Optimized culturing and nucleic acid-based methods for the detection of Salmonella enterica in poultry environments.

The main objective of this trial was to set up a series of assays following quantified inoculation of Salmonella samples in 2 initial enrichment methods to ultimately determine the most effective and fastest detection method for recovery of Salmonella in a poultry environment matrix. Samples were randomly split into 2 different containers containing either buffered peptone water (BPW) + yeast extract, or tetrathionate broth (TT) with added iodine and Brilliant Green solution 0.1%. A frozen stock Salmonella culture was thawed and serially diluted 10-fold to inoculate 100 µL of the dilution into the enriched samples. The samples were incubated at 42 and 37°C, respectively, for 24 h and secondarily enriched in modified semi-solid Rappaport Vassiliadis (MSRV) incubated at 42°C. All samples then were reincubated under the same conditions. After secondary enrichment, the samples were streaked onto Chromogenic agar/ XLT4 bi-plates and incubated under the same conditions. After initial inoculation and each 24-h incubation, a portion of the enriched samples was analyzed using a real-time PCR assay. The results of this trial indicate that recovery of Salmonella in a culture-based assay may be enhanced by up to 3 logs by using the TT as the initial enrichment media compared with BPW. The incorporation of MSRV as a secondary cultural selective media after the TT gave the best recovery of Salmonella. These data indicate that considerable time can be saved by using TT as an initial media for Salmonella recovery.

Detection of Salmonella spp. survival and virulence in poultry feed by targeting the hilA gene.

AIMS: The objectives of this work were to evaluate immunomagnetic beads and a reverse transcriptase (RT)-PCR method for the detection of Salmonella inoculated into feed. In addition, a reverse transcriptase (RT)-PCR method was evaluated for quantifying virulence gene hilA expression of Salmonella ssp. in poultry feed matrices and utilized to determine the influence of poultry feed environmental factors on Salmonella hilA expression. METHODS AND RESULTS: An immunomagnetic separation technique was evaluated for increased recovery of Salmonella from feed. Salmonella cultures were inoculated into feed samples and exposed to heat treatments of 70°C and sampled periodically. From these samples, RNA was collected and hilA gene expression was measured relative to the housekeeping 16S rRNA gene. The immunomagnetic bead protocol increased recovery by 1 log. The up-regulation of hilA was demonstrated after 5 and 10 min of inoculated feed samples being exposed to heat treatment. CONCLUSIONS: From this work, the data indicate that the ability to detect live Salmonella cells in feed samples may be increased by targeting the hilA gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Foodborne salmonellosis originating from poultry is a major problem, and feed is a leading source of contamination in poultry, but detection in feed is complicated by low concentrations. The assays and experiments in this study examine possible improvements to recovery and detection of Salmonella in feed.

Campylobacter biofilm phenotype exhibits reduced colonization potential in young chickens and altered in vitro virulence.

In this study, we evaluated the ability of different Campylobacter phenotypes (biofilm versus planktonic) to colonize young poultry. It has been suggested that a persistent Campylobacter biofilm reservoir may be involved in the initial contamination of poultry flocks. Campylobacter jejuni cultured adherent to agar was utilized as the biofilm model and C. jejuni cultured in broth was evaluated as the planktonic model. In 2 independent trials, 1-d-old broiler chicks were given 1 of 3 treatments: 1) 10(5) cfu.mL(-1) of C. jejuni cultured in broth, 2) 10(5) cfu.mL(-1) of C. jejuni cultured adherent to agar, or 3) no C. jejuni (negative control). Cecal contents of all birds were evaluated by culturing 12 d after the initial challenge with C. jejuni. In both trials, birds challenged with C. jejuni cultured in broth had approximately 3 to 4 log higher cecal Campylobacter concentration than birds challenged with C. jejuni cultured adherent to agar. Using 2 cell lines (INT 407 and DF1), virulence of C. jejuni cultured in broth versus adherent to agar also was evaluated by challenging monolayers of eukaryotic cells with 1 of 3 treatments: 1) 10(5) cfu.mL(-1) of C. jejuni cultured in broth, 2) 10(5) cfu.mL(-1) of C. jejuni cultured adherent to agar, or 3) no C. jejuni (negative control). The virulence study also showed differences of C. jejuni cultured in broth or agar in attachment and invasion abilities to tissue culture cells, but differences were not as consistent as with the chick colonization study. This study indicates that phenotype may play a role in colonization of chickens and virulence by C. jejuni.

Campylobacter jejuni as a secondary colonizer of poultry biofilms.

AIMS: The objective of this study was to determine if survival of culturable Campylobacter jejuni outside the host was increased by entrapment in pre-established biofilms. METHODS AND RESULTS: Campylobacter jejuni was inoculated into four biofilm populations isolated from poultry environments and cultured at three temperatures. Survival of culturable Camp. jejuni in some pre-established biofilms was extended vs survival of culturable Camp. jejuni in broth. But some biofilms were detrimental to survival of culturable Camp. jejuni. Denaturing gradient gel electrophoresis analysis indicated differences in bacterial profiles depending on initial source and temperature of culturing, which may have had impacts on survival of culturable Camp. jejuni. Further investigation showed no evidence of interspecies cell signalling indicating that secondary colonization was only physical. CONCLUSIONS: The results of this study show Camp. jejuni's attachment to surfaces is facilitated by pre-established biofilms and survival of culturable Camp. jejuni may be extended in some pre-established biofilms, but these biofilms do not fully explain long-term survival of culturable Camp. jejuni outside hosts. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information concerning survival of culturable Camp. jejuni outside the host and shows biofilms may be important in transmission and prevalence of Camp. jejuni.

[Experiences with early weaning of calves in Central America]. / Experiencias sobre destete precoz de terneros en Centroamérica.

This study presents a review of traditional feed calves systems used in Central American specialized dairy farms. The systems discussed are considered costly, due to the high quantity of milk and concentrate required, besides causing shortage of milk for human consumption. The early weaning calf system developed by INCAP is also discussed, as well as its implementation with slight modifications oriented towards the application of research findings to achieve a reduction of the milk volume commonly used in the traditional rearing system. The development of appropriate concentrate formulations for animals of that age, using local feed ingredients, is also presented.

[Effects of the treatment of coffee pulp, fresh or ensilaged, with calcium hydroxide, on its nutritive value]. / Efectos del tratamiento de la pulpa de café, fresca o ensilada, con hidróxido de calcio, sobre su valor nutritivo.

This study was carried out to determine the effects of the addition of calcium hydroxide on the chemical composition and nutritive value of fresh or ensilaged coffee pulp. Fresh or ensilaged pulp were mixed with 1, 2 and 3% of calcium hydroxide. The process was carried out during 0 and 16 hr, after which time the treated pulp was sun-dried for 36 hr until moisture content reached 12%. These samples were then analyzed for their proximate chemical composition and for some minerals (Ca, P, Na, K), as well as for caffeine, tannins and chlorogenic and caffeic acids content. Diets were then prepared from these materials, containing 15% protein and 15 or 30% fresh or ensilaged coffee pulp, and offered to weanling rats during six weeks. Information required on weight gain, food conversion, apparent digestibility and toxicity of the diets was recorded. Results of the chemical analysis revealed that the main changes found in both types of pulp as a result of the calcium hydroxide treatment were the following: a decrease in ether extract (from 4.0 to 2.5 g/100 g), crude fiber (from 18.3 to 11.9 g/100 g) and protein content (from 12.3 to 8.6 g/100 g) in an inverse relation to the amount of calcium hydroxide used. The amount of ash increased, fluctuated between 5.5 and 15.4%, depending on the amount of calcium hydroxide used. The latter affected the Ca:P ratio in the diets, where an average ratio of 7.2:1 was found in the control pulp (0% calcium hydroxide) and 59.0:1 in those treated with the highest amount of calcium hydroxide (3%). Regarding the caffeine, tannins and chlorogenic and caffeic acids contents, calcium hydroxide was effective in decreasing only tannins, more so in the fresh than in the ensilaged pulp; the decrease was in direct proportion to the amount of calcium hydroxide added and to the length of the Ca(OH)2 treatment. The results of the biological assays showed that the addition of Ca(OH)2 in either of the two time periods used and at either of the concentrations studied, did not improve the nutritive value of coffee pulp. There was always a better performance in the animals that consumed ensilaged pulp than in those fed fresh pulp. The animals fed 15% coffee pulp either fresh or ensilaged performed better than those consuming 30% coffee pulp.(ABSTRACT TRUNCATED AT 400 WORDS)

[Preliminary trial of the cultivation of winged beans (Psophocarpus tetragonolobus) in Guatemala and variations in their chemical composition]. / Prueba preliminar del cultivo de frijol alado (Psophocarpus tetragonolobus) en Guatemala y variación en composición química.

There is no reliable information concerning the cultivation practices of winged bean cultivars in the area; therefore, it was considered of interest to participate in the First International Winged Bean Trial, which was peformed according to a standardized procedure. The planting site was located at 90 degrees longitude and 15 degrees latitude, and 250 meters above sea level, in a hot-humid environment. Sowing was performed in the middle of November, under continuous irrigation until April. During the growth period no diseases or major pests were observed. At the initiation of harvesting in February, there were differences among varieties regarding time to reach maturity. The most promising varieties were Thailand, TPT 1, UPS 121 and Indonesia 1 with respect to seed yield. The highest protein value, 36.5%, corresponded to TPT 1, and the lowest value, 31.7% to UPS 62. Oil content in seeds of all varieties ranged from 14.0% to 19.2%. The five higher protein content varieties were analyzed for their amino acid content, and all varieties for their trypsin inhibitor activity. Leaves, roots and threshed mature pods were also analyzed for their protein content.