REP-PCR tracking of the origin and spread of airborne Staphylococcus aureus in and around chicken house.

UNLABELLED: Staphylococcus aureus was used as an indicator to study the origin and spread of microbial aerosol in and around chicken houses. Air samples indoor, upwind (10 and 50 m), and downwind (10, 50, 100, 200, and 400 m) of four chicken houses were collected using Andersen-6 stages sampler. The concentrations of S. aureus were determined for every sample site. Isolation of S. aureus from chicken feces was performed according to the standard method. The genetic relationship among the isolates was determined by profiles of PCR-amplified repetitive extragenic palindromic (REP-PCR) elements. The results showed that the concentrations of S. aureus indoor of four chicken houses were higher than those upwind and downwind sites (P < 0.05 or P < 0.01), but there were no significant concentration differences among downwind sites (P > 0.05). The fingerprints and the phylogenetic tree indicated that a part of the S. aureus (55.6%, 10/18) isolates from indoor air had the same REP-PCR fingerprints as feces isolates. Consequently, most isolates (57.1%, 20/35) from downwind 10, 50, 100, 200, even 400 m had the same REP-PCR fingerprints as those from indoor or feces. These data indicated that some isolates from downwind and indoor originated from the chicken feces. However, those isolates from upwind had low similarity (similarity index 0.6-0.87) to those from indoor or feces. Therefore, the isolates upwind were not from the chicken feces or indoor. These results suggest that microbes in chicken feces can be aerosolized and spread indoor and outdoor, especially to downwind of the chicken houses. It should have an important epidemiological and public health significance. PRACTICAL IMPLICATIONS: Thus, the use of S. aureus as an indicator to study the origin and spread of airborne pathogens from chicken houses is potentially useful for enhancing public health and understanding the airborne epidemiology of this pathogen. Meanwhile it can provide evidence for studying the spreading model of airborne pathogens.

Clostridium perfringens toxin types from freshwater fishes in one water reservoir of Shandong Province of China, determined by PCR.

Four hundred and twenty intestinal content samples (not including intestinal tissues) of freshwater fishes (60 silver carps, 100 carps, 100 crucian carps, 60 catfishes and 100 zaieuws) caught from one water reservoir were examined bacteriologically for the occurrence of C. perfringens. Isolates were examined by polymerase chain reaction (PCR) for genes encoding the four lethal toxins (alpha, beta, epsilon and iota) for classification into toxin types and for genes encoding enterotoxin and the novel beta2 toxin for further subclassification. C. perfringens could be isolated in 75 intestinal contents samples (17.9%) from freshwater fish including: 13 silver carps, 2 carps, 12 crucian carps, 40 zaieuws, and 8 catfishes. In 75 isolates, 58 strains (77.3%) were C. perfringens toxin type C (alpha and beta toxin positive), 13 strains (17.3%) were toxin type A (alpha toxin positive) and 4 strains (5.3%) were toxin type B (alpha, beta and epsilon toxin positive). In addition, the gene encoding for beta2 toxin was found in 47 strains (62.7%) of all the isolates, seven from type A, two from type B, and 38 from type C. The gene encoding for enterotoxin was not found in any isolate. These amplified toxin gene fragment were cloned and sequenced and compared with reference strains, the identity varied from 98.15% to 99.29%. This is the first report of C. perfringens alpha, beta, epsilon, beta2 toxins in freshwater fish and of beta, epsilon toxins in fish in general, and is the first discovery that the beta2 toxin could be detected in strains of type B. The origin of this bacterium and its importance to human food poisoning in freshwater fish is discussed.

Isolation and characterization of Clostridium perfringens from apparently healthy animals of the Shandong province of China.

In a pilot study the presence and frequency of Clostridium (C.) perfringens was investigated among apparently healthy farm animals in the Shandong province of China. 748 faecal samples were collected from 9 pig-, 4 sheep-, 7 cattle- and 5 rabbit farms. C. perfringens was isolated from 124 samples (16.6%). The isolates were classified into major toxin types by using PCR analysis detecting the genes encoding these toxins. All isolates were identified as C perfringens toxin type A. There are also some reports from different regions in China linking C. perfringens toxin type A strains to gastrointestinal diseases. Therefore further investigations about the epidemiologic role of C perfringens toxin type A strains in the Shandong region are necessary. Currently, cases of enterotoxemia from this region are investigated for the presence of C perfringens.

Detection of strain-specific antigenic epitopes on the lipo-oligosaccharide of Haemophilus parasuis by use of monoclonal and polyclonal antibodies.

OBJECTIVE: To investigate the antigenic diversity of lipo-oligosaccharides of Haemophilus parasuis. PROCEDURES: Immunoblot assays were done with monoclonal and polyclonal antibodies on whole-cell lysates. Individual colonies of H parasuis strains H 54, H 53, and H 128 were tested for reactivity with lipo-oligosaccharide-specific monoclonal antibodies after a single passage on chocolate agar, and colonies of strain H 54 were analyzed after 10 passages. Colony blot tests were used to screen H parasuis strains for spontaneously occurring antigenic variation in their lipo-oligosaccharides. RESULTS: Eight H parasuis strains were separated into 4 lipo-oligosaccharide serovars on the basis of immunoblot reactions with 3 polyclonal rabbit antisera. Nine monoclonal antibodies against lipo-oligosaccharides of a lipo-oligosaccharide-serovar I strain reacted with all tested serovar I strains but failed to react with other H parasuis strains. CONCLUSIONS: Variations in the antigenic reactivity after 1 or 10 passages on chocolate agar were not observed. The serovar I lipo-oligosaccharide strains included virulent as well as avirulent H parasuis strains, indicating that these epitopes do not correlate directly with virulence properties of H parasuis.

Determination of the inflammatory potential of bioaerosols from a duck-fattening unit by using a limulus amebocyte lysate assay and human whole blood cytokine response.

Inhalation of bioaerosols from animal houses can induce acute inflammatory reactions in the respiratory tract. Determination of the concentration of airborne endotoxins is widely used to characterize this risk. In this study, the activity of bioaerosol samples from a duck-fattening unit to induce interleukin-1beta (IL-1beta) in human blood and to react with Limulus Amebocyte Lysate (LAL) was investigated. The activity detected in the whole blood assay correlated well with the endotoxic activity found in the LAL assay (Spearmen's rho = 0.902). However in all samples, the inflammation-inducing potential was overestimated by the LAL assay. It is assumed that this overestimation could be, in part, a result of an overestimation of the inflammatory potential of endotoxins originating from Pseudomonadaceae by the LAL assay. Pseudomonadaceae were regularly isolated from the air of the duck-fattening unit. The results presented here indicate that the whole blood assay can be used besides the LAL assay as an additional method to characterize the inflammation-inducing potential of bioaerosols.

[Investigations on airborne microorganisms in animal stables. 3: Relationship between inhalable endotoxin, inhalable dust and airborne bacteria in a hen house]. / Untersuchungen zum Luftkeimhaushalt in Tierställen 3. Mitteilung: Beziehungen zwischen einatembarem Endotoxin, einatembarem Staub und luftgetragenen Bakterien in einer Legehennenbatterie.

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.

[Air microorganisms in animal housing--4. Airborne gram-negative bacteria and airborne endotoxin in pig houses]. / Untersuchungen zum Luftkeimhaushalt in Tierställen--4. Mitteilung: Luftgetragene gramnegative Bakterien und luftgetragenes Endotoxin in Schweineställen.

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.

[Effect of transport stress on the content of endotoxin in blood of slaughter pigs]. / Auswirkungen von Transportbelastungen auf den Endotoxingehalt im Blut von Schlachtschweinen.

Stress during transportation of slaughter pigs could lead to an increased level of translocation of endotoxin from the gut. In particular, during longtime transportation increased concentrations of endotoxin in blood are likely. Since nonlethal doses of endotoxin could trigger translocation of bacteria from the gut to systemic organs an endogenous contamination of the carcasses also with human pathogens may occur. Therefore, it is necessary to evaluate transportation regimes of slaughter animals also with regard to food-borne infections and intoxications.

[Airborne microorganisms in animal stables. 1. Anaerobic airborne bacteria in a calf stable with special regard to Clostridium perfringens]. / Untersuchungen zum Luftkeimhaushalt in Tierställen. 1. Mitteilung: Der anaerobe Luftkeimgehalt in einem Kälberstall unter besonderer Berücksichtigung von Clostridium perfringens.

The total number of airborne anaerobic bacteria with special regard to Clostridium perfringens (C. perfringens) as well as the total number of airborne aerobic bacteria was estimated indoor and outdoor of a calf stable. 65 indoor samples and 63 outdoor samples were collected by using a 6 stage Andersen sampler. The total number of airborne anaerobic bacteria of the indoor samples ranged from 3,216 to 24,222 CFU/m3 (Colony Forming Units) with a part of 4 to 1179 CFU/m3 of C. perfringens. 734 to 1588 CFU/m3 of airborne anaerobic bacteria were found in the outdoor samples. The number of C. perfringens CFU varied between 2 and 15 CFU/m3. The total number of airborne aerobic bacteria indoor ranged from 9,166 to 53,932 CFU/m3, outdoor from 2,443 to 21,205 CFU/m3. Furthermore the results suggested that C. perfringens is suitable for indicating the bacterial contamination of air by faeces of animals.

Airborne gram-negative bacterial flora in animal houses.

The concentration and the species composition of airborne gram-negative bacteria were studied in four cattle houses, one pig house and one poultry barn. On average only between 0.02 and 5.2% of the total number of culturable aerobic bacteria were identified as gram-negative bacteria. Obligate anaerobic gram-negative bacteria were not isolated at all. In the airborne gram-negative bacterial flora the following bacterial families dominated: the Enterobacteriaceae, the Pseudomonadaceae and the Neisseriaceae. Within the family of the Enterobacteriaceae the species Escherichia coli and Enterobacter agglomerans were predominant. In animal houses using straw as bedding material Ent. agglomerans was most frequent, whereas in animal houses without litter E. coli was mainly found. Airborne Neisseriaceae were isolated very frequently in cow barns with Acinetobacter lowffii as the primary species. Airborne Pseudomonadaceae were found in high concentrations during periods of high air humidity. The results presented may also give some indications on the origin and sources of airborne endotoxins in animal housing.

Investigations on airborne microorganisms in animal stables. 2. Report: further characterization of airborne Clostridium perfringens.

Toxovars of 97 airborne C. perfringens isolates and 10 C. perfringens isolates from fecal samples of a calf stable were determined by an EIA procedure. Most airborne and fecal isolates belonged to toxovar A (88.7% and 80.0% respectively). Eight point two% of airborne C. perfringens were identified as toxovar C and 3.1% as toxovar D. Toxovar B was not found in the airborne state. Twenty% of fecal C. perfringens belonged to toxovar D. Toxovar B and C was not isolated from fecal samples. In addition, all fecal and air-borne isolates of C. perfringens toxovar D strains were analyzed in SDS-PAGE for their polypeptide pattern. All isolates from both sources exhibited the same polypeptide pattern after electrophoretic analysis in SDS-PAGE. Both results, determination of toxovars as well as polypeptide pattern analysis in SDS-PAGE, suggest that a major source of airborne C. perfringens in animal stables is animal feces.

Short communication: bovine paraplegic syndrome (Mal de Aquidabàn) in Paraguay caused by C. perfringens toxovar D.

In the vicinity of the river Aquidabán in Eastern Paraguay occurred in the last years an endemic disease within cattle herds which killed thousands of heads (Mal de Aquidabán [MdA]). The disease was seen in form of an afebrile, paralytic condition characterized by muscle tremors, swaying of the hind quarters, recumbence and sudden death of the animals. Therefore it was described as "Bovine Paraplegic Syndrome". Comparable symptoms in adult cattle have also being seen in Eastern and Central Venezuela. After we found in the peritoneal fluid of 2 animals, which died under typical symptoms, toxins of C. perfringens toxovar D, we started to investigate the occurrence of C. perfringens in the feces of clinical healthy animals on farms, where MdA was diagnosed before. There we found 73% of the isolated strains belonging to toxovar D. In herds with no case of the disease toxovar A was isolated up to 90%. The next step was the production of a vaccine (anaculture) with a high toxin producing strain of C. perfringens toxovar D. 2000 cattle were vaccinated for 3 years twice a year. None of the vaccinated animals diseased. In 2 control-herds some cases of MdA occurred. But we became quite sure that MdA was an infectious disease, when we started to vaccinate cattle of unvaccinated herds where MdA suddenly occurred. One or two animals died in the next 2 days and after this period the endemic disease was immediately stopped.