Detection and molecular characterization of major enteric pathogens in calves in central Ethiopia

Abstract

Background: Calf diarrhea is a major cause of morbidity and mortality in the livestock sector worldwide and it can be caused by multiple infectious agents. In Ethiopia, cattle are the most economically important species within the livestock sector, but at the same time the young animals suffer from high rates of morbidity and mortality due to calf diarrhea. However, studies including both screening and molecular characterization of bovine enteric pathogens are lacking. Therefore, we aimed to both detect and molecularly characterize four of the major enteric pathogens in calf diarrhea, Enterotoxigenic Escherichia coli (E. coli K99 +), Cryptosporidium spp., rotavirus A (RVA), and bovine coronavirus (BCoV) in calves from central Ethiopia. Diarrheic and non-diarrheic calves were included in the study and fecal samples were analyzed with antigen-ELISA and quantitative real-time PCR (qPCR). Positive samples were further characterized by genotyping PCRs.

Results: All four pathogens were detected in both diarrheic and non-diarrheic calves using qPCR and further characterization showed the presence of three Cryptosporidium species, C. andersoni, C. bovis and C. ryanae. Furthermore, genotyping of RVA-positive samples found a common bovine genotype G10P[11], as well as a more unusual G-type, G24. To our knowledge this is the first detection of the G24 RVA genotype in Ethiopia as well as in Africa. Lastly, investigation of the spike gene revealed two distinct BCoV strains, one classical BCoV strain and one bovine-like CoV strain.

Conclusions: Our results show that Cryptosporidium spp., E. coli K99 + , RVA and BCoV circulate in calves from central Ethiopia. Furthermore, our findings of the rare RVA G-type G24 and a bovine-like CoV demonstrates the importance of genetic characterization.

Keywords: Cryptosporidium; E. coli K99 +; Bovine coronavirus; Calf diarrhea; Ethiopia; Rotavirus A.

Fig. 1

qPCR based detection rates of each pathogen based on diarrheal status. Numbers above bars indicate number of qPCR positive animals (A). Percentage of calves testing positive for one, two, three or no pathogens with qPCR (B)

Fig. 2

Maximum likelihood trees of the VP7 gene (A) and partial VP4 gene (nt 11–887, VP8* segment) (B) rooted at the midpoint. G10 and P[11] genotypes are highlighted with a blue background and G24 genotypes with a purple background. Strains identified in the current study are indicated by bold text. Bootstrap values > 0.7 are displayed

Fig. 3

Maximum likelihood tree of the hypervariable region of the S1 subunit (nt: 1332–1778 of Mebus, U00735) rooted at the midpoint. Classical BCoV strains are highlighted with a blue background and bovine-like CoV strains with a purple background. Strains identified in the current study are indicated by bold text. Bootstrap values > 0.7 are displayed

Fig. 4

Map showing the location of the towns in Ethiopia where sampling was conducted. The towns Sebeta, Holeta, Sululta and Bishoftu are represented by diamonds. The capital Addis Ababa is represented by a black circle. Maps were generated using R software (version 4.2.2)