Investigations on airborne microorganisms in animal stables. 3: Relationship between inhalable endotoxin, inhalable dust and airborne bacteria in a hen house

The relationship between inhalable endotoxin, inhalable dust and airborne bacteria was studied in a hen house. Neither the concentration of inhalable dust nor the concentration of airborne bacteria are suitable to reflect the concentration of airborne endotoxin. Furthermore it was found that the endotoxic activity can persist over a long period of time in dust samples. Therefore an accumulation of endotoxin in different environments is possible. Airborne endotoxin seems to be a suitable marker to characterize exposure to airborne organic dust, since this toxin is responsible for different respiratory diseases (e.g. toxic pneumonitis, airway obstructions) and the toxic activity of endotoxin in dust samples is known to persist for a long time.     

Airborne microorganisms in animal stables: stability of endotoxins in the environment

The stability of endotoxins was investigated in potential sources (feed-stuff, litter, water, excrements, surface dust) of that air contaminant and in the airborne state. No changes in the endotoxic activity were found in materials with high dry matter (straw, hay, dried faeces) during 84 days. In manure samples stages of increasing and decreasing endotoxic activity were observed. In water a continuous decline of the endotoxic activity was found. However this process was characterised by half-life periods on a weekly scale. Bacterial degradation seems to be responsible for that loss of endotoxic activity.     

Susceptibility of Clostridium perfringens of animal origin to fifteen antimicrobial agents

The minimal inhibitory concentrations of fifteen antibacterial agents were determined by agar-dilution method against 121 strains of Clostridium perfringens isolated from pigs, cattle and poultry. All strains, regardless of host of origin, were susceptible to avoparcin, furazolidone, monensin, nitrovin, penicillin G, ronidazole and tiamulin and resistant to flavomycin. Poultry strains were also susceptible to carbadox, chloramphenicol, erythromycin and virginiamycin. Bacitracin-resistant poultry strains were susceptible to all tested antibacterial agents except tetracycline, but the bacitracin-resistant cattle strains were polyresistant. Porcine strains were susceptible to bacitracin and bovine strains to carbadox. Carbadox-resistant porcine strains were resistant to erythromycin, lincomycin and tetracycline and susceptible to chloramphenicol. Resistance to erythromycin was associated with resistance to lincomycin. High level erythromycin-lincomycin-resistant strains were susceptible to virginiamycin, but the intermediate level erythromycin-lincomycin-resistant cattle strains were resistant to virginiamycin. Resistance to chloramphenicol or erythromycin-lincomycin was always associated with resistance to tetracycline but the reverse was not always true.     

Genotypic and phenotypic characterization of Clostridium perfringens and Clostridium difficile in diarrheic and healthy dogs

The objectives of this study were to examine the potential roles of Clostridium difficile and enterotoxigenic Clostridium perfringens in diarrhea in dogs by comparison of isolation, determination of toxin status via enzyme-linked immunosorbent assay (ELISA), and application of multiplex polymerase chain reaction (PCR). These techniques were used to evaluate fecal specimens in 132 healthy and diarrheic dogs. These dogs were prospectively evaluated by grouping them into the following 3 categories: hospitalized dogs with diarrhea (n = 32), hospitalized dogs without diarrhea (n = 42), and apparently healthy outpatient dogs without diarrhea (n = 58). All fecal specimens were cultured using selective media for C difficile, Salmonella spp., and Campylobacter spp. and selective media after heat shock for C perfringens. No significant difference was found in the isolation of C perfringens or C difficile among the 3 groups. A significant association was found between the presence of diarrhea and detection of C perfringens enterotoxin (CPE) or toxin A via ELISA for both C perfringens and C difficile, respectively. PCR performed on C difficile isolates for toxin A and toxin B genes revealed no significant differences among the 3 groups, but diarrheic dogs were significantly more likely to be positive for the enterotoxin gene of C perfringens. Based on the results of this study, the use of ELISA for detection of CPE in feces combined with the detection of enterotoxigenic fecal isolates obtained via heat shock provides the strongest evidence for the presence of C perfringens-associated diarrhea.     

Air microorganisms in animal housing--4. Airborne gram-negative bacteria and airborne endotoxin in pig houses

The species composition of airborne gram-negative bacteria and the relationship between inhalable endotoxin, inhalable dust and airborne bacteria were studied in 4 pig houses. The airborne gram-negative bacterial flora was dominated by Enterobacteriaceae. Within the Enterobacteriaceae the species E. coli and Enterobacter agglomerans were predominant. Significant correlation were found between the concentration of inhalable dust and inhalable endotoxin as well as between the concentration of airborne gram-negative bacteria and inhalable endotoxin. However these correlation were not very strong. With respect to the characterization of the potential hazard of organic dust exposure, measurements of the concentration of airborne dust or airborne bacteria should not be used for the estimation of the concentration of airborne endotoxin.     

The majority of atypical cpb2 genes in Clostridium perfringens isolates of different domestic animal origin are expressed

This study examined the prevalence and expression of the "consensus" and the "atypical"cpb2 genes in Clostridium perfringens isolates from cattle, chickens, dogs, goats, horses, pigs and sheep using polymerase chain reaction (PCR), sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting. Almost all porcine isolates (12/14) carried and expressed the consensus form of cpb2 but, when present in 108 non-porcine isolates, the gene was usually the atypical form (40 atypical versus 9 consensus). Western blotting showed expression in 30 of 40 (75%) atypical cpb2-positive isolates, considerably more frequently than reported previously. CPB2 was expressed by almost all (20/21) the consensus cpb2-positive isolates, regardless of source.     

波尔山羊产气荚膜梭菌并发大肠杆菌的诊断报告

波尔山羊是近年来各地广泛引种,进行繁殖育种和杂交的优质肉羊品种。波尔山羊与当地山羊杂交,杂种羊表现出较强的杂种优势,生长快、抗病力强、饲料转化率高,成为养羊户的热门首选羊,给波尔山羊种羊饲养者带来了极高的利润,普通肉羊饲养户也从中获得了效益。     

Prevalence of Clostridium perfringens in commercial broiler hatcheries.

Clostridium perfringens, a cause of human foodborne and poultry disease, has been isolated from the intestinal tract of poultry and from the processed carcass. Little is known about the incidence and sources of this pathogen in the poultry production environment. To determine if the broiler hatchery is a possible source of C. perfringens, we collected samples from three hatcheries, each operated by a different poultry integrator, and the presence of C. perfringens in these samples was determined. For each sampling period, eggshell fragments, chick fluff from the hatcher, and paper pads stored in the hatchery before use with chicks and after placement beneath chicks for 1 hr were evaluated. Clostridium perfringens was found in eggshell fragments, fluff, and paper pads in each of the three hatcheries. The percentages of C. perfringens-positive samples from the three hatcheries ranged from 13% to 23%, with an overall incidence of 20%. Positive samples were consistently found, i.e., detected on each of the nine sampling days (three sampling days for each of three hatcheries). These results suggest that the hatchery is a potential source/reservoir for C. perfringens in the integrated poultry operation.     

Isolation and characterization of antibodies to Clostridium perfringens epsilon toxin from hyperimmune horse serum.

Antibodies against epsilon toxin were isolated from hyperimmune horse serum by affinity chromatography. Purified epsilon prototoxin covalently bound to Affigel 202 was used as immunosorbent, and antibodies were eluted with 6.0 M guanidine chloride. In a single run 80 mg of antibody could be recovered from a 20 microliter column of immunosorbent. The antibody was shown to belong to the IgG(T) class of immunoglobulins.     

Real-time multiplex PCR assays for reliable detection of Clostridium perfringens toxin genes in animal isolates

Typing of Clostridium perfringens strains by PCR-based determination of toxin genes proved to be a reliable method for diagnosis of enterotoxaemia in various animal species. We report the establishment and validation of three real-time fluorogenic (TaqMan) multiplex PCRs for the detection of C. perfringens alpha-, beta-, beta2-, epsilon-, entero- and iota-toxin genes. The composition of the PCRs was chosen with regard to robustness of the assays and in order to increase sensitivity compared to the conventional simplex PCRs. The combination of probe dyes selected for the real-time assays (FAM/TAMRA, Cy-5/BHQ-2 and VIC/TAMRA) as well as the designation of the chromosome-borne alpha-toxin as internal positive control allowed the creation of highly specific and sensitive, as well as time and cost effective PCRs. One hundred and three strains of C. perfringens isolated in Switzerland derived from clinical or suspected cases of enterotoxaemia in 10 different animal species were tested. The toxin genotypes were in agreement in both the conventional PCRs and the newly designed multiplex PCRs. Furthermore, the real-time PCR carried out as simplex allows to quantitate the copy numbers of plasmid-borne toxin genes in relation to the chromosomally located alpha-toxin gene.     

Recurrent diarrhea associated with enterotoxigenic Clostridium perfringens in 2 dogs

Two dogs were diagnosed with enterotoxigenic Clostridium perfringens-associated diarrhea. Diarrhea was responsive to antimicrobial therapy, but recurred after treatment was ceased. Clostridium perfringens enterotoxin was present in feces during diarrheic episodes but not when feces were normal. Both dogs responded to a prolonged course of oral cephalexin and dietary modification.     

产气荚膜梭菌α毒素的研究进展

产气荚膜梭菌 (Clostridiumperfringens)的致病作用一般由它所产生的胞外酶或毒素诱导。主要有α -毒素、β -毒素、θ -毒素及其他的一些胞外酶。本文针对产气荚膜梭菌α毒素的组成、结构、理化性质、作用机理及分子遗传等方面的研究进展作了简要综述。     

禽产气荚膜梭菌研究进展

产气荚膜梭菌（Clostridium perfringens,CP）是重要的食源性病原体之一,可对人类和动物健康造成严重威胁。CP在家禽中主要引起禽坏死性肠炎,严重影响了全球肉鸡贸易与生产。关于禽CP的研究当前主要集中于检测及分型方法、疫苗和耐药性方面。目前禽CP疫苗多处于实验室研发阶段,因此禽CP感染的防治主要依靠抗生素,而抗生素的频繁使用又导致了菌株耐药性问题,防治效果有限。因此,CP耐药性研究以及临床诊断技术完善及疫苗研发推进迫在眉睫。本文简述了CP的病原特点、致病机理及禽间的优势流行分型,梳理了禽CP不同检测方法的优点及分型方法,阐述了禽CP耐药现状以及疫苗研发方向及进展,提出了禽CP感染辅助防治措施,以期为CP感染防控提供参考。     

Detection and characterization of Clostridium perfringens in the feces of healthy and diarrheic dogs

Clostridium perfringens has been implicated as a cause of diarrhea in dogs. The objectives of this study were to compare 2 culture methods and to evaluate a multiplex polymerase chain reaction (PCR) assay to detect C. perfringens toxin genes alpha (α), beta (β ), beta 2 (β2), epsilon (ɛ), iota (ι), and C. perfringens enterotoxin (cpe) from canine isolates. Fecal samples were collected from clinically normal non-diarrheic (ND) dogs, (n = 105) and diarrheic dogs (DD, n = 54). Clostridium perfringens was isolated by directly inoculating stool onto 5% sheep blood agar (SBA) and enrichment in brain-heart infusion (BHI) broth, followed by inoculation onto SBA. Isolates were tested by multiplex PCR for the presence of α, β, β2, ɛ, ι, and cpe genes. C. perfringens was isolated from 84% of ND samples using direct culture and from 87.6% with enrichment (P = 0.79). In the DD group, corresponding isolation rates were 90.7% and 93.8% (P = 0.65). All isolates possessed the α toxin gene. Beta (β), β2, ɛ, ι, and cpe toxin genes were identified in 4.5%, 1.1%, 3.4%, 1.1%, and 14.8% of ND isolates, respectively. In the DD group, β and β2 were identified in 5%, ɛ and ι were not identified, and the cpe gene was identified in 16.9% of isolates. Enrichment with BHI broth did not significantly increase the yield of C. perfringens, but it did increase the time and cost of the procedure. C. perfringens toxin genes were present in equal proportions in both the ND and DD groups (P ≤ 0.15 to 0.6). Within the parameters of this study, culture of C. perfringens and PCR for toxin genes is of limited diagnostic usefulness due to its high prevalence in normal dogs and the lack of apparent difference in the distribution of toxin genes between normal and diarrheic dogs.     

The partial purification of Clostridium perfringens beta toxin.

An attempt was made to purify Clostridium perfringens Beta toxin. Crude toxin prepared by ammonium sulphate precipitation of culture supernatants was purified by chromatography on Sephadex G50, Sephadex G100 and DEAE cellulose. This material, although highly purified was not homogeneous on polyacrylamide gel electrophoresis. It had a toxicity of 800 000 mouse MLDs/mg N, a typical protein absorption spectrum in the UV region, an iso-electric point of 5, 6 and the main component had a molecular mass of 42 000 +/- 2 000 (estimated by electrophoresis in sodium dodecyl sulphate containing polyacrylamide gels).