Probiotic potential of Weissella strains isolated from horse feces.

In this study, we isolated four Weissella confusa strains from the healthy horse feces to test their potential as equine probiotics. The identification and characteristics of these isolates were determined as per standard methods. Resistance and susceptibility of the isolated strains were tested to low pHs, different heat treatments, commonly used antibiotics and against the pathogenic strains of Salmonella, Pasteurella, Staphylococcus aureus, and Escherichia coli. After 3 h cultural in different pH medium, the 4 strains still had a certain amount of survival above pH 3.0. WH2 and WH4 were still viable at pH2.5. All the isolated strains showed proper growth at 60 °C while no strain survived at 80 °C. The inhibition of α-amylase, the scavenging ability of free radical DPPH· and hydroxyl free radical HO·were also investigated. The results showed that WH4 had highest inhibition rate of α-amylase activity and DPPH· free radical scavenging rate, and the inhibition rate of α-amylase activity was 24.09% and the DPPH· free radical scavenging rate was 35.78%. The inhibition rate ofα-amylase activity and DPPH· scavenging rate of free radicals in the other three strains were about 10%. The clearance rate of hydroxyl radical (HO·) in 4 strains was between 12% and 15%. The antibiotic susceptibilities varied for these four Weisella strains but all of them showed resistance against the frequently used equine antibiotics. All the four strains successfully suppressed the growth of standard strains in in vitro bacteriostasis experiment, which included Salmonella enteritidis (NTNC13349), Escherichia coli (C83902) and Staphylococcus aureus (BNCC186335). they also successfully suppressed the growth of state key laboratory isolating pathogens, which are Pasterurella multocida and Salmonella. Our findings suggest that the isolated strains of Weissella confusa can act as potential equine probiotics and should be explored further.

Pathogenicity of Pasteurella sp. in lumpsuckers (Cyclopterus lumpus L.).

The incidence of disease caused by Pasteurella sp. in farmed lumpsuckers in Norway has been steadily increasing in recent years, causing significant economic losses and fish welfare issues. The disease affects all life stages, both in hatcheries and after release into salmon cages. Therefore, it is important to establish robust challenge models, to be used for vaccine development. Exposure experiments via intramuscular and intraperitoneal injection underlined the high virulence of the bacteria, whereas the cohabitation and bath models allowed the chronic symptoms of the disease to be studied more accurately. Skin lesions and haemorrhage at the base of fins were observed in the more acute cases of the disease. Symptoms including white spots over the skin, especially around the eyes, characterized the chronic cases. The latter were most prominent from the bath challenge model. Histopathology indicated a systemic pattern of disease, whereas qPCR analysis from head kidney showed that bacteria may be present in survivor fish at the end of the challenges. In all the challenge models investigated, Pasteurella sp. was re-isolated from the fish, thus fulfilling Koch's postulates. These findings highlight the importance of screening of lumpsuckers prior to transfer to minimize the risks of carrying over asymptomatic carriers.

Bibersteinia trehalosi inhibits the growth of Mannheimia haemolytica by a proximity-dependent mechanism.

Mannheimia (Pasteurella) haemolytica is the only pathogen that consistently causes severe bronchopneumonia and rapid death of bighorn sheep (BHS; Ovis canadensis) under experimental conditions. Paradoxically, Bibersteinia (Pasteurella) trehalosi and Pasteurella multocida have been isolated from BHS pneumonic lungs much more frequently than M. haemolytica. These observations suggest that there may be an interaction between these bacteria, and we hypothesized that B. trehalosi overgrows or otherwise inhibits the growth of M. haemolytica. Growth curves (monoculture) demonstrated that B. trehalosi has a shorter doubling time ( approximately 10 min versus approximately 27 min) and consistently achieves 3-log higher cell density (CFU/ml) compared to M. haemolytica. During coculture M. haemolytica growth was inhibited when B. trehalosi entered stationary phase (6 h) resulting in a final cell density for M. haemolytica that was 6 to 9 logs lower than expected with growth in the absence of B. trehalosi. Coculture supernatant failed to inhibit M. haemolytica growth on agar or in broth, indicating no obvious involvement of lytic phages, bacteriocins, or quorum-sensing systems. This observation was confirmed by limited growth inhibition of M. haemolytica when both pathogens were cultured in the same media but separated by a filter (0.4-microm pore size) that limited contact between the two bacterial populations. There was significant growth inhibition of M. haemolytica when the populations were separated by membranes with a pore size of 8 mum that allowed free contact. These observations demonstrate that B. trehalosi can both outgrow and inhibit M. haemolytica growth with the latter related to a proximity- or contact-dependent mechanism.

Fever and reduced iron: their interaction as a host defense response to bacterial infection.

When rabbits are infected with Pasteurella multocida, the concentration of iron in their plasma decreases and their rectal temperature rises. To determine whether the rise in body temperature (fever) and the fall in plasma iron may be a coordinated host defense response, Pasteurella multocida were grown in vitro at various temperatures and iron concentrations. At afebrile temperatures the bacteria grew equally well at low or high concentrations of iron. However, when the temperature of the bath was raised to a febrile temperature the growth of the bacteria was inhibited by the low, but not the high, iron concentrations. These data support the hypothesis that one of the mechanisms behind the adaptive (or beneficial) role of fever is the reduced ability of pathogenic bacteria to grow well at elevated temperatures in an iron-poor medium.

Variation in Pasteurella (Bibersteinia) and Mannheimia spp. following transport and antibiotic treatment in free-ranging and captive Rocky Mountain bighorn sheep (Ovis canadensis canadensis).

Morbidity and mortality associated with respiratory disease following capture and translocation of bighorn sheep (Ovis canadensis canadensis) is a significant concern, particularly when establishing new or augmenting existing bighorn populations. Administration of prophylactic antibiotics at the time of capture is often done to minimize the risk of respiratory disease, but the efficacy of this practice is unknown. The effects of oxytetracycline and florfenicol on the Pasteurella (Bibersteinia) and Mannheimia spp. isolated from samples collected from the oropharynx at the time of capture and 3 or 42 day later were evaluated in two groups of bighorn sheep. The most evident change in the isolation rates or types of Pasteurella (Bibersteinia) spp., Mannheimia spp., or both was an increase of beta-hemolytic strains isolated from bighorn sheep 3 day following oxytetracycline treatment. Both groups of bighorn sheep carried Pasteurella (Bibersteinia) trehalosi identified as the same biovariants, but they did not share biovariants of Mannheimia spp. No animals had signs of respiratory disease. Isolates representative of all biovariants present in cultures from the two bighorn sheep groups were sensitive to in vitro tests to both oxytetracycline and florfenicol and the majority were also sensitive to seven other antibiotics tested. The administration of neither oxytetracycline nor florfenicol eliminated Pasteurella (Bibersteinia) or Mannheimia from the oropharyngeal mucosa. Resistance to either antibiotic used in these animals was not noted. Although the prophylactic benefits of these drugs in preventing disease are uncertain, therapeutic levels of antibiotics in lung tissue during times of stress may reduce the risk of disease. Representative sampling of the oropharyngeal microflora of bighorn sheep source and recipient populations prior to being intermingled should be considered as one of the tools to minimize exposure of naive populations to potentially pathogenic bacteria.

Serotypes A1 and A2 of Mannheimia haemolytica are susceptible to genotypic, capsular and phenotypic variations in contrast to T3 and T4 serotypes of Bibersteinia (Pasteurella) trehalosi.

Mannheimia haemolytica and Bibersteinia (Pasteurella) trehalosi are the most common bacterial isolates that cause pulmonary diseases in ruminants worldwide. The disease is determined by specific serotypes found in cattle and small ruminants. The molecular epidemiology of strains involved in disease is important in the control of outbreaks as well as in the preparation of vaccines. This study aimed to detect the instability and variations of bacterial strains that may affect the analysis of epidemic strains, or the stability of vaccinal strains. Eight strains of M. haemolytica belonging to serotypes A1 and A2 and three B. trehalosi strains of the T3 and T4 serotypes were used. Strains were subjected to pulsed field gel electrophoresis (PFGE) and capsular and phenotypic typing at each round of a total of 50 successive subcultures. Remarkable stability was found in all selected strains of B. trehalosi in contrast to M. haemoltyica, in which strains of both serotypes showed pattern variations produced by PFGE and capsular and phenotypic analysis. Objective criteria for M. haemolytica and B. trehalosi typing are consequently addressed.

Isolation of a spermatozoa motility inhibiting factor from chicken seminal plasma with antibacterial property.

A 78-kDa spermatozoa motility inhibiting factor (SMIF) was purified from chicken (Gallus domesticus) seminal plasma by anion exchange (DE-53) followed by affinity chromatography on concanavalin A-Sepharose. The factor is thermostable and inhibited the spermatozoa motility in a dose dependent manner. In addition, SMIF inhibited the growth of gram negative bacteria, Pasteurella multocida but not gram positive Streptococcus equi. The factor lost its spermatozoa immobilizing property after treatment with trypsin, chymotrypsin or pepsin. The inhibition of SMIF by beta-mercaptoethanol suggest the involvement of disulfide bonds in its activity. Similarly, this property was lost in presence of chicken seminal plasma or incubating SMIF with anti-SMIF antibodies. Evidence is provided for the presence of a high molecular weight protein (> 100 kDa) in chicken seminal plasma that neutralizes the motility inhibiting property of SMIF. No significant decrease in spermatozoa ATP was observed in presence of SMIF suggesting that the loss of spermatozoa motility was due to factors other than depletion in cell's energy. Using anti-SMIF antibodies, a cross-reactive protein was identified in the blood, liver and reproductive tissues of chicken and the seminal plasma of cattle and buffalo. However, the cross-reactive protein failed to inhibit chicken spermatozoa motility. The significance of SMIF in chicken seminal plasma is discussed.

Isolation of Pasteurella caballi from an infected wound on a veterinary surgeon.

Comparison of phenotypical characters obtained from the type strain of Pasteurella caballi and a previously unclassified P.sp., isolated from an infected wound on a veterinary surgeon, allowed classification of the P.sp. with P. caballi. The P.sp. was isolated in a mixed culture with Escherichia coli and probably represents the first human isolate of P. caballi.

A selective medium for Pasteurella multocida and its use with animal and human specimens.

A selective medium (CGT medium), containing clindamycin, gentamicin, potassium tellurite and amphotericin B in 5% horse-blood agar, allowed unimpaired growth of almost all strains of Pasteurella multocida, and P pneumotropica, while inhibiting other bacteria that might be encountered in upper respiratory tract secretions. With its use, P multocida was readily detected in oral swabs from four of 23 dogs, and 10 of 25 cats, but not detected in oral swabs from 47 human subjects. One of 500 sputum specimens yielded P pneumotropica.

Toxin production by Pasteurella granulomatis.

Pasteurella granulomatis (Pg) is a recently identified bacterium associated with proliferative fibrogranulomatous panniculitis (also called "lechiguana") in Brazilian cattle. Recent attempts to experimentally reproduce this disease have only been partially successful. We hypothesized that Pg may produce hemolysin(s) and/or cytotoxin(s) which could contribute to its pathogenicity in susceptible cattle. The objective of this study was to determine the presence and degree of hemolytic and leukotoxic activity of selected isolates of Pg. Either ovine or bovine blood agar plates were streaked with 1 of 7 Pg isolates, incubated at 37 degrees C +/- 1 C for 48 hours, and examined for hemolysis. Two of seven isolates showed hemolytic activity on bovine plates, while all seven showed hemolytic activity on ovine plates. By use of the CAMP reaction, involving simultaneous intersecting cultures of Staphylococcus aureus and Pg, all seven Pg isolates showed enhanced (positive CAMP) hemolysis within 24 hours on bovine blood agar plates. Preliminary results using tetrazolium (MTT) dye reductions with bovine neutrophils showed leukotoxicity in 13 of 16 Pg cultures. Alamar blue tests indicate leukotoxic activity for all 7 Pg isolates. We conclude that some Pg isolates have variable hemolytic and/or leukotoxic properties and that this variability (presence and/or degree) of these 2 properties may affect the relative pathogenicity of Pg in susceptible cattle.

Capsular polysaccharide expressed by Pasteurella piscicida grown in vitro.

Pasteurella piscicida grown in a glucose-rich medium produces a capsule that can be see under light and electron microscopy. The capsular polysaccharide was purified and characterized by chemical and HPLC analysis. The polymer has the composition glucose/mannose/N-acetylgalactosamine/galacturonic acid/acetic acid in the molar ratios of approximately 2.5:1.3:0.5:0.4:2.5. The polysaccharide was immunogenic in rabbits and did not cross-react with antibodies against the O-antigen lipopolysaccharide.

Effects of antipyresis on bacterial numbers in infected rabbits.

A previous investigation demonstrated that infusion of an antipyretic drug into the preoptic anterior hypothalamus (PO/AH) of rabbits reduced the fever usually seen during the initial stages of infection. This was followed by an increased fever and an increased mortality rate [32]. The work reported here investigated the hypothesis that the increased mortality was the result of decreased killing and/or increased multiplication of bacteria during the initial, attenuated phase of the febrile course in the antipyretic-treated rabbits. Rabbits were injected intravenously with Pasteurella multocida and either sodium salicylate or a control solution was infused directly into the PO/AH. Infusion of sodium salicylate reduced the mean fever 4 hours after injection of bacteria from 2.07 +/- 0.28 degrees C (S.E.M.) to 0.62 +/- 0.43 degrees C. Rabbits with reduced fevers had decreased blood leucocyte counts and greater numbers of bacteria in lung and liver samples. No differences were seen in reticuloendothelial clearance of carbon, hematocrit, or intracellular viability of bacteria when antipyretics were administered. This increase in bacterial numbers corresponds well to the increased mortality found in previous studies in animals with reduced fevers.

The influence of growth conditions of Pasteurella multocida on its ability to colonise the nasal mucosa of SPF piglets.

Colonisation of type D Pasteurella multocida was studied in five groups of seven SPF piglets each. Piglets of Group 1 were kept together with seven 5-week-old piglets obtained from a large herd infected with toxigenic P. multocida for 16 weeks (contact infection). These piglets were made free from toxigenic Bordetella bronchiseptica by local immunisation. Piglets of Group 2 were inoculated with 5 x 10(7) colony-forming units (cfu) of P. multocida washed from the nasal mucosa of piglets free from toxigenic B. bronchiseptica with fetal calf serum. Piglets of Group 3 were inoculated intranasally with 5 x 10(7) cfu of P. multocida washed from yeast-extract proteose-peptone cystine (YPC)-blood agar with fetal calf serum. Piglets of Group 4 were inoculated with 5 x 10(7) cfu of P. multocida grown in a YPC-based broth without blood. Piglets of Group 5 served as controls. The piglets of Group 1 did not contract P. multocida infection from infected contact piglets. After a single inoculation one of four, while after three inoculations two of three piglets of Group 2 became infected by P. multocida. After a single inoculation none of four, while after three inoculations one of three piglets of Group 3 were colonised by P. multocida. Both single and repeated inoculation failed in piglets of Group 4.

The airborne survival of Pasteurella haemolytica and its deposition in and clearance from the mouse lung.

Pasteurella haemolytica A1 was aerosolised by a Collison nebuliser in a Henderson apparatus and its survival in air was measured. The organism was fragile in aerosol and survived best at high humidity and warm temperature. Mice were exposed to the aerosol and clearance from the lung measured. Deposition in the mouse lung showed a good linear correlation with bacterial concentration in the spray suspension fluid. Clearance from the lung was rapid over 24 h although some bacteria could be detected 2 and 4 days after exposure. Mice which received a second exposure 2 weeks later exhibited accelerated clearance from the lung whereby no bacteria could be detected after 12 h. This was associated with serum IgG antibody production, and local and splenic lymphocyte responses to bacterial antigen in vitro.

Colonisation by Pasteurella multocida in atrophic rhinitis of pigs and immunity to the osteolytic toxin.

Gnotobiotic pig antisera to purified toxoid from a capsule type A or D strain of Pasteurella multocida contained large quantities of antitoxin but comparatively little antibody to a crude lysate of P. multocida. These sera given intraperitoneally to further pigs were almost completely protective against turbinate atrophy after intranasal inoculation of dilute acetic acid and infection with type D toxigenic P. multocida. In contrast, antisera to a crude lysate or bacterin of toxigenic P. multocida which contained large titres of antibody to P. multocida lysate, but no detectable antitoxin, were not protective. Colonisation by toxigenic P. multocida was significantly reduced in protected pigs and was similar to colonisation by nontoxigenic P. multocida in pigs untreated or treated with dilute acetic acid. These results indicated (1) that antitoxin was protective and cross protective between toxins from different capsule types; and (2) that the toxin was the main colonisation factor produced by toxigenic bacteria in the acetic acid model of infection and that immunity to it did not eliminate infection.

Relationship between the iron regulated outer membrane proteins and the outer membrane proteins of in vivo grown Pasteurella multocida.

The SDS-PAGE patterns of the outer membrane protein (OMP) extracts of Pasteurella multocida strain P1059, grown under iron-restricted, iron-replete and in vivo conditions, were examined. The results showed that the iron-regulated outer membrane proteins (IROMPs) with molecular masses of 76 kDa, 84 kDa, and 94 kDa were expressed by bacteria grown in iron-restricted media. They were also expressed by in vivo grown P. multocida. Convalescent-phase sera, obtained from turkeys which had survived pasteurellosis, contained antibodies that reacted intensly with th three IROMPs. This indicated that these proteins were expressed in vivo. Bacteria expressing the IROMPs showed greater binding to Congo Red when compared to cells not expressing IROMPs. Cells expressing the IROMPs or its OMP extracts grown in iron-restricted media also showed greater binding to 59Fe-pasteurella siderophore (multocidin) when compared to bacteria or its extracts not expressing IROMPs. Convalescent-phase sera, which contained antibodies against the IROMPs, blocked this specific 59Fe-multocidin binding to IROMPs. Autoradiography was used to determine which of these IROMPs functioned as a receptor for the iron-multocidin complex. The results suggested that these three IROMPs have specific epitopes for binding to the iron multocidin complex.

Carbon source assimilation and fermentation tests: study of 77 animal Pasteurella strains by a microtechnique.

77 strains of Pasteurella from various animal species were tested using the API micromethod for their ability to assimilate 147 carbon substrates and ferment 49 carbohydrates. Typical profiles of P. multocida and P. haemolytica were determined. This micromethod should prove useful in establishing the biotypes of Pasteurella species and in studying their epidemiology and pathogenicity.

Pasteurella haemolytica is highly sensitive to ultraviolet irradiation.

The response of Pasteurella haemolytica to ultraviolet irradiation was determined. The results for survival show that P. haemolytica is very sensitive to UV-irradiation. This UV-sensitivity is similar to E. coli strains defective in UV repair mechanism(s). Analysis of the distribution of TCA insoluble versus TCA soluble [3H]thymine dimers in UV-irradiated DNA of P. haemolytica during a 2-h post-irradiation period indicates that the bacterium is deficient in an excision-repair system. These data suggest that P. haemolytica lacks some of the important mechanisms to repair UV-induced damage.

Phagocytosis and intracellular killing of Pasteurella multocida by porcine alveolar macrophages after infection with pseudorabies virus.

The purpose of this study was to determine if pseudorabies virus (PrV) interfered with normal alveolar macrophage phagocytic functions. Porcine alveolar macrophages (PAM) obtained by pulmonary lavage were exposed to PrV. At 1 hour postinfection, cells were challenged with Pasteurella multocida labeled with 3[H] thymidine. The phagocytosis assay was performed by measuring total radioactivity 1 hour after Pm challenge in a soft-beta spectrophotometer. Intracellular killing was measured by counting viable bacteria 3 hours after P. multocida challenge. Phagocytic values of PrV-infected and control PAM ranged from 11% to 20%, a non-significant difference. Values for intracellular killing for PrV-infected PAM ranged from 7.1 X 10(5) to 1 X 10(6) in contrast to 5.1 X 10(1) to 1.8 X 10(2) for the control PAM. This difference in killing function was significantly lower in PrV-infected PAM than in control cells (P less than 0.01). This alteration of macrophage function may be a factor in the pathogenesis of PrV-Pm mediated pneumonia in pigs.

An aerogenic Pasteurella-like organism isolated from horses.

Thirteen strains of a gram-negative, fermentative bacterium that produced gas from glucose were isolated from horses with a variety of clinical conditions. The morphological and biochemical characteristics of this bacterium are similar to those described for the family Pasteurellaceae. These strains appear to constitute a new taxon within the genus Pasteurella; however, the final taxonomic position of this group depends upon more detailed genetic studies. Case histories indicate that this bacterium may be a primary respiratory pathogen and may play a secondary role in various other disease conditions.