Investigation of Trehalose Supplementation Impacting Campylobacter jejuni and Clostridium perfringens from Broiler Farming.

In 2006, the European Commission banned the use of antibiotic promoters in animal feed. However, there is a new situation in poultry disease where it is necessary to study feed additives, which can overcome the diseases that were previously controlled through the addition of antibiotics and antimicrobial growth promoters in the feed. Therefore, trehalose was investigated to determine whether it impacts the growth performance and pathogenic bacteria (C. jejuni and C. perfringens) inoculation in broilers. In the first experiment, the tolerance of broilers to the addition of trehalose to their feed was investigated. There was no significant difference (p > 0.05) in body weight changes, daily weight gain, feed intake or feed conversion ratio during the feeding period. Within a 35-day feeding period, it was concluded that a trehalose dosage up to 10% does not exert a negative effect on broiler farming. Moreover, there was no significant difference (p > 0.05) in the broilers' growth performance, as well as C. jejuni and C. perfringens counts in the intestines and feces of broilers observed over a 5-week feeding period. However, Lactobacillus counts significantly increased in these groups with 3% and 5% trehalose supplementation. The findings indicate that trehalose supplementation in the feed cannot directly decrease C. jejuni and C. perfringens counts but may enhance gut health by raising Lactobacillus counts in chicken gut, particularly when enteropathogenic bacteria are present.

Incidence and Antimicrobial Susceptibility to Clostridium perfringens in Premarket Broilers in Taiwan.

Clostridium perfringens infection causes subclinical and clinical necrotic enteritis in poultry flocks, and it is estimated to result in US$2 billion of losses worldwide every year. The aims of this study were to determine the incidence, toxin types, and antimicrobial resistance levels to C. perfringens isolated from premarket, 5-wk-old, clinically healthy broiler chickens in Taiwan, and to examine the relationships between intestinal lesions and the numbers of C. perfringens in intestinal contents. In total, 435 samples of chicken ileum contents were collected from 98 broiler farms during June 2012 to February 2013. The C. perfringens isolation rate was 9.9% (43/435). The positive rate of tested farms was 29.6% (29/98). All the isolates were C. perfringens type A, only possessing the cpa gene encoding for toxin α. No netB gene encoding NetB toxin associated with necrotic enteritis, and no cpe gene encoding for the C. perfringens enterotoxin causing human intestinal disorder were detected. A quantitative PCR analysis revealed that the mean C. perfringens number in the intestinal contents was 3.9 × 10(6) colony-forming units (CFU)/g, ranging from 6.85 × 10(2) to 1.61 × 10(7) CFU/g. The gross and histopathologic lesions revealed a positive correlation (p < 0.05) between lesion score and C. perfringens number in the ilea of C. perfringens -positive chickens. Antimicrobial susceptibility tests of all C. perfringens isolates indicated that the minimum inhibitory concentration inhibiting 50% of isolates (MIC50) for amoxicillin, bacitracin, chlortetracycline, enrofloxacin, erythromycin, florfenicol, and lincomycin was ≤0.125, 0.5, 128, 0.25, ≥256, 2, and ≥256 µg/ml, respectively. Most of the C. perfringens isolates were susceptible to amoxicillin, bacitracin, and enrofloxacin but resistant to chlortetracycline, erythromycin, and lincomycin. Interestingly, C. perfringens isolated from chickens with severe lesions had higher MIC50 for erythromycin and lincomycin than those isolates from chickens with mild lesions. Conclusively, reductions in both the incidence of C. perfringens infection on farms and the concentrations of C. perfringens in intestines to improve broiler health are still needed in Taiwan.

The impact of Salmonella Enteritidis on lipid accumulation in chicken hepatocytes.

Salmonella enterica serovar Enteritidis (SE) is a public health concern and infected chickens serve as a reservoir that potentially transmits to humans through food. Although SE seldom causes systemic disease in chickens, virulent SE strains can colonize in intestines and lead a persistent infection of the liver. The liver is the primary organ for lipid metabolism in chickens and the site for production and assembly of main components in yolk. We performed a time-course experiment using LMH-2A cells that were infected with SE and co-incubated with ß-oestradiol to evaluate if SE infection affected lipid metabolism and subsequently changed lipoprotein formation for egg yolk. The results indicated that lipid accumulation significantly increased in infected LMH-2A cells while the viability of these cells was only slightly decreased. The mRNA expressions of lipid transportation and most lipogenetic genes including sterol regulatory element binding protein 1, acetyl-CoA carboxylase, fatty-acid synthase, long-chain-fatty-acid-CoA ligase 1, peroxisome proliferator-activated receptor-γ, and very-low-density lipoproteins (VLDLs) II were significantly up-regulated while the expression of lipogenetic-related stearoyl-CoA denaturase 1 was down-regulated. Moreover, decline in lipid transportation of hepatocytes was evidenced by the down-regulation of oestrogen receptor α which promotes VLDLy formation, an increase of intra-cellular accumulation of Apoprotein B (ApoB) protein, and a decrease of cellular excretion of VLDL protein. Conclusively, SE infection could elevate lipid synthesis and reduce lipid transportation in the chicken hepatocytes. These changes may lead excessive lipid accumulation in liver and slower lipoprotein deposition in yolk.

Prevalence and Persistence of Listeria monocytogenes in Ready-to-Eat Tilapia Sashimi Processing Plants.

A 2-year study was performed at two ready-to-eat tilapia sashimi processing plants (A and B) to identify possible routes of contamination with Listeria monocytogenes during processing. Samples were collected from the aquaculture environments, transportation tanks, processing plants, and final products. Seventy-nine L. monocytogenes isolates were found in the processing environments and final products; 3.96% (50 of 1,264 samples) and 3.86% (29 of 752 samples) of the samples from plants A and B, respectively, were positive for L. monocytogenes . No L. monocytogenes was detected in the aquaculture environments or transportation tanks. The predominant L. monocytogenes serotypes were 1/2b (55.70%) and 4b (37.97%); serotypes 3b and 4e were detected at much lower percentages. At both plants, most processing sections were contaminated with L. monocytogenes before the start of processing, which indicated that the cleaning and sanitizing methods did not achieve adequate pathogen removal. Eleven seropulsotypes were revealed by pulsed-field gel electrophoresis and serotyping. Analysis of seropulsotype distribution revealed that the contamination was disseminated by the processing work; the same seropulsotypes were repeatedly found along the work flow line and in the final products. Specific seropulsotypes were persistently found during different sampling periods, which suggests that the sanitation procedures or equipment used at these plants were inadequate. Plant staff should improve the sanitation procedures and equipment to reduce the risk of L. monocytogenes cross-contamination and ensure the safety of ready-to-eat tilapia products.

Residue Depletion Study of Danofloxacin in Cultured Tilapia (Oreochromis mossambicus).

Danofloxacin is an antibacterial drug of the fluoroquinolone group developed for therapeutic purposes in veterinary medicine. The studies described here include investigations of the residues following a single dose or multiple doses of danofloxacin. Residue depletion studies were performed to determine residues in plasma and tissues of saltwater tilapia fish (Oreochromis mossambicus) after a single oral administration of danofloxacin at the dose of 10 mg/kg body weight and also after daily dose of 10 mg/kg body weight for five consecutive days. Danofloxacin residues were analyzed by HPLC with fluorescence detection. Following a single oral dose, danofloxacin residues in 6 h postdosing tilapia were at a maximum of 1.44, 12.48, and 13.18 µg/g in serum, liver, and kidney samples, respectively, while a peak muscle concentration of 2.15 µg/g was reached at 12 h. From single-dose data, the concentration of danofloxacin in serum, muscle, liver, and kidney samples declined with half-lives of 29, 34, 49, and 44 h, respectively. Based on the maximum residue level (MRL) of 0.1 µg/g in edible tissue for fin fish, the withdrawal times of danofloxacin in muscle were estimated to fall below the MRL after a withdrawal period of 21 days following the multiple-dose administration. These results may be helpful to regulatory agencies as they determine what tissues should be monitored to ensure that the established residue safety tolerance levels are not exceeded.

Salmonella Enteritidis infection slows steroidogenesis and impedes cell growth in hen granulosa cells.

Infection by Salmonella Enteritidis (SE) causes decreased egg production in laying hens. Immunoresponse, steroidogenesis, and cell proliferation by chicken granulosa cells (cGCs) are of particular interest because these changes are involved in follicular growth, atresia, and ovulation. To elucidate the possible mechanisms underlying these changes, transcriptional alterations in cGCs at distinct stages of follicular maturity were studied. Luteinizing hormone (LH)-and follicle-stimulating hormone (FSH) were applied to the cGCs isolated from hierarchical and prehierarchical follicles, respectively, to imitate the effects of gonadotropin during in vitro SE infection. Results showed that the expression of Toll-like receptor 15 was dependent on the follicular maturity, with mature cells having a more significant and progressively stronger immunoresponse. Attenuated responses to LH and FSH as well as retardant steroidogenesis due to down-regulated LH receptor, FSH receptor, and the P450 side-chain cleavage system were observed and may have led to delayed hierarchical follicular growth. Deteriorated cell viability of prehierarchical follicles may occur, as the proliferation of stimulator heparin-binding epidermal growth factor was reduced significantly. Furthermore, the infection led to a higher probability of cGCs from the smaller follicles undergoing apoptosis than those from F1 follicles. Collectively, the data provide evidence of a tendency toward pathogen elimination in F1 follicles by induction of a strong immune response and cell apoptosis in smaller follicles to avoid bacterial transovarian infection. It is our speculation that slowed steroidogenesis and impeded follicular growth may play essential roles in decreased ovulation rate as well as further decreased egg production during SE infection.