Comparison of the Pathogenicity of Five Clostridium perfringens Isolates Using an Eimeria maxima Coinfection Necrotic Enteritis Disease Model in Commercial Broiler Chickens.

Clostridium perfringens (CP) is the etiologic agent of necrotic enteritis (NE) in broiler chickens that is responsible for massive economic losses in the poultry industry in response to voluntary reduction and withdrawal of antibiotic growth promoters. Large variations exist in the CP isolates in inducing intestinal NE lesions. However, limited information is available on CP isolate genetics in inducing NE with other predisposing factors. This study investigated the ability of five CP isolates from different sources to influence NE pathogenesis by using an Eimeria maxima (EM) coinfection NE model: Str.13 (from soil), LLY_N11 (healthy chicken intestine), SM101 (food poisoning), Del1 (netB+tpeL-) and LLY_Tpel17 (netB+tpeL+) for NE-afflicted chickens. The 2-wk-old broiler chickens were preinfected with EM (5 × 103 oocysts) followed by CP infection (around 1 × 109 colony-forming units per chicken). The group of the LLY_Tpel17 isolate with EM coinfection had 25% mortality. No mortality was observed in the groups infected with EM alone, all CP alone, or dual infections of EM/other CP isolates. In this model of EM/CP coinfections, the relative percentages of body weight gain showed statistically significant decreases in all EM/CP groups except the EM/SM101 group when compared with the sham control group. Evident gut lesions were only observed in the three groups of EM/LLY_N11, EM/Del1, and EM/LLY_Tpel17, all of which possessed an essential NE pathogenesis locus in their genomes. Our studies indicate that LLY_Tpel17 is highly pathogenic to induce severe gut lesions and would be a good CP challenge strain for studies investigating pathogenesis and evaluating the protection efficacy for antibiotic alternative approaches.

Eimeria maxima-induced transcriptional changes in the cecal mucosa of broiler chickens.

BACKGROUND: Apicomplexan protozoans of the genus Eimeria cause coccidiosis, one of the most economically relevant parasitic diseases in chickens. The lack of a complete understanding of molecular mechanisms in the host-parasite interaction limits the development of effective control measures. In the present study, RNA sequencing (RNA-Seq) was applied to investigate the host mRNA profiles of the cecal mucosa collected at day 5 post-infection with Eimeria maxima (EM). RESULTS: Total RNA from cecal samples of the uninfected naïve control and the EM groups was used to make libraries, generating 354,924,372 and 356,229,250 usable reads, respectively, which were assembled into a total of 386,088 high-quality unigenes (transcripts) in Trinity software. RNA-Seq analysis of cecal samples in the two groups revealed 332 upregulated and 363 downregulated genes with significant differences (P ≤ 0.05), including several significant immune-related gene families, such as the major histocompatibility complex (MHC) class I alpha chain, granzyme A and immunoglobulin subtype genes among upregulated differentially expressed genes. In addition, a total of 60 clusters of differentiation (CD) molecular genes and 570 novel genes were found. The completeness of the assembled transcriptome was further assessed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, Gene ontology (GO), eggNOG and CAZy for gene annotation. The broad gene categories represented by the highly differentiated host genes suggested enrichment in immune responses, and downregulation in the metabolic pathway, MARK signaling pathway, vascular smooth muscle contraction, and proteins processing in endoplasmic reticulum after EM infection. CONCLUSIONS: Eimeria maxima induced statistically significant differences in the cecal mucosal gene expression of infected chickens. These findings provide new insights into the host-parasite interaction and enhance our understanding of the molecular mechanism of avian coccidiosis.

Characterization of Virulent netB+/tpeL+Clostridium perfringens Strains from Necrotic Enteritis-Affected Broiler Chicken Farms.

Clostridium perfringens (CP) type A and newly created type G strains are the key etiological factors in the induction of necrotic enteritis (NE), an important enteric disease that is responsible for the annual loss of $6 billion in the worldwide poultry industry. Several CP toxin genes were found to be critical in NE pathogenesis in chickens, but limited information is available on the CP lethal toxin tpeL gene. In this study, 19 CP strains isolated from NE-affected chicken farms were characterized microbiologically and molecularly and evaluated for their pathogenicity in commercial broiler chickens. Toxin typing by PCR revealed that all strains tested were positive for the netB toxin gene, but only five strains were positive for the tpeL toxin gene (LLY-TpeL 13, -TpeL 15, -TpeL 17, -TpeL 18, and -TpeL 19, simplified as TpeL 13, TpeL 15, TpeL 17, TpeL 18, and TpeL 19). High levels of TpeL proteins were detected in the concentrated culture supernatant from strains TpeL 13, 15, 17, and 19 by western blotting. Quantitative PCR showed that strains TpeL 13, 15, 17, 18, and 19 harbored a high number of copies of tpeL genes, while TpeL 18 had the highest number of copies of the tpeL gene among all CP strains tested when normalized with copy numbers of 16S rRNA gene as a housekeeping gene marker. The in vivo NE challenge test using multiple oral CP inoculations combined with a high-protein diet showed that TpeL 17 was the most virulent in inducing typical NE lesions, followed by TpeL 19 as the next most virulent, when tested in commercial broiler chickens. Infection with TpeL 17 reduced the growth rate significantly, as shown by reduced relative body weight gain percentage at day 5 postinfection. Availability of the virulent netB+tpeL+ CP strains is essential for the development of a CP-alone NE challenge model that could provide significant tools for understanding CP pathogenesis and for development of alternative to antibiotics.

Caracterización de cepas virulentas de Clostridium perfringens netB+/tpeL+ de granjas de pollos de engorde con enteritis necrótica. Clostridium perfringens (CP) tipo A y los tipos nuevos G son los factores etiológicos clave en la inducción de enteritis necrótica (NE), una enfermedad entérica importante que es responsable de pérdidas anuales de $6 mil millones en la industria avícola mundial. Se ha determinado que varios genes de toxinas de C. perfringens son críticos en la patogénesis de la enteritis necrótica en pollos, pero se dispone de información limitada sobre el gene tpeL de la toxina letal de C. perfringens. En este estudio, se caracterizaron microbiológicamente y molecularmente 19 cepas de C. perfringens aisladas de granjas avícolas afectadas por enteritis necrótica y se evaluaron en su patogenicidad en pollos de engorde comerciales. La tipificación de toxinas por PCR reveló que todas las cepas analizadas fueron positivas para el gene de la toxina netB, pero solo cinco fueron positivas para el gene de la toxina tpeL (LLY-TpeL 13, -TpeL 15, -TpeL 17, -TpeL 18, y -TpeL 19, simplificado como TpeL 13, TpeL 15, TpeL 17, TpeL 18 y TpeL 19). Los altos niveles de proteínas TpeL se detectaron en el sobrenadante del cultivo concentrado de las cepas TpeL 13, 15, 17 y 19 mediante inmunoelectrotransferencia tipo Western. La PCR cuantitativa mostró que las cepas TpeL 13, 15, 17, 18 y 19 albergaban un alto número de copias de los genes tpeL, mientras que TpeL 18 mostró el mayor número de copias del gene tpeL entre todas las cepas de C. perfringens analizadas cuando se normalizó con los números de copias del gene 16S rRNA como un marcador genético constitutivo. La prueba in vivo de desafío de enteritis necrótica utilizando múltiples inoculaciones orales de C. perfringens combinadas con una dieta alta en proteínas mostró que TpeL 17 fue la más virulenta en la inducción de lesiones típicas de enteritis necrótica, seguida de TpeL 19 como la siguiente más virulenta cuando se inoculó en pollos de engorde comerciales. La infección con TpeL 17 redujo significativamente la tasa de crecimiento, como lo demuestra la reducción del porcentaje de aumento de peso corporal relativo en el día cinco posterior a la infección. La disponibilidad de las cepas netB+ tpeL+ de C. perfringens virulentas es esencial para el desarrollo de un modelo de desafío enteritis necrótica únicamente con C. perfringens que podría proporcionar herramientas significativas para comprender la patogénesis de C. perfringens y para el desarrollo de alternativas a los antibióticos.