Survival of avian influenza and Newcastle disease viruses in compost and at ambient temperatures based on virus isolation and real-time reverse transcriptase PCR.

In four composting experiments, survival of avian influenza (AI) and Newcastle disease (ND) viruses was assessed by virus isolation in embryonated chicken eggs (ECEs) and by real-time reverse transcriptase-polymerase chain reaction. Specimens contained in nylon mesh bags consisted of 20-g samples of chicken manure, used litter, or feed that had been inoculated with allantoic fluid containing an AI virus (H6N2, Expt. 1) or an ND vaccine virus (Expt. 2). Other specimens consisted of 20-g samples of infected ECEs that had been homogenized and mixed with corn silage. As a control, allantoic fluid diluted in phosphate-buffered saline was contained in sealed vials. Except for the feed, in which the AI virus was inactivated soon after the specimen was inoculated, on day 0 the specimens buried in compost or placed outside at ambient temperatures contained at least 5.0 log10 of virus and 7.7 log10 of viral RNA. By day 7, temperatures in compost ranged from 50 C to 65 C, and viruses had been killed in all specimens in bags. In comparison, viruses in sealed vials remained viable to day 10. Viral RNA in mesh-bag specimens had been degraded to nondetectable levels by day 10, but it was still detected in sealed vials on day 21. In specimens that were held at ambient temperatures (13 C-28 C), the viruses in mesh-bag specimens were inactivated by day 21, but their RNA was still detected. In comparison, the viruses in sealed vials survived to day 21. In Expts. 3 and 4, viruses were inactivated in carcass specimens and in whole ECEs during composting. In an in vitro experiment, the time required for a 1-log10 reduction of viruses was significantly shorter (P < 0.05) in water extracts from compost than in phosphate buffers at temperatures of 25 C to 45 C. This study provided evidence that microbial activity during composting contributed to the rapid killing of AI and ND viruses and to the degradation of their viral RNA.

Development of methods for detection and quantification of avian influenza and Newcastle disease viruses in compost by real-time reverse transcription polymerase chain reaction and virus isolation.

Composting has been used for disposal of poultry carcasses and manure following outbreaks caused by avian influenza virus (AIV) and Newcastle disease virus (NDV), but methods are needed to test for survival of the viruses in compost to ensure biosecurity. Methods developed in the present study include extracting viruses from compost and purifying viral RNA. The extracted viruses were detected by virus isolation using embryonated chicken eggs, and the purified RNA was detected by real-time reverse transcription PCR (RRT-PCR). The virus isolation and the RRT-PCR methods were evaluated with 3 compost preparations that were produced from chicken manure mixed with corn silage, wood shavings, or wheat straw. The detection limits of both methods were 1,700 and 1,000 embryo lethal doses of AIV and NDV per gram of compost, respectively. The copy number of viral RNA quantified by RRT-PCR was highly correlated with the amount of virus in compost. The results suggested that the RRT-PCR method may be used as an alternative to the virus isolation method for rapid detection and accurate quantification of AIV and NDV in compost.

The effect of hen-egg antibodies on Clostridium perfringens colonization in the gastrointestinal tract of broiler chickens.

We evaluated the ability of hen-egg antibodies (HEA) to reduce intestinal colonization by Clostridium perfringens in broiler chickens. Antibodies against C. perfringens or cholera toxin (negative control) were obtained from the eggs of laying hens hyperimmunized using a C. perfringens bacterin or cholera toxin. Eggs were collected, pooled, and egg antibodies were concentrated by polyethylene-glycol precipitation. An initial experiment was conducted to determine the in vivo activity of the administered antibody along the length of the intestine. Thereafter, two feeding trials were performed to assess the efficacy of feed amended with the egg antibodies in reducing the level of colonization of C. perfringens in challenged birds. Antibody activity declined from proximal to distal regions of the intestine but remained detectable in the cecum. In the first experiment there was no significant reduction in the number of C. perfringens in the birds fed the diet amended with the anti-C. perfringens egg antibody, compared to the birds that received the anti-cholera toxin egg antibody (n=10), at any of the sampling times. In the second experiment there was a significant decrease in C. perfringens intestinal populations 72 h after treatment (n=15) as assessed by culture-based enumeration, but there was no decrease as measured by quantitative PCR based on the C. perfringens phospholipase C gene. Intestinal-lesion scores were higher in the birds that received the anti-C. perfringens HEA. Our work suggests that administration of HEA did not reduce the level of C. perfringens intestinal colonization and conversely might exacerbate necrotic enteritis.

Dietary glycine concentration affects intestinal Clostridium perfringens and lactobacilli populations in broiler chickens.

Previous studies have reported that intestinal populations of Clostridium perfringens, the causative agent of necrotic enteritis (NE), are correlated with diets high in glycine. To establish a direct causative link, 3 trials were conducted to examine the effect of dietary glycine levels on gut populations of C. perfringens, alpha-toxin production, and NE lesion scores in broiler chickens. In trials 1 and 2, 12 groups of 4 birds were fed 4 different ideal protein-balanced diets formulated to contain 0.75, 1.58, 3.04, or 4.21% glycine from d 14 to 28 of age. In trial 3, 24 groups of 4 birds were given 6 different ideal protein-balanced diets formulated to contain 0.50, 0.75, 1.00, 1.50, 2.00, or 4.00% glycine. All birds were orally challenged with a broth culture of C. perfringens type A on d 1 and between d 14 and 21 of age and killed on d 28. The majority of birds showed clinical signs of NE with 4.16 to 8.33% mortality in the 3 trials. The highest mortality and intestinal lesion scores were observed in chickens receiving 3.04% glycine in trials 1 and 2, and 4.00% glycine in trial 3. Clostridium perfringens populations in the cecum varied quadratically with increasing dietary glycine, with the maximal response seen at 3.30,3.89, and 3.51% dietary glycine in trials 1, 2, and 3, respectively. Numbers of lactobacilli in cecum declined significantly (P < 0.05) with increasing levels of glycine. The results suggest that dietary glycine level has a significant effect on C. perfringens and lactobacilli populations and may be a predisposing factor for NE in broiler chickens.

Germ theory vs. community theory in understanding and controlling the proliferation of biofilms.

Germ theory and pure culture methods have provided invaluable information concerning the role of bacteria in diseases resulting from a single organism which bypasses a host's defenses. However, they do not provide sufficient information concerning the synergisms which allow the members of biofilm communities to proliferate more effectively as communities rather than as individuals. The mechanisms of these synergies are potential targets for antimicrobial agents as well as potential mechanisms of resistance to antimicrobial agents. Understanding community-level phenomena in oral biology requires the culture, identification, and classification of functional plaque communities as well as new methods of identifying and quantifying communal relationships. Cultured biofilm communities also provide ideal models of bacterial self-organization in which information related to adaptive strategies arises not only through the recombination of genes within genomes, but also through the recombination of organisms within communities.