http://www.aclad.org/

Craig L. Franklin

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

ACLAD Officers 2000:

Greg P. Boivin

(E-mail: boivingp@email.uc.edu)

Diane J. Gaertner

(E-mail: gaertner@aecom.yu.edu)

Glenn Otto

(E-mail: gotto@leland.stanford.edu)

Benjamin J. Weigler (Webmaster)

(E-mail: bweigler@fhcrc.org)

Jerry Davis

(E-mail: jkdavis@nersp.nerdc.ufl.edu)

CONTENTS FOR THIS ISSUE:

1. Request for newsletter submissions

2. Program for annual AALAS meeting

3. Featured Contributions

4. Dues Notice

As always, ACLAD is in need of contributions for the newsletter. 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.

The ACLAD General Business Meeting is scheduled for Sunday, October 20, 2001, from 4:00 - 5:00 pm in the Baltimore Room of the Hyatt Hotel. Please plan to attend!

The ACLAD will also sponsor a full slate of programs on Tuesday, October 23, 2001 at the Annual meeting of the American Association for Laboratory Animal Medicine.

1. In the morning, ACLAD will sponsor a seminar session entitled "Molecular Diagnostics in Laboratory Animal Medicine." This session will run from 8:00 a.m. to 10:45 a.m. in Room 308 of the Baltimore Convention Center. The following is the abstract and speaker list for this session from the AALAS Meeting Program:

This seminar will feature laboratory animal veterinarians and research scientists working with advanced molecular diagnostic techniques for infectious diseases. Current techniques rely extensively on the ability to detect a serological response in affected sentinel animals. Molecular methods allow detection of infection in animals that do not mount serological responses. With the developing information on the genomics of both infectious organisms and their host, molecular methods also may allow differentiation between virulent and nonvirulent organisms and determined effective host responses that will allow better understanding of pathogenesis. This seminar should be of special interest to comparative medicine scientists, veterinarians with interest in laboratory diagnosis of infectious diseases, postdoctoral trainees and research scientists. Participants will learn uses and problems of modern molecular diagnostic methods.

Leaders: Jerry K. Davis, Gregory P. Boivin

Speakers/Topics:

William R. Shek Current Methods of Diagnosis Success and Failures

Lela K. Riley Overview of Molecular Diagnostic Methods

Susan R. Compton Development of Molecular Diagnostic Methods

Deborah F. Talkington Molecular Diagnosis in Epidemiological Studies

David N. Fredericks Use of PCR and other Molecular Diagnostic Methods in Human Studies,

including Discovery of Novel Microbes

All Panel Discussion

Editor's Note: As of 10/15/01, Drs. Talkington and Fredericks have had to

cancel their appearances at this seminar, but the other presentations will proceed as planned.

2. The Wally Rowe Lecture

Following the morning session, ACLAD and BioReliance are proud to once again sponsor the Wallace P. Rowe Lecture. This year’s lecture will be given at 11:00 in Room 307 of the Baltimore Convention Center by Stephen W. Barthold, DVM, PhD, Diplomate, ACVP. Dr. Barthold is the Director of the Center for Comparative Medicine and the University of California-Davis Mouse Biology Program, the Associate Dean of Comparative Medicine for the School of Veterinary Medicine, and a Professor of Pathology at the University of California-Davis. His seminar is entitled "Mouse Genomics: Who Needs Veterinarians?"

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 year we want to encourage all members of ACLAD, including those that are not affiliated with a training program, to attend." The luncheon will be held on Tuesday, October 23 in the Baltimore Hyatt Hotel immediately after the Wallace P. Rowe Lecture. The guest speakers, Ravi Tolwani (Stanford University), Craig Franklin (University of Missouri), and Dave Besselsen (University of Arizona) will present "Opportunities in Laboratory Animal Disease Investigation and Comparative Medicine."

4. Rodent Pathology Quiz Bowl 2001

ACLAD is also proud to sponsor the Rodent Pathology Quiz Bowl on Wednesday, October 24 in Room 303 of the Baltimore Convention Center. This ACLAD tradition was reestablished in 2000 and will once again be offered at this years AALAS meeting. This round-table will consist of informal review of the pathology of rodents in the form of a Kodachrome slide quiz lead by Drs. Craig Franklin, Jeff Everitt, Greg Boivin and Cynthia Besch-Williford. Topics will include lesions of well-described infectious diseases, pathological manifestations of emerging diseases, lesions associated with the murine environment, as well as selected phenotypic manifestations of important genetically altered rodent models. The slides will be educations and challenging to laboratory animal scientists at all levels of pathology expertise. Participation is required and fabulous prizes will be awarded.

Minute virus of mice (MVM) and mouse parvovirus-1 (MPV) are among the most prevalent infectious agents found in contemporary laboratory mouse colonies . Although the immunosuppressive strain of MVM (MVMi) can induce a potentially lethal renal hemorrhagic disease when experimentally inoculated into neonatal mice [Brownstein, 1991], clinical disease and histologic lesions have not been reported for mice naturally infected with MVM. Similarly, clinical disease and histologic lesions have not been observed in mice naturally or experimentally infected with MPV [Jacoby, 1995; Smith, 1993]. Despite the absence of clinical disease and histopathology, murine parvoviruses can have significant deleterious effects on research due to their immunomodulatory effects both in vivo and in vitro [Bonnard, 1976; Engers, 1981; McKisic, 1993; McKisic, 1996; McKisic, 1998; McMaster, 1981]. In addition, MVM is a common contaminant of cell cultures, tissues, and other specimens of mouse origin [Bonnard, 1976; Collins, 1972; Nicklas, 1993], and MPV has the potential to be a contaminant of biological materials as demonstrated by its initial isolation from mouse splenocyte cultures [McKisic, 1993]. Finally, there is significant potential for MVM and MPV to be transmitted among animal facilities and stocks of biological materials due to a high degree of environmental stability. Therefore, identification of infected laboratory mice and contaminated biological materials is critical to minimize the impact of murine parvoviruses on research. Although numerous techniques can potentially be used to diagnose murine parvovirus infections (immunohistochemistry, virus isolation, etc.), this article will focus on serologic and PCR assays as these are currently the most commonly used diagnostic procedures.

Serological evaluation for the presence of anti-parvovirus antibodies has typically been used to diagnose MVM and MPV infections in mice [Jacoby, 1996], although the type of immunoassay and the source of diagnostic antigen vary significantly among rodent diagnostic laboratories. The most common methods include the enzyme-linked immunosorbent assay (ELISA), the indirect fluorescent antibody assay (IFA), and the hemagglutination inhibition assay (HAI). The ELISA is often preferred by larger laboratories due to its high throughput capability, with the IFA and HAI assays considered more labor intensive. The ELISA and IFA are considered slightly more sensitive than the HAI, while the HAI is considered more specific. The enhanced sensitivity of the ELISA and IFA assays is likely due to the abundance of a variety of viral epitopes in these preparations, while the slightly decreased specificity is reflective of adherence of non-specific antibodies to cellular proteins in the antigen preparation. The HAI is considered more specific since it detects only the interaction of antibodies directed against a specific conformational epitope, the parvovirus hemagglutinin, which is distinct for each parvovirus species. Therefore, most laboratories screen sera with an ELISA or IFA assay to take advantage of the increased sensitivity of these assays, then perform confirmatory testing with the HAI assay to ensure the result is truly specific for the targeted parvovirus.

More important than methodology is the choice of parvovirus antigen, especially for the detection of MPV. For several decades MVM antigens have been generated by infecting a susceptible cell line (A9_2L, BHK-21, NB324K, etc.) with the prototype strain of MVM (MVMp), which replicates extremely well in cell culture and produces highly concentrated preparations of MVM antigen. This antigen has worked (and continues to work) extremely well for the detection of MVM infections in mice by the ELISA, IFA, and HAI formats [Kraft, 1986; Smith, 1983]. MVMp antigen also displays some cross-reactivity with antibodies directed against MPV, and indeed provided evidence for the existence of MPV even prior to the initial isolation of MPV in the early 1990s. This cross-reactivity is primarily the result of antibodies generated to the nonstructural (NS) proteins of MPV binding to the NS proteins of MVMp in the antigen preparation, reflecting the high degree of homology among the NS proteins of the rodent parvoviruses [Besselsen, 1996]. However, the serodiagnosis of MPV infections with MVM antigen-based assays has several disadvantages. The primary disadvantage is that the diagnosis of MPV is made by default on the basis of an MVM HAI negative result coupled with an MVM ELISA and/or IFA positive result, especially if only nuclear fluorescence (reflective of nuclear localization of NS protein) is observed with the IFA assay. Since this approach detects antibodies directed against NS proteins, which as stated previously are highly conserved among all rodent parvoviruses, this approach does not necessarily detect MPV-specific (e.g. capsid) antigens, and one cannot rule out the possibility that a parvovirus distinct from MVM and MPV is causing the infection. In addition, the relative abundance of NS proteins may vary between MVM antigen preparations for the ELISA and IFA formats, which can impact the sensitivity of each of these assays in their ability to detect MPV. This is because cell culture infections for ELISA antigen preparations are allowed to go to complete cytopathic effect (CPE), while IFA antigen preparations are harvested after only a few days to keep the cells intact so that antibody localization (nuclear and/or cytoplasmic) can be visualized. Since the temporal production of parvovirus proteins favors nonstructural (NS) proteins early in the infection and capsid proteins later [Schoborg, 1991], the relative abundance of NS protein is likely higher in IFA preparations as compared to ELISA preparations.

As a result of the disadvantages of using MVM antigen to detect seroconversion to MPV, several approaches have been taken to improve serologic detection of MPV. Shortly after its initial isolation, cell culture-propagated MPV was utilized as antigen in both IFA and HAI formats [Smith, 1993]. However, high titered stocks of MPV are extremely difficult and expensive to obtain via cell culture propagation, and this has largely precluded the use of cell culture propagated MPV in a high throughput ELISA screening assay. Therefore, at this time MPV IFA and HAI assays are used mainly for confirmatory testing. To overcome the variability in the amount of NS protein in MVM ELISA antigen preparations, the major NS protein of MVM (NS1) was cloned into a baculovirus expression system to provide abundant purified NS1 protein for use as ELISA antigen [Riley, 1996]. This assay significantly improved the sensitivity of detection of MPV antibodies as compared to the MVM ELISA assay and has been used as the serologic screening assay by several laboratories for the past few years. However, direct comparison of NS1 ELISA, MVM ELISA, MVM IFA, MPV IFA, MPV HAI in their ability to detect seroconversion in mice experimentally inoculated with MPV revealed a potential problem with assays which rely upon NS antigens. Notably, the NS1 ELISA and MVM IFA often failed when mice were infected with MPV after 12 weeks of age, whereas seroconversion in the same mice was readily detected by the MPV IFA and MPV HAI assays which detect antibodies directed against MPV capsid proteins [Besselsen, 2000]. Coupled with previous reports that suggest MPV is shed for only a few weeks after exposure [Shek, 1998; Smith, 1993], these findings indicate MPV could go undetected for significant periods of time in animal facilities that use sentinels to detect MPV in their mouse colonies. This would be of concern in facilities that replace sentinel mice on a quarterly or semi-annual basis (e.g. mice may be older than 12 weeks when exposed) in combination with serologic assays that detect antibodies to parvoviral nonstructural antigens (e.g. the NS1 ELISA and the MVM IFA). To overcome this problem and the limited ability to generate cell culture propagated MPV antigen, ELISA assays that utilize baculovirus-expressed recombinant MPV capsid protein (VP2) as antigen have recently been developed for use as a screening assay for MPV. Preliminary data in both naturally and experimentally infected mice suggest this MPV VP2 ELISA significantly improves the sensitivity for MPV detection as compared to the NS1 ELISA (Tables 1 and 2). Similarly, an ELISA utilizing a baculovirus-expressed recombinant MVM VP2 antigen has been developed and also displays improved sensitivity as compared to the NS1 ELISA in detecting mice naturally and experimentally infected with MVM.

Although serologic assays will certainly remain the mainstay for diagnosis of murine parvovirus infections, there are several applications for which polymerase chain reaction (PCR) based assays are required or preferred. For example, PCR assays can be used to detect parvoviruses directly in immunodeficient strains of mice that do not generate a humoral immune response or in environmental samples such as swipes of cages or air filters. PCR assays also provide an attractive alternative to the mouse antibody production (MAP) test for detection of murine parvovirus contamination in biological materials. As compared to MAP testing, PCR confers the significant advantages of a greatly reduced turnaround time and cost while also providing an alternative to whole animal testing. The most critical aspects to the success of accurate PCR detection of murine parvovirus DNA are tissue selection, prevention of false positive results secondary to contamination, and prevention of false negative results due to PCR inhibitors. Since both MVM and MPV can be detected in the enteric tract, display lymphotropism, and MPV has been shown to persist for at least 9 weeks in the mesenteric lymph nodes [Jacoby, 1995], this tissue is considered the best suited as a diagnostic sample from mice for PCR analysis. Other tissue samples that can be analyzed include spleen and small intestine, although detection of MPV DNA in small intestine appears to be inconsistent. Prevention of false positive results secondary to contamination with DNA template or PCR product requires appropriate decontamination of instruments with bleach between tissue acquisition, excellent benchtop laboratory technique, and separate areas for DNA isolation, PCR set up, and PCR product analysis. In our experience, false negative results secondary to the presence of PCR inhibitors have been greatly minimized by the use of commercial DNA isolation kits (e.g. Qiagen kits), which generally minimize PCR inhibition. However, to ensure that inhibitors are not present, it is recommended that sample DNA is spiked with a detectable amount of positive control DNA in addition to evaluating the non-spiked sample DNA.

Similar to the existence of multiple serologic assays for the detection of murine parvoviruses, several PCR assays that detect these viruses have also been published [Besselsen, 1998]. In general, assays either target regions in the NS genes that are highly conserved among rodent parvoviruses (genus-specific) or target heterologous regions in the viral capsid genes (species-specific). The genus-specific assays provide a useful screening assay for the presence of any parvovirus, such as detection of parvovirus contamination in biological materials as an alternative to MAP testing. Species-specific assays are useful to discriminate which parvovirus species is present. Recently, genus-specific and species-specific assays for the detection of murine parvoviruses have been developed with fluorogenic nuclease PCR technology [Kendall, 2000], also known as real-time PCR or TaqMan PCR. These assays inherently confer several advantages over the existing gel detection PCR assays through the use of an internal fluorogenic hybridization probe detection system. These advantages include a quantitative, closed-tube detection system that eliminates post-PCR processing and carry-over contamination and potentially imparts improved specificity via the internal probe. When directly compared to the published gel detection assays, the fluorogenic nuclease PCR assays also displayed enhanced sensitivity, with the most significant difference between the genus-specific assays for which the fluorogenic nuclease PCR assay was able to detect 5 logs less virus than the corollary gel detection PCR assay [Besselsen, 2001].

In conclusion, serologic and PCR assays provide useful diagnostic tools for the detection of murine parvoviruses in infected mice and contaminated biological materials. Serologic assays that utilize cell culture propagated MVM or recombinant MVM VP2 protein work well as both screening and confirmatory diagnostic test procedures for the detection of MVM infections in mice. Serologic assays that utilize antigen preparations that contain MPV capsid proteins appear to provide significantly improved sensitivity for the detection of MPV as compared to those that rely upon MVM NS antigens alone, and are therefore recommended as assays to screen for the presence of MPV. Confirmatory testing can then be performed by other MPV-specific serologic assays or by MPV-specific PCR analysis of an appropriate target tissue. PCR assays are also useful for the detection of murine parvoviruses in immunodeficient mice, environmental samples, and in biological materials that are to be inoculated into mice.

¹ Number of sera positive divided by number of sera tested (%)

Bpeters@bioreliance.com, Ph: 301 610 2225, Fax: 301 838 0371

In August of 1999 birds of various species such as herons, eagles, hawks and American crows started dying in and around the Bronx Zoo and NYC. Later in August an infectious disease physician in NY reported an increase in encephalitis with an unusual muscle weakness pattern among patients at a hospital in Queens, NY. The two events seemed unrelated, initially. Sera from these patients were sent to the Centers for Disease Control (CDC) to determine if an infectious agent was involved. The answer came back as St. Louis Encephalitis virus (SLE), and spraying for mosquitoes, the vector of this virus, began in the Queens area. Because SLE does not kill birds, the two events were not tied together. A pathologist for the Wildlife Conservation Society undertook an investigation into the cause of death of the birds at the Bronx Zoo. Sections of bird tissues were sent to USAMRIID at Ft. Detrick, MD and the CDC Arbovirus group at Ft. Collins CO for identification of the infecting agent. CDC acknowledged the virus infecting the birds was West Nile Virus (WNV) in mid September and then that in humans in late September. This was surprising because WNV had only been identified in Africa, the Middle East and Eurasia, but never in this hemisphere. This story has captured the interest of the media and general public now that WNV has spread to most states on the East Coast and beyond and will eventually spread across the country. In 1999, 62 persons with WNV illness, including 7 deaths, were detected in New York City (NYC) and nearby New York counties. In 2000, 21 persons were identified with acute WNV infection (14 in New York, 6 in New Jersey, and one in Connecticut), including 2 deaths (one each in New York and New Jersey) (CDC MMWR, Apr. 2001). Fatality following WNV infection is actually somewhat less than that seen for SLE which has been in this country for a long time. The biggest concern, beyond human infection, is the effect this virus is having on wildlife and particularly birds in this country. Hundreds of thousands of birds have died and the mortality rate in horses has been about 40%. Dead crows seem to be the hallmark of the virus' entry into a given region because of the crows' high sensitivity to the virus and their large numbers. Spread of the virus was initially, relatively slow, due to the restricted range of most of the birds that are being infected. However, migration of susceptible birds last Fall has resulted in spread of the virus throughout a wider area of the U.S. with reports coming from as far west as Indiana and Wisconsin and south to Florida, Louisiana and Mexico. Many other species are susceptible with one mortality in a pet cat. Approximately 11% of dogs sampled in Queens were seropositive, some with high viremias. Many mammalian and avian species are at risk. Infection of the rhesus monkey has been demonstrated with some neurologic sequelae. The human infection toll is estimated to have exceeded 8000 in the northeastern U.S. WNV is spread by several species of mosquito, including Culex (the predominant species), and Aedes. All of the index human cases were associated with evening outdoor activity and standing water around the home. Early warnings were for night feeding mosquitoes, but it was later found that day-feeding mosquitoes were also carrying the virus. A license to produce a vaccine for horses has been issued under an emergency status and horses are now being vaccinated. A human vaccine is estimated to be 2 to 3 years away.

Abstracted from Pritt, S, and C Schiller. Exotic DVM 3.2: 8-9, 2001 (www.exoticdvm.com)

A two-year old, female, pedigreed Peruvian guinea pig was presented for a suspected abnormal pregnancy. Previous medical problems included a herd-wide conjunctivitis and upper respiratory disease caused by Pseudomonas and Streptococcus spp. Radiographs revealed one large, fully formed fetus lying horizontally in the cranial abdominal cavity. At necropsy, after an attempted ovariohysterectomy, it was observed that the fetus was lying in an omental pouch, attached to the left cranial abdominal wall. Grossly, the uterus and ovaries appeared normal, and there was fatty infiltrate noted in the liver.

The dead fetus was 5 inches long and 2 inches wide, weighing 151 g (normal is 50-100 g). It is theorized that expulsion of the fetus through a thin-walled diverticulum into the abdominal cavity during parturition. The large fetal size could account for the severe stretching and subsequent compromise of the uterine horn wall after which the fully developed fetus was expelled into the abdominal cavity. The fetus was most likely in the abdominal cavity for 4 months beyond the typical guinea pig gestation time. Implantation of the fetus within the abdominal cavity did not occur. In addition to hepatic lipidosis, acute adrenal necrosis and erosive gastritis was also present.

Spontaneous ectopic pregnancy in guinea pigs has been reported in two laboratory guinea pigs and ectopic embryo implantation has been performed experimentally. Mummified fetuses (which this fetus would have become) have been anecdotally reported.

A similar report to this one was documented in a gold hamster that was found to have 5 fetuses in the abdominal cavity after giving birth to 5 normal pups. It could not be determined if there was primary ectopic implantation or if the fetuses were expulsed into he abdominal cavity during parturition.

A recent report about a similar occurrence in a primate adds to information about ectopic pregnancies due to possible ruptured fetuses in laboratory species. In a poster presentation to be given at this year's American Association for Laboratory Animal Science, RE Wolf will discuss a "Mummified Fetus Associated with Uterine Rupture in a Baboon (Papio sp.) (Cont. Top. Lab. Anim. Sci. 40: 85-86, 2001). In this case, abdominal palpation revealed a large mass that was found to be a mummified fetus attached to the uterus by a pedicle.