Progression of the faecal microbiome in preweaning dairy calves that develop cryptosporidiosis

Abstract

Background: Cryptosporidiosis is a diarrheal disease that commonly affects calves under 6 weeks old. The causative agent, Cryptosporidium parvum, has been associated with the abundance of specific taxa in the faecal microbiome during active infection. However, the long-term impact of these microbiome shifts, and potential effects on calf growth and health have not yet been explored in depth.

Methods: Three hundred and forty-six (346) calves from three dairy farms had one faecal swab collected during the first week of life (W1). Thereafter, sampled calves were monitored for diarrhoeal disease and those that suffered a diarrhoea event were tested for C. parvum by lateral flow testing (LFT). Calves that experienced diarrhoea and tested positive for C. parvum by LFT were assigned to the Cryptosporidium-positive (Cp+) group (n = 32). Matched healthy (H) controls with no history of diarrhoea were selected from the remaining cohort (n = 33). The selected subset of calves (n = 65) was observed until weaning, collecting a faecal swab, at approximately Week 5 (W5) and Week 10 (W10) after birth, resulting in a total of 191 samples (W1; n = 65, W5; n = 64, W10; n = 62). 16S rRNA gene amplicon sequencing was performed on all extracted samples.

Results: Analysis of the longitudinal microbiome showed significant changes in the microbial diversity and composition across all three time-points. Whilst Firmicutes were elevated in the Cp+ group at W5 compared to the H group, no other significant differences were detected between H and Cp+ groups. Whilst the core microbiota showed some taxa were exclusive to each group, the role of these taxa in health and disease has yet to be determined. Antibiotics were also found to have an impact on the relative abundance of some taxa. Though healthy calves received a significantly higher body condition score than Cp+ calves at W5, the difference did not reach significance at W10, suggesting that Cp+ calves may catch up to their healthy counterparts once the infection has resolved.

Conclusions: The findings of this study illustrated the changes in the microbial diversity and composition during the preweaning period in dairy calves. The results also indicated that the faecal microbiome is not predictive of cryptosporidiosis and implied that cryptosporidiosis doesn't cause long-term gut dysbiosis. This study furthered our understanding of the parasite-microbiome relationship and its impact on the bovine host.

Keywords: Cryptosporidium parvum; 16S rRNA gene amplicon sequencing; Bovine cryptosporidiosis; Calf diarrhoea/diarrhea; Faecal/fecal microbiome; Longitudinal.

Fig. 1

Alpha diversity increased with age but was not influenced by Cryptosporidium parvum. A Observed OTUs between healthy control and Cryptosporidium-positive groups at Week 1, 5, and 10. A pairwise Wilcoxon test shows differences in observed OTUs between time-points. The red dots indicate the mean. B Shannon diversity between healthy control and Cryptosporidium-positive groups at Week 1, 5, and 10. A pairwise Wilcoxon test shows differences in Shannon indices between time-points. The red dots indicate the mean. C Local regression with locally estimated scatterplot smoothing (LOESS) of observed OTUs between healthy control and Cryptosporidium-positive groups stratified by day of sampling shows within time-point variation. D Local regression with LOESS of Shannon diversity between healthy control and Cryptosporidium-positive groups stratified by day of sampling shows within time-point variation

Fig. 2

Beta diversity altered with age but was not impacted by Cryptosporidium parvum. A Weighted Bray-Curtis PCoA plot shows similarity between healthy control and Cryptosporidium-positive groups at Week 1, 5, and 10, and dissimilarity between sample time-points (pairwise PERMANOVA: H1 vs. Cp+1, p = 0.49; H5 vs. Cp+5, p = 0.40; H10 vs. Cp+10, p = 0.89; W1 vs. W5 vs. W10, p = 0.001). B Unweighted Jaccard PCoA plot displays similarity between healthy control and Cryptosporidium-positive groups at Week 1, 5, and 10, and dissimilarity between sample time-points (pairwise PERMANOVA: H1 vs. Cp+1; p = 0.20, H5 vs. Cp+5; p = 0.69, H10 vs. Cp+10; p = 0.90; W1 vs. W5 vs. W10, p = 0.001)

Fig. 3

The pre-/post-infection taxonomic composition and core microbiome were minimally impacted by Cryptosporidium parvum infection. A Top 5 most abundant phyla at Week 1, 5, and 10 in healthy control and Cryptosporidium-positive groups. B Top 15 most abundant genera at Week 1, 5, and 10 in healthy control and Cryptosporidium-positive groups. C Venn diagrams of the core microbiota in healthy control and Cryptosporidium-positive groups at Week 1, 5, and 10 time-points. Genera were detected at 0.01% abundance in at least 80% of the samples

Fig. 4

The most abundant genera experienced changes in relative abundance over time. Relative abundance of the top 30 most abundant genera for healthy control and Cryptosporidium-positive groups at Week 1, 5, and 10. The red dot shows the mean relative abundance

Fig. 5

Body condition scores were impacted by past Cryptosporidium parvum infection, but daily live weight gain and respiratory disease scoring was not associated with Cryptosporidium parvum infection. A Body condition scores of healthy control and Cryptosporidium-positive groups at Week 1, 5, and 10. B Daily live weight gain in kilograms per day of healthy control and Cryptosporidium-positive groups at Week 5 and 10 on Farm 2. C Total Wisconsin scores for healthy control and Cryptosporidium-positive groups at Week 1, 5, and 10. D Lung scores for healthy control and Cryptosporidium-positive groups at Week 10. The red dots indicate the mean