The immunoglobulin M-degrading enzyme of Streptococcus suis (IdeSsuis) leads to long-lasting inhibition of the activation of porcine IgM-secreting B cells.

Streptococcus suis (S. suis) is one of the most important porcine pathogens, causing severe pathologies such as meningitis or polyarthritis. It is also a very successful colonizer of mucosal surfaces. The IgM-degrading enzyme of S. suis (IdeSsuis) specifically cleaves porcine IgM, which results in complement evasion. On the basis of our previous finding that IdeSsuis also cleaves the IgM B cell receptor in vitro, we verified IgM B cell receptor cleavage ex vivo in whole regional lymph nodes and investigated the working hypothesis that this IgM B cell receptor cleavage results in a long-lasting impaired B cell function. The number of IgM-secreting cells was determined via ELISpot analysis after porcine peripheral blood mononuclear cells had initially been treated with different recombinant S. suis proteins and subsequently stimulated with interleukin-2 and the toll-like receptor 7/8 ligand R848. Compared with treatment with medium or recombinant muramidase-released protein, treatment with rIdeSsuis but also with a cleavage-deficient variant led to a reduction in the number of IgM-secreting cells as well as the level of secreted IgM. Flow cytometry analysis confirmed that the IgM B cell receptor was cleaved only by rIdeSsuis, and the receptor recovered to pretreatment levels on day 2 after treatment. Flow cytometry analysis of B and T cells incubated with fluorescein-labelled recombinant proteins revealed that different rIdeSsuis variants bind specifically to B cells, most prominently the cleavage-deficient variant. Our results indicate that in vitro interference of rIdeSsuis with the IgM B cell receptor results in long-lasting impaired IgM secretion by B cells after toll-like receptor activation. Further studies are warranted to prove that the modulation of B cell function by IdeSsuis could play a role in vivo.

Brachyspira in dogs: risk factors of shedding in central Germany and longitudinal study of an infected kennel.

BACKGROUND: Brachyspira (B.) pilosicoli is a zoonotic pathogen, able to infect different animal species such as pigs, poultry, and rodents, causing intestinal spirochetosis. An association of gastrointestinal clinical signs, such as diarrhea, with the isolation of B. pilosicoli from fecal samples or rectal swabs has not been proven in dogs. Other Brachyspira species commonly isolated from dogs, such as "B. canis" and "B. pulli", are considered commensals. This study investigated the occurrence of different Brachyspira species in rectal swabs and fecal samples in an independent canine cohort in central Germany. These included samples from shelter dogs, hunting dogs, and dogs presenting at regional small animal practices with various clinical signs. Data about the dogs, including potential risk factors for Brachyspira isolation, were obtained using a standardized questionnaire. The study also longitudinally investigated a colony of Beagle dogs for Brachyspira over 5 years. RESULTS: The rate of Brachyspira spp. isolation was 11% and included different Brachyspira species ("B. canis", "B. pulli", and B. pilosicoli). "B. canis" was detected in 18 dogs, whereas B. pilosicoli was only isolated from 1 dog in the independent cohort (not including the Beagle colony). Risk factors for shedding Brachyspira and "B. canis" were being less than 1 year of age and shelter origin. Gastrointestinal signs were not associated with the shedding of Brachyspira. B. pilosicoli and "B. canis" were isolated from several dogs of the same Beagle colony in 2017 and again in 2022, while Brachyspira was not isolated at multiple sampling time points in 2021. CONCLUSIONS: Shedding of B. pilosicoli in dogs appears to be uncommon in central Germany, suggesting a low risk of zoonotic transmission from dogs. Commensal status of "B. canis" and "B. pulli" is supported by the results of this study. Findings from the longitudinal investigation of the Beagle colony agree with an asymptomatic long-term colonization of dogs with "B. canis" and B. pilosicoli and suggest that introducing new animals in a pack can trigger an increased shedding of B. pilosicoli.

Detection of subtilisin 3 and 6 in skin biopsies of cattle with clinically manifested bovine ringworm.

Trichophyton (T.) verrucosum is a highly pathogenic dermatophyte causing zoonotic bovine ringworm that is transmissible to humans. The virulence factors subtilisin (Sub)3 and Sub6 are discussed to contribute to disease manifestation but no protein expression study is available for T. verrucosum. We used customized antibodies (against Trichophyton-species, Sub3 and Sub6) to examine skin biopsies of infected cattle via immunofluorescence stainings. Both virulence factors Sub3 and 6 were solely expressed by conidia and not only found in epidermal but also in dermal and hair structures. The anti-T-antibody reliably detected the fungus and proved more sensitive compared to histological stains. LAY SUMMARY: We examined the zoonotic dermatophyte Trichophyton (T.) verrucosum in bovine skin and studied two important virulence factors called subtilisin (Sub)3 and Sub6 that T. verrucosum produces and secretes using immunolabeling.

Survival of Streptococcus suis in Porcine Blood Is Limited by the Antibody- and Complement-Dependent Oxidative Burst Response of Granulocytes.

Bacteremia is a hallmark of invasive Streptococcus suis infections of pigs, often leading to septicemia, meningitis, or arthritis. An important defense mechanism of neutrophils is the generation of reactive oxygen species (ROS). In this study, we report high levels of ROS production by blood granulocytes after intravenous infection of a pig with high levels of S. suis-specific antibodies and comparatively low levels of bacteremia. This prompted us to investigate the working hypothesis that the immunoglobulin-mediated oxidative burst contributes to the killing of S. suis in porcine blood. Several S. suis strains representing serotypes 2, 7, and 9 proved to be highly susceptible to the oxidative burst intermediate hydrogen peroxide, already at concentrations of 0.001%. The induction of ROS in granulocytes in ex vivo-infected reconstituted blood showed an association with pathogen-specific antibody levels. Importantly, inhibition of ROS production by the NADPH oxidase inhibitor apocynin led to significantly increased bacterial survival in the presence of high specific antibody levels. The oxidative burst rate of granulocytes partially depended on complement activation, as shown by specific inhibition. Furthermore, treatment of IgG-depleted serum with a specific IgM protease or heat to inactivate complement resulted in >3-fold decreased oxidative burst activity and increased bacterial survival in reconstituted porcine blood in accordance with an IgM-complement-oxidative burst axis. In conclusion, this study highlights an important control mechanism of S. suis bacteremia in the natural host: the induction of ROS in blood granulocytes via specific immunoglobulins such as IgM.

Visualising virulence factors: Trichophyton benhamiaes subtilisins demonstrated in a guinea pig skin ex vivo model.

BACKGROUND: Dermatophytoses rank among the most frequent communicable diseases in humans, and the zoonotic transmission is increasing. The zoophilic dermatophyte Trichophyton (T.) benhamiae is nowadays one of the main causes of tinea faciei et corporis in children. However, scientific data on molecular pathomechanisms and specific virulence factors enabling this ubiquitous occurrence are scarce. OBJECTIVES: To study tissue invasion and the expression of important virulence factors of T. benhamiae, isolates that were recovered from two groups of hosts (humans vs. guinea pigs (GP)) using an ex vivo skin model. METHODS: After confirmation of species identity by ITS sequencing, CFU suspensions of dermatophyte isolates (n = 20) were applied to the skin infection model and cultured. Employing specific immunofluorescence staining techniques, the expression of subtilisin 3 and 6 and metallocarboxypeptidase A was analysed. The general mode of invasion was explored. Results were compared with biopsies of naturally infected GP. RESULTS: All isolates were successfully recovered and proliferated well after application to the infection model. Progressive invasion of hyphae through all skin structures and destruction of explants were observed with early events being comparable to natural infection. An increasing expression of the examined virulence factors towards the end of culture was noticed but no difference between the two groups of isolates. CONCLUSIONS: For the first time, important in vivo markers of dermatophytosis were visualised immunohistochemically in an ex vivo skin infection model and in skin biopsies of GP naturally infected with T. benhamiae. More research on the underlying pathomechanisms of dermatophyte infection is urgently needed.

Identification of hypocrealean reptile pathogenic isolates with MALDI-TOF MS.

Biotyper analysis of Nannizziopsis guarroi, a fatal fungal pathogen in lizards, was described recently. Hypocrealean fungal infections in captive reptiles appear with an increasing frequency during the last decade. Therefore, the aim of this study was to proof Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) as diagnostic tool for the identification of reptile pathogenic hypocrealean fungi. Ten fungal isolates obtained from nine reptiles with fungal glossitis, disseminated visceral mycosis, pneumomycosis, and fungal keratitis were analyzed. Phylogeny consisted of fragments of the large subunit of nuclear encoded ribosomal DNA (D1/D2, LSU) and the internal transcribed spacer region 1 of nuclear encoded ribosomal DNA (ITS1) as well as the protein coding gene translation elongation factor 1 alpha (TEF). Results revealed unanimously two Metarhizium granulomatis genotypes in a total of three isolates, various M. viride genotypes (n = 3), two different Purpureocillium lilacinum isolates as well as one isolate of each P. lavendulum and Beauveria bassiana. Purpureocillium lilacinum and B. bassiana are likewise frequently employed as a mycoinsecticide and mycoacaricide in agriculture on a worldwide scale and have occasionally been reported in man, causing fungal keratitis, sclerokeratitis, nosocomial infections in immunosuppressed patients, as well as cavitary pulmonary disease and cutaneous hyalohyphomycosis in immunocompetent patients. According to the results establishment of Biotyper analysis for faster differentiation of reptile-associated fungal pathogens is entirely justified.

Comparative analysis of humoral immune responses and pathologies of BALB/c and C57BL/6 wildtype mice experimentally infected with a highly virulent Rodentibacter pneumotropicus (Pasteurella pneumotropica) strain.

BACKGROUND: Mice are a natural host for Rodentibacter (R.) pneumotropicus. Despite specific monitoring, it is still one of the most important infectious agents in laboratory animals. The objective of this study was to determine the virulence of a prevalent pathotype of R. pneumotropicus and characterize the host response in a new animal model. RESULTS: Intranasal infection of C57BL/6 and BALB/c mice with a R. pneumotropicus strain (JF4Ni) bearing the genes of the three known repeats in toxin (RTX) toxins resulted in an unprecedented high mortality and morbidity above 50 and 80%, respectively. Morbidity was associated with severe weight loss as well as conjunctivitis and dyspnea. A main pathology was a catarrhal purulent to necrotic bronchopneumonia. Specific immune globuline (Ig) A was detected in tracheonasal lavages of most surviving mice which were still colonized by R. pneumotropicus. Furthermore, all surviving animals showed a distinct production of IgG antibodies. To differentiate T-helper cell (Th) 1 and Th2 immune responses we used subclasses of IgGs as indicators. Mean ratios of IgG2b to IgG1 were below 0.8 in sera drawn from both mice strains prior infection and from BALB/c mice post infection. In contrast, C57BL/6 mice had a mean IgG2b/IgG1 ratio of 1.6 post infection indicating a Th1 immune response in C57BL/6 versus a Th2 response in BALB/c mice associated with a tenfold higher bacterial load in the lung. In accordance with a Th1 response high antigen-specific IgG2c titers were detected in the majority of surviving C57BL/6 mice. CONCLUSIONS: R. pneumotropicus JF4Ni is a highly virulent strain causing severe pneumonia and septicemia after intranasal infection of C57BL/6 and BALB/c mice. Persisting infections in the two mice strains are associated with Th1 and Th2 immune responses, respectively, and differences in the bacterial burden of the lung. The described model is ideally suited for future vaccination studies using the natural host.

MALDI-TOF MS analysis of bovine and zoonotic Trichophyton verrucosum isolates reveals a distinct peak and cluster formation of a subgroup with Trichophyton benhamiae.

The zoophilic dermatophyte Trichophyton verrucosum is the most important causative agent of bovine dermatophytosis. Additionally, it causes profound and poorly healing skin infections in humans indicating the high zoonotic potential. The objective of this study was to establish differentiation of T. verrucosum from other dermatophytes by mass spectrometry and to identify distinct features of the mass spectra. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was successful for identification of this pathogen only after extension of the database of the manufacturer with spectra from T. verrucosum strains, which were identified as such by sequencing of the internal transcribed spacer (ITS) region. MALDI-TOF MS analysis was conducted with 46 field isolates from cattle, two live vaccine strains, and 10 isolates from humans identified as T. verrucosum by sequence analysis of the ITS region. The results suggest a very good agreement of both methods. Comparison with the mass spectra of 68 strains of other keratinophilic fungi revealed that most T. verrucosum wild-type isolates showed a characteristic peak at 7950-7954 m/z, which was missing in the spectra of other keratinophilic fungi and the live vaccine strains. The spectra of T. verrucosum were most similar to the spectra of T. benhamiae, an emerging zoophilic dermatophyte. In summary, MALDI-TOF MS is a powerful and reliable tool to identify T. verrucosum.

Characterization of Nannizziopsis guarroi with genomic and proteomic analysis in three lizard species.

Fungal infections in captive as well as in free-living reptiles caused by emerging obligate pathogenic fungi appear with increasing frequency and give occasion to establish new and fast methods for routine diagnostics. The so-called yellow fungus disease is one of the most important and common fungal dermatomycoses in central bearded dragons (Pogona vitticeps) and green iguanas (Iguana iguana) and is caused by Nannizziopsis guarroi. The aim of this study was to prove reliability in identification of N. guarroi with Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in comparison to molecular biological analysis of ribosomal DNA genes. In seven lizards from three different species, including central bearded dragons, green iguanas, and a European green lizard (Lacerta viridis), dermatomycoses caused by N. guarroi were diagnosed by isolation of the fungal pathogen as well as histopathological confirmation of the granulomatous inflammatory reaction in deep skin biopsies. With this survey, we proved that MALDI-TOF MS is a diagnostic tool for accurate identification of N. guarroi. Besides small subunit 18S rDNA (SSU) and internal transcribed spacer (ITS)1-5.8S rDNA, a large fragment of the large subunit of the 28S rDNA (LSU), including the domain (D)1 and D2 have been sequenced, for phylogenetical analysis. Large fragment of the LSU from N. guarroi has been sequenced for the first time. Yellow fungus disease in a European lizard species is described for the first time to our knowledge as well, which could be of importance for free-ranging populations of European lizards.

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.

Release kinetics of tumor necrosis factor-α and interleukin-1 receptor antagonist in the equine whole blood.

BACKGROUND: Horses are much predisposed and susceptible to excessive and acute inflammatory responses that cause the recruitment and stimulation of polymorphnuclear granulocytes (PMN) together with peripheral blood mononuclear cells (PBMC) and the release of cytokines. The aim of the study is to develop easy, quick, cheap and reproducible methods for measuring tumor necrosis factor alpha (TNF-α) and interleukin-1 receptor antagonist (IL-1Ra) in the equine whole blood cultures ex-vivo time- and concentration-dependently. RESULTS: Horse whole blood diluted to 10, 20 and 50 % was stimulated with lipopolysaccharide (LPS), PCPwL (a combination of phytohemagglutinin E, concanavalin A and pokeweed mitogen) or equine recombinant TNF-α (erTNF-α). TNF-α and IL-1Ra were analyzed in culture supernatants, which were collected at different time points using specific enzyme-linked immunosorbent assays (ELISA). Both cytokines could be detected optimal in stimulated 20 % whole blood cultures. TNF-α and IL-1Ra releases were time-dependent but the kinetic was different between them. PCPwL-induced TNF-α and IL-1Ra release was enhanced continuously over 24-48 h, respectively. Similarly, LPS-stimulated TNF-α was at maximum at time points between 8-12 h and started to decrease thereafter, whereas IL-1Ra peaked later between 12-24 h and rather continued to accumulate over 48 h. The equine recombinant TNF-α could induce also the IL-1Ra release. CONCLUSIONS: Our results demonstrate that similar to PCPwL, LPS stimulated TNF-α and IL-1Ra production time-dependently in whole blood cultures, suggesting the suitability of whole blood cultures to assess the release of a variety of cytokines in health and diseases of horse.

Agonist-induced ß2-adrenoceptor desensitization and downregulation enhance pro-inflammatory cytokine release in human bronchial epithelial cells.

It is not clear whether increased asthma severity associated with long-term use of ß2-adrenoceptor (ß2-AR) agonists can be attributed to receptor degradation and increased inflammation. We investigated the cross-talk between ß-AR agonist-mediated effects on ß2-AR function and expression and cytokine release in human bronchial epithelial cells. In 16HBE14o(-) cells grown in the presence and absence of ß-AR agonists and/or antagonists, the ß2-AR density was assessed by radioligand binding; the receptor protein and mRNA was determined using laser scanning cytometer and RT-PCR; cAMP generation, the cytokines IL-6 and IL-8 release were determined using AlphaScreen Assay and ELISA, respectively. Isoprenaline (ISO) and salbutamol (Salbu) induced a concentration- and time-dependent significant decrease in ß2-AR density. Both Salbu and ISO reduced cAMP generation in a concentration-dependent manner while in same cell culture the IL-6 and IL-8 release was significantly enhanced. These effects were antagonized to a greater extent by ICI 118.551 than by propranolol, but CGP 20712A had no effect. Reduction of the ß2-AR protein and mRNA could be seen when cells were treated with ISO for 24 h. Our findings indicate a direct link between cytokine release and altered ß2-AR expression and function in airway epithelial cells. ß2-AR desensitization and downregulation induced by long-term treatment with ß2-AR agonists during asthma may account for adverse reactions also due to enhanced release of pro-inflammatory mediators and should, thus, be considered in asthma therapy.

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.

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.

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.

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.

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.

Detection of Serum IgG Specific for Brachyspira pilosicoli and "Brachyspira canis" in Dogs.

Brachyspira pilosicoli (B. pilosicoli) is a pathogen in pigs, poultry, and humans causing colitis, diarrhea, and poor growth rates. Its role as a canine pathogen is controversial, and the seroprevalence of specific IgG antibodies against B. pilosicoli in dogs is unknown. A further, not yet officially recognized Brachyspira species in dogs is "Brachyspira canis" ("B. canis"), which is proposed to be apathogenic. This study evaluates enzyme-linked immunosorbent assays (ELISAs) measuring serum IgG antibodies specific for B. pilosicoli or "B. canis" and investigates levels of specific IgG antibodies against B. pilosicoli and "B. canis" in a cohort of clinical patients presented at an animal referral clinic. These ELISAs use detergent-extracted antigens from B. pilosicoli and "B. canis". To increase analytic specificity, we precipitated the antigens with trichloroacetic acid (TCA) to isolate and concentrate the respective protein fraction. Our results indicate that a large number of serum IgG antibodies bind to shared epitopes of detergent-extracted antigens of the two spirochaetes. Our data also suggest that dogs might not only carry B. pilosicoli but also have "B. canis"-specific serum IgG antibodies.

Immunoproteomics enable broad identification of new Aspergillus fumigatus antigens in severe equine asthma.

Introduction: Severe equine asthma (SEA) is a common chronic disease of adult horses with characteristic recurrent airway obstruction and similarities to neutrophilic asthma in humans. As an extrinsic stimulus, hay dust exposure is a major risk factor and induces acute exacerbation in susceptible horses. However, single inducing agents of SEA have hardly been identified on a molecular basis. Aspergillus fumigatus (A. fumigatus) is a common mold species in hay and has been described as a major provoking agent of SEA. Methods: Aiming to identify disease-relevant antigens, we analyzed A. fumigatus using an immunoproteomics approach on two-dimensional immunoblots of A. fumigatus protein probed with serum from environmentally matched asthmatic and healthy horses (n=5 pairs). A. fumigatus binding serum immunoglobulins (Pan-Ig), and the isotypes IgG4/7 and IgG3/5 were quantified for each protein spot and then compared between asthmatic and healthy horses. Results and discussion: For 21 out of 289 spots serum immunoglobulin (Ig) binding was different between the two groups for Pan-Ig or the isotypes. If differences were detected, Pan-Ig and IgG4/7 binding to the proteins were lower, while IgG3/5 binding was higher in asthmatic than healthy horse sera. Proteins were extracted from the 21 spots of interest and analyzed by liquid chromatography mass spectrometry. Eight prioritized proteins (candidate antigens) were expressed as recombinant proteins. Some of these have been previously described as major or minor A. fumigatus allergens, alongside other proteins, most with hydrolase activity. Recombinant candidate antigens were tested on 1D immunoblots to confirm their relevance as antigens by serum antibody binding. Four proteins (beta-hexosaminidase, class II aldolase/adducin domain protein, glucoamylase, peptide hydrolase B0XX53) showed different antibody binding characteristics between asthmatic and healthy horses and are likely relevant antigens in SEA. Their identification can provide the basis for innovative diagnostics, prevention, or therapeutic approaches. Additionally, a more profound understanding of SEA and its potential underlying mechanisms can be established. Elevated serum IgG3/5 antibodies correlate with T helper cell 2 responses in other equine pathologies, and the recombinant SEA antigens developed here can become instrumental in analyzing the involvement of SEA-specific T cell responses and Ig responses in future studies.

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.