Serological Response to Pasteurella multocida NanH Sialidase in Persistently Colonized Rabbits

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CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Sept. 2004, p. 825–834 1071-412X/04/$08.00⫹0 DOI: 10.1128/CDLI.11.5.825–834.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Vol. 11, No. 5

Serological Response to Pasteurella multocida NanH Sialidase in Persistently Colonized Rabbits Susan Sanchez,1 Shaikh Mizan,2 Charlotte Quist,1 Patricia Schroder,2 Michelle Juneau,1 Donald Dawe,2 Branson Ritchie,3 and Margie D. Lee2* Athens Diagnostic Laboratory,1 Department of Infectious Diseases,2 and Department of Small Animal Medicine,3 College of Veterinary Medicine, University of Georgia, Athens, Georgia Received 1 March 2004/Returned for modification 5 May 2004/Accepted 21 May 2004

Pasteurella multocida is a mucosal pathogen that colonizes the upper respiratory system of rabbits. Respiratory infections can result, but the bacteria can also invade the circulatory system, producing abscesses or septicemia. P. multocida produces extracellular sialidase activity, which is believed to augment colonization of the respiratory tract and the production of lesions in an active infection. Previously, it was demonstrated that some isolates of P. multocida contain two unique sialidase genes, nanH and nanB, that encode enzymes with different substrate specificities (S. Mizan, A. D. Henk, A. Stallings, M. Meier, J. J. Maurer, and M. D. Lee, J. Bacteriol. 182:6874-6883, 2000). We developed a recombinant antigen enzyme-linked immunosorbent assay (ELISA) based on the NanH sialidase of P. multocida and demonstrated that rabbits that were experimentally colonized with P. multocida produce detectable anti-NanH immunoglobulin M (IgM) and IgG in serum, although they demonstrated no clinical signs of pasteurellosis. In addition, clinically ill pet rabbits infected with P. multocida possessed IgM and/or IgG antibody against NanH. The NanH ELISA may be useful for the diagnosis of P. multocida infections in sick rabbits as well as for screening for carriers in research rabbit colonies.

P. multocida isolates vary in their abilities to produce disease in animals; some are associated primarily with upper respiratory disease, while others cause septicemia, abscesses, and pneumonia (11, 15). However, in order to initiate infection, the bacteria must colonize the respiratory mucosa, and organisms that inhabit mucosal surfaces frequently produce sialidases (12, 52). These enzymes have been shown to exhibit glycolytic activity on mucin, which releases terminal sialic acid residues that can then be used as a bacterial carbon source (12, 43, 52). Sialidase is the only extracellular glycolytic enzyme produced by P. multocida, suggesting that this enzyme probably plays a major role in the ability of P. multocida to colonize animals (16, 35, 50). Many P. multocida isolates possess two sialidase genes that encode enzymes with different substrate specificities, and NanH sialidase-deficient mutants of P. multocida have a reduced ability to replicate with host glycoconjugants as carbon sources (43). Expression of sialidase has been shown to occur during infection; therefore, the animal host is likely to have been exposed to the protein during mucosal colonization by the bacteria (51, 58). Except for substrate-binding residues, the Pasteurella enzyme exhibits little homology with other sialidases (43), suggesting that this antigen may be useful for the serological diagnosis of pasteurellosis. In this study we report on the use of a NanH enzyme-linked immunosorbent assay (ELISA) to detect P. multocida infection in healthy and clinically ill rabbits.

Pasteurella multocida can be a virulent pathogen of rabbits, producing fatal septicemia, pneumonia, chronic rhinitis, and otitis media as well as multiple abscesses; however, some animals are persistently colonized and exhibit no apparent signs of disease (37, 55). Many rabbits become colonized with Pasteurella soon after birth, and after weaning more than 75% of rabbits that nurse from infected dams become culture positive (22). The prevalence of P. multocida in clinically healthy rabbits ranges from 20 to 90%, depending on the methods used for detection, as well as the age and health status of the rabbit (19, 37, 55). Laboratory rabbits colonized with P. multocida often develop clinical disease after being shipped to a research facility, but persistently colonized, asymptomatic rabbits have been shown to produce aberrant results if they are used in research (18, 48). The effect of pasteurellosis on biomedical research is so profound that continuous screening of research rabbit colonies is recommended (54). Culture of nasal swab specimens has been shown to be unreliable for screening, since up to 30% of infected animals may not be detected by this method (23, 24). In addition, serological screening has not been effective in identifying all persistently colonized rabbits because most of the serological tests use uncharacterized antigen mixtures that may not detect the multitude of serotypes that colonize rabbits (11, 13, 26, 30, 32, 38, 39, 41, 49, 59). Vaccination is not commercially available because of a lack of efficacy, and furthermore, antibiotics may be effective for resolving the symptoms in sick animals but usually do not clear the bacteria from colonized animals (17, 27, 40, 56).

MATERIALS AND METHODS Bacterial strains and growth conditions. The Pasteurella isolates used for analysis in this study are described in Table 1. Escherichia coli strain M15(pREP4) (Qiagen, Chatsworth, Calif.) and P. multocida isolates were stored at ⫺70°C in a solution of 0.1% peptone–15% glycerol. Bacterial strains and isolates were grown in brain heart infusion (BHI) broth or on agar (Difco

* Corresponding author. Mailing address: Department of Avian Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602. Phone: (706) 583-0797. Fax: (706) 542-5630. Email: [email protected] 825

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CLIN. DIAGN. LAB. IMMUNOL. TABLE 1. Pasteurella isolates used in study

P. multocida isolate

Characteristics

Reference(s) or source

86-1913 P-1059 X-73 P-1662 P-1702 P-2192 P-1997 P-1581 P-2095 P-2100 P-903 P-1573 P-1591 P-2225 P-2237 P-2723 ATTC 7228 SS-1 SS-2

Turkey isolate; LPS serotype A:3,4 Reference type strain, LPS serotype 3 Reference type strain, LPS serotype 1 Reference type strain, LPS serotype 4 Reference type strain, LPS serotype 5 Reference type strain, LPS serotype 6 Reference type strain, LPS serotype 7 Reference type strain, LPS serotype 8 Reference type strain, LPS serotype 9 Reference type strain, LPS serotype 10 Reference type strain, LPS serotype 11 Reference type strain, LPS serotype 12 Reference type strain, LPS serotype 13 Reference type strain, LPS serotype 14 Reference type strain, LPS serotype 15 Reference type strain, LPS serotype 16 Rabbit isolate used for colonization study Rabbit isolate used for colonization study Rabbit isolate used for colonization study

35, 43 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 American Type Culture Collection This study This study

Laboratories, Detroit, Mich.) at 37°C. For the isolation of P. multocida from clinical samples, swabs were streaked onto plates of blood, chocolate, Knight’s (33), and Avril’s (6) agars and then incubated at 37°C for 48 h in ambient atmosphere; and duplicate plates were incubated in a candle jar. The sialidase activities of the P. multocida isolates were qualitatively assayed by using 2⬘-(4methylumbelliferyl)-␣-D-N-acetylneuraminic acid in a filter paper spot test (43). Unless otherwise indicated, chemicals were obtained from Sigma Chemical Co. (St. Louis, Mo.). DNA sequence analysis. An internal portion of nanH was amplified from P. multocida genomic DNA by PCR with primers F1 (5⬘-GCT TTG ATG GCA GTT TAT ATG TG-3⬘) and R2 (5⬘-TGA AGG AGC CGC TGT AGT CG-3⬘) by denaturation for 1 min at 94°C, renaturation for 1 min at 55°C, and primer extension for 1 min at 72°C in a 30-cycle program with an Amplitron II thermocycler (Fisher Scientific, Pittsburgh, Pa.). The reaction mixture contained 2 mM MgCl2, 50 mM Tris (pH 7.4), 0.1 mM primer, 0.2 mM nucleotides, and 1 U of Taq polymerase per 20 ␮l. The DNA sequence of the 512-bp amplicon was determined by dideoxy termination in an Applied Biosystems automated sequencer at the Molecular Genetics Instrumentation Facility at the University of Georgia. DNA sequences were aligned with the DNA sequence of nanH (GenBank accession number AF274869) by using the AlignX program of Vector NTi software (Informax, North Bethesda, Md.). DNA-DNA hybridization. The nanH-specific probe was created from isolate 86-1913 genomic DNA by PCR, as described above, except that digoxigeninlabeled nucleotides (Boehringer Mannheim, Indianapolis, Ind.) were used. Free nucleotides were removed from the amplicon by purification with a Wizard DNA Clean-up kit (Promega, Madison, Wis.). The amplicon was eluted in 50 ␮l of distilled water and added to 50 ml of sterile hybridization buffer (750 mM sodium chloride, 75 mM sodium citrate, 0.1% N-lauryl sarcosine, 0.02% sodium dodecyl sulfate [SDS], 1% Blocking Reagent [pH 7.0; Boehringer Mannheim]). Genomic DNA from the P. multocida isolates was extracted from the cell suspensions by the cetyltrimethylammonium bromide method described in Current Protocols in Molecular Biology (5). The DNA was digested with HindIII and separated on a 0.7% agarose gel. DNA was transferred and hybridized by the protocol for Southern blotting on nylon membranes described in Current Protocols in Molecular Biology (5). High-stringency washes were performed with 0.1⫻ SSC (1⫻ SSC is 0.15 M NaCl plus 0.015 M sodium citrate) containing 0.1% SDS at 68°C; low-stringency washes were performed at 55°C. Cloning and purification of the recombinant sialidase. The 5⬘ portion of the nanH sialidase gene (GenBank accession number AF274869) corresponding to base pairs 175 to 1390 was fused to an amino-terminal histidine tag by using the vector pQE30, according to the directions of the manufacturer (Qiagen). The recombinant nanH construct, pMZ1, was predicted to produce a 47.6-kDa protein (432 amino acids), which was visualized by SDS-polyacrylamide gel electrophoresis (PAGE) on 10% polyacrylamide slab gels by the protocol of Laemmli (34). Purification of NanH was achieved with a nickel affinity column. Fractions containing the protein were pooled, and the protein concentration was determined with the bicinchoninic acid protein assay reagent (Pierce, Rockford, Ill.).

The purity of the NanH preparation was analyzed by SDS-PAGE. For Western blotting analysis, the proteins were transferred to nitrocellulose membranes and probed with NanH antiserum (5). Production of NanH polyclonal antibodies. Rabbit NanH antiserum was produced by intradermal injection of 200 ␮g of purified antigen in 500 ␮l of complete Freund’s adjuvant in 20 different sites on a New Zealand White male rabbit. The rabbit was revaccinated twice subcutaneously with 100 ␮g of antigen in Freund’s incomplete adjuvant at 3-week intervals. A preimmune blood sample was collected before the first immunization; serum was collected 14 days after each revaccination. Rabbit challenge. Nineteen male New Zealand White rabbits (weight, 2.7 to 3.6 kg) were acquired from a certified Pasteurella-free vendor (Myrtle’s Rabbits, Thompson Station, Tenn.). The rabbits were housed individually in stainless steel cages with stainless steel slatted floors. The lighting consisted of 12-h cycles of illumination and darkness. Approximately, 150 g of commercial pelleted feed was provided daily, and water was available ad libitum. The room temperature was maintained at 20 to 22°C, the relative humidity was 40 to 60%, and the room was ventilated at 12 air changes/h. Blood collection, bacterial challenge, and euthanasia were done under Institutional Animal Care and Use Committee-approved protocols. Serum samples were collected from 12 control rabbits, 2 weeks after they were housed, in order to establish a negative baseline for the NanH ELISA. Five rabbits, housed separately from the controls, were administered intranasally 10 ␮l of phosphate-buffered saline containing approximately 105 CFU of isolate P-1059. These rabbits became systemically ill within 30 h postexposure and were administered 10 mg of enrofloxacin per kg of body weight subcutaneously twice daily for 5 days. Serum was collected from the rabbits prior to challenge and weekly thereafter for the next 5 weeks, after which the animals were euthanatized. In addition, two rabbits were administered intranasally a cocktail of three lapine isolates of P. multocida (Table 1). Serum was collected from the rabbits prior to challenge and weekly thereafter for the next 11 weeks. On week 14, the rabbits were euthanatized with an overdose of sodium pentobarbital; and then samples were taken from the deep nasal turbinates, pharynx, trachea, ear bullae, and cervical lymph nodes for both culture and PCR. A full necropsy was performed on all of the animals in order to detect gross indications of infection. Samples of lung, liver, kidney, and brain were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin-eosin for histologic evaluation. Clinical samples. Veterinary clinics and laboratory animal facilities were solicited to supply rabbit serum and lesion samples for the study. Forty-two swabs of lesions or nasal exudates with accompanying serum samples were acquired from rabbits with clinical signs suggestive of pasteurellosis. These were streaked onto selective (Knight’s and Avril’s agar) and nonselective (blood and chocolate) agar plates for P. multocida culture and then placed in BHI broth and incubated at 37°C overnight to be processed for PCR. The clinical signs of the affected rabbits from which samples were submitted included sneezing, congestion, chronic nasal discharge, chronic ocular discharge, otitis, ataxia, head tilt, anorexia, recumbency, and abscesses in various locations. The swabs had been used

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to sample the nostrils, ocular discharge, inner ear, or abscesses. The swabs were placed in the transport medium routinely used in the particular veterinary practice (in most cases, Culturette). In addition, 76 serum samples were acquired from rabbits examined by veterinarians in private practice. These samples were not accompanied by clinical histories. We also obtained 86 serum samples from rabbits from eight different laboratory animal facilities which either were believed to be P. multocida-free or possessed rabbit colonies with a history of individuals exhibiting very mild signs of pasteurellosis (upper respiratory infection). The storage and mailing of serum samples were done by the routine methods for each facility. Samples took an average of 3 days to arrive at the laboratory. Future samples are to be submitted to Patricia Schroder (phone: (706) 542-5812 or (706) 542-5494; e-mail: [email protected]). ELISA. Secondary antibody conjugates were acquired from Accurate Antibody (Westbury, N.Y.). A whole-cell-lysate ELISA was performed as described by Kawamoto et al. (29, 30) (the Kawamoto ELISA) in order to confirm the infection status of some of the rabbits. The NanH ELISA was performed by using a modification of a previously described ELISA protocol (53). Briefly, 50 ␮l of 50 mM carbonate-bicarbonate buffer (pH 9.6) containing 0.5 ␮g of NanH was used to coat flat-bottom microtiter wells. After overnight incubation at 4°C the plates were stored at ⫺70°C. Before use, unbound antigen was removed by washing with 200 ␮l of phosphate-buffered saline (pH 7) containing 0.05% Tween 20 (washing buffer). Antigen-coated wells were exposed to rabbit sera of various dilutions (range, 1:4 to 1:128). Fifty microliters of serum, diluted in phosphatebuffered saline containing 0.1% Tween 20 and 5% nonfat dry milk (conjugate buffer), was placed into duplicate microtiter wells; and the plates were incubated at 37°C for 45 min. The wells were washed three times with 200 ␮l of washing buffer. Fifty microliters of horseradish peroxidase-conjugated goat anti-rabbit secondary antibody was added at a dilution previously determined by checkerboard titration; anti-(whole-molecule) rabbit immunoglobulin G (IgG) was diluted 1:10,000, and anti-(␮-chain-specific) rabbit IgM was diluted 1:2,000 in conjugate buffer. The plates were incubated at 37°C for 45 min, and then the wells were washed three times with 200 ␮l of washing buffer. The plates were developed by adding 50 ␮l of horseradish peroxidase substrate (50 mM citricphosphate buffer [pH 5.0] containing 1 mg of o-phenylenediamine per ml and 0.03% H2O2). After 10 min of incubation at 37°C, the reaction was stopped by the addition of 12.5 ␮l of 2 M HCl. The plates were read at 490 nm with a microplate reader (Bio-Tek Instruments, Inc., Winooski, Vt.). The mean optical density (OD) was calculated from the readings from the duplicate wells. Since goat anti-(whole-molecule) rabbit IgG was used as the secondary antibody, the NanH IgG ELISA actually detected nonisotype-specific IgG; therefore, in the Results section, any reference to IgG titers actually refers to nonisotype-specific NanH IgG. PCR detection of P. multocida. The template for PCR was prepared by incubating sample swabs or control strains in BHI broth at 37°C overnight. P. multocida isolate P-1059 was used as the positive control. Bacteria were collected by centrifugation (5,000 ⫻ g for 5 min) and were washed twice with 1 ml of sterile distilled water. The pellets were resuspended in 50 ␮l of sterile distilled water, boiled for 10 min, and then diluted 1/10 in water for use in the amplification cocktail. PCR was performed as described by Kasten et al. (28) with primers Pslf (5⬘-ATG AAA AAA CTA ACT AAA GTA-3⬘) and Pslr (5⬘-TTA GTA TGC TAA CAC AGC ACG ACG-3⬘) to amplify 453 bp of the psl gene, which encodes the P6-like protein. This gene has been shown to be unique to P. multocida and Haemophilus influenzae and has been used for the detection of pasteurellosis in turkeys (28). The identities of the amplicons were confirmed by DNA-DNA hybridization by a PCR-ELISA (44). This consisted of a psl PCR master mixture containing digoxigenin-labeled nucleotides (Roche Molecular Biochemicals, Indianapolis, Ind.) and 0.1 fM of the 3⬘-biotinylated psl-specific probe 5⬘-GAT GCA CAT GCG GCG TTC TTA A-3⬘. After 30 cycles the amplicons were denatured by incubation for 1 min at 96°C, with probe annealing for 15 min at 60°C. Probe-amplicon hybrids were captured in streptavidin-coated wells and detected with antidigoxigenin antibody-conjugate, as described by the manufacturer (Roche Molecular Biochemicals). Nucleotide sequence accession numbers. The nanH sequences were deposited in GenBank under accession numbers AY153793 to AY153804.

RESULTS Distribution of nanH among Pasteurella spp. All of the reference serotypes and lapine isolates of P. multocida tested produced sialidase activity in the filter paper spot test. We did not possess isolates of serotype 2 or 6; therefore, these sero-

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types were not tested. It was previously determined (43) that some isolates contain at least two sialidases, NanH and NanB. In order to determine whether NanH was conserved among isolates, an internal portion of nanH from isolates of the reference serotypes was amplified by PCR and the DNA was sequenced. The DNA sequences that were amplified demonstrated 81% identity; however, the derived amino acid sequences demonstrated 96% similarity and 79% identity, suggesting that the NanH sialidase is well conserved among isolates of different serotypes (Fig. 1). However, since repeated PCR attempts failed to amplify nanH from serotypes 1 and 14, DNA-DNA hybridization was performed to determine whether nanH was present in these isolates. Genomic DNA from 12 of the 14 serotypes tested hybridized with the nanHspecific probe under high-stringency conditions (68°C wash) (data not shown). When the stringency was lowered (55°C), all isolates hybridized (data not shown), including those of serotypes 1 and 14, suggesting that nanH is widely distributed among the serotypes of P. multocida. In addition, serotypes 1 and 14 were also reactive with NanH antiserum when they were used in a whole-cell ELISA, suggesting that all serotypes tested produce an antigenically conserved NanH sialidase (data not shown). These results suggest that NanH may be a useful antigen for the serological screening of animals infected with P. multocida. NanH ELISA. NanH has been shown to be difficult to purify from P. multocida because it is membrane associated (43). In order to produce a protein that would be more amenable to purification, it was cloned to allow its expression with a histidine affinity tag. The recombinant NanH protein was isolated by affinity chromatography in essentially pure form with no contaminating proteins, as detected by SDS-PAGE and Coomassie blue staining (data not shown). When the NanH protein was used to vaccinate a rabbit, high-titer anti-NanH serum was acquired, and its reactivity was confirmed by Western blotting at a 1/18,000 dilution. The anti-NanH serum was used as the positive control serum in the ELISA. All 19 rabbits in our research group were nasal swab culture and PCR negative for P. multocida and demonstrated no symptoms of disease prior to testing by ELISA or challenge. Sera from 12 control rabbits were tested for cross-reactive antibodies against NanH by ELISA. Detection of cross-reactive IgG yielded ODs at 490 nm that ranged from 0 to 0.379 (mean ⫽ 0.11, standard deviation ⫽ 0.07) at a 1/4 dilution, 0 to 0.238 (mean ⫽ 0.06, standard deviation ⫽ 0.06) at a 1/8 dilution, and 0 to 0.136 (mean ⫽ 0.03, standard deviation ⫽ 0.04) at a 1/16 dilution. Detection of cross-reactive IgM yielded ODs that ranged from 0.06 to 0.125 (mean ⫽ 0.09, standard deviation ⫽ 0.02) at a 1/4 dilution and 0.04 to 0.108 (mean ⫽ 0.06, standard deviation ⫽ 0.02) at a 1/8 dilution. Since cross-reactive antibodies were most apparent at the lower dilutions, we chose dilutions of 1/16 (IgG) and 1/8 (IgM) as the minimal dilutions for the detection of NanH antibody titers. Net absorbance values of 0.2 at the 1/16 dilution for IgG and 0.13 at 1/8 dilution for IgM were calculated to be the threshold values for a positive titer in serum, according to method of Balfour and Harford (7). This value was determined by adding the mean absorbance value for the sera from the 12 negative control rabbits plus 4 times the standard deviation. The serum from two negative rabbits was pooled to provide

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FIG. 1. Derived amino acid sequence of P. multocida NanH sialidase acquired by PCR amplification of reference serotype strains (except those of serotypes 1 and 14, which were not amplified, and serotypes 2 and 6, which were not tested). The sequences exhibit 96.5% similarity and 78.8% identity among isolates; the sequence identity (dots) with the sequence of isolate 86-1913 is shown.

a stock of negative control serum for quality control (QC) in subsequent assays. Serum from the NanH-vaccinated rabbit, which was exsanguinated 2 weeks after the third immunization, served as the positive control for the NanH IgG ELISA; serum removed from this rabbit 2 weeks after the first immunization served as the positive control serum for the IgM ELISA. In order to establish the QC range for the negative and the positive control sera, each was used in multiple ELISA trials. In 125 IgG ELISA trials the negative control serum yielded ODs at 490 nm that ranged from 0 to 0.161 (mean ⫽ 0.046, standard deviation ⫽ 0.036) at a 1/16 dilution. The IgG QC OD range for the negative control serum was determined to be 0 to 0.11 [equal to the mean OD at 490 nm ⫾ (standard deviation ⫻ 2)]. By using these QC criteria, the results for 94.4% of the 125 trials were within the acceptable range. Similarly, detection of NanH IgG by using the positive control serum in 129 ELISA trials yielded ODs at 490 nm that ranged from 0.669 to 1.961 (mean ⫽ 1.317, standard deviation ⫽ 0.225) at a 1/1,000 dilution and 0.391 to 1.298 (mean ⫽ 0.825, standard deviation ⫽ 0.176) at a 1/10,000 dilution. Therefore,

the NanH IgG QC readings for the positive control serum were determined to be ⬎0.87 at the 1/1,000 dilution and ⬎0.47 at the 1/10,000 dilution [mean OD at 490 nm ⫺ (standard deviation ⫻ 2)]. By using these QC criteria, the results for 98.4% of the 129 trials with the positive control serum were within the acceptable ranges. Sera from two negative rabbits were also used as the negative control for the IgM ELISA. In six IgM ELISA trials the negative control serum yielded ODs at 490 nm that ranged from 0.019 to 0.101 (mean ⫽ 0.062, standard deviation ⫽ 0.027) at a 1/8 dilution. The IgM QC range for the negative control serum was determined to be 0 to 0.116 [mean OD at 490 nm ⫾ (standard deviation ⫻ 2)]. By using these QC criteria, the results for all six trials were within the acceptable range. Similarly, detection of NanH IgM by using the positive control serum in 11 ELISA trials yielded ODs at 490 nm that ranged from 0.233 to 0.932 (mean ⫽ 0.6, standard deviation ⫽ 0.206) at a 1/16 dilution. Consequently, the IgM QC reading for the positive control serum was established to be ⬎0.187 at a 1/16 dilution (mean OD 490 nm ⫺ [the standard deviation ⫻

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FIG. 2. Phases of colonization of the respiratory system and infection of rabbits by P. multocida. After aerosol exposure, the replication of low-virulence strains may be limited to the mucus layer and the epithelial cell surface of the upper respiratory tract. Heavy colonization of the nasal turbinates and trachea can result without overt signs of illness. These animals may exhibit anti-NanH antibodies if they are persistently colonized. However, depression of mucosal defenses may enable bacterial invasion of the respiratory mucosa and consequent clinical symptoms of disease. Highly virulent strains of P. multocida may rapidly invade host cells and replicate in deep tissue, resulting in acute symptoms of localized or systemic disease. Clinically ill animals may possess anti-NanH antibodies if they are persistently colonized with P. multocida but probably exhibit antibodies against whole-cell lysates during chronic infection and during the convalescent period.

2]). By using these QC criteria, the results for all 11 trials were within the acceptable ranges. These QC criteria were used to evaluate whether the results of the trials with the test sera were valid. Serological testing of rabbits experimentally challenged with P. multocida. We hypothesized that sialidase expression by P. multocida would facilitate nutrient acquisition during colonization and infection of host respiratory mucosal surfaces. Therefore, the immune systems of colonized animals may have become exposed to P. multocida surface proteins during colonization and bacterial cells may also have been phagocytosed by antigen-processing cells present in the mucosal tissue (Fig. 2). In order to determine whether NanH antibody could be detected in colonized rabbits, we challenged rabbits intranasally with several P. multocida isolates. Five rabbits that were administered highly virulent isolate P-1059 exhibited symptoms of septicemia on the day after challenge. The animals were treated with an antibiotic that resolved the infection, and all of the rabbits recovered. Serum from the rabbits did not display NanH antibodies, and P. multocida was not detected posttreatment by PCR or culture. However, two of the three

rabbits that were tested by the Kawamoto whole-cell-lysate ELISA produced positive titers 2 to 3 weeks postchallenge, confirming their Pasteurella infection status. The rapid onset of disease, coupled with therapeutic treatment, probably resulted in inadequate exposure to P. multocida surface sialidase. In contrast, two rabbits that were administered a cocktail of low-virulence isolates produced NanH IgM antibodies, and one rabbit demonstrated IgG antibodies 6 weeks after inoculation (Fig. 3). Since we used anti-whole-molecule IgG for detection, the NanH IgG titer was actually nonisotype-specific IgG. NanH IgG levels continued to rise in both of these rabbits, but the second rabbit exhibited a positive titer only after 11 weeks postexposure. IgM titers were transient and undetectable in the samples collected at 9 weeks postexposure. P. multocida was detected in both rabbits at 14 weeks postinoculation by PCR of tracheal and nasal swab specimens collected at necropsy. P. multocida was also isolated by culture from a tracheal swab specimen of the rabbit that demonstrated the highest IgG levels postinoculation. Neither rabbit exhibited any clinical signs of P. multocida infection, nor were lesions detected by gross pathology or histopathology, suggesting that

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FIG. 3. Serological response to NanH sialidase by two rabbits experimentally infected with a cocktail of three low-virulence P. multocida isolates. Colonization of the animals was confirmed by PCR detection of the bacteria in the trachea 14 weeks after exposure. Serum samples were tested for NanH (nonisotype-specific) IgG (at a 1:16 dilution) and IgM (at a 1:8 dilution) by ELISA. Serum samples (obtained 5 to 11 weeks postinfection) were tested by a whole-cell-lysate ELISA, as described by Kawamoto et al. (29). (A) Changes in the ODs of the samples, as determined by ELISA, over 11 weeks. Asterisks, positive readings; star, the sample positive by the Kawamoto ELISA. (B) The highest dilution (titer) that resulted in a positive OD.

the animals were colonized but not adversely affected by the bacteria. While serum samples from these rabbits produced increasing OD values by the Kawamoto ELISA over the course of the experiment, only one serum sample from one rabbit had a positive titer (Fig. 3). These results suggest that NanH sialidase is sufficiently expressed by P. multocida during surface colonization of the respiratory mucosal system to stimulate serum antibody production. Serological testing of rabbits housed in research facilities. Since we demonstrated that respiratory colonization by P. multocida could be detected by the NanH ELISA, we sought to determine if the ELISA could identify asymptomatic carriers in colonies of research animals. While the prevalence of P. multocida may vary among research facilities, a single rabbit carrier in a facility can result in colony-wide exposure. The results of these tests are shown in Table 2. We received serum samples from two research facilities where the facility veterinarian reported no recent history of P. multocida infections among the rabbits. None of these 18 samples produced either anti-NanH IgG or anti-NanH IgM antibodies. However, six facilities which reported a history of endemic pasteurellosis contained asymptomatic individuals that did exhibit antibodies. Twenty to 50% of these samples had anti-NanH IgG at titers as high as 1:128. However, some of the rabbit samples had only

anti-NanH IgM antibodies. Samples from two of these facilities were also tested by the Kawamoto ELISA, which revealed that 36 to 57% of the samples were positive for antibodies. Three serum samples were negative by the Kawamoto ELISA but exhibited high titers by the NanH ELISA, results comparable to our findings in the experimental colonization study. Apparently, these facilities contained several carrier animals that served as a source of bacteria for naïve rabbits in the colony. Our results suggest that the NanH ELISA may be useful for identifying healthy carrier rabbits in a research facility. Serological testing of rabbits seen by veterinarians in private clinical practice. In order to determine whether the NanH ELISA could identify P. multocida-infected clinically ill animals, we identified the infected rabbits by detecting the bacteria in nasal discharges or lesions (Table 3). We successfully cultured P. multocida from only 1 of 44 swab samples; however, we detected the pathogen in 8 swabs (17.4%) by PCR. Six of the eight PCR-positive rabbits were also positive for serum anti-NanH IgG at titers up to 1:128. However, the two PCRpositive, NanH ELISA-negative sick rabbits were also negative by the Kawamoto whole-cell-lysate ELISA, suggesting that they may have been acutely infected. One of these rabbits was reported by the veterinarian to exhibit an ocular discharge,

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TABLE 2. NanH ELISA screening of rabbits housed in research facilities Sourcea d

Facility 1 Facility 2 Facility 3 Facility 4 Facility 5d Facility 6 Facility 7 Facility 8d Total

No. of rabbits negative/total no. tested (%)

IgMb

IgGb

IgM and IgGc

Total

3/3 (100) 15/15 (100) 6/11 (55) 3/21 (14) 9/15 (60) 0/4 (0) 3/7 (43) 3/10 (30) 42/86 (49)

0/3 (0) 0/15 (0) 1/11 (9) 0/21 (0) 2/15 (13) 1/4 (25) 1/7 (14) 2/10 (20) 7/86 (8)

0/3 (0) 0/15 (0) 4/11 (36) 6/21 (29) 3/15 (20) 2/4 (50) 3/7 (43) 4/10 (40) 22/86 (26)

0/3 (0) 0/15 (0) 0/11 (0) 12/21 (57) 1/15 (7) 1/4 (25) 0/7 (0) 1/10 (10) 15/86 (17)

0/3 (0) 0/15 (0) 5/11 (45) 18/21 (86) 6/15 (40) 4/4 (100) 4/7 (57) 7/10 (70) 44/86 (51)

No. of rabbits positive for the following/total no. tested (%):

a

All research facilities except facilities 1 and 2 reported a recent history of pasteurellosis among rabbits in the same area. Sample positive only for IgG or IgM but not both. To be considered positive for NanH antibody, samples exhibited an OD at 490 nm of 0.2 or above at a 1:16 dilution by the NanH IgG assay or an OD of 0.13 or above at a dilution of 1:8 by the NanH IgM assay. The NanH IgG ELISA detected nonisotype-specific IgG. c Samples positive for both IgM and IgG NanH antibodies. d Some samples from facilities 1, 5, and 8 were also tested by the whole-cell-lysate ELISA as described by Kawamoto et al. (29). Samples from facility 1 did not produce antibodies; however, both facilities 5 and 8 possessed rabbits positive for antibodies to whole-cell lysates. b

while the other was believed to have a retrobulbar abscess. Either of these infections could have been very rapid in onset, leading to a veterinary visit and, possibly, antibiotic treatment before the rabbits produced detectable antibody titers. However, some of the cases of suspected pasteurellosis may have been caused by other organisms, such as Bordetella bronchiseptica, Pseudomonas spp., or Staphylococcus spp. (14, 25). In order to determine the sensitivity and specificity of the NanH IgG ELISA for the detection of P. multocida, the 8 PCR-positive rabbits were considered infected; the 12 healthy, negative control rabbits were considered uninfected because they were PCR negative. Testing of the sera from these groups by the NanH IgG ELISA indicated that the assay is 100% specific and 75% sensitive. However, infections in acutely ill animals may not be accurately diagnosed by either the NanH IgG or the NanH IgM ELISA. Although few of the rabbits (17.4%) were PCR positive, 21 of the 44 serum samples (48%) were positive for antibodies by the anti-NanH IgG ELISA. Some of these ELISA-positive rabbits probably had clinical infections caused by unrelated bacteria but may have had respiratory colonization with P. multocida, resulting in the NanH antibodies. Other ELISApositive rabbits were probably chronically infected with P. multocida but may have cleared most of the bacteria or may have been treated with antibiotics or the swab samples were poorly

preserved during shipping. While many of these ELISA-positive animals exhibited symptoms of deep infection (nasal discharge, head tilt, torticollis, mandibular abscess, and ataxia), some of the swab samples from the PCR-negative rabbits could not have been obtained from the lesions without euthanatizing the pet. Many of the swabs submitted were probably used to sample the nares and may have contained too few bacteria for detection by the PCR. Table 4 shows the titers from several of the anti-NanH IgG-positive rabbits for which we obtained a description of symptoms. Some of the PCR-negative sick rabbits possessed high serum antibody titers, suggesting that the NanH IgG ELISA may be more sensitive than PCR for the identification of chronically infected animals. However, it was difficult to determine which animals exhibited high titers as a result of respiratory colonization and which rabbits were truly sick from a P. multocida infection. To interpret the NanH titers, it would be useful to know the general prevalence of titers among pet rabbits. In order to determine the prevalence of anti-NanH antibodies among rabbits visiting veterinarians in private practice, we screened 76 serum samples by the NanH IgG and the NanH IgM ELISAs. These samples were not accompanied by clinical histories; however, we were interested in determining the frequencies of antibodies among a mixed population of healthy and sick rabbits. Forty-five percent (34 of 76) of the samples

TABLE 3. Correlation among the results of NanH IgG ELISA, PCR, and culture for diagnosis of P. multocida infection in rabbits No. of samples with the indicated result/total no. tested (%): Test and result

ELISA Positive

ELISA positive PCR positive Culture⫹ Total a

14/25 (56) 1/25 (4) 23/25 (92)

a

PCRb Negative

2/25 (8) 0/25 (0) 2/25 (8)

Culturec

Positive

Negative

Positive

Negative

14/25 (56)

9/25 (36)

1/25 (4) 16/25 (64)

0/25 (0) 9/25 (36)

1/25 (4) 1/25 (4)

22/25 (88) 15/25 (60)

1/25 (4)

24/25 (96)

To be considered positive for NanH IgG antibody, samples exhibited an OD at 490 nm of 0.2 or greater at a 1:16 dilution. The NanH IgG ELISA detected nonisotype-specific IgG. b PCR was performed with DNA extracted from nasal or lesion swab specimens incubated overnight in brain heart infusion broth by the method of Kasten et al. (28), and then the results were confirmed by DNA hybridization with a biotinylated psl-specific probe (44). c Swab specimens of nasal extracts or lesions were inoculated onto blood and chocolate agars and selective media, consisting of Knight’s agar (33) and Avril’s agar (6), and were then incubated at 37°C for 48 h in ambient atmosphere. Duplicate plates were then incubated in a candle jar. Presumptive positive colonies (mucoid, gray, entire) were confirmed to be P. multocida by PCR by the method of Kasten et al. (28).

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CLIN. DIAGN. LAB. IMMUNOL.

TABLE 4. Screening of clinically ill rabbits for P. multocida infection by the NanH (nonisotype-specific) IgG ELISA and PCR Sample no.

Symptom or condition exhibited

IgG ELISA titera

PCR resultb

117 119 125 130 131 132 140 141 143 150 156

Nasal discharge Abscess Upper respiratory, nasal discharge Nasal discharge Nasal discharge Mandibular abscess Upper respiratory Abscess Upper respiratory, head tilt Ocular discharge, head tilt Ataxia

1:64 ⬎1:128 1:128 1:128c 1:64 1:128 1:128c 1:128 1:64 1:128 1:128

⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

a To be considered positive for NanH IgG; samples exhibited an OD at 490 nm of 0.2 or greater at a 1:16 dilution; the dilutions for the positive samples are presented as the highest dilution (titer) that was positive. b PCR was performed with DNA extracted from nasal or lesion swab specimens incubated overnight in BHI broth by the method of Kasten et al. (28), and then the result was confirmed by DNA hybridization with a biotinylated pslspecific probe (44). c Samples 130 and 140 were tested by a whole-cell-lysate ELISA, as described by Kawamoto et al. (29); both samples were positive for antibodies.

were negative by both assays, suggesting that about half of the pet rabbits were not colonized with P. multocida. This prevalence was comparable to the 52% negative results for antibodies detected for sick rabbits. However, only 23% of these surveillance samples had anti-NanH IgG antibodies, whereas 48% of the sick animals had anti-NanH IgG antibodies. Fifty percent of the samples were positive for anti-NanH IgM, with 24 of 76 (32%) positive for IgM alone. If it is assumed that the NanH IgM ELISA was not producing a high number of falsepositive reactions, the data suggest that nearly a third of these 76 animals have only IgM antibodies. DISCUSSION Because of its virulence, early diagnosis of P. multocida infection in pet rabbits is critical. Figure 2 illustrates the putative phases of P. multocida respiratory colonization and potential infection of rabbits. Exposure to virulent, invasive strains may result in rapid penetration of the respiratory mucosa, leading to peracute or acute disease (1, 3, 36, 46). In the challenge with isolate P-1059 described in this report, we observed a rapid infection phenomenon in which the rabbits were clinically ill within 30 h after intranasal exposure to a low bacterial dose. Similar results have been reported by others using P1059 and other virulent P. multocida isolates in rabbits (1, 3, 4). These infections might be detectable by PCR or culture because of the high replication rate of the bacteria in the host tissue (35). Although culture is considered the “gold standard” for detection, culture techniques for the isolation and identification of P. multocida are time-consuming and often fail because some transport media, including commonly used commercial transport swabs, such as Transwab and Culturette, do not maintain P. multocida viability for more than 1 day at room temperature (31). In some clinical cases, the organism cannot be cultured from obviously diseased organs because the animal may have received antibiotics prior to sam-

ple submission, but PCR should be adequate for identifying the presence of the bacteria postmortem. In sick animals that exhibit peracute disease, an ELISA may not be useful for the detection of convalescent-phase antibodies postinfection if the animals have been treated with antibiotics. It is also important to determine that healthy research animals are P. multocida free so that they can be used as breeders or research subjects. The bacteria can be present at low numbers deep in the nasal turbinates of carrier rabbits, rendering their detection by culture impossible without killing the animal (24, 54). Several research groups have investigated ELISA as a method for the detection of colonization of healthy rabbits with P. multocida (23, 26, 30, 32, 39, 45, 59). However, rabbits exposed to low-virulence strains may become persistently colonized (13), but the bacteria may be primarily associated with the mucus layer or may adhere to the surfaces of mucosal epithelial cells (2, 20). The NanH ELISA can detect this colonization state in chronically colonized animals. This rabbit response is comparable to the serological responses of young cystic fibrosis patients persistently colonized with Pseudomonas aeruginosa (57). The bacteria are poorly invasive but can reach high densities on the mucosae of children with cystic fibrosis (47). West et al. (57) demonstrated that serology with extracellular proteins or cell lysates could detect colonization 6 to 12 months before culture detection, analogous to the screening of rabbits for P. multocida infection. Stress or shipping can reduce rabbit mucosal defenses and allow the bacteria to invade the mucosal surface, with subsequent host exposure to other bacterial antigens (Fig. 2). Lowvirulence strains would be more likely to produce chronic infections, which would result in high anti-P. multocida antibody titers in serum that could be detected by most serological methods. Unfortunately, the detection methods used to screen research animals are not standardized, and as a result serology is not widely used to detect P. multocida exposure. Most serological tests use boiled whole P. multocida cells, heat-stable cell lysates, or purified lipopolysaccharides (LPSs) as the antigen, which can result in both high background levels and significant numbers of false-positive or false-negative results (10, 26, 38, 41, 42). For example, the Kawamoto ELISA uses a heat-stable whole-cell lysate of P. multocida that is primarily composed of LPSs (9, 21, 29). Infected rabbits may make high levels of LPS-specific antibody (10, 38, 41, 42), but the diversity of P. multocida serotype-specific LPS ensures that some strains may not be reactive by all assays (8, 11, 49). In addition, the extraction process may alter antigenic specificity, so that animals that are negative by a LPS ELISA assay may actually be infected (10, 41, 42). A better target for P. multocida detection would be a homogeneous antigen that could be easily purified and that is present in all isolates. In order to obtain a more homogeneous antigen for serological testing, we purified a recombinant NanH sialidase. In a past study (43) it was shown that NanH is associated with the P. multocida outer membrane and does not exhibit a high degree of homology to other sialidases that have been characterized. In the present study, we demonstrated that the gene is ubiquitous in P. multocida isolates that cause disease in domestic animals. Many organisms that colonize the respiratory system produce sialidase, and this enzyme functions in the removal of sialic acid from mucus, which allows

SEROLOGICAL DETECTION OF P. MULTOCIDA COLONIZATION

VOL. 11, 2004

sialidase-producing bacteria such as P. multocida to access sialic acid as an energy source (12, 43, 52). We have shown that expression of sialidase during colonization or chronic infection of deep host tissue elicits a humoral response, which enables detection of persistent colonization by NanH serology. ACKNOWLEDGMENTS This work was made possible by the National Institutes of Health and the UGARF Animal Health Foundation. Many thanks go to veterinarians nationwide for sending samples and histories for the study. REFERENCES 1. Al-Haddawi, M. H., S. Jasni, M. Zamri-Saad, A. R. Mutalib, and A. R. Sheikh-Omar. 1999. Ultrastructural pathology of the upper respiratory tract of rabbits experimentally infected with Pasteurella multocida A:3. Res. Vet. Sci. 67:163–170. 2. Al-Haddawi, M. H., S. Jasni, M. Zamri-Saad, A. R. Mutalib, I. Zulkifli, R. Son, and A. R. Sheikh-Omar. 2000. In vitro study of Pasteurella multocida adhesion to trachea, lung and aorta of rabbits. Vet. J. 159:274–281. 3. Al-Haddawi, M. H., S. Jasni, D. A. Israf, M. Zamri-Saad, A. R. Mutalib, and A. R. Sheikh-Omar. 2001. Ultrastructural pathology of nasal and tracheal mucosa of rabbits experimentally infected with Pasteurella multocida serotype D:1. Res. Vet. Sci. 70:191–197. 4. Al-Lebban, Z. S., L. B. Corbeil, and E. H. Coles. 1988. Rabbit pasteurellosis: induced disease and vaccination. Am. J. Vet. Res. 49:312–316. 5. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1993. Current protocols in molecular biology. Greene Publishing Associates Inc. and John Wiley & Sons, Inc., Boston, Mass. 6. Avril, J., P. Donnio, and P. Pouedras. 1990. Selective medium for Pasteurella multocida and its use to detect oropharyngeal carriage in pig breeders. J. Clin. Microbiol. 28:1438–1440. 7. Balfour, A. H., and J. P. Harford. 1990. Quality control and standardisation, p. 36–47. In T. G. Wreghitt and P. Morgan-Capner (ed.), ELISA in the clinical laboratory. Public Health Service Laboratory, London, United Kingdom. 8. Brogden, K. A., and R. A. Packer. 1979. Comparison of Pasteurella multocida serotyping systems. Am. J. Vet. Res. 40:1332–1335. 9. Brogden, K. A., and P. A. Rebers. 1978. Serologic examination of the Westphal-type lipopolysaccharides of Pasteurella multocida. Am. J. Vet. Res. 39:1680–1682. 10. Cary, C. J., G. K. Per, C. E. Chrisp, and D. F. Keren. 1984. Serological analysis of five serotypes of Pasteurella multocida of rabbit origin by use of an enzyme-linked immunosorbent assay with lipopolysaccharide as antigen. J. Clin. Microbiol. 20:191–194. 11. Chengappa, M. M., R. C. Myers, and G. R. Carter. 1982. Capsular and somatic types of Pasteurella multocida from rabbits. Can. J. Comp. Med. 46:437–439. 12. Corfield, T. 1992. Bacterial sialidases—roles in pathogenicity and nutrition. Glycobiology 2:509–521. 13. DeLong, D., P. J. Manning, R. Gunther, and D. L. Swanson. 1992. Colonization of rabbits by Pasteurella multocida: serum IgG responses following intranasal challenge with serologically distinct isolates. Lab. Anim. Sci. 42: 13–18. 14. DeLong, D., and P. J. Manning. 1992. Bacterial diseases, p. 131–162. In P. J. Manning, D. H. Ringler, and C. E. Newcomer (ed.), The biology of the laboratory rabbit, 2nd ed. Academic Press, Inc., San Diego, Calif. 15. DiGiacomo, R. F., Y. M. Xu, V. Allen, M. H. Hinton, and G. R. Pearson. 1991. Naturally acquired Pasteurella multocida infection in rabbits: clinicopathological aspects. Can. J. Vet. Res. 55:234–238. 16. Drzeniek, R., W. Scharmann, and E. Balke. 1972. Neuraminidase and Nacetylneuraminate pyruvate-lyase of Pasteurella multocida. J. Gen. Microbiol. 72:357–368. 17. Gaertner, D. J. 1991. Comparison of penicillin and gentamicin for treatment of pasteurellosis in rabbits. Lab. Anim. Sci. 41:78–80. 18. Gilman, A. P., D. C. Villeneuve, V. E. Secours, A. P. Yagminas, B. L. Tracy, J. M. Quinn, V. E. Valli, and M. A. Moss. 1998. Uranyl nitrate: 91-day toxicity studies in the New Zealand white rabbit. Toxicol. Sci. 41:129–137. 19. Glass, L. S., and J. N. Beasley. 1989. Infection with and antibody response to Pasteurella multocida and Bordetella bronchiseptica in immature rabbits. Lab. Anim. Sci. 37:406–410. 20. Glorioso, J. C., G. W. Jones, H. G. Rush, L. J. Pentler, C. A. Darif, and J. E. Coward. 1982. Adhesion of type A Pasteurella multocida to rabbit pharyngeal cells and its possible role in rabbit respiratory tract infections. Infect. Immun. 35:1103–1109. 21. Heddleston, K. L., and P. A. Rebers. 1975. Properties of free endotoxin from Pasteurella multocida. Am. J. Vet. Res. 36:573–574. 22. Holmes, H. T., N. M. Patton, and P. R. Cheebe. 1984. The occurrence of

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