Fall 1999 Vol. 20, No. 1
Editor for This Issue:
Craig L. Franklin, DVM, PhD
Research Animal Diagnostic and
Investigative Laboratory
University of Missouri
Columbia, MO 65211
Telephone: (573) 882-6623
FAX: (573) 884-7521
E-mail: franklinc@missouri.edu
* Items for the Newsletter, general comments
Editorial Assistant:
Joan Bailie*
Yale Animal Resource Center
Yale University
220 South Frontage Road
New Haven, CT 06510
Telephone: (203) 785-7256
FAX: (203) 785-3099
E-mail: joan.bailie@yale.edu
* Address changes, memeber dues
ACLAD Officers 1999:
PRESIDENT
Diane J. Gaertner (E-mail: gaertner@aecom.yu.edu)
IMMEDIATE PAST PRESIDENT
John Strandberg (E-mail: jstrand@welchlink.welch.jhu.edu)
VICE-PRESIDENT (PRESIDENT-ELECT)
(Open)
NEWSLETTER EDITOR
Craig L. Franklin (E-mail: franklinc@missouri.edu)
TREASURER
Glenn Otto (interim - E-mail: gotto@leland.stanford.edu)
COUNCILORS
Jerry Davis (E-mail: jkd.anires@mail.health.ufl.edu)
Julia K. Hilliard (E-mail: biojkh@panther.gsu.edu)
CONTENTS FOR THIS ISSUE:
1. Request for newsletter submissions
2. ACLAD Elections
3. Program for annual AALAS meeting
4. Featured Contributions:
5. ACLAD Luncheon Invitation and Registration Form
6. Dues Notice
7. Ballot for Treasurer and Councilor
NEWSLETTER SUBMISSIONS
As always, ACLAD is in need of contributions for the newsletter. The next issue will be published following the AALAS meeting. Please consider contributing!
The Newsletter publishes 1-2 page articles on topics such as research relating to laboratory animal diseases, disease reviews, case reports, information on disease incidence and serosurveys, issues in standardization and improvement of diagnostic methodologies, new animal models for disease, and special problems in transgenic animals. This newsletter is a perfect place to present contemporary material, and is an especially good forum for quality research at stages prior to formal publication.
ACLAD ELECTIONS
ACLAD is in need of a Treasurer and Councilor for the term commencing January 1, 2000 and ending December 31, 2000. A ballot is attached for this election.
AALAS PROGRAM
The ACLAD will sponsor a full slate of programs on Tuesday, November 9, 1999 at the Annual meeting of the American Association for Laboratory Animal Medicine.
1. Seminar
In the morning, ACLAD will sponsor a seminar session entitled "Genetically Engineered Mice: Phenotyping, Cryopreservation and Health Maintenance. This session will run from 8:00 a.m. to 10:45 a.m. in the Sagamore Ballwoom #3. The following is the abstract and speaker list for this session from the AALAS Meeting Preliminary Program:
Populations of transgenic and gene-targeted mice are growing explosively as scientists begin to use them to answer a myriad of important questions that could not previously be answered in vivo. This seminar presents expert advice from working scientists regarding state-of-the-art techniques for characterizing the phenotypic effects of genetic modifications. Speakers will draw from their own experience and research and will suggest directions for future research in these areas. This seminar will be of special interest to postdoctoral trainees, comparative medical scientists, research veterinarians, clinical veterinarians and program directors.
Leaders: Diane J. Gaertner, Abigail L. Smith
Speakers / Topics:
Philip Wood...........Phenotype Assessment: Are You Missing Something?
Charles Graves.......Oocyte and Embryo Cryopreservation-Present Status and Future
Prospects
John Critser............Cryopreservation of Sperm-Present Status and Future
Prospects
William Shek..........Special Diagnostic Issues Relating to Genetically Altered
Rodents
2. The Wally Rowe Lecture
Following the morning session, ACLAD is proud to once again sponsor the Wallace P. Rowe Lecture. This year's lecture will be given at 11:00 in the Sagamore Ballroom #3 of the Indianapolis Convention Center by Richard D. Palmiter, Ph.D., Professor, Department of Biochemistry and Investigator, Howard Hughes Medical Institute, University of Washington, Seattle. Dr. Palimiter's seminar is entitled "Genetics of Behavior: Future Directions of Mammalian Genetic Research." The following is the abstract for this lecture from the AALAS Meeting Preliminary Program.
It is now possible to alter the expression of any gene in the mouse that has been cloned. Infinite alterations are possible including complete gene inactivation, inactivation in only some cells or only at a particular developmental stage. It is also possible to redirect the expression of a gene to different cells, to visualize the cells that express a particular gene, or to kill cells that express a particular gene. These techniques offer tremendous opportunity to examine how particular genes influence development, various physiological processes and disease. Genetic manipulation of mice is reaffirming many tenets of physiology, endocrinology, metabolism, etc., but they occasionally challenge basic ideas when the genetic results do not coincide with predictions. The resolution of these differences will bring better understanding of important principles in various disciplines. Because our understanding of the role of specific genes is limited, these genetic approaches often produce surprising results. Most of the tools are in place for manipulating the mouse genome. The next decade will witness greater refinement of the genetic lesions, improved ease of genetic manipulation, and application of these techniques to other species. Dr. Palmiter's genetic studies on the role of neuromodulators (catecholamines and neuropeptides) will be used to illustrate these techniques and potential applications.
3. The Annual Luncheon for Trainees
ACLAD will continue the tradition of sponsoring a luncheon for trainees and their mentors at the national meeting. This luncheon will be held on Tuesday, November 9 in Room 101 of the Indianapolis Convention Center immediately after the Wallace P. Rowe Lecture. The guest speaker will be Jeff Roberts from the California Regional Primate Research Center whose topic will be "Careers in Nonhuman Primate Medicine." For more information, please see the letter of invitation from Dr. Jacoby and registration form on pages 13 and 14 of this newsletter.
4. Platform Session
In the afternoon, ACLAD will sponsor the Platform Session "Postdoctoral Fellows' Research and Comparative Medicine: Selected Hot Topics." This session will feature research presentations by individuals currently in comparative medicine training. Presentations will be followed by question and answer sessions lead by the trainee's mentor.
Earl K. Steffen, Ph.D.
University of Missouri, Research Animal Diagnostic and Investigative Laboratory,
Columbia, MO
Werner Nicklas, DVM,
Deutsches Krebsforschungzentrum, Heidelberg, Germany
Introduction
The family Pasteurellaceae presently includes the genera Pasteurella, Actinobacillus, and Haemophilus. Members of this family are usually small (0.2-0.3 x 0.3-2.0 um), gram-negative coccobacilli or bacilli that produce acid from glucose, usually without the production of gas. Nitrate reductase, oxidase, catalase, and alkaline phosphatase are almost always positive, although the oxidase reaction may be weak or delayed (1). The mol% G + C content of the DNA is 38-47 and the genome size range is 1.2-2.2 x 10^9 daltons (2).
Some species of this family are among the most fastidious of bacterial pathogens, requiring the use of chocolate agar (CA) to support growth. Commonly required growth factors, especially among Haemophilus species, include V (NADH) and X (hemin) (3). Recently, it has become apparent that strains of the other two genera may also have X and/or V factor growth requirements (4,5). It is likely that additional genera will soon be added to this family and various organisms will be renamed to achieve a more valid taxonomy. For example, members of the Pasteurella haemolytica complex have been renamed and now form several species of the newly established genus Mannheimia (6).
While the Pasteurellaceae are considered to be infrequent, although potentially serious, human pathogens that are typically transmitted by animal bite wounds (3), they are of keen interest to rodent diagnosticians because of the frequency of their colonization of the upper respiratory tracts and other mucosal surfaces of rats and mice. The species of widest distribution and greatest concern in rodents is undoubtedly Pasteurella pneumotropica. The role of P. pneumotropica in rodent medicine remains quite controversial. Estimates of its pathogenic potential run the entire spectrum from those who consider it to be no more pathogenic than any of the other members of this family to those who consider it to be a significant primary pathogen. The most common viewpoint regarding this organism is that it is a major opportunistic pathogen or a co-pathogen capable of contributing to serious disease by acting synergistically with other infectious agents of mice and rats, especially in the respiratory tract. As such, it should be excluded from rodent colonies wherever possible, although its presence does not necessarily mean that colonized rodents are unacceptable research subjects. A detailed review on P. pneumotropica and its role in rodent health can be accessed on the Internet (7).
Isolation and Identification of Pasteurellaceae using classical bacteriology techniques
Isolation Media. The most commonly employed medium for the isolation of Pasteurellaceae from the rodent nasopharynx is an enriched agar base supplemented with 5% sheep erythrocytes, commonly referred to as blood agar (BA). BA affords a good level of differentiation among various colony types because of the readily apparent colony color contrast against the red background and the detection of all types of hemolysis. Unfortunately, the majority of the Pasteurellaceae, including P. pneumotropica, produce a small, non-hemolytic, low, convex, entire, gray colony on BA; it is therefore impossible to differentiate species or even genera based on this unremarkable colonial appearance (1).
Historically, CA has been the medium of choice for the isolation of species of the genus Haemophilus because most are known to require X and/or V factors for growth. These factors are both provided in CA by the erythrocytes that have undergone the mild heat lysis used during its preparation. All primary isolation agars for the Pasteurellaceae should be incubated at 35-37oC in a moist, 5-10% CO2 atmosphere. Fastidious strains require a relatively moist agar for growth; agar with a wrinkled surface is therefore unacceptable (3). Some strains may require an incubation period in excess of 24 hours to produce a clearly visible colony (1).
As mentioned in the Introduction, it has been reported that "atypical" strains of Pasteurellaceae, most closely related to the genus Haemophilus, isolated from the lungs and upper respiratory tracts of rodents may have a V factor growth requirement (4,5). Such reports obviously hold serious implications for the current practice of rodent clinical microbiology in that they raise the legitimate concern that BA is inadequate for the recovery of all potentially pathogenic strains of rodent Pasteurellaceae. Because hemolysis cannot be determined on CA, the level of differentiation between various colony types is poorer on CA than BA, and CA will support the growth of more types of bacteria than BA (the majority of which prove to be harmless commensals), a principal concern from the viewpoint of the clinical microbiologist is that the routine use of CA versus BA will lead to a large increase in the numbers of colonies indistinguishable from those typical of the Pasteurellaceae, particularly P. pneumotropica (1). This increase will inevitably lead to more difficulty finding true Pasteurellaceae on the agar plate as their relative percentage of the total growth on the plate decreases. This, in turn, will necessitate the examination of many more suspect colonies that will prove not to be members of the Pasteurellaceae. This is quite significant because, as will be described in the next section, species identification within the Pasteurellaceae is not a trivial task for the clinical microbiologist.
Identification of rodent Pasteurellaceae
Once a suspect colony is identified on primary isolation agar, the process of identification can begin. It should be noted at this point that some rodent isolates of this family do not survive for more than five days on BA, even when refrigerated at 4oC (1). Therefore, it is important that suspect colonies be subcultured at least every third day during the identification process to maintain viability. Freezing fresh cultures in an appropriate cryopreservation medium at -80oC may be used to permanently store isolates.
A gram stain of a pure, fresh subculture on BA of a suspect colony is performed. If the isolate is a gram-negative coccobacillus or short bacillus, a portion of the colony is removed for oxidase testing. All of the Pasteurellaceae are considered to be oxidase positive, but in practice this reaction in strains of rodent origin is often weak or delayed. Thus, any detectable degree of reactivity with the oxidase reagent should be considered sufficient to warrant further characterization of an isolate (1). A colony yielding gram-negative, oxidase positive coccobacilli or bacilli is used to inoculate the following media: Triple Sugar Iron Agar, Christensen's Urea Agar, L-tryptophane Indole Broth, Ornithine Decarboxylase Broth and Base Control (both with sterile mineral oil overlay), Dextrose Phenol Red Broth (with Durham tube for the detection of gas production), Phenol Red Mannitol Broth, Phenol Red Maltose Broth, Phenol Red Xylose Broth, Phenol Red Trehalose Broth, Phenol Red Sucrose Broth, Phenol Red Lactose Broth, MacConkey Agar, and Esculin Iron Agar. The use of these media and characteristics allows presumptive identification of six species of Actinobacillus and nine species of Pasteurella, including P. pneumotropica (3). Confirmation of a presumptive identification of P. pneumotropica can be performed using commercially available identification kits such as the API NH™ (5), the API NFT™ (8), and the Remel RapID NF Plus™ (1).
In practice, clinical microbiologists often isolate strains of Pasteurellaceae that cannot be reliably identified to the species level using classical microbiological techniques. Among the many factors that contribute to this are: the fastidious nature of many strains of the Pasteurellaceae (e.g., false negative fermentation results due to lack of sufficient growth), the fact that the taxonomic status of the Pasteurellaceae is in flux (9), and the observation that commercially available identification kits incorporate tests that have been chosen to maximize discrimination among the most important human pathogens and therefore do not emphasize the Pasteurellaceae (10).
The high level of interest in P. pneumotropica versus other species in the family Pasteurellaceae has prompted the adoption of a "rapid" screening protocol for P. pneumotropica at the University of Missouri Research Animal Diagnostic and Investigative Laboratory. This protocol emphasizes the use of a primary immediate screen using characteristics and reactions with a very high positive percentage. A secondary overnight screen, again incorporating high positive percentage reactions but using a small test battery that requires an overnight incubation, follows the primary immediate screen. If any one of these characteristics or reactions is inconsistent with P. pneumotropica at any point in the screening protocol, the isolate is reported out as a member of the 'PAH (Pasteurella/Actinobacillus, Haemophilus) complex'. This screening protocol, which is still undergoing clinical evaluation, is summarized below:
Isolate: P. pneumotropica (primary isolation medium - BA)
Incubation conditions: 48 hours at 35oC in 7% carbon dioxide
Colony size: punctiform to 3 mm diameter
Colony description: round, entire, slightly gray to gray
Primary immediate screen:
Gram reaction/morphology negative rod
TSI (24 hours) w / neutral or w / w
Oxidase + or w
Rapid urease (30 minute reaction; footnote #1) +
Secondary overnight screen:
Subculture to BA
Glucose + / no gas (with Durham tube and serum; footnote #2)
Ornithine decarboxylase +
Final identification: Remel RapID NF Plus™ using BA overnight subculture
for the inoculum.
Reporting:
Report P. pneumotropica if and only if kit profile is consistent.
Report 'PAH complex' if kit profile is inconsistent with P. pneumotropica.
Footnote #1: This technique is performed by applying a very heavy inoculum from a loop upon and into a slightly cut surface of a Christensen's Urea agar slant over a very small area and checking for any shade of pink after 30 minutes of incubation at 35C. While the efficacy of this procedure compared to conventional overnight incubation is still undergoing evaluation, it appears to correlate very highly for the Pasteurellaceae.
Footnote #2: Because of the fastidious nature of some isolates, growth
in unsupplemented carbohydrate fermentation broth may be so weak that false
negative reactions may result. To preclude this possibility, make a heavy bacterial
suspension from a pure, overnight culture in 1.25 ml of freshly-thawed, sterile
fetal bovine serum. Using a sterile dropper, add 3 drops of this suspension
to each tube of fermentation broth. Since some batches of fetal bovine serum
contain unacceptably high levels of glucose, each batch should be tested using
a tube of fermentation broth base (no added carbohydrate) supplemented with
3 drops of the fetal bovine serum and inoculated with a strain of P.
pneumotropica. If acid production occurs in the test tube as determined
by comparison to an identically inoculated control using a batch of fetal bovine
serum with an acceptably low level of glucose, the test batch cannot be used
for this application.
Molecular techniques
16S rDNA sequencing. The use of molecular methods, especially 16S rDNA sequencing, has now clearly shown that many of the classical phenotypic criteria used to speciate the Pasteurellaceae are of dubious significance and do not truly reflect taxonomic relationships. For example, dependency on growth factors has been used historically as the major criterion to separate the genus Haemophilus from other Pasteurellaceae. However, it has become obvious from the phylogenetic tree based on 16S rDNA sequences that bacteria with growth requirements for NADH and/or hemin are scattered among species throughout the family (11,12). Furthermore, this phylogenetic tree demonstrates that some current classifications in the other two genera Pasteurella and Actinobacillus will also be transient and that additional genera will have to be established (11,12).
Based on 16S rDNA sequences, bacterial strains of the Pasteurellaceae that infect rodents belong to two clusters (13). One cluster consists of V factor dependent bacteria that exhibit a high DNA homology with reference strains of Haemophilus parainfluenzae. The remaining sequenced isolates form a separate cluster unique to rodents. Within this cluster, there are two separate subclusters. One subcluster consists of P. pneumotropica biotype Heyl and P. pneumotropica biotype Jawetz. While P. pneumotropica biotype Heyl is apparently homogeneous, P. pneumotropica biotype Jawetz actually consists of several species. Some V factor dependent isolates also appear within this subcluster. The second subcluster consists of Actinobacillus muris, (the so-called "Haemophilus influenzaemurium") and a heretofore unnamed species referred to as Pasteurella sp. rat (14).
PCR techniques. While 16S rDNA sequences have been used to establish phylogenetic trees of the Pasteurellaceae, a number of PCR assays have been used to demonstrate the presence of Pasteurellaceae, particularly P. pneumotropica, in rodents. Wang et al. (15) established their PCR based on the sequence of the type strain of P. pneumotropica biotype Jawetz. However, due to the genetic heterogeneity of P. pneumotropica, their primer sets do not detect P. pneumotropica biotype Heyl or other Pasteurellaceae infecting or colonizing rodents. Kodjo et al. (16) have developed a PCR which detects P. pneumotropica biotypes Jawetz and Heyl. Bootz et al. (17) selected primers on the basis of 16S sequences of various rodent isolates representing different phenotypic groups together with all sequences from Genebank and EMBL. This PCR was established with the goal of detecting all Pasteurellaceae known to colonize mice and rats. The 530 bp PCR product can be cut by restriction endonucleases to further identify organisms to the species level.
Future trends
Since the rodent Pasteurellaceae must be considered among the most difficult bacteria that clinical microbiologists are routinely called upon to cultivate and identify, molecular techniques will certainly play an increasingly important diagnostic role in the future. As progress continues in sequencing rodent isolates, a clearer picture of the true taxonomic relationships among these organisms will emerge. Knowledge of these DNA sequences will enable molecular biologists to devise new PCR primer sets with even more highly defined specificities. The trend toward more rapid and cheaper molecular diagnostic assays that lend themselves to automated procedures will undoubtedly contribute to the more routine use of PCR-based technology. Direct detection of target organisms from likely sites of colonization or infection will not only speed up the process of diagnosis, but may very well also prove to be more sensitive and reliable than classical culture techniques.
It is clear that certain biotypes of the Pasteurellaceae have more potential than others to contribute to disease in rodents. One of the long-term goals of researchers in this area should be to define the virulence factors of the rodent Pasteurellaceae. When this is accomplished, it may be possible to devise molecular diagnostic procedures that will detect gene sequences that code for these virulence factors; the particular species involved will be of only secondary importance.
A basic, underlying question for rodent producers, users, and diagnosticians is whether a given isolate of the Pasteurellaceae has the potential to contribute to disease in rodents. Armed with powerful molecular assays, diagnosticians may someday be able not only to detect the more virulent strains of Pasteurellaceae, but also to determine if strains isolated from sites harboring a normal flora, such as the nasopharynx, contain the genetic sequences which code for virulence factors.
References
1. E. Steffen, personal observation.
2. Bergey's Manual of Systematic Bacteriology, Vol. 1. 1984. N.R. Krieg, Editor.
Williams and Wilkins Press, Baltimore, MD, USA.
3. Manual of Clinical Microbiology. 1995. P.R. Murray, Editor in Chief. 6th
Edition. American Society for Microbiology Press, Washington, D.C., USA.
4. Nicklas, W., M. Staut, and A. Benner. 1993. Prevalence and biochemical properties
of V factor-dependent Pasteurellaceae from rodents. Zentralbl. Bakteriol. 279:114-124.
5. Boot, R, H. Thuis, and M.A. Koedam. 1995. Infection by V factor dependent
Pasteurellaceae (Haemophilus) in rats. J. Exp. Anim. Sci. 37:7-14.
6. Angen, O., R. Mutters, D. A. Caugant, J. E. Olsen, and M. Bisgaard. 1999.
Taxonomic relationship of the Pasteurella haemolytica complex as evaluated by
DNA-DNA hybridizations and 16S rRNA sequencing with proposal of Mannheimia haemolytica
gen. nov., comb. nov., Mannheimia granulomatis comb. nov., Mannheimia glucosida
sp. nov., Mannheimia ruminalis sp. nov. and Mannheimia varigena sp. nov. Int.
J. Syst. Bacteriol. 49:67-86.
7. http://www.criver.com/techdocs/past-1.html
8. Goelz, M.F., J.E. Thigpen, J. Mahler, W.P. Rogers, J. Locklear, B.J. Weigler,
and D.B. Forsythe. 1996. Efficacy of various therapeutic regimens in eliminating
Pasteurella pneumotropica from the mouse. Lab. Anim. Sci. 46:280-284.
9. Boot, R. and M. Bisgaard. 1995. Reclassification of 30 Pasteurellaceae strains
isolated from rodents. Lab. Anim. 29:314-319.
10. E. Steffen, personal communication.
11. Dewhirst, F.E., B.J. Paster, I. Olsen, and G.J. Fraser. 1992. Phylogeny
of 54 representative strains of species in the family Pasteurellaceae as determined
by comparison of 16S sequences. J. Bacteriol. 174:2002-2013.
12. Dewhirst, F.E., B.J. Paster, I. Olsen, and G.J. Fraser. 1993. Phylogeny
of the Pasteurellaceae as determined by comparison of 16S ribosomal ribonucleic
acid sequences. Zentralbl. Bakteriol. 279:35-44.
13. W. Nicklas, unpublished data.
14. Schulz, S., S. Pohl, and W. Mannheim. 1977. Mischinfektion von Albinoratten
durch Pasteurella pneumotropica und eine neue pneumotrope Pasteurella species.
Zentralbl. Vet. Med. B24:476-485.
15. Wang, R., W. Campbell, W. Cao, C. Summage, R.S. Steele, and C.E. Cerniglia.
1996. Detection of Pasteurella pneumotropica in laboratory mice and rats by
polymerase chain reaction. Lab. Anim. Sci. 46(1):81-85.
16. Kodjo, A., L. Villard, F. Veillet, F. Escande, E. Borges, F. Maurin, J.
Bonnod, and Y. Richard. 1999. Identification by 16S rDNA fragment amplification
and determination of genetic diversity by random amplified polymorphic DNA analysis
of Pasteurella pneumotropica isolated from laboratory rodents. Lab. Anim. Sci.
49(1):49-53.
17. Bootz,F., S. Kirschnek, W. Nicklas, S.K. Wyss, and F.R. Homberger. 1998.
Detection of Pasteurellaceae in rodents by polymerase chain reaction analysis.
Lab. Anim. Sci. 48:542-546.
Lela K. Riley, Joe H. Simmons, Greg Purdy, Robert S. Livingston,
Craig L. Franklin, and Cynthia L. Besch-Williford
Research Animal Diagnostic & Investigative Laboratory
University of Missouri, Columbia, MO
Robert J. Russell
Harlan Sprague Dawley, Inc.
Indianapolis, IN
Idiopathic pulmonary lesions in rats have been described in several recent publications (Farrar and LaRegina, 1997; Riley et al., 1997; Elwell et al., 1997); however, little is known about the progression of lesion development or the etiologic agent. Our laboratory is actively investigating these lesions as a collaborative project with Harlan Sprague Dawley, Inc. This document is designed to provide an update of research activities by our laboratory aimed at characterization of this newly recognized disease, elucidation of the etiologic agent, and development of a diagnostic assay to detect infected rats.
Characterization of the Disease
To gain a better understanding of the lesion progression and pathogenesis, rats of various ages were obtained from a facility in which pulmonary lesions had been reported. Groups of five rats at 2, 6, 8, 10, 12 and 18 weeks of age were evaluated. All rats evaluated were asymptomatic. Gross lesions were seen in 8-, 10-, 12-, and 18-week-old rats. Lesions were small, gray to white, raised, multifocal, and randomly distributed on all lung lobes (Figure 1). Histologically, no lesions were seen in two-week-old rats. In 6-week-old rats, histologic lesions consisted of mild multifocal perivascular lymphoid infiltrates. In 8-week-old rats, lesions were more severe consisting of multiple perivascular lesions characterized by lymphocytic and neutrophilic infiltrates. Lesions were most severe in rats 10-12 weeks of age. These rats had multifocal perivascular lesions of moderate severity that were often accompanied by foci of interstitial pneumonia. Pneumonic foci consisted of alveolar infiltrates of lymphocytes, neutrophils and macrophages and occasional hyperplasia of Type II pneumocytes. Advanced lesions resulted in localized consolidation of the lung (Figure 2). In 18-week-old rats, fewer lesions were seen and those lesions that were present consisted of alveolar septal thickening with associated luminal macrophage infiltrates with minimal consolidation. Collectively, the decreased number and lesser severity of lesions suggested that lesions were resolving.
To determine the prevalence within an affected colony, multiple groups of twenty 10-12 week-old rats were evaluated for lesions. In some groups, lesions were found in 2-4 rats; in other groups no rats exhibited lung lesions. These findings indicate that the prevalence of histologic disease is sporadic. No differences were seen in lesion severity or number between males and females. Histologic evaluation of 10-12-week-old rats from several strains/stocks indicated that the disease occurs in a wide variety of rat strains/stocks.
Identification of the Etiologic Agent
Identification of the etiology is critical to understanding the impact of the
disease on biomedical research utilizing laboratory rats and to controlling
and preventing transmission of the disease. Therefore, extensive efforts have
been made by our laboratory to identify the etiologic agent. Efforts include
examination of lungs for foreign bodies and screening for bacterial, fungal
and viral pathogens by histology, culture, and molecular assays.
Foreign body evaluations. To ascertain whether pulmonary lesions were due to inhalation of foreign bodies, lungs from affected rats were examined histologically with polarized light for birefringent material, indicative of the presence of foreign bodies. Results were negative.
Bacterial screening. To assess whether a bacterial agent was responsible
for pulmonary lesions, sera from rats in affected colonies were screened for
antibodies to known respiratory pathogens and tissues from affected rats were
screened by histology and culture and by several polymerase chain reaction (PCR)
assays. Serologic evaluations showed no antibodies against the two known murine
bacterial respiratory pathogens, Mycoplasma pulmonis and the cilia-associated
respiratory (CAR) bacillus. Histologic evaluation of lungs with hematoxylin-eosin,
modified Steiner's silver stain, tissue gram stain, and acid-fast stain failed
to demonstrate the presence of bacteria. Culture of lung homogenates on blood
and chocolate agars, media that are rich and allow growth of many bacterial
species, yielded no growth. Additional culture on Dutch agar for Mycoplasma
pulmonis, and Lowenstein-Jensen agar for Mycobacteria spp. were also
negative for growth. PCR assays that detect CAR bacillus and all species of
Mycoplasma were negative. Negative results were also obtained with a
PCR assay that uses conserved primers within the 16S rRNA gene and is capable
of detecting all known bacterial species.
Fungal and Pneumocystis carinii screening. Lung sections from affected rats
were stained with Gomori methenamine silver stain to detect fungal agents and
P. carinii. Histologic evaluation of stained sections was negative.
Additionally, PCR assays specific for P. carinii were negative.
Viral screening. To determine whether pulmonary lesions were caused by previously identified murine viral pathogens, serum from affected rat colonies were screened for antibodies to known viral respiratory agents of rats and mice including sialodacryoadenitis virus (SDAV), Sendai virus and pneumonia virus of mice (PVM). All results were negative. Serologic screenings for non-respiratory pathogens, including parvoviruses, reovirus-3, Theiler's murine encephalomyelitis virus (TMEV), adenovirus, and lymphocytic choriomeningitis virus (LCMV), were also negative. Culture of a putative viral agent was accomplished by incubating lung homogenates from affected rats on 15 different cell lines. After multiple blind passages, a limited cytopathic effect was observed in two cell lines. Indirect fluorescence assays (IFAs) in which affected cell lines were probed with serum from rats with documented lung lesions showed cytoplasmic fluorescence. Similar experiments with sera from colonies with no history of lung lesions were negative. These results suggested the presence of a viral organism in affected rats that is not present in unaffected rats. Isolation and culture of this virus has been successfully repeated with lung tissue obtained from a number of affected rats. No virus has been identified from identical experiments using rat lung tissue from colonies with no history of pulmonary disease.
Our laboratory has continued to propagate the virus in order to characterize and identify the virus and perform experimental infection studies. These studies have been tremendously hampered by the difficulties associated with in vitro propagation of the virus. Viral titers in cell culture are extremely low; thus, we have expended a great deal of time and effort optimizing in vitro viral culture and virus purification procedures. Recently we have achieved modest success in these efforts allowing us to initiate experimental infection studies in naïve rats to determine whether the virus we have propagated in vitro can cause pulmonary disease. These experiments are on going.
Diagnostic Test Development
Availability of a sensitive and specific diagnostic assay for detection of infected
rats will be critical to control and prevention of this disease within laboratory
animal facilities. To this end, our laboratory is testing the in vitro
propagated virus as a potential antigen on which to base a serologic assay.
Currently, an IFA assay has been developed and is being tested on a research
basis. Preliminary serologic test results correlate well with histologic findings
from affected and unaffected rat colonies. We anticipate making the serologic
assay available to the biomedical community when it has been fully validated
and we have fulfilled Koch's postulates.
Prevalence
Serologic screening of rats routinely submitted to our diagnostic laboratory from rat users worldwide suggests that the virus is widespread. Of 26 facilities tested by IFA, 6 facilities yielded positive results for antibodies to the virus.
Summary
Recently identified pulmonary lesions in laboratory rats occur in multiple
strains and stocks of rats and appear to be prevalent within laboratory animal
facilities. Lesions are transient, being most severe in 10-12-week-old rats.
There appears to be no gender bias as the severity and incidence of lesions
is similar in males and females. While we have not yet identified the etiologic
agent, our laboratory ruled out foreign bodies, bacteria and fungal etiologies.
We are currently investigating a virus that we have cultured from the lungs
of affected rats as the putative etiologic agent. Rats from affected colonies
appear to have antibody to the virus, whereas rats from unaffected colonies
do not. Experimental infection studies with this virus are currently underway.
References Cited
Riley L, G Purdy, J Dodds, C Franklin, C Besch-Williford, R Hook Jr, and J Wagner. Idiopathic lung lesions in rats: search for an etiologic agent. Cont. Top. Lab. Anim. Sci. 36:46. Presented at the 48th Annual Meeting of the American Association for Laboratory Animal Science in Anaheim, California, November, 1997.
Farrar P, and M LaRegina. Diagnostic Exercise: Interstitial pneumonia in viral
and mycoplasmal antibody-free Sprague Dawley Rats. ACLAD Newsletter 18:5-9,1997.
Elwell M, and J Mahler. "Have you seen this?" Inflammatory lesions
in the lungs of rats. Tox. Pathol. 25:529-531, 1997.
A letter from Neil Lipman, Hai Nguyen, Scott Perkins, Joe Scott
We are posting this notice to inform you that we have recently detected mousepox in one of our mouse holding rooms at the Cornell University Medical College. At this time we have not confirmed the source of the virus, however we speculate and are confirming the following possibilities: 1) Contaminated cell line; however, the cell line administered to the affected animals had been MAP tested twice by 2 different institutions and was determined to be negative for ectromelia. In addition, the line has been used here repeatedly, without problems, for ~ 5 years; 2) Commercial mouse serum; however, its inclusion in media used for cells which would eventually be inoculated into mice would have been an accident.
Mice were found dead or depressed ~ 6 days after immunization with killed cells. Pathology was consistent with previous reports and numerous acidophilic intracytoplasmic inclusions were observed in the skin overlying the immunization sites.
This outbreak appears to be markedly similar to that reported by the Naval Medical Research Institute, Bethesda, MD (LAS, 1996 46:602-611) which was determined to be associated with mouse serum.
Follow-up memo: Fri, 23 Apr 1999
From: Neil S. Lipman, VMD
Director and Associate Professor of Pathology
Cornell University Medical College
and the Memorial Sloan-Kettering Cancer Center
1275 York Avenue Box 270 New York, New York 10021
Phone: 212 639 8901 fax: 212 794 4330
E-mail: lipmann@mskmail.mskcc.org
Subject: Mousepox update: A real threat!
Dear Colleagues: As you may have already been informed, we detected mousepox in one of our mouse colonies at the Weill Medical College of Cornell University. The outbreak involved a group of 20 mice maintained in a single animal holding room, housing one investigator's animals. Infection was confirmed by histopathology, immunohistochemistry, and PCR. Commercial pooled mouse serum was confirmed as the source of virus by MAP testing and PCR. Although the serum was not intended to be used in vivo, the investigator accidentally supplemented media used to feed cells that were subsequently inoculated into mice. We were fortunate that the mouse strain (CAF1) inoculated with mousepox was extremely sensitive. The animals died or were sacrificed before skin lesions and viral shedding occurred. We eliminated the virus by sacrificing all replaceable mice and isolating unique strains. Infection did not spread beyond the room in which it was originally detected. As we were confident that the infection was contained and eliminated, we recently removed the ban we had implemented restricting shipment of our mice to other institutions. We have contacted the vendor supplying the contaminated serum and they were of considerable assistance. We were able to determine that the serum aliquot we received (11/98) originated from China. The serum was imported in early 1995 as part of a batch of at least 43 liters. The importer sold the serum to a company which blended the material into a single lot and filtered it (0.2 micron) before distributing the serum to other major suppliers between April '95 through December '96. The vendor from which our aliquot originated received the serum from one of these suppliers. The identification of the serum source has been eye opening. As we are certain you recognize, it requires > 80,000 mice to yield a serum batch of 43 liters. Therefore, the risk of contamination is quite high. For all intensive purposes, the importation of this material is unregulated. The USDA only monitors the material to ensure it is not contaminated with biologics of livestock origin. As mouse serum is generally sold and used in relatively small quantities, thousands of aliquots of this batch of serum could have been distributed throughout the country. We suspect this material was/is being sold by many vendors. Obviously, there is great risk for domestic mouse colonies. We are aware of at least one other mousepox outbreak currently. Perhaps there are or will be more. The regulation of imported rodent materials is something in which we all must be concerned.
With best regards. Sincerely, Neil S. Lipman Hai Nguyen Scott Perkins
__________________________________________________________________________________________________________________________________________________________________________
An invitation to the ACLAD Luncheon
Dear Colleagues:
I am pleased to tell you that the American Committee on Laboratory Animal Disease (ACLAD) will continue its sponsorship of the lunch for trainees and their mentors at the national AALAS meeting. This year's event will be held at noon on Tuesday, November 9 in Room 101 of the Indianapolis Convention Center immediately after the Wallace P. Rowe Lecture. The guest speaker will be Jeff Roberts from the California Regional Primate Research Center whose topic will be "Careers in nonhuman primate medicine." Those of you who have attended the lunch before know that it has been highly successful, with attendance averaging about 100 people. It provides trainees with an excellent opportunity to meet with peers from other programs as well as with more senior members of the laboratory animal medicine and comparative medicine communities. We hope that you and your trainees will attend. To that end, please return the enclosed reservation form together with a check(s) for the lunch. The deadline for receipt of reservation and check is October 1, so that we can confirm arrangements with the hotel and mail tickets to you.
We look forward to seeing you in Indianapolis.
Cordially and for ACLAD,
Robert O. Jacoby, DVM, PhD
ACLAD membership dues support ACLAD-sponsored programs at the National AALAS meeting
and underwrite the cost of reproducing and mailing the ACLAD Newsletter.
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