Comparative Medicine Vol 58, No 5 Copyright 2008 October 2008 by the American Association for Laboratory Animal Science Pages 440–446

Study of a hinzii Isolate from a Laboratory Mouse

Nobuhito Hayashimoto,* Masahiko Yasuda, Kazuo Goto, Akira Takakura, and Toshio Itoh

Bordetella hinzii isolated from the trachea and lungs of a laboratory mouse with a respiratory infection was identified based on its phenotypic and genetic traits. The mouse showed sneezing with a chattering sound but without nasal discharge, and histopathologic examination revealed rhinitis, tracheitis, and bronchopneumonia. The isolate was a gram-negative, oxidase- and -positive, short rod-shaped organism that produced alkali from malonate. The results of biochemical identification, an alkali production test from malonate, and partial sequence analysis of the 16S rRNA gene (1523 bp) were consistent with those reported previously for B. hinzii. The isolate induced sneezing in ICR mice and sneezing and slight to severe dyspnea in NOD-SCID mice after experimental infection. Histopathologic examination revealed catarrhal rhinitis and bronchopneumonia in both strains of mice and interstitial in NOD-SCID mice. In light of these findings,B. hinzii was deemed to be a novel causative agent of respiratory disease in mice. This report describes the first isolation ofB. hinzii from a mouse and confirms the organism’s pathogenicity in mice.

The genus Bordetella consists of 8 named species and 1 that is chattering sound but without nasal discharge. The mouse was still awaiting formal description (Bordetella ansorpii sp. nov.).16 euthanized by exsanguination from the axillary artery and vein Some species in the genus are known pathogens of both humans under isoflurane anesthesia and underwent routine serologic, and animals. B. pertussis and B. parapertussis are the causative bacteriologic, and parasitologic examinations as described pre- agents of in humans,19 and B. bronchiseptica is a viously11 and additional tests. Organisms under investigation respiratory pathogen in some animal species, including dogs,2,23 included Bordetella bronchiseptica, Citrobacter rodentium, Corynebac- guinea pigs,24 pigs,14 rabbits,26 and laboratory rats.3 B. avium is a terium kutscheri, Mycoplasma pulmonis, Pasteurella pneumotropica, respiratory pathogen in poultry,4,12,13 whereas B. hinzii is a com- , Salmonella spp., and Staphylococcus aureus mensal organism of the respiratory tracts of poultry20,25 and an by culture; B. bronchiseptica, cilia-associated respiratory bacillus, opportunistic pathogen in immunocompromised humans.7,9 Al- Clostridium piliforme, Ectromelia virus, hantavirus, lymphocytic though some human clinical cases associated with B. hinzii infec- choriomeningitis virus, minute virus of mice, mouse adenovirus, tion have been associated with bacteremia,5,7,25 fatal septicemia,15 mouse cytomegalovirus, mouse encephalomyelitis virus, mouse chronic cholangitis,1 and infectious pulmonary exacerbation in a hepatitis virus, mouse parvovirus, Mycoplasma pulmonis, pneumo- patient with ,8 no cases of overt clinical infection in nia virus of mice, rotavirus, reovirus type 3, and Sendai virus by nonhuman mammals have been reported previously. Although 1 serology; and intestinal protozoa, pinworms, and ectoparasites strain of B. bronchiseptica from rabbits was reidentified asB. hinzii, by microscopic observation. Pneumocystis carinii was examined thus leading to the first reported isolation of this species from a by PCR assay of the left lobe of the lung as described previously.10 nonhuman mammal, a clinical description of B. hinzii infection in Respiratory were cultured by using tracheal swabs with rabbits was not provided.20 5% horse blood agar (Poa Media, Eiken Chemical, Tokyo, Japan) We recently isolated B. hinzii from the trachea and lungs of a and were assessed by observation of the surface of agar plates mouse with a respiratory infection. Here, we report this first case after incubation for 48 h at 37 °C. In addition, the cut surface of of B. hinzii infection in a mouse and present the results of experi- the accessory lobe of the lung was streaked directly on 5% horse mental infection of ICR and NOD-SCID mice with the isolate to blood agar and incubated for 48 h at 37 °C. investigate its significance as a murine pathogen. Microbiologic investigations. Biochemical tests of the isolate were performed (Api 20NE test kit, bioMérieux Japan, Tokyo, Ja- Materials and Methods pan) after Gram staining and oxidase and catalase tests. An alkali production test from malonate was performed by using malonate Index case. A female C57 BL/6 mouse of unknown substrain broth (Eiken Chemical) according to the manufacturer’s instruc- and unknown age was delivered to the ICLAS Monitoring Center tions. Confirmation of growth of the isolate was confirmed us- (Kawasaki, Japan) to investigate the cause of its sneezing with a ing DHL agar (Poa Media, Eiken Chemical), which is commonly used for isolation of Bordetella bronchiseptica from contaminated samples from laboratory rodents and rabbits in Japan.17 In addi- Received: 20 Dec 2007. Revision requested: 3 Feb 2008. Accepted: 2 March 2008. International Council for Laboratory Animal Science (ICLAS) Monitoring Center, Central tion, a slide agglutination test with a rabbit antiserum to B. bron- Institute for Experimental Animals, 1430 Nogawa, Miyamae, Kawasaki, Japan chiseptica strain BB1 (generated inhouse) was performed for the *Corresponding author. Email: [email protected]

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isolate. Briefly, 1 bacterial colony was mixed with 20 μl of rabbit cages and kept in a microisolation rack system (FRP BIO2000, antiserum on a slide glass and immediately was assessed by mac- CLEA Japan) that consist of 16 individually ventilated boxes with roscopic observation. glass fiber filters (FG50, American Air Filter, Louisville, KY) and Sequencing of the 16S rRNA gene and phylogenetic analysis. an exhaust unit with a HEPA filter. The boxes accommodated Organisms grown on 5% horse blood agar were collected and each cage as an isolated environment. This microisolation rack washed with PBS by centrifugation (2300 × g for 5 min at 4 °C), system was used exclusively for this study throughout the experi- and bacterial DNA was extracted (MagExtractor Genome DNA mental period. Sterilized commercial food pellets (CA1, CLEA Kit, Toyobo, Osaka, Japan) according to the manufacturer’s in- Japan) and autoclaved tap water were provided ad libitum. Each structions. The 16S rRNA gene of the isolate was amplified by cage was changed once weekly. The animals were maintained at using universal primers A and H6 in 50-μl reaction mixtures con- room temperature of 23 to 25 °C and relative humidity of 50% to taining 100 ng template DNA, 200 μM each dNTP (Takara Bio, 60% under a 12:12-h light:dark cycle.

Shiga, Japan), 10 μl 5× PCR buffer containing MgCl2 (Takara Bio), Ten mice of each strain were divided into 2 experimental 0.3 μM each primer and 1.25 U PrimeSTAR HS DNA Polymerase groups inoculated with either a high or low dose of the bacterial (Takara Bio) on a GeneAmp PCR System 9700 thermal cycler (Ap- suspension (5 × 107 CFU/ml or 5 × 103 CFU/ml, respectively). plied Biosystems, Foster City, CA). The amplification program Four mice of each strain served as negative controls. The mice comprised 98 °C for 1 min, followed by 30 cycles of 98 °C for 10 in the experimental groups were inoculated intranasally with s, 55 °C for 5 s, and 72 °C for 1.5 min, and a final extension step 25 μl of each bacterial stock. The bacterial cells for the inocula at 72 °C for 5 min. The amplified 16S rRNA gene of the isolate were cultured on blood agar for 48 h at 37 °C, collected with ster- was cloned into a pUC118 cloning vector (Takara Bio) by using ilized swabs, and suspended in sterile PBS to the appropriate the Mighty Cloning Kit (Takara Bio). The nucleotide sequence of concentration. The inocula were confirmed as pure cultures by recombinant plasmid was determined by using the Taq DyeDe- back-titration culture on horse blood agar. Control animals were oxy Terminator Cycle Sequencing kit (Applied Biosystems) and inoculated with 25 μl sterile PBS only. All mice were euthanized an automated DNA sequencer (model 310, Applied Biosystems) as described earlier and examined on postinoculation day 28. The with M13M4 and RV primers (Takara Bio) and sequence primers nasal concha, lungs and cecum of all mice underwent histopatho- designed for this study: F1, 5′ GCA GGC GGT TCG GAA AGA logic examination after hematoxylin and eosin staining. Samples AA 3′; F2, 5′ TCT AAT GAG ACT GCC GGT GA 3′; R1, 5′ GCA from nasal and tracheal swabs, the cut surface of the caudal lobe CCC TAC GTA TTA CCG CG 3′; and R2, 5′ ACA GCC ATG CAG of the right lung, and 100 μl blood drawn from the heart were in- CAC CTG TG-3′. oculated onto 5% horse blood agar for isolation of bacteria; cecal Sequences were assembled by using sequence analysis soft- samples were grown on DHL agar. Recovered bacterial colonies ware (Genetyx version 7, Genetyx, Tokyo, Japan). This software were assessed by using Gram staining, oxidase and catalase tests, also was used for multiple alignment and construction of a phy- biochemical tests, and malonate broth and were confirmed as iso- logenetic tree of the sequences. The sequence of the isolate was late 3224 by compliance with reported biochemical characteristics compared with the GenBank/EMBL/DDBJ data banks by us- of B. hinzii.8,15 Mice that died during the incubation period did not ing the NCBI FASTA program.22 The phylogenetic tree was con- undergo histopathologic or bacteriologic examinations because of structed from a total of 1432 bp of the 16S rRNA gene sequence extensive postmortem changes, such as autolysis. data of the isolate and deposited data of other Bordetella species in Health status of animals in experimental study. Vendor and in- GenBank/EMBL/DDBJ data banks by using the neighbor-joining house monitoring reports indicated that the mice were free of method.21 The branching pattern was tested with 1000 bootstrap bacterial, viral, and parasitic pathogens, as follows. On the basis replications. of serologic test results, mice were free of cilia-associated respi- Histopathologic examination for the index mouse. Histopatho- ratory bacillus, Cl. piliforme, Ectromelia virus, hantavirus, lym- logic examinations of the nasal cavity mucosa, trachea, right and phocytic choriomeningitis virus, minute virus of mice, mouse accessory lobes of the lung, liver, spleen, and kidney were per- adenovirus, mouse cytomegalovirus, mouse encephalomyelitis formed. These organs were fixed in 10% buffered formalin and virus, mouse hepatitis virus, M. pulmonis, pneumonia virus of stained with hematoxylin and eosin according to standard pro- mice, rotavirus, reovirus type 3, and Sendai virus. Mice were free cedures. The nasal concha was decalcified with 5% formic acid of B. bronchiseptica, B. hinzii, Ci. rodentium, Co. kutscheri, M. pulmo- prior to staining. nis, Pa. pneumotropica, Ps. aeruginosa, Salmonella spp., S. aureus and Experimental study. The protocol for the animal experiments dermatophytes were not detected by culture methods. Parasite was approved by the institutional animal care and use committee screening indicated that the animals were free of ectoparasites, according to the Regulations for Animal Experimentation of the intestinal protozoa, and pinworms. Pn. carinii, mouse parvovi- Central Institute for Experimental Animals. Sixteen 4-wk-old SPF rus, Helicobacter hepaticus and H. bilis were not detected by PCR female Jcl:ICR (ICR) mice and sixteen 5-wk-old SPF female NOD/ assay. ShiJic-scidJcl (NOD-SCID) mice were obtained from a commercial Slide agglutination test of serum from experimentally infected supplier (CLEA Japan, Tokyo, Japan). Prior to the study, 2 mice of ICR mice. each strain were euthanized by exsanguination from the axillary To investigate crossreactivity between B. hinzii and B. bron- artery and vein under isoflurane anesthesia and were examined chiseptica in serologic assays, serum samples from 10 experimen- for the following organisms, which were not included among tally infected ICR mice were obtained on postinoculation day those evaluated by the commercial supplier: mouse parvovirus, 28 and were tested against B. bronchiseptica strain BB1 in slide B. bronchiseptica, B. hinzii, S. aureus, and Pn. carinii for ICR mice agglutination tests. Briefly, 20 µl of each serum sample was mixed and mouse parvovirus, B. bronchiseptica, and B. hinzii for NOD- with 1 colony of B. bronchiseptica on a slide glass and immediately SCID mice. The animals were housed in sterilized polycarbonate assessed by macroscopic observation.

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Nucleotide sequence accession number. The sequence data of the isolate have been deposited in the GenBank/EMBL/DDBJ data banks under accession no. AB371725.

Results The mouse in the index case. The index mouse arrived at our center after approximately 2 h transport by car. No emaciation or abnormal behavior was observed. Consolidation in the accessory lobe of the lung was found at necropsy. No gross lesions were apparent in other organs. Histopathologic examination revealed rhinitis, tracheitis, and bronchopneumonia. Rhinitis (Figure 1 A ) and tracheitis were characterized by mild infiltration of inflam- matory cells (mainly neutrophils) in the submucosa, slight epithe- lial necrosis, and mild mucus secretion. Bronchopneumonia was characterized by hyperplasia of peribronchial lymph nodes and infiltration of inflammatory cells, mainly neutrophils, in the peri- bronchial interstitium (Figure 2 A, B). Bronchopneumonia also was present in the other lobes of the lung but to a lesser degree than in the accessory lobe. All microbiologic monitoring items tested were negative for the mouse. Microbiologic investigation. Round, convex, glistening, small (2 to 3 mm), grayish bacterial colonies were isolated in pure cul- ture on 5% horse blood agar inoculated with tracheal swabs and lung samples after 48 h of incubation at 37 °C. Gram staining of this isolate (assigned number 3224 at our institution) revealed gram-negative short rod-shaped bacteria. Isolate 3224 produced catalase and oxidase. Biochemical identification revealed a profile consistent with Bordetella avium (percent identification, 96.6%). The isolate yielded a positive alkali production test from ma- lonate. Together these findings were suggestive ofB. hinzii.8,15 Iso- late 3224 grew on DHL agar and was not agglutinated by a rabbit antiserum to B. bronchiseptica in a slide agglutination test. Sequencing of the 16S rRNA gene and phylogenetic analysis. Partial sequencing (1523 bp) of the 16S rRNA gene of isolate 3224 by using universal primers A and H and FASTA searches of the GenBank/EMBL/DDBJ data banks revealed the closest similar- ity (99.9%) with the sequence of B. hinzii LMG 13051T (accession number, AF177667). The sequence of our isolate differed from B. hinzii LMG 13051T at only 2 of 1523 nucleotides. In phylogenetic analysis, isolate 3224 formed a monophyletic cluster with B. hinzii LMG 13501T (Figure 3) with a high bootstrap value (98%). This cluster occupied a position intermediate between the clusters of 4 Bordetella species and B. avium; these results were consistent with the previously reported phylogenetic position of B. hinzii.15 Experimentally infected mice. By postinoculation day 3, all in- fected NOD-SCID mice showed sneezing with a chattering sound and slight dyspnea, as did all infected ICR mice by postinocula- tion day 10. The symptoms lasted throughout the experimental period (28 d after inoculation) in all mice. The sneezing in ICR mice gradually became less frequent over time. Whereas none of the ICR mice died, 4 of the NOD-SCID mice died or were eu- Figure 1. Histopathology of nasal cavities of the mouse in the index thanized for humane reasons due to severe dyspnea. One NOD- case and experimentally infected mice. (A) The index case. (B) Experi- mentally infected ICR mouse in the high-dose group on postinoculation SCID mouse in the high-dose group died on postinoculation day day 28. (C) Experimentally infected NOD-SCID mouse in the high-dose 10, and 1 in the low-dose group died on postinoculation day 21. group on postinoculation day 28. These histopathologic changes were Two NOD-SCID mice in the high-dose group were euthanized on characterized by mild infiltration of inflammatory cells (mainly neu- postinoculation days 11 and 15 (1 on each day). The NOD-SCID trophils) in the submucosa, slight epithelial necrosis, and mild mucus mice that died spontaneously had shown only slight dyspnea secretion. Hematoxylin and eosin stain; scale bar, 50 μm. prior to their sudden death. Because their condition did not ap- pear serious, euthanasia was not deemed necessary with regard to either severe disease or anticipation of spontaneous death.

442 Bordetella hinzii in a laboratory mouse

Figure 2. Histopathology of lungs of the mouse in the index case and experimentally infected mice. (A, B) The index case. (C, D) Experimentally infected ICR mouse in the high-dose group on postinoculation day 28. (E, F) Experimentally infected NOD-SCID mouse in the high-dose group on postinoculation day 28. Bronchopneumonia in the mouse in the index case (A, B) and ICR mice (C and D) was limited to the peribronchiolar region and characterized by hyperplasia of peribronchial lymph nodes. In contrast, bronchopneumonia in NOD-SCID mice (E, F) was spread to deep branches of bronchioles, including terminal and respiratory bronchioles. Pulmonary changes in NOD-SCID mice were characterized by hypertrophy of alveolar septa due to infiltration of neutrophils (interstitial pneumonia) and infiltration of macrophages into the alveolar lumen. The mice shown in this figure are the same as those in Figure 1. Hematoxylin and eosin stain; scale bar, 50 μm (A, C, and E) or 10 μm (B, D, and F).

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Isolate 3224 was recovered from the nasal cavity, trachea, and lungs in pure culture and with some species of intestinal bacterial flora from the cecum in all infected animals on postinoculation day 28 (Table 1). The mice that were euthanized on postinocula- tion days 11 and 15 had the same bacterial isolation results as those from animals on postinoculation day 28 (data not shown). No bacteria were isolated from the blood samples of any of the mice. Among negative control mice, no bacteria were isolated from the nasal cavity, trachea, or lungs, but some species of intes- tinal bacterial flora were isolated from cecum. Slide agglutination test of serum from experimentally infected ICR mice. All of the serum samples from the 10 experimentally infected ICR mice showed negative results for B. bronchiseptica in the slide agglutination test.

Discussion According to the biochemistry profile, the isolate from the in- dex case was tentatively identified as B. avium; this same profile previously was attributed also to B. hinzii.1,7,8,9,15 The production of alkali from malonate, a characteristic of our isolate, is a biochemi- cal feature useful for distinguishing B. hinzii from B. avium.8,15 Figure 3. Maximum likelihood consensus dendrogram based on 1432 Although the characteristic cellular fatty acid composition of B. consecutive positions of the 16S rRNA of the isolate and other Borde- hinzii has been used for identification and discrimination from tella species. Numbers at branching nodes are percentage of 1000 boot- related species,5,9 the relative proportions of fatty acids can vary strap replications. The scale bar represents 0.01 substitutions per nucle- depending on whether bacterial cells are harvested after 24 or 48 otide position. The GenBank accession numbers of the sequences used h of incubation.15 Discrimination of B. hinzii from the phenotypi- to construct the phylogenetic tree are: B. ansorpii, AY594190; B. avium, cally related species B. avium by cellular fatty acid analysis is not AF177666; B. bronchiseptica, U04948; B. parapertussis, U04949; B. pertussis, possible after 48 h of culture.15 In addition, cellular fatty acid com- AF142326; B. petrii, AJ249861; B. hinzii, AF177667; B. holmesii, U04820; B. 15 trematum; AJ277798; and Pseudomonas aeruginosa (used as the outgroup), position can vary somewhat between B. hinzii strains. For these Z76651. reasons, we did not use cellular fatty acid analysis for identifica- tion of our isolate in this study. Partial 16S rRNA sequencing re- cently was reported to be sufficient for identification ofB. hinzii.15 On necropsy, mucus was present in the nasal cavities of all in- The sequence of our isolate showed the closest similarity (99.9%) fected mice. No other gross lesions were noted in any of these with B. hinzii LMG13501T (AF177667) in the GenBank/EMBL/ animals, including the NOD-SCID mice that were euthanized DDBJ data banks. On the basis of these observations, isolate 3224 on postinoculation days 11 and 15. Histopathologic examination ultimately was identified as B. hinzii. revealed catarrhal rhinitis and bronchopneumonia in the lungs In the experimental infection study, all infected mice showed of all infected mice. Interstitial pneumonia was present in all in- sneezing, and the symptoms were similar to those of the mouse in fected NOD-SCID mice but not in the ICR mice. Rhinitis of mice the index case. The frequency of sneezing in ICR mice decreased of both strains was characterized by mild infiltration of inflamma- with time, and careful observation was necessary to confirm the tory cells (mainly neutrophils) in the submucosa, slight epithelial symptoms in the latter half of the experimental period. There- necrosis, and mild mucus secretion (Figure 1 B, C). Bronchopneu- fore, detection of chronic B. hinzii infection in immunocompetent monia in ICR mice was limited to the peribronchiolar region and mice solely by general observation may be difficult in advanced was characterized by hyperplasia of peribronchial lymph nodes stages. and infiltration of inflammatory cells, mainly neutrophils, in peri- In routine microbiologic monitoring of laboratory mice, the bronchial interstitium (Figure 2 C, D). In contrast, bronchopneu- trachea and cecum typically are used as culture samples to detect monia in NOD-SCID mice was not limited to the peribronchiolar respiratory and intestinal bacterial pathogens, respectively.18 In region but spread to deep branches of bronchioles such as termi- the present study, the isolate was recovered from these sites in all nal and respiratory bronchioles. Pulmonary changes in the NOD- experimentally infected mice. Therefore, these sites similarly may SCID mice were characterized by hypertrophy of alveolar septa be useful as culture samples to detect B. hinzii in microbiologic due to infiltration of neutrophils (interstitial pneumonia), and in- monitoring of laboratory mice. filtration of macrophages into the alveolar lumen (Figure 2 E, F). In this study, the C57BL/6 mouse in the index case showed NOD-SCID mice that were euthanized on postinoculation days 11 negative results in the agglutination test for B. bronchiseptica, the and 15 showed similar histopathologic changes to those of NOD- Bordetella species most frequently associated with laboratory ani- SCID mice on postinoculation day 28 (data not shown). Neither of mals. All 10 experimentally infected ICR mice likewise showed the strains of mice had any histopathologic changes in the cecum. negative results for B. bronchiseptica in the agglutination test on No clinical symptoms, gross lesions, and histopathologic changes postinoculation day 28, and isolate 3224 was not agglutinated by were noted in any of the negative control animals. a rabbit antiserum to B. bronchiseptica. Therefore, these 2 Bordetella

444 Bordetella hinzii in a laboratory mouse

Table 1. Number of experimentally infected mice positive for Bordetella hinzii on postinoculation day 28 Source of sample Nasal cavity Trachea Lung Blood Cecum High dose ICR mice (n = 5) 5 5 5 0 5 NOD-SCID mice (n = 2)a 2 2 2 0 2

Low dose ICR mice (n = 5) 5 5 4 0 5 NOD-SCID mice (n = 4)b 4 4 4 0 4 aOne mouse died on postinoculation day 10. One mouse each was euthanized on postinoculation days 11 and 15. bOne mouse died on postinoculation day 21. species appear to differ antigenically, and this difference may be hinzii from a patient with myelodysplastic syndrome. J Med Micro- useful for a serologic test for B. hinzii. biol 56:1700–1703. B. hinzii is a commensal organism of the avian respiratory tract, 8. Funke G, Hess T, von Graevenitiz A, Vandamme P. 1996. Char- and poultry have been suggested as important sources of human acteristics of Bordetella hinzii strains isolated from a cystic fibrosis 7,9 patient over a 3-year period. J Clin Microbiol 34:966–969. infection. However, some clinical cases lacked avian exposure, 9. Gadea I, Cuenca-Estrella M, Benito N, Blanco A, Fernández- and the importance of poultry as a source of infection is not yet Guerrero ML, Valero-Guillén PL, Soriano F. 2000. Bordetella hinzii, a 20 clear. With the exception of 1 case of B. hinzii infection in a rabbit, “new” opportunistic pathogen to think about. J Infect 40:298–299. no previous reports have clearly indicated that mammals are po- 10. Hunter JA, Wakefield AE. 1996. Genetic divergence at the mito- tential hosts of this bacterium. However, the results of the present chondrial small subunit ribosomal RNA gene among isolates of study suggest that mice may be a host of B. hinzii and therefore an Pneumocystis carinii from five mammalian host species. J Eukaryot important source of B. hinzii infection in humans. Microbiol 43:24S–25S. In conclusion, B. hinzii was isolated from the trachea and lung 11. Itoh T. 1999. Quality testing system for SPF animals in Japan and problems in the management of such systems. 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