexan parasites, but the role of CaM in C. parvum remains unknown. In this study, the CaM of C. parvum encoded by the cgd2_810 gene was expressed in Escherichia coli, and the biological functions of CpCaM were preliminarily investigated. The transcriptional level of the cgd2_810 gene peaked at 36 h post infection (pi), and the CpCaM protein was mainly located around the nucleus of the whole oocysts, in the middle of sporozoites and around the nucleus of merozoites. Anti-CpCaM antibody reduced the invasion of C. parvum sporozoites by 30.69%. The present study indicates that CpCaM is potentially involved in the growth of C. parvum. Results of the study expand our knowledge on the interaction between host and Cryptosporidium.
Asunto(s)
Criptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Animales , Humanos , Cryptosporidium parvum/genética , Cryptosporidium/genética , Criptosporidiosis/parasitología , Oocistos/metabolismo , Esporozoítos/metabolismoRESUMEN
Severe diarrhea was reported in goat kids in Chungcheongbuk-do, Korea, from 2021 to 2023, and Cryptosporidium infection was suspected. To confirm the cause of this outbreak, fecal samples were collected from goat farms where diarrhea had been reported and analyzed for Cryptosporidium infection using a molecular assay. A total of 65 fecal samples, including 37 from goats with diarrhea and 28 from goats without diarrhea, were collected from six goat farms. Forty-eight of the goats were kids (<2 months) and 17 were adults (>1 year). Cryptosporidium was identified in 53.8% (35/65) of total samples. Overall, 86.5% (32/37) of the diarrheic fecal samples tested positive; however, Cryptosporidium was not detected in any fecal sample from non-diarrheic adult goats. Therefore, cryptosporidiosis was significantly associated with diarrhea in goat kids, and adult goats were not responsible for transmission of Cryptosporidium to them. Phylogenetic analysis and molecular characterization revealed two Cryptosporidium species, namely, C. parvum (n = 28) and C. xiaoi (n = 7). In the C. parvum-positive samples, gp60 gene analysis revealed three zoonotic subtypes-IIaA18G3R1, IIdA15G1, and IIdA16G1. To the best of our knowledge, this study is the first to identify C. parvum IIaA18G3R1 and IIdA16G1 in goats, as well as the first to identify C. xiaoi in goats in Korea. These results suggest that goat kids play an important role as reservoir hosts for different Cryptosporidium species and that continuous monitoring with biosecurity measures is necessary to control cryptosporidiosis outbreaks.
Asunto(s)
Criptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Enfermedades de las Cabras , Enfermedades de las Ovejas , Animales , Ovinos , Criptosporidiosis/epidemiología , Cryptosporidium parvum/genética , Cabras , Filogenia , Enfermedades de las Cabras/epidemiología , Enfermedades de las Ovejas/epidemiología , Cryptosporidium/genética , Diarrea/epidemiología , Diarrea/veterinaria , Heces , Brotes de Enfermedades/veterinaria , República de Corea/epidemiología , GenotipoRESUMEN
Cryptosporidium is a highly pathogenic water and food-borne zoonotic parasitic protozoan that causes severe diarrhea in humans and animals. Apicomplexan parasites invade host cells via a unique motility process called gliding, which relies on the parasite's microfilaments. Actin depolymerizing factor (ADF) is a fibrous-actin (F-actin) and globular actin (G-actin) binding protein essential for regulating the turnover of microfilaments. However, the role of ADF in Cryptosporidium parvum (C. parvum) remains unknown. In this study, we preliminarily characterized the biological functions of ADF in C. parvum (CpADF). The CpADF was a 135-aa protein encoded by cgd5_2800 gene containing an ADF-H domain. The expression of cgd5_2800 gene peaked at 12 h post-infection, and the CpADF was located in the cytoplasm of oocysts, middle region of sporozoites, and cytoplasm of merozoites. Neutralization efficiency of anti-CpADF serum was approximately 41.30%. Actin sedimentation assay revealed that CpADF depolymerized but did not undergo cosedimentation with F-actin and its ability of F-actin depolymerization was pH independent. These results provide a basis for further investigation of the roles of CpADF in the invasion of C. parvum.
Asunto(s)
Criptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Humanos , Animales , Cryptosporidium parvum/genética , Cryptosporidium parvum/metabolismo , Actinas/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Destrina/metabolismo , Criptosporidiosis/parasitología , Proteínas de Microfilamentos/metabolismoRESUMEN
New Zealand's endemic reptile fauna is highly threatened and pathogens causing infectious diseases may be a significant risk to already endangered species. Here, we investigate Cryptosporidium infection in captive endemic New Zealand reptiles. We found two mammal-related Cryptosporidium species (C. hominis and C. parvum) and six subtypes from three gp60 families (Ib, Ig and IIa) in 12 individuals of captive endemic Tuatara, Otago and Grand skinks, and Jewelled and Rough geckos. Cryptosporidium serpentis was identified in two Jewelled geckos using 18S. In New Zealand, C. hominis and C. parvum are associated with infections in humans and introduced domestic animals but have also been recently found in wildlife. Our finding of Cryptosporidium infection in endemic reptiles can help inform strategies to monitor the conservation of species and manage potential introductions of pathogens to in-situ and ex-situ populations.
Asunto(s)
Criptosporidiosis , Cryptosporidium , Lagartos , Humanos , Animales , Criptosporidiosis/epidemiología , Cryptosporidium/genética , Nueva Zelanda/epidemiología , Mamíferos , Genotipo , Heces , ADN ProtozoarioRESUMEN
Cryptosporidium parvum is the second-most prevalent Cryptosporidium species that infects humans worldwide. In European countries, it is the most prevalent species in sheep, suggesting that these animals are a source of zoonotic infection. Preweaned lambs and goats are particularly susceptible to infection by the parasite and may suffer from severe diarrhea whilst excreting large quantities of infectious oocysts. Fifty fecal samples from preweaned lambs and goats with diarrhea from 35 farms across Israel, found to be Cryptosporidium-positive by microscopy, were tested by PCR and sequence analyses to determine the infective species and subtypes. Cryptosporidium parvum DNA was detected in most samples from both lambs and goats (46/50). Cryptosporidium xiaoi DNA was detected in three samples from kids, with co-infection detected in a single sample. Eleven different C. parvum subtypes were found, 10 in lambs and 5 in goats. All subtypes were from the IIa and IId subtype families, with subtypes IIdA20G1 and IIaA15G2R1 being the most prevalent and widespread. These subtypes were previously found in calves and humans in Israel and are considered the most prevalent C. parvum subtypes in small ruminants globally. These results underline the zoonotic potential of C. parvum from small ruminants and the high subtype diversity compared to previous reports from other Middle Eastern countries. In addition, this is the first report of C. xiaoi in Israel.
Asunto(s)
Enfermedades de los Bovinos , Criptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Animales , Humanos , Ovinos , Bovinos , Cryptosporidium parvum/genética , Cryptosporidium/genética , Criptosporidiosis/epidemiología , Criptosporidiosis/parasitología , Cabras/parasitología , Israel/epidemiología , Heces/parasitología , Diarrea/epidemiología , Diarrea/veterinaria , Diarrea/parasitología , GenotipoRESUMEN
Animals with a chronic infection of the parasite Toxoplasma gondii are protected against lethal secondary infection with other pathogens. Our group previously determined that soluble T. gondii antigens (STAg) can mimic this protection and be used as a treatment against several lethal pathogens. Because treatments are limited for the parasite Cryptosporidium parvum, we tested STAg as a C. parvum therapeutic. We determined that STAg treatment reduced C. parvum Iowa II oocyst shedding in gamma interferon knockout (IFN-γ-KO) mice. Murine intestinal sections were then sequenced to define the IFN-γ-independent transcriptomic response to C. parvum infection. Gene Ontology and transcript abundance comparisons showed host immune response and metabolism changes. Transcripts for type I interferon-responsive genes were more abundant in C. parvum-infected mice treated with STAg. Comparisons between phosphate-buffered saline (PBS) and STAg treatments showed no significant differences in C. parvum gene expression. C. parvum transcript abundance was highest in the ileum and mucin-like glycoproteins and the GDP-fucose transporter were among the most abundant. These results will assist the field in determining both host- and parasite-directed future therapeutic targets.
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Criptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Animales , Cryptosporidium/genética , Inmunidad , Interferón gamma , Ratones , Ratones Endogámicos C57BL , TranscriptomaRESUMEN
Cryptosporidiosis was shown a decade ago to be a major contributor to morbidity and mortality of diarrheal disease in children in low-income countries. A serious obstacle to develop and evaluate immunogens and vaccines to control this disease is the lack of well-characterized immunocompetent rodent models. Here, we optimized and compared two mouse models for the evaluation of vaccines: the Cryptosporidium tyzzeri model, which is convenient for screening large numbers of potential mixtures of immunogens, and the Cryptosporidium parvum-infected mouse pretreated with interferon gamma-neutralizing monoclonal antibody.
Asunto(s)
Criptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Animales , Criptosporidiosis/prevención & control , Diarrea , Modelos Animales de Enfermedad , RatonesRESUMEN
Cryptosporidium parvum is an obligate protozoan parasite invading epithelial cells of small intestine of human and animals, and causing diarrheal disease. In apicomplexan parasites, calcium signaling can regulate many essential biological processes such as invasion and migration. As the main intracellular receptor for calcium ions, calmodulins control the activities of hundreds of enzymes and proteins. Calmodulin-like protein (CML) is an important member of the calmodulin family and may play a key role in C. parvum, however, the actual situation is still not clear. The present study aimed to identify the parasite interaction partner proteins of C. parvum calmodulin-like protein (CpCML). By constructing the cpcml bait plasmid, 5 potential CpCML - interacting proteins in C. parvum oocyst were screened by yeast-two-hybrid system (Y2H). Bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation (Co-IP) were performed as subsequent validations. Fibrillarin RNA methylase (FBL) was identified via this screening method as CpCML interacting protein in C. parvum. The identification of this interaction made it possible to get a further understanding of the function of CpCML and its contribution to the pathogenicity of C. parvum.
Asunto(s)
Criptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Animales , Calmodulina/genética , Calmodulina/metabolismo , Proteínas Cromosómicas no Histona , Criptosporidiosis/parasitología , Cryptosporidium/genética , Cryptosporidium parvum/genética , Cryptosporidium parvum/metabolismo , Humanos , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , ARNt MetiltransferasasRESUMEN
Cryptosporidium parvum is a major cause of diarrheal disease in immature or weakened immune systems, mainly in infants and young children in resource-poor settings. Despite its high prevalence, fully effective and safe drugs for the treatment of C. parvum infections remain scarce, and there is no vaccine. Meanwhile, curcumin has shown protective effects against C. parvum infections. However, the mechanisms of action and relationship to the gut microbiota and innate immune responses are unclear. Immunosuppressed neonatal mice were infected with oocysts of C. parvum and either untreated or treated with a normal diet, curcumin or paromomycin. We found that curcumin stopped C. parvum oocysts shedding in the feces of infected immunosuppressed neonatal mice, prevented epithelial damage, and villi degeneration, as well as prevented recurrence of infection. Curcumin supplementation increased the relative abundance of Bacteroidetes and decreased the relative abundance of Firmicutes and Proteobacteria in mice infected with C. parvum as shown by 16S rRNA gene sequencing analysis. The relative abundance of Lactobacillus, Bacteroides, Akkermansia, Desulfovibrio, Prevotella, and Helicobacter was significantly associated with C. parvum infection inhibited by curcumin. Curcumin significantly (P < 0.01) suppressed IFN-γ and IL -18 gene expression levels in immunosuppressed neonatal C. parvum-infected mice. We demonstrate that the therapeutic effects curcumin are associated with alterations in the gut microbiota and innate immune-related genes, which may be linked to the anti-Cryptosporidium mechanisms of curcumin.
Asunto(s)
Criptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Curcumina , Microbioma Gastrointestinal , Animales , Animales Recién Nacidos , Criptosporidiosis/tratamiento farmacológico , Criptosporidiosis/prevención & control , Cryptosporidium parvum/fisiología , Curcumina/farmacología , Curcumina/uso terapéutico , Heces , Inmunidad Innata , Ratones , ARN Ribosómico 16S/genéticaRESUMEN
Until now, no completely effective parasite-specific drugs or vaccines have been approved for the treatment of cryptosporidiosis. Through the separation and identification of the sporozoite membrane protein of Cryptosporidium parvum (C. parvum), 20 related proteins were obtained. Among them, a calmodulin-like protein (CML) has a similar functional domain-exchange factor hand (EF-hand) motif as calmodulin proteins (CaMs), so it may play a similarly important role in the invasion process. A 663 bp full gene encoding the C. parvum calmodulin-like protein (CpCML) was inserted in pET28a vector and expressed in Escherichia coli. An immunofluorescence assay showed that CpCML was mainly located on the surface of the sporozoites. Three-week-old female BALB/c mice were used for modelling the immunoreactions and immunoprotection of recombinant CpCML (rCpCML) against artificial Cryptosporidium tyzzeri infections. The results indicated a significantly increased in anti-CpCML antibody response, which was induced by the immunized recombinant protein. Compared to rP23 (recombinant P23), GST6P-1 (expressed by pGEX-6P-1 transfected E. coli), GST4T-1 (expressed by pGEX-4T-1 transfected E. coli), glutathione (GSH), adjuvant and blank control groups, rCpCML-immunized mice produced specific spleen cell proliferation in addition to different production levels of IL-2, IFN-γ, TNF-α, IL-4 and IL-5. Additionally, immunization with rCpCML led to 34.08% reduction of oocyst shedding in C. tyzzeri infected mice faeces which was similar to rP23. These results suggest that CpCML may be developed as a potential vaccine candidate antigen against cryptosporidiosis.
Asunto(s)
Criptosporidiosis , Cryptosporidium parvum , Proteínas de la Membrana , Proteínas Protozoarias , Animales , Anticuerpos Antiprotozoarios , Calmodulina , Criptosporidiosis/prevención & control , Cryptosporidium parvum/genética , Escherichia coli/genética , Femenino , Proteínas de la Membrana/genética , Ratones , Proteínas Protozoarias/genética , EsporozoítosRESUMEN
AIMS: The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii are identified as public health priorities and are present in a wide variety of environments including the marine ecosystem. The objective of this study was to demonstrate that the marine bivalve blue mussel (Mytilus edulis) can be used as a tool to monitor the contamination of marine waters by the three protozoa over time. METHODS AND RESULTS: In order to achieve a proof of concept, mussels were exposed to three concentrations of G. duodenalis cysts and Cryptosporidium parvum/T. gondii oocysts for 21 days, followed by 21 days of depuration in clear water. Then, natural contamination by these protozoa was sought for in wild marine blue mussels along the northwest coast of France to validate their relevance as bioindicators in the field. Our results highlighted that: (a) blue mussels bioaccumulated the parasites for 21 days, according to the conditions of exposure, and parasites could still be detected during the depuration period (until 21 days); (b) the percentage of protozoa-positive M. edulis varied under the degree of protozoan contamination in water; (c) mussel samples from eight out of nine in situ sites were positive for at least one of the protozoa. CONCLUSIONS: The blue mussel M. edulis can bioaccumulate protozoan parasites over long time periods, according to the degree of contamination of waters they are inhabiting, and can highlight recent but also past contaminations (at least 21 days). SIGNIFICANCE AND IMPACT OF THE STUDY: Mytilus edulis is a relevant bioaccumulators of protozoan (oo)cysts in laboratory and field conditions, hence its potential use for monitoring parasite contamination in marine waters.
Asunto(s)
Criptosporidiosis , Cryptosporidium , Mytilus edulis , Animales , Ecosistema , Biomarcadores Ambientales , Laboratorios , AguaRESUMEN
BACKGROUND: Cryptosporidium parvum (C. parvum) is a cosmopolitan parasite that infects various livestock animals including cattle. Microsatellite typing tools for identification of C. parvum subtypes are currently employed to better understand the species-specific epidemiology of cattle cryptosporidiosis. The aim of this study was to analyse the population genetics of C. parvum strains infecting cattle and recognise geographical distribution and time-span correlations in subtype prevalence in Poland. In total, 1601 faecal samples were collected from 2014 to 2018 from healthy cattle from dairy, meat and mixed breeds at the age of 1 week to 4 months. The 267 farms visited were randomly selected and represented all Polish provinces. PCR-RFLP based identification of C. parvum at the 18 small subunit ribosomal RNA (SSU rRNA) locus was performed, followed by strain subtyping by GP60-PCR. RESULTS: The overall prevalence of C. parvum in Polish cattle was estimated at 6.2% (100/1601). Animals below the age of 1 month were the major host for this parasite. Excluding one breed, that of dairy-meat mixed, there were no significant differences observed between breed and presence of C. parvum infections (95% TPIAll breeds: 1.67-73.53%; POPR = 0.05-0.95). Infected animals were detected in 15 out of 16 Polish provinces, with significant regional prevalence diffrences (Kruskal-Wallis rank sum test, Kruskal-Wallis χ2 = 13.46, p < 0.001). When the population genetics of C. parvum strains were analysed, 11 parasite subtypes from the IIa and IId genetic families were identified. Compared to other parasite strains, IIaA17G1R1 and IIaA17G2R1 appeared at statistically significantly higher frequency (F-test, F = 3.39; p = 0.0003). The prevalence of C. parvum subtypes in cattle was breed-related (Chi-squared test, χ2 = 143.6; p < 0.001). CONCLUSIONS: The analysis of the population genetics of C. parvum subtypes showed that strains from the IIa subtype family predominated in the tested cattle population. However, relations in changes of subtype prevalence and circulation over time were observed. They were associated with the disappearance of some strains and emergence of new variants from the same genetic family in different geographical locations.