Laboratory Animal Science Vol 48, No 5 Copyright 1998 October 1998 by the American Association for Laboratory Animal Science

Detection of Pasteurellaceae in Rodents by Polymerase Chain Reaction Analysis

Frank Bootz,1* Susanne Kirschnek,2 Werner Nicklas,2 Stefanie K. Wyss,1 and Felix R. Homberger1

The family Pasteurellaceae presently consists of the gen- sidering P. pneumotropica the only relevant species of era , , and . Infection Pasteurellaceae is, therefore, short sighted at best. Although with Pasteurellaceae is found in all mammalian and avian there is no universal agreement, we believe that other species. These are the most common organisms members of the Pasteurellaceae may also be of importance isolated from contemporary conventional and barrier-main- for laboratory animal medicine. If not principally as agents tained rodent colonies (1, 2). A number of them have been causing overt disease, they may influence biomedical re- documented to be opportunistic pathogens and have been search by causing subclinical infection. This is based on implicated as causative agents of various diseases. Infec- our interpretation of the information available, either pub- tions are usually asymptomatic and persistent. Clinical lished or from personal experiences. In addition, we believe manifestation of infection may include pneumonia; conjunc- that the name P. pneumotropica, due to a lack of sufficiently tivitis; metritis; cystitis; pleural, peritoneal, and orbital sensitive differentiation methods, has been used ambigu- abscesses; dermatitis; and panopthalmitis (3). ously in the past. This should be investigated further. Newer Pasteurellaceae also are known to affect the immune sys- methods, such as polymerase chain reaction (PCR) assays tem and alter the immune response (4). Owing to the bio- (12, 13), will help to answer this question by differentiat- chemical diversity of the Pasteurellaceae, identification of ing Pasteurella strains on a genetic level. Therefore, to not isolates often has been challenging (5). Animals infected miss an organism that might be potentially detrimental to with these organisms are usually asymptomatic carriers biomedical research, a state-of-the-art health-monitoring harboring few bacteria in their respiratory or genital tract. program in laboratory rodents needs to include all mem- The organisms sometimes are difficult to culture because bers of the Pasteurellaceae in its screening spectrum (14). they are present in small numbers or may be overgrown Monitoring for bacterial agents is usually performed by by other bacteria; some are even growth factor dependent culture methods. Another method used to detect infection (6). Actually, this phenomenon has historically been used with Pasteurellaceae in rodents is serologic testing, which for taxonomic purposes. All factor-dependent Pasteurellaceae may be unreliable due to lack of cross-reactivity between have been assigned to the Haemophilus genus. However, different isolates or may yield false-positive results due to recent studies involving comparison of the 16S rRNA gene unpredictable cross-reactivities. Owing to the diversity of have suggested that classical done on the basis of the Pasteurellaceae, a number of antigens are required to factor dependency and biochemical profiling may not be cover all species in the family. In our hands at least, we adequate. Factor-dependent bacteria have been found in all have found a high number of false-positive reactions, most three genera of Pasteurellaceae, and the biochemical profile often due to the age of the animals from which the sera is not directly linked with phylogenetic relationship (6). were obtained. To avoid false-positive reactions, it has been Pasteurella pneumotropica is considered the most (often recommended that animals older than 12 weeks (15) not only) pathogenic member of the Pasteurellaceae in rodents. be tested. It was specifically looked for in routine health moni- The most promising approach for detecting infection with toring; all other members of this family were disre- Pasteurellaceae might be PCR. This method, as well as se- garded (7). However, other members of this family, such rologic testing, was documented to detect P. pneumotropica as H. influenzaemurium (8, 9), P. ureae (later classified as in culture-negative animals (16–18). This recently described Actinobacillus muris [10]), and a not yet definitely described PCR is based on the 16S rRNA sequence of the type strain Pasteurella species found in rats (11), have been described and is therefore unlikely to detect all biotypes that are ex- as causing clinical disease. Also, different isolates from the pected to infect laboratory rodents. On the basis of this same species of Pasteurellaceae may have different bio- background, we decided to develop a PCR assay that is quick logical traits such as pathogenicity. Pathogenicity of an iso- and easy to perform, and is able to detect all strains of late does not correlate with its biochemical profile. Con- Pasteurellaceae that might be infective for rodents. The investigation was carried out using animals from Institute of Laboratory Animal Science, University of Zurich, Switzerland1 our routine health-monitoring program. A total of 35 mice, and Central Animal Laboratories, German Cancer Research Center, Heidel- 23 rats, 2 gerbils, 10 Syrian hamsters, and 1 rabbit of vari- berg, Germany2 *Address correspondence to Dr. Frank Bootz, Institute of Laboratory Ani- ous ages and either sex were examined. The animals were mal Science, University of Zurich, Winterthurerstrasse 190, CH-8057 derived from different experimental colonies of the Uni- Zurich, Switzerland.

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Table 1. Phenotypic properties of Pasteurellaceae isolates that tested positive by polymerase chain reaction (PCR) Pasteurella pneumotropica isolates ATCC NCTC 12555a I198012 E409011 8141b H199011 K272011 E323021 G019021 J426011 F443021 F065011 K205031 Heyl Heyl Heyl Jawetz Jawetz Jawetz 1a 1a 1a 20 5 6 NAD dependency ------Mannitol ------Urease + +++++ ++ ++++ Phosphatase + +++++ ++ ++++ Indole - +++++ - - -+++ Arabinos + + + ------+ Xylose + +++++ ++ + -- + Trehalose + +++++ ++ ++- - Melibiose + + + - - - + + + - - - Raffinose + + + - - - + + + - - - Ribose + +++++ ++ ++++ Esculin ------Origin of isolate Mouse Rat Mouse Mouse Mouse Mouse Rat Mouse Rat Mouse Rat Rat NCTC NCTC NCTC HIM K227011 K048012 11051c 11146d K063022 K091017 K026021 J384012 G106012 I159022 7857e 931-8f Pp21 Pp22 P. sp.(rat) H. infl. m. H. infl. m. P. sp. HW Act. murisActino 11 Actino 12 Hae. para. Hae. para. Hae. sp. NAD dependency ------++ Mannitol ------+ ++-- Urease + + ---- -+ ++-+ Phosphatase ------++ Indole - + ------++-+ Arabinose ------Xylose ------+ Trehalose + +++++ ++ ++ -- Melibiose + + ---- +- ++ -- Raffinose + + ---- ++ ++ +- Ribose + + - + + + + + + + - + Esculin - + ---- ++ +- -- Origin of isolate Mouse Mouse Rat Mouse Mouse Mouse Mouse Mouse Mouse Mouse Human Rat aPasteurella pneumotropica biotype Heyl bP. pneumotropica biotype Jawetz through this incision into the nasopharynx from which cPasteurella sp. (rat) specimens were collected for culture. Pharynx, trachea, and dHaemophilus influenzaemurium eH. parainfluenzae lung specimens were collected and stored at -70ЊC before fHaemophilus sp. taxon B Kilian HK 447 being submitted to DNA extraction. The bacterial strains tested were chosen to include a Table 2. Bacterial strains that tested negative by PCR member of each genus and subgroup or phenotype of all subgroup 1 hitherto detected rodent biotypes and to cover the broad spectrum of the family. We also tested a number of bacte- E. sakazakii ria not of the Pasteurellaceae family, either closely related A 189/2/83 to the target organism or often present in diagnostic samples, such as Enterobacteriaceae (average phylogenetic Salmonella spp. difference of 12%) (19), , Pseudomonadaceae, A 329/2/95 Vibrionaceae subgroup 2 and Mycoplasmataceae. A complete list of bacterial strains Aeromonas sobria B 6/2/95 used in this study is given in Tables 1 and 2. Pseudomonaceae Twenty-four strains of Pasteurellaceae isolated or main- Bordetella bronchiseptica rat 437/95 tained at the German Cancer Research Center (DKFZ) (5) Mycoplasmataceae were grown on sheep blood agar. Three strains of Mycoplasma pneumoniae Micrococcaceae Pasteurellaceae (phenotypic properties not shown) and Staphylococcus aureus those not Pasteurellaceae were maintained at the Insti- tute of Laboratory Animal Science of the University of versity of Zurich animal facility. Cages, feeders, and water Zurich as reference strains on various media. One-day-old bottles were changed each week. All animals had access to colonies were picked and diluted in 100 ␮l of phosphate- feed (no. 890 mouse and rat maintenance diet; NAFAG, buffered saline for DNA extraction. Gossau, Switzerland) and water ad libitum. Animal rooms Swab specimens were plated directly onto 5% sheep blood were kept at 22 Ϯ 2ЊC with a 12/12-h light/dark cycle and and chocolate agars, and incubated in an oxygen-reduced 55 Ϯ 10% relative humidity. Quarterly health monitoring atmosphere in a candle jar at 37ЊC. After 24 and 48 h, the of rodents included comprehensive ante- and postmortem plates were examined for colonies morphologically typical diagnostic evaluations for viral, mycoplasmal, bacterial, for the Pasteurellaceae. The bacterial colonies were deter- fungal, and parasitic agents. mined to belong to the Pasteurellaceae, using the combined Animals were euthanized by use of carbon dioxide. The results of morphologic (colony morphology, small gram- trachea (immediately distal to the pharynx) was laid bare negative coccobacilli or rods) and biochemical evaluations, aseptically, and an incision was made. A swab was inserted including the API-NE test system (BioMérieux, Marcy-

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510 bp

Figure 1. Detection of various Pasteurellaceae isolates by polymerase chain reaction (PCR) analysis, using a 1% agarose gel. Lanes: Marker, 1-kb DNA molecular size marker; 1, Pasteurella pneumotropica (isolate E323021); 2, P. pneumotropica biotype Heyl (E409011); 3, P. pneumotropica biotype Jawetz (NCTC 8141); 4, P. pneumotropica biotype Heyl (I198012); 5, Pasteurella pneumotropica biotype Heyl (ATCC 12555); 6, P. pneumotropica biotype Jawetz (K272011); 7, P. pneumotropica (F065011); 8, P. pneumotropica (K205031); 9, Pasteurellaceae 20 (F443021); 10, Pasteurellaceae 21 (K227011); 11, Pasteurellaceae 22 (K048012); 12, Pasteurellaceae rat (NCTC 11051); 13, Actinobacillus sp. 12 (I159022); 14, Actinobacillus sp. 11 (G106012); 15, A. muris (J384012); 16, Haemophilus influenzaemurium (K091017); 17, H. influenzaemurium (K063022); 18, H. influenzaemurium (NCTC 11146); Control, mixture of pharynx, trachea, and lung specimens from a specific-pathogen-free mouse; and 510 bp on the right side is the size of the amplified fragment. l’Etoile, France). Minimal characteristics of the family database to confirm that there is no identity with any other Pasteurellaceae are lack of motility, formation of small known DNA sequence outside the Pasteurellaceae (Fast A, amounts of glucose under aerobic (oxidative) and anaero- GCG, HUSAR). bic (fermentative) conditions, weak oxidase reaction, reduc- Reaction mixtures in a total volume of 25 ␮l contained 1 ␮l tion of nitrate to nitrite, and no metabolization of citrate of sample DNA and 24 ␮l of PCR MasterMix (10 ␮l of PCR and arginine. Species requiring growth factors (e.g., NAD, buffer, 0.8 ␮l of 25 mM dNTPs, 1 ␮l of forward primer, 1 ␮l of haemin) do not grow on blood agar. They can be cultured reverse primer, 0.5 ␮l of Taq DNA polymerase [Boehringer on chocolate agar and are classified as Haemophilus. Mannheim, Mannheim, Germany], and 87 ␮l of H2O). The DNA from tissue specimens was extracted, using a Amplifications were performed in a Perkin Elmer DNA extraction kit (QiAmp tissue kit; Qiagen, Hilden, GeneAmp PCR System 2400 thermocycler (Perkin Germany). Cultured bacteria were treated with sodium Elmer, Foster City, Calif.). The program consisted of one dodecyl sulfate, and DNA was isolated by phenol/chloro- cycle of 95ЊC for 4 min, then 35 cycles of 94ЊC for 1 min, form extraction and ethanol precipitation. The DNA was 55ЊC for 1 min, 72ЊC for 1 min, and finally one cycle of pelleted by centrifugation, washed in 70% ethanol, and re- 72ЊC for 4 min followed by subsequent storage at 4ЊC. suspended in 10␮l of H2O. The PCR products were separated by gel electrophore- A set of primers was selected on the basis of 16S rRNA sis in 1% agarose in Tris-borate/EDTA electrophoresis sequences of rodent isolates representing five pheno- buffer containing ethidium bromide, and were visual- typic groups (20) and the type strains of P. ized under UV light. pneumotropica (P. pneumotropica Jawetz NCTC 8141 Using the described primer set, a 510-bp DNA fragment obtained from the EMBL nucleotide sequence database) was amplified from all Pasteurellaceae, but not from 13 and A. muris (21). The aim was to detect all hitherto other bacterial species tested. The diagnostic band is re- sequenced rodent Pasteurellaceae, including those iso- producibly identical in all cases. The specific PCR prod- lates found in the EMBL database. uct could be found in all test-positive samples. All The forward primer, 5'-CATAAGATGAGCCCAAG-3', a Haemophilus strains yielded an additional band approxi- 17-base segment, was chosen between bases 215 and 231. mately 1,200 bp long (Figure 1). This additional band was It has five base differences, compared with the same se- not found in any other isolate. The identity of this larger quence of Escherichia coli. The reverse primer, 5'- fragment has not yet been investigated. GTCAGTACATTCCCAAGG-3', is located between bases Our PCR was able to detect all strains of Pasteurellaceae 747 and 730 and differs by six bases from that of E. coli. tested, even strains with slight sequence differences from Base positions are given according to P. pneumotropica those of our previously sequenced strains. Recently obtained NCTC 8141 numbering (accession no. M75083). Primers additional sequences of rodent Pasteurellaceae, including were checked against the whole EMBL nucleotide sequence growth factor-dependent rodent isolates belonging to the

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Table 3. Comparison of PCR and culture detection of Pasteurellaceae Pasteurellaceae-free on the basis of culture results. Revers- from various sites in naturally infected animals ing the issue, we used our more sensitive PCR as the “gold a b N Species Sample PCR Culture results standard” to calculate the diagnostic sensitivity of the cul- 17 Mouse ph/tra/lu - - - Negative ture isolation method to be only 69%. 2 Mouse ph/tra/lu + + + Negative 1 Mouse ph/tra/lu / + + Negative In our study, we used tissue specimens from pharynx, tra- 1 Mouse ph/tra/lu + - - Negative chea, and lungs for DNA isolation. As expected, we most of- 1 Mouse ph/tra/lu / + - Negative 5 Mouse ph/tra/lu + + + P. pneumotropica ten found that we were able to detect Pasteurellaceae in the 1 Mouse ph/tra/lu + + - P. pneumotropica pharynx. However, in some instances only the lung speci- 7 Mouse ph/tra/lu - - + P. pneumotropica mens were test positive. In our limited study, this was most 6 Rat ph/tra/lu - - - Negative 1 Rat ph/tra/lu + - - Negative often the case for animals that had been infected with the V 1 Rat ph/tra/lu - - + Negative factor-dependent organisms. It is known that these bacteria 1 Rat ph/tra/lu + - - P. pneumotropica most frequently colonize the lungs and the trachea (24), and 2 Rat ph/tra/lu + + - P. pneumotropica 1 Rat ph/tra/lu + + Haemophilus sp. 80 to 90% of all strains were isolated from these sites. Cul- 2 Rat ph/tra/lu + + - Haemophilus sp. ture of specimens from the deep part of the respiratory tract 2 Rat ph/tra/lu + - - Haemophilus sp. 7 Rat ph/tra/lu - - + Haemophilus sp. is therefore advisable. Thus, we recommend routine testing 1 Gerbil ph/tra/lu + + + Negative of at least pharynx and lung, using our PCR assay. 1 Gerbil ph/tra/lu + + - Negative The fact that this new PCR assay works without prior 1 Hamster ph/tra/lu + - - Negative 3 Hamster ph/tra/lu - - + Negative culture of the organism not only increases sensitivity of de- 6 Hamster ph/tra/lu - - - Negative tection, but also significantly reduces the turnaround time 1 Rabbit ph/tra/lu + + + P. multocida of the assay. Typical culture isolation requires a minimum aPharynx/trachea/lung of three days until the organism is identified. Detection us- b- = Negative PCR result; + = positive PCR result; / = no specimen tested ing this PCR takes a mere 5 h after specimen collection. The H. parainfluenzae complex, and the hitherto unclassified actual work time remains about the same, and costs are taxon B (22), had substitutions of up to two bases near the equivalent. For laboratories familiar with the methods used 5' end of primer 1. Because these strains were also test in molecular biology, the PCR assay is easy to perform and positive by our PCR assay, we conclude that these substi- does not require expertise in identifying bacterial colonies. tutions have no influence on sensitivity of the PCR assay. The major advantage of this novel PCR assay is that it The sensitivity of the new PCR assay was tested by com- detects all members of the Pasteurellaceae at the same time. parison with bacterial culture (Table 3). All culture-posi- If an animal or a colony tests negative, further work-up is tive samples also were test positive by use of the PCR not required. A colony can be declared Pasteurellaceae-free method. The diagnostic sensitivity of a test is the percent- with the highest degree of confidence. If an animal tests age of samples with true-positive results (in this instance, positive, one may choose to attempt to identify the strain. determined by culture) that tested positive (in this instance, This may be done by use of traditional methods, such as by PCR) (23). For our new PCR assay, summary of the com- culture and biochemical characterization, keeping in mind parison lets us calculate this to be 100%. that pathogenicity of a strain may not correlate with its bio- The diagnostic specificity of a test traditionally is the chemical profile. A more straightforward option is sequenc- percentage of samples with true-negative results that tested ing of the PCR product generated and comparing it with the negative (23). However, the PCR assay was able to detect published sequences of other strains. If one is interested only more test-positive samples than was the culture method in P. pneumotropica, this strain may be selectively identi- (Table 3). This brings us now to limitations of these com- fied by randomly amplified polymorphic DNA-PCR (12, 13). parison tests. Using culture isolation as the “gold standard,” On the basis of this information, the laboratory animal vet- it seems that the PCR yielded 13 false-positive results. This erinarian, together with the investigator, can then decide on would mean that the diagnostic specificity of the assay is the best course of action. Issues to be considered are mani- <70%. We therefore decided to look at the analytic specific- festation of clinical disease (especially in immunodeficient ity of our PCR assay. This was tested by amplification of 24 and/or genetically altered animals) and the potential of in- strains of Pasteurellaceae and 13 strains that were not terference with the research projects involved. Pasteurellaceae. The expected 510-base pair DNA fragment On the basis of these findings, we conclude that our was amplified from all Pasteurellaceae tested (Table 1), but PCR assay will greatly facilitate and improve detec- not from any other bacteria (Table 2). From these results, tion of Pasteurellaceae in rodents. It is 30% more sen- the analytic specificity was calculated to be 100%. sitive than the culture isolation method and more spe- On the basis of this finding, we believe it is safe to as- cific than serologic testing, without limiting testing to sume that the 13 samples for which culture and PCR re- a certain age group. The PCR assay is the ideal screen- sults disagreed were indeed false-negative results on the ing method for routine testing. It will detect most mem- basis of the culture method. Interestingly, all specimens bers of the Pasteurellaceae with high sensitivity and that were test positive by PCR and culture negative were specificity. A positive PCR result may then be followed from colonies where samples from other animals were test up by isolation and biochemical characterization if desired. positive. There was never a positive PCR result for speci- mens taken from a colony that was determined

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12. Weigler, B. J., J. P. Thigpen, M. F. Goelz, et al. 1996. Ran- Acknowledgement domly amplified polymorphic DNA polymerase chain reaction We thank Maria Hagmann for help with the cultural testing of assay for molecular epidemiologic investigation of Pasteurella the clinical samples. pneumotropica in laboratory rodent colonies. Lab. Anim. Sci. 46:386–392. 13. Weigler, B. J., L. A. Wiltron, S. I. Hancock, et al. 1998. References Further evaluation of a diagnostic polymerase chain reac- tion assay for Pasteurella pneumotropica. Lab. Anim. Sci. 1. Saito, M., M. Nakagawa, K. Kinoshita, et al. 1978. Etio- 48:193–196. logical studies on natural outbreaks of pneumonia in mice. 14. Rehbinder, C., P. Baneux, D. Forbes, et al. 1996. FELASA Jpn. J. Vet. Sci. 40:283–290. recommendations for the health monitoring of mouse, rat, 2. Sparrow, S. 1976. The microbiological and parasitological hamster, gerbil, guinea pig and rabbit experimental units. Lab. status of laboratory animals from accredited breeders in the Animals 30:193–208. United . Lab. Animals 10:365–373. 15. Boot, R. Personal communication. 3. National Research Council. 1991. Infectious diseases of 16. Wang, R-F., W. Campbell, W. W. Cao, et al. 1996. Detection mice and rats. National Academy Press, Washington, D.C. of Pasteurella pneumotropica in laboratory mice and rats by 4. Czuprynski, C. J., and A. K. Sample. 1990. Interactions of polymerase chain reaction. Lab. Anim. Sci. 46:81–85. Haemophilus-Actinobacillus-Pasteurella bacteria with phago- 17. Boot, R., H. Thuis, and M. A. Koedam. 1994. Infection by V cytic cells. Can. J. Vet. Res. 54:36–40. factor dependent Pasteurellaceae (Haemophilus) in rats. J. 5. Nicklas, W., A. Benner, and P. Mauter. 1995. Computer- Exp. Anim. Sci. 37:7–14. assisted classification of Pasteurella pneumotropica biotypes 18. Boot, R., H. Thuis, J. Veenema, et al. 1994. Colonization on the basis of biochemical criteria. Contemp. Top. Lab. Anim. and antibody response in mice and rats experimentally in- Sci. 34:68. fected with Pasteurellaceae from different rodent species. Lab. 6. Nicklas, W. 1989. Haemophilus infection in a colony of labo- Animals 28:130–137. ratory rats. J. Clin. Microbiol. 27:1636–1639. 19. Dewhirst, F. E., B. J. Paster, I. Olsen, et al. 1992. Phylog- 7. Kraft, V., A. Deeny, H. M. Blanchet, et al. 1994. Recom- eny of 54 representative strains of species in the family mendations for the health monitoring of mouse, rat, hamster, Pasteurellaceae as determined by comparison of 16S rRNA guinea pig and rabbit breeding colonies. FELASA report no. sequences. J. Bacteriol. 174:2002–2013. 1. Lab. Animals 28:1–12. 20. Nicklas, W., and S. Kirschnek. 1996. Phenotypic and geno- 8. Csukas, Z. 1976. Reisolation and characterization of typic relationship of Pasteurella pneumotropica biotypes and -murium. Acta Microbiol. Acad. Sci. additional selected rodent Pasteurellaceae. Contemp. Top. Lab. Hung. 23:89–96. Anim. Sci. 35:52. 9. Kirschnek, S., M. Ryll, J. Busse, et al. 1997. Identification 21. Dewhirst, F. E. Personal communication. and characterization of Haemophilus influenzaemurium iso- 22. Nicklas, W., M. Staut, and A. Benner. 1993. Prevalence and lated from laboratory mice, p. 66–68. In P. N. O’Donoghue (ed.), biochemical properties of V factor dependent Pasteurellaceae Harmonization of laboratory animal husbandry. Royal Soci- from rodents. Zentralbl. Bakteriol. 279:114–124. ety of Medicine Press, London. 23. Meyer, D. J., and J. W. Harvey. 1998. Interpretation and 10. Ackermann, J. I., and J. G. Fox. 1981. Isolation of Pas- diagnosis. p. 17-19. In W. B. Saunders, Veterinary laboratory teurella ureae from reproductive tracts of congenic mice. medicine. W. B. Saunders Co., Philadelphia. J. Clin. Microbiol. 13:1049–1053. 24. Nicklas, W., and A. Benner. 1994. Prevalence of V factor 11. Schulz, S., S. Pohl, and W. Mannheim. 1977. Mischinfektion dependent Pasteurellaceae in laboratory rodents and their von Albinoratten durch Pasteurella pneumotropica und eine phenotypic classification. In J. Bunyan (ed.), Welfare and sci- pneumotrope Pasteurella species. Zentralbl. Vet. Med. B ence. Royal Society of Medicine Press, London. 24:476–485.

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