The effectiveness of peppermint and thyme essential oil mist in reducing bacterial contamination in broiler houses.

The antimicrobial properties of essential oils have been demonstrated by various in vitro studies, whereas their effect on poultry farm hygiene has not been thoroughly investigated, in particular with reference to aerial treatment. The present study aims to assess the antibacterial effects of natural essential oils in broiler houses. Two experimental rooms were fogged with aqueous solutions of peppermint and thyme oils. The control room was sprayed with pure water. The experiment was conducted on broilers aged 1 to 42 d. The rooms were fogged every 3 d. One day after fogging, the total counts of mesophilic aerobic bacteria, Enterobacteriaceae, and mannitol-positive staphylococci were determined. Samples were collected from the air, litter, walls, and drinkers. The results of the study demonstrate that essential oil mist may improve hygiene standards in broiler farms. During broiler growth, the mean total counts of mesophilic bacteria in the rooms treated with essential oils were lower (P < 0.01 or P < 0.05) in comparison with the control. Enterobacteriaceae and staphylococci counts were also higher in the control group. A single exception was noted in a litter sample where the mean count of Enterobacteriaceae in the room fogged with peppermint oil was higher than in the control. Both oils reduced bacterial counts, but thyme oil was more effective in reducing coliform bacteria, whereas peppermint oil had a higher inhibitory effect on the proliferation of staphylococci. These promising results encourage further research to determine the optimal doses and the effects of essential oils and their combinations on the living conditions and health status of broiler chickens.

Volatile gas concentrations in turkey houses estimated by Fourier Transform Infrared Spectroscopy (FTIR).

1. The aim of the present study was to estimate gas concentrations in commercial turkey houses by Fourier Transform Infrared Spectroscopy (FTIR). 2. The experiment was conducted in 5 buildings of a commercial turkey farm. The measurements of gases were carried out every 3 weeks of the growth cycle. 3. The results demonstrate that ammonia and carbon dioxide are the prevalent gases released during the entire production cycle in turkey houses. The mean concentrations of the above compounds ranged between 4-31 ppm and 220-2058 ppm, respectively. Thiols, nitriles, amines, aldehydes, hydrocarbons and other organic and inorganic compounds also occurred in turkey houses, but they were emitted periodically and their mean concentrations were significantly lower in comparison with CO2 and NH3. 4. Lower ventilation ratio and higher moisture of excreta in the first half of the growth period accelerated the release of some gases, whereas gradual faeces and urine accumulation contributed to an increase in the concentration of selected organic compounds. 5. A portable FTIR analyser is a useful device for measuring gas concentrations in commercial turkey farms, and it supports determinations of tolerable emission limits in turkey production.

Toll-like receptor gene polymorphism and its relationship with somatic cell concentration and natural bacterial infections of the mammary gland in sheep.

Possible correlation between Toll-like receptor (TLR)-gene mutations and the susceptibility of the mammary gland to bacterial infections and also the associate breed-dependent aspects of somatic cell concentration (SCC), bacterial infection and TLR-gene mutations in sheep are described. In Polish Lowland Sheep (PLS), milk samples exceeding the level of 500/microL (i.e. 5 x 10(5) per mL) of SCC were recorded almost twice more frequently than in Polish Heath Sheep (PHS) (40 and 22.3%, respectively). The frequency of bacterial infections was also found in a similar ratio (20 and 12.7%, respectively). During detection of the TLR-gene mutation we recorded 2 alleles of TLR1, 6 alleles of TLR2 and 10 alleles of TLR4 genes in PHS sheep, while PLS sheep possessed 2, 4 and 6 alleles, respectively. Statistical analyses revealed a relationship between the specified TLR alleles, SCC and the frequency of incidence of bacterial inflammations of mammary gland. The data may serve as a benchmark for further study of TLR-gene mutation-dependent predisposition of mammary gland defensive cells to recognize the pathogen properly and initiate the immunological response, and may help in identifying one of the markers of natural resistance against sheep mastitis.

Calcium binding to recoverin: implications for secondary structure and membrane association.

Recoverin is an EF-hand calcium-binding protein reportedly involved in the transduction of light by vertebrate photoreceptor cells. It also is an autoantigen in a cancer-associated degenerative disease of the retina. Measurements by circular dichroism presented here demonstrate that the binding of calcium to recoverin causes large structural changes. increasing the alpha-helical content of the protein and decreasing its beta-turn, beta-sheet and 'other' structures. The maximum helical content (67%) was observed at 100 microM free calcium and, unlike calmodulin, decreased as the calcium concentration was modulated in either direction from this value. Fluorescence measurements indicated that recoverin may aggregate or undergo structural changes independent of calcium binding as the calcium concentration is increased above 100 microM. EGTA also appeared to affect the structure of recoverin independent of its chelation of calcium. While calcium-induced conformational changes have been proposed to alter the membrane binding of recoverin through association of its myristoylated amino terminus, in the experiments presented here the partitioning of recoverin between the cytoplasmic and membrane compartments of the rod photoreceptor outer segment was unaffected by the concentration of calcium, therefore it appears unlikely that a calcium-myristoyl switch acts alone to anchor recoverin directly to the membrane. These experiments were conducted with native recoverin which is heterogeneously acylated, but mass spectrometry confirmed that simple chromatographic methods could be devised to isolate the different forms of recoverin for further studies.

Serological and structural features of Hafnia alvei lipopolysaccharides containing D-3-hydroxybutyric acid.

The serological heterogeneity of Hafnia alvei lipopolysaccharides from strains ATCC 13337, 1187, 1221, 114/60, 1211 and 1216, that contain D-3-hydroxybutyric acid, was analyzed by rocket immunoelectrophoresis, immunoblotting and passive hemagglutination. The significance of D-3-hydroxybutyric acid component for their cross-reactivity has been discussed. The results obtained allowed us to place four H. alvei strains (ATCC 13337, 1187, 1221 and 114/60) in one serotype (A) and to consider two other strains (1211 and 1216) as separate serotypes (B and C, respectively).

Lipopolysaccharide core region of Hafnia alvei: serological characterization.

Covalent glycoconjugates containing, as a ligand lipopolysaccharide core, oligosaccharides of Hafnia alvei standard strain ATCC 13337 and R mutant 1 M were used to produce anti-H. alvei core antibodies. The sera obtained were tested in rocket immunoelectrophoresis, immunoblotting and ELISA using H. alvei lipopolysaccharides of various strains. The experiments were carried out to study the antigenic relationships between lipopolysaccharide core regions in the H. alvei genus.

Studies on virulence of Shigella flexneri.

The antigenic structure and some biological properties were compared in virulent S. flexneri strains of differences in: 1) antigenic composition; 2) biochemical activity; 3) sensitivity to a set of Shigella phages and 4) susceptibility to phagocytosis. The only difference found concerned the LD50 for mice; it was 10 to 100 times larger for avirulent than for virulent strains of S. flexneri. The toxic products of the strains were also compared. The lipopolisaccharide and free endotoxin purified from virulent and avirulent variants behaved similarly when tested for: LD50, pyrogenicity and the local Shwartzman reaction. A striking difference was demonstrated in the ultrastructure of lipopolisaccharide and free endotoxin isolated from virulent and avirulent variant of S. flexneri 3a.

Effect of outer membrane proteins of Shigella on delayed hypersensitivity in mice to sheep red blood cells.

Small doses of outer membrane proteins (OMP) of Shigella flexneri injected intraperitoneally into mice 1 to 3 days before or 3 days after sensitization of animals with sheep erythrocytes were found to increase delayed hypersensitivity as measured by the footpad reaction. In contrast, administration of higher doses of OMP resulted in suppression of hypersensitivity response. Cell transfer experiments showed that the spleen cells from sensitized and OMP treated mice transferred stimulating and suppressing activity to normal recipients. Suppression of hypersensitivity was also observed when recipients were injected with OMP 24 h before they were infused with spleen cells obtained from donor mice sensitized with sheep erythrocytes.

Some biological properties of outer membrane proteins of Shigella.

Outer membrane proteins (OMP) derived from two antigenically different representatives of Shigella (Sh. flexneri 3a and Sh. sonnei phase I) were tested for the toxicity, pyrogenicity, ability to induce Shwartzman reaction as well as for their influence on the leukocyte system. LD50 dose determined on mice was 28 mg/kg for OMP of Sh. flexneri and 23 mg/kg for OMP of Sh. sonnei. Both preparations injected intravenously to rabbits caused moderate increase of body temperature, expressed by the value 1.8 degrees C. Intravenous administration of protein preparations to rabbits, induced at first leukopenia and then transient leukocytosis. When injected subcutaneously in the dose of 500 micrograms and after 24 h intravenously in the dose 100 micrograms, they produced slight hemorrhagic changes at the site of administration.

Phagocytic and bactericidal properties of macrophages of mice immunized with outer membrane proteins (OMP) of Shigella.

Macrophages obtained from peritoneal exudates of mice immunized with the single doses (1 and 5 micrograms) of OMP were shown to have stronger phagocytic as well as bactericidal properties in relation to Shigella flexneri bacilli than nonactivated macrophages. Macrophages from the animals immunized with 10, 20 and 30 micrograms OMP doses showed phagocytic and bactericidal properties similar to those of nonactivated macrophages while the immunization with the dose 50 micrograms resulted in their suppression. Likewise, activity of macrophages from mice immunized twice or three times with various doses of OMP did not differ much from that obtained after immunization with single OMP doses. On the other hand, immunization of mice with a sublethal dose of live Shigella flexneri did not activate either phagocytic or bactericidal properties of macrophages. Besides, phagocytic and bactericidal activity of macrophages of mice immunized with OMP of Shigella was determined in relation to Salmonella typhimurium. The doses 1 and 5 micrograms of OMP resulted in slight activation of macrophages which manifested itself by a little increase in their phagocytic and bactericidal ability. When used in the dose 10 micrograms, OMP remained without any effect on the above activity of macrophages. Only the dose 50 micrograms, slightly suppressed their phagocytic properties.

Outer membrane protein composition of Hafnia alvei.

The outer membrane proteins (OMP) obtained from 33 strains of Hafnia of different origin, were analysed by SDS-polyacrylamide gel electrophoresis. About 21 protein bands could be distinguished in OMP of each strain. The major proteins of the outer membrane were of apparent molecular weight 34.000 (34K), 37.000 (37K), 39.000 (39K) and 41.000 (41K). On the basis of the electrophoregrams obtained, four groups of strains possessing different composition of major OMP were distinguished: (1) 34K and 37K, (2) 34K, 37K and 39K, (3) 34K, 37K and 41K, (4) 34K, 39K and 41K. The major OMP of 34K present in all strains examined was susceptible to proteolytic enzymes and was found to be heat modifiable. The remaining major OMP of 37K, 39K and 41K were identified as peptidoglycan associated proteins.

Protective properties of peptidoglycan associated protein of Shigella flexneri.

Groups of CH3/Hej mice were immunized with peptidoglycan associated protein (PGP) from Shigella flexneri 3a administered in a single intraperitoneal injection (5 micrograms per mouse). After various periods post immunization the animals were challenged intraperitoneally with homologous bacteria. PGP proved to be strongly immunogenic. As early as one week after immunization all the animals survived challenge with 100 X LD100, and one third of the animals survived challenge with a dose of 500 X LD100. This high state of immunity maintained at almost unaltered intensity for 12 weeks after immunization (the end of the observations period).

Induction of tumor necrosis factor and interleukin 6 by outer membrane proteins of Shigella in spleen cells and macrophages of mice.

The ability of outer membrane proteins (OMP) of Shigella flexneri to induce tumor necrosis factor (TNF) and interleukin 6 (IL-6) production by spleen cells and macrophages of mice was investigated. Treatment of spleen cells with OMP resulted in the release of only traces of TNF activity. In contrast, macrophages treated with OMP produced moderate levels of TNF. OMP was also found to be an inducer of IL-6. Both spleen cells and macrophages, treated with OMP, were found to produce substantial levels of this cytokine. The effect of OMP on the release of TNF and IL-6 was dose and time dependent, maximal production being reached at 10 micrograms of OMP after 20 h. The ability of OMP to induce production of these cytokines may explain part of the previously described immunomodulatory effects of this preparation on the immune system.

Humoral response in mice immunized with outer membrane proteins of Shigella flexneri.

Intraperitoneal immunization of mice with outer membrane proteins (OMP) of Sh. flexneri induced in the animals a synthesis of specific antibodies. Their level determined by ELISA test was found to be relatively low in the sera of animals immunized with a single dose (10 micrograms) of OMP; it was markedly higher in mice immunized with two doses of OMP, and very high after three fold immunization. The specific antibodies maintained in the animals for 8-16 weeks after immunization. Anti-OMP sera given to normal mice by intraperitoneal route protected them not only against challenge with homologous Shigella but also against Proteus and Escherichia.

Characterization of outer membrane proteins of virulent phase I Shigella sonnei strains and their avirulent phase I derivatives.

Comparison of polyacrylamide gel electrophoretic protein profiles of four isogenic sets of virulent phase I Sh. sonnei strains and their avirulent phase I cells revealed no differences in outer membrane protein composition between virulent and avirulent derivatives. However, significant qualitative and quantitative differences were found in composition of major outer membrane proteins between strains of different origin. All four strains tested contained one major protein of 33K. This protein was susceptible to proteolytic enzymes and was found to be heat modifiable. Other major proteins of 35K and 37K present in three strains and 36K present in one strain were identified as peptidoglycan associated proteins.

Studies on specificity of protection induced by immunization with outer membrane proteins of Shigella.

Immunization of C3H/HeJ mice with outer membrane proteins (OMP) and with peptidoglycan associated proteins (PGP) isolated from Shigella flexneri 3a and from Shigella sonnei phase I protected the animals against lethal dose of homologous and heterologous bacteria and against various serotypes of Shigella flexneri. Neither of the protein preparations protected the animals against challenge with Escherichia, Klebsiella, Citrobacter, Salmonella, Serratia, Proteus and Pseudomonas. OMP preparations however, isolated from these species protected the animals not only against challenge with homologous bacteria but also against Shigella flexneri 3a.

Transfer of immunity by means of spleen cells from mice immunized with outer membrane proteins of Shigella flexneri.

Intraperitoneal immunization of mice with a single dose (5 micrograms) of outer membrane proteins (OMP) of Shigella flexneri was found to evoke in the spleen the appearance of cells by means of which immunity to lethal dose of Shigella could be transferred into other mice. Active cells capable of transferring immunity appeared in the spleen of the animals as early as on day 3, reached the strongest protective activity on day 4 and disappeared on day 8 after immunization. Active cells from animals immunized with two doses of OMP maintained in the spleens for 19 days. The experiments revealed that immunity to Shigella could be transferred only with lymphocytes; macrophages were found to be inactive.

Hypersensitivity to Shigella outer membrane proteins in mice.

Mice sensitized with living Sh. flexneri 3a or with outer membrane proteins (OMP) derived from these bacteria showed Arthus and delayed type hypersensitivity (DTH) when injected in the footpad with OMP. The delayed reactivity to OMP was transferable to normal mice by spleen cells obtained from donor animals which were previously sensitized with living bacteria.

Studies on immunity in mice immunized with outer membrane proteins of Hafnia.

Nonspecific protection induced in mice after administration outer membrane proteins of Hafnia alvei against infection with homologous and heterologous bacteria was transferred into other mice with lymphocytes isolated from spleens of mice immunized with outer membrane proteins. It was also found that mice sensitized with outer membrane proteins derived from H. alvei or with living bacteria induced in animals delayed hypersensitivity (DTH) in homologous and heterologous systems. The observed type of hypersensitivity was transferable to normal mice by lymphocytes obtained from donor animals which were previously sensitized with OMP. The experiments revealed that immunity induced with outer membrane proteins of Hafnia alvei is cell-mediated.

Effect of outer membrane proteins from Shigella on humoral immunity induced in mice by SRBC.

Shigella flexneri outer membrane proteins (OMP) which had been earlier found to exert immunomodulatory effect on cell mediated immune response were also found to act as immunomodulator of the humoral immune response. Effects of OMP were investigated in the experiments in vitro and in vivo, where the level of humoral immune response, measured as the number of plaque-forming cells (PFC) to SRBC in the spleen was evaluated. We demonstrate that small doses of OMP (1-5 micrograms) stimulate, whereas higher doses (10-50 micrograms) suppress the humoral immunity.