Pasteurella spp. Infections in Atlantic salmon and lumpsucker.

The use of cleaner fish as a delousing method in Norwegian salmonid aquaculture has increased tremendously over the last few years. This has led to the emergence of a new large industry of farming lumpsuckers (Cyclopterus lumpus L.). The use of lumpsuckers as cleaner fish has, however, not been problem-free. Bacterial diseases cause high mortalities with pasteurellosis as one of the major emerging diseases. During the past few years, outbreaks of pasteurellosis in farmed Atlantic salmon (Salmo salar L.) have become more frequent. This has led to an increasing concern that this disease will become common in salmon farming as well. The purpose of this study was to investigate the susceptibility of Atlantic salmon to Pasteurella spp. infection and the possibility of lumpsuckers transmitting pasteurellosis to Atlantic salmon. Atlantic salmon were experimentally challenged, either by bath or by cohabitation with challenged lumpsuckers, using two different strains of Pasteurella spp. (originating from lumpsucker and Atlantic salmon, respectively). No clinical signs of pasteurellosis were observed on any of the Atlantic salmon. The lumpsuckers were, however, equally susceptible to both isolates. In addition, clear differences in histopathological changes were observed between individuals challenged with the two isolates.

Impact of meteorological factors on the prevalence of porcine pasteurellosis in the southcentral of Mainland China.

Using data collected from 2006 to 2014, we applied geographic information system (GIS) mapping and spatial clustering analysis to evaluate prevalence of porcine pasteurellosis in all 31 provinces of Mainland China. All provinces have been affected, but our results show that there is a very high incidence in provinces of the southcentral of Mainland China. Six provinces comprise the area and account for 14082 outbreaks or 74.66% of the total 18862 number: Guangxi (4574), Sichuan (3493), Chongqing (2443), Guangdong (1584), Guizou (1041) and Yunnan (947). This study aims to evaluate the relation between meteorological factors and number of cases of porcine pasteurellosis in the southcentral of Mainland China. Local meteorological variables and case data of porcine pasteurellosis were provided by authorities. Spearman rank correlation analysis and cross-correlation analysis were used to control for collinearity and lag effects. A zero-inflated Poisson model was used to estimate the probability of an impact of meteorological factors in the epidemiology of porcine pasteurellosis. The results of this model indicated that ENSO have a positive effect on the occurrence of the disease. And there is a positive correlation between mean monthly temperature, relative humidity of the current and previous month and the number of cases of the disease. In contrast, average wind speed of the current month negatively correlated to the number of newly reported cases. Our findings indicate that there may exist meteorological conditions in the southcentral of Mainland China that increase the risk for the appearance of porcine pasteurellosis. Moreover, these meteorological variables may be used to estimate the number of disease' cases in this region.

Evaluation of the Biolog system for the identification of certain closely related Pasteurella species.

The zoonotic impact of Pasteurella species in human wounds caused by cats and dogs has increased recently. In this study, the effectiveness of the Biolog Microstation ID System (Biolog, Hayward, CA) for the identification of certain species of Pasteurella sensu stricto was analysed. Thirty-eight isolates originating from dogs and cats were studied by pheno- and genotypic methods. The classical biochemical tests identified these isolates as P. multocida, P. dagmatis, and P. canis, while the Biolog system distinguished only 2 categories: P. multocida and P. dagmatis. The sodA gene sequence-based phylogenetic analysis revealed that the isolates identified as P. dagmatis by the Biolog system were either P. dagmatis, P. canis, or P. dagmatis-like genomospecies. The low discrimination power of the Biolog system in the case of these closely related Pasteurella species draws attention to the need of continuously improving the database of automated microbial identification systems.

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.

Comparative studies on [Pasteurella] testudinis and [P.] testudinis-like bacteria and proposal of Chelonobacter oris gen. nov., sp. nov. as a new member of the family Pasteurellaceae.

A collection of 12 strains, isolated from diseased tortoises and tentatively identified as [Pasteurella] testudinis-like based on phenotypic characters, was compared with three reference strains of [P.] testudinis. All strains could be separated from the reference strains with respect to 16S rRNA gene sequences, partial sequences of the rpoB housekeeping gene and by phenotypic characters. Based upon differences in 16S rRNA and rpoB gene sequences, the new isolates are suggested to represent a novel species in a new genus of the family Pasteurellaceae Pohl 1981, for which the name Chelonobacter oris gen. nov., sp. nov. is proposed. The type strain is 1662(T) (=CCUG 55632(T)=DSM 21392(T)). beta-Haemolysis and acid production from (+)-l-arabinose, dulcitol, (-)-d-mannitol, (+)-d-mannose, trehalose and salicin separated the new strains from members of existing genera of the family Pasteurellaceae, in addition to the beta-galactosidase, urease and alpha-glucosidase reactions. Differences in indole production, phosphatase, beta-glucosidase and production of acid from dulcitol and trehalose separated C. oris from [P.] testudinis. Several phenotypic characters separated C. oris from Bisgaard's taxa 14 and 32.

Plasmid-mediated florfenicol resistance in Pasteurella trehalosi.

OBJECTIVES: A florfenicol-resistant Pasteurella trehalosi isolate from a calf was investigated for the presence and the location of the gene floR. METHODS: The P. trehalosi isolate 13698 was investigated for its in vitro susceptibility to antimicrobial agents and its plasmid content. A 14.9 kb plasmid, designated pCCK13698, was identified by transformation into Pasteurella multocida to mediate resistance to florfenicol, chloramphenicol and sulphonamides. The plasmid was sequenced completely and analysed for its structure and organization. RESULTS: Plasmid pCCK13698 exhibited extended similarity to plasmid pHS-Rec from Haemophilus parasuis including the region carrying the parA, repB, rec and int genes. Moreover, it revealed similarities to plasmid RSF1010 in the parts covering the mobC and repA-repC genes and to plasmid pMVSCS1 in the parts covering the sul2-catA3-strA gene cluster. Moreover, the floR gene area corresponded to that of transposon TnfloR. In addition, two complete insertion sequences were detected that were highly similar to IS1593 from Mannheimia haemolytica and IS26 from Enterobacteriaceae. Several potential recombination sites were identified that might explain the development of plasmid pCCK13698 by recombination events. CONCLUSIONS: The results of this study showed that in the bovine pathogen P. trehalosi, floR-mediated resistance to chloramphenicol and florfenicol was associated with a plasmid, which also carried functionally active genes for resistance to sulphonamides (sul2) and chloramphenicol (catA3). This is to the best of our knowledge the first report of resistance genes in P. trehalosi and only the second report of the presence of a florfenicol-resistance gene in target bacteria of the family Pasteurellaceae.

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.

Vibrio sp. strain NM 10, isolated from the intestine of a Japanese coastal fish, has an inhibitory effect against Pasteurella piscicida.

Vibrio sp. strain NM 10 with an inhibitory activity against Pasteurella piscicida K-III was isolated from the intestine of a spotnape ponyfish (Leiognathus nuchalis). This bacterium efficiently produced an antibacterial substance after growth at 20 degrees C for 24 h on 1/5 PYBG agar prepared with 50% seawater at pHs of 7.5 to 9.0. The antibacterial substance was heat labile and proteinaceous, with a molecular mass of less than 5 kDa, possibly a bacteriocin or a bacteriocin-like substance.

Bacteria associated with enzootic pneumonia in goats.

A histological and microbiological study of lung samples from 83 slaughtered goats (33 kids and 50 adults) drawn from a flock with a history of pleuropneumonia caused by mycoplasmas of the M. mycoides group was carried out. A total of 82% (27/33) of kids and 36% (18/50) of adult goats presented pulmonary lesions characteristic of enzootic pneumonia: lesions took the form of bronchointerstitial pneumonia with peribronchial and peribronchiolar proliferation of lymphocytes. Microbiological analysis confirmed a range of mycoplasma species, including Mycoplasma mycoides ssp. mycoides Large Colony (MmmlC) (3.70%; 1/27), Mycoplasma mycoides ssp. capri (Mmc) (7.40%; 2/27), Mycoplasma putrefaciens (22.2%; 6/27), Mycoplasma arginini (3.70%; 1/27) and Mycoplasma sp. (7.40%; 2/ 27), as well as Pasteurella multocida (14.8%; 4/27), associated with enzootic pneumonia lesions in younger animals, whereas Mycoplasma sp. was associated with enzootic pneumonia in adult goats (22.0%; 4/18). Cilia-associated respiratory (CAR) bacillus found by histochemical examination was associated with enzootic pneumonia in kids (25.9%; 7/27) and goats (44.4%; 8/18), being the first description of this bacterium in adult goats.

Adherence and invasive capacities of the fish pathogen Pasteurella piscicida.

Pasteurella piscicida strains were weakly or moderately adherent to cell lines, the levels of attachment being variable depending on the cells employed. All the isolates exhibited the highest binding capacity to CHSE-214 cells. Adhesive capacities were affected by heat and sugars but not by proteinase K or by treatment with antisera raised against the lipopolysaccharides of P. piscicida, implicating components of glycoprotein(s) as ligands in the adhesion process. The isolates showed a great binding capacity to intestines from the marine fish hosts gilthead sea bream, sea bass and turbot, with values ranging from 10(4) to 10(5) bacteria/g. Although the P. piscicida strains showed a weak invasiveness in the poikilothermic cell lines employed as in vitro model, the bacteria remained viable inside the infected cells at least for 2 days. The invasion process was inhibited by cytochalasin D indicating the active participation of the host cytoskeleton in the internalization of P. piscicida.

In vitro efficacy of cefquinome (INN) and other anti-infective drugs against bovine bacterial isolates from Belgium, France, Germany, The Netherlands, and the United Kingdom.

The in vitro antibacterial activity of cefquinome (INN), an aminothiazolyl-cephalosporin of the fourth generation of cephalosporins, was investigated by determining the minimal inhibitory concentration (MIC, microgram/ml) for 714 bacterial isolates of bovine origin and comparing it with those of amoxicillin, amoxicillin and clavulanic acid, ceftiofur, cephapirin, enrofloxacin, gentamicin, kanamycin and oxytetracycline. Drug resistance was determined by using break-points, which consider the dosage regimen and pharmacokinetics of the veterinary antimicrobials investigated. Cefquinome demonstrated a very high in vitro activity against bacterial isolates of Pasteurella spp., Escherichia coli and Salmonella spp. Overall, the level of resistance against the different anti-infectives tested was lowest for cefquinome. For the remaining substances examined, in vitro activity and the level of resistance showed considerable differences. The chemical and pharmaceutical features of cefquinome are discussed.

Physiological characteristics, antimicrobial susceptibility and serotypes of Pasteurella anatipestifer isolated from ducks in Thailand.

Twenty isolates of Pasteurella (Moraxella) anatipestifer from ducks with serositis and septicemia in Thailand between 1988 and 1989 were characterized by various tests. Eighteen isolates fermented glucose and maltose, 3 fructose and 1 each mannose, arabinose, trehalose or sorbitol. All isolates produced gelatinase but not urease, while 2, 3, 5 and 6 produced indole, were CAMP positive, and were proteolytic for milk and coagulated serum respectively. Seven enzymes, phosphatase alkaline, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, phosphatase acid and phosphoamidase were detected from all the isolates. The isolates were highly susceptible to ampicillin, erythromycin, penicillin G and tylosin. Gel-diffusion precipitin tests demonstrated that serotype 1 was most prevalent (60%) and serotype 6 followed (5%). Seven isolates (35%) were untypable. These results indicated that P. anatipestifer of serotype 1 played an important role in recent outbreaks of the disease in Thailand.

Classification of Pasteurella field strains isolated from farms in Germany using traditional methods and DNA-DNA hybridization.

410 Pasteurella (P.) field strains isolated from calves and piglets were classified according to Bisgaard et al. (1). 376 strains were assigned to P. multocida ssp. multocida, 34 of them were ornithine- and trehalose+, and 61 of them ornithine- and trehalose-. 4 strains belonged to P. multocida ssp. septica, 4 to P. multocida ssp. gallicida, 6 to P. avium biovar 2 and 20 to P. canis biovar 2. There was no difference in the prevalence of the species in calves and pigs. The fact that strains belonging to P. multocida ssp. septica were isolated only from calves and P. multocida ssp. multocida ornithine- and trehalose- were mostly isolated from piglets could indicate a certain host specificity of these isolates. In genotypic investigations 20 field isolates of P. multocida belonging to different Carter serotypes, as well as serologically negative strains were compared to reference strains in terms of deoxyribonucleic acid (DNA) relatedness. The data obtained by filter hybridization revealed a considerable degree of genotypic intraspecies heterogeneity within P. multocida. No correlations to the respective serotypic classification could be detected.

Ecology and significance of Pasteurellaceae in man--an update.

Within the last decade new knowledge has emerged concerning the significance of Pasteurellaceae in man; the classification has undergone some changes, and new taxa were described. Haemophilus influenzae serotype b was shown to have a clonal distribution that is related to demographic patterns of the human host. Brazilian purpuric fever is caused by a special clone of Haemophilus aegyptius. H. influenzae biotype IV seems to be a genital pathogen, and may deserve species rank. New Pasteurella species have been described, that occur in well known pathological foci in man, e.g. bite wounds. Toxigenic P. multocida may occur in man also; the significance of toxigenicity in man is not known. The real actinobacilli of man, A. ureae and A. hominis are still very rarely reported. In order to avoid wrong epidemiological conclusions, correct diagnosis is emphasized.

Ecology and significance of Pasteurellaceae in animals.

The reservoir of eighty-one taxa/groups classified with the family Pasteurellaceae Pohl 1981 is reviewed based upon published data and own investigations. With the exception of certain strains of P. multocida, A. pleuropneumoniae and [H.] paragallinarum organisms belonging to this family are usually regarded as opportunistic, secondary invaders which under normal conditions coexist peacefully with the animal host on mucosal membranes of the upper respiratory- and lower genital tracts. Very little is known about factors that govern the ecological preferences that certain members of this family show for specific surfaces and hosts. Mechanisms of colonization, survival and multiplication, invasion and pathogenic action are incompletely understood. The significance of Pasteurellaceae in animals and man has recently been reviewed. Subsequent publications have underlined the significance of biovars 2 of P. canis and P. avium and ornithine negative P. multocida in pneumonia in cattle. In addition, differences in pathogenicity have been demonstrated for different serovars of [H.] parasuis. The disease potential of many taxa/groups is only incompletely known.

Hemagglutination by Pasteurellaceae isolated from rodents.

Pasteurellaceae notably P. pneumotropica, have been associated with severe outbreaks of respiratory disease in several species of rodents. Host-specific parasitism of Pasteurellaceae in rodents has hardly been studied. Since host tropism in many bacteria involves adhesive mechanisms, we examined the hemagglutinating (HA) properties of 44 isolates from different rodent species (mouse (15) rat (8), hamster (9), gerbil (10) and Mastomys (2)). Only 13 mouse isolates and the 2 Mastomys isolates hemagglutinated human (type O Rh+) and canine red blood cells (RBCs). No HA was found using RBCs from 10 other animal species. HA was not inhibited by simple sugars and glycoconjugates, but was completely inhibited by heating of bacterial cells for 10 min at 80 or 100 degrees C, partially inhibited by glutaraldehyde and inhibited in a dose-dependent mode by NaIO4, suggesting the involvement of bacterial polysaccharide structures in the HA process. Enrichment procedures did not reveal the presence of HA- subpopulations in HA+ isolates or the presence of HA+ subpopulations in HA- isolates. Electron microscopy revealed the presence of fimbriae both in HA+ and HA- isolates. A regularly structured (RS) layer was detected on cells of part of the HA+ isolates only. Our results suggest that Pasteurellaceae of mice and Mastomys may be related and differ from isolates isolated from other rodent species.

Spondylitis in turkeys associated with experimental Pasteurella anatipestifer infection.

Nine previously vaccinated turkeys were inoculated intravenously with Pasteurella anatipestifer, and blood samples were taken periodically to evaluate the potential of chronically infected turkeys to serve as reservoirs of infection for blood-feeding arthropod vectors. Vertebral osteomyelitis (spondylitis), as yet unreported in the literature in association with infection with the organism, was found in the thoracic vertebrae of five out of nine inoculated turkeys, and P. anatipestifer was isolated from the thoracic vertebrae of three of the five. The organism was isolated from the peripheral blood of six turkeys 24 hours postinoculation and from the peripheral blood of one turkey 7 days postinoculation. The organism was also isolated from the heart blood of two birds at necropsy--from one at 21 days and, following an intramuscular injection of dexamethasone, from the other turkey at 38 days postinoculation.

The role of leukocytes in the pathogenesis of fibrin deposition in bovine acute lung injury.

The peculiarly fibrinous nature of bovine acute lung injury due to infection with Pasteurella haemolytica A1 suggests an imbalance between leukocyte-directed procoagulant and profibrinolytic influences in the inflamed bovine lung. Calves with experimental pneumonia produced by intratracheal inoculation with P. haemolytica A1 developed acute locally extensive cranioventral fibrinopurulent bronchopneumonia. Pulmonary alveolar macrophages (PAM) recovered by segmental lavage from affected lung lobes were 30 times more procoagulant than PAM obtained from unaffected lung lobes and 37-fold more procoagulant than PAM from control calf lungs. Unlike the enhancement of procoagulant activity, profibrinolytic activity (plasminogen activator amidolysis) of total lung leukocytes (PAM and plasminogen activator neutrophils [PMN]) was decreased 23 times in cells obtained from affected lung lobes and also was decreased four times in cells obtained from unaffected lobes of infected animals. This marked imbalance in cellular procoagulant and fibrinolytic activity probably contributes significantly to enhanced fibrin deposition and retarded fibrin removal. In addition, PAM from inflamed lungs were strongly positive for bovine tissue factor antigen as demonstrated by immunocytochemistry. Intensely tissue factor-positive PAM enmeshed in fibrinocellular exudates and positive alveolar walls were situated such that they were likely to have, in concert, initiated extrinsic activation of coagulation in the acutely inflamed lung. These data collectively suggest that enhanced PAM-directed procoagulant activity and diminished PAM- and PMN-directed profibrinolytic activity represent important modifications of local leukocyte function in bovine acute lung injury that are central to the pathogenesis of lesion development with extensive fibrin deposition and retarded fibrin removal.

Determination of affinity of Pasteurella multocida isolates for porcine respiratory tract mucus, and partial characterization of the receptors.

The ability of 25 Pasteurella multocida isolates to adhere in vitro to porcine respiratory tract mucus was examined. Microplate wells were coated with crude mucus preparation, then bacteria were added. After incubation, unbound bacteria were removed by washing, and the number of mucus-bound bacteria was estimated by quantitation of the adherent colony-forming units and by use of an ELISA. Pasteurella multocida had affinity to respiratory tract mucus, although significant differences were not observed in affinity of capsular type-A and type-D isolates. Preliminary characterization, using ultrafiltration, gel filtration chromatography, electrophoresis, and enzymatic treatments, indicated that the receptors may be a class of protein molecules of low molecular weight (less than 25,000). The origin of these receptors, however, is not known at this time.