Phylogenetic Analysis of Isolates of Pasteurella pneumotropica from Laboratory Animals Based on the gyrB Gene Sequence.

Phylogenetic analysis using the gyrB sequence was performed to investigate the genetic relevance among 49 isolates of P. pneumotropica. In the phylogeny, the isolates were clearly classified into three groups as follows: group A for the isolates of biotype Jawetz derived from mice, group B for the isolates of biotype Jawetz derived from rats, and group C for the isolates of biotype Heyl. These results suggest that the gyrB sequence of P. pneumotropica differs between the isolates of two biotypes, and also between the isolates derived from mice and rats in the biotype Jawetz.     

Identification by 16S rDNA fragment amplification and determination of genetic diversity by random amplified polymorphic DNA analysis of Pasteurella pneumotropica isolated from laboratory rodents

Pasteurella pneumotropica is an opportunistic bacterium frequently isolated from colonies of various laboratory rodents. Identification of this species, including its differentiation into two distinct biotypes (Jawetz and Heyl), is usually based on the use of conventional bacteriologic methods. In this study, a 16S rDNA fragment amplification procedure was developed for use as an alternative method for identification and differentiation of P. pneumotropica. Polymerase chain reaction (PCR) products were two distinctive fragments of 937 and 564 bp specific for biotypes Jawetz and Heyl, respectively. Specificity of PCR products could be achieved by EcoRI cleavage, leading to 596 plus 341-bp and 346 plus 218-bp fragments for each of the amplification products. Use of this procedure confirmed identification of 34 field isolates and allowed definitive identification of some strains that could not have been done by use of bacteriologic examinations. Field isolates subjected to random amplified polymorphic DNA (RAPD) analysis had high genetic diversity among biotype Jawetz strains in contrast to biotype Heyl strains. In conclusion, RAPD could represent an additional means for identification of ambiguous strains of biotype Heyl and a valuable epidemiologic tool for identification of biotype Jawetz strains of P. pneumotropica.     

Evaluation of antigen panels for ELISA monitoring of mouse colonies for antibodies to Pasteurellaceae

Pasteurellaceae infection in mice may be monitored by the detection of serum antibody using enzyme-linked immunosorbent assay (ELISA). We re-evaluated our standard antigen panel comprising Pasteurella pneumotropica and a V-factor requiring Haemophilus species (strain H21) by studying their serological relationship with Actinobacillus muris and 'Haemophilus influenzae-murium'. Serologically, A. muris and 'H. influenzae-murium' were found to be unrelated and to differ from P. pneumotropica and Haemophilus strain H21. These four antigens were used for monitoring breeding and experimental mouse colonies for a period of four years. The addition of 'H. influenzae-murium' antigen to the standard panel of antigens significantly increased the proportion of sera and serum panels showing anti-Pasteurellaceae antibody activity, but the addition of A. muris antigen did not.     

An enzyme-linked immunosorbent assay for detection of chronic subclinical Pasteurella pneumotropica infection in mice.

Serum samples from seventy-five, 3- to 12-week-old and 16 retired breeder male Swiss mice from a conventional colony with enzootic chronic subclinical Pasteurella pneumotropica infection were tested by enzyme-linked immunosorbent assay (ELISA) and Western blots for IgG antibodies to whole cell (WC) and lipooligosaccharide (LOS) antigens of P. pneumotropica. In 3- to 12-week-old mice, serum antibody levels to LOS exceeded those to the WC preparation. Western blots of sera from mice in this age group substantiated that a major component of the early IgG antibody response was directed against LOS antigens. Higher antibody levels to both antigen preparations in 3-week-old mice compared to mice 4 and 6 weeks old were interpreted as reflecting a decline in antibodies acquired from the dam. Active immunity indicative of infection was first detected at 8 weeks of age. Serum samples from retired breeder mice (28 weeks of age) also had substantial antibody titers to LOS but, in contrast to sera from mice in the younger age groups, retired breeders had significantly greater IgG reactivity to WC preparations than to LOS antigens. The superior specificity of the LOS antigen compared to the WC preparation in the ELISA was demonstrated by testing serum samples from retired breeder mice against WC and LOS antigens from P. ureae, P. multocida, and P. hemolytica. The reactivity of IgG against LOS antigens from these organisms was negligible, whereas substantial titers were evident to WC antigens. This ELISA, using LOS preparations as antigen, is a useful serologic assay for the detection of subclinical P. pneumotropica infection in mice.     

嗜肺巴氏杆菌外膜蛋白和脂多糖抗原在血清学诊断中的意义

目的评价嗜肺巴氏杆菌外膜蛋白(OMP)和脂多糖(LPs)作为血清学诊断抗原的敏感性和特异性.方法用OMP、LPS和全菌(WC)作为Western blot和ELISA的诊断抗原检测自然感染和实验感染嗜肺巴氏杆菌小鼠相应的IgG抗体滴度,同时测定3种抗原与实验动物常见致病菌的交叉反应.结果与嗜肺巴氏杆菌自然感染和实验感染小鼠血清的ELISA反应中,不同时期,LPS作为诊断抗原时血清抗体阳性率最高,WC次之,OMP最低.自然感染小鼠群中,出生4周LPS抗体阳性率即可达80%,而同期的WC和OMP仅为25%和20%,故LPS敏感性最高.与实验动物常见致病菌免疫血清和阴性种鼠血清的ELISA反应中,WC抗原表现出较高的吸光度(A)值,经Western blot证实,其反应为非特异性反应,LPS抗原特异性最强,OMP抗原次之.结论混合多株具有型或种特异性的OMP或LPS作为ELISA的诊断抗原,无论从特异性和敏感性上均高于全菌抗原.     

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.     

Implementation of a PCR assay of Pasteurella pneumotropica to accurately screen for contaminated laboratory mice

The authors implemented a PCR protocol to rapidly screen for Pasteurella pneumotropica and to accurately identify contaminated laboratory mice in a clinical setting. This protocol was implemented in response to a severe outbreak of P. pneumotropica in their animal facility. Although a sentinel program was in place to routinely screen for P. pneumotropica, it was inadequate for the identification of contaminated animals. As a result, several additional strains of mice were contaminated and developed clinical signs of infection. The authors implemented a screening method using PCR with reported primer pairs previously developed to identify the biotype isolates of P. pneumotropica in laboratory mice. Throat culture swabs were collected from live mice and placed in a bacterial culture. The DNA from these cultures was isolated and screened by PCR. This procedure enabled the authors to eliminate P. pneumotropica from several animal housing rooms. The assay can be easily applied in most animal facilities.     

Assessment of rpoB and 16S rRNA genes as targets for PCR-based identification of Pasteurella pneumotropica

Diagnosis of Pasteurella pneumotropica in laboratory animals relies on isolation of the organism, biochemical characterization, and, more recently, DNA-based diagnostic methods. 16S rRNA and rpoB gene sequences were examined for development of a real-time PCR assay. Partial sequencing of rpoB (456 bp) and 16S rRNA (1368 bp) of Pasteurella pneumotropica isolates identified by microbiologic and biochemical assays indicated that either gene sequence can be used to distinguish P. pneumotropica from other members of the Pasteurellaceae family. However, alignment of rpoB sequences from the Pasteurella pneumotropica Heyl (15 sequences) and Jawetz (16 sequences) biotypes with other Pasteurellaceae sequences from GenBank indicated that although rpoB DNA sequencing could be used for diagnosis, development of diagnostic primers and probes would be difficult, because the sequence variability between Heyl and Jawetz biotypes is not clustered in any particular region of the rpoB sequence. In contrast, alignment of 16S rRNA sequences revealed a region with unique and stable nucleotide motifs sufficient to permit development of a specific fluorogenic real-time PCR assay to confirm P. pneumotropica isolated by culture and to differentiate Heyl and Jawetz biotypes.     

Naturally acquired Pasteurella multocida subsp. multocida infection in a closed colony of rabbits: characteristics of isolates

Twelve litters, comprising 41 rabbits aged 35 to 60 days old, in a closed university colony, were monitored for acquisition of nasal Pasteurella multocida subsp. multocida infection. Isolates from 11 infected rabbits were characterized by colonial morphology, capsular type, biotype and antibiotic resistance. Selected isolates were further characterized by somatic antigen typing. Two major strains of P. multocida subsp. multocida were detected in the colony. One strain had mucoid colonies, fermented few carbohydrates and was serotype A:5, whereas, the other strain had smooth iridescent colonies, non-typeable capsular antigen, type 3 somatic antigen and fermented more than twice as many carbohydrates.     

Ten years-long survey on pathogen status of mouse and rat breeding colonies

Eleven pathogens including P. aeruginosa, Salmonella spp., E. coli O115a, c: K(B), P. pneumotropica, B. bronchiseptica, C. kutscheri, Tyzzer's organism, M. pulmonis, Sendai virus, MHV and Syphacia spp. were surveyed in 217 mouse and rat breeding colonies during 1972-1981. In conventional animals, P. pneumotropica and/or Syphacia spp. were detected in nearly 90% of 89 mouse and 64 rat colonies. Sendai virus, M. pulmonis, P. aeruginosa and MHV were positive in 51.7 to 23.6% of the colonies, and Tyzzer's organism, B. bronchiseptica and probably SDA virus were also detected in more than 10% of the rat colonies. Salmonella spp., E. coli O115a, c: K(B) and C. kutscheri were found in a few colonies. In SPF animals, P. aeruginosa was isolated from about one third of 33 mouse and 31 rat colonies, and P. aeruginosa was isolated from about one third of 33 mouse and 31 rat colonies, and P. pneumotropica was also positive in 3 rat colonies. Infection rates of P. pneumotropica, M. pulmonis, Sendai virus and Syphacia spp. were usually higher than 40% of animals sampled from colonies contaminated with them. Accidental contaminations of SPF colonies were usually caused by P. pneumotropica and Syphacia spp.     

一株新分离巴氏杆菌的初步鉴定

对清洁级昆明小鼠常规监测中分离的一株可疑嗜肺巴氏杆菌进行了有关遗传学和生物表型特征的检定。该菌形态学及一般培养特性与嗜肺巴氏杆菌相似 ,与嗜肺巴氏杆菌参考血清发生凝集反应 ,生化试验不符合嗜肺巴氏杆菌典型特征 ,染色体DNA的G -Cmol%测定不能排除巴斯德氏菌属细菌的可能 ,进一步生化检定不符合国外报道的嗜肺巴氏杆菌Jawetz和Hey 1 .2个生物型 ,但与国外报道的Pasteurella .sp组细菌相似。     

Diagnosis of murine infections in relation to test methods employed

Comparative investigations of Sendai virus, pneumonia virus of mice (PVM), mouse encephalomyelitis virus (mouse polio), minute virus of mice (MVM), and reovirus type 3 (Reo 3) infected murine colonies revealed a 30% higher incidence of positive sera when enzyme-linked immunosorbent assay (ELISA) was employed instead of hemagglutination inhibition (HI) tests. Equivalent sensitivity as in the ELISA was obtained when the same sera were investigated by indirect immunofluorescence (IF) tests. The virus purification techniques described resulted in highly suitable antigens for all indirect ELISA established. Since IIF requires no purified antigens, this test is recommended as an alternative to ELISA as well as to HI and complement fixation (CF) tests for laboratories lacking the necessary equipment for high speed centrifugation. A high incidence of false positive HI reactions was found particularly in Reo 3 routine serology. An updated survey of seromonitoring showed that European murine colonies appeared to be infected far less with Reo 3 if ELISA or IIF tests were employed. During 1982-1984, only 13% of the mouse colonies screened possessed Reo 3 positive sera whereas no natural Reo 3 infection was found in rat colonies. Mouse hepatitis virus (MHV) and the coronaviruses of rats exhibited the highest incidence in murine colonies. A total of 60% of mouse and 41% of rat colonies were found to be infected by these viruses. In comparison with earlier serological surveys, the relative incidence of other murine infections was similar. Antibodies against Bacillus piliformis (Tyzzer's disease) were detected by the IIF test in 41% of the rat colonies screened.     

Pasteurella multocida toxin type D serological assay as an alternative to the toxin neutralisation lethality test in mice.

The objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Pasteurella multocida toxin type D, that correlated to a mouse lethality test. Currently, the mouse lethality test is one of several tests used world-wide to evaluate serological responses in animals immunised with vaccines containing toxoids. The mouse lethality test involves injecting mice with a mixture of toxin and test serum sample (from animals that have been vaccinated with a toxoid), and then determining antibody titre of the test serum from the number of mice that survive. Thus, the titre calculated is based on the neutralising activity of the test serum. The mouse lethality test requires large numbers of animals and causes severe distress to the animals. Organisations world-wide are working towards alternatives to animals in the development and control of biological products for human and veterinary use. Additionally, the mouse lethality test is labour-intensive, costly and lacks robustness and may be difficult to reproduce between different technicians. We have developed a double sandwich ELISA to measure anti- P. multocida toxoid type D antibodies in swine serum. Sera from swine immunised with vaccines containing type D toxoid showed good correlation to the mouse lethality assay (Spearman analysis=0.94 and Pearson analysis=0.84). When compared to the mouse lethality test, titres obtained using the ELISA format had higher correlation with protective immunity (i.e., lower turbinate atrophy) following challenge with virulent P. multocida. The ELISA assay is more robust, reproducible and costs less than the mouse lethality assay; and it complements efforts to reduce the use of animals in testing.     

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.     

Screening rabbit colonies for antibodies to Pasteurella multocida by an ELISA.

Rabbit serum samples from eleven different research facilities were evaluated for the presence of immunoglobulin G against Pasteurella multocida by using an enzyme-linked immunosorbent assay (ELISA). Each facility which submitted serum samples also provided a brief history of each rabbit colony tested. Rabbits from colonies reported to have endemic P. multocida or of undetermined status had 83 (58.9%) of 141 rabbits that were positive. Colonies reported to be free from P. multocida had 110 (92.4%) of 119 rabbits that were negative by ELISA. The ELISA test described here showed a high degree of agreement (92-94%) with two other P. multocida ELISAs at different diagnostic facilities. This study confirms that an ELISA testing for serum antibodies against the P. multocida is a reliable diagnostic tool to screen colonies for P. multocida.     

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.     

Ultrastructural characteristics of the external surfaces of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits

The surface structures of the cells of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits were examined by transmission electron microscopy after ruthenium red staining and polycationic ferritin labelling. P. pneumotropica strains ATCC 35149 and K 79114 had slight extracellular fibrous materials associated with cell walls with ruthenium red staining. Ferritin labelling method revealed thick strands or sparsely ferritin-labelled materials on the cell surface of the strains. P. multocida strains Pm-78 and P-2440 had ferritin-labelled capsules surrounded with the cell wall. Strain Pm-78, which was serotyped as A:12, had a thick capsule, whereas serotype -:3 strain P-2440 had a thin and irregular capsule.     

Bacteriological and serological studies on Pasteurella multocida infection in rabbits

Bacteriological and serological examinations were made on Pasteurella multocida infection in two rabbit-breeding colonies. The organism was localized in the paranasal sinuses of 53 of 54 infected rabbits, excreting to the external nares of 49 rabbits. It was also isolated from the trachea and middle and inner ear of half of the infected animals and occasionally from the conjunctiva, lung and heart. The infection rate of the organism was very low (4.3%) in sucklings younger than a month old, but increased with advancing age, reaching nearly 100% in adults more than five months of age. A rapid increase of the infection rate was observed at two to three months of age. Serum antibody against somatic antigen of P. multocida was demonstrated in infected rabbits by use of the tube agglutination test, showing a close correlation with isolation of the organism in adults. Sucklings younger than one month of age from infected dams were significantly resistant to experimental nasal infection of P. multocida.     

Serological relationship of some V-factor dependent Pasteurellaceae (Haemophilus sp.) from guineapigs and rabbits

Culture of guinea pig and rabbit respiratory tracts for bacteria using X- (haemin) and V- (NAD) factor in agar media detected infection by V-factor dependent Pasteurellaceae (Haemophilus sp.) in three colonies of guinea pigs and a group of rabbits. The 12 Haemophilus strains comprised three API NH codes classed as Haemophilus parainfluenzae and two codes classed as Haemophilus aphrophilus/paraphrophilus. Six cell wall lipid profiles were detected, but these were not related to API NH codes. Both bacteriological properties were used to select strains for serological studies but any relationship between bacteriological and serological properties of the Haemophilus strains was not evident. Varying serological relationships occurred between the newly isolated Haemophilus strains, [Pasteurella] pneumotropica NCTC 8284 and Haemophilus strains previously isolated from rats.     

Microbiological monitoring in inbred mouse foundation stocks in Japan

Microbiological monitoring on 128 inbred mouse foundation stocks consisted of common 10 inbred strains and inbred strains originated from outbred dd mice was performed by cooperation of 24 organizations. A total of 881 mice were divided into 647 conventional animals from 95 colonies and 234 barrier-sustained animals from 33 colonies. Three viral, one mycoplasmal, 6 bacterial, one fungal and 3 parasitic agents selected as monitoring microbes according to the proposed selection standards. Among conventional colonies, 84.2% were positive for at least one agent. The highest detection rate was 44.2% for S. obvelata, followed by P. pneumotropica and S. muris, P. aeruginosa, G. muris, Sendai virus, M. pulmonis, MHV and E. coli O115a, c: K (B). Of these agents, only one microbe, P. aeruginosa, was detected in barrier-sustained colonies (36.4%), thus the efficacy of barrier system for the microbiological quality control of the inbred mouse foundation stocks was actually demonstrated. The positive rates of MHV (6.3%) and Sendai v. (16.8%) were significantly low compared with those in experimental mouse colonies. Positivity for parasites was rather high and they were infested together with other pathogens in many cases. Thus parasites including G. muris, S. muris and S. obvelata were regarded as useful indicators to see microbiological contaminations in conventional mice. There observed no strain difference in susceptibility to pathogens except for C57BL/6 and AKR mice which seemed to be high antibody responders to MHV.