ACLAD NEWSLETTER
American Committee on Laboratory Animal Diseases

www.aclad.org

Fall 1998 Vol. 19, No. 2

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 1998:

PRESIDENT
John D. Strandberg (E-mail: jstrand@welchlink.welch.jhu.edu)

VICE-PRESIDENT (PRESIDENT-ELECT)
Diane J. Gaertner (E-mail: gaertner@aecom.yu.edu)

SECERETARY AND WEB SITE PROGRAMMER
Benjamin J. Weigler (E-mail: bweigler@bart.rprc.washington.edu)

NEWSLETTER EDITOR
Craig L. Franklin (E-mail: franklinc@missouri.edu)

TREASURER
Kimberly Waggie (E-mail: waggiek@zgi.com)

COUNCILORS
Stephen W. Barthold (E-mail: swbarthold@ucdavis.edu)
Julia K. Hilliard (E-mail: juliah@icarus.sfbr.org)

WEB SITE RELOCATED:

The ACLAD Web Site has moved!

Please update your browser's bookmark to the following:

http://www.rprc.washington.edu/aclad/index.html

CONTRIBUTORS THIS ISSUE:

1. Gregory P. Boivin. The Role of the Comparative Pathologist in Examination of Transgenic Mice.

2. Craig L. Franklin, Cynthia L. Besch-Williford, and Lela K. Riley. Chronic Typhlitis in Immunocompetent Mice with Helicobacter hepaticus.

NEWSLETTER SUBMISSIONS:

As always, ACLAD is in need of contributions for the newsletter. The next issue will be published in April. 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 high quality research at stages prior to formal publication.

AALAS MEETING ANNOUNCEMENTS:

1. The Wally Rowe Lecture

ACLAD is proud to once again sponsor the Wallace P. Rowe Lecture. This year's lecture will be given on Tuesday, October 20, 1998 at 11:00 in Ballroom A of the Cincinnati Convention Center by Dr. Thomas Doetschman, Associate Professor, Department of Molecular Genetics, Biochemistry and Microbiology, College of Medicine, University of Cincinnati. Dr. Doetschman is a highly respected member of the biomedical community and his work on postimplantation embryogenesis using targeted gene modification "knockout" techniques is well known and highly regarded. Dr. Doetschman's seminar, "Genetic Engineering in the Mouse," will focus on the importance of genetic engineering of mouse genes to discovering gene function.

2. The Annual Luncheon for Trainees

ACLAD will also continue the tradition of sponsoring a luncheon for trainees and their mentors at the national meeting. This year's event will be held at noon on Tuesday, October 20 in the Bronze Ballroom A, Regal Cincinnati immediately after the Wallace P. Rowe Lecture, and will include lunch and a program entitled "Applying for an NIH Grant: Strategies and Opportunities." Panel members will include Drs. Leo Whitehair and Neal West. If you would like to attend, please return the attached registration form by October 2. If you have any questions, please contact Valeria Krizsan by phone at (203) 785-2525, by fax at (203) 785-7499 or by email: valeria.krizsan@yale.edu.

THE ROLE OF THE COMPARATIVE PATHOLOGIST IN EXAMINATION OF TRANSGENIC MICE

by Gregory P. Boivin,
Department of Pathology and Laboratory Medicine,
University of Cincinnati, Cincinnati, Ohio

The advent of the technology to make transgenic and knockout mice has lead to a remarkable increase in the number of animal models of human disease. This new opportunity is not only an exciting area of exploration into gene function, but also opens doors for studying the pathogenesis of diseases, and to better understand the underlying phenomena we see histologically. Unfortunately, mice may go uncharacterized because the lead investigator is unaware of the expertise or availability of comparative pathologists. At the University of Cincinnati (UC) we have developed core facilities to assure the adequate analysis of these mice. The Division of Comparative Pathology provides pathology support for the gross, histologic, immunohistochemical, morphometric, and ultrastructural analysis of transgenic and knockout mice. Thus, I have had the opportunity to examine many of the mice developed at UC, and have been asked to provide some general suggestions on the analysis of tissues from these animals.

Because of the large number of genes being manipulated there is a vast array of potential changes that can occur which makes the analysis of tissues particularly complex. Despite the increased diversity of lesions that are seen there is nothing new to learn to adequately analyze tissues from transgenic and knockout mice. In other words, the lesion patterns seen in mice or any other mammal in response to an insult do not differ in mutant mice. However, unlike disease diagnosis and health monitoring there is often a need to be more thorough in the analysis of an organ, or to examine all tissues. In truth, the analysis of transgenic and knockout mice more closely parallels toxicological analysis because of the thoroughness needed to quantify even the most subtle changes.

Training and familiarity with developmental pathology, geriatric changes, morphometry and stereology have been an integral part of the understanding of the changes induced by gene addition or disruption. A few suggestions for performing analysis of the tissues are provided below. One of the more interesting results of the analysis of genetically manipulated mice is the discovery of an unexpected change. For many of the genes that are manipulated, the tissue expression is known. This often leads the investigator to suggest targeting the analysis to the particular organ of interest. However, this is not always the wisest of decisions. One example of this can be seen in the NHE2 (Na+/H+ exchanger isoform 2) knockout mouse (1). This gene is heavily expressed in the small intestine, kidney and to a lesser extent the stomach and other tissues. Despite the high expression in the intestine and kidney, no lesions were observed in these tissues. In contrast, there was marked alteration to the glandular mucosa of the stomach characterized by a severe depletion of parietal cells with an associated inflammatory infiltrate. This was not completely a surprise since there is moderate expression of NHE2 in the stomach. However, these mice also consistently developed multiple cystic lesions in the anterior pituitary (unpublished data). There was no previous knowledge of NHE2 activity in the pituitary so this was a very unexpected finding. Without a complete analysis of all tissues of these mice, the pituitary lesion would have been missed.

In a second transgenic mouse, an investigator targeted gene expression to the lung. Upon examination of these mice, we found severe hyperplasia and dysplasia of the glandular mucosa of the stomach, with gross thickening of the stomach wall to 20 times normal size. In a few of the mice, we also observed a severe dilation of the cecum. There was no known expression of the promoter in these tissues, so this was a totally unexpected finding for this pulmonary biologist. Currently two hypotheses for the gastrointestinal lesions are being explored, both of which have significant consequence to analysis of mutant mice. The first is possible expression of the gene outside of the lung. Although many of the promoters are presumed to be tissue specific, there may be expression at different times and locations during development. It is also possible that low level expression in other tissues had previously gone undetected. A second possibility is that the local expression in the lungs of the targeted gene could have induced a systemic response. This is not an unexpected finding for genes that control endocrine functions (although the gene manipulated in this mouse is not known to have systemic effects). Thus, there is further justification for performing a complete tissue analysis.

These two examples highlight the need for a thorough examination of all tissues in genetically engineered mice. Although in the second example the gross distension of the abdomen clinically with obvious enlargement of the stomach needed no prodding for further analysis, certainly this was not the case for the first example. It is important to remember that the location of a gene expression is not always an indicator of where lesions will occur. The thorough histologic analysis of these two animals has provided more information on location of the gene expression (or systemic effects of the expression). Thus, the comparative pathologist can provide very valuable contributions in addition to lesion diagnosis.

Examination of target tissues also presents unique challenges to the comparative pathologist. As with any disease condition, lesion severity in targeted tissues may be extremely variable, ranging from very subtle to grossly apparent. The latter lesions typically present no problem in diagnosis; however, the more subtle lesions can prove to be challenging. In these cases, even knowing the location of the potential lesion is insufficient for detection. A 10% change in the size of a cell or structure is undetectable when scanning a tissue. More sophisticated methods are often required to adequately characterize the organ. We have found that morphometric analysis of tissues to determine alterations in several cell variables is valuable. Cardiac myocyte size, jejunal villous height, skin thickness, fibrocyte density, and other measurements all lend themselves to morphometric analysis. Measurements of these items can prove valuable in determining hypertrophy and/or hyperplasia of tissues. Imaging equipment and software is readily available for performing morphometric analysis. Our system includes a Sony DXC930 color video camera, PowerMac 9600/350, Scion Image 1.60 and NIH Image 1.60 software, and a Scion CG7 RGB PCI frame grabber. Scion Image software is a variant of the public domain NIH Image program (developed at the U.S. National Institutes of Health and available at http://rsb.info.nih.gov/nih-image/). Adobe Photoshop 4.0 is used to manipulate images and Excel 4.0 is used for data collection and analysis. The tissues are examined on an Olympus BH-2 microscope with lens magnification of 1-100X dry. With this equipment, we are able to perform quantitative analysis of the tissues allowing interpretation of changes that may not be perceived by routine histologic analysis. We have also extended these measurements in some models to examining the tissues using stereologic principles. Stereology is the three dimensional interpretation of a structure based on area measurements. This technique yields even more data on tissues.

A common question that is asked is whether this level of analysis is really necessary, or if there is any biological significance to these changes. In fact, there are occasions when an investigator reports physiologic changes in a tissue, in which, using conventional histology, we are unable to detect any differences. Fortunately, the use of morphometric analysis has improved our ability to detect subtle lesions allowing us to more accurately correlate physiologic perturbations with changes in tissues.

Finally, there is one general area that is of particular importance in the examination of transgenic and knockout mice. That is the histologic examination of the fetus and neonatal mouse. When a gene being altered plays a critical role in development, it is common for there to be fetal or neonatal pathologic changes, if not death. This is in spite of the fact that many of the development processes have overlapping genetic protection. Because of the rapid changes occurring in the development of the fetus, determining age of death of the fetus, and tissue abnormalities is challenging. Developing a collection of normal organ histology from the fetus and neonatal mouse is very beneficial to these analyses.

In general, examination of transgenic and knockout mice parallels traditional analysis of mice in many ways. However, because of the diversity of lesions and the very subtle changes in some mutants, a more extensive study of the tissues is usually warranted. Traditionally training of veterinarians in comparative pathology has focused on assuring our ability to adequately diagnose disease conditions that could adversely effect rodents, and thus meets the basic service needs of health monitoring. This is reflected by a strong awareness of infectious diseases that may effect rodent colonies. This training is augmented with analysis of aging animals, yielding additional background in the diagnosis of neoplasia. Further training in mouse developmental histology is essential for preparation of a comparative pathologist to support mutant mouse studies. In addition, it is important to develop a familiarity with morphometric analysis techniques to detect minimal alterations.

REFERENCES:

1. Schultheis, P. J., L. L. Clarke, P. Menton, M. Harline, G. P. Boivin, G. Stemmermann, J. J. Duffy, T. Doetschman, M. L. Miller, and G. E. Shull. 1998. Targeted disruption of murine Na+/H+ exchanger isoform 2 gene causes reduced viability of gastric parietal cells and loss of net acid secretion. J Clin Invest. 101:1243-1253.

CHRONIC TYPHLITIS IN IMMUNOCOMPETENT MICE INFECTED WITH HELICOBACTER HEPATICUS

by Craig L. Franklin, Cynthia L. Besch-Williford and Lela K. Riley,
Research Animal Diagnostic and Investigative Laboratory,
College of Veterinary Medicine, University of Missouri, Columbia, Missouri

In 1990, the word "helicobacter" was only familiar to most of us as the genus of bacteria capable of causing gastric ulcers. In the laboratory animal community, discussions about helicobacters usually centered on animal models for gastric disease such as those in ferrets and cats. However, in 1992, with the discovery of Helicobacter hepaticus, this genus took on a new image in our community (2). "Helicobacter" is now a household word and carries a new meaning of pathogen of laboratory rodents. H. hepaticus is the best known of the helicobacters; it was the first species of Helicobacter recognized as a pathogen and remains the most troublesome to laboratory animal colony managers. H. hepaticus has been associated with chronic active hepatitis in some strains of mice and proliferative inflammatory bowel disease in immunodeficient mice (6-8). These disease manifestations are well described in the literature and have become engrained in the repertoire of lesions recognized by laboratory animal diagnosticians. In this report, we will not focus on these manifestations, but rather will discuss a feature of H. hepaticus infection that has not received as much press: enteric lesions in immunocompetent mice.

A discussion of enteric lesions caused by H. hepaticus often centers on the proliferative typhlocolitis seen in immunodeficient mice. This disease manifestation was first described by Ward et al. in mice with either the nu or scid mutation on a variety of genetic backgrounds, and has since been described in several lines of genetically altered mice that were likely rendered in some part immunodeficient (1, 7). In contrast, enteric lesions of immunocompetent mice infected with H. hepaticus have not received much attention. Queries of individuals submitting animals to the University of Missouri Research Animal Diagnostic and Investigative Laboratory (MURADIL) often reveal that they are familiar with hepatic manifestations of H. hepaticus and enteric disease of immunocompromised rodents but are unaware that H. hepaticus may cause enteric disease in immunocompetent mice.

Fox et al. first described non-suppurative typhlitis in 3-18 month old A/JCr mice naturally infected with H. hepaticus, but enteric disease was not a focus of the manuscript (3). The disease was reproduced in germfree Swiss mice orally inoculated with H. hepaticus (4). In addition to necrotizing hepatitis, these mice developed mild typhlitis by 10 to 24 weeks post-inoculation. The typhlitis was characterized by mixed cell infiltrates consisting of lymphocytes, plasmacytes and occasional macrophages. Discrete lymphoid aggregates with follicle formation were evident by 33 weeks post-inoculation. Mucosal epithelial hyperplasia associated with this inflammation and colitis were also seen in some mice. Recently, Whary et al described a similar chronic typhlitis in 12-month old A/JCr mice experimentally inoculated with H. hepaticus (9).

In our laboratories, we have experimentally inoculated A/JCr mice with H. hepaticus by oral gavage, and followed the progression of inflammatory bowel disease over a 12-month period. Briefly, H. hepaticus organisms were grown overnight in broth cultures and 108 organisms were inoculated into groups of A/JCr mice by gastric gavage. At 2 weeks post-inoculation, mice were positive for H. hepaticus by PCR evaluation of feces, indicating that colonization of the intestinal tract had occurred. At 3, 6, 9, and 12 months post-inoculation, PCR analyses confirmed the presence of H. hepaticus, documenting that persistent H. hepaticus infections were established in 100% of experimentally inoculated mice. Histologic examination of infected A/JCr mice revealed moderate chronic typhlitis in several of the mice examined. A milder chronic typhlitis was also present in control mice in the 12-month post-inoculation group. Cecal lesions were similar to those described previously (3, 4, 9) and characterized by predominantly mononuclear cell infiltrates in the lamina propria, hyperplasia of mucosal associated lymphoid tissue with follicle formation and mild mucosal epithelial hyperplasia (Figure 1. A & B). A lesion scoring system based on the one described by Mohammadi et al. for analysis of chronic gastritis in H. felis-infected mice was used to objectively grade lesions (5). Briefly, lesions were scored for intensity of inflammation, longitudinal extent of inflammation and vertical extent of inflammation. In addition, lesions were scored for hyperplasia using the following criteria: hyperplasia was defined as the presence of basophilic staining "crypt" epithelial cells in at least the lower 2/3 of gland or at least doubling of the height of the mucosal epithelium. Focal hyperplasia was given a score of one and diffuse hyperplasia was given a score of two. Scores were added for each animal and the total score used for comparisons.

Intestinal lesions were consistently more severe and more prevalent in female mice. (Figure 2). Statistical analysis using a Mann-Whitney Rank Sum test revealed statistically significant differences in the following comparisons: control females vs. experimental females at 48 weeks post-inoculation; pooled (all time points) control females vs. pooled experimental females; and pooled experimental females vs. pooled experimental males. These observations suggest that there may be a gender bias in the development of typhlitis induced by H. hepaticus with female mice developing the most significant disease. Curiously, this is in contrast to the gender bias seen with hepatic lesions due to this bacterium; male mice inoculated with H. hepaticus develop more severe hepatic lesions of earlier onset (6, 8). The mechanisms of these phenomena await further study.

We also occasionally observe chronic typhlitis in immunocompetent mice submitted to the MURADIL and in many cases, these mice are either seropositive for antibodies to H. hepaticus or colonized with this bacterium as diagnosed by fecal PCR examinations. Chronic typhlitis associated with H. hepaticus colonization has been observed in several strains or stocks of mice including BALB/c, Swiss Webster and ICR. The latter observations suggest that H. hepaticus is capable of inducing enteric lesions in immunocompetent mice in a natural setting.

Collectively, these results indicate that H. hepaticus causes not only hepatic disease but also disease of the intestinal tract, especially the cecum. Furthermore, enteric disease is not restricted to immunocompromised mice but can also be seen in immunocompetent mice.

Figure 1. (A) Histopathologic findings from the cecum and liver of Helicobacter hepaticus infected A/JCr mouse characterized by predominantly mononuclear cell infiltrates and mild mucosal epithelial hyperplasia. (B) A section of cecum at the same magnification from an uninfected A/JCr mouse is included for comparison.

Figure 2. Chronic typhlitis scores in mice inoculated with Helicobacter hepaticus and from uninoculated controls.

REFERENCES:

1. Foltz, C. J., J. G. Fox, R. Cahill, J. C. Murphy, L. Yan, B. Shames, and D. B. Schauer. 1998. Spontaneous inflammatory bowel diseae in multiple mutant mouse lines: association with colonization by Helicobacter hepaticus. Helicobacter. 3:69-78.
2. Fox, J. G., F. E. Dewhirst, J. G. Tully, B. J. Paster, L. Yan, N. S. Taylor, M. J. Collins, Jr., P. L. Gorelick, and J. M. Ward. 1994. Helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice. J Clin Microbiol. 32:1238-45.
3. Fox, J. G., X. Li, L. Yan, R. J. Cahill, R. Hurley, R. Lewis, and J. C. Murphy. 1996. Chronic proliferative hepatitis in A/JCr mice associated with persistent Helicobacter hepaticus infection: a model of Helicobacter-induced carcinogenesis. Infect Immun. 64:1548-1558.
4. Fox, J. G., L. Yan, B. Shames, J. Campbell, J. C. Murphy, and X. Li. 1996. Persistent hepatitis and enterocolitis in germfree mice infected with Helicobacter hepaticus. Infect Immun. 64:3673-81.
5. Mohammadi, M., R. Redline, J. Nedrud, and S. Czinn. 1996. Role of the host in pathogenesis of Helicobacter-associated gastritis: H. felis infection of inbred and congenic mouse strains. Infect Immun. 64:238-45.
6. Ward, J. M., M. R. Anver, D. C. Haines, and R. E. Benveniste. 1994. Chronic active hepatitis in mice caused by Helicobacter hepaticus. Am J Pathol. 145:959-68.
7. Ward, J. M., M. R. Anver, D. C. Haines, J. M. Melhorn, P. Gorelick, L. Yan, and J. G. Fox. 1996. Inflammatory large bowel disease in immunodeficient mice naturally infected with Helicobacter hepaticus. Lab Anim Sci. 46:15-20.
8. Ward, J. M., J. G. Fox, M. R. Anver, D. C. Haines, C. V. George, M. J. Collins, Jr., P. L. Gorelick, K. Nagashima, M. A. Gonda, R. V. Gilden, and et al. 1994. Chronic active hepatitis and associated liver tumors in mice caused by a persistent bacterial infection with a novel Helicobacter species. J Natl Cancer Inst. 86:1222-7.
9. Whary, M. T., T. J. Morgan, C. A. Dangler, K. J. Gaudes, N. S. Taylor, and J. G. Fox. 1998. Chronic active hepatitis induced by Helicobacter hepaticus in A/JCr mice is associated with a Th1 cell-mediated immune response. Infect Immun. 66:3142-3148.

ACLAD is an Affiliate Organization of AALAS.

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ACLAD Luncheon Registration
(in conjunction with the 49th AALAS Annual Meeting)

PLACE: Bronze Ballroom A, Regal Cincinnati

DATE: Tuesday, October 20, 1998

TIME: 12:00-2:00

CUISINE: Regal Delicatessen Buffet @ $18.00 per person (includes tax and gratuity)

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