Unravelling the prevalence and impact of Cryptosporidium parvum on the gut microbiome, metabolome, and health status of neonatal calves

Cryptosporidium parvum (C. parvum), colonises and infects the gastrointestinal tract (GIT) of a diverse range of vertebrate hosts, resulting in the development of cryptosporidiosis, a clinical condition characterised by profuse watery diarrhoea. Additional symptoms in infected hosts include dehydration, weight loss, fever, and abdominal pain. This disease has a global distribution, affecting millions of individuals each year and disproportionately impacting children in developing countries, where it stands as a prominent cause of mortality. Furthermore, cryptosporidiosis is notably prevalent among young livestock, particularly neonatal calves, exhibiting symptoms similar to those seen in human infants. This prevalence leads to significant economic losses in the dairy and beef industry. Moreover, infected animals can excrete and disseminate millions of infectious parasites daily into the environment, perpetuating the cycle of infection and posing a continued threat to human health. Addressing the global challenge of cryptosporidiosis necessitates an exploration of the diversity of Cryptosporidium species (spp.), their zoonotic potential, and the dynamics of transmission within dairy farms across various countries. Additionally, it is essential to recognise the pivotal role of the gut microbiota in maintaining gut health, bolstering defences against harmful pathogens, facilitating energy production, and preserving the integrity of the gut barrier. Disruptions in the normal gut microbiota have been linked to various diseases, but the precise molecular and biochemical mechanisms through which C. parvum disrupts these intricate systems remain poorly understood. Therefore, a comprehensive investigation into the interaction between the parasite and the gut microorganisms and metabolites of the host holds the potential to reveal novel therapeutic strategies and identify potential biomarkers for detecting the presence of C. parvum. This research could provide invaluable insights into mitigating the impact of C. parvum on gut health and its associated diseases. To achieve these objectives, I first conducted an extensive analysis of Cryptosporidium spp. prevalence and transmission dynamics in cattle farms spanning a broad geographic area encompassing Belgium, France, the Netherlands, and Cyprus. In addition, I also delved into the genetic variation of C. parvum to evaluate the potential circulation of zoonotic subtypes within dairy farms. This project required the development and implementation of a robust protocol for deoxyribonucleic acid (DNA) extraction from faecal samples. Subsequently, a two-step nested polymerase chain reaction (PCR) was utilised to amplify and sequence specific fragments of the 18S ribosomal rRNA (18S rRNA) and 60-kDa glycoprotein (gp60) genes of the targeted organism. Furthermore, I employed a combination of bioinformatics and phylogenetic analyses to characterise the obtained sequences. The findings of this research shed light on the widespread prevalence of Cryptosporidium spp. within dairy farms across different countries. Notably, the research also raised concerns regarding cattle serving as potential carriers of C. parvum zoonotic subtypes, suggesting a potential threat to human health.

For the second part of my PhD, I pioneered a multiomics approach to understand the impact of C. parvum propagation in the calves GIT. This research aimed to monitor how the parasite infection modulates the gut microbiome and metabolome of the host by first characterising the impact of C. parvum infection on the taxonomic composition of the neonatal calves' gut microbiota and then assess the collective change of metabolites in the gut during C. parvum infection. To accomplish this, a cross-sectional study was conducted on 23 cattle farms in the Lille region of France. A total of 134 faecal samples were collected from neonatal calves aged between four to 21 days.

In this study, I employed both a nested PCR and qPCR protocol for accurate detection and quantification of the parasite. After molecular detection and faecal scoring, the calves were classified into three groups: non-Cryptosporidium infected (HH), Cryptosporidium infected without diarrhoea (IH), and Cryptosporidium infected with diarrhoea (DH). Out of the total 134 calves, 73 were found to be negative for C. parvum while the remaining 61 calves tested positive for the parasite (IH = 41 and DH = 20). The collected faecal samples were subjected to 16S rRNA gene sequencing, while 102 out of the 134 faecal samples were analysed through One-dimensional proton nuclear magnetic resonance (1D 1H-NMR) metabolomics. The resulting data underwent thorough analysis using various bioinformatics tools. The 16S rRNA analysis, revealed a significant impact of C. parvum on the gut microbiota of infected calves (particularly for the DH group) when compared to the non-infected group (HH). Notably, opportunistic bacterial genera like Fusobacterium_A, Clostridium_P and Gallibacterium exhibited increased relative abundance in the DH group. Conversely, bacterial genera associated with a healthy gut, such as Bifidobacterium_388775 and Parabacteroides_B_862066 had higher relative abundances in the HH group. Metabolomic profiles between these groups also showed marked differences, specifically between the DH versus the HH group. DH calves exhibited a significant decrease in the abundance of five metabolites, with special focus on the short-chain fatty acid (SCFA) Valerate, in contrast with the HH group. Conversely, the DH calves displayed a significant increase in 10 metabolites, including Ethanol, Formate, Lactate and Succinate, which are intermediates for SCFAs. The observed accumulation of these metabolites coupled with the decrease in valerate might be closely related to an increase in harmful bacteria and a decrease in commensal bacteria in the DH group in response to C. parvum infection. The findings from this study underscore the substantial impact of C. parvum infections on the composition of faecal microbiota in calves, leading to changes in faecal metabolites that might compromise the calves' ability to resist C. parvum infection. This research lays a crucial foundation for further investigations into the role of intestinal microbiota and their metabolism in neonatal calf diarrhoea associated with C. parvum, providing insights that can potentially inform future strategies for managing and preventing cryptosporidiosis. Moreover, I've established, for the first time, an NMR-based metabolomics approach to quantify metabolite changes in faeces post-C. parvum infection in calves. This method could be employed for future investigations examining the interplay between gut flora and metabolites in the context of other enteric protozoan infections.