Diagnosis of ecto- and endoparasites in laboratory rats and mice.

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

Diagnostic necropsy and selected tissue and sample collection in rats and mice.

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

Simian parvoviruses: biology and implications for research.

The simian parvoviruses (SPVs) are in the genus Erythrovirus in the family Parvoviridae and are most closely related to the human virus B19. SPV has been identified in cynomolgus, rhesus, and pigtailed macaques. All of the primate erythroviruses have a predilection for erythroid precursors. Infection, which is common in macaques, is usually clinically silent. Disease from SPV is associated with immunosuppression due to infection with various retroviruses (SIV, simian retrovirus, and simian-human immunodeficiency virus), surgery, drug toxicity studies, and posttransplantation immunosuppressive treatment and therefore is of concern in studies that use parvovirus-positive macaques.

Polyomaviruses of nonhuman primates: implications for research.

Polyomaviruses are a family of small nonenveloped DNA viruses that infect birds and mammals. At least 7 nonhuman primate polyomaviruses that occur in macaques, African green monkeys, marmosets baboons, and chimpanzees have been described, as well as 4 polyomaviruses that occur in humans. Simian virus 40 (SV40), which infects macaques, was the first nonhuman primate polyomavirus identified as a contaminant of early polio vaccines. Primate polyomaviruses cause inapparent primary infections but persist in the host and can cause severe disease in situations of immunocompromise. This review describes the primate polyomaviruses, and the diseases associated with the viruses of macaques. In macaques, the greatest current concerns are the potential confounding of study results by polyomavirus infections and the zoonotic potential of SV40.

Naturally occurring Tyzzer's disease in cotton-top tamarins (Saguinus oedipus).

We noted naturally occurring infection with Clostridium piliforme (Tyzzer's disease) in 2 captive-reared cotton-top tamarins (Saguinus oedipus). Spontaneous Tyzzer's disease has been reported in multiple species of laboratory, domestic, and wild animals but is extremely rare in humans and nonhuman primates. Distinct from idiopathic colitis, which is common in cotton-top tamarins, these 2 tamarins had severe, transmural, necrotizing typhlocolitis accompanied by myocarditis and hepatitis. Abundant bacteria compatible with C. piliforme, the etiologic agent of Tyzzer's disease, were present adjacent to lesions in the cecum-colon, liver, and heart. Therefore, colitis caused by C. piliforme, although rare, should be included as a differential diagnosis in cotton-top tamarins and as a cause of postnatal mortality in this species.

Systemic arteriopathy in SIV-infected rhesus macaques (Macaca mulatta).

BACKGROUND: Severe disseminated vasculopathy was observed in two simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta). These animals developed clinical signs of AIDS, including lymphadenopathy, weight loss, diarrhea and collapse. RESULTS AND DISCUSSION: Grossly, both animals showed emaciation, lymphadenopathy, vegetations on the mitral valve, renal infarcts and a dilated intestine; one animal had multifocal hemorrhages in multiple organs. Histologically, both cases had disseminated arteriopathy characterized by intimal thickening and fibrosis with varying degrees of vasculitis. The lesion was prominent in the kidney, intestine, pancreas, liver, heart, lymph nodes, spleen and testis. Occasional venules had intimal thickening. Both cases had cytomegalovirus (CMV) infection with intranuclear inclusions, CMV antigen and nucleic acid; some inclusions were observed in endothelial cells within some of the vascular lesions in one of the two. These data suggest that CMV caused the unusual lesions.

Cell tropism of simian immunodeficiency virus in culture is not predictive of in vivo tropism or pathogenesis.

SIVmac239/316 is a molecular clone derived from SIVmac239 that differs from the parental virus by nine amino acids in env. This virus, unlike the parental SIVmac239, is able to replicate well in alveolar macrophages in culture. We have not however, observed macrophage-associated inflammatory disease in any animal infected with SIVmac239/316. Therefore, we sought to examine the cell tropism of this virus in vivo in multiple tissues using in situ hybridization combined with immunohistochemistry and multilabel confocal microscopy for viral nucleic acid and multiple cell-type-specific markers for macrophages and T lymphocytes. Tissues examined included brain, heart, lung, lymph nodes, spleen, thymus, and small and large intestine. Matched tissues from macaques infected with the parental SIVmac239 and uninfected macaques were also examined. Many infected cells were detected in the tissues of animals infected with SIVmac239 and SIVmac239/316 although there appeared to be fewer positive cells in animals infected with SIVmac239/316. Surprisingly, in light of the cell culture observations, nearly every simian immunodeficiency virus-infected cell in animals inoculated with SIVmac239/316 was a T lymphocyte rather than a macrophage. This was true both during early infection (first 2 months) and in terminal disease. In contrast, as previously described, SIVmac239 was found in both T cells and macrophages in tissues as early as 21 days after infection. These studies indicate that during both acute and chronic SIVmac239/316 infection T lymphocytes rather than macrophages are the principal targets in vivo. These data combined with the absence of macrophage-associated lesions in SIVmac239/316-infected animals indicate that in vitro cell tropism is not predictive of in vivo tropism or disease pathogenesis.

Effect of humoral immune responses on controlling viremia during primary infection of rhesus monkeys with simian immunodeficiency virus.

Cellular immune responses mediated by CD8+ lymphocytes exert efficient control of virus replication during primary simian immunodeficiency virus (SIV) infection. However, the role that antibodies may play in the early control of virus replication remains unclear. To evaluate how antibody responses may affect virus replication during primary SIVmac infection, we depleted rhesus monkeys of B cells with anti-CD20 antibody. In normal rhesus monkeys immunized with tetanus toxoid, anti-CD20 treatment and resulting depletion of B cells inhibited the generation of antitetanus antibodies, while tetanus-specific T-cell responses were preserved. During the first 4 weeks after inoculation with SIVmac251, development of SIV-specific neutralizing antibody was delayed, and titers were significantly lower in B-cell-depleted monkeys than control-antibody-treated monkeys. Despite the lower neutralizing antibody titers, the levels of plasma SIV RNA and the linear slope of the decline seen in B-cell-depleted monkeys did not differ from that observed in monkeys treated with control antibody. However, beginning at day 28 after SIV infection, the B-cell-depleted monkeys showed a significant inverse correlation between neutralizing antibody titers and plasma virus level. These results suggest that the rapid decline of peak viremia that typically occurs during the first 3 weeks of infection was not significantly affected by SIV-specific antibodies. However, the inverse correlation between neutralizing antibodies and plasma virus level during the postacute phases of infection suggests that humoral immune responses may contribute to the control of SIV replication.

Decreased frequency of cytomegalovirus (CMV)-specific CD4+ T lymphocytes in simian immunodeficiency virus-infected rhesus macaques: inverse relationship with CMV viremia.

The frequency of cytomegalovirus (CMV)-specific CD4+ T lymphocytes was determined in CMV-seropositive rhesus macaques with or without simian immunodeficiency virus (SIV) infection by using the sensitive assays of intracellular cytokine staining and gamma interferon ELISPOT. Both techniques yielded 3- to 1,000-fold-higher frequencies of CMV-specific CD4+ T lymphocytes than traditional proliferative limiting dilution assays. The median frequency of CMV-specific CD4+ T lymphocytes in 23 CMV-seropositive SIV-negative macaques was 0.63% (range, 0.16 to 5.8%). The majority of CMV-specific CD4+ T lymphocytes were CD95(pos) and CD27(lo) but expressed variable levels of CD45RA. A significant reduction (P < 0.05) in the frequency of CMV-specific CD4+ T lymphocytes was observed in pathogenic SIV-infected macaques but not in macaques infected with live attenuated strains of SIV. CMV-specific CD4+ T lymphocytes were not detected in six of nine pathogenic SIV-infected rhesus macaques. CMV DNA was detected in the plasma of four of six of these macaques but in no animal with detectable CMV-specific CD4+ T lymphocytes. In pathogenic SIV-infected macaques, loss of CMV-specific CD4+ T lymphocytes was not predicted by the severity of CD4+ T lymphocytopenia. Neither was it predicted by the pre-SIV infection frequencies of CD45RA(neg) or CCR5(pos) CMV-specific CD4+ T lymphocytes. However, the magnitude of activation, as evidenced by the intensity of CD40L expression on CMV-specific CD4+ T lymphocytes pre-SIV infection, was three- to sevenfold greater in the two macaques that subsequently lost these cells after SIV infection than in the two macaques that retained CMV-specific CD4+ T lymphocytes post-SIV infection. Future longitudinal studies with these techniques will facilitate the study of CMV pathogenesis in AIDS.