Metagenomic analysis of 16S Clostridium perfringens amplicons corroborates C. perfringens counts on select agar and C. perfringens PCR analyses of bacteria in broiler farm litter

Authors: Jenkins, Mark; Parker, Carolyn; OBRIEN, CELIA; CAMP, MARY; Vinyard, Bryan; HEEDER, CARL - MOUNTAIRE FARMS, INC.; Proszkowiec-Weglarz, Monika

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/6/2021
Publication Date: 11/12/2021
Citation: Jenkins, M.C., Parker, C.C., Obrien, C.N., Camp, M.J., Vinyard, B.T., Heeder, C., Proszkowiec-Wegla, M.K. 2021. Metagenomic analysis of 16S Clostridium perfringens amplicons corroborates C. perfringens counts on select agar and C. perfringens PCR analyses of bacteria in broiler farm litter. Avian Diseases. 65:554-558. https://doi.org/10.1637/aviandiseases-D-21-00054.
DOI: https://doi.org/10.1637/aviandiseases-D-21-00054

Interpretive Summary: Avian coccidiosis is an intestinal disease of poultry caused by protozoa in the genus Eimeria. One important outcome of coccidiosis infection is increased incidence of necrotic enteritis (NE) which leads to acute death in chicks 3-9 weeks of age. NE is caused by Clostridium perfringens, bacteria that harbor toxin genes that upon invasion of intestinal tissue damaged by Eimeria infection, release toxins that cause increased morbidity and mortality in afflicted chickens. The purpose of this study was two-fold: to determine if new technology based on sequencing ribosomal RNA genes (Cp-16S) of the entire bacterial population in litter corroborated findings from traditional, yet more labor-intensive assays, such as culturing on select agar or polymerase chain reaction (Cp-PCR), for relative number of C. perfringens. The second goal was to determine if the abundance of C. perfringens by Cp-16S assay or assays for netB- or Tpel-toxin genes were correlated with chick mortality. The results showed that Cp-16S assay was highly correlated with both standard plate counts and Cp-PCR. Thus, Cp-16S assay offers not only a way to determine the relative level of C. perfringens in a sample, but also all the other bacterial genera present. However, no correlation was observed between Cp-16S assay and chick mortality. A strong correlation was found between both netB or Tpel assay and chick mortality. These findings suggest that not all C. perfringens harbor toxin genes, such as netB or Tpel, that are known to cause NE in chickens. Assaying for netB or Tpel toxin genes may be a useful predictor of chick mortality during broiler growout.

Technical Abstract: The purpose of this study was two-fold: first, to determine if analysis of bacterial 16S rRNA in poultry litter corroborated standard Clostridium perfringens counts and PCR assay. Second, to find if there was a correlation between 16S rRNA analysis, netB-toxin, or Tpel-toxin PCR intensity with chick mortality. At 3 timepoints of growout (0, 2, and 4 wk), litter samples were collected from 23 broiler houses representing 8 farms during a coccidiosis vaccine control program. DNA extracted from these samples was used for microbiota determination by sequencing the hypervariable V3-V4 region of bacterial 16s rRNA (Illumina). Obtained sequences were analyzed using QIIME2 and Greengenes database for taxonomic composition and relative abundance of C. perfringens in the litter bacterial population. Clostridium perfringens counts on select agar and semi-quantitative PCR for C. perfringens were compared to 16S analysis for equivalence testing. Relative abundance of C. perfringens estimated by 16S analysis and semi-quantitave PCR for netB- and Tpel-toxin DNA were analyzed by Pearson linear correlation and statistical equivalence analyses with cumulative chick mortality at 4 and 9 wk growout. When data from all time-points were combined, abundance estimates by C. perfringens 16S (Cp-16S) were statistically equivalent to both C. perfringens PCR and C. perfringens counts (P < 0.05). Yet, no correlations were observed between any estimate of C. perfringens abundance and cumulative % chick mortality at 4- or 9-wk growout. However, correlation analyses revealed a significant linear relationship between netB-signal at 0 wk (r=0.55) and 4 wk (r=0.46) and cumulative mortality at 9 wk growout (P < 0.05). Similarly, abundance of Tpel at 0 and 2 wk showed a linear relationship with cumulative % mortality at both 4- and 9-wk growout (0.44 < r < 0.54, P < 0.05). No correlations were observed between any other genera/species determined by 16S and cumulative % chick mortality.