In silico prediction and expression analysis of vaccine candidate genes of Campylobacter jejuni.

Campylobacter jejuni (C. jejuni) is the most common food-borne pathogen that causes human gastroenteritis in the United States. Consumption of contaminated poultry products is considered as the major source of human Campylobacter infection. An effective vaccine would be a promising alternative to antibiotic supplements to curb C. jejuni colonization in poultry gastrointestinal (GI) tract. However, the genetic diversity among the C. jejuni isolates makes vaccine production more challenging. Despite many attempts, an effective Campylobacter vaccine is not yet available. This study aimed to identify suitable candidates to develop a subunit vaccine against C. jejuni, which could reduce colonization in the GI tract of the poultry. In the current study, 4 C. jejuni strains were isolated from retail chicken meat and poultry litter samples and their genomes were sequenced utilizing next-generation sequencing technology. The genomic sequences of C. jejuni strains were screened to identify potential antigens utilizing the reverse vaccinology approach. In silico genome analysis predicted 3 conserved potential vaccine candidates (phospholipase A [PldA], TonB dependent vitamin B12 transporter [BtuB], and cytolethal distending toxin subunit B [CdtB]) suitable for the development of a vaccine. Furthermore, the expression of predicted genes during host-pathogen interaction was analyzed by an infection study using an avian macrophage-like immortalized cell line (HD11). The HD11 was infected with C. jejuni strains, and the RT-qPCR assay was performed to determine the expression of the predicted genes. The expression difference was analyzed using ΔΔCt methods. The results indicate that all 3 predicted genes, PldA, BtuB, and CdtB, were upregulated in 4 tested C. jejuni strains irrespective of their sources of isolation. In conclusion, in silico prediction and gene expression analysis during host-pathogen interactions identified 3 potential vaccine candidates for C. jejuni.

Riboflavin and Bacillus subtilis effects on growth performance and woody-breast of Ross 708 broilers with or without Eimeria spp. challenge.

This study was conducted to assess the effects of the dietary supplementation of riboflavin (as a bile salt hydrolase [BSH] inhibitor) and Bacillus subtilis on growth performance and woody breast of male broilers challenged with Eimeria spp. Intestinal bacteria, including supplemented probiotics, can produce BSH enzymes that deconjugate conjugated bile salts and reduce fat digestion. A 3 × 2 × 2 (riboflavin × Bacillus subtilis × Eimeria spp. challenge) factorial arrangement of treatments in randomized complete block design was used. On d 14, birds were gavaged with 20× doses of commercial cocci vaccine (CoccivacR-B52, Merck Animal Health, Omaha, NE). Dietary treatment of riboflavin and B. subtilis did not affect body weight (BW), body weight gain (BWG), and feed conversion (FCR) d 0 to 14 and overall d 0 to 41. Eimeria spp challenge reduced BWG, feed intake (FI), and increased FCR between d 14 to 28, but increased BWG and lowered FCR between d 28 to 35. There were no effects of the Eimeria spp. challenge on the overall d 0 to 41 FCR and FI, but BWG was reduced. Eimeria spp. challenge increased the abdominal fat pad weight and slight woody breast incidences on processed birds on d 42. Dietary inclusion of B. subtilis and riboflavin at tested levels did not help birds to mitigate the negative impact of Eimeria spp. challenge to enhance the growth performance.

Effects of riboflavin and Bacillus subtilis on internal organ development and intestinal health of Ross 708 male broilers with or without coccidial challenge.

In a companion study, we found that inclusion of different doses of riboflavin affected growth performance of Ross 708 male broilers' responses to coccidial challenge (by 5 Eimeria spp on day 14 of age) and dietary Bacillus subtilis (B. subtilis) supplementation. The current study was conducted to further test whether supplementation of B. subtilis and riboflavin will reduce negative impact and inflammation caused by Eimeria spp proliferation and help proper function of internal organs. A total of 1,248 Ross × Ross 708 male broiler chicks were randomly placed in 96 floor pens (8 blocks, 12 treatments). Treatments were arranged in a 3 (riboflavin) × 2 (B. subtilis) × 2 (Coccidial challenge) factorial arrangement in a randomized complete block design. Coccidial challenge reduced the weight of sampled birds on day 27 and day 36 and increased the relative weights of the internal organs of proventriculus, duodenum, jejunum, ileum, and spleen to BW on day 27, which may be because of inflammation caused by proliferation of Eimeria spp. The increased relative weights of duodenum, jejunum, ileum, and spleen on coccidial challenged birds were lost on day 36. Correlation analysis also indicated that the jejunum weight was positively related to villus height, Eimeria acervulina, and Eimeria maxima on day 27 but was not on day 36. The loss of the positive relationships may be because of recovery of the birds from coccidiosis on day 36. Even though the coccidial challenge and riboflavin interactively affected feed conversion ratio and BW gain and supplementation of dietary B. subtilis reduced mortality from day 35 to 42 in the companion study, the same response of internal organs was not observed in the current study. Coccidial challenge compromised development of internal organs of Ross 708 male broilers at an early age, but the negative effects subsided with age of birds rather than supplementation of riboflavin and B. subtilis at current tested levels under our experimental set up.