Characterization of some previously unclassified Pasteurellaceae isolated from hamsters

Bacteria isolated from purulent processes on the jaws of European hamsters ( Cricetus cricetus ) and from intestinal inflammatory processes in Syrian hamsters ( Mesocricetus auratus ), bred as laboratory animals have been shown to be phenotypically similar but not identical with Pasteurella pneumotropica . Deoxyribonucleic acid (DNA)–DNA hybridization studies indicate that with one exception, the strains represent two new species of the family Pasteurellaceae. In the absence of a close genomic relatedness to members of the genera Actinobacillus or Pasteurella or allied organisms, however, the two new taxa are described without any formal designation. The one exception was identified as Actinobacillus capsulatus , a species not previously isolated from hamsters.     

Pasteurella pneumotropica and the prevalence of the AHP (Actinobacillus, Haemophilus, Pasteurella)-group in laboratory animals

134 bacterial isolates originally identified as Pasteurella pneumotropica were cultured from healthy, ill or dead mice, rats, hamsters, guinea pigs, rabbits and cats originating from 7 conventional laboratory animal facilities. They occurred seldom in pure culture and were found in a variety of organs. Thorough identification (41 criteria) revealed that only 83 isolates (62%) were P. pneumotropica and could be subdivided into 3 biotypes. 3 isolates were P. aerogenes, 1 was P. ureae, 11 (8%) were qualified as Actinobacillus spp. and 13 (10%) as Haemophilus spp. Close relationship of the 3 genera--the 'AHP-group' --made the differentiation difficult. 23 atypical cultures were discarded at the beginning of the study as not belonging to the 'AHP-group'. Two-thirds of isolates were associated with inflammation or suppuration; Haemophilus spp. seemed to be more pathogenic than Pasteurella and Actinobacillus species.     

Actinobacillus rossii sp. nov., Actinobacillus seminis sp. nov., nom. rev., Pasteurella bettii sp. nov., Pasteurella lymphangitidis sp. nov., Pasteurella mairi sp. nov., and Pasteurella trehalosi sp. nov

Evidence from numerical taxonomic analysis and DNA-DNA hybridization supports the proposal of new species in the genera Actinobacillus and Pasteurella. The following new species are proposed: Actinobacillus rossii sp. nov., from the vaginas of postparturient sows; Actinobacillus seminis sp. nov., nom. rev., associated with epididymitis of sheep; Pasteurella bettii sp. nov., associated with human Bartholin gland abscess and finger infections; Pasteurella lymphangitidis sp. nov. (the BLG group), which causes bovine lymphangitis; Pasteurella mairi sp. nov., which causes abortion in sows; and Pasteurella trehalosi sp. nov., formerly biovar T of Pasteurella haemolytica, which causes septicemia in older lambs.     

Analysis of 16S-23S rRNA internal transcribed spacer regions in Pasteurellaceae isolated from laboratory rodents

The internal transcribed spacer (ITS) regions of members of Pasteurellaceae isolated from rodents, including the [Pasteurella] pneumotropica biotypes Jawetz and Heyl, [Actinobacillus] muris, "Hemophilus influenzaemurium" and Bisgaard taxon 17 were studied and their feasibility to discriminate these species was analyzed. The reference strains of all species analyzed showed unique species-specific ITS patterns which were further present in 49 clinical isolates of [P.] pneumotropica biotypes Jawetz and Heyl and [A.] muris allowing their identification by comparison to the reference strains pattern. Sequence analysis of the amplified fragments revealed in all species, with exception of "H. influenzaemurium", a larger ITS(ile+ala) which contained the genes for tRNA(Ile(GAU)) and tRNA(Ala(UGC)) and a smaller ITS(glu) with the tRNA(Glu(UUC)) gene. "H. influenzaemurium" revealed two each of the larger and respectively the smaller ITS fragments. Both the length and the sequence of each ITS type were highly conserved within the [P.] pneumotropica biotypes Jawetz and Heyl and [A.] muris strains tested. On the contrary, ITS sequences revealed significant interspecies variations with identity levels ranging from 61.2 to 89.5% for ITS(ile+ala) and 56.5 to 86.8% for ITS(glu). Sequences regions with significant interspecies variation but highly conserved within the species were identified and might be used to design probes for the identification of rodent Pasteurellaceae to the species level.     

Application of multivariate analyses of enzymic data to classification of members of the Actinobacillus-Haemophilus-Pasteurella group.

Outer membrane vesicles and fragments from Actinobacillus actinomycetemcomitans, Actinobacillus lignieresii, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Haemophilus parainfluenzae, Pasteurella haemolytica, and Pasteurella multocida were isolated and examined semiquantitatively for 19 enzyme activities by using the API ZYM micromethod. The enzyme contents of vesicles and fragments were compared with the enzyme contents of whole cells of the same organisms. Enzymic data were analyzed by using principal-component analysis and soft independent modeling of class analogy. This technique allowed us to distinguish among the closely related organisms A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus. A. actinomycetemcomitans was divided into two groups of strains. A. lignieresii fell outside or on the border of the A. actinobacillus class. A. ureae, H. influenzae, H. parainfluenzae, P. haemolytica, and P. multocida fell outside the A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus classes.     

Prevalence of a Novel Capsule-Associated Lipoprotein Among Pasteurellaceae Pathogenic in Animals

Capsular serotype A strains of Pasteurella multocida of avian origin express a 40-kDa lipoprotein (Plp-40) thought to attach the extracellular polysaccharide to the cell surface. The objective of the present study was to assess the prevalence of Plp-40 in P. multocida strains of disparate serotypes and host origins, as well as other pathogenic members of the family Pasteurellaceae. Exponential-phase reference and clinical isolates were radiolabeled with [³H]-palmitate, lysed to obtain whole-cell protein fractions, and analyzed using SDS-PAGE and fluorography to assess lipoprotein content. The ability to produce Plp-40 was found to be conserved among certain P. multocida reference and clinical strains of different host origins including avian, human, porcine, bovine, feline, canine, ovine, and cervine, but not rabbit. Production of a 40-kDa lipoprotein was exhibited by all clinical isolates of Pasteurella aerogenes, Pasteurella pneumotropica, Actinobacillus suis, Actinobacillus suis-like organism, and Actinobacillus pleuropneumoniae examined, but not Pasteurella (Mannheimia) haemolytica, Actinobacillus lignieresii, or Haemophilus spp. These data suggest that, while not all Pasteurellaceae are able to produce a 40-kDa lipoprotein under the present experimental conditions, expression is somewhat conserved among diverse isolates of disparate host origins. Received: 28 September 2001 / Accepted: 15 October 2001     

Sharing of Pasteurella spp. between free-ranging bighorn sheep and feral goats

Pasteurella spp. were isolated from feral goats and free-ranging bighorn sheep (Ovis canadensis canadensis) in the Hells Canyon National Recreation Area bordering Idaho, Oregon, and Washington (USA). Biovariant 1 Pasteurella haemolytica organisms were isolated from one goat and one of two bighorn sheep found in close association. Both isolates produced leukotoxin and had identical electrophoretic patterns of DNA fragments following cutting with restriction endonuclease HaeIII. Similarly Pasteurella multocida multocida a isolates cultured from the goat and one of the bighorn sheep had D type capsules, serotype 4 somatic antigens, produced dermonecrotoxin and had identical HaeIII electrophoretic profiles. A biovariant U(beta) P.haemolytica strain isolated from two other feral goats, not known to have been closely associated with bighorn sheep, did not produce leukotoxin but had biochemical utilization and HaeIII electrophoretic profiles identical to those of isolates from bighorn sheep. It was concluded that identical Pasteurella strains were shared by the goats and bighorn sheep. Although the direction of transmission could not be established, evidence suggests transmission of strains from goats to bighorn sheep. Goats may serve as a reservoir of Pasteurella strains that may be virulent in bighorn sheep; therefore, goats in bighorn sheep habitat should be managed to prevent contact with bighorn sheep. Bighorn sheep which have nose-to-nose contact with goats should be removed from the habitat.     

Genetic Diversity on 16S rDNA Sequence and Phylogenic Tree Analysis in Pasteurella pneumotropica Strains Isolated from Laboratory Animals

To reveal the genetic diversity of Pasteurella pneumotropica, the 16S rDNA sequence and multiple alignments were performed for 35 strains (from 17 mice, 13 rats, 3 hamsters, 1 rabbit, and 1 guinea pig) identified as P. pneumotropica using a commercial biochemical test kit or PCR test and two reference strains (ATCC 35149 and CNP160). Each strain showed a close similarity with one of the following organisms: P. pneumotropica (M75083), Bisgaard taxon22 (AY172726), Pasteurella sp. MCCM00235 (AF224300), Pasteurellaceae gen. sp. Forsyth A3 (AF224301), and Actinobacillus muris (AF024526) on GenBank, and were divided into six clusters on a phylogenic tree. Two reference strains, P. pneumotropica biotype Jawetz and Heyl, were classified at both ends of the clusters. Our conclusion is that P. pneumotropica should be reclassified because of the very wide genetic diversity that exists.     

A numerical taxonomic study of Actinobacillus, Pasteurella and Yersinia

A numerical taxonomic study of strains of Actinobacillus, Pasteurella and Yersinia, with some allied bacteria, showed 23 reasonably distinct groups. These fell into three major areas. Area A contained species of Actinobacillus and Pasteurella: A. suis, A. equuli, A. lignieresii, P. haemolytica biovar A, P. haemolytica biovar T, P. multocida, A. actinomycetemcomitans, 'P. bettii', 'A. seminis', P. ureae and P. aerogenes. Also included in A was a composite group of Pasteurella pneumotropica and P. gallinarum, together with unnamed groups referred to as 'BLG', 'Mair', 'Ross' and 'aer-2'. Area B contained species of Yersinia: Y. enterocolitica, Y. pseudotuberculosis, Y. pestis and a group 'ent-b' similar to Y. enterocolitica. Area C contained non-fermenting strains: Y. philomiragia, Moraxella anatipestifer and a miscellaneous group 'past-b'. There were also a small number of unnamed single strains.     

A cryptic plasmid from Pasteurella multocida has a predicted protein nearly identical to a transport protein from Actinobacillus actinomycetemcomitans

Several plasmids from Pasteurella multocida have been shown to carry antibiotic resistance genes but no other genes possibly related to the organism's pathogenesis. We report here that sequence from the plasmid pLEM from a fowl isolate of P. multocida, strain 1059, contained one open reading frame that had significant identity with a predicted protein from pVT745, a plasmid that was isolated from a human oral isolate of Actinobacillus actinomycetemcomitans. This predicted protein had significant homology at the amino acid level to cation transport proteins.     

Genomic fingerprints, ARDRA profiles and quinone systems for classification of Pasteurella sensu stricto

In order to investigate the relationships between species of the genus Pasteurella sensu stricto such as Pasteurella multocida, Pasteurella canis, Pasteurella stomatis, Pasteurella dagmatis, Pasteurella avium, Pasteurella volantium, Pasteurella gallinarum, Pasteurella species A, Pasteurella species B and "Pasteurella leonis" MCCM 00659 their genomic fingerprints and ARDRA profiles were compared and their quinone systems were analysed. Visual comparison of band patterns from rep-PCR (ERIC-, REP- and BOX-PCR) and the analyses of the combined band patterns by UPGMA (unweighted pair group method with averages) dendrogram derived from the combined fingerprint profiles demonstrated that each strain displays a distinct genomic fingerprint. In members of the same species several similarities in the band patterns were observed. Combined ARDRA profiles, obtained after digestion of amplified 23S rRNA coding genes with the enzymes DdeI, MseI and RsaI, revealed a dissection of the members of the genus Pasteurella sensu stricto into two groups which was in agreement with the two groups obtained from our analyses of the quinone systems. These two groups corresponded with the two phylogenetically determined subclusters 3A and 3B described previously. The species of subcluster 3A displayed a quinone system with ubiquinone Q-7 (32-56%) and ubiquinone Q-8 (44-63%) as major compounds. Members of subcluster 3B had a quinone system with ubiquinone Q-8 (86-97%) as the major compound. Based on these results it can be suggested that the genus Pasteurella sensu stricto should be restricted to the species of subcluster 3B including the species Pasteurella multocida, Pasteurella canis, Pasteurella stomatis, Pasteurella dagmatis and Pasteurella species B. In addition, evidence was found which would indicate that: 1) Pasteurella canis MCCM 00927 is misnamed and should be reclassified with Pasteurella multocida; 2) Pasteurella multocida subsp. septica may be classified as a separate species; and 3) "Pasteurella leonis" MCCM 00659 represents a separate species within subcluster 3B and thus could be described as a species of Pasteurella sensu stricto (also in a redefined genus) when more strains become available.     

Multivariate analysis of quantitative chemical and enzymic characterization data in classification of Actinobacillus, Haemophilus and Pasteurella spp

Chemotaxonomic data for strains of Actinobacillus, Haemophilus and Pasteurella spp. were analysed using three multivariate statistical strategies: principal components, partial least squares discriminant, and soft independent modelling of class analogy. The species comprised Actinobacillus actinomycetemcomitans. Haemophilus aphrophilus, H. paraphrophilus, H. influenzae, Pasteurella multocida, P. haemolytica and P. ureae. Strains were characterized by cell sugar and fatty acid composition, lysis kinetics during EDTA and EDTA plus lysozyme treatment, and methylene blue reduction. In total 23 quantitative variables were compiled from chemotaxonomic analyses of 25 strains. A. actinomycetemcomitans and H. aphrophilus formed distinct classes which differed from those of H. paraphrophilus, H. influenzae and Pasteurella spp. All characterization variables, except those describing fatty acid content, contributed significantly to inter-species discrimination.     

Phylogeny of 54 representative strains of species in the family Pasteurellaceae as determined by comparison of 16S rRNA sequences.

Virtually complete 16S rRNA sequences were determined for 54 representative strains of species in the family Pasteurellaceae. Of these strains, 15 were Pasteurella, 16 were Actinobacillus, and 23 were Haemophilus. A phylogenetic tree was constructed based on sequence similarity, using the Neighbor-Joining method. Fifty-three of the strains fell within four large clusters. The first cluster included the type strains of Haemophilus influenzae, H. aegyptius, H. aphrophilus, H. haemolyticus, H. paraphrophilus, H. segnis, and Actinobacillus actinomycetemcomitans. This cluster also contained A. actinomycetemcomitans FDC Y4, ATCC 29522, ATCC 29523, and ATCC 29524 and H. aphrophilus NCTC 7901. The second cluster included the type strains of A. seminis and Pasteurella aerogenes and H. somnus OVCG 43826. The third cluster was composed of the type strains of Pasteurella multocida, P. anatis, P. avium, P. canis, P. dagmatis, P. gallinarum, P. langaa, P. stomatis, P. volantium, H. haemoglobinophilus, H. parasuis, H. paracuniculus, H. paragallinarum, and A. capsulatus. This cluster also contained Pasteurella species A CCUG 18782, Pasteurella species B CCUG 19974, Haemophilus taxon C CAPM 5111, H. parasuis type 5 Nagasaki, P. volantium (H. parainfluenzae) NCTC 4101, and P. trehalosi NCTC 10624. The fourth cluster included the type strains of Actinobacillus lignieresii, A. equuli, A. pleuropneumoniae, A. suis, A. ureae, H. parahaemolyticus, H. parainfluenzae, H. paraphrohaemolyticus, H. ducreyi, and P. haemolytica. This cluster also contained Actinobacillus species strain CCUG 19799 (Bisgaard taxon 11), A. suis ATCC 15557, H. ducreyi ATCC 27722 and HD 35000, Haemophilus minor group strain 202, and H. parainfluenzae ATCC 29242. The type strain of P. pneumotropica branched alone to form a fifth group. The branching of the Pasteurellaceae family tree was quite complex. The four major clusters contained multiple subclusters. The clusters contained both rapidly and slowly evolving strains (indicated by differing numbers of base changes incorporated into the 16S rRNA sequence relative to outgroup organisms). While the results presented a clear picture of the phylogenetic relationships, the complexity of the branching will make division of the family into genera a difficult and somewhat subjective task. We do not suggest any taxonomic changes at this time.     

Epidemic pasteurellosis in a bighorn sheep population coinciding with the appearance of a domestic sheep

A pneumonia epidemic reduced bighorn sheep (Ovis canadensis) survival and recruitment during 1997-2000 in a population comprised of three interconnected wintering herds (Kenosha Mountains, Sugarloaf Mountain, Twin Eagles) that inhabited the Kenosha and Tarryall Mountain ranges in central Colorado, USA. The onset of this epidemic coincided temporally and spatially with the appearance of a single domestic sheep (Ovis aires) on the Sugarloaf Mountain herd's winter range in December 1997. Although only bighorns in the Sugarloaf Mountain herd were affected in 1997-98, cases also occurred during 1998-99 in the other two wintering herds, likely after the epidemic spread via established seasonal movements of male bighorns. In all, we located 86 bighorn carcasses during 1997-2000. Three species of Pasteurella were isolated in various combinations from affected lung tissues from 20 bighorn carcasses where tissues were available and suitable for diagnostic evaluation; with one exception, beta-hemolytic mannheimia (Pasteurella) haemolytica (primarily reported as biogroup 1(G) or 1(alphaG)) was isolated from lung tissues of cases evaluated during winter 1997-98. The epidemic dramatically lowered adult bighorn monthly survival in all three herds; a model that included an acute epidemic effect, differing between sexes and with vaccination status, that diminished linearly over the next 12 mo best represented field data. In addition to the direct mortality associated with epidemics in these three herds, lamb recruitment in years following the pneumonia epidemic also was depressed as compared to years prior to the epidemic. Based on observations presented here, pasteurellosis epidemics in free-ranging bighorn sheep can arise through incursion of domestic sheep onto native ranges, and thus minimizing contact between domestic and bighorn sheep appears to be a logical principle for bighorn sheep conservation.     

Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes.

The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance.     

Head tilt in rabbits caused by pasteurellosis and encephalitozoonosis

Rabbits (Oryctolagus cuniculus) of non-dwarf (Group A) and dwarf (Group B) strains showing a common clinical sign of head tilt (torticollis) were examined. With 1 exception, all rabbits of group A had otitis and empyema of either one or both middle ears. Pasteurella multocida was isolated from pus and from the nose of all but 1 of these rabbits, and on occasion was also isolated from the brain. By contrast in all dwarf rabbits the presence of the protozoan Encephalitozoon cuniculi was confirmed both histologically and serologically. This parasite did not affect the ears of the animals but rather the central nervous system. We assume that the different exposure to both agents, rather than the degree of susceptibility, was responsible for the differences found between the 2 types of rabbits.     

Elimination of Pasteurella pneumotropica from a contaminated mouse colony by oral administration of Enrofloxacin.

Enrofloxacin, a fluoroquinolone bactericidal antibiotic, was administered in an attempt to eradicate Pasteurella pneumotropica (P. pneumotropica) from a contaminated mouse colony. Contaminated mice, maintained within 4 animal rooms, were administered Enrofloxacin in drinking water at a daily dosage of 25.5 mg/kg for 2 weeks. Following one week of Enrofloxacin treatment, mice were selected randomly from each room and examined for P. pneumotropica. This procedure was repeated two or three times until all mice examined tested negative for the Pasteurella strain. With the exception of one room, treated mice consistently tested negative for P. pneumotropica for up to 45 weeks following completion of Enrofloxacin treatment. Thus, oral administration of Enrofloxacin significantly eliminated P. pneumotropica from a contaminated mouse colony.     

Asymmetric learning to avoid heterospecific males in Mesocricetus hamsters

If a female mates with a male of a closely related species, her fitness is likely to decline. Consequently, females may develop behavioral mechanisms to avoid mating with heterospecific males. In some species, one such mechanism is for adult females to learn to discriminate against heterospecific males after exposure to such males. We have previously shown that adult, female Syrian hamsters (Mesocricetus auratus) learn to discriminate against male Turkish hamsters (Mesocricetus brandti) after exposure to a single heterospecific male during 8 days across a wire-mesh barrier. Here we repeated that experiment but this time we exposed female Turkish hamsters to a male Syrian hamster for 8 days and then measured sexual and aggressive behaviors towards that heterospecific male and towards a conspecific male. In contrast to female Syrian hamsters, female Turkish hamsters did not differ in their latency to go into lordosis or in any measure of aggression towards either type of male. Female Turkish hamsters spent less time in lordosis with the heterospecific male, but the percentage of trials in which females copulated with conspecific and heterospecific males did not differ. When comparing females from both species that had been exposed to a heterospecific male for 8days, female Syrian hamsters copulated less and were more aggressive towards the heterospecific male compared to the behavior of female Turkish hamsters. We discuss how this asymmetric response between females of the two species may be due to the much larger geographical range of Turkish hamsters compared to Syrian hamsters.     

Tn 7 inserts in both orientations at a single chromosomal location and apparently forms cointegrates in Pasteurella multocida

Pasteurella multocida transconjugants isolated after mating with Escherichia coli strains that carry one or the other of two Tn7-containing suicide plasmids, pRKTV5 and pUW964 (pRKTV5::Tn5), were analysed. These plasmids have the ColE1 replication origin and were thus expected to deliver transposons but not be maintained as free replicons in Pasteurella. Five out of six transconjugants selected for acquisition of Tn7 from E. coli (pRKTV5) had simple insertions of the transposon, in either orientation, at a single chromosomal location, while the sixth had pRKTV5 integrated at the same location. By contrast, all of 27 transconjugants selected for acquisition of either Tn7 or Tn5 from E. coli (pUW964) maintained pUW964. Of seven subsequently examined at the molecular level, all had pUW964 (in one case, a deletion derivative) integrated at the same location as the Tn7 insertions obtained with pRKTV5. A copy of Tn7 was present at each boundary between the integrated plasmids (pRKTV5 or pUW964) and the chromosome in each strain. The two copies of Tn7 at either end of an integrated plasmid were either in the same (six cases) or in opposite (two cases) orientations with respect to each other. These seem to be products of replicative transposition by Tn7 but can also derive from conservative mechanisms.