A combined Clostridium perfringens/Trueperella pyogenes inactivated vaccine induces complete immunoprotection in a mouse model.

Clostridium perfringens (C. perfringens) and Trueperella pyogenes (T. pyogenes) are two bacterial pathogens frequently associated with wound infections and following lethal complications in livestock. However, prudent use of antimicrobial agents is highly required given the emergence of multidrug-resistant strains of both bacteria and need for food safety. In the current study, a combined vaccine, composed of inactivated C. perfringens and T. pyogenes, was prepared. The amount of formaldehyde being used to inactivate two bacteria was optimized to retain the immunogenicity of antigens. Three adjuvants were tested for their potency in improving specific immune responses against the candidate antigens. Then inactivated combined C. perfringens/T. pyogenes vaccine was prepared using inactive cultures of two organisms. The ratio of inactive cultures of two organisms for preparation of combined vaccine was optimized to gain effective protective immunity against the two pathogens. Results revealed that combined C. perfringens/T. pyogenes inactive vaccine can elicit high level of exotoxins and cell-associated antigen-specific antibodies and induce complete protection against C. perfringens and T. pyogenes infections in mice. The combined vaccine could be used as an alternative of antibiotics for prevention of C. perfringens and T. pyogenes infections in animals.

Subcutaneous immunization with inactivated bacterial components and purified protein of Escherichia coli, Fusobacterium necrophorum and Trueperella pyogenes prevents puerperal metritis in Holstein dairy cows.

In this study we evaluate the efficacy of five vaccine formulations containing different combinations of proteins (FimH; leukotoxin, LKT; and pyolysin, PLO) and/or inactivated whole cells (Escherichia coli, Fusobacterium necrophorum, and Trueperella pyogenes) in preventing postpartum uterine diseases. Inactivated whole cells were produced using two genetically distinct strains of each bacterial species (E. coli, F. necrophorum, and T. pyogenes). FimH and PLO subunits were produced using recombinant protein expression, and LKT was recovered from culturing a wild F. necrophorum strain. Three subcutaneous vaccines were formulated: Vaccine 1 was composed of inactivated bacterial whole cells and proteins; Vaccine 2 was composed of proteins only; and Vaccine 3 was composed of inactivated bacterial whole cells only. Two intravaginal vaccines were formulated: Vaccine 4 was composed of inactivated bacterial whole cells and proteins; and Vaccine 5 was composed of PLO and LKT. To evaluate vaccine efficacy, a randomized clinical trial was conducted at a commercial dairy farm; 371 spring heifers were allocated randomly into one of six different treatments groups: control, Vaccine 1, Vaccine 2, Vaccine 3, Vaccine 4 and Vaccine 5. Late pregnant heifers assigned to one of the vaccine groups were each vaccinated twice: at 230 and 260 days of pregnancy. When vaccines were evaluated grouped as subcutaneous and intravaginal, the subcutaneous ones were found to significantly reduce the incidence of puerperal metritis. Additionally, subcutaneous vaccination significantly reduced rectal temperature at 6±1 days in milk. Reproduction was improved for cows that received subcutaneous vaccines. In general, vaccination induced a significant increase in serum IgG titers against all antigens, with subcutaneous vaccination again being more effective. In conclusion, subcutaneous vaccination with inactivated bacterial components and/or protein subunits of E. coli, F. necrophorum and T. pyogenes can prevent puerperal metritis during the first lactation of dairy cows, leading to improved reproduction.

Interview: Investigating immunomodulators among the Actinomycetales. Interview by Hannah Wilson.

John L Stanford speaks to Hannah Wilson, Assistant Commissioning Editor John L Stanford is Chief Scientific Officer at BioEos Ltd (Kent, UK). Dr Stanford began his career as a senior lecturer and then reader in microbiology at Middlesex Hospital Medical School (London, UK), then University College London Medical School, where he became Professor in Medical Microbiology and Head of Department in 1997. He retired and became Professor Emeritus in 2004. Dr Stanford's career has been devoted to research into mycobacteria, the diseases that they cause and the practical uses of this research. His special interest in recent years has been the development of bacterial immunotherapeutics for a range of diseases including tuberculosis and cancer. Dr Stanford was one of the founding directors of Stanford Rook Ltd (London) and of BioEos Ltd, where he remains a director. He also played a part in the founding of Immodulon Therapeutics Ltd (London) and of a new company, ActinoPharma Ltd (London), and has published more than 200 peer-reviewed scientific papers.

Beneficial effects of immunotherapy with extracts derived from Actinomycetales on rats with spontaneous obesity and diabetes.

AIMS: To determine whether immunotherapy with heat-killed, selected Actinomycetales species could influence the progression of spontaneous Type 2 diabetes mellitus and obesity in a rat model. MATERIALS & METHODS: Preparations of either Gordonia bronchialis, Tsukamurella inchonensis or a saline placebo were given by three subcutaneous injections, 30 days apart, starting when rats were aged 120 days, just before development of Type 2 diabetes mellitus, and at day 440, when the disease was well established. Bodyweight, blood sugar, cholesterol, triglycerides and insulin levels were measured to determine the effects and at the end of the experiments, animals were subjected to necropsy. RESULTS: The development of Type 2 diabetes mellitus was prevented by both reagents, most effectively by T. inchonensis. In the treatment experiment, the effects of the disease were reduced by both treatments, markedly so by T. inchonensis. In both experiments obesity was reduced in treated animals. The possible mechanisms of action are discussed. CONCLUSION: Our findings suggest that Type 2 diabetes mellitus in the studied rats is associated with obesity, and that both diabetes and obesity can be prevented or improved by treatment with Actinomycetales immune modulators.

Successful immunotherapy of canine flea allergy with injected Actinomycetales preparations.

AIMS: Can heat-killed, borate-buffered suspensions of Gordonia bronchialis, Rhodococcus coprophilus or Tsukamurella inchonensis be used to treat canine flea allergy? MATERIALS & METHODS: Organisms cultured on Sauton's medium into stationary phase were autoclaved in borate-buffered saline and stored at 10 mg wet weight/ml. Intradermal injections of 0.1 ml containing 1 mg of bacilli were administered on the first and 20th days of the study. G. bronchialis and R. coprophilus were most effective in a pilot study of a small number of dogs with flea allergy. A larger number of affected dogs were then randomized to receive placebo or either of the two selected reagents. The extent and severity of allergic signs and symptoms were scored and blood samples were collected just before the first injection and 28 days after the second. RESULTS: Both selected reagents reduced the extent and severity of lesions (p < 0.001) and reduced scratching. Eosinophil numbers were reduced (p < 0.0001) between the first and second assessment. CONCLUSIONS: Injections of G. bronchialis or R. coprophilus effectively reduce the signs and symptoms of flea allergy in dogs.

Maternal immunization with actinomycetales immunomodulators reduces parasitemias in offspring challenged with Trypanosoma cruzi.

This article describes the first use of heat-killed, borate-buffered preparations of aerobic actinomycetales to immunize pregnant animals in order to determine the effect on their pregnancy and fertility and the survival coefficients of their offspring. Pregnant rats received three injections of Gordonia bronchialis, Rhodococcus coprophylus or physiological saline and a proportion of their offspring were challenged with live Trypanosoma cruzi at the time of weaning. Levels of parasitemia and, in some animals, of the cytokines IFN-γ and IL-10 were measured. The progress of pregnancy, fertility and survival of offspring were unaffected by the maternal immunizations. The offspring of rats immunized with G. bronchialis displayed significantly reduced parasitemias, with increased levels of IFN-γ and reduced levels of IL-10, 4 days after challenge. The offspring of rats immunized with R. coprophylus displayed greater parasitemias than did those of the control group. These unexpected results are discussed and their causation considered.

Assessment of Dietzia subsp. C79793-74 for treatment of cattle with evidence of paratuberculosis.

The objective of the present investigation was to determine whether the bacterium Dietzia subsp. C79793-74, previously shown to inhibit growth of Mycobacterium subsp. paratuberculosis under in vitro culture conditions, has therapeutic value as a probiotic for adult cattle with paratuberculosis.  Animals were obtained from several herds with evidence of disease based on seropositivity and/or fecal shedding.  Sixty-eight cows with initial evidence of Stage II or III paratuberculosis and 2 with an initial Stage IV disease were evaluated longitudinally.  Animals were either treated daily with variable, disease-dependent doses of Dietzia (n = 48) or left untreated (n = 22).  Clinical aspects of disease (diarrhea, emaciated, cachectic and appetite) were recorded until the animal recovered or required euthanasia due to advanced clinical paratuberculosis or other severe conditions.  Paratuberculosis parameters-antibody serology (ELISA, AGID) and fecal culture-were longitudinally monitored over the lifetime of each animal.  The results indicated that daily treatment with Dietzia was therapeutic for paratuberculosis cows based on: (a) longitudinal decline in ELISA values only occurred in animals that were treated; (b) prolonged survival was dependant upon treatment--the length being directly associated with low initial ELISA values; and (c) treated animals were the only ones cured of disease.  Further investigations are envisaged to determine optimal, long-term dosages that may result in even better therapeutic outcomes as well as to evaluate potential application for therapy of the Johne's disease, human-counterpart, Crohn's disease.

Comparison of specificity and sensitivity of immunochemical and molecular techniques for determination of Clavibacter michiganensis subsp. michiganensis.

Detection of Clavibacter michiganensis subsp. michiganensis (Cmm), causing bacterial canker of tomato, was verified using PTA-ELISA and IFAS with PAbs of Neogen Europe Ltd. (UK), and with published and also laboratory-generated PCR primers from the Cmm tomatinase gene. The specificity of this technique was determined with 15 plant-pathogenic and 4 common, saprophytic bacteria. With IFAS, crossreactions were found for Pantoea dispersa, P. agglomerans and Rahnella aquatilis, and with PTA-ELISA for Curtobacterium flaccumfaciens, Pectobacterium atrosepticum and Dickeya sp. Cross-reactions with subspecies other than michiganensis were also found using both methods. Molecular methods were optimized by verification of annealing temperatures and times for both primers. Conditions were finally adjusted to 30 s at 65 degrees C for Dreier's and 10 s at 69 degrees C for our primer set. After this optimization, both primer pairs produced positive reaction only with Cmm. By means of PTA-ELISA and IFAS, Cmm strains were detected at a concentration up to 10(5) CFU/mL and 10(3) CFU/mL, respectively. The PCR test with bacterial cell suspensions reached a sensitivity of 10(3) CFU/mL with our designed primers and 104 CFU/mL with Dreier's primer pair.

Immunotherapy for the prevention of myointimal hyperplasia after experimental balloon injury of the rat carotid artery.

We assessed the effect of novel immunotherapeutic heat-killed bacterial (Actinomycetales) preparations on the development of myointimal hyperplasia (MIH) in a rat carotid balloon trauma model and the effect on the immune response by measuring the expression of interferon gamma (IFN-gamma; (Th1) and interleukin 4 (IL-4; Th2). There was a significant reduction (P < .001) in intima/media ratios (mean +/- SEM) in the rats treated by immunomodulation (0.52 +/- 0.03 Gordonia bronchialis, 0.60 +/- 0.03 Rhodococcus coprophilus, 0.43 +/- 0.03 Tsukamurella inchonensis, 0.37 +/- 0.03 Mycobacterium vaccae), in comparison with untreated controls (0.91 +/- 0.05). Postballoon trauma G bronchialis increased messenger RNA (mRNA) IFN-gamma (P < .02) and reduced mRNA IL-4 (P < .05). R coprophilus, T inchonensis, and M vaccae significantly increased production of mRNA IFN-gamma (P < .001). R coprophilus and M vaccae also decreased production of mRNA IL-4 (P < .05, P < .01). Treatment with heat-killed Actinomycetales inhibits MIH through a combination of enhanced Th1 and attenuated Th2 response. Immunomodulation may provide a novel therapeutic option to prevent restenosis.

Fleece rot and dermatophilosis in sheep.

Fleece rot and dermatophilosis reduce health and production of sheep and predispose them to blow fly strike. This paper reviews aetiology, prevalence, pathogenesis, resistance, attempts to develop vaccines and prospects for new control strategies to these important skin diseases. Although the severity of fleece rot is associated with the abundance of Pseudomonas aeruginosa on skin, microbial ecology studies are providing new insights into the contribution of other bacteria to the disease. Wool traits and body conformation traits that predispose sheep to fleece rot and dermatophilosis are heritable and have been used as indirect selection criteria for resistance for many years. Selection against BoLA-DRB3-DQB class II haplotype in cattle can substantially reduce the prevalence of dermatophilosis and holds promise for identification of gene markers for resistance to these bacterial diseases in sheep. Immune responses in skin and systemic antibody responses to bacterial antigens are acquired through natural infection and contribute to resistance; however, prototype antibacterial vaccines have to date failed to provide protection against the diversity of isolates of Dermatophilus congolensis and Pseudomonas species present in the field. Opportunities for future control through breeding for resistance, vaccines and non-vaccine strategies for controlling the microbial ecology of fleece are discussed. In combination, control strategies need to reduce the risk of transmission, minimise exposure of animals to stressors that enhance the risk of infection, and enhance resistance though genetics or vaccines.

Improved outcome of Trypanosoma cruzi infection in rats following treatment in early life with suspensions of heat-killed environmental Actinomycetales.

The well-established model of Chagas' disease in "l" rats was used to evaluate the effects of three injections of heat-killed Gordonia bronchialis, Rhodococcus coprophilus or saline on Trypanosoma cruzi parasitaemia and acute and chronic myocarditis, sequelae of the infection. Two vaccinating injections were given prior to challenge with T. cruzi, and the third, immunotherapeutic, injection was given 7 days after challenge. Treatment with either actinomycete significantly reduced acute parasitaemia (p<0.04), modified cellular infiltration during acute myocarditis and limited chronic myocarditis (p<0.03) in comparison with the saline-treated control animals. Immunological investigations showed that both bacterial preparations achieved their results through different mechanisms. The relevance of our findings to human Chagas' disease is discussed.

Reduced peripheral and mucosal Tropheryma whipplei-specific Th1 response in patients with Whipple's disease.

Whipple's disease is a rare infectious disorder caused by Tropheryma whipplei. Major symptoms are arthropathy, weight loss, and diarrhea, but the CNS and other organs may be affected, too. The incidence of Whipple's disease is very low despite the ubiquitous presence of T. whipplei in the environment. Therefore, it has been suggested that host factors indicated by immune deficiencies are responsible for the development of Whipple's disease. However, T. whipplei-specific T cell responses could not be studied until now, because cultivation of the bacteria was established only recently. Thus, the availability of T. whipplei Twist-Marseille(T) has enabled the first analysis of T. whipplei-specific reactivity of CD4(+) T cells. A robust T. whipplei-specific CD4(+) Th1 reactivity and activation (expression of CD154) was detected in peripheral and duodenal lymphocytes of all healthy (16 young, 27 age-matched, 11 triathletes) and disease controls (17 patients with tuberculosis) tested. However, 32 Whipple's disease patients showed reduced or absent T. whipplei-specific Th1 responses, whereas their capacity to react to other common Ags like tetanus toxoid, tuberculin, actinomycetes, Giardia lamblia, or CMV was not reduced compared with controls. Hence, we conclude that an insufficient T. whipplei-specific Th1 response may be responsible for an impaired immunological clearance of T. whipplei in Whipple's disease patients and may contribute to the fatal natural course of the disease.

Autoimmunohistochemistry: a new method for the histologic diagnosis of infective endocarditis.

BACKGROUND: Although the pathologic examination of cardiac valves remains the reference standard for the diagnosis of infective endocarditis, the detection of microorganisms often poses a challenge for pathologists. This can be done by use of nonspecific histochemical stains or by immunohistochemical analysis, but specific antibodies are often not available. We describe a novel method for the detection of microorganisms in valve specimens from patients with infective endocarditis. METHODS: Detection of microorganisms was performed in valve specimens from patients with endocarditis caused by gram-positive cocci (25 specimens), blood culture-negative endocarditis (15 specimens: 6 cases caused by Coxiella burnetii, 5 caused by Tropheryma whipplei, and 4 caused by Bartonella species), or noninfective degenerative damage (30 specimens, used as negative controls), using the patients' own serum. This technique, called "autoimmunohistochemistry," is an immunohistochemical peroxidase-based method that we compared with results of culture and polymerase chain reaction (PCR) assay. RESULTS: Bacteria were detected by autoimmunohistochemistry in 20 (80%) specimens from patients with endocarditis caused by gram-positive cocci and in 15 (100%) specimens from patients with blood culture-negative endocarditis but in no control specimens. The rate of detection of bacteria by autoimmunohistochemistry was significantly higher than that by culture but was similar to that by PCR. CONCLUSIONS: Autoimmunohistochemistry may be useful for the detection of microorganisms in samples of valves from patients with infective endocarditis. This new diagnostic tool may be particularly useful in cases of blood culture-negative endocarditis.

Structural elucidation of the predominant motifs of the major cell wall arabinogalactan antigens from the borderline species Tsukamurella paurometabolum and Mycobacterium fallax.

Tsukamurella paurometabolum and Mycobacterium fallax are members of the suprageneric actinomycete group Corynebacterineae that possesses a cell wall skeleton composed of a peptidoglycan to which an arabinogalactan is covalently attached. This polysaccharide is further modified by esterification with C60-C80 mycolic acid residues in mycobacteria and T. paurometabolum. However, M. fallax and T. paurometabolum produce polyenoic (up to six double bonds) mycolic acids whereas the most common type of mycobacterial mycolates, called alpha-mycolates, are mono- and di-enoic or -cyclopropanated mycolic acids. To determine whether this difference also applied to the structures of cell wall arabinogalactans, competitive inhibition experiments using antibodies raised against the cell wall from Mycobacterium bovis and the arabinogalactans from T. paurometabolum and M. fallax were performed. They demonstrated the structural identity between the polysaccharide of M. fallax and those of mycobacteria and showed a strong similarity between the latter polysaccharides and that of T. paurometabolum. Structural analyses of the per-O-alkylated alditol fragments derived from the polysaccharides by gas chromatography-mass spectrometry (GC-MS) and 13C nuclear magnetic resonance (NMR) spectroscopy of the intact solubilized polysaccharides demonstrated that the polysaccharides from the two species analyzed contained all the major structural features previously characterized in mycobacterial arabinogalactans. These include (1) the homogalactan of alterning 5-linked galactofuranosyl (Galf) and 6-linked Galf residues, (2) a linear 5-linked arabino furanosyl (Araf), (3) a beta-Araf-(1-->2)-alpha-Araf disaccharide branched on both position 3 and position 5 of an alpha-Araf unit, and (4) a 5-linked-alpha-Araf unit branched on both position 3 and position 5 of an alpha-Araf residue. The polysaccharide from T. paurometabolum possesses additional structural domains composed of a terminal (t) Araf directly linked to either a 5-linked-alpha-Araf or to both position 3 and position 5 of a 3,5-linked alpha-Araf unit. Both the remarkable similarity of arabinogalactans from Corynebacterineae and their genus- and/or species-specificities are reflected in their 13C NMR spectra that may be used as a valuable help in the identification of members of the actinomycete group.

The ERK MAP kinase cascade mediates tail swelling and a protective response to rectal infection in C. elegans.

The nematode Caenorhabditis elegans is proving to be an attractive model organism for investigating innate immune responses to infection. Among the known pathogens of C. elegans is the bacterium Microbacterium nematophilum, which adheres to the nematode rectum and postanal cuticle, inducing swelling of the underlying hypodermal tissue and causing mild constipation. We find that on infection by M. nematophilum, an extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase cascade mediates tail swelling and protects C. elegans from severe constipation, which would otherwise arrest development and cause sterility. Involvement in pathogen defense represents a new role for ERK MAP kinase signaling in this organism.

Poisoning of livestock in oregon in the 1940s to 1960s attributed to corynetoxins produced by Rathayibacter in nematode galls in chewings fescue (Festuca nigrescens).

Tunicaminyluracil antibiotics, similar to the corynetoxins produced by Rathayibacter toxicus in Australia and South Africa, were found in old nematode seed-galls from Festuca nigrescens from New Jersey (USA) and New Zealand (NZ). The toxin profiles from the NZ and USA galls were similar to each other, but differed from those produced by R toxicus from Australia and South Africa, suggesting that a geographical variant of R toxicus or closely related species may be involved. The NZ galls gave a positive response to a R toxicus-specific monoclonal antibody assay, albeit a considerably weaker response than that seen with Australian R toxicus galls, but the older USA galls were negative, possibly due to deterioration of the antigen. From these findings, it is postulated that livestock deaths associated with the feeding of nematode and bacterial infected screenings of F nigrescens in Oregon, USA, in the 1940s to 1960s were caused by corynetoxin-like toxins produced by the bacterium.

Mucosal immune response to Tropheryma whipplei.

Whipple's disease is a rare infectious disease caused by the ubiquitously occurring Tropheryma whipplei in predisposed persons. Genetic or acquired defects in the mucosal and peripheral immune system become apparent as diminished Th1 immune functions with decreased production of IL-12 and IFN-gamma accompanied by an increased secretion of IL-4. These defects may enable T. whipplei to survive and replicate. The recently established cultivation of the bacterium in HEL cells and the isolation from infected intestinal biopsies enable a multitude of experimental possibilities which may lead to an improved diagnosis as well as understanding of the etiology and pathogenesis of Whipple's disease.

Caenorhabditis elegans as a host for the study of host-pathogen interactions.

Recently, pathogenicity models that involve the killing of the genetically tractable nematode Caenorhabditis elegans by human pathogens have been developed. From the perspective of the pathogen, the advantage of these models is that thousands of mutagenized bacterial clones can be individually screened for avirulent mutants on separate petri plates seeded with C. elegans. The advantages of using C. elegans to study host responses to pathogen attack are the extensive genetic and genomic resources available and the relative ease of identifying C. elegans mutants that exhibit altered susceptibility to pathogen attack. The use of Caenorhabditis elegans as the host for a variety of human pathogens is discussed.

[Detection of Arcanobacterium haemolyticum phospholipase D neutralizing antibodies in patients with acute tonsillitis]. / Prukaz neutralizacních protilátek proti fosfolipáze D Arcanobacterium haemolyticum u nemocných s akutní tonzilitidou.

Neutralization test carried on a non-nutrient blood agar prepared from sheep erythrocytes sensibilized with equi factor of Rhodococcus equi was used for the detection of antibodies against phospholipase D (PLD) of Arcanobacterium haemolyticum. Altogether, 433 sera from 404 patients aged 1 to 25 years with the signs of acute tonsillitis were examined. Antibodies against PLD were found in 28 patients (6.9%). In 116 sera from persons without the signs of tonsillitis the antibodies against PLD were detected in one case (0.9%). The titres reached the values from 4 to 64. Out of 84 paired sera, the significant change of the titre, i.e. at least the four-fold one, was observed five times: three times as the seroconversions, twice as the declines of the titre. The antibodies were found most often in children aged 11 to 15 years, 10 times out of 97 (10.3%). In 253 patients with the tonsillitis, the result of the parallel bacteriologic examinations of throat swabs was at the disposal. A. haemolyticum was successfully isolated in 3 cases, but only owing to the inhibition of staphylococcal hemolysis around its colonies. All three cases were also serologically positive. Consequently, our results show that neutralizing antibodies against PLD of A. haemolyticum can be detected in patients with tonsillitis.

Response of sawmill workers to work-related airborne allergens.

The aim of this work was to determine the reactivity of sawmill workers to biological allergens associated with wood dust. Allergological examinations by skin and precipitin tests were performed in 43 workers employed in a sawmill processing coniferous wood (pine), in 90 workers employed in two sawmills processing deciduous wood (oak), and in 32 healthy urban dwellers not exposed to organic dusts (referents). The skin test was performed by the intradermal method with the saline extracts of wood dust and of the cultures of three microbial species (Rahnella sp., Brevibacterium linens and Penicillium citrinum) isolated from the air polluted with wood dust. Sawdust from pine was used for testing of the pine processing workers and referents while sawdust from oak was used for testing of the oak processing workers. Skin reactions were recorded after 20 minutes, 8 hours and 24 hours. The agar-gel test for the presence of precipitins in serum was performed with the extract of pine wood dust and extracts of 17 microbial isolates. The workers processing pine showed a very high frequency of positive skin reactions to the extract of wood dust at all time intervals, significantly greater compared to the workers processing oak and referents (p < 0.001). The early skin reactions to the extracts of dust-borne bacteria and fungi were very common among sawmills workers and showed a significant relationship with the degree of exposure. The frequency of reactions to Gram-negative bacterium Rahnella sp. was significantly greater in the pine processing workers than in the oak processing workers and referents (p < 0.001). By contrast, the oak processing workers reacted significantly more frequently to Penicillium citrinum, compared to the pine processing workers and referents (p < 0.01). These results conform to the prior study of airborne microflora in which the dominancy of Gram-negative bacteria was stated in the pine processing sawmill while mould fungi were most common in the oak processing sawmills. The antibody response of sawmill workers to work-related antigens was much weaker compared to skin reactions. As many as 41 sawmill workers reported the occurrence of work-related symptoms. A significant relationship was found between the occurrence of symptoms and frequency of allergic reactions, but only with a limited number of antigens. The obtained results suggest that early allergic reactions to coniferous wood and to microorganisms associated with wood dust are common among sawmill workers, posing a potential risk of work-related disease in this occupational group.