Overview of Plasmodium spp. and Animal Models in Malaria Research.

Malaria is a parasitic disease caused by protozoan species of the genus Plasmodium and transmitted by female mosquitos of the genus Anopheles and other Culicidae. Most of the parasites of the genus Plasmodium are highly species specific with more than 200 species described affecting different species of mammals, birds, and reptiles. Plasmodium species strictly affecting humans are P. falciparum, P. vivax, P. ovale, and P. malariae. More recently, P. knowlesi and other nonhuman primate plasmodia were found to naturally infect humans. Currently, malaria occurs mostly in poor tropical and subtropical areas of the world, and in many of these countries it is the leading cause of illness and death. For more than 100 y, animal models, have played a major role in our understanding of malaria biology. Avian Plasmodium species were the first to be used as models to study human malaria. Malaria parasite biology and immunity were first studied using mainly P. gallinaceum and P. relictum. Rodent malarias, particularly P. berghei and P. yoelii, have been used extensively as models to study malaria in mammals. Several species of Plasmodium from nonhuman primates have been used as surrogate models to study human malaria immunology, pathogenesis, candidate vaccines, and treatments. Plasmodium cynomolgi, P. simiovale, and P. fieldi are important models for studying malaria produced by P. vivax and P. ovale, while P. coatneyi is used as a model for studying severe malaria. Other nonhuman primate malarias used in research are P. fragile, P. inui, P. knowlesi, P. simium, and P. brasilianum. Very few nonhuman primate species can develop an infection with human malarias. Macaques in general are resistant to infection with P. falciparum, P. vivax, P. malariae, and P. ovale. Only apes and a few species of New World monkeys can support infection with human malarias. Herein we review the most common, and some less common, avian, reptile, and mammal plasmodia species used as models to study human malaria.

Contributions of Diet and Age to Ulcerative Dermatitis in Female C57BL/6J Mice.

C57BL/6J (B6) mice are commonly affected by ulcerative dermatitis (UD), a disease of unknown etiology with poor response to treatment. To study the possible role of diet in UD, we compared skin changes in B6 female mice fed a high-fat diet with those of mice fed a control diet. In addition, skin samples from mice with no, mild, moderate, and severe clinical signs of UD were examined by light and transmission electron microscopy (TEM). Mice fed a high-fat diet for 2 mo had more skin mast cell degranulation than did mice fed the control diet for the same period. Regardless of diet, older mice had more skin mast cells and more of these cells were degranulating as compared with younger mice. Microscopic changes in very early lesions were characterized by an increase in dermal mast cells and degranulation with focal areas of epidermal hyperplasia with or without hyperkeratosis. As the condition progressed, a mixed but predominantly neutrophilic inflammatory cell infiltrate appeared in the dermis, with or without epidermal erosion and scab formation. TEM showed that dermal mast cell membranes had disrupted and released of large number of electron dense granules, whereas degranulated mast cells were filled with isolated and coalescing empty spaces due to fusion of granule membranes. Ulceration appeared to occur very quickly, probably as result of intense scratching due to the pruritogenic properties of the histamine released from mast cell granules. This study showed a direct correlation between dietary fat and skin mast cell degranulation in female B6 mice. In addition, the number of skin mast cells and degranulation rates was higher in older mice. Treatments directed at preventing mast cell degranulation may result in better outcomes when applied early in UD cases. As noted previously in studies using caloric restriction, lower fat content in rodent diets may help prevent UD.

A Review of the Effects of Some Extrinsic Factors on Mice Used in Research.

Animals have been used in research for over 2,000 y. From very crude experiments conducted by ancient scholars, animal research, as a science, was refined over hundreds of years to what we know it as today. However, the housing conditions of animals used for research did not improve significantly until less than 100 years ago when guidelines for housing research animals were first published. In addition, it was not until relatively recently that some extrinsic factors were recognized as a research variable, even when animals were housed under recommended guidelines. For example, temperature, humidity, light, noise, vibration, diet, water, caging, bedding, etc., can all potentially affect research using mice, contributing the inability of others to reproduce published findings. Consequently, these external factors should be carefully considered in the design, planning, and execution of animal experiments. In addition, as recommended by others, the housing and husbandry conditions of the animals should be described in detail in publications resulting from animal research to improve study reproducibility. Here, we briefly review some common, and less common, external factors that affect research in one of the most popular animal models, the mouse.

Identification and characterization of the pathogenic potential of phenol-soluble modulin toxins in the mouse commensal Staphylococcus xylosus.

In contrast to the virulent human skin commensal Staphylococcus aureus, which secretes a plethora of toxins, other staphylococci have much reduced virulence. In these species, commonly the only toxins are those of the phenol-soluble modulin (PSM) family. PSMs are species-specific and have only been characterized in a limited number of species. S. xylosus is a usually innocuous commensal on the skin of mice and other mammals. Prompted by reports on the involvement of PSMs in atopic dermatitis (AD) and the isolation of S. xylosus from mice with AD-like symptoms, we here identified and characterized PSMs of S. xylosus with a focus on a potential involvement in AD phenotypes. We found that most clinical S. xylosus strains produce two PSMs, one of the shorter α- and one of the longer ß-type, which were responsible for almost the entire lytic and pro-inflammatory capacities of S. xylosus. Importantly, PSMα of S. xylosus caused lysis and degranulation of mast cells at degrees higher than that of S. aureus δ-toxin, the main PSM previously associated with AD. However, S. xylosus did not produce significant AD symptoms in wild-type mice as opposed to S. aureus, indicating that promotion of AD by S. xylosus likely requires a predisposed host. Our study indicates that non-specific cytolytic potency rather than specific interaction underlies PSM-mediated mast cell degranulation and suggest that the previously reported exceptional potency of δ-toxin of S. aureus is due to its high-level production. Furthermore, they suggest that species that produce cytolytic PSMs, such as S. xylosus, all have the capacity to promote AD, but a high combined level of PSM cytolytic potency is required to cause AD in a non-predisposed host.

Detection of early myocardial cell death in owl monkeys (Aotus nancymai) using complement component C9 immunohistochemistry in formalin-fixed paraffin-embedded heart tissues: A retrospective study.

BACKGROUND: Owl monkeys are commonly used in biomedical research which is affected by the high incidence of cardiomyopathy in this species. Occasionally, owl monkeys with no clinical signs of heart disease are found dead and at necropsy show no, or very mild, cardiomyopathy. A possible explanation for sudden death is acute myocardial infarction; however, early myocardial changes may be difficult to assess by conventional stains and light microscopy. METHODS: Complement component C9 immunohistochemistry was performed in paraffin-embedded heart tissue samples from owl monkeys who died suddenly, or were euthanized due to sickness, to determine whether these animals suffered from acute myocardial infarcts. RESULTS AND CONCLUSION: C9 deposits were found in the myocardium of 19 out of 20 (95%) animals. The findings in this study suggest owl monkeys suffer from acute myocardial infarcts, and complement component C9 immunohistochemistry may be a useful diagnostic tool.

Retrospective Study of Intercalated Disk Defects Associated with Dilated Cardiomyopathy, Atrial Thrombosis, and Heart Failure in BALB/c Mice Deficient in IL4 Receptor α.

An increased incidence of dilated cardiomyopathy and atrial thrombosis was noted in a breeding colony of BALB/c mice deficient in IL4 receptor α. The condition affected mice of both sexes and of various ages, and extensive testing (microbiology, serology, histopathology) failed to ascertain the cause. Transmission electron microscopy of heart samples showed structural defects in the myocardial intercalated disks, characterized by unorganized and heavily convoluted arrangement with lower density and less prominent desmosomes and adherens junctions, widening of the intercellular space, myofibrillar lysis adjacent to intercalated disks, occasional sarcomere lysis with marked myofiber degeneration, vacuolation, accumulation of cell debris, and myelin figures. The intercalated disk contains cell adhesion molecules that form cell junctions, allowing contraction coupling of cardiomyocytes and the electrical and mechanical connection between cardiac fibers. Thus, defects at this level result in poor myocardial contraction, intracardiac blood stagnation, and consequently cardiac dilation with clinical signs of heart failure. The background strain or, potentially, the Cre-loxP-mediated recombination system used to create these mice may have contributed to the elevated incidence of cardiomyopathy and atrial thrombosis in this colony. Due to the backcrossing breeding scheme used, we cannot discount the emergence and colonywide dissemination of a spontaneous mutation that affects the intercalated disk. This report underscores the importance of carefully monitoring genetically modified mice colonies for unexpected phenotypes that may result from spontaneous or unintended mutations or enhanced strain background pathology.

Eosinophilic aortitis with thoracic aortic aneurysm and rupture in a captive-born owl monkey.

Eosinophilic aortitis is a rare condition in animals and humans, and it has been occasionally reported associated with parasitic migration and with a poorly understood complex group of autoimmune vasculitides. Here, we describe a case of eosinophilic aortitis with thoracic aortic aneurysm and rupture in a captive-born owl monkey and discuss the differential diagnoses.

Acute Occlusion of the Abdominal Aorta with Sudden Paraplegia in a Captive Mustached Tamarin (Saguinus mystax).

A wild-caught, research-naïve, adult male mustached tamarin (Saguinus mystax) experienced sudden onset of bilateral hindlimb paresis. Physical examination established the presence of paralysis and the lack of femoral pulses and deep pain in both legs. There were no signs of external trauma and, due to a poor prognosis, euthanasia was elected. Necropsy findings included pleural effusion, partial pulmonary atelectasis and congestion, dilatatory cardiomyopathy, a renal hemorrhagic infarct, and a thromboembolus located at the trifurcation of the distal abdominal aorta. The clinical and histologic findings were indicative of an aortic-iliac thrombosis.

Genetic diversity of Klebsiella pneumoniae isolates during an outbreak in a non-human primate research colony.

BACKGROUND: Klebsiella pneumoniae can be a serious pathogen in non-human primates, particularly Neotropical monkeys. METHODS: During a K. pneumoniae outbreak in an owl monkey research colony, 13 K. pneumoniae isolates were DNA fingerprinted by automated repetitive extragenic palindromic-polymerase chain reaction and the profiles compared to isolates obtained from other non-human primate species during the same time period and isolates from previous outbreaks. RESULTS: Eleven different types of K. pneumoniae were circulating in the owl monkey colony at the time of the outbreak. When comparing owl monkey isolates relatedness to previous colony outbreak isolates and squirrel monkey and capuchin monkey isolates, all were different. CONCLUSIONS: These results agree with recent reports where K. pneumoniae nosocomial isolates in hospital settings can have high genetic diversity, and multiple strains can be circulating simultaneously. This potential genetic diversity should be considered when designing strategies for controlling K. pneumoniae outbreaks in captive non-human primate colonies.

Comparison of 3 Topical Treatments against Ulcerative Dermatitis in Mice with a C57BL/6 Background.

Ulcerative dermatitis (UD) is a common condition in C57BL/6 mice and strains with this background. The etiology of UD is unclear but appears to have a genetic component associated with the C57BL/6 strain and has been reported as secondary to a variety of conditions. Treatment is unrewarding, resulting in euthanasia in many cases. In the present study we compared 3 topical treatments against spontaneous UD in mice with a C57BL/6 background. In total, 301 mice of both sexes were included in this study, and the tested treatments comprised bacitracin-neomycin sulfate-polymixin B sulfate ointment twice daily, 10% povidone-iodine ointment plus 1% silver sulfadiazine cream once daily, and 0.005% sodium hypochlorite once daily. Lesion healing was defined as complete skin reepithelialization with or without hair regrowth. Sex, age, lesion location, and type and length of treatment were analyzed by using univariate and multivariate logistic regression. Of the 79 mice treated with triple-antibiotic ointment, 27 (34%) healed, compared with 43 of the 125 (34%) treated with povidone-iodine and sulfadiazine and 69 of the 97 (71%) treated with hypochlorite. Lesion size and treatment with 0.005% sodium hypochlorite were the only significant predictors of healing; all other variables were not statistically significant in multivariate analysis. We conclude that 0.005% sodium hypochlorite is an effective topical treatment alternative for UD in C57BL/6 mice and strains on this background, and a favorable prognosis depends on the early identification and treatment of those lesions.

Pathobiology of hepatitis E: lessons learned from primate models.

Like the other hepatitis viruses, hepatitis E virus (HEV) has been difficult to study because of limitations in cell culture systems and small animal models. Much of what we know has come from epidemiological studies in developing countries and, more recently, in industrialized countries. However, the epidemiology is very different in these two settings: hepatitis E in developing countries is epidemic as well as sporadic, principally water-borne, most likely to cause disease in older children and young adults and relatively severe, especially in pregnant women; in industrialized countries the disease is sporadic, principally food-borne, most common in the elderly and probably associated with mostly inapparent infections. These differences are believed to be genotypically determined. To examine the biological parameters of hepatitis E, we have studied HEV infections in nonhuman primates, which are surrogates of man. Infections with HEV genotypes 1-3 were compared in rhesus and cynomolgus macaques and chimpanzees. In general, the biological characteristics of the different HEV genotypes mirrored their epidemiological characteristics.

Select agent and toxin regulations: beyond the eighth edition of the Guide for the Care and Use of Laboratory Animals.

In the interval between the publication of the seventh and eighth editions of the Guide for the Care and Use of Laboratory Animals (Guide), much has changed with regard to the regulation and funding of highly pathogenic biologic agents and toxins (Select Agents). Funding of research involving highly pathogenic agents has increased dramatically during this time, thus increasing the demand for facilities capable of supporting this work. The eighth edition of the Guide briefly mentions Select Agents and provides a limited set of references. Here we provide some background information regarding the relevant laws and regulations, as well as an overview of the programmatic requirements pertaining to the use of Select Agents, with a focus on use in animals.

Intracardiac thrombosis and aortic dissecting aneurysms in mustached tamarins (Saguinus mystax) with cardiomyopathy.

Spontaneous intracardiac thrombosis is rarely reported in animals, particularly nonhuman primates. The finding of 2 cases of intracardiac thrombi in mustached tamarins (Saguinus mystax) that died as a consequence of congestive heart failure prompted us to do a retrospective study to determine the frequency of this condition. Clinical records, necropsy reports, and tissues from 60 mustached tamarins that died or were euthanized between 1996 and 2009 were reviewed. Of the 60 monkeys whose cases were reviewed, 10 (16.6%) had intracardiac thrombi, and 4 (6.6%) had dissecting aortic aneurysms. Of the 10 animals with intracardiac thrombosis, 3 had left ventricular involvement alone; 4 monkeys had thrombi only in the right ventricle, and the remaining 3 animals exhibited thrombi in both ventricles. Myocardial fibrosis and chronic renal disease were common findings in affected animals. The causes of the intracardiac thrombosis in the tamarins in the present study are not known, but the clinical signs and gross and microscopic lesions suggest that congestive heart failure secondary to cardiomyopathy is the primary contributor. In addition, the cause of the aortic dissecting aneurysms in the tamarins in this study is not known. Further studies are required to determine whether factors including aortic curvature, genetic background, or hypertension-alone or in combination-play a role. To our knowledge, the current retrospective study is the first report of intracardiac thrombosis and aortic aneurysms in mustached tamarins.

Spontaneous Staphylococcus xylosus infection in mice deficient in NADPH oxidase and comparison with other laboratory mouse strains.

Staphylococcus xylosus typically is described as a nonpathogenic common inhabitant of rodent skin. Reports of S. xylosus as a primary pathogen in human and veterinary medicine are scarce. Here we report 37 cases, affecting 12 strains of laboratory mice, of spontaneous infections in which S. xylosus was isolated and considered to be the primary pathogen contributing to the death or need for euthanasia of the animal. Infection with S. xylosus was the major cause of death or euthanasia in 3 strains of mice deficient in the production of phagocyte superoxide due to defects in NADPH oxidase. NADPH-oxidase-deficient mice (n = 21) were most susceptible to spontaneous S. xylosus infections. The infections were characterized by abscesses and granulomas in soft tissues, with bacterial migration to internal organs (primarily regional lymph nodes and lungs and, to a lesser degree, muscle, bone, and meninges). In contrast, 9 strains of phagocyte-superoxide-producing mice (n = 16) also had S. xylosus infections, but these were largely confined to eyelids, ocular conjunctiva, and skin and rarely involved other tissues or organs. Because exhaustive bacterial culture and isolation may not be performed routinely from mouse abscesses, S. xylosus infections may be underdiagnosed. S. xylosus should be considered in the differential diagnosis in laboratory mice with abscesses and other skin lesions. This report expands the range of mouse strains and tissues and organs susceptible to spontaneous S. xylosus infection and compares the pathology among various mice strains.

Splenic angioleiomyoma in an owl monkey (Aotus nancymae).

BACKGROUND: An adult male owl monkey (Aotus nancymae) underwent a splenectomy. When the spleen was removed, a small, nodular mass slightly bulging over the splenic surface was noted. METHODS: The mass was examined by light and transmission electron microscopy and by immunohistochemistry. RESULTS: On light microscopy, the mass was well-circumscribed, non-encapsulated, and composed of haphazardly arranged smooth muscle bundles admixed with numerous small capillary-like structures containing blood. Immunohistochemical (IHC) staining revealed the tumor was strongly positive for smooth muscle actin yielding vascular smooth muscle bundles, and for Factor VIII, staining endothelial cells within the smooth muscle bundles. Transmission electron microscopy (TEM) showed a large portion of the cells to be atypical appearing smooth muscle and a few cells had structures resembling Weibel-Palade bodies indicating endothelial cells. CONCLUSIONS: Based on cell morphology, by light and TEM, and IHC a final diagnosis of splenic angioleiomyoma was made. This is, to our knowledge, the first report of an angioleiomyoma in a non-human primate.

Management and care of African dormice (Graphiurus kelleni).

African dormice (Graphiurus spp.) are small nocturnal rodents that currently are uncommon in laboratory settings. Their use may increase as they have recently been shown to develop an infection with monkeypox virus and may prove to be a valuable animal model for infectious disease research. Because African dormice are not commercially available, an extensive breeding colony is required to produce the animals needed for research use. Husbandry modifications that increased the production of offspring were the use of a high-protein diet, increased cage enrichment, and decreased animal density. To optimize consumption of a high-protein diet, we tested the palatability of several high-protein foods in a series of preference trials. Dormice preferred wax worm larva, cottage cheese, roasted soy nuts, and canned chicken. Issues related to medical management of Graphiurus kelleni include potential complications from traumatic injury. The development of a program for the husbandry and care of African dormice at our institution typifies the experiences of many laboratory animal facilities that are asked to support the development of animal models using novel species.

Multisystemic eosinophilia resembling hypereosinophilic syndrome in a colony-bred owl monkey (Aotus vociferans).

In animals, multisystemic eosinophilic disease is a rare condition characterized by eosinophilic and lymphoplasmacytic infiltrates in various organs. This disorder resembles the human disease known as hypereosinophilic syndrome, a condition defined by prolonged peripheral eosinophilia in the absence of recognizable etiology and associated with end-organ damage. In this report we describe a research-naïve, colony-born, juvenile female owl monkey (Aotus vociferans) who presented clinically with severe respiratory distress and histologically with multiple end-organ infiltration with phenotypically mature eosinophils, plasma cells, and lymphocytes. No tumors or infectious agents were noted either macroscopically or microscopically. Cultures from lung samples revealed no bacteria or fungi. Histologic examination of lung, heart, thymus, liver, spleen, kidney, adrenal, pancreas, stomach, small intestine, and colon revealed no migrating nematode larvae, other parasites, or foreign material that might trigger eosinophilia, nor was there any evidence of or history consistent with an allergic etiology. Given that we ruled out most exogenous and endogenous triggers of eosinophilia, the signs, symptoms, and pathologic findings support the diagnosis of multisystemic eosinophilic disease. To our knowledge, this report is the first description of presumptive hypereosinophilic syndrome in a nonhuman primate.

Suboptimal ability of dirty-bedding sentinels to detect Spironucleus muris in a colony of mice with genetic manipulations of the adaptive immune system.

Spironucleus muris is an unacceptable infectious agent for most rodent colonies. Exposure of sentinel mice to dirty bedding and examination of sentinel intestinal smears was not sufficient for identification of the extent of spironucleosis within 1 mouse room. Clinical abnormalities were not reported in the animals housed in the room despite extensive breeding and a preponderance of mice genetically engineered to have nonfunctional T and B cells. In addition, researchers reported that the infection had not altered their research data. During investigation of the outbreak, direct intestinal smears performed on related animals (conspecifics, offspring, or siblings) revealed that immunodeficient mice often tested negative whereas the immunocompetent cohort tested positive. In this study, we used culled colony animals and compared direct intestinal exam test results with histologic results. The comparison showed the extent of false negatives that may occur when direct intestinal exam alone is used to detect this protozoon. Sensitivity of the direct intestinal exam for detection of S. muris was calculated to be 71%, while histology sensitivity was 91%. In light of the study results and an extensive literature review, we revised our health surveillance plan so that the age and duration of exposure to dirty bedding among sentinel mice is varied at the time of testing.

Detection of Spironucleus muris in unpreserved mouse tissue and fecal samples by using a PCR assay.

The purpose of this study was to develop a rapid DNA isolation method and a sensitive and specific PCR assay for detecting Spironucleus muris in mouse tissue and fecal samples. A PCR assay based on the carboxy terminus of the elongation factor 1a gene was developed; the PCR product was confirmed by nucleic acid sequencing and nested PCR. The new PCR assay then was used to test feces from animals that had been screened for S. muris by using direct intestinal examination and histology. The PCR assay was determined to be a more sensitive test than either direct intestinal examination or intestinal histology. To our knowledge, this assay represents the first use of a PCR-based diagnostic screening method to confirm the presence of S. muris in murine tissue and fecal samples.

Visceral and neural larva migrans in rhesus macaques.

Large ascarid larvae within granulomas were noted histologically in the mesenteric and pancreatic lymph nodes of 13 of 21 rhesus macaques (Macaca mulatta) euthanized as part of an experimental viral pathogenesis study. In addition, 7 of the 13 monkeys had cerebral granulomas, which in 4 animals contained nematode larvae similar to those within the lymph nodes. Despite the lesions, the animals did not show clinical signs associated with the parasitic infections. Characteristics of the larvae included, on cross-section, a midbody diameter of approximately 60 to 80 mum, a centrally located and slightly compressed intestine flanked on either side by large triangular excretory columns, and prominent single lateral cuticular alae. The morphology of the larvae was compatible with Baylisascaris spp. Baylisascariasis is a well-described infection of animals and humans that is caused by migrating larvae of the raccoon roundworm, Baylisascaris procyonis. A similar species, B. columnaris, is found in skunks and can cause cerebrospinal nematodiasis, but most reported cases of baylisascariasis have been due to B. procyonis. Our macaques were born free-ranging on an island in the southeastern United States where raccoons, but not skunks, were found to be common inhabitants, indicating that B. procyonis was the most likely parasite involved. These cases are similar to the low-level or covert cases of Baylisascaris infection described to occur in humans and provide further evidence of the existence of this parasite in the southeastern United States.