Vol. 22, No.

4 April 2000

CE

Refereed Peer Review

Pasteurellosis in Rabbits
FOCAL POINT #Serology combined with
polymerase chain reaction–based assays can be used to rapidly and accurately document pasteurellosis in rabbits. University of Georgia

Susan Sanchez, PhD Branson W. Ritchie, DVM, PhD

Shaikh Mizan, PhD Margie D. Lee, DVM, PhD

ABSTRACT: Pasteurella multocida infections in rabbits may remain subclinical or can cause a variety of acute or chronic diseases in most organs, particularly tissue in the repiratory and genital tracts. Although clinical signs are frequently used to diagnose pasteurellosis, other organisms can cause similar clinical signs. The status of a rabbit with respect to P. multocida can be best determined using a polymerase chain reaction–based assay in conjunction with a sensitive and specific ELISA.

KEY FACTS
I Pasteurella multocida is one of the most common diseasecausing agents in rabbits. I Culture accuracy of P. multocida depends on the transport medium, storage temperature of the swab, previous antibiotic treatment, and laboratory culture conditions. I A polymerase chain reaction–based assay is a more sensitive test than is culture, partially because the former detects the presence of both viable and nonviable bacteria. I Serology can be used to detect P. multocida antibodies, particularly in chronically infected rabbits. I Nasopharyngeal swabs are recommended for obtaining culture samples to detect the presence of P. multocida; however, sample collection from conscious rabbits is difficult.

P

asteurella multocida can cause various clinical changes in rabbits. As one of the most common disease-causing agents in this genus, P. multocida has been associated with rhinitis (snuffles), abscessation, pneumonia, otitis, conjunctivitis, genital tract infections, abortions, neonatal mortality, and septicemia.1,2 Although this organism is highly infectious, many rabbits are subclinically infected until stress or concomitant problems precipitate recognizable disease. The reported prevalence of P. multocida in clinically stable rabbits ranges from 20% to 90%, depending on the animal’s age, sex, and health status and the chosen testing method.3,4 Based on studies by Webster and Smith, it is generally believed that rabbits infected with P. multocida can resist or clear infection, become chronically but subclinically infected, or develop acute or chronic disease. The type of infection is believed to be influenced by the strain of infecting Pasteurella and the specific host resistant to that strain.5 Generally, Pasteurellaceae should be considered commensal organisms that may be pathogenic in immunosuppressed rabbits. Peracute pasteurellosis usually causes septicemia and death in seemingly healthy rabbits. Upper respiratory tract disease (e.g., snuffles) associated with P. multocida infection starts as a serous nasal discharge that usually turns mucopurulent (Figure 1). Hair on the animal’s forelimbs is usually moist from repeated attempts to clean the muzzle. Epiphora, sneezing, and coughing may accompany the upper respiratory tract form of pasteurellosis. Anorexia, weight loss, dyspnea, pleuropneumonia, and, in the most severe cases, death may occur if untreated rhinitis progresses to more generalized disease. P. multocida is a common cause of abscessation in rabbits, irrespective of the presence or absence of respiratory tract disease. These abscesses are filled with thick, yellow–gray mucopurulent exudate and can occur in any location on or within the body (Figures 2 and 3). Microscopic examination of material collected from these abscesses usually reveals small gram-negative coccobacilli suggestive of P. multocida infection (Figure 4). P. multocida can also cause otitis externa and otitis media. If the vestibular system is involved, torticollis with the head tilted to

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Figure 1—Mucopurulent rhinitis suggestive of progressive

pasteurellosis.

Figure 2—Pasteurella multocida infection can cause abscessation in any organ. In this rabbit, the scrotum is ulcerated and its necrotic abscessed testicle was surgically removed.

the affected side and head shaking are common (Figure 5). Pasteurella-associated metritis, which also occasionally affects rabbits, typically follows breeding or pseudopregnancy and is manifested by chronic purulent discharge from the vulva. Surviving rabbits are frequently sterile.3,6

DIAGNOSIS Suggestive clinical signs are frequently used to diagnose pasteurellosis. However, other organisms alone or in combination with viruses and parasites can cause clinical signs that are characteristic of pasteurellosis.5 Accurate diagnosis and treatment of pasteurellosis are important to reduce the spread of P. multocida infection, particularly in multiple-rabbit households or rabbitries. Isolating the organism from the rabbit’s nostrils, conjunctiva, ears, or abscessed tissue has been the most common method for confirming pasteurellosis. In a classic study5 involving a rabbit colony with endemic pasteurellosis, 58 of 100 rabbits had gross upper respiratory changes at necropsy. P multocida was recovered . from the nares of 55 of these rabbits and from 8 rabbits with no signs of upper respiratory disease.5 However, culture of P. multocida in living rabbits can be problematic because even deep nasal cultures frequently fail to confirm upper respiratory tract infection when an organism in subclinically infected rabbits is present in low numbers. Localized infection in the tympanic bullae, caudal oropharynx, genital tract, and other internal organs renders ante. mortem sampling for culture difficult.7,8 In other cases, P multocida cannot be cultured from obviously diseased organs because the rabbit has received antibiotics. In addition to problems with sample collection, the cul-

Figure 3—Mucopurulent, necrotizing pneumonia in a rabbit with chronic pasteurellosis. The affected lung was surgically removed.

ture process can produce false-negative results. Immediate transfer of samples to culture medium is not always possible, and transport medium must preserve the organism until samples reach the laboratory. Compounding this problem is the fact that P multocida is extremely delicate . and does not survive well in most common transport media (e.g. Stuart’s, Amies), in some instances surviving less than 24 hours at room temperature.9 The organism may survive for several days in Cary-Blair medium. Because refrigeration can reduce survival of P multocida,9 samples for . culture should be maintained at room temperature or

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Figure 4—Pasteurella species are gram-negative coccobacilli

Figure 5—Torticollis secondary to Pasteurella-associated otitis

that can frequently be detected in cytologic preparations of secretions or exudates.

media/interna.

frozen. Use of fresh blood agar plates containing 5% rabbit blood can increase the sensitivity of P multocida isola. tion, but this modification of routine cultures is not common in commercial bacteriology laboratories usually used . by exotic animal clinicians.5 In addition, some strains of P multocida grow best at 34˚C to 35˚C,5 which approximates the temperature of rabbit nares but is lower than that used for routine cultures (37˚C). As a rule, polymerase chain reaction (PCR)–based assays are more sensitive than are those for cultures, because PCR assays detect target nucleic acid sequences even from nonviable organisms. Cultures and the most sensitive PCR assays can produce false–negative results if the sample does not contain viable organism (cultures) or sufficient quantities of nucleic acid (PCR assays). Serology can be a useful adjunct to cultures or PCR assays to establish whether a rabbit has active infection (rising antibody titer in paired serum samples tested in the same laboratory at the same time) and is the only method currently available for demonstrating previous infection. ELISAs are considered reliable for epidemiology studies in rabbit colonies.10–12 Rabbits with detectable antibodies may have cleared an infection in the immunologically detectable past or may be seropositive yet remain subclinically infected. In general, high levels of anti-Pasteurella immunoglobulin (Ig) G correlate with chronic infection.13 Routinely used ELISAs may not detect acute infection because it generally takes rabbits 2 to 3 weeks to develop detectable IgG antibody titers.10 In rabbits younger than 8 weeks of age, detectable antibodies are probably derived from the female. Antibodies from normal bacteria found

in rabbits cross-react with the ELISA at low levels. Thus it has been suggested that the ideal antibody assay would detect antibody to an epitope unique to P multocida.5 .

CLINICAL TRIALS A clinical study14 compared routine culture, a recently developed PCR assay,a and a recently developed serologic testb for confirming P. multocida infection in 58 rabbits with clinical signs suggestive of pasteurellosis. Clinical signs in tested rabbits included sneezing, congestion, chronic nasal discharge, chronic ocular discharge, otitis, ataxia, head tilt, anorexia, recumbency, or the presence of abscesses in various locations. All samples were submitted to the laboratory by regular mail, which is typical for routine culture.c A summary of results for each test as an independent assay is shown in Table I. Although samples from a total of 58 rabbits were used in the study, not all samples were submitted for each rabbit. For testing purposes, rabbits were placed in subgroups based on the samples
a P. multocida PCR, Infectious Diseases Laboratory, College of Veterinary Medicine, The University of Georgia, Athens, Georgia. b P. multocida ELISA, Infectious Diseases Laboratory, College of Veterinary Medicine, The University of Georgia, Athens, Georgia. c For each hospital, swabs for culture and PCR evaluation were taken from the nostrils, ocular discharge, inner ear, or abscesses; placed in transport medium for storage; and shipped. Samples took an average of 3 days to arrive at the laboratory. For P. multocida isolation, swabs were plated onto primary (blood and chocolate agar) and selective (modified Knight’s and Avril’s)13,14 media maintained in aerobic and microaerophilic conditions at 37˚C for 48 hours.

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TABLE I Summary of the Test Results to Diagnosis Pasteurellosis Total Tested Culture Polymerase chain reaction assay Serology 55 38 55 Positive 2 (3.6%) 11 (29%) 25 (45%) Negative 53 (96.4%) 27 (71%) 30 (55%)

TABLE II Results of Serology and Culture Testing in 52 Rabbits with Clinical Signs Suggestive of Pasteurellosis No. of Rabbits 27 (52%) 23 (44%) 2 (4%) Serology – + + Culture – – +

TABLE III Results of Serology and Polymerase Chain Reaction Testing in 35 Rabbits with Clinical Signs Suggestive of Pasteurellosis No. of Rabbits 16 (46%) 10 (29%) 8 (23%) 1 (3%) Polymerase Chain Reaction – + – + Serology – + + –

TABLE IV Results of Polymerase Chain Reaction and Culture Testing in 38 Rabbits with Clinical Signs Suggestive of Pasteurellosis No. of Rabbits 27 (71%) 9 (24%) 2 (5%) Culture – – + Polymerase Chain Reaction – + +

available for each animal. In total, swabs from 55 rabbits were cultured. P. multocida was recovered from only two (3.6%) of the swabs (Table I). The serum from these two rabbits contained anti-Pasteurella antibodies that could be detected using an ELISA with recombinant neuraminidase as an antigen.14 These results suggest that the selective media (Avril and modified Knight’s media containing antibiotics that inhibit fungi and other bacteria) were effective in preventing the growth of other microorganisms in the swabs, thereby reducing the possibility of other organisms overgrowing P. multocida.16,17 Samples from 52 rabbits were used to compare serology and culture results (Table II). Serum and swabs from 27 rabbits (52%) had negative results on both culture and serology. Samples from 23 of 52 rabbits (44%) tested serologically positive but had negative culture results, and two of the 52 rabbits (4%) tested positive on both culture and serology. Samples from 35 rabbits were used to compare serology and PCR assay results (Table III). Serum and swab results for 16 rabbits (46%) were negative for both

PCR assay and serology. Samples from eight rabbits (23%) were serologically positive but negative on the PCR assay. Ten rabbits (29%) were positive on both PCR assay and serology. One rabbit (3%) tested positive on PCR assay and negative on serology. Samples from 38 rabbits were available for testing using culture and PCR assay (Table IV). Twenty-seven swabs (71%) had negative results by both PCR assay and culture; nine swabs (24%) tested positive on PCR assay but negative on culture. Results of two swabs (5%) from which P. multocida was cultured were also positive on PCR assay. Samples from 35 rabbits were available for testing using serology, culture, and PCR assay (Table V). Sixteen rabbits (46%) tested negative and only two rabbits (6%) tested positive on all three tests. The results of eight of 35 rabbits (23%) were positive on both PCR assay and serology. The same number of rabbits also had negative results on PCR assay but had detectable levels of serum antibodies. P. multocida nucleic acid was detected on a swab from one rabbit (3%) that tested serologically negative; the remaining 16 rabbits also tested negative on PCR assay. The results of this study showed that rabbits in which

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TABLE V Results of Culture, Serology, and Polymerase Chain Reaction Testing in 35 Rabbits with Clinical Signs Suggestive of Pasteurellosis No. of Rabbits 16 (46%) 2 (6%) 8 (23%) 8 (23%) 1 (3%) Culture – + – – – Serology – + + + – Polymerase Chain Reaction – + + – +

than half of the sera submitted (30 of 55) tested negative for anti-Pasteurella IgG antibodies by ELISA. Although samples were submitted for rabbits with clinical signs suggestive of pasteurellosis, the results of this study suggest that many cases of suspected pasteurellosis may be caused by other organisms alone or in combination. Candidate organisms include Bordetella bronchiseptica, Pseudomonas species, Staphylococcus species or other Pasteurella species.5 Thus although P. multocida is a ubiquitous bacteria, it may be an overstated cause of respiratory disease in rabbits when clinical signs alone are used for diagnosis. The clinical trial reviewed in this manuscript suggests that PCR testing in combination with serology using a neuraminidase– based ELISA can help confirm pasteurellosis as the cause of disease in rabbits with suspicious clinical signs.
ACKNOWLEDGMENTS

P. multocida was detected by culture and/or PCR assay also tested positive on a recently developed ELISA.14 Furthermore, positive serology results indicated that eight rabbits (23%) had been infected with P. multocida in their immunologically detectable past even though the organism was not demonstrated on are nasal swabs for PCR assay or culture. Most of the submitted swabs were obtained from the nasal passages, which may have reduced the number of bacteria available for detection on culture or PCR assay. P. multocida has an affinity for the mucosal epithelium of the nasopharynx.8,15 Nasopharyngeal swabs are reportedly 36% more sensitive than are nasal swabs in detecting P. multocida; however, collecting a sample from the nasopharynx is difficult in a conscious rabbit.8 The use of nasal swabs in this study, instability of P. multocida during shipment, and ability of PCR assay to detect nucleic acid from inactive organisms likely contributed to the higher sensitivity of PCR assay compared with that of culture. To screen for bacteria in rabbits with rhinitis, a No. 4 calcium alginate swab can be inserted 1 to 4 cm into the nares along the nasal septum on both sides.5 The ELISA used in this study detected anti-Pasteurella IgG in 25 of 55 or 45.5% of the sera samples tested. By comparison, only 11 of 38 rabbits (29%) had positive results on PCR assay, and only two of 55 rabbits (3.64%) had positive culture results. All except one of the rabbits with positive results on PCR assay and/or culture also had positive results on serology. Because no additional sample was available from the one rabbit with positive PCR results and negative serology results, whether the serology was a false-negative reaction, the swab was contaminated with target nucleic acid, or the rabbit had an early infection to which the immune system had not mounted a detectable immune-response cannot be determined. Serology detected more positive results than did any of the other methods, but more

This research was supported by the University of Georgia Research Foundation Animal Health Fund and the Veterinary Medical Experiment Station. The authors thank Drs. William Chavez, Joe Deck, Cheryl Greenacre, Heather McClure, Masako Mori, Robert Ness, Richard Nye, Robert Owen, Pamela Slack, Samuel Vaughn, and Heather Wilson for submitting samples used in this project.

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Pasteurella multocida to rabbit pharyngeal cells and its possible role in rabbit respiratory tract infections. Infect Immun 35:1103–1109, 1982. 16. Knight DP, Paine JE, Speller DCE: A selective medium for Pasteurella multocida and its use with animal and human specimens. J Clin Pathol 36:591–594,1983. 17. Avril J, Donnio P, Pouedras P: Selective medium for Pasteurella multocida and its use to detect oropharyngeal carriage in pig breeders. J Clin Microbiol 28:1438–1440, 1990.

About the Authors
Dr. Sanchez is affiliated with the Athens Diagnostic Laboratory, Drs. Mizan and Lee with the Department of Medical Microbiology and Parasitology, and Dr. Ritchie with the Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, The University of Georgia, Athens, Georgia.