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We compared the efficacies of two different individually ventilated cage systems (Allentown and Tecniplast) for health monitoring (HM) of murine infectious agents using exhaust air particle (EAP) capture and real-time PCR. After three months of monitoring, both EAP capture media allowed detection of Helicobacter, Pasteurella and Entamoeba. Use of the EAP real-time PCR for HM reduces the number of mice used.

Monitoring of rodents for Pasteurellaceae infection may be carried out by the polymerase chain reaction (PCR). We tested which of 17 rodent Pasteurellaceae strains were detected by three PCR primer sets. By phylogenetic analysis, 12 strains were assigned to the Rodent cluster and five strains to other clusters, namely the Somnus cluster, Pasteurella sensu stricto, Actinobacillus sensu stricto, the Mannheimia and Rossii cluster. A primer set developed to detect biotype Heyl [ Pasteurella] pneumotropica produced amplicons from three strains and appeared specific for this taxon. A primer set developed to detect biotype Jawetz [ P.] pneumotropica produced amplicons from the [ P.] pneumotropica type strain and two other strains within the Rodent cluster. A primer set as described by Bootz and his co-workers (Bootz F, Kirschnek S, Nicklas W, Wyss SK, Homberger FR. Detection of Pasteurellaceae in rodents by polymerase chain reaction analysis. Lab Anim Sci 1998;48:542–6) for the detection of all Pasteurellaceae indeed detected all bacterial strains examined. Bootz's primer set should be used to monitor rodents for Pasteurellaceae infection by PCR as FELASA recommends the monitoring of rodents for all Pasteurellaceae taxa. Health monitoring reports should specify the primer set(s) used for PCR testing rodents for Pasteurellaceae infection.

Survival has been determined for Pasteurella pneumotropica on various surfaces found in an animal room at 23 ± 1°C and 50 = 10% relative humidity. Longest survival (120 min) was found on mouse hair, shortest (< 30 min) on laboratory coat fabric. Transmission experiments were performed using sentinel animals in order to evaluate the efficiency of their use for the detection of P. pneumotropica in quarantined mice. In sentinels exposed to infected mice by close contact, P. pneumotropica was detected by culture 2 weeks post-exposure and seroconversion 3 weeks after contact. Transfer of soiled bedding from Pasteurella-infected mice did not infect sentinels within a period of 12 weeks as tested by cultivation or serum antibodies.

Culture of guineapig 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 guineapigs 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.

Thirty Pasteurellaceae strains isolated from gerbil, guineapig, hamster, mouse, muskrat and rat were reinvestigated and reclassified after comparison with reference strains. Strains originally described as Pasteurella pneumotropica were reclassified as [ Pasteurella] pneumotropica Heyl biotype (7), [ P.] pneumotropica Jawetz biotype (1), Pasteurella dagmatis (1) or Taxon 22 (2). Strains previously reported as Actinobacillus sp. were reclassified as [ P.] pneumotropica biotype Jawetz (3), P. dagmatis (3) or Taxon 6 (7). Strains earlier described as Pasteurella gallinarum were renamed as SP group pasteurellas (4) or Taxon 25 (2). Some of these reclassified Pasteurellaceae have not been reported previously in rodents. The present findings underline the importance of extended characterization of isolates and comparison with reference strains to avoid misclassification within the family Pasteurellaceae Pohl 1981.

Cross-reactivity studies with Pasteurellaceae from guineapigs revealed 5 serologically distinct groups, comprising Pasteurella multocida, Sp group bacteria, SP-like bacteria, Pasteurella pneumotropica and an actinobacillus-like bacterium. Guineapig Pasteurellaceae differed serologically from mouse-derived P. pneumotropica NCTC 8284. An enzyme-linked immunosorbent assay (ELISA) using SP group antigen, developed to monitor 'natural' infections by SP group Pasteurellaceae in guineapigs, detected significantly more infection than did cultivation, and was found superior to an ELISA performed with P. pneumotropica NCTC 8284.

Antiobiogrammes were made of a number of isolates of Staphylococcus aureus, Escherichia coli and Pasteurella pneumotropica derived from rodent, rabbit or minipig colonies never treated with antibiotics. For S. aureus no differences between rats and mice were found in the percentage of resistant isolates. Gentamicin and erythromycin were found to be the most efficient, while the highest percentages of resistance were found to be against penicillins and sulphonamides. In general, the results from antibiogrammes on E. coli were rather uniform, with only slight differences between isolates from different species, except that more vancomycin and tetracycline-resistant minipig isolates were found. In almost all isolates of E. coli, resistance was shown against penicillin, fucidin, macrolides, lincosamides and tiamulin. For a number of antibiotics, mouse isolates of P. pneumotropica were more frequently found to be sensitive than rat isolates. The resistance patterns of E. coli from the minipigs were quite similar to resistance patterns found in farm pigs, but apart from this, the resistance patterns of the bacterial species tested did not resemble human or farm animal patterns in any of the animal species, and, therefore, these studies do not support the theory that S. aureus and E. coli in laboratory animal colonies derive from the normal flora of the human caretakers. The fact that rodent species of E. coli, in contrast to human and farm animal species, are sensitive to ampicillin, tetracyclines, and the combination of sulphonamides and trimethoprim, might be due to the fact that these antibiotics are not used in rodent populations.

Purpose-bred minipigs, are often used as the non-rodent species in toxicology. Infections may interfere with animal experiments, and there are no scientific reasons why the non-rodent species should be of a lower microbiological quality than the rodent species. Therefore, a system for health monitoring of pigs was developed in order to raise the quality of laboratory pigs to the level of laboratory rodents. This system, which includes screening for several viruses, bacteria and ecto- and endoparasites, was used for monitoring minipigs from a barrier unit with the same standards applied to rodents units. In these pigs only rotaviruses are found, which was shown by both serological antibody detection and by detection of rotaviral antigen in faeces. In minipigs from another unit with far less hygienic protection rotaviruses were also found along with certain influenza- and coronaviruses, as well as Pasteurella spp. It is concluded, that it is possible to raise pigs of a microbiological quality comparable to the quality of rats and mice, and that advanced microbiological monitoring in pigs will reveal useful information.

Cholera toxin (CT) is a potent adjuvant for the mucosal immune system. The purpose of this study was to determine if coadministration of CT with a potassium thiocyanate extract of Pasteurella multocida (PTE) leads to enhanced anti-PTE antibody activity and increased protection of rabbits against infection with P. multocida and associated disease. Groups of rabbits were immunized intranasally on days 0, 7, and 14, with phosphate buffered saline (PBS), 200 µg of CT, 1.0 mg of PTE, or 1.0 mg PTE with 200 ±g CT. Nasal lavage and serum samples were collected over 28 days after initial immunization and evaluated by ELISA for specific antibody directed against PTE. Marked increases in serum (IgG) and nasal lavage (IgA) anti-PTE antibody activity were found beginning after day 14 in rabbits immunized with PTE. Rabbits immunized with PTE and CT demonstrated further increases in this activity. Tracheobronchial lavage samples collected at the time of necropsy demonstrated a significant level of anti-PTE IgA activity in animals immunized with PTE, and coadministration with CT stimulated a further significant increase in this activity. Groups of similarly immunized rabbits were challenged 16 days after initial immunization with 5 × 107 CFUs of P. multocida. Nasal lavage samples were cultured for P. multocida over the next 10 days. Rabbits were euthanized within 10 days after challenge, tissues cultured for P. multocida, and histopathologic lesion severity graded using a numeric scale. Rabbits immunized with PTE survived longer, had less severe lesions of the lungs, pleura, and liver, and fewer P. multocida CFUs cultured from samples than PBS or CT controls. Coadministration of CT led to further reductions in lesion severity of those tissues and numbers of P. multocida CFUs cultured from samples. Increased nasal turbinate atrophy of rabbits immunized with PTE with or without CT was associated with increased mean survival time. In summary, coadministration of CT with PTE enhanced protective immunity to P. multocida disease and infection in rabbits.

The stability of pathogenic bacteria from laboratory animals was investigated in various transport media at different temperatures. Bordetella bronchiseptica and Salmonella typhimurium survived for 8 days in phosphate-buffered saline (PBS, pH 7.0) at 37, 24, 4 and -20 degrees C; Brucella canis at 24, 4 and -20 degrees C; Corynebacterium kutscheri at 4 and -20 degrees C; and Pseudomonas aeruginosa at all but -20 degrees C. A marked decrease in numbers of Pasteurella multocida and Past. pneumotropica was observed in PBS at all temperatures. Skimmed milk in PBS improved the survival of Pasteurella spp. and Ps. aeruginosa at -20 degrees C. Neither glycerin, ascorbic acid nor sodium thioglycollate improved survival. The numbers of viable B. canis, Ps. aeruginosa and S. typhimurium were maintained in blood or faecal specimens held for 8 days at 4 degrees C. These results indicated that transport in PBS at 4 degrees C was the only method satisfactory for all species of pathogenic organisms tested, but Pasteurella spp. were the most difficult to maintain.

2 groups of 44 specified-pathogen-free (SPF) mice, caged in groups of 4, were housed at weaning either in isolation in a building housing conventionally-reared animals, or in contact with those animals. 4 mice from each group were examined for microbiological flora and 4 for parasites at intervals for up to 90 days after transfer. Proscribed bacteria were isolated from the 'in contact' group after 42 days and endoparasites were observed by 16 days and thereafter. No proscribed bacteria were isolated from the other group, although some endoparasites were observed by 16 days. Proscribed bacteria were shown to be present in the conventionally-reared animals at weaning and ecto- and endoparasites by day 16 and thereafter. It is suggested that it may be possible to maintain SPF animals outside a SPF Unit for short periods of time, and that with great care the animals remain free of proscribed microflora.

In the course of post-mortem bacteriological examination of conventional guineapigs, 88 isolates belonging to the Pasteurella-Actinobacillus-group were recovered from 69 of 279 animals (25%). Most isolates were recovered from pneumonic lung, enteritic jejunum and inflamed mammary gland. No relationship was found between biotype and source of isolation. About 50% of isolates were recovered in pure culture or as the predominant micro-organism. It is concluded that members of the Pasteurella-Actinobacillus-group must be considered potentially pathogenic for guineapigs.

Organs of 10 mice or 5 rats were pooled and homogenized. A portion of the homogenate was cultured on several selective and enrichment media. The method was tested by adding various pathogenic organisms to organ homogenates. 103-104 microorganisms in homogenates of 10 organs were readily detected. This procedure allows even a small microbiological laboratory to detect a low frequency of infected animals in a colony.

Using a colony of Wistar-Imamichi rats contaminated with P. pneumotropica, the vaginal microflora was qualitatively and quantitatively investigated by swabbing. P. pneumotropica was the most dominant organism in the majority of rats examined. The population of P. pneumotropica and indigenous bacteria increased significantly higher at oestrus than in other oestrous stages. By the vaginal flushing technique changes in the population of P. pneumotropica and total bacteria, and changes in vaginal cell type and bacterial counts adhering to vaginal epithelial cells were consecutively investigated. The populations of P. pneumotropica and total bacteria were maximal at oestrus. The increase was correlated with an increase in cornified non-nucleated cells, with large numbers of adherent Gram-negative coccobacilli. The findings indicate that the vagina is a suitable site for colonization by P. pneumotropica in adult female rats, and that proliferation of P. pneumotropica may be due to increased affinity of the organism for cornified non-nucleated cells.

Pasteurella pneumotropica was isolated from cases of clinical conjunctivitis in mice. No isolates were obtained from normal mice.

46 feral rodents, including a common vole ( Microtus arvalis), house mice ( Mus musculus), muskrats ( Ondatra zibetica), house rats ( Rattus rattus) and brown rats ( R. norvegicus) were examined for bacteria of the Haemophilus- Pasteurella- Actinobacillus group. Haemophilus spp. (only M. musculus examined) were not obtained. All animal species were found contaminated by P. pneumotropica and/or Actinobacillus spp. Almost all M. musculus (96%) and most Rattus spp. (76%) were contaminated by P. pneumotropica and/or Actinobacillus spp. These bacteria were obtained most frequently from the upper respiratory tract, to a lesser extent from the lung and rarely from caecal contents, It is concluded that feral rodents might constitute an important source of contamination of laboratory rodents by members of the HPA-group.

134 bacterial isolates originally identified as Pasteurella pneumotropica were cultured from healthy, ill or dead mice, rats, hamsters, guineapigs, 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.

The efficiency of a commercially-available filter rack in providing a barrier to cross-infection between animals housed within it was evaluated by physical and biological methods. Although the rack provided ventilation to each animal box, the pattern of air movement within and around the rack encouraged rather than discouraged the transfer of certain pathogens between boxes. The use of air-barrier systems to provide locally clean environments is discussed.

A method for obtaining antemortem nasopharyngeal cultures is described. Its usefulness for detecting the carrier state of Pasteurella multocida in clinically normal, apparently healthy nasal-culture-negative rabbits is also discussed.

The effects of ovarian hormones on the vaginal population of Pasteurella pneumotropica in rats were investigated. Specified-pathogen-free adult female Wistar-Imamichi rats with a 4 day oestrous cycle were inoculated with P. pneumotropica in the vagina. Cyclic changes in the vaginal population of P. pneumotropica were not observed in ovariectomized rats and the bacterial population was at a similar level to that at normal dioestrus. Administration of oestrogen to ovariectomized rats caused an increase in the numbers of P. pneumotropica and total bacteria in the vagina nearly equal to that at oestrus in intact rats. The numbers of these organisms in the vagina of ovariectomized rats treated with progesterone did not change and were similar to those of control ovariectomized rats treated with sesame oil. Vaginal smears of ovariectomized rats treated with oestrogen were characterized by abundant cornified non-nucleated epithelial cells with many adherent Gram-negative coccobacilli and were similar to smears from intact rats at oestrus. These findings suggest that the proliferation of P. pneumotropica at oestrus in rat vagina may be primarily due to the environment provided by the degeneration of vaginal epithelial cells promoted by oestrogen secretion from the ovaries.

Hooded Lister rats were found to exhibit symptoms of Mycoplasma pulmonis infection. During the course of this chronic respiratory disease they developed ophthalmia and conjunctivitis which proved to be an opportunist infection with Pasteurella multocida. The severity of disease varied in individual rats, newborn rats entirely escaping infection. All cases responded to treatment with appropriate topical antibiotic and no sequelae were observed.

An unnamed Mycoplasma sp. (58B), Pasteurella pneumotropica and Streptobacillus moniliformis were isolated from the conjunctivas of rats, some of which were affected with conjunctivitis. There was no evidence to suggest that any of these organisms were the primary cause of conjunctivitis.

Experimental inoculation of organisms isolated from the conjunctiva of rats with clinical signs of conjunctivitis did not produce any disease. Natural transmission of these organisms from infected parents to their young diminished with succeeding generations.

Pasteurella multocida was isolated from 4 conventional rabbit colonies. Amongst 51 isolates, 48 (94%) belonged to a group of strains with similar morphological, cultural, biochemical and antigenic characteristics; they were named for convenience Lo strains and gave rise to local or ascending infections. All Lo strains were found to be a new serotype, but 8 of them had additional antigenic factors characteristic of known serotypes. The other 3 isolates (6%) were identified as serotype 3, known to have a wide host range: 2 strains were associated with septicaemia and haemotogenic spread, and 1 with snuffles. Diagnostic agglutinating antibodies were present in the sera of adult rabbits. Maternal antibodies were in the sera of rabbits of 3 and 4 weeks old, but by the age of 8 weeks the titres had fallen to an undetectable level.

The possible routes by which Pasteurella pneumotropica could have gained access to the animals in a strictly barrier-maintained unit, where it established an infection of the upper respiratory tract, are discussed. Details of management before and after the breakdown and the methods of restocking the same building as rapidly as possible with uninfected animals are described.

SUMMARY Clinical pasteurellosis was eliminated by administration of sulphamezathine in the drinking water (0'13 %) to pregnant females for 4 days before the expected date of parturition. 39 Pasteurella multocida infections were found to be causing problems in a small rabbit colony breeding 200 rabbits per annum. Of the first 20 does culled from the colony, 9 were culled because of Pasteurella infection, either mastitis, snuffles, otitis media or pyometron. Intermittent losses from pneumonia also occurred in sucking and recently weaned rabbits, caused by Pasteurella multocida. Following a report by Smith, Cross & Parker (1969) on the control of Pasteurella pneumonia in lambs using prenatal medication of the ewe with sulphamezathine, prekindling medication was commenced in the rabbit colony. The pregnant does were given medicated water (0·13 % sulphamezathine) commencing 4 days prior to the expected parturition date ad libitum. Does were not allowed access to other water in this period, to ensure intake of the medicated water. Medication was removed when the doe kindled. Since these control measures were instituted no further cases of otitis media, snuffles or pyometron have been seen. A further outbreak of mastitis did occur, associated with a change in staff servicing the colony. Investigation revealed that does were gaining access to non-medicated water in the prekindling period and were not drinking the medicated water. After this was corrected, no further cases were observed. Hagen (1966) used medication of the food in an attempt to control Pasteurella multocida infection. Sulphamezathine medicated feed (100 g/ton food) reduced neither pneumonia deaths nor Pasteurellaisolations. In a second observation (Hagen, 1967), sulphamezathine reduced Pasteurella isolations from mature animals.

A simple barrier system was used successfully to exclude Mycoplasma pulmonis and Sendai virus from a conventional mouse room. 49 weeks after introduction of the barrier system of management Pasteurella pneumotropica was first isolated from animals in the room.