Different virulence levels of Enterococcus cecorum strains in experimentally infected meat-type chickens

In recent years, pathogenic strains of Enterococcus cecorum (EC) have emerged as a causing agent of septicemia and skeletal infection in broiler chickens with a high economic impact worldwide. Although research has been conducted, many aspects of the pathogenesis of the EC-associated disease are still unknown. In the present study, an experimental infection model was established in broiler chickens. Two different EC strains (EC14 and EC15) were compared in two different concentrations of each strain (2 × 106 and 2 × 108 colony-forming units per milliliter (CFU/mL)) after oral infection of one-day-old chicks. Clinical signs and gross lesions of the EC-associated disease were monitored in the following seven weeks. Although both EC strains were originally isolated from clinical disease outbreaks and had a high embryonic lethality, only EC14 successfully induced the typical course of the EC-associated disease with characteristic clinical signs and gross lesions. In total, 23% of the birds in the two EC14-groups were EC-positive in extraintestinal organs on culture, and no differences were found between the two infectious doses. EC14 was frequently detected via real-time PCR in the free thoracic vertebra (FTV) and femoral heads without any detectable gross lesions. The number of EC positive spleens from infected broilers was comparable using bacterial isolation and a specific real-time PCR. Interestingly, EC15 was not detected in extraintestinal organs, although birds in the EC15 groups were colonized by EC in the ceca after experimental infection. The present study represents first proof that virulence differs among EC strains in experimentally infected chickens, and emphasizes the need to further characterize virulence factors and pathogenic mechanisms of EC. The strain EC14 at a dose of 106 CFU is suitable for reproduction of the EC-associated disease. The experimental infection model reported here provides the basis for further research on the EC pathogenesis and possible prevention and intervention strategies.

Partridge and embryonated partridge egg as new preclinical models for candidiasis

Candida albicans (C. albicans) is the most common cause of candidiasis in humans and animals. This study was established to a new experimental infection model for systemic candidiasis using partridge and embryonated partridge egg. First, we tested the induction of systemic candidiasis in partridge and embryonated partridge egg. Finally, interaction between virulence factors of C. albicans and Bcl-2 family members was predicted. We observed that embryonic infection causes a decrease in survival time and at later embryonic days (11–12th), embryos showed lesions. Morphometric analysis of the extra-embryonic membrane (EEM) vasculature showed that vascular apoptotic effect of C. albicans was revealed by a significant reduction in capillary area. In immunohistochemistry assay, low expression of Bcl-2 and increased expression of Bax confirmed apoptosis. The gene expression of Bax and Bcl-2 was also altered in fungi-exposed EEM. Ourin silico simulation has shown an accurate interaction between aspartic proteinase, polyamine oxidase, Bcl-2 and BAX. We observed that the disease was associated with adverse consequences, which were similar to human candidiasis. Acquired results support the idea that partridge and embryonated partridge egg can be utilized as appropriate preclinical models to investigate the pathological effects of candidiasis.

Whip-LAMP: a novel LAMP assay for the detection of Trichuris muris-derived DNA in stool and urine samples in a murine experimental infection model

Background Trichuris trichiura (human whipworm) infects an estimated 477 million individuals worldwide. In addition to T. trichiura, other Trichuris species can cause an uncommon zoonosis and a number of human cases have been reported. The diagnosis of trichuriasis has relied traditionally on microscopy. Recently, there is an effort to use molecular diagnostic methods, mainly qPCR. LAMP technology could be an alternative for qPCR especially in low-income endemic areas. Trichuris muris, the causative agent of trichuriasis in mice, is of great importance as a model for human trichuriasis. Here, we evaluate the diagnostic utility of a new LAMP assay in an active experimental mouse trichuriasis in parallel with parasitological method by using stool and, for the first time, urine samples. Methods Stool and urine samples were collected from mice infected with eggs of T. muris. The dynamics of infection was determined by counting the number of eggs per gram of faeces. A LAMP based on the 18S rRNA gene from T. muris was designed. Sensitivity and specificity of LAMP was tested and compared with PCR. Stool and urine samples were analysed by both LAMP and PCR techniques. Results Trichuris muris eggs were detected for the first time in faeces 35 days post-infection. LAMP resulted specific and no cross-reactions were found when using 18 DNA samples from different parasites. The detection limit of the LAMP assay was 2 pg of T. muris DNA. When testing stool samples by LAMP we obtained positive results on day 35 p.i. and urine samples showed amplification results on day 20 p.i., i.e. 15 days before the onset of T. muris eggs in faeces. Conclusions To the best of our knowledge, we report, for the first time, a novel LAMP assay (Whip-LAMP) for sensitive detection of T. muris DNA in both stool and urine samples in a well-established mice experimental infection model. Considering the advantages of urine in molecular diagnosis in comparison to stool samples, should make us consider the possibility of starting the use urine specimens in molecular diagnosis and for field-based studies of human trichuriasis where possible. Further studies with clinical samples are still needed.

Direct evidence of preventive effect of severe diarrhea in calf by milk replacer-based probiotic feeding

Background Diarrhea is one of the major causes of death in calves directly linked to economic loss in cattle industry. Fermented milk has been widely used for calf feeding to reduce the diarrhea in clinical settings. However, the fermentation of raw milk has many difficulties, such as unstable fermentation due to large variances of nutrient composition in raw milk and contamination with coliform. Practical use of fermented milk replacer (FMR) has a strong potential to overcome these difficulties. However, the clinical efficacy of FMR on calf diarrhea is still unclear. Result In this study, we developed a stable condition of fermentation for FMR and verified the preventive effects of FMR feeding on calf diarrhea by an experimental infection model of bovine rotavirus (BRV) in newborn calf and a field study in dairy farms with calf diarrhea. Additionally, the clinical efficacy of lactic acid bacteria (LAB)-supplemented milk replacer (LAB-MR) was also evaluated in the experimental infection model. In the experimental infection model, control calves fed a milk replacer showed severe watery diarrhea after BRV challenge. Two out of three control calves were died after rapid decline of milk intake. In contrast, calves fed FMR or high-concentrated LAB-MR showed diarrhea, but the water content of feces was low and stable. In addition, the amount of milk intake decreased temporarily, but recovered immediately in the FMR- or LAB-MR-fed calves. Histopathological analysis of intestinal tracts revealed mucosal lesions were slighter in the FMR- or LAB-MR-fed calves than in the control calves. In a field study of FMR feeding in dairy farms with mixed infection with BRV and Cryptosporidium parvum, the incidence of enteritis and mortality of calf by enteritis were significantly reduced by the allocation of FMR. Additionally, the FMR-fed calves exhibited shorter duration of treatment, fewer consultations, and lower cost of medical care for enteritis compared with control calves. Conclusion These results suggest that feeding of milk replacer-based probiotics to calves reduces severity of diarrhea and tissue damage to intestinal tracts caused by BRV infection and provides significant clinical benefits for the prevention and treatment of calf diarrhea.