INDIGENOUS POULTRY FEED MICROFLORA RESPONSE TO ETHYL ALCOHOL AND BUFFERED PROPIONIC ACID ADDITION

The present study was designed to compare ethyl alcohol with buffered propionic acid feed treatment on the survival of indigenous poultry feed bacteria and fungi. The aerobic bacterial poultry feed populations were not substantially reduced by either ethyl alcohol or buffered propionic acid treatments. Likewise, indigenous poultry feed fungal populations also were not markedly reduced by buffered propionic acid treatment of the feed but fungal poultry feed populations exposed to ethyl alcohol treatments were significantly lower (P<0.05) than fungal populations recovered from either control and buffered propionic acid treated feeds. Ethyl alcohol treatment may have potential for reducing fungal contamination in poultry feed.     

SURVIVAL OF AN UNIRRADIATED SALMONELLA TYPHIMURIUM MARKER STRAIN INOCULATED IN POULTRY FEEDS AFTER IRRADIATION

The present study was designed to compare unirradiated Salmonella typhimurium survival during storage after inoculation in either irradiated or unirradiated poultry feed. The effects of irradiation (5 kGy) on the indigenous feed microflora and on the survival of marker strain of S. typhimurium contaminated after irradiation treatment were determined during 56 days of storage of either soybean meal (SBM) or meat and bone meal (MBM) based feeds. The initial aerobic bacterial populations were reduced more than 90% in both SBM (4.96 to 4.08 ± 0.03 log₁₀ CPU/g feed) and MBM (5.12 to 3.90 ± 0.03) by irradiation. Irradiation treatment reduced the average fungal counts during 56 days of storage in both SBM (4.24 to 2.74 ± 0.03) and MBM (4.38 to 2.15 ± 0.03) containing feeds. However, unirradiated S. typhimurium populations inoculated after irradiation of the feed were not different in either irradiated or nonirradiated SBM and MBM based feeds. Therefore, the differences in fungal versus bacterial sensitivity among the feed types and storage times suggests that gamma irradiation can alter the makeup of indigenous microbial populations in feed but this does not appear to have a discernible influence on subsequent survival of unirradiated S. typhimurium added as a dry inoculum after irradiation.     

EFFECTIVE METHOD FOR DRY INOCULATION of BACTERIAL CULTURES

An effective way for inoculation of bacteria into dry foods/ingredients that gives a uniform mixture was developed. In the first part of this experiment, Salmonella typhimurium was successfully inoculated into chalk. Chalk tubes were weighed then soaked in a broth with S. typhimurium and allowed to dry back to their original weight. the dried chalk was made into a powder form. A viable cell count of this chalk, using a selective media for S. typhimurium , showed that the organism survived the drying while entrapped in the chalk. the "charged" chalk was used in an experiment as a dry inoculum where it was mixed with a low-moisture poultry feed. In comparison to a liquid inoculum, the "charged" chalk was a superior way of inoculating into dry particles because it created a more homogenous mixture with the feed without altering any properties of the feed itself. the second part of this experiment entailed a shelf-life study of the "charged" chalk. the same procedures for inoculating chalk were done using ten different cultures including Bacillus cereus, Clostridium perfringens, Escherichia coli, Enterobacter aerogenes, Lactobacillus plantarum, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus , and Streptococcus faecalis. Data showed that the cultures are stable in the chalk for at least six months.     

Effect of the raw extract of Arthrinium strains (Hyphomycetes, Dematiaceae) on the growth of pathogenic bacteria in poultry feed

Poultry feed contains a significant reservoir of bacteria and is a possible source of Salmonella typhimurium, Staphylococcus aureus and Escherichia coli, which can potentially infect farm animals and humans. The objective of this study was to determine whether the extract obtained from the culture of some Arthrinium species was able to inhibit the growth of these bacteria. The results obtained showed that the raw extracts of Arthrinium aureum, Arthrinium serenensis and Arthrinium phaeospermum inhibited the growth of Escherichia coli in poultry feed. In some cases the percentage inhibition of the growth of Escherichia coli was > 80%. With the raw extract of Arthrinium in poultry feed, the rate of growth of S. typhimurium fell to between 50% and 80%. The raw extract of A. serenensis had the lowest inhibitory activity. S. aureus counts were not affected by any Arthrinium raw extracts.     

Survival of Escherichia coli O157:H7 and Salmonella typhimurium inoculated on chicken by aqueous chlorine dioxide treatment

Inactivation of Escherichia coli O157:H7 and Salmonella typhimurium was evaluated on inoculated chicken by aqueous chlorine dioxide (ClO2) treatment. Chicken samples were inoculated with 6-7 log CFU/g of Escherichia coli O157:H7 and Salmonella typhimurium, respectively. The chicken samples were then treated with 0, 50, and 100 ppm of ClO2 solution and stored at 4 +/- 1 degrees C. Aqueous ClO2 treatment decreased the populations of the pathogenic bacteria on the chicken breast and drumstick. In particular, 100 ppm ClO2 treatment on the chicken breast and drumstick reduced Escherichia coli O157:H7 and Salmonella typhimurium by 1.00-1.27 and 1.37-1.44 log CFU/g, respectively. Aqueous ClO2 treatment on the growth of the bacteria was continuously in effect during storage, resulting in the decrease of the populations of Escherichia coli O157:H7 and Salmonella typhimurium. These results suggest that aqueous ClO2 treatment should be useful in improving the microbial safety of chicken during storage.     

Comparative study of the effect of ferrocyanide and EDTA on the production of ethyl alcohol from molasses by Saccharomyces cerevisiae

The effects of potassium ferrocyanide and EDTA on ethyl alcohol production from molasses by Saccharomyces cerevisiae were investigated on simulated batch pilotplant-scale conditions for alcoholic fermentation of molasses. Ethyl alcohol production was more sensitive to ferrocyanide than to EDTA. When ferrocyanide was introduced into the cultures at the time of inoculation, there was stimulation of ethyl alcohol production, with 261 ppm ferrocyanide producing the maximum effect, which was 3.0% more than in control cultures. When added during the propagation of the yeast, ferrocyanide depressed ethyl alcohol production by 4.0% maximum whereas EDTA stimulated ethyl alcohol production by 2.0%. Addition of ferrocyanide during the fermentation stage produced no significant effect on alcohol production, whereas over a wide range of EDTA concentration there was a steady increase in alcohol yield.     

Litter and aerosol sampling of chicken houses for rapid detection of Salmonella typhimurium contamination using gene amplification

Rapid screening of poultry houses for contamination is critical for Salmonella control. Use of air filter sampling has great potential for efficient and reliable monitoring of Salmonella spp., as it could represent an entire poultry house and solve sample-size problems. Two sampling methods (litter and air filter) were compared for detection in four chicken pens inoculated with a S. typhimurium antibiotic resistant strain. Salmonella levels in both litter and air filter samples were determined by PCR amplification and by conventional enrichment. Although amplified DNA was not directly detected, amplified DNA could be detected using a dual probe hybridization sensor. The ratio of the positive samples to total samples determined by gene amplification was much lower than that obtained by conventional enrichments (29/128 versus 102/128 samples). However, the ratio obtained by gene amplification with air filter samples was greater than that with litter samples (26/64 versus 3/64). These results demonstrate that the air filter sampling method is an alternative method of Salmonella detection in poultry house using PCR gene amplification protocol. Journal of Industrial Microbiology & Biotechnology (2000) 24, 379–382. Received 12 August 1999/ Accepted in revised form 17 February 2000     

INFLUENCE OF OXIDATION-REDUCTION REDUCTANTS ON SALMONELLA TYPHIMURIUM GROWTH RATES AFTER INITIAL pH ADJUSTMENT AND ZINC COMPOUND ADDITION

The objective of this study was to examine the separate influence of acidic pH, reductants (cysteine and sulfide) and zinc compounds (Zn acetate and Zn sulfate) during anaerobic growth of a S. typhimurium poultry isolate in rich or minimal media. The anaerobic growth of a S. typhimurium poultry isolate in TSB medium was significantly inhibited by either acidic initial pH or higher concentrations of Zn acetate. S. typhimurium anaerobic growth in M9 minimal medium was significantly inhibited by either acidic pH or higher concentrations of Zn acetate or Zn sulfate. Most anaerobic growth rates of the S. typhimurium poultry isolate in acidic media and with higher concentrations of Zn acetate or Zn sulfate were less than 0.10 h^-1. The overall anaerobic growth rates of S. typhimurium were inhibited more in the presence (0.089 h^-1) of reductants than in the absence (0.102 h^-1) of reductants in M9 medium. Results in this study suggest that either increasing Zn concentration or decreasing initial pH can reduce growth rate of foodborne Salmonella under anaerobic growth conditions.     

Efficacy of a commercial polymerase chain reaction-based assay for detection of Salmonella spp. in animal feeds

Salmonellosis is a cyclic problem in the food industry, to which animal feed has been contributory. Current conventional methods of Salmonella spp. detection require 96 h for detection and confirmation. With modern and just-in-time production schedules, a 96-h hold represents a significant expense in storage and decontamination. The commercially available assay, 'BAX for Screening/Salmonella' (BAX), is based on the principle of the polymerase chain reaction and may represent a significant decrease in assay time. Seven fresh feed formulations, two fresh feed ingredients, seven stored feeds and two stored feed ingredients were artificially contaminated with a primary poultry isolate of Salmonella typhimurium and analysed by conventional and BAX methodology. The results of BAX agreed with conventional plating results for 16 of 18 samples spiked with 1200 cfu 10 g(-1) of feed and 13 of 18 samples spiked with 40 cfu 10 g(-1) of feed. Indigenous Salmonella spp. were detected in five of eight samples of poultry diets by conventional methods. With BAX, Salmonella spp. could not be detected in any of the samples after only 7 h of enrichment but could be detected in two dietary samples after 13 h of enrichment and four dietary samples after 24 h of enrichment. Specific sequences of salmonella DNA that were extracted from poultry diets could be detected with BAX.     

Salmonella typhimurium strains carrying independent mutations display similar virulence phenotypes yet are controlled by distinct host defense mechanisms

The outcome of Salmonella infection in the mammalian host favors whoever succeeds best in disturbing the equilibrium between coordinate expression of bacterial (virulence) genes and host defense mechanisms. Intracellular persistence in host cells is critical for pathogenesis and disease, because Salmonella typhimurium strains defective in this property are avirulent. We examined whether similar host defense mechanisms are required for growth control of two S. typhimurium mutant strains. Salmonella pathogenicity island 2 (SPI2) and virulence plasmid-cured Salmonella mutants display similar virulence phenotypes in immunocompetent mice, yet their gene loci participate in independent virulence strategies. We determined the role of TNF-alpha and IFN-gamma as well as different T cell populations in infection with these Salmonella strains. After systemic infection, IFN-gamma was essential for growth restriction of plasmid-cured S. typhimurium, while SPI2 mutant infections were controlled in the absence of IFN-gamma. TNFRp55-deficiency restored systemic virulence to both Salmonella mutants. After oral inoculation, control of plasmid-cured bacteria substantially relied on both IFN-gamma and TNF-alpha signaling while control of SPI2 mutants did not. However, for both mutants, ultimate clearance of bacteria from infected mice depended on alphabeta T cells.     

猪源乳酸杆菌对鼠伤寒沙门氏菌DT104在猪肠道上皮细胞IPEC-J2上粘附的影响

采用猪肠道上皮细胞株IPEC-J2体外培养模型,考察9株猪源乳酸杆菌对IPEC-J2细胞的粘附特性,以及对鼠伤寒沙门氏菌DT104粘附的竞争、排斥、置换和抗侵袭作用.结果显示,9株乳酸杆菌均能粘附IPEC-J2细胞,粘附率在0.1%～10%之间,具有菌株特异性和浓度效应.乳酸杆菌和沙门氏菌同时加入细胞培养,能竞争性抑制沙门氏菌的粘附,并具有浓度效应,高浓度(109 CFU/mL)添加K30、K67和K16时,抑制率可达80%以上.乳酸杆菌预处理细胞后再加入沙门氏菌,高浓度乳酸杆菌可降低沙门氏菌粘附率40%～70%,而中浓度(108 CFU/mL)乳酸杆菌能抑制23%～33%沙门氏菌对细胞的侵入.但是,只有高浓度添加乳酸杆菌能置换已经粘附的沙门氏菌,置换率在12%～84%之间.该结果为临床上筛选乳酸杆菌,有效防治猪沙门氏菌病提供了一条新的途径.     

The destruction of Salmonella typhimurium in chicken exudate by different freeze-thaw treatments

Antibacterial treatments for frozen poultry, including holding at -5°C and slow thawing at 4°C to which exponential phase cells of Salmonella typhimurium were susceptible, were found to be relatively ineffective against stationary phase cells. Exposure of the latter, however, to a pre-freezing triple stress treatment of cold-shock exposure at 5°C to a solution containing 5 mg/l of free available chlorine in 1% succinic acid (pH 2.5) for 20 min substantially lowered the resistance of the cells to subsequent freezing, storage and thawing in poultry flesh exudate. Cell survival was further decreased by storage of exudate at - 18°C for 28 d and this reduced the proportion of stationary phase cells to less than 1% of initial numbers, with a concomitant increase in sensitivity to deoxycholate. Such a combined pre-treatment may have practical potential for salmonella decontamination in the production of frozen poultry.     

Protection of chicks against environmental challenge with Salmonella enteritidis by 'competitive exclusion' and acid-treated feed

'Competitive exclusion' treatment protected chicks against an environmental challenge with Salmonella enteritidis phage type 4, whether or not feed containing antimicrobial organic acids was also used. Treatment reduced the incidence of Salmonella -positive caecal samples from 82 to 8%, with a corresponding reduction in liver infection from 52 to 13%. Although acid-treated feed had no influence on an environmental challenge with salmonellas, its compatibility with 'competitive exclusion' suggests an advantage in their combined use to maximize protection of poultry against Salmonella infection.     

Salmonella typhimurium poultry isolate growth response to propionic acid and sodium propionate under aerobic and anaerobic conditions

Propionic acid and its sodium salt have long been used as additives in poultry feed to reduce microbial populations, including Salmonella spp. Propionic acids in poultry feed may have a potential role in inhibiting growth of Salmonella in the chicken intestine. In this study, we determined growth response of a Salmonella typhimurium poultry isolate to propionic acid and sodium propionate under aerobic and anaerobic conditions. Growth rate consistently decreased with the addition of greater concentrations of either propionic acid or sodium propionate. The extent of growth inhibition was much greater with propionic acid than the sodium form. Media pH decreased only with addition of propionic acid. Growth inhibition was more effective under anaerobic growth conditions with either propionic acid or sodium propionate. When determined at the same pH level, growth rate was significantly lowered by addition of 25 mM of either propionate or sodium propionate alone, and also by the decrease in pH levels (P<0.05). These results showed that growth inhibition of S. typhimurium by propionic acid or sodium propionate is greatly enhanced by pH decrease, and to lesser extent by anaerobiosis. We also found that sodium propionate was more inhibitory for growth of S. typhimurium than propionic acid when compared at the same pH levels.     

Molecular Epidemiology of Nontyphoidal Salmonella in Poultry and Poultry Products in India: Implications for Human Health

Human infections with non-typhoidal Salmonella (NTS) serovars are increasingly becoming a threat to human health globally. While all motile Salmonellae have zoonotic potential, Salmonella Enteritidis and Salmonella Typhimurium are most commonly associated with human disease, for which poultry are a major source. Despite the increasing number of human NTS infections, the epidemiology of NTS in poultry in India has not been fully understood. Hence, as a first step, we carried out epidemiological analysis to establish the incidence of NTS in poultry to evaluate the risk to human health. A total of 1215 samples (including poultry meat, tissues, egg and environmental samples) were collected from 154 commercial layer farms from southern India and screened for NTS. Following identification by cultural and biochemical methods, Salmonella isolates were further characterized by multiplex PCR, allele-specific PCR, enterobacterial repetitive intergenic consensus (ERIC) PCR and pulse field gel electrophoresis (PFGE). In the present study, 21/1215 (1.73 %) samples tested positive for NTS. We found 12/392 (3.06 %) of tissue samples, 7/460 (1.52 %) of poultry products, and 2/363 (0.55 %) of environmental samples tested positive for NTS. All the Salmonella isolates were resistant to oxytetracycline, which is routinely used as poultry feed additive. The multiplex PCR results allowed 16/21 isolates to be classified as S. Typhimurium, and five isolates as S. Enteritidis. Of the five S. Enteritidis isolates, four were identified as group D Salmonella by allele-specific PCR. All of the isolates produced different banding patterns in ERIC PCR. Of the thirteen macro restriction profiles (MRPs) obtained by PFGE, MRP 6 was predominant which included 6 (21 %) isolates. In conclusion, the findings of the study revealed higher incidence of contamination of NTS Salmonella in poultry tissue and animal protein sources used for poultry. The results of the study warrants further investigation on different type of animal feed sources, food market chains, processing plants, live bird markets etc., to evaluate the risk factors, transmission and effective control measures of human Salmonella infection from poultry products.     

The destruction of Salmonella typhimurium in chicken exudate by different freeze-thaw treatments

Antibacterial treatments for frozen poultry, including holding at -5 degrees C and slow thawing at 4 degrees C to which exponential phase cells of Salmonella typhimurium were susceptible, were found to be relatively ineffective against stationary phase cells. Exposure of the latter, however, to a pre-freezing triple stress treatment of cold-shock exposure at 5 degrees C to a solution containing 5 mg/l of free available chlorine in 1% succinic acid (pH 2.5) for 20 min substantially lowered the resistance of the cells to subsequent freezing, storage and thawing in poultry flesh exudate. Cell survival was further decreased by storage of exudate at -18 degrees C for 28 d and this reduced the proportion of stationary phase cells to less than 1% of initial numbers, with a concomitant increase in sensitivity to deoxycholate. Such a combined pre-treatment may have practical potential for salmonella decontamination in the production of frozen poultry.     

Efficiency of various bacterial suspensions derived from cecal floras of conventional chickens in reducing the population level of Salmonella typhimurium in gnotobiotic mice and chicken intestines

The antagonistic effect exerted towards Salmonella typhimurium by the flora issued from conventional chickens was studied in gnotobiotic animals. In germfree chickens and mice inoculated with S. typhimurium, the highest bacterial counts were observed in ceca, and were not significantly different in either host. The protection afforded by the inoculation of cecal flora issued from a conventional chicken was more effective when this flora was inoculated first into germfree chickens than when it was given only after inoculation with S. typhimurium. Administration of a cecal flora from a 15-day-old chick to gnotobiotic mice and chicken resulted in the inhibition of a further intestinal colonization by S. typhimurium in both hosts. Sixteen strains were isolated among the predominant populations of the fecal flora from chicken flora recipient mice. Association of 14 strains of strictly anaerobic bacteria with 2 strains of Escherichia coli and Streptococcus faecium only decreased the number of S. typhimurium in the ileum of gnotobiotic mice, but not in their cecum. Anaerobe cultures were obtained from 10(-6) and 10(-8) dilutions prepared from the fecal flora of gnotobiotic recipient mice. Antagonistic bacteria were present only in cultures from the 10(-6) dilution. Cecal concentrations of volatile fatty acids were shown not to be the sole factor implicated in the antagonistic effect against S. typhimurium.     

Mutagens in the feces of 3 South-African populations at different levels of risk for colon cancer.

The incidence of mutagens in the feces of 3 South-African populations at different risk levels for colon cancer has been determined. Lyophilized fecal samples were extracted with ether and the mutagenicity of the extracts determined using the Salmonella/mammalian microsome mutagenicity test. 19% of the samples from urban white South-Africans, a population at a high risk for colon cancer, were mutagenic using Salmonella typhimurium strain TA100. This incidence was significantly greater (p less than 0.001) than the incidence of mutagen excretion in the low-risk populations of urban blacks (2%) and rural blacks (0%). This pattern was also obtained using Salmonella typhimurium strain TA98. The incidence of mutagen excretion for urban whites was 10%, as compared to 5% and 2% for urban and rural blacks, respectively.     

Continous ethyl acetate production by Kluyveromyces fragilis on whey permeate

The synthesis of ethyl acetate by Kluyveromyces fragilis on diluted whey permeate was studied. Ethanol, lactose and O₂ are the direct precursors for ethyl acetate synthesis by this yeast. Ethyl acetate production is affected by many parameters, particularly the carbon/nitrogen (C/N) ratio, Tween 80 and iron. Ethyl acetate synthesis is optimum for C/N = 45. Tween 80 lowered slightly the level of ethyl acetate whereas iron completely stopped ethyl acetate production. The level of ethanol in the feed, the dissolved O₂ (DO) and dilution rate (D) were also optimised. Thus at D = 0.24 h ^–1, for 4 g/l of ethanol in the feed and 40% DO, the productivity of ethyl acetate was optimal (0.7 g/l per hour). Correspondence to: A. Miclo     

Intracellular replication of Salmonella typhimurium strains in specific subsets of splenic macrophages in vivo

We used flow cytometry and confocal immunofluorescence microscopy to study the localization of Salmonella typhimurium in spleens of infected mice. Animals were inoculated intragastrically or intraperitoneally with S. typhimurium strains, constitutively expressing green fluorescent protein. Independently of the route of inoculation, most bacteria were found in intracellular locations 3 days after inoculation. Using a panel of antibodies that bound to cells of different lineages, including mononuclear phagocyte subsets, we have shown that the vast majority of S. typhimurium bacteria reside within macrophages. Bacteria were located in red pulp and marginal zone macrophages, but very few were found in the marginal metallophilic macrophage population. We have demonstrated that the Salmonella SPI-2 type III secretion system is required for replication within splenic macrophages, and that sifA ⁻ mutant bacteria are found within the cytosol of these cells. These results confirm that SifA and SPI-2 are involved in maintenance of the vacuolar membrane and intracellular replication in vivo .