| Sheep coccidiosis is a kind of self-limited illness caused by Eimeria coccidiosis.Sheep are susceptible to coccidiosis at all stages,especially in weaned lambs.Coccidia infection can lead to varying degrees of intestinal inflammation in lambs.The clinical manifestations are lethargy,loss of appetite,diarrhea,dehydration,weight loss,and even death of lambs.E.ovinoidalis is considered to be the most pathogenic and harmful.Because sheep coccidia are often co-infected with several kinds of coccidiosis,there is a lack of research on the pathogenicity of E.ovinoidalis.In this experiment,E.ovinoidalis was isolated and purified and infected with weaned lambs,and its effects on mental state,growth performance,blood index,microbiome and metabolites of weaned lambs were analyzed,and its endogenous development was explored to provide basic data for the prevention and control of sheep coccidiosis and guiding clinical medication.1 Isolation and amplification of E.ovinoidalisThrough morphological identification,purebred E.ovinoidalis oocysts were obtained by single oocyst isolation technique,purebred offspring were collected by 3-day-old lambs,and were orally inoculated to 1-mouth-old lambs for reduplication.At the same time,the regularization of oocyst amplification and subculture amplification,the morphology and structure of oocysts were studied,and the SSU r RNA and COI sites of oocysts were amplified and analyzed by PCR.The morphological and molecular biological characteristics of E.ovinoidalis were consistent with those of its parents and reported in the literature.However,when 3-day-old lambs were used to amplify coccidia,it was found that the regularization of oocyst excretion of lambs was slightly different from that reported in the literature,and the latent period was slightly longer than that reported in the literature.2 Study on the Pathogenicity of E.ovinoidalisSelect 50 day-old weaned lambs that have been treated with insect repellent,divided into four groups,except for the control group(group C),5 × 104 oocysts,3 × 104 oocysts and 1 × 104 oocysts respectively in high(H),medium(M)and low(L)dose groups.The clinical symptoms of lambs in each group after artificial infection were observed,Samples were collected regularly to detect the regularity of oocysts,growth-performance,the blood of lambs in group C and H was collected to detect blood indexes and cytokines.Pathological sections were made on the intestines of infected lambs at different time points and the endogenous development of E.ovinoidalis was observed.The results showed that the prepatent period of weaned lambs infected with E.ovinoidalis was 11 days,the patent period was 5 ~ 6 days,and the peak period was 13 ~ 15 dpi.The lambs infected with E.ovinoidalis had loss of appetite,depression,diarrhea,dehydration and weight loss.The severity of symptoms was positively correlated with the dose of infection.Infection with 3 × 104 oocysts could cause lamb death.Blood index analysis showed that the increase of erythrocyte and hemoglobin in group H was significantly higher than that in group C,and reached the highest in 14 dpi,the increase of hemoglobin concentration in group H was always higher than that in group C and significantly higher in 14 dpi than that in group C,and the increase of platelet in group H was higher than that in group C and very significantly higher than that in group C at 14 dpi.There was no significant difference in leukocyte concentration between group H and group C,but during 0 ~ 14 dpi,the number of leukocytes in group H was lower than that in group C,and the concentration of 14 ~ 21 dpi began to increase.Compared with group C,the levels of IFN-γ,IL-6 and IL-17 in the serum of lambs in group H were significantly higher than those in group C at 21dpi(P < 0.05),and IL-17 in group H was the significantly highest than that in group C at 7dpi(P < 0.01).The expression of anti-inflammatory cytokine IL-10 in group H began to decrease from 7dpi,and there was significant difference between the two groups in 21dpi(P <0.05).The intestinal pathological sections of lambs in control group and infection group showed that coccidia infection could lead to tissue exfoliation of the intestinal mucosa,the epithelial cells are destroyed,incomplete crypt structure,severe bleeding and so on.At the same time,it was found that the first generation merozoites of E.ovinoidalis were mainly parasitized in lamb ileum crypt epithelial cells and lamina propria cells.while the second generation merozoites,gametophytes and zygotes were mainly parasitized in cecal and colonic crypt epithelial cells.The above results showed that E.ovinoidalis infection could cause severe clinical symptoms and acute inflammation in lambs,A large number of Eimeria destroyed intestinal structure,resulting in reduced absorption of nutrients and affected the weight gain of lambs.3 E.ovinoidalis modulates the gut microbiome and metabolism in lambRectal fecal samples of lambs in group C and H were collected at different time points to analyze the effects of E.ovinoidalis infection on gut microbiome and metabolism in lamb.The results showed that E.ovinoidalis infection destroyed the balance of gut microbiome and decreased the richness and diversity of gut microflora in lambs.Especially during the peak period of excrete oocysts,it promoted the reproduction of some harmful bacteria in Proteobacteria and Actinobacteriota,and reduced the richness of beneficial bacteria such as lachnospiraceae and Rikenellaceae.In the later stage of the patent period,the richness of harmful bacteria in the intestine decreased,the richness of beneficial bacteria which could produce anti-inflammatory substances began to increase,and the richness and diversity of intestinal flora also tended to group C.The main metabolites of lambs in group H and C were Fatty Acyls,Organooxygen compounds,Steroids and steroid derivatives,and Metabolism of terpenoids and polyketides was the main metabolic pathway.Coccidia infection could lead to the increase of differential metabolites and metabolic pathways between group H and group C,but the difference decreased with time.During the peak period of excrete oocysts,although the antimicrobial metabolites such as Lividamine were up-regulated,the excess of these metabolites could still induce the production of endotoxin,while Butanoic acid and other anti-inflammatory metabolites decreased significantly.After the peak period of excrete oocysts,the content of butyric acid began to increase,and the metabolites such as chenodeoxycholic acid which can promote diarrhea in lambs began to decrease significantly.However,it was found that the levels of Sulfur metabolism and Nitrogen metabolism which are closely related to life were down-regulated.Although the immunity of the body was improved in the later stage of the patent period,the effect of coccidia on the body still existed.E.ovinoidalis infection can cause severe clinical symptoms and induce acute inflammation in lambs.It destroys the homeostasis of intestinal environment and the metabolic function of the body,which affects the normal growth and development of lambs,reduces the product quality,and seriously hinders the development of industry. |
PDF Full Text Request