First identification of Cryptosporidium parvum virus 1 (CSpV1) in various subtypes of Cryptosporidium parvum from diarrheic calves, lambs and goat kids from France.

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.

Cryptosporidium parvum Infection Depletes Butyrate Producer Bacteria in Goat Kid Microbiome.

Cryptosporidium parvum is an important apicomplexan parasite infecting ruminants and humans. We characterized the impact of C. parvum infection on the goat kid microbiome. C. parvum was orally administered to parasite-naïve goats, and infection was monitored for 26 days in fecal samples using immunofluorescence assay and qPCR tests. Age-matched goats served as uninfected controls. A reduction in body weight gain, diarrhea, and dehydration were observed in infected goats compared to the uninfected controls. Infection decreased the bacterial diversity 5 days post-infection (dpi), but this parameter recovered at 15 dpi. The infection altered the relative abundance of several taxa. A total of 38 taxa displayed significant differences in abundance between control and infected goats at both 5 and 15 dpi. Co-occurrence network analysis revealed that the infection resulted in a differential pattern of taxa interactions and that C. parvum infection increased the relative abundance of specific taxa. The 16S data set was used for metagenome predictions using the software package PICRUSt2. As many as 34 and 40 MetaCyc pathways (from 387 total) were significantly affected by the infection at 5 and 15 dpi, respectively. Notably, C. parvum decreased the abundance of butyrate-producing pathways in bacteria. Low levels of butyrate may increase mucosal inflammation and tissue repair. Our results suggest that the gut inflammation induced by C. parvum infection is associated with the reduction of butyrate-producing bacteria. This insight could be the basis for the development of novel control strategies to improve animal health.

First identification of Cryptosporidium parvum zoonotic subtype IIaA15G2R1 in diarrheal lambs in France.

To date, no information is available about the presence of Cryptosporidium spp. in French sheep, nor their potential role as zoonotic reservoirs. A total of 23 fecal samples were collected from diarrheic lambs (<11 days old) from seven randomly selected farms. Cryptosporidium-oocysts were detected microscopically with Direct Immunofluorescence Assays (DFA) in 23/23 (100%) of fecal samples. PCR-RFLP of the 18S rRNA gene was used to determine species in all samples, and only Cryptosporidium parvum was identified. Isolates were subtyped by sequencing the 60 kDa glycoprotein (gp60) gene. Two zoonotic subtypes within the IIa subtype family were identified, including IIaA15G2R1 (22/23) and IIaA16G3R1 (1/23). This study reports for the first time the identification and genotyping of zoonotic C. parvum subtypes from lambs in France. Sheep could thus play an important role as potential reservoirs for this zoonotic protist.

Molecular characterization of Cryptosporidium isolates from diarrheal dairy calves in France.

Cryptosporidium is an obligate intracellular protist parasite infecting a wide range of vertebrate hosts and causes significant intestinal disease in both animals and humans, as some species are zoonotic. Cattle and especially calves have been identified as one of the most common reservoirs of this protist. However, little is known about the genetics of Cryptosporidium in calves in some regions of France. The aim of this study was to detect and isolate Cryptosporidium spp. in faecal samples from naturally infected pre-weaned calves (≤45 days-old) in France. A total of 35 diarrhoeic pre-weaned calf faecal samples were collected from 26 dairy cattle farms in six departments (French administrative provinces). Cryptosporidium presence was established by microscopically screening samples for oocystes with an immunofluorescent (DFA) staining method. DFA-positive samples were then analysed by PCR-RFLP and 18S rRNA gene sequencing to determine species. Cryptosporidium parvum-positive samples were subtyped via nested PCR analysis of a partial fragment of the 60 kDa glycoprotein (gp60) gene product. Data were then integrated into phylogenetic tree analysis. DFA revealed the presence of Cryptosporidium oocysts in 31 out of 35 (88%) samples. Combined with 18S rRNA gene analysis results, C. parvum was detected in 30 samples. Subtyping analysis in 27/30 samples (90%) of the C. parvum isolates revealed two zoonotic subtype families, IIa (24/27) and IId (3/27). Four subtypes were recognised within the subtype family IIa, including the hypertransmissible IIaA15G2R1 subtype that is the most frequently reported worldwide (21/27), IIaA17G3R1 (1/27), IIaA17G1R1 (1/27), and IIaA19G1R1 (1/27). Two subtypes were recognised within the IId subtype family including IIdA22G1 (2/27) and IIdA27G1 (1/27). These findings illustrate the high occurrence of Cryptosporidium in calves in dairy herds and increase the diversity of molecularly characterised C. parvum isolates with the first description of IIaA17G3R1, IIaA19G1R1, and IId subtypes in France. The presence of zoonotic C. parvum subtype families (IIa, IId) in this study suggests that pre-weaned calves are likely to be a significant reservoir of zoonotic C. parvum, and highlights the importance of animal to human cryptosporidiosis transmission risk. Further molecular studies in calves and small ruminants from other French regions are required to better understand the epidemiology of cryptosporidiosis in France.

Molecular characterization of zoonotic Cryptosporidium spp. and Giardia duodenalis pathogens in Algerian sheep.

Little is known about the presence of Cryptosporidium spp. and Giardia duodenalis in Algerian sheep, nor their potential role as zoonotic reservoirs. This study aimed to investigate the occurrence and distribution of these two protists in lambs. A total of 83 fecal samples were collected from lambs (< 40 days old) from 14 different farms. Samples were screened for Cryptosporidium spp. and Giardia duodenalis presence using immunofluorescent techniques (IF). Nested PCR of the small subunit ribosomal RNA (rRNA) gene, followed by restriction fragment length polymorphism (PCR-RFLP) and sequence analyses were used to identify Cryptosporidium species. C. parvum was further subtyped by sequencing the highly polymorphic 60 kDa glycoprotein (gp60) gene. For G. duodenalis, nested PCR of the glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) genes was performed and then PCR-RFLP was used to identify G. duodenalis assemblages. Cryptosporidium oocysts and Giardia cysts were detected in 36/83 (43%) and 23/83 (28%) of fecal samples, respectively. Of the 21/36 (58%) Cryptosporidium samples that were positive with IF, 16/21 (76%) were identified as C. parvum, and 5/21 (24%) as C. ubiquitum. From 15C. parvum isolates, 2 subtypes were identified within the IIa subtype family, including IIaA21G2R1 (3/15) and IIaA13G2R1 (1/15), while IIdA16G1 (11/15) was the only subtype identified from the IId subtype family. Of the 16/23 (69%) G. duodenalis IF-positive samples, the most frequent assemblage was ruminant-specific assemblage E (10/16), followed by assemblage D (4/16), and A + E mixed assemblages (2/16). This study is the first to identify and genotype both Cryptosporidium spp. and Giardia duodenalis in Algerian lambs, and is also the first to describe G. duodenalis assemblage D in small ruminants. The presence of zoonotic C. parvum subtype families (IIa, IId), C. ubiquitum, as well as G. duodenalis assemblage A + E, indicates that sheep could play an important role as a potential reservoir for protists.

Cryptosporidium parvum-Infected Neonatal Mice Show Gut Microbiota Remodelling Using High-Throughput Sequencing Analysis: Preliminary Results.

BACKGROUND: During the last decade, the scientific community has begun to investigate the composition and role of gut microbiota in normal health and disease. These studies have provided crucial information on the relationship between gut microflora composition and intestinal parasitic infection, and have demonstrated that many enteric pathogen infections are associated with altered gut microflora composition. In this study, we investigated the effects of Cryptosporidium parvum infection (zoonotic protozoan affecting a large range of vertebrates) on both qualitative and quantitative composition of gut microbiota in a CD-1 neonatal mouse model. METHODS: 5-day-old neonate mice were experimentally infected with 105Cryptosporidium parvum Iowa oocysts by oesophageal gavage. The intestinal microbiota of both infected (Cp+) and uninfected (Cp-) mice groups was examined by high-throughput sequencing of the bacterial 16S rDNA gene V3-V4 hypervariable region. RESULTS: The most consistent change in the microbiota composition of Cp+ mice was the increased proportion of bacterial communities belonging to the Phylum Bacteroidetes. In contrast, the microbiota of Cp- mice was associated with increased proportions of several Firmicutes and Actinobacteria phyla members. CONCLUSION: For the first time, our study provides evidence of an association between cryptosporidial infection and gut dysbiosis, thus contributing valuable knowledge to the as-yet little-explored field of Cryptosporidium-microbiota interactions in a neonatal mouse model.

Efficacy of chitosan, a natural polysaccharide, against Cryptosporidium parvum in vitro and in vivo in neonatal mice.

Cryptosporidiosis is a zoonotic disease caused by species in the genus Cryptosporidium. In young ruminants, Cryptosporidium parvum causes economically significant disease with mild to severe clinical signs and occasional death. The typical clinical course in animals aged 1-3 weeks old is acute diarrhoea. Currently there are no available treatments that are fully effective against cryptosporidiosis in either humans or animals. Therefore there is a critical need for the development of new therapeutic agents. We adapted two in vitro culture systems (HCT-8 and Caco-2 cell lines) for C. parvum infection to investigate the "anticryptosporidial" activity of two chitosans; Chitosan NAG and Chitosan Mix. Chitosan-a naturally-occurring polysaccharide compound-has been found to be active against a variety of diseases, possessing both antimicrobial and anticancer properties. We investigated both chitosan's toxicity and effects on C. parvum in the two in vitro models. To evaluate chitosan's effects on oocyst shedding in vivo, CD-1 neonate mice were orally inoculated with C. parvum oocysts (Iowa strain), treated with chitosan, and compared to infected non-treated animals. Paromomycin, a classical drug used in veterinary medicine, was used as a reference compound. Immunofluorescence techniques were used to analyse the parasites. Our results showed significant reductions in Cryptosporidium oocyst viability (>95%) after oocyst pre-incubation with either paromomycin (P < 0.001), Chitosan Mix or Chitosan NAG (P < 0.001), for 24 h at 37 °C. Additionally, paromomycin, Chitosan Mix, and Chitosan NAG significantly inhibited C. parvum multiplication in HCT-8 and Caco-2 cell lines (P < 0.005). These effects were dose-dependent. In in vivo studies, treatment with both chitosans (Chitosan NAG, Chitosan Mix) or paromomycin sulfate significantly reduced parasite shedding in infected treated newborn mice (-56%, -34.5% and -58%, respectively). In conclusion, these findings provide the first in vitro and in vivo evidence of the anticryptosporidial activities of this natural polysaccharide.