The plate counting method widely used at present to discern viable from non-viable Brucella in the host or cell is time-consuming and laborious. Therefore, it is necessary to establish a rapid, simple method for detecting and counting viable Brucella organisms. Using propidium monoazide (PMA) to inhibit amplification of DNA from dead Brucella, a novel, rapid PMA-quantitative PCR (PMA-qPCR) detection method for counting viable Brucella was established. The standard recombinant plasmid with the target BCSP31 gene fragment inserted was constructed for drawing a standard curve. The reaction conditions were optimised, and the sensitivity, specificity, and repeatability were analysed. The optimal exposure time and working concentration of PMA were 10 min and 15 μg/mL, respectively. The correlation coefficient (R²) of the standard curve was 0.999. The sensitivity of the method was 10³ CFU/mL, moreover, its specificity and repeatability also met the requirements. The concentration of B. suis measured by the PMA-qPCR did not differ significantly from that measured by the plate counting method, and the concentrations of viable bacteria in infected cells determined by the two methods were of the same order of magnitude. In this study, a rapid and simple PMA-qPCR counting method for viable Brucella was established, which will facilitate related research.

Bovine mastitis is an inflammatory disease of the udder that causes important economic losses in the animal breeding and dairy product industries. Nowadays, the conventional livestock antibiotic treatments are slowly being replaced by alternative treatments. In this context, the main aim of this study was to evaluate the efficacy of natural products in alternative treatment of bovine mastitis. Two natural formulations with previously suggested in vitro antimicrobial effect were tested in vivo on mastitic cows. Animals with a positive diagnosis for mastitis (n = 20) were divided into three treatment groups: two groups (n = 8) were administered formulations of propolis, alcoholic extracts of Brewers Gold and Perle hops, plum lichen, common mallow, marigold, absinthe wormwood, black poplar buds, lemon balm, and essential oils of oregano, lavender, and rosemary designated R4 and R7 (differing only in the latter being more concentrated) and one group (n = 4) a conventional antibiotic mixture. In vivo efficacy of treatments was evaluated by somatic cell and standard plate counts, the treatment being considered efficacious when both parameters were under the maximum limit. R7 was effective in the most cases, being therapeutically bactericidal in six out of eight cows, while R4 gave good results in three out of eight cows, and conventional antibiotics cured one out of four. These results suggest the possible therapeutic potential of these natural products in bovine mastitis.

Introduction: The purpose of the study was to determine and model the growth rates of L. monocytogenes in cooked cured ham stored at various temperatures. Material and Methods: Samples of cured ham were artificially contaminated with a mixture of three L. monocytogenes strains and stored at 3, 6, 9, 12, or 15°C for 16 days. The number of listeriae was determined after 0, 1, 2, 3, 5, 7, 9, 12, 14, and 16 days. A series of decimal dilutions were prepared from each sample and plated onto ALOA agar, after which the plates were incubated at 37°C for 48 h under aerobic conditions. The bacterial counts were logarithmised and analysed statistically. Five repetitions of the experiment were performed. Results: Both storage temperature and time were found to significantly influence the growth rate of listeriae (P < 0.01). The test bacteria growth curves were fitted to three primary models: the Gompertz, Baranyi, and logistic. The mean square error (MSE) and Akaike’s information criterion (AIC) were calculated to evaluate the goodness of fit. It transpired that the logistic model fit the experimental data best. The natural logarithms of L. monocytogenes’ mean growth rates from this model were fitted to two secondary models: the square root and polynomial. Conclusion: Modelling in both secondary types can predict the growth rates of L. monocytogenes in cooked cured ham stored at each studied temperature, but mathematical validation showed the polynomial model to be more accurate.

In this study, a genetically engineered live attenuated Salmonella Enteritidis (SE) vaccine was evaluated for its ability to protect against Salmonella Typhimurium (ST) infection in chickens. The birds were orally primed with the vaccine on the 1st day of life and given an oral booster at 5 wk of age. Control birds were orally inoculated with phosphate-buffered saline. Both groups of birds were orally challenged with a virulent ST strain at 9 wk of age. Compared with the control chickens, the vaccinated chickens had significantly higher levels of systemic IgG and mucosal IgA against specific ST antigens and a significantly greater lymphoproliferative response to ST antigens. The excretion of ST into the feces was significantly lower in the vaccinated group than in the control group on days 9 and 13 d after challenge. In addition, the vaccinated group had significantly fewer pronounced gross lesions in the liver and spleen and lower bacterial counts in the internal organs than the control group after challenge. These data indicate that genetically engineered live attenuated SE may induce humoral and cellular immune responses against ST antigens and may confer protection against virulent ST challenge.

Objective—To evaluate whether the application of steam to a variety of surface types in a veterinary hospital would effectively reduce the number of bacteria. Sample—5 surface types.Procedures—Steam was applied as a surface treatment for disinfection to 18 test sites of 5 surface types in a veterinary hospital. A pretreatment sample was obtained by collection of a swab specimen from the left side of each defined test surface. Steam disinfection was performed on the right side of each test surface, and a posttreatment sample was then collected in the same manner from the treated (right) side of each test surface. Total bacteria for pretreatment and posttreatment samples were quantified by heterotrophic plate counts and for Staphylococcus aureus, Pseudomonas spp, and total coliforms by counts on selective media. Results—Significant reductions were observed in heterotrophic plate counts after steam application to dog runs and dog kennel floors. A significant reduction in counts of Pseudomonas spp was observed after steam application to tub sinks. Bacterial counts were reduced, but not significantly, on most other test surfaces that had adequate pretreatment counts for quantification. Conclusions and Clinical Relevance—Development of health-care–associated infections is of increasing concern in human and veterinary medicine. The application of steam significantly reduced bacterial numbers on a variety of surfaces within a veterinary facility. Steam disinfection may prove to be an alternative or adjunct to chemical disinfection within veterinary practices.

The objective of this study was to evaluate the bactericidal effect of shock waves (SWs) on gram-negative or gram-positive monocultured biofilms grown on an orthopedic implant in vitro. Cortical bone screws were individually cultured with Escherichia coli or Staphylococcus epidermidis to produce a biofilm. In each run of 8 screws, 6 screws were treated with shock waves and then sonicated to disrupt the biofilm. One screw was sonicated only and one was not shock waved or sonicated before sampling for plate count dilutions. Post-treatment serial dilutions and plate counts were done on an aliquot from the vial containing each screw to obtain the number of colony-forming units (CFUs). Shock waves were at a constant energy of 0.15 mJ/mm2. Pulse number and screw orientation were varied. A linear mixed-effects model was used with “treatment” as a fixed effect and “run” as a random effect. Pairwise comparisons of treatments were performed with Tukey-Cramer’s adjustment for P-values. Sonicated plate counts were greater than nonsonicated counts for each run. When all sonicated screws were compared to all nonsonicated screws, the counts were significantly increased (P = 0.0091). For each paired comparison between sonicated and shock wave treatment, the only significant difference was in the S. epidermidis biofilm treated at 2000 pulses in a horizontal position, which increased the post-treatment count (P = 0.0445). No bactericidal effects were seen on monocultured biofilms on cortical bone screws treated with shock waves.

Objective—To determine antimicrobial effects of caprylic acid and its derivatives, monocaprylin and sodium caprylate, on Dermatophilus congolensis and to determine effects of caprylic acid on the ultrastructure of D congolensis by use of transmission electron microscopy (TEM). Sample—3 strains of D congolensis (33411, 33413, and 14639). Procedures—Strains of D congolensis were incubated separately under anaerobic conditions at 37°C for up to 48 hours in brain heart infusion (BHI) broth that was supplemented with various concentrations of caprylic acid (7.5, 12.5, 15, 17.5, or 20mM), monocaprylin (2.5, 5, 7.5, or 10mM), or sodium caprylate (15, 50, 60, 70, 100, or 120mM) or contained no antimicrobial treatment. After incubation, bacterial counts were determined by means of plating in triplicate on BHI-agar plates. Caprylic acid-treated or untreated D congolensis samples were embedded in epoxide resin for TEM; cross sections were examined for structural damage. Results—Minimum inhibitory concentrations of caprylic acid, monocaprylin, and sodium caprylate against D congolensis were 7.5, 2.5, and 15mM, respectively. Minimum bactericidal concentrations of caprylic acid, monocaprylin, and sodium caprylate against D congolensis were 15, 5, and 70mM, respectively. Examination via TEM revealed that a 15-mM concentration of caprylic acid disintegrated the plasma membrane of D congolensis. Conclusions and Clinical Relevance—Results indicated that caprylic acid, monocaprylin, and sodium caprylate could potentially be used to treat D congolensis infections. However, in vivo studies should be undertaken to determine whether these compounds can be considered as treatment options.

Holstein steers were fed carbohydrate-rich, short-fiber basal diets with and without added sodium sulfate. Steers fed the high-sulfate diet developed the CNS disorder polioencephalomalacia (PEM). The onset of signs of PEM was associated with increased sulfide concentration in the rumen fluid. Over the course of the disease, anaerobic rumen bacteria were enumerated in roll tubes by use of the Hungate method to determine the effect of dietary sulfate on sulfate-reducing bacterial numbers. Media used included a general type for total counts and sulfate-containing media with and without cysteine to assess sulfate-reducing bacteria. Changes in total and sulfate-reducing bacterial numbers attributable to dietary sulfate content were not observed. The capacity to generate hydrogen sulfide from sulfate in fresh rumen fluid in vitro was substantially increased only after steers had been fed the high-sulfate diet for 10 to 12 days, which coincided with the onset of signs of PEM. The low capacity for hydrogen sulfide production of rumen fluid taken at earlier times in the feeding period suggests that rumen microorganisms must adapt to higher dietary sulfate content before they are capable of generating potentially toxic concentrations of sulfide.

This study compared a new live attenuated Salmonella Enteritidis vaccine candidate secreting Escherichia coli heat-labile enterotoxin B subunit (SE-LTB) with a commercial Salmonella Enteritidis (SE) vaccine for efficacy of protection against SE infection in laying hens. Chickens were divided into 3 groups of 20 each. Group A chickens were inoculated orally with phosphate-buffered saline and served as controls, group B chickens were inoculated orally with the vaccine candidate, and group C chickens were inoculated intramuscularly with a commercial vaccine, the primary inoculation in groups B and C being at 10 wk of age and the booster at 16 wk. Groups B and C showed significantly higher titers of plasma immunoglobulin G, intestinal secretory immunoglobulin A, and egg yolk immunoglobulin Y antibodies compared with the control group, and both vaccinated groups showed a significantly elevated cellular immune response. After virulent challenge, group B had significantly lower production of thin-shelled and/or malformed eggs and a significantly lower rate of SE contamination of eggs compared with the control group. Furthermore, the challenge strain was detected significantly less in all of the examined organs of group B compared with the control group. Group C had lower gross lesion scores only in the spleen and had lower bacterial counts only in the spleen, ceca, and ovary. These findings indicate that vaccination with the SE-LTB vaccine candidate can efficiently reduce internal egg and internal organ contamination by Salmonella and has advantages over the commercial vaccine.

An experimental chlorate product (ECP) developed by the United States Department of Agriculture has been shown to have bactericidal effects against enteropathogens such as Escherichia coli. In studies with broilers and pigs, the bactericidal activity of ECP was enhanced by prior adaptation of gastrointestinal microflora to nitrate. The objective of this study was to examine the effects of nitrate adaptation on the bactericidal activity of ECP against E. coli in Holstein steers. Results indicate that ECP was effective at reducing E. coli. However, ECP did not reduce E. coli in a dose-dependent manner, indicating that the highest ECP dose provided in this study exceeded that needed to be efficacious. Adapting gastrointestinal microflora with nitrate prior to feeding ECP did not improve efficacy of ECP against E. coli. Rapid reduction of nitrate in the rumen is implicated as a possible explanation for why adaptation to nitrate did not enhance the bactericidal effects of ECP in cattle.