Control of Plaque and Gingivitis by an Herbal Toothpaste - A Randomised Controlled Study.

PURPOSE: To compare the efficacy of an herbal toothpaste with two other chemically active toothpastes regarding plaque and gingivitis control. MATERIALS AND METHODS: Seventy-six (27 females and 49 males, mean age 47.8 years, range 40-58 years) of 84 initial participants with slight and moderate chronic periodontitis used standardised manual toothbrushes and their usual technique for daily manual mechanical plaque control for 24 weeks of supportive periodontal therapy. The volunteers were randomly assigned to one of 3 groups: group 1 used the herbal toothpaste, group 2 a triclosan/copolymer toothpaste, and group 3 an amine/stannous fluoride toothpaste. OHI, API, SBI, BOP, PD and AL were recorded at baseline and after 6, 12 and 24 weeks (PD and AL only at baseline). The Kruskal-Wallis, Mann-Whitney U-, Friedman, and Wilcoxon tests were used for statistical analysis. RESULTS: Moderate changes occurred in API and OHI in all groups. The herbal toothpaste resulted in significantly lower API and OHI in comparison to the fluoride toothpaste during the study period (p = 0.001 and 0.049, minimum and maximum of cases, respectively). SBI was significantly improved in all groups starting after 12 weeks (p = 0.001 and 0.033). BOP remained largely unchanged in all groups and was always significant lower in the herbal toothpaste group (p = 0.001 and 0.036). CONCLUSION: During the study period of 24 weeks, the herbal toothpaste was as good as the control toothpastes. No side effects were seen. In terms of improving periodontal conditions, the tested herbal toothpaste could be a suitable alternative to conventional toothpastes with artificial chemical ingredients.

Impact of humic acids on the colonic microbiome in healthy volunteers.

AIM: To test the effects of humic acids on innate microbial communities of the colon. METHODS: We followed the effects of oral supplementation with humic acids (Activomin®) on concentrations and composition of colonic microbiome in 14 healthy volunteers for 45 d. 3 × 800 mg Activomin® were taken orally for 10 d followed by 3 × 400 mg for 35 d. Colonic microbiota were investigated using multicolor fluorescence in situ hybridization (FISH) of Carnoy fixated and paraffin embedded stool cylinders. Two stool samples were collected a week prior to therapy and one stool sample on days 10, 31 and 45. Forty-one FISH probes representing different bacterial groups were used. RESULTS: The sum concentration of colonic microbiota increased from 20% at day 10 to 30% by day 31 and remained stable until day 45 (32%) of humic acid supplementation (P < 0.001). The increase in the concentrations in each person was due to growth of preexisting groups. The individual microbial profile of the patients remained unchanged. Similarly, the bacterial diversity remained stable. Concentrations of 24 of the 35 substantial groups increased from 20% to 96%. Two bacterial groups detected with Bac303 (Bacteroides) and Myc657 (mycolic acid-containing Actinomycetes) FISH probes decreased (P > 0.05). The others remained unaffected. Bacterial groups with initially marginal concentrations (< 0.1 × 109/mL) demonstrated no response to humic acids. The concentrations of pioneer groups of Bifidobacteriaceae, Enterobacteriaceae and Clostridium difficile increased but the observed differences were statistically not significant. CONCLUSION: Humic acids have a profound effect on healthy colonic microbiome and may be potentially interesting substances for the development of drugs that control the innate colonic microbiome.

Phenotypic and Genotypic Characterization of Bacteriocinogenic Enterococci Against Clostridium botulinum.

The present study aimed to characterize Enterococcus faecalis (n = -6) and Enterococcus faecium (n = 1) isolated from healthy chickens to find a novel perspective probiotic candidate that antagonize Clostridium botulinum types A, B, D, and E. The isolated enterococci were characterized based on phenotypic properties, PCR, and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF). The virulence determinants including hemolytic activity on blood agar, gelatinase activity, sensitivity to vancomycin, and presence of gelatinase (gelE) and enterococcal surface protein (esp) virulence genes were investigated. Also, the presence of enterocin structural genes enterocin A, enterocin B, enterocin P, enterocin L50A/B, bacteriocin 31, enterocin AS48, enterocin 1071A/1071B, and enterocin 96 were assessed using PCR. Lastly, the antagonistic effect of the selected Enterococcus spp. on the growth of C. botulinum types A, B, D, and E was studied. The obtained results showed that four out of six E. faecalis and one E. faecium proved to be free from the tested virulence markers. All tested enterococci strains exhibited more than one of the tested enterocin. Interestingly, E. faecalis and E. faecium significantly restrained the growth of C. botulinum types A, B, D, and E. In conclusion, although, the data presented showed that bacteriocinogenic Enterococcus strains lacking of virulence determinants could be potentially used as a probiotic candidate against C. botulinum in vitro; however, further investigations are still urgently required to verify the beneficial effects of the tested Enterococcus spp. in vivo.

Ethyl Pyruvate: An Anti-Microbial Agent that Selectively Targets Pathobionts and Biofilms.

The microbiota has a strong influence on health and disease in humans. A causative shift favoring pathobionts is strongly linked to diseases. Therefore, anti-microbial agents selectively targeting potential pathogens as well as their biofilms are urgently demanded. Here we demonstrate the impact of ethyl pyruvate, so far known as ROS scavenger and anti-inflammatory agent, on planktonic microbes and biofilms. Ethyl pyruvate combats preferably the growth of pathobionts belonging to bacteria and fungi independent of the genera and prevailing drug resistance. Surprisingly, this anti-microbial agent preserves symbionts like Lactobacillus species. Moreover, ethyl pyruvate prevents the formation of biofilms and promotes matured biofilms dissolution. This potentially new anti-microbial and anti-biofilm agent could have a tremendous positive impact on human, veterinary medicine and technical industry as well.

Hygienisation and nutrient conservation of sewage sludge or cattle manure by lactic acid fermentation.

Manure from animal farms and sewage sludge contain pathogens and opportunistic organisms in various concentrations depending on the health of the herds and human sources. Other than for the presence of pathogens, these waste substances are excellent nutrient sources and constitute a preferred organic fertilizer. However, because of the pathogens, the risks of infection of animals or humans increase with the indiscriminate use of manure, especially liquid manure or sludge, for agriculture. This potential problem can increase with the global connectedness of animal herds fed imported feed grown on fields fertilized with local manures. This paper describes a simple, easy-to-use, low-tech hygienization method which conserves nutrients and does not require large investments in infrastructure. The proposed method uses the microbiotic shift during mesophilic fermentation of cow manure or sewage sludge during which gram-negative bacteria, enterococci and yeasts were inactivated below the detection limit of 3 log10 cfu/g while lactobacilli increased up to a thousand fold. Pathogens like Salmonella, Listeria monocytogenes, Staphylococcus aureus, E. coli EHEC O:157 and vegetative Clostridium perfringens were inactivated within 3 days of fermentation. In addition, ECBO-viruses and eggs of Ascaris suum were inactivated within 7 and 56 days, respectively. Compared to the mass lost through composting (15-57%), the loss of mass during fermentation (< 2.45%) is very low and provides strong economic and ecological benefits for this process. This method might be an acceptable hygienization method for developed as well as undeveloped countries, and could play a key role in public and animal health while safely closing the nutrient cycle by reducing the necessity of using energy-inefficient inorganic fertilizer for crop production.

Detection of Clostridium botulinum in liquid manure and biogas plant wastes.

Biogas plants have been considered as a source for possible amplification and distribution of pathogenic bacteria capable of causing severe infections in humans and animals. Manure and biogas wastes could be sources for spore-forming bacteria such as Clostridium botulinum. In the present study, 24 liquid manure and 84 biogas waste samples from dairies where the majority of the cows suffered from chronic botulism were investigated for the presence of botulinum neurotoxins (BoNT) and C. botulinum spores. The prevalence of BoNT/A, B, C, D, and E in biogas wastes was 16.6, 8.3, 10.7, 7.1, and 10.8 %, respectively, while in manure, the prevalence was 0.0, 0.0, 0.0, 8.3, and 4.1 %, respectively. After enrichment of samples in reinforced cultural medium, they were tested for C. botulinum BoNT/A, B, C, D, and E using ELISA (indirect C. botulinum detection). The prevalence of C. botulinum type A, B, C, D, and E samples in biogas wastes was 20.2, 15.5, 19, 10.7, and 34.8 %, respectively, while the prevalence in liquid manure was 0.0, 0.0, 0.0, 8.3, and 12.5 %, respectively. In conclusion, the occurrence of BoNT and C. botulinum spores in biogas waste of diseased animals indicates an increased and underestimated hygienic risk. Application of digestates from biogas fermentations as fertilizers could lead to an accumulation of long lifespan spores in the environment and could be a possible health hazard.

The influence of glyphosate on the microbiota and production of botulinum neurotoxin during ruminal fermentation.

The aim of the present study is to investigate the impact of glyphosate on the microbiota and on the botulinum neurotoxin (BoNT) expression during in vitro ruminal fermentation. This study was conducted using two DAISY(II)-incubators with four ventilated incubation vessels filled with rumen fluid of a 4-year-old non-lactating Holstein-Friesian cow. Two hundred milliliter rumen fluid and 800 ml buffer solution were used with six filter bags containing 500 mg concentrated feed or crude fiber-enriched diet. Final concentrations of 0, 1, 10, and 100 µg/ml of glyphosate in the diluted rumen fluids were added and incubated under CO2-aerated conditions for 48 h. The protozoal population was analyzed microscopically and the ruminal flora was characterized using the fluorescence in situ hybridization technique. Clostridium botulinum and BoNT were quantified using most probable number and ELISA, respectively. Results showed that glyphosate had an inhibitory effect on select groups of the ruminal microbiota, but increased the population of pathogenic species. The BoNT was produced during incubation when inoculum was treated with high doses of glyphosate. In conclusion, glyphosate causes dysbiosis which favors the production of BoNT in the rumen. The global regulations restrictions for the use of glyphosate should be re-evaluated.

Characterisation of the aerobic bacterial flora of boid snakes: application of MALDI-TOF mass spectrometry.

The aim of this study was to identify aerobic bacterial isolates from the respiratory tract of boids with matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). From 47 boid snakes, swabs from the oral cavity, tracheal wash samples and, in cases in which postmortem examination was performed, pulmonary tissue samples were taken. Each snake was classified as having inflammation of the respiratory tract and/or oral cavity, or without evidence of inflammation based on combination of clinical, cytological and histopathological findings. Samples collected from the respiratory tract and oral cavity were inoculated onto routine media and bacteria were cultured aerobically. All morphologically distinct individual colonies obtained were analysed using MALDI-TOF MS. Unidentified isolates detected in more than three snakes were selected for further 16S rDNA PCR and sequencing. Among all examined isolates (n=243), 49 per cent (n=119) could be sufficiently speciated using MALDI-TOF MS. Molecular biology revealed several bacterial species that have not been previously described in reptiles. With an average of 6.3 different isolates from the respiratory tract and/or oral cavity, boids with inflammatory disease harboured significantly more bacterial species than boids without inflammatory disease (average 2.8 isolates).

Detection of pathogenic clostridia in biogas plant wastes.

As the number of biogas plants has grown rapidly in the last decade, the amount of potentially contaminated wastes with pathogenic Clostridium spp. has increased as well. This study reports the results from examining 203 biogas plant wastes (BGWs). The following Clostridium spp. with different frequencies could be isolated via a new enrichment medium (Krüne medium) and detected by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS): Clostridium perfringens (58 %) then Clostridium bifermentans (27 %), Clostridium tertium (23 %) and Clostridium butyricum (19 %), Clostridium cadaveris (15 %), Clostridium parapurificum (6 %), Clostridium glycolicum (5 %), Clostridium baratii (4 %), Clostridium sporogenes (2 %), Clostridium sordellii (1 %) and Clostridium subterminale (0.5 %). The mean most probable number (MPN) count of sulfite reducing bacteria was between 10(3) and 10(4)/mL, and the higher the MPN, the more pathogenic Clostridium spp. were present. Also, real-time PCR was used to be compared with culture method for C. perfringens, C. bifermentans, C. butyricum, C. sporogenes/Clostridium botulinum and C. sordellii. Although real-time PCR was more sensitive than the culture method, both systems improve the recovery rate but in different ways and are useful to determine pathogenic clostridia in biogas plants. In conclusion, BGWs could present a biohazard risk of clostridia for humans and animals.

Possible effects of glyphosate on Mucorales abundance in the rumen of dairy cows in Germany.

Glyphosate (N-phosphonomethyl glycine) is registered as a herbicide for many food and non-food crops, as well as non-crop areas where total vegetation control is desired. Glyphosate influences the soil mycobiota; however, the possible effect of glyphosate residues in animal feed (soybean, corn, etc.) on animal mycobiota is almost unknown. Accordingly, the present study was initiated to investigate the mycological characteristics of dairy cows in relationship to glyphosate concentrations in urine. A total of 258 dairy cows on 14 dairy farms in Germany were examined. Glyphosate was detected in urine using ELISA. The fungal profile was analyzed in rumen fluid samples using conventional microbiological culture techniques and differentiated by MALDI-TOF mass spectrometry. LPS-binding protein (LBP) and antibodies (IgG1, IgG2, IgA, and IgM) against fungi were determined in blood using ELISA. Different populations of Lichtheimia corymbifera, Lichtheimia ramosa, Mucor, and Rhizopus were detected. L. corymbifera and L. ramosa were significantly more abundant in animals containing high glyphosate (>40 ng/ml) concentrations in urine. There were no significant changes in IgG1 and IgG2 antibodies toward isolated fungi that were related to glyphosate concentration in urine; however, IgA antibodies against L. corymbifera and L. ramosa were significantly lower in the higher glyphosate groups. Moreover, a negative correlation between IgM antibodies against L. corymbifera, L. ramosa, and Rhizopus relative to glyphosate concentration in urine was observed. LBP also was significantly decreased in animals with higher concentrations of glyphosate in their urine. In conclusion, glyphosate appears to modulate the fungal community. The reduction of IgM antibodies and LBP indicates an influence on the innate immune system of animals.

Efficacy of an anticoccidial live vaccine in prevention of necrotic enteritis in chickens.

Necrotic enteritis (NE) is an important disease in poultry caused by Clostridium perfringens combined with predisposing factors, mainly eimeriosis. In the present study, we investigated the protective effect of a commercial attenuated anticoccidial live vaccine against NE in a clinical infection model using 60 day-old chicks. Vaccination was performed on study day (SD) 1 with natural booster-infections for 4 weeks from Eimeria spp. oocysts present in litter. On SD 28, five groups were formed (n=12): group V+/C-E- (vaccinated, uninfected), group V+/C-E+ (vaccinated, infected with Eimeria spp.), group V+/C+E+ (vaccinated, infected with clostridia and Eimeria spp.), group V-/C+E+ (unvaccinated, infected with clostridia and Eimeria spp.), and group NC (negative control). Efficacy was measured by clinical parameters, pathogen multiplication, and pathological parameters assessed during two necropsies on SD 34 and SD 40, respectively. Additionally, cytokine expression was measured in gut and spleen tissues at necropsy. Clinical signs of NE were observed only in the coinfected groups, mainly in group V-/C+E+. Accordingly, lowest body weight gain was observed in group V-/C+E+ (301.8 g from SD 28 to SD 40; group NC: 626.2 g). Oocyst excretion varied significantly (P<0.01) between all Eimeria spp. infected groups and was highest in group V-/C+E+, followed by V+/C+E+, and lowest in group V+/C-E+. NE typical intestinal lesions showed only in groups V+/C+E+ and V-/C+E+. The intestinal mucosa featured partly severe lesions in the jejunum, C. perfringens colonization was histologically visible. Upregulation of IFN-γ, was observed in the jejunal tissue of group V-/C+E+ (P<0.01 (SD 34) or P<0.05 (SD 40) compared to all other groups). IL-10 and IL-12 were upregulated in group V-/C+E+, IL-10 also in group V+/C+E+ (SD 40) while IL-2 expression remained unaltered. In conclusion, vaccination against coccidiosis was effective in preventing NE in a mixed infection comparable to field situations.

Chronic botulism in a Saxony dairy farm: sources, predisposing factors, development of the disease and treatment possibilities.

The aim of this study is to investigate Clostridium botulinum at a Saxony dairy farm with 159 cows and 18 heifers. The animals exhibited clinical symptoms of chronic botulism. To determine the source of the infection, feces, blood, organs, and gastrointestinal fluids of dead or euthanized cows; as well as soil, water, silage and manure were tested for C. botulinum spores and BoNTs using ELISA. BoNT/C and C. botulinum type C were detected in 53% and 3% of tested animals, respectively, while BoNT/D and C. botulinum type D were detected in 18% of the animals. C. botulinum also was detected in organs, gastrointestinal fluids, drinking water and manure. To evaluate possible treatments, animals were given Jerusalem artichoke syrup (JAS), Botulism vaccine (formalinised aluminum hydroxide gel adsorbed toxoid of C. botulinum types C and D) or a suspension of Enterococcus faecalis. After four weeks treatment with JAS, BoNT/C and C. botulinum type C were not detected in feces. In contrast, BoNT/D and C. botulinum type D were not significantly influenced by the JAS treatment. Vaccination with botulism vaccine and the E. faecalis suspension significantly decreased BoNT/D and C. botulinum type D. A significant increase of Enterococci was detected in animals treated with E. faecalis. Interestingly, there was a negative correlation between the detection of both BoNT and C. botulinum with the concentration of Enterococci in feces. Although C. botulinum C and D antibodies increased significantly (p < 0.0001) after vaccination with the botulism vaccine, the reduction of C. botulinum and BoNT in feces did not result in recovery of the animals because they were deficient of trace elements [manganese (Mn), cobalt (Co), copper (Cu) and selenium (Se)]. Animals treated with trace elements recovered. It appears that intestinal microbiota dysbiosis and trace element deficiency could explain the extensive emergence of chronic Botulism.

Relationship between gastrointestinal dysbiosis and Clostridium botulinum in dairy cows.

The gastrointestinal tract is a balanced ecosystem that can get out of balance and predisposed to clostridial diseases or other pathological conditions. The objective of the present study was to evaluate the gut microbiota in dairy cows suffering from chronic botulism. Cows were investigated for Clostridium (C.) botulinum in faeces and rumen fluids. In order to study the relationship between botulism and gastrointestinal microbiota, faeces and rumen fluid were tested for bacterial composition using conventional microbiological culture techniques and fluorescence in situ hybridization (FISH). Protozoa were analyzed in rumen fluid microscopically. The presence of C. botulinum was associated with specific changes in the faecal microbiota, especially a significant reduction of total aerobic bacteria, total anaerobic bacteria, enterococci, Clostridium perfringens and yeast and fungi. Also C. botulinum positive rumen fluid had significantly more Bacteroides spp., C. histolyticum group, Alfa- proteobacteria, Gammaproteobacteria, and sulfate-reducing bacteria; as well as significantly fewer Euryaracheota, and the protozoa Epidinium spp. Dasytricha spp., Diplodiniinae spp. and Ophryoscolex spp. In conclusion, C. botulinum is common in dairy cows in Germany but the incidence of botulism is associated with microbial changes and composition in the gastrointestinal tract. Bacteria, yeast and protozoa appear to be crucial in the colonization process; however, the chronology of these events and role of each microbial group needs further evaluation.

Effects of feeding different roughage components to sows in gestation on bacteriological and immunological parameters in colostrum and immune response of piglets.

In piglet production, Escherichia coli (E. coli) and the presence of lipopolysaccharides (LPS) were considered as predominant causative agents in the aetiology of important diseases of sows and piglets. The aim of the study was to assess the effects of feeding different roughage sources to sows in gestation on the microbiota and endotoxin concentration in colostrum and on the specific immune response of their piglets. In two trial runs, a total of 144 sows were assigned to one of the six dietary treatments: the Control group receiving a concentrate diet and five groups with a restrictive supply of the control diet in combination with straw (S), hay (H), clover grass silage (GS), maize silage (MS) or Jerusalem artichoke (JA). During lactation no roughage was offered. Colostrum samples were analysed for bacteriological and immunological parameters. Blood samples were taken from two piglets per corresponding litter to examine the concentration of C-reactive protein (CRP) and of specific antibodies to LPS of E. coli (strain J5). Roughage feeding had an effect neither on the total bacterial count nor on the content of E. coli in colostrum of sows. The concentration of LPS in colostrum was reduced in Group MS. The concentration of CRP in the colostrum samples was low and independent of the feeding regime (max. 9.3 µg/ml). However, the administration of roughage components rich in crude fibre (Groups H and S) decreased the level of CRP in colostrum significantly. The analysis of the specific immunoglobulin to LPS of E. coli (strain J5) showed a higher concentration of IgG-anti-LPS in blood serum of piglets from sows of Group JA. The results suggest that the inclusion of selected roughage components may have the potential to affect the immunocompetence of sows and their corresponding piglets.

Neutralization of the antimicrobial effect of glyphosate by humic acid in vitro.

In the present study, the neutralization ability of the antimicrobial effect of glyphosate by different humic acids was investigated. The minimal inhibitory concentrations of glyphosate for different bacteria such as Bacillus badius, Bifidobacterium adolescentis, Escherichia coli, E. coli 1917 strain Nissle, Enterococcus faecalis, Enterococcus faecium, Salmonella enteritidis and Salmonella typhimurium were determined in the presence or absence of different concentrations of humic acid (0.25, 0.5 and 1.0 mg mL(-1)). Our findings indicated that humic acids inhibited the antimicrobial effect of glyphosate on different bacteria. This information can help overcome the negative impact of glyphosate residues in feed and water.

The effects of stress hormones on growth of selected periodontitis related bacteria.

The focus of this study was to examine in vitro the effects of stress hormones (catecholamines: epinephrine, norepinephrine, dopamine and hydrocortisone: cortisol) on the growth of four anaerobic species of periodontitis-related bacteria (Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythia) and one facultative anaerobic species (Eikenella corrodens). Bacterial growth was determined by two different methods: fluorescence in situ hybridization (FISH), and the viable count by culture method. To simulate stress, each single strain was grown in a special growth medium with three different concentrations of each hormone, using an anaerobic chamber at 37 °C. Growth of F. nucleatum increased in the presence of all stress hormones. Growth of P. gingivalis was not significantly influenced by any hormone. Growth of P. intermedia and E. corrodens was inhibited by almost all stress hormones tested. Both methods of analysis revealed that the highest concentrations of norepinephrine and cortisol increased the growth of T. forsythia. Different hormones have a different effect on the growth of periodontitis-related bacteria in vitro. It appears that bacterial viability is more strongly influenced than is bacterial metabolic activity. The growth of F. nucleatum particularly and partially of T. forsythia is increased by several stress hormones and may have an additional negative impact on periodontal disease.

Efficacy of early treatment with toltrazuril in prevention of coccidiosis and necrotic enteritis in chickens.

In the present study, efficacy of the toltrazuril treatment for prevention of coccidiosis and necrotic enteritis was tested. Ninety-six 14-day-old commercial broiler chickens were caged and divided into eight groups (n=12), designated groups 1 to 8. Chickens of groups 1 to 6 were inoculated orally at 18 days of age with 25,000 oocysts of Eimeria tenella and 75,000 oocysts of Eimeria brunetti. At 22 days of age, chickens of groups 1 to 6 were infected with 10(9) colony-forming unit Clostridium perfringens. Chickens of group 1 were treated with 75 parts/10(6) toltrazuril in drinking water for 8 h on two consecutive days up to 12 h before Eimeria infection, while chickens of groups 2 to 5 were treated with the same dose of toltrazuril at 12 h, 36 h, 60 h and 84 h after Eimeria infection, respectively. The non-treated group 6 served as a positive control. Chickens in group 7 were treated with toltrazuril at 17 and 18 days of age, and those of group 8 remained uninfected and non-treated as a negative control. The feed conversion ratio was higher in the positive control compared with other groups. The mortality rates were 16.8% and 41.7% in the late toltrazuril-treated (at 84 h) and infected non-treated chickens, respectively. Lesions scores of necrotic enteritis or coccidiosis in infected, non-treated chickens were significantly more severe compared with negative controls (P<0.01) and late toltrazuril-treated (at 84 h) chickens (P<0.05). In conclusion, application of toltrazuril before Eimeria challenge protected chickens from coccidiosis and indirectly from successive necrotic enteritis caused by C. perfringens infection.

Efficacy of Clostridium botulinum types C and D toxoid vaccination in Danish cows.

In the present study the efficacy Botulism vaccine (formalinised aluminum hydroxide gel adsorbed toxoid of Clostridium botulinum types C and D) was evaluated in four Danish dairy cows under field conditions. Other four dairy herds were unvaccinated. Blood serum of all animals was analyzed for specific C. botulinum types A, B, C, D and E antibodies using a developed ELISA. Feces of all animals were analyzed for botulinum neurotoxins (BoNTs) and C. botulinum spores. C. botulinum types C and D antibodies were significantly (p < 0.05) increased in vaccinated animals. Vaccination with botulism vaccine significantly reduced (p < 0.001) BoNTs and C. botulinum spores in cattle feces. Our findings represent that C. botulinum vaccination increases specific blood serum antibodies and reduces free BoNTs and C. botulinum spores in feces.

Safety and efficacy of a metabolic drift live attenuated Salmonella Gallinarum vaccine against fowl typhoid.

Fowl typhoid (FT), a systemic disease that results in septicemia in poultry, is caused by Salmonella enterica serovar Gallinarum biovar Gallinarum (SG). Mortality and morbidity rates from FT can reach up to 80%. Attenuated live Salmonella vaccine candidates have received considerable attention because they confer solid immunity, and they can produce systemic and mucosal immunity in the gut when administered orally. In the present study, five metabolic drift (MD) mutants with a single-(designated SG-Rif1, SG-Sm6) or double-attenuating marker (designated SG-Rif1-Sm4, SG-Sm6-Rif10, and SG-Rif1-Sm10) were isolated. The relative colony sizes to wild-type strain after 24 hr at 37 C incubation were 50%, 40%, 30%, 30%, and 20%, respectively. The probability of a back mutation can almost be excluded because the reduced colony sizes were stable after at least 50 passages on culture media. The safety and immunogenicity were evaluated in susceptible 1-day-old commercial layer chickens. After oral administration of 10(8) colony-forming units (CFU), all developed MD mutants proved to be safe and did not cause death of any infected birds during 15 days postvaccination, whereas chickens receiving 10(6) CFU SG wild-type strain showed a high mortality rate (40%). Vaccination of commercial layer chicks with SG-Rif1, SG-Sm6, SG-Rif1-Sm4, and SG-Sm6-Rif10 MD mutants could protect chickens against challenge by homologous wild-type strain; however, SG-Rif1-Sm10 could not protect against challenge, indicating hyperattenuation. In conclusion, vaccination with SG MD mutant vaccine appears to be safe and offers protection against FT in chickens.

Development of the genital microflora in stallions used for artificial insemination throughout the breeding season.

An important factor influencing stallion fertility is the microbial contamination of semen. Aims of this study were to investigate changes in the microbiological population of the genital mucosa and semen in artificial insemination stallions (n=16) from before to after one breeding season (February-August). MALDI-TOF-MS (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) was used for identification of microbial agents. For bacteriology, swabs from the urethral opening, urethral fossa and penile sheath as well as semen were collected at 4-week-intervals. For semen motility and percentage of morphologically normal spermatozoa, changes over time (P<0.001) occurred. In 14.3% of genital swabs and 25.0% of ejaculates no microbial growth was found. Intensity of total microbial growth increased throughout the breeding season (P<0.001). From the penile sheath, between 1.4±0.1 microbial species in February and 3.3±0.4 in August were identified. From semen, 1.1±0.3 microbial species in February and 2.9±0.6 in August were obtained. The number of microbial species isolated from the sheath of the penis (2.0±0.1) and urethral fossa (2.0±0.1) was greater (P<0.01) compared with the urethral opening (1.6±0.1) and semen (1.5±0.1). The microbial flora consisted of aerobic and anaerobic bacteria, dominated by coagulase-negative staphylococci, alpha-haemolytic streptococci and coryneforms. Only occasionally potentially pathogenic agents (E. coli, Ps. aeruginosa) were found. The microbial flora was not related to seminal characteristics.