Histopathology of incidental findings in beagles used in toxicity studies

The purpose of our publication is to widely communicate the pictures of spontaneous findings occurring in beagles. Spontaneous arteritis occurs commonly in beagles. Frequent sites of arteritis are the heart, spleen, pancreas, epididymis and spinal cord. Morphological similarities between spontaneous and drug-induced arterial lesions may cause confusion when evaluating vascular toxicity of chemicals such as vasodilating agents. Focal and minimal inflammatory lesions are occasionally seen in the lung and may be associated with aspiration of food particles or of unknown causes. A cystic change with copious mucin production occurs occasionally in the mucosal epithelium of the gall bladder. Nesidioblastosis is seen rarely in the pancreas of beagles. C-cell complex and lymphocytic thyroiditis are common thyroid lesions. Spontaneous focal hypospermatogenesis and lobular Sertoli-cell-only seminiferous tubules occurring frequently in beagles must be distinguished from drug-induced damage of the seminiferous tubules in toxicity studies. The morphological differences of the female genital system in each cycle need to be understood; therefore, we present the normal features of the cyclic changes of the female genital organs. Further, we provide more information on spontaneous findings in beagles for exact diagnoses in toxicity studies.     

Quantitative analyses of lymphoid tissue in the spleen, lymph nodes and Peyer's patches in cynomolgus monkeys

To clarify the morphological characteristics of the cynomolgus monkey immune system, we analyzed quantitative data on their lymphoid organs. Spleens, major lymph nodes and Peyer's patches were sampled from cynomolgus monkeys, and the lymphoid follicle and germinal center areas and percentages of CD3- and CD20-positive areas were calculated. All the organs analyzed showed large interindividual variations in the sizes of lymphoid follicles and germinal centers. Lymphoid follicle in the spleen, submandibular lymph nodes and Peyer's patches showed no marked difference in size. Germinal center size in the mesenteric lymph nodes and Peyer's patches were significantly smaller than those in the spleen. Areas containing T cells were largest in the lymph nodes, while those containing B cells were largest in the spleen and Peyer's patches. The mean size of the splenic lymphoid follicle in cynomolgus monkeys is larger than that in rats and similar to that in humans. Based on the large individual variation and the characteristics of lymphoid organs, it is important to use cynomolgus monkeys in standard toxicity studies. Taking advantage of the characteristics of each species enables reliable evaluation of the immunologic system in standard toxicity studies.     

The Incidence of Lymphoplasmacytic Gastritis in the Fundus and Antrum of Cynomolgus Monkey ( Macaca fascicularis ) Stomachs

Lymphoplasmacytic gastritis is a concern for toxicological pathologists reading preclinical, non-human primate toxicity studies because subtle gastric changes which could be treatment-related may be masked and gastritis lesions may be confused with treatment-related effects and thus a gastric finding may be incorrectly assigned as a treatment-related lesion. This paper discusses the incidence of lymphoplasmacytic gastritis in cynomolgus monkeys at a contract research organization. The incidence of lymphoplasmacytic gastritis in the fundus and antrum of control cynomolgus monkeys on 18 non-gastric compound studies, was scored. The average fundus score ranged from 0.3 to 1.5 and the average antral score ranged from 0.9 to 3.5 in the cynomolgus monkey stomachs examined. The number of affected control animals in a study ranged from 0 to 5 control animals. No correlation between the route of vehicle administration and the severity or incidence of the lesions was noted. The percentage incidence of affected animals ranged from 0 to 100%. An increased incidence lymphoplasmatic gastritis from 2000 to 2004 was noted. The implications of lymphoplasmacytic gastritis in cynomolgus monkeys used for acute toxicity studies are discussed.     

Uterine infarctions in cynomolgus monkeys (Macaca fascicularis)

Uterine infarctions have not been reported in domestic animals, and there are few reports in the human medical literature. In a retrospective study, uterine infarctions were identified in 9 of 323 (2.8%) female cynomolgus monkeys (Macaca fascicularis) necropsied over a 13-year period. The infarctions were grossly visible, after fixation, on the serosal surface of the uterus in 2 monkeys; the remainder were first recognized in histologic sections. Histologically, the lesions consisted of well-demarcated regions of endometrial and myometrial necrosis and of hemorrhage. All affected monkeys had histologic evidence of a previous pregnancy, which included enlarged myometrial vessels with an expanded perivascular matrix. In all monkeys with uterine infarctions, there was clinical evidence of severe systemic illness, which included trauma, diarrhea, hypovolemia, or septicemia. The major pathologic findings in affected monkeys included cutaneous or skeletal muscle necrosis (n = 5), enterocolitis (n = 4), pulmonary edema or diffuse alveolar damage (n = 3), and intestinal amyloidosis (n = 1). Histopathologic evidence of intravascular fibrin thrombi in multiple organs of 5 monkeys was consistent with a diagnosis of disseminated intravascular coagulopathy (DIC). Based on these findings, it appears that uterine infarction is an uncommon finding in cynomolgus monkeys and may occur secondary to a severe systemic illness, predisposing to DIC.     

Two cases of spontaneous pseudohermaphroditism in Cynomolgus monkeys (Macaca fascicularis)

Disorders of genital development occur in all mammals. Hermaphroditism is a condition where the subject has genital organs of both sexes. True hermaphrodites have both ovarian and testicular tissue. Pseudohermaphrodites have only one type of gonadal tissue according to which they are classified as male or female pseudohermaphrodites. Two cases of spontaneously occurring pseudohermaphroditism in Cynomolgus monkeys (Macaca fascicularis) were seen here during 1999-2001. Both animals had female external genitalia, but each was found to have testicular tissue.     

Malaria in cynomolgus monkeys used in toxicity studies in Japan

Plasmodium spp. protozoa cause malaria and are known to infect humans and a variety of animal species including macaque monkeys. Here we report both our experience with malaria recrudescence in cynomolgus monkeys (Macaca fascicularis) in a toxicity study and the results of a survey on Plasmodium infection in cynomolgus monkeys imported to Japan for laboratory use. A cynomolgus monkey from the toxicity study presented with severe anemia and Plasmodium protozoa in erythrocytes on a thin blood smear and was subsequently diagnosed with symptomatic malaria. In this animal, congestion and accumulation of hemozoin (malaria pigment) in macrophages were noted in the enlarged and darkly discolored spleen. As a follow-up for the experience, spleen sections from 800 cynomolgus monkeys in toxicity studies conducted between 2003 and 2013 were retrospectively examined for hemozoin deposition as a marker of Plasmodium infection. The origin of the animals included Cambodia, China, Indonesia, and Vietnam. Hemozoin deposition was confirmed in 44% of all examined monkeys. Monkeys from Indonesia showed the highest incidence of hemozoin deposition (approx. 80%). A high prevalence of Plasmodium infection in laboratory monkeys was also confirmed with polymerase chain reaction (PCR) by using Plasmodium genus-specific primers. Although Japan is not a country with endemic malaria, it is important to be aware of the prevalence and potential impact of background infection with Plasmodium spp. and recrudescence of symptomatic malaria in imported laboratory monkeys on pharmaceutical toxicity studies.     

Microfilarial granulomas in the spleens of wild-caught cynomolgus monkeys (Macaca fascicularis)

Splenic nodules from 38 cynomolgus monkeys (Macaca fascicularis) which were captured in Malaysia and Indonesia were studied histologically. The lesions were characterized by well-circumscribed focal fibrosis, accumulation of eosinophils and histiocytes, hemorrhage or hemosiderosis, and loss of normal splenic architecture. Small arteries in the lesion frequently had intimal thickening and narrowing of the lumen in addition to the presence of microfilariae. Microfilariae were also seen in the extravascular area of the lesion, and were occasionally engulfed by multinucleated giant cells. The splenic lesion was thought to have been initiated by incomplete infarction caused by intimal thickening and microfilarial occupation of the small arteries.     

Hyperplastic and neoplastic lesions of the mammary gland in macaques

Macaques provide an important animal model for the study of hormonal agents and their effects on risk biomarkers for breast cancer. A common criticism of this model is that spontaneous breast cancer has rarely been described in these animals. In this report, we characterize 35 mammary gland lesions ranging from ductal hyperplasia to carcinoma in situ and invasive ductal carcinoma in cynomolgus and rhesus macaques. Based on a retrospective analysis, we estimated the lifetime incidence of mammary gland neoplasia in aged female macaques to be about 6%. Hyperplastic lesions (n = 19) occurred segmentally along ducts and included such features as columnar alteration, micropapillary atypia, and fibroadenomatous change. In situ carcinomas (n = 8) included solid, comedo, cribriform, and micropapillary elements, encompassing 4 of the major architectural patterns seen in human lesions. Invasive ductal carcinomas (n = 8) were generally solid, with prominent central necrosis and mineralization, often on a background of micropapillary ductal hyperplasia and in situ carcinoma. Cytologic changes of invasive lesions included increased mitoses, nuclear pleomorphism, extensive microinvasion, and stromal desmoplasia. Axillary lymph-node metastases were confirmed in 5 of the 8 invasive carcinomas. On immunohistochemistry, intraductal and invasive carcinomas had increased Ki67/MIB1 and HER2 expression and selective loss of estrogen and progesterone receptors. These findings suggest that breast cancer is an underreported lesion in macaques and highlight unique morphologic and molecular similarities in breast cancer between human and macaque species.     

A case of spontaneous autoimmune skin disease in a cynomolgus monkey (Macaca fascicularis)

Pemphigus is an autoimmune blistering disease characterized by lesions on the skin and mucous membranes. To date, no spontaneous cases of this disease have been reported in cynomolgus monkeys. This report describes the histopathological characteristics of spontaneous pemphigus in a cynomolgus monkey. Macroscopically, redness and scaling with pruritus were observed on the skin of the entire body. Histopathologically, the epidermis showed intercellular edema, and eosinophils and mononuclear cells infiltrated the epidermis. There was no obvious acantholysis in the epidermis. The perivascular area showed edema, and eosinophils and mononuclear cells infiltrated the vessels in the dermis. Immunohistochemically, the intercellular area in the epidermis was positive for Immunoglobulin G and Complement component 3. Serologically, anti-desmoglein 1 and desmoglein 3 antibodies in the serum were negative. From these findings, this case was diagnosed as an autoimmune skin disease, suspected to be pemphigus, and concluded as lesions being similar to those in human “pemphigus herpetiformis”.     

Collagenofibrotic glomerulonephropathy in a cynomolgus macaque (Macaca fascicularis)

Collagenofibrotic glomerulonephropathy (CFGN) is characterized by the deposition of type III collagen within the mesangial matrix and the absence of mesangial cell proliferation. A case of CFGN in a 2.7-year-old female cynomolgus macaque was investigated in the present study. Clinically, the animal was shown to have severe systemic edema along with hypoproteinemia. At necropsy, the kidneys were swollen and pale. The glomerular lesions were characterized by massive diffuse and global accumulation of fibrous materials in the mesangial areas. Neither mesangial cell proliferation nor changes in other organs were found. The fibrous materials were confirmed by the results of immunohistochemical and electron microscopic findings to consist mainly of randomly arranged, curve-shaped, twisted, and entwined type III collagen. This is the first case report of CFGN in nonhuman primates to date.     

Pathology of human influenza A (H5N1) virus infection in cynomolgus macaques (Macaca fascicularis)

Infection with influenza A (H5N1) virus, which has not been associated with respiratory disease in humans previously, caused clinical signs of acute respiratory distress syndrome and multiple-organ dysfunction syndrome with high mortality in humans in Hong Kong in 1997. To study the pathogenesis of this disease, we infected four cynomolgus monkeys (Macaca fascicularis) with 2.5 x 104 median tissue culture infectious dose (TCID50) of influenza virus A/Hong Kong/156/97 (H5N1) and euthanatized them 4 or 7 days after infection. The main lesion was a necrotizing broncho-interstitial pneumonia (4/4) similar to those found in primary influenza virus pneumonia in humans, with desquamation of respiratory epithelium (4/4), intra-alveolar hemorrhage (4/4), hyaline membrane formation (2/4), and infiltration with neutrophils and macrophages (4/4). Lesions in other organs consisted of a suppurative tonsillitis (2/4) and necrosis in lymphoid organs (1/4), kidney (1/4), and liver (1/4). By immunohistochemistry, influenza virus antigen was limited to pulmonary tissue (4/4) and tonsils (2/4). Based on these results, we suggest that the cynomolgus monkey is a suitable animal model for studying the pathogenesis of human H5N1 virus infection and that multiple-organ dysfunction syndrome in this disease may be caused by diffuse alveolar damage from virus replication in the lungs alone.     

Cryptosporidium muris-like infection in stomach of cynomolgus monkeys (Macaca fascicularis)

Abstract. Protozoa were present in routine sections of the gastric fundus of 15 cynomolgus monkeys (Macaca fascicularis) that were being studied in three toxicity studies with novel immunosuppressive agents. Upon detailed light microscopic and ultrastructural evaluation, all stages of parasite development (trophozoites, schizonts, gamonts, and oocysts) were seen and they structurally resembled Cryptosporidium muris, which normally is found in stomachs of rodents. Cryptosporidia were primarily present in the upper one third of fundic glands that were often concurrently colonized by a Helicobacter heilmannii-like organism; however, no clear correlation was found between bacterial burden and the number of protozoa. The primarily mononuclear cellular infiltrate appeared to coincide with the presence of protozoa only in a few animals. Changes in mucous epithelial cells mainly occurred in animals that were part of a 39-week study. Mucous epithelial cells in affected glands contained an increased amount of mucus composed of predominantly acid mucosubstances compared to the normally present neutral mucosubstances. C. muris-like protozoa are newly recognized etiologies for opportunistic infections in the stomach of immunocompromized nonhuman primates. This is the first report of C. muris-like parasite in stomachs of monkeys.     

Thirteen-week Intravenous Toxicity Study of a Novel Humanized Anti-Human Death Receptor 5 Monoclonal Antibody, CS-1008, in Cynomolgus Monkeys

CS-1008, a humanized monoclonal antibody that is agonistic to human death receptor 5, was intravenously administered to cynomolgus monkeys twice a week for 13 weeks at 3 different dose levels (5, 15 and 42 mg/kg) in order to evaluate its potential toxicity. A control group received phosphate buffered saline containing 0.01% polysorbate 80. Each of the 4 groups consisted of 3 male and 3 female cynomolgus monkeys. No animal in any group died during the dosing period. No toxic changes in clinical signs, food consumption, body weight, electrocardiography, ophthalmology, urinalysis, hematology, blood chemistry, gross pathology, organ weights or histopathology were noted in any group during the dosing period. In the toxicokinetic analysis, the values for the maximum concentration of CS-1008 in plasma and the area under the curve generally increased with increasing dose. No clear differences in the toxicokinetic parameters or profiles were observed between the sexes. Development of anti-CS-1008 antibodies was not detected in any sample. The no-observed adverse-effect level (NOAEL) of CS-1008 in cynomolgus monkeys under the conditions of this study was concluded to be 42 mg/kg in both sexes, when administered intravenously twice a week for 13 weeks. This study supports the development of CS-1008 as a therapeutic biopharmaceutical.     

Obstructive protein cast nephropathy in cynomolgus monkeys treated with small organic molecules

We have observed a renal toxicity consistent with an obstructive protein cast nephropathy in cynomolgus macaques but not in other species treated with different therapeutic candidates having a common carboxylic acid moiety, suggesting a species-specific sensitivity. Here, we present renal toxicity findings consistent with a protein cast nephropathy in a 2-week safety study in cynomolgus monkeys. Light microscopic changes consisted of intratubular cast formation, tubular dilatation, interstitial inflammation, and expansion of the medullary interstitium. Tubular cast material was identified as Tamm-Horsfall protein (THP) and, on ultrastructure, crystalloid material was present in vacuoles of tubular epithelium. It is hypothesized that microcrystal formation in the urinary tubular spaces induces aggregation of THP protein and cast formation in monkeys. Drug-induced obstructive nephropathy is not identified as a major problem in humans; thus, the clinical relevance of the above findings in monkeys is not clear.     

Disseminated oesophagostomiasis in the rhesus monkey.

Disseminated oesophagostomiasis was diagnosed at necropsy of 5 rhesus monkeys (Macaca mulatta). Nodular lesions were in visceral organs as well as in the colon and mesentery. Lesions varied greatly with respect to degree of organ involvement and damage due to parasite invasion. In 2 monkeys, nodular lesions were in the colon, mesentery, liver, kidneys, abdominal wall, urinary bladder, and uterus; in 1 of these 2 monkeys, a nodular lesion inhabited by a parasite was in a lung. The ectopic nodules in the less severely affected monkeys were scattered in the liver, kidneys, and abdominal wall.     

Lentivirus-induced pulmonary lesions in rhesus monkeys (Macaca mulatta) infected with simian immunodeficiency virus.

Necropsy reports from 28 rhesus monkeys that had been experimentally infected with simian immunodeficiency virus (SIV) and that were free of cytomegalovirus were reviewed. Lung sections from 24 of these monkeys that had no etiologic agent other than SIV detected in the lung were studied in detail by histopathologic, immunohistochemical, and electron microscopic examination and by in situ hybridization. Fourteen of the monkeys were part of a serial euthanasia study, while others were euthanatized after they became moribund. The following lesions were detected: perivascular inflammation, vasculitis, interstitial pneumonia, syncytial cells, hemorrhage, fibrin exudation, and pleural fibrosis. Perivascular inflammation was the most frequent lesion and occurred as early as 2 weeks after inoculation. Severe pneumonia and numerous syncytial cells were seen only in animals euthanatized because they had become moribund. The lesions appeared to be directly due to SIV infection. SIV antigens, RNA, and virions were detected in syncytial cells and macrophages by immunohistochemical examination, in situ hybridization, and transmission electron microscopic examination, respectively. The amount of virus present was correlated with the severity of the lesions. The SIV-induced lesions were different from those of the lymphocytic interstitial pneumonia, which occurs in human immunodeficiency virus-infected children and in ovine lentivirus-infected sheep and goats.     

Cyclooxygenase-2 expression in inflammatory lung lesions of nonhuman primates

Mammalian cells contain two related but unique isoforms of cyclooxygenase (COX-1 and COX-2). COX-1 is expressed constitutively in a majority of tissues and is involved in the production of prostaglandins (PGs) that modulate normal physiologic functions. COX-2 is inducible by various stimuli and is involved in the production of PGs that modulate physiologic events in development, cell growth, and inflammation. With the exception of peribronchial glands and chondrocytes of peribronchial cartilage, COX-2 is not detectable in the normal lung of nonhuman primates. We evaluated COX-2 expression by immunohistochemical methods in the inflammatory lesions of two cynomolgus monkeys (Macaca fascicularis) with acute severe pneumonia. Both monkeys exhibited acute severe bronchopneumonia; histologically, lung lesions were characterized by infiltration of large numbers of neutrophils and fewer macrophages, mild bronchial epithelial hyperplasia, and slight type-2 pneumocyte hyperplasia. In both monkeys, mild to marked COX-2 immunoreactivity was detected within the cytoplasm of macrophages, bronchial epithelial cells, type-2 pneumocytes, and endothelial cells of blood vessels. No COX-2 immunoreactivity was detectable in the neutrophils that constituted >90% of the inflammatory cells. These observations suggest that in acute inflammatory lung lesions in nonhuman primates 1) COX-2 is induced in the bronchial and alveolar epithelial cells, 2) macrophages are the primary inflammatory cells that exhibit COX-2, and 3) neutrophils do not express COX-2.     

Pathology of Mycoplasma agalactiae induced granular vulvovaginitis (GVV) in goats.

Granular vulvovaginitis (GVV) was experimentally produced in female kids by topical application of M. agalactiae on the scarified vulvar mucosa. Grossly visible GVV lesions were seen in 25 out of 30 infected kids, yet all were positive for microscopic lesions. Microscope lesions that appeared by 7 days post infection (DPI) were comprised of stromal oedema, lymphocytic infiltration into the lamina propria and perivascular accumulation of a few lymphocytes. The lesions observed between 28 and 49 DPI were comparable to those of spontaneous cases (severe). The changes seen in kids euthanized between 56 to 70 DPI were suggestive of the chronic stage of the disease. M. agalactiae was reisolated from all the infected kids from 7 to 70 days after inoculation. The pathology and pathogenesis of this condition have been described and discussed.     

Measles in the cynomolgus monkey (Macaca fascicularis)

The measles virus is known to infect several species of monkeys. A group of 87 cynomolgus monkeys (Macaca fascicularis) was screened to observe whether there was an association between measles and the cold symptoms seen in most of the animals. Another 23 monkeys were vaccinated with attenuated measles vaccine and their antibody titres monitored to ascertain whether the vaccine would protect them against measles.     

Genital lesions in an outbreak of caprine contagious agalactia caused by Mycoplasma agalactiae and Mycoplasma putrefaciens

This paper reports on the genital lesions observed in adult male and female goats from a commercial flock in the Extremadura region of southwestern Spain, following an outbreak of contagious agalactia syndrome caused by Mycoplasma agalactiae and M. putrefaciens. Although both species were isolated from several organs, M. putrefaciens was the only agent isolated from the genital lesions reported here, characterized by desquamative salpingitis and cystic catarrhal metritis in females and by testicular degeneration in males. Mycoplasma putrefaciens was isolated from the testes of only one of the males examined.