Pathologic characteristics of infectious diseases in macaque monkeys used in biomedical and toxicologic studies

Abstract

Nonhuman primates (NHPs), which have many advantages in scientific research and are often the only relevant animals to use in assessing the safety profiles and biological or pharmacological effects of drug candidates, including biologics. In scientific or developmental experiments, the immune systems of animals can be spontaneously compromised possibly due to background infection, experimental procedure-associated stress, poor physical condition, or intended or unintended mechanisms of action of test articles. Under these circumstances, background, incidental, or opportunistic infections can seriously can significantly complicate the interpretation of research results and findings and consequently affect experimental conclusions. Pathologists and toxicologists must understand the clinical manifestations and pathologic features of infectious diseases and the effects of these diseases on animal physiology and experimental results in addition to the spectrum of infectious diseases in healthy NHP colonies. This review provides an overview of the clinical and pathologic characteristics of common viral, bacterial, fungal, and parasitic infectious diseases in NHPs, especially macaque monkeys, as well as methods for definitive diagnosis of these diseases. Opportunistic infections that can occur in the laboratory setting have also been addressed in this review with examples of cases of infection disease manifestation that was observed or influenced during safety assessment studies or under experimental conditions.

Introduction

Nonhuman primates (NHPs) have important applications in scientific research owing to their genetic similarities to humans¹. The safety profiles and biological or pharmacological effects of drug candidates, including biologics, can be often evaluated only in NHPs. Hence, the demand for NHPs in drug development research^{1, 2} and scientific research in the fields of biology, medicine, and pharmaceutics is increasing in the United States of America (USA), the European Union, and Asia^{3, 4, 5}.

Cynomolgus (Macaca fascicularis) and rhesus (Macaca mulatta) macaques are the most commonly used NHPs in scientific research⁵. Macaques must be transferred and maintained in the laboratory setting for applications in biomedical and toxicological research. In Japan, monkeys are allowed to be imported only from the USA, the People’s Republic of China, the Republic of Indonesia, the Republic of the Philippines, the Socialist Republic of Viet Nam, the Republic of Suriname, the Co-operative Republic of Guyana, and the Kingdom of Cambodia according to the Regulations Concerning the Importation of Animals That May Convey Pathogens of Infectious Diseases under Regulations Concerning the Importation of Animals That May Convey Pathogens of Infectious Diseases (Order of MHLW and MAFF No. 2 of 1999). Monkeys are subject to import and export regulations under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES, commonly known as the Washington Convention, Ministry of Economy, Trade, and Industry), while macaque monkeys are subject to import regulations under the Law Concerning the Prevention of Damage to Ecosystems from Specified Invasive Alien Species (commonly known as the Invasive Alien Species Act, Ministry of the Environment). Therefore, additional prior procedures/permissions are required for importing monkeys into Japan.

The spread of Ebola hemorrhagic fever-causing and Marburg fever-causing viruses must be strictly prevented by quarantining monkeys based on Article 55 of the Act on Prevention of Infectious Diseases and Medical Care for Infectious Patients (Act No. 114 of 1998, as amended). Additionally, each facility strives to prevent unexpected outbreaks of infections by performing various tests during the quarantine and acclimatization periods.

Although animal care and handling procedures have markedly progressed, the immune system of animals can be spontaneously compromised due to background infection and experimental condition-induced stress, poor physical condition, or the intended or unintended mechanism of the test articles^{6, 7}. For example, simian retrovirus (SRV) and measles affect both humoral and cellular immune systems^{8, 9}, while simian immunodeficiency virus (SIV) compromises cellular immunity^{10, 11, 12} in infected macaques. Background, incidental, or opportunistic infections can seriously affect the interpretation of the experimental results. To prevent the effects of infectious diseases on experimental results, specific pathogen-free colonies of NHPs can be used. However, eliminating infectious diseases in NHP colonies is challenging when compared with that in several other laboratory animal species. This is because macaques need parental and social interactions for physiological development and additionally considerable numbers of NHP colonies are raised outdoors^{13, 14}. Furthermore, the elimination of all infectious agents, including those that are not pathogenic in healthy monkeys, is not ideal because the recurrence of infectious diseases may provide good evidence for assessing or revealing the safety profiles of therapeutic drugs, biomedical agents, or experimental procedures. Therefore, it is important for pathologists to understand the spectrum of infectious diseases in normal NHP colonies, as well as the clinical, gross pathologic, and histopathologic characteristics of these diseases and their effects on physiology and experimental outcomes^{14, 15}.

This review provides an overview of the clinical and pathologic characteristics of known or common viral, bacterial, parasitic, and fungal infectious diseases in NHPs (Table 1) (represented by macaque monkeys in this study), as well as diagnostic approaches to identify these diseases. Additionally, potential opportunistic infections in laboratory settings have been discussed (Table 1). Furthermore, some examples of cases of infectious disease manifestation that were observed or influenced during safety assessment studies or under experimental conditions are included.

Table 1. Viral, Bacterial, Fungal, and Parasitic Infectious Agents That Can Affect Studies in Macaques.

Viral Infection

Simian retrovirus (SRV): synonymous names: simian betaretrovirus, Type D (SRV/D) retrovirus (Serotype: SRV1–5, 7 and SRV-T are reported in Asian macaques)¹⁶

Overview. The prevalence of seropositivity and/or virus positivity in macaques is known to be relatively high but varies depending on the efforts of the facility. In a facility in Tsukuba, Japan, the prevalence of SRV infection in cynomolgus macaques was 95% in 2010¹⁷. SRV can be transmitted via contact with saliva, urine, and/or feces of infected individuals as determined during the spread of SRV/D-Tsukuba infection within the facility¹⁸. SRV is one of the causes of simian acquired immune deficiency (SAIDS), which affects both humoral and cellular immune functions^{8, 19}. The hematocrit, white blood cell, lymphocyte, and neutrophil values in seropositive or viremic animals are lower than those in normal animals, However, changes that fit the definition of SAIDS are not commonly found in those seropositive and viremic animals²⁰. In one study, only one of the 24 experimentally infected cynomolgus macaques exhibited clinical signs consistent with SAIDS²⁰. A CD8⁺ T-cell-depleted rhesus monkey transfused with whole blood from a monkey positive for SRV1 antibody and polymerase chain reaction (PCR)-positive for viremia did not exhibit clinical or marked hematological or pathologic changes indicative of SRV-related SAIDS within a few months²¹. SRV-4 and SRV-5 are reported to infect Japanese macaques with different pathogenicity^{231, 232, 237, 238}.

Clinical and anatomical pathologic features. Clinical signs in cynomolgus monkeys include nonspecific anemia, weight loss, and diarrhea. The hallmark of SRV-induced SAIDS is the reactivation of opportunistic infections. Various bacterial infections have been reported in SRV-infected macaques²² in addition to cytomegalovirus (CMV) infection, oral and esophageal candidiasis, and intestinal cryptosporidiosis, which are relatively common in macaques experimentally infected with the SIV^{16, 22, 24}. Histological changes are characterized by increased nonspecific infection and increased incidence or severity of focal lymphoid infiltration (predominantly in the kidney, pancreas, salivary glands, bone marrow, and brain) with occasional germinal center formation, abscess, arthritis, or myositis along with lymphoid hyperplasia in the spleen and splenomegaly due to reactions in the germinal centers and paracortex of the lymph nodes^{8, 18, 20}. Lymphoid depletion due to poor conditions associated with the infection may be observed. Some macaques infected with SRV exhibit proliferative lesions of retroperitoneal fibromatosis, which is characterized by the formation of masses with infiltrative growth of fibroblastic cells with collagen fibers^{8, 23}. When infected animals exhibit reactivation of opportunistic infections due to SAIDS, the pathologic features discussed in the sections on individual opportunistic infections, including CMV, Candida, and Cryptosporidium infections, should be referred. In Japanese macaques, SRV-4 or SRV-5 infection is reported to induce related severe thrombocytopenia^{231, 232, 237}.

Diagnosis. In addition to serological examinations (including commercially available enzyme-linked immunosorbent assay (ELISA) kits), PCR for proviruses, reverse transcription (RT)-PCR, immunohistochemistry (IHC), and in situ hybridization (ISH) methods can be used to identify SRV infection^{18, 231}. Some detection methods may not exhibit effective performance due to the stage of infection²³¹. Hence, a combination of these procedures may be useful. IHC and ISH analyses reveal the viral antigen and genome in the ductal epithelium located between the acinar epithelium of the salivary gland¹⁸.

Simian immunodeficiency virus (SIV) [No natural infection reported in Asian macaques]

Overview. Large proportions of African primates, which are natural hosts of SIV, are serologically positive for SIV. However, clinical disease, which is characterized by the loss of CD4⁺ T cells and immunosuppression, is not observed in these monkeys. In cases of cross-species transmission to Asian macaques, SIV infection can decrease the CD4⁺ T-cell count and compromise the immune system, which are similar to the effects of human immunodeficiency virus (HIV) and to associated acquired immunodeficiency syndrome (AIDS). SIV is not endemic to Asian macaques, and natural cross-species transmission of SIV from African NHPs to Asian macaques is rare^{16, 24}. Hence, the probability of SIV infection in Asian laboratory macaques is low. Rhesus monkeys experimentally infected with SIV are frequently used as a model of AIDS. SIV infection in cynomolgus monkeys is reported to be less pathogenic when compared with that in Indian rhesus monkeys²³³.

Clinical and anatomical pathologic features. The SIV-specific pathologic features include selective downregulation of CD4⁺ lymphocyte counts, lymphoma^{10, 16}, and the occurrence of opportunistic infections, including manifestations of CMV infection, oral and esophageal candidiasis, and intestinal cryptosporidiosis^{16, 24}. For the pathologic features of opportunistic infections, refer to the sections below for each infectious agent. Nonspecific findings that are potentially associated with the manifestations of immune deficiency include decreased body weight, diarrhea, generalized lymphadenopathy, and splenomegaly.

Diagnosis. Antibody responses are induced within weeks of SIV infection. SIV genomes can be detected in peripheral blood CD4⁺ cells, sometimes within days of infection^{25, 26}. A combination of serological and molecular assays can be used to eliminate the risk of SIV-related undesirable immune compromise from colonies used in scientific studies.

Measles virus

Overview. Humans are the only natural hosts that sustain measles virus transmission²⁷. Most NHP species are susceptible to measles infections from their infected human handlers²⁸. Measles belongs to the family Paramyxoviridae.

Clinical and anatomical pathologic features. The clinical, gross, and histopathologic hallmark of measles in macaques is maculopapular skin rash, which is histologically characterized by multinucleated giant cells in the epidermis and hair follicles with mild inflammation. Although measles is associated with lymphocytopenia, the depletion of peripheral lymphocytes, which as a cause of immune suppression, is often undetectable because lymphocyte counts rapidly return to control levels after virus clearance. Therefore, measles-induced lymphocytopenia may be missed. However, the suppression of humoral and cellular immune functions can last several weeks to months, resulting in mortality predominantly due to secondary infections in the respiratory and digestive tracts⁹. In the lung, the lesions range from mild bronchiolar pneumonia to severe interstitial pneumonia; These changes are associated with the presence of multinucleated giant cells and minimal alveolar edema. Giant cells and intranuclear and cytoplasmic inclusion bodies can also be observed in reticular or phagocytic cells of the lymphoid system and in epithelial cells of the respiratory system, gastrointestinal tract, salivary glands, thyroid gland, liver, pancreas, and urinary system^{29, 239}. Representative eosinophilic intranuclear and cytoplasmic inclusion bodies of measles in the infected bronchial epithelium are shown in Fig. 1a and 1b.

Fig. 1.

Measles virus. (a) Eosinophilic intranuclear inclusion bodies in the measles-infected cells surrounded by inflammatory cells, including neutrophils. (b) Eosinophilic cytoplasmic inclusion bodies in the infected bronchiolar epithelium. Photographs are kindly provided by Dr. Shinichiro Nakamura, Azabu University.

Diagnosis. In addition to commercially available detection kits for measles-specific IgM and IgG³⁰, RT-PCR can also be used for measles virus detection^{31, 32}.

Herpes B virus (HBV, Cercopithecine herpesvirus 1)

Overview. Most naturally captured Asian macaques are seropositive for HBV. HBV infection is latent and is not associated with clinical signs¹⁶. In humans, HBV infection is rare but has been sporadically reported³³. HBV infection can be fatal in humans³⁴. To avoid fatal infection from handlers and potential effects on the experiments, HBV seronegative animals are usually used³⁵. However, to ensure handler protection, we should be aware that serological testing for antibodies or PCR testing for the virus usually shows negative results in latently infected animals without viremia. HBV infection in macaques is persistent and remains latent in the trigeminal and spinal ganglia. Immunosuppressive conditions, such as under stress can reactivate the infection, leading to the shedding or transmission of virus with increased antibody titers^{33, 36, 37}.

Clinical and anatomical pathologic features. In macaques, HBV infection is clinically and pathologically characterized by small to large (sometimes erosive) vesicles on the oral and genital mucosa together with a multifocal necrotizing hepatitis with multinucleated syncytial cells and intranuclear inclusion bodies in the epithelial cells of affected organs and tissues^{16, 37, 38}.

Diagnosis. Serological examination to diagnose HBV infection is challenging owing to the close genetic relationship between herpes simplex virus and HBV and antibodies in the serum can cross react^{39, 40}. PCR detection of HBV-specific DNA or viral isolation is used for diagnosis^{39, 40, 41, 42}.

Simian T-cell leukemia viruses (STLVs)

Overview. STLVs can infect Asian macaques^{16, 43} although the seroprevalence of STLVs is rare in macaques maintained under laboratory settings³⁵. Clinical signs are usually not apparent even in cases of STLV infection is confirmed¹⁶.

Clinical and anatomical pathologic features. Characteristic pathologic changes include lymphoproliferative lesions with changes in cytokine profiles, which are most apparent in baboons (Papio spp.)¹⁶ and the African green monkey (Chlorocebus sabaeus)⁴⁴. The common findings associated with these lymphomas in NHPs are decreased bodyweight, weakness, lethargy, dyspnea with pneumonia, leukemia with or without multilobulated lymphocytes in the peripheral blood, generalized lymphadenopathy, hepatosplenomegaly, and nodular skin lesions with diffuse infiltration of neoplastic lymphocytes^{44, 45, 46} and multilobulated lymphocytes⁴³.

Diagnosis. Antibodies against STLV-1 antigens can be detected in the peripheral blood of animals with suspected infections. The lymph node section containing proliferative lesions can be subjected to IHC to demonstrate the neoplastic increase in T-cell lineages²⁵.

Cytomegalovirus (CMV): Betaherpesvirinae Macacine herpesviruses 3 (rhesus CMV) and 8 (cynomolgus CMV)

Overview. CMV infects captive rhesus and cynomolgus macaques without clinical signs and seroprevalence of CMV is high (more than 90% in all populations tested and almost 100% in adults^{16, 28, 35, 47, 48}). Latent CMV activation is one of the most commonly observed opportunistic infections in immunocompromised macaques, which can be attributed to the high prevalence of CMV (similar to that in humans)⁴⁹. CMV is a highly species-specific virus owing to its long evolutionary history with its host. Therefore, cross-species infection is restricted. Additionally, cross-species infection is rare in primates even under experimental conditions^{50, 51}. In cases of CMV activation in monkeys immunosuppressed for tissue transplantation, CMV DNA has been detected in animals with the white blood cell (WBC) counts decreasing to less than 4,500/μL and the lymphocyte counts decreasing to less than 1,800/μL⁵². However, in our experience, a clear decrease in WBC parameters has not been detected before the manifestation of CMV-related clinical signs. Thus, functional alterations in the immune system without marked changes in peripheral WBC counts can also cause CMV reactivation.

Clinical and anatomical pathologic features. In immunosuppressed animals, CMV infection most commonly affects the lungs. Inflammation of the brain, gastrointestinal tract, reproductive organs, and lymphoid system can also be observed^{16, 28}. In our experience with opportunistic systemic CMV infection accompanied by clinical manifestations, a severely affected animal exhibits decreased food consumption and decreased activity. At necropsy there was pulmonary effusion with diffuse dark red discoloration of all lung lobes. In the lung, diffuse infiltration of inflammatory cells (predominantly foamy macrophages), serous or fibrinous exudate, and hemorrhage (Fig. 2a) were histologically observed. Enlargement of alveolar epithelial cells and vascular endothelial cells were also observed. These cells frequently contained intracytoplasmic and intranuclear inclusion bodies with or without clear hollows (owl’s eyes) (Fig. 2b and 2c). Perivascular edema and inflammatory cell infiltration with hemorrhagic necrosis of blood vessels along with hypertrophy of alveolar epithelial cells, and syncytial cells were also observed. Lymphoid depletion in the bronchiole-associated lymphoid tissue, various lymph nodes with foamy macrophages with intranuclear inclusions, gastric ulceration and inflammation, and inclusion bodies in the systemic vascular endothelium are also frequently observed.

Fig. 2.

Cytomegalovirus (CMV). (a) Diffuse interstitial pneumonia with hemorrhage and fibrinous exudate in the alveoli were observed in the lung of CMV-infected cynomolgus macaque. (b), (c) Intranuclear inclusion bodies with characteristics of CMV (so-called owl’s eyes, with a distinct halo around the inclusion bodies) were found mainly in the enlarged endothelial cells of small vessels and macrophages in the alveoli (bars=50 μm).

Diagnosis. Electron microscopy revealed the presence of intracytoplasmic virus particles with envelope and core characteristics of herpes virus. Immunohistochemical staining with commercially available antibodies against human CMV did not work well in our experience. CMV DNA can be quantified in peripheral blood using real-time PCR with the primers and probes described by Han et al.⁵² and Kaur et al⁵³.

Lymphocryptovirus (LCV): Gammaherpesvirinae, Macacine herpesvirus 4 (rhesus LCV), Epstein-Barr virus (EBV)

Overview. LCV is a genus of viruses in the order Herpesvirales, in the family Herpesviridae, in the subfamily Gammaherpesvirinae which includes Human gammaherpesvirus 4 (EBV). EBV infection, which is usually asymptomatic but may be symptomatic in humans infected after puberty, is associated with lymphoproliferative lesions in immunocompromised patients. The seroprevalence of LCV in cynomolgus and rhesus macaques is high: almost 100% of adult monkeys in the laboratory are positive for LCV (or EBV)^{54, 55, 56}. Similar to EBV infection in humans, primary LCV infection in macaque monkeys is not associated with clinical signs. The clinical and histopathologic changes are reportedly limited to macaques immunocompromised by immunosuppressive viral infection, immunosuppression for tissue transplantation, or treatment with immune-modulating drugs^{57, 58, 59, 60}. LCV has been detected in most malignant lymphomas (non-Hodgkin lymphomas) in macaques infected with SIV⁶¹. One case report on cynomolgus monkeys demonstrated LCM-related rapidly progressive B-cell lymphoma without marked evidence of immunosuppression in a 3-month toxicity study²⁴². In this case, close evaluation using immunophenotyping revealed a low natural killer (NK) cell count before the initiation of the study.

Clinical and anatomical pathologic features. Enlarged lymph nodes were detected as masses in rhesus or cynomolgus monkeys experimentally inoculated with SIV and LCV. The histologic features of these monkeys involve the infiltration of immunoblastic cells, predominantly comprising CD20-positive B cells, and decreased numbers of CD8⁺ T cells⁵⁸. In Japanese monkeys, lymphomas derived from other lymphocytes, including CD4+ T cells and NK cells, have also been reported^{240, 241}. Infected animals with immunosuppression for tissue transplantation can exhibit lymphadenopathy along with lymphoid infiltration of non-lymphoid organs, including the liver, lungs, heart, and kidney⁶². In a monkey treated with immunomodulatory biologics, enlargement of lymph nodes was clinically noted at week 28 of treatment. In a case of B-cell lymphoma observed in a 3-month toxicity study, which was characterized by large unclassified cells with lymphocytosis, monocytosis, and mild neutrophilia in hematology examination and a high cellularity was noted in a bone marrow smear evaluation that mostly consisted of a polymorphic lymphoid cell population²⁴². Enlargement of the spleen and various lymph nodes observed at necropsy were histologically associated with loss of the normal lymphoid architecture, which was replaced by neoplastic lymphocytes; the diagnosis was lymphoma⁵⁹. EBV-associated (LCV-associated) focal proliferative lesions have been found on the squamous epithelium of the tongue, esophagus, or penis, or on the haired skin of the lip, hand, or thorax of experimentally SIV-infected and immunocompromised monkeys. The skin lesions consisted of hyperkeratosis, parakeratosis, and acanthosis. Ballooning degeneration of keratinocytes was noted in the tongue, esophagus, and penis, with intranuclear inclusion bodies in cells in the middle and superficial layers of the stratified epithelium⁵⁸. The surface of epithelial lesions is frequently accompanied by colonies of Candida species or gram-positive cocci⁶¹.

Diagnosis. By transmission electron microscopy, herpesvirus particles with envelope and core can be found in inclusion-bearing cells are. The virus also can be observed by IHC for LMP-1 (latent membrane protein 1) or EBNA-2 (Epstein-Barr nuclear antigen 2) and ISH for EBV-encoded RNA^{60, 61}. In lymphoproliferative lesions, increased numbers of LCV-infected lymphocytes can be observed using ISH⁵⁹.

Simian adenovirus

Overview. More than 50 adenovirus serotypes have been identified in NHPs, including macaques. and adenovirus is highly species specific^{16, 28}. Generally, simian adenovirus infection is not clinically apparent and can be detected in healthy animals. Infection may target the respiratory and gastrointestinal tracts and conjunctiva (conjunctivitis). The liver, pancreas, and kidney were less frequently affected^{63, 64, 65}. Adenoviral infection in the intestine is relatively common in macaque monkeys⁶⁶. Infected macaques can shed adenoviruses in the stool⁶⁷. Aerosols from sneezes or coughs and feces from animals exhibiting clinical manifestations or latent infections can be sources of viral transmission.

Clinical and anatomical pathologic features. Gastrointestinal tract infections may be associated with diarrhea with no visible gross lesions but with microscopic enteritis, erosion or ulceration, or villus atrophy of the small intestine with intranuclear inclusion bodies^{24, 68, 69, 70}. In the infected respiratory tract, necrosis of epithelial cells of the trachea, bronchi, bronchioles, and alveoli with basophilic intranuclear inclusion bodies, is characteristic and accompanied by neutrophil and macrophage infiltration¹⁶. The intranuclear inclusion bodies of adenovirus are generally large and smudged as reported in cases of pancreatic and renal infections^{64, 65}.

Diagnosis. Antigens of serotypes 40 and 41 in feces can be detected using commercially available Adenoclone 40/41 kits (Meridian Diagnostics, Cincinnati, OH, USA)⁶⁶. PCR can be performed with fecal or tissue samples using generic primate adenovirus^{71, 72}. Meanwhile, the presence of viral structures in tissues with intranuclear inclusion bodies can be confirmed using electron microscopy^{63, 73}.

Simian virus 40 (SV40): Closely related to cynomolgus polyomavirus

Overview. SV40 and cynomolgus polyomaviruses are a family of small, non-enveloped DNA viruses that commonly cause latent infections in Asian monkeys, including rhesus and cynomolgus macaques. They can cause severe disease in situations of immunocompromised conditions^{16, 74}.

Clinical and anatomical pathologic features. Polyomaviruses-related lesions are observed in the central nervous system and kidney, and less frequently in the lung^{16, 28}. The following two types of SV40-related central nervous system lesions have been reported: lesions similar to those in human progressive multifocal leukoencephalopathy (PML) and meningoencephalitis with renal and pulmonary lesions^{74, 75}. Both the PML type and the meningoencephalitis type have the common etiology of latent infection in oligodendrocytes and astrocytes, as detected by ISH⁷⁶. The difference of manifestation can be attributed to the age or duration of infection in animals infected with SV40 before immunosuppression induced by SIV infection. PML type noted in rhesus macaques is characterized by multifocal demyelination, predominantly in the white matter (most commonly near the boundary with the gray matter), with microgliosis, large astrocytes, and oligodendrocytes with swollen, atypical nuclei containing marginated chromatin and intranuclear inclusions⁷⁶. In meningoencephalitis, lesions are observed in the meninges and superficial gray matter and are characterized by inflammation with infiltration of lymphocytes, eosinophils, and macrophages that spread from the perivascular area to the parenchyma. Enlargement of nuclei with smudgy amphophilic intranuclear inclusions in glial cells and/or multinucleation of large, bizarre, gemistocytic astrocytes have also been observed as well as PML-type, but without substantial demyelination⁷⁵.

The histological characteristics of nephritis associated with polyomavirus in experimentally immunosuppressed cynomolgus macaques include nuclear enlargement (sometimes with nuclear inclusion bodies), cellular apoptosis and detachment, and destruction of the basement membrane, primarily in the collecting ducts, associated with patchy interstitial infiltration of lymphoplasmacytic inflammatory cells^{74, 77}. Involvement of the vascular endothelium or glomeruli is not observed. Mild renal dysfunction with elevated serum creatinine level is sporadically noted.

Diagnosis. The virus can be identified in the affected brain and kidney and sometimes in the ureter using IHC and DNA amplification specific for the large T antigen of the monkey polyomavirus family. Polyomaviruses can also be detected using electron microscopy in the nuclei of infected tubular epithelial cells or astrocytes as a characteristic sheet-like arrangement of non-enveloped virions^{16, 75, 77}.

Simian parvovirus (SPV)

Overview. SPV is a small, non-enveloped DNA virus with 65% sequence similarity to human parvovirus B19^{16, 78}. In humans, the serological prevalence of parvovirus B19 is high, especially in elderly people. However, viremia is rare, and the disease is unapparent or has mild, nonspecific symptoms in immunocompetent hosts⁷⁸. In one facility, a serologic screening test was conducted to prevent SPV outbreaks and all cynomolgus monkeys tested were SPV seronegative without any case of unexpected anemia from 2001 to 2006⁷⁹. Therefore, the prevalence of SPV may not be high. SPV outbreaks are thought to be preventable by examining seronegativity during the initial quarantine period⁷⁹. SPV identified in cynomolgus, rhesus, and pigtailed macaques is usually latent and is activated under immunosuppressive conditions caused by viral infection (e.g. SRV infection) or under experimental conditions, including treatment in preparation for transplantation^{78, 80}.

Clinical and anatomical pathologic features. Clinically apparent SPV infection is characterized by severe normocytic, normochromic, and non-regenerative anemia (decrease in red blood cells by −80%⁸³), whereas the WBC count remains within the normal range. The histologic characteristics involve decreased counts of erythroid and myeloid lineages and intranuclear parvoviral inclusions in erythroid precursors (predominantly in normoblasts and sometimes in pronormoblasts) in the bone marrow^{78, 79}.

Diagnosis. SPV can be confirmed using electron microscopy based on the presence of a cluster of parvovirus-like particles in erythroid precursor cells in the bone marrow of infected animals. Additionally, SPV can be diagnosed using PCR analysis of DNA isolated from the sera of affected monkeys⁸⁰.

Papillomavirus (PV)

Overview. PVs are a diverse family of small, non-enveloped DNA viruses. The prevalence of PV in cynomolgus monkeys is approximately 30% in adults aged >10 years⁸¹. Recently, 75.2% of wild captive clinically healthy rhesus macaques tested positive for PV DNA next-generation sequencing amplicon assays, most commonly from genital swabs, then less commonly from oral and perianal swabs⁸². In humans it is well known that the incidence and severity of PV-related proliferative lesions are increased in immunocompromised patients⁸³.

Clinical and anatomical pathologic features. Opportunistic activation of PV can result in proliferative or neoplastic lesions. PV can cause various proliferative lesions in the squamous epithelium, including cutaneous papilloma or cancers of the uterine cervix, oral mucosa, or other epithelia^{84, 85, 86}. However, PV infection may not manifest with overt clinical symptoms. Cervical and vaginal neoplasms have been observed in only 5.2% (20 out of 385) of cynomolgus monkeys in a facility. The neoplasms were intraepithelial and were detected in routine histological specimens with no or less than obvious gross finding that needs additional sampling⁸⁷. Cancer may require considerable time to develop after PV infection and thus mortality resulting from PV infection in macaques is rare in toxicity studies of pharmaceutical drugs. Cervical and vaginal neoplastic lesions can take the form of intraepithelial dysplasia, benign papilloma, or invasive cervical carcinoma, with common morphological features, including epithelial dysplasia, epithelial pearls, koilocytosis (cells with vacuoles around nuclei), nuclear atypia, and expansion of the basal epithelium⁸⁷.

In cynomolgus monkeys, beta PV-related papilloma has been observed on the skin of the hand and foot^{85, 86}. The lesions are histologically characterized by diffuse thickening of the epidermis with foci of large and pale superficial cells and rarely with eosinophilic intranuclear inclusion bodies⁸⁶.

Diagnosis. IHC analysis of AU-1, which is an antigen of the genus-specific PV epitope, can be performed to diagnose PV. Additionally, other antibodies that cross-react with PV in monkeys can be used to detect PV capsid proteins expressed in the nuclei of surface epithelial cells of lesions. PCR^{85, 87} and whole-genome sequencing can also be used⁸⁶.

Rhesus rhadinovirus (RRV): Gammaherpesvirinae, Macacine herpesvirus 5

Overview. RRV is highly endemic in socially housed and captive rhesus macaques, and the seroprevalence of RRV is almost 100% in adults^{88, 89}. Viral DNA is detected in the blood or saliva, or both, in clinically healthy rhesus monkeys, and the detection rates of viral DNA are high in young (aged <2 years) monkeys^{88, 90}. Infection is not usually associated with clinical signs but is associated with lymphoproliferative changes, resembling those of non-Hodgkin’s lymphoma or multicentric Castleman’s disease^{91, 92}. Kaposi’s sarcoma-associated herpesvirus (KSHV) and primate retroperitoneal fibromatosis-associated herpesvirus (RFHV), which can cause mesenchymal proliferative lesions, are closely associated with RRV¹⁶. RRV has been detected in areas of retroperitoneal fibromatosis in SIV-infected rhesus macaques and is considered one of the causative agents of mesenchymal proliferative lesions in macaques⁹¹.

Clinical and anatomical pathologic features. In macaques experimentally infected with SIV and RRV, lymphoproliferative lesions resembling those of multicentric Castleman disease have been reported. In these animals, marked peripheral lymphadenopathy was observed with pronounced splenomegaly 10 weeks after RRV infection; and in the animals with severe hemolytic anemia was observed 30 weeks after RRV infection⁹². The affected lymph nodes and spleen there are many lymph follicles with large reactive germinal centers, which are often irregular in shape and have indistinct mantle zones. In enlarged follicles of the spleen, hyalinized cell-poor areas with increased numbers of blood vessels were observed⁹². The presence of plasma cell clusters in the spleen and lymph nodes may also be a characteristic feature. Enlargement of the liver and spleen along with lymphadenopathy and increased erythropoiesis in the bone marrow has been observed^{91, 92}.

Diagnosis. In addition to the characteristic histopathologic features, detection of RRV can be done using PCR for definitive diagnosis^{91, 92}.

Simian varicella virus (SVV): Cercopithecine herpesvirus 9

Overview. Simian varicella epizootics occur sporadically in NHP facilities, including Japanese facilities^{93, 94}. After natural infection in primates, SVV becomes latent and latent viral DNA can be detected in the ganglia⁹⁵. SVV becomes latent in ganglionic neurons and can be reactivated spontaneously or opportunistically in monkeys immunosuppressed by social or environmental stress^{96, 97} or by experimental treatment with irradiation or immunosuppressive agents^{98, 99, 100, 101}. The seroprevalence of SVV may not be high. The reported incidence of SVV antibodies in newly captured wild pigtailed and cynomolgus macaque monkeys in Malaysia is only 0.8%⁹³. However, SVV can spread in the colonies at research facilities. The seroprevalences at facilities where clinical manifestations of SVV were observed were 35% and 20% in rhesus macaques¹⁰¹ and pigtailed macaques¹⁰⁰, respectively. In one rhesus macaque facility in which 57 animals received total body irradiation, SVV activation was observed in 2 animals¹⁰¹.

Clinical and anatomical pathologic features. SVV causes skin erythematous lesions in macaques and other monkeys. The outbreaks of SVV infections can result in high morbidity and mortality⁹³. The most characteristic cutaneous changes are diffuse multifocal papules/vesicles often associated with hemorrhage (maculopapulovesicular rashes). Many vesicles can appear successively and form crusts^{16, 100}. Macroscopically, hemorrhage or ulcerative lesions may also be observed in other squamous epithelium including the oral cavity and mucosal and serosal surfaces of thoracic and abdominal organs. Skin vesicles are histologically characterized as intra-epidermal vesicles containing cellular debris and/or erythrocytes. In the epithelial cells, eosinophilic intranuclear inclusion bodies consistent with herpesvirus infection (Cowdry type A) can be observed. Visceral organs, including the lung and liver, and gastric mucosa, can be infected and manifest necrosis with hemorrhage and intranuclear inclusion bodies in infected epithelial cells^{93, 100}. In a case of cynomolgus monkey in which anti-SVV antibody was detected by serological evaluation, eruption was observed whole body surface especially on the neck, chest and inner thighs and were histologically vesicles in the skin with intranuclear inclusion bodies in the basal cells of epidermis (Fig. 3a and 3b). Necrotizing inflammation with hemorrhage was observed in the lungs of this monkey (Fig. 3c). The presence of a bacterial colony was indicative of immunosuppression in this animal.

Fig. 3.

Simian varicella virus (SVV). (a) A vesicle in the skin with cell debris. (b) An image of higher magnification of the area denoted using a square in Fig. 3a. Intercellular edema and intranuclear inclusion bodies are identified based on hematoxylin and eosin staining. Photographs were kindly provided by Dr. Junko Sato and Dr. Takuya Doi, LSIM Safety Institute Corporation.

Diagnosis. Serological detection of anti-SVV antibodies is one of the most reliable diagnostic methods^{93, 98}. IHC can also be used to detect herpesviruses in vesicles^{100, 101}.

Hepatitis A virus (HAV)

Overview. HAV, an RNA virus, infects humans and NHPs naturally, and there are species-specific variations in its sequences^{102, 103, 104, 105}. Rhesus and cynomolgus macaques are the natural hosts of HAV¹⁰². Infection is usually self-limiting in immunologically normal animals, and baseline prevalence may be low. As HAV infection can be transmitted via the fecal-oral route¹⁰⁵, the infection may spread, especially under group-housing conditions.

Clinical and anatomical pathologic features. In cynomolgus macaques experimentally infected with human HAV, no clinical manifestations of the disease are observed. Histopathologic changes in the liver may be characterized by inflammatory cell infiltration in the portal area and parenchyma with microvesicular vacuolation of the hepatocytes. Inflammation associated with infection is characterized by the presence of diffuse and scattered inflammatory cells (predominantly macrophages, lymphocytes, and a few neutrophils)^{106, 107}. In immunocompromised animals, the infection may be serious. The livers of severely affected humans and chimpanzees are characterized by hepatocellular swelling, ballooning, and necrosis. Additionally, associated elevation of serum liver enzymes and the manifestation of jaundice have been reported¹⁰⁷.

Diagnosis. As the histopathologic characteristics and clinical manifestations are usually nonspecific, a definitive diagnosis can be performed using a combination of determining the presence of hepatitis, serological examination, and RT-PCR analysis of the serum, feces, saliva, or tissues¹⁰⁶.

Canine distemper virus (CDV)

Overview. CDV belongs to the genus Morbillivirus and the family Paramyxoviridae. Paramyxoviridae includes various highly pathogenic viruses, such as measles virus, rinderpest, and peste-des-petits-ruminants virus. In addition to canines, CDV is known to infect several carnivorous and non-carnivorous species, including Japanese rhesus and cynomolgus monkeys, but does not infect humans^{223, 224, 225, 226, 227}. In macaque monkeys, CDV infection outbreaks have been reported in China and Japan^{224, 225, 226, 227}. CDV infection can be lethal with a mortality rate of approximately 10%²²⁴, which is less than that in canines (approximately 50%).

Clinical and anatomical pathologic features. Clinical signs of infected macaque monkeys include fever, conjunctivitis and rhinitis with mucous discharge, cough, anorexia, diarrhea, generalized red rash, and periocular or plantar swelling^{224, 226, 228}. Necropsy of severely affected monkeys revealed focal red or dark-red discolored areas on the lung or focal or diffuse hemorrhage on the brain surface^{225, 227}. Histopathologic changes in cynomolgus monkeys sacrificed moribund were predominantly observed in the lungs, brain, and lymphoid organs²²⁴. Lung pneumonia with syncytial giant cell formation in the alveoli was observed (Fig. 4a and 4c). Gliosis (Fig. 4g) and/or demyelination were focally observed in the cerebrum and/or cerebellum²²⁴. Severe lymphoid depletion in the lymphoid organs suggesting immune suppression, has also been reported²²⁴. In such cases, characteristic perivascular cuffing by lymphocytes may not be observed. Giant cells may be observed in other organs, including the skin, intestines, kidneys, salivary glands, and testes^{224, 229}. Eosinophilic inclusion bodies are observed in epithelial cells of these affected organs in other animal species^{229, 230}. The causes of death could be severe pneumonia or neurological clinical signs^{224, 227}.

Fig. 4.

Canine distemper virus (CDV). (a) The lung of a cynomolgus macaque with CDV infection sacrificed moribund. Cellular infiltration in the alveoli and thickening of the alveolar wall are noted. (b) Same area of “a” immunohistochemically stained for CDV. Cells in the alveoli and bronchial epithelium are positive for CDV. (c) Higher magnification of “a.” Inflammatory cell infiltration and syncytial giant cell formation in the alveoli. (d) Syncytial giant cells and some cells in the alveolar wall are immunohistochemically positive for CDV. (e) Syncytial cells in the bronchiolar epithelium. (f) Syncytial cells in the bronchiolar epithelium are also immunohistochemically positive for CDV. (g) The brain of the same animal. Slight focal gliosis is noted. (h) The focus of gliosis is immunohistochemically positive for CDV. (a, c, e, and g) Hematoxylin and eosin staining. (b, d, f, and h) Immunohistochemical staining of CDV antigen. Photographs of sections obtained from animals sacrificed moribund are kindly provided by Dr. Noriyo Nagata, National Institute of Infectious Diseases and Dr. Shigeru Morikawa, Okayama University of Science²²⁴.

Diagnosis. CDV can be diagnosed using IHC analysis of viral antigens in mononuclear cells in lymphoid organs, glial cells in the brain, and giant cells in the lung. Additionally, CDV can be diagnosed based on characteristic histopathologic changes (Fig. 4a–4h). Furthermore, CDV antigen detection kits can also be used to diagnose animals with clinical signs.

Bacterial Infection

Campylobacter, Shigella, and Yersinia (causes of bacterial diarrhea): Campylobacter spp., S. flexneri, Y. enterocolitica, or Y. pseudotuberculosis

Overview. Campylobacter spp. (Campylobacter coli and Campylobacter jejuni), Shigella flexneri, and Yersinia enterocolitica have been identified as causative infectious agents as well as adenovirus and the parasite Strongyloides fuelleborni in samples collected from macaque monkeys with chronic diarrhea^{108, 109}. Among the bacteria that can cause diarrhea in macaque monkeys, Campylobacter spp. (especially C. jejuni and C. coli) and S. flexneri are the most commonly isolated bacteria, while Y. enterocolitica, Y pseudotuberculosis, and Salmonella spp. are less commonly isolated^{24, 110}. Routine quarantine procedures in which only clinical signs are evaluated do not guarantee freedom from either shigella or salmonella¹¹⁰. Campylobacter jejuni and C. coli, which are the main organisms responsible for infectious enteritis, are difficult to eradicate because simple isolation or treatment with antibiotics does not prevent recurrent infections with diarrheal bacteria in a colony¹¹⁰. Diligent serial testing and treatment are needed to control infection with these bacteria.

Clinical and anatomical pathologic features. Diarrhea caused by Shigella flexneri can be observed during quarantine within the first month after import from the country of origin. Shigella flexneri-induced diarrhea often exhibits antibiotic resistance and causes lethality¹¹⁰. The clinical signs of shigellosis include mucus and bloody diarrhea, abdominal pain, vomiting, and fever¹¹¹. Shigellosis-related lesions, which are primarily observed in the cecum and colon, may be focal or diffuse and are characterized by edema, hemorrhage, erosions, and ulceration often accompanied by crypt abscesses but less commonly accompanied by pseudomembrane formation. Shigella occasionally causes periodontitis in monkeys^{111, 112}.

Campylobacter spp.-related diarrhea can be observed later in the quarantine period. In contrast to S. flexneri-related diarrhea, Campylobacter spp.-related diarrhea is chronic. Clinical signs associated with experimental C. jejuni infection include fever and stool changes (soft feces or diarrhea). Campylobacter spp.-related lesions and evidence of direct damage in the intestine are not evident although lymphoreticular cells can infiltrate the lamina propria of the ileum, cecum, and colon¹¹³. The clinical syndrome of bacterial diarrhea can be complicated at all stages by the presence of helminths or viral infections¹¹⁰. Histologically, gram-negative S-shaped or spiral shaped bacteria may be observed but identification of Campylobacter spp. is difficult because of the presence of many other organisms in the intestinal flora.

Limited number of studies have reported Y. enterocolitica infections in macaque monkeys. In one case of yersiniosis in cynomolgus monkeys, both Y. pseudotuberculosis and Y. enterocolitica were detected. At necropsy, that enterocolitis was occasionally accompanied by enlargement of the mesenteric lymph nodes and presence of necrotic foci in the liver and spleen¹¹⁴. Gastrointestinal lesions comprise superficial erosions or ulcerations with masses of gram-negative coccobacilli and acute inflammatory exudate¹¹⁵.

Diagnosis. Diagnosis of shigellosis is based on clinical signs and isolation of the organism from deep rectal swabs and fresh stool specimens. Bacterial strains can be identified by isolation culture on selective media or based on biochemical characterization and PCR analysis^{234, 235}.

Campylobacter spp.-related diarrhea can be definitively diagnosed based on the recovery of the organism in samples obtained from animals with clinical signs^{113, 116}.

Yersinia spp. infection can be definitively identified by recovering organisms from samples collected from animals with clinical signs and analyzing the histopathologic characteristics, including necrotic foci in the liver and spleen.

Mycobacterium tuberculosis: aerobic gram-positive rods, Mycobacterium avium complex

Overview. Tuberculosis (TB) caused by M. tuberculosis and its complex (Mycobacterium tuberculosis complex: M. tuberculosis, M. africanum, M. bovis, M. canettii, M. microti, and others, together known as MTBC) is an insidious disease¹¹⁷ that can be latent in some animals and exacerbated by immunosuppressive condition¹¹⁸. In macaque monkeys from Asian countries and Gibraltar, PCR testing revealed a 32% positivity rate for MTBC. Generally, the prevalence of MTBC is high among NHPs in countries where the World Health Organization has reported an increased prevalence of human MTBC infection¹¹⁹. Facilities conducting transplant experiments perform repeated tests to detect MTBC owing to its high prevalence¹¹⁸.

Clinical and anatomical pathologic features. In cynomolgus monkeys experimentally infected with M. tuberculosis, clinical signs include anorexia, weight loss, cachexia, tachypnea, and dyspnea. X-ray examination can reveal the signs of bronchopneumonia. At necropsy, disseminated miliary nodules (0.5–3 mm in diameter) with a caseous cut surface are observed throughout the lung lobes. Lymphadenopathy with caseation and necrosis along with the involvement of the liver, spleen, and mesenteric tissues have been observed¹²⁰. Animals with the active progression of disease exhibit macroscopic pulmonary changes comprising nodules of various sizes with caseous cut surfaces (Fig. 5a–5b). The histopathologic characteristics include multifocal caseous and non-necrotizing granulomas consisting of epithelioid macrophages with a surrounding peripheral infiltration of lymphocytes and neutrophils (Fig. 5c). Granulomas were observed in histologic sections of various organs, including the liver and lymph nodes (Fig. 5d and 5e). In granulomas with active necrosis or inflammation, acid-fast bacilli can be observed, which can be confirmed using Ziehl-Neelsen staining^{118, 121}. In latently infected monkeys, TB-related lesions may be limited to mineralization of the hilar lymph nodes or small sclerotic granulomas in the lung.

Fig. 5.

Mycobacterium tuberculosis. (a) Lung of a cynomolgus macaque exhibiting active tuberculosis. Multifocal yellowish-white nodules are apparent. (b) Caseous cut surface of the nodules. Granulomas in the (c) lung, (d) lymph node, and (e) liver. (bars=200 μm). (c, d, e) Hematoxylin and eosin staining. Photographs are kindly provided by Dr. Takuya Doi, LSIM Safety Institute Corporation.

Diagnosis. Macaques with TB are diagnosed based on the characteristic macroscopic and microscopic features, including caseous nodules indicative of granulomatous inflammation with the presence of acid-fast positive bacilli, which can be confirmed using Ziehl-Neelsen staining^{118, 121} A positive tuberculin skin test (TST or Mantoux test) can be evidence of a history of TB infection¹¹⁸. The detection of TB based on clinical signs is challenging, especially during the early phase of infection or in the case of latent infection. TST yields positive results in NHPs from week 4 to week 8 post-infection^{120, 122}. Serological assays based on the detection of MTBC-specific antibodies and the measurement of cytokine (TB-specific IFNγ) levels using ELISA are available for diagnosis. However, these methods are not effective owing to false-positive and false-negative results²³⁶. PCR methods have been suggested to detect the presence of MTBC DNA in bronchoalveolar lavage, gastric aspirate, and, for humans, sputum samples¹¹⁹.

Helicobacter pylori and Helicobacter heilmannii

Overview. Helicobacter pylori is a curved, spiral-shaped, flagellated gram-negative bacterium commonly detected in rhesus and cynomolgus monkeys¹¹⁷. Natural H. pylori infections do not cause clinical disease, but microscopic gastric lesions are sometimes obvious and may obscure, or cover, the mild effects of test articles on the stomach of monkeys in preclinical safety or pharmacological studies. Rhesus monkeys are used as a model for human H. pylori infection owing to the similarity in pathologic changes¹²³. In rhesus monkeys, H. pylori infection occurs in about 40% of socially housed NHPs by 12 weeks of age and increased up to 90% by 1 year of age¹²⁴. A high prevalence has also been reported in cynomolgus monkeys; the prevalence likely depends on the source country of origin (up to 93% in adolescent cynomolgus monkeys from the Philippines, 20% in monkeys bred in Japan, and 0% in those from Indonesia^{125, 126, 127}).

Clinical and anatomical pathologic features. In the stomachs of cynomolgus monkeys infected with H. pylori, inflammatory cell infiltration of the lamina propria of the fundus or pylorus¹²⁸ was the most characteristic finding. This is consistent with chronic inflammation characterized by lymphoplasmacytic infiltration with frequent loss of parietal cells and reactive proliferation of the mucosal epithelium, resulting in mucosal thickening in severe cases^{125, 127} (Fig. 6a). Clusters of bacteria may be observed in the mucosal pits and upper glands with hematoxylin and eosin (H&E) staining alone although they are difficult to detect in regenerative proliferative lesions. Warthin-Starry staining can reveal the presence of curved or spiral-shaped bacteria, i.e. H. pylori and H. heilmannii-like organisms, on the surfaces and in the lumens of the gastric glands, as well as in the parietal cells^{126, 129, 130} (Fig. 6b–6e).

Fig. 6.

Helicobacter pylori. (a) Chronic inflammation of the pyloric mucosa of a cynomolgus macaque infected with H. pylori (hematoxylin and eosin (H&E) staining). (b) Bacilli in the glandular lumen (H&E and Giemsa staining). (c) Higher magnification of bacilli in the lumen. Spiral shapes can be seen. Bacilli in the glandular lumen stained with (d) Giemsa and (e) Warthin-Starry stains, and (f) subjected to immunohistochemistry for H. pylori.

Diagnosis. The presence of curved or spiral-shaped bacteria detected by Warthin-Starry staining and electron microscopy can be evidence. Small comma-shaped bacteria are considered H. pylori, while large bacteria with 6 to 8 spirals were considered H. heilmannii-like organisms¹²⁷. H. pylori can also be diagnosed using IHC (Fig. 6f). Coinfection with several species of Helicobacter bacteria (the so-called H. heilmannii-like organisms) may yield positive results for H. pylori in IHC analysis^{129, 130, 131, 132}. For the precise identification of Helicobacter species, PCR and other molecular techniques can be used.

Moraxella catarrhalis (M. macacae in macaques)

Overview. Moraxella catarrhalis is a gram-negative aerobic diplococcus formerly known as Branhamella catarrhalis^{28, 117}. M. catarrhalis is a common inhabitant of the nasopharynx in humans and NHPs, and is an opportunistic pathogen associated with otitis media in infants and children. In macaques, a similar Moraxella sp., possibly M. macacae, causes epistaxis (nasal hemorrhage, known as bloody nose syndrome)^{133, 134, 135}. Moraxella macacae can be transmitted in closed facilities. Epistaxis caused by M. macacae can be treated with antibiotics¹³⁴.

Clinical and anatomical pathologic features. M. macacae infection in monkeys is associated with clinical signs including sneezing, epistaxis (clear to serosanguineous, mucohemorrhagic, or bloody nasal discharge), and periocular swelling^{133, 135}. Examination of nasal swab smears revealed WBCs and red blood cells and large diplococcal organisms¹³⁵.

Diagnosis. In addition to nasal swab cultures, real-time PCR can be used to identify the infecting species¹³⁴.

Rhodococcus equi

Overview. Rhodococcus equi is a gram-positive coccoid or bacillary facultative anaerobe that is found in large numbers on dry soil surfaces. R. equi is an important pathogen in foals and is also pathogenic in immunocompromised animals and humans^{136, 137, 138}.

Clinical and anatomical pathologic features. Infection usually manifests as pneumonia but can also as wound infection^{136, 137, 139}. In the lungs of infected foals, gross lesions are characterized by multifocal to coalescing nodules ranging from a few millimeters to 10 cm in diameter. The Histopathologic features include multifocal pyogranulomatous inflammation characterized by the infiltration of neutrophils and macrophages, occupying the alveolar spaces and bronchioles. Macrophages occasionally contain myriad coccobacillary basophilic gram-positive bacteria. Lung lesions may also include multinucleated giant cells, multifocal thrombosis, interstitial and alveolar edema, and tissue necrosis¹⁴⁰. In humans treated with immunosuppressants for organ transplantation, R. equi-related lung lesions include inflammation of the lung parenchyma, with foamy histiocytic infiltration. In these foamy macrophages, intracellular bacteria that are positive for periodic acid Schiff or Gram staining can be observed¹⁴¹. Pyogranulomatous inflammation can also be observed in the liver, kidney, spleen, and nervous tissue¹³⁹. Previously, one case of R. equi isolation from monkeys has been reported¹⁴².

Diagnosis. In addition to the characteristic histopathologic findings of pyogranulomatous pneumonia with intracytoplasmic bacteria in the macrophages infiltrating the lesions, positive staining for R. equi in intralesional macrophages is a potential tool for definitive diagnosis¹⁴⁰. However, obtaining an appropriate antibody for IHC analysis is sometimes difficult. Various PCR assays have been developed^{143, 144, 145} and can be used to detect R. equi in lesions.

Enteropathogenic Escherichia coli (EPEC)

Overview. Diarrheagenic E. coli strains are the most common etiological agents for diarrhea in mammals. E. coli strains are categorized as follows based on their specific virulence factors and phenotypic traits: enteropathogenic E. coli (EPEC), causes diarrhea in children and animals; enterotoxigenic E. coli (ETEC), causes traveler’s diarrhea and porcine and bovine diarrhea; enterohemorrhagic E. coli (EHEC), causes hemorrhagic colitis and hemolytic uremic syndrome and includes verotoxin-producing/Shiga toxin-producing E. coli (VTEC/STEC); enteroinvasive E. coli (EIEC), causes watery diarrhea and dysentery; enteroaggregative E. coli (EAEC), causes persistent diarrhea in humans; diffusely adherent E. coli (DAEC), a subclass of EAEC that causes diarrhea in children^{146, 147}. EPEC strains are important agents that can cause chronic diarrhea and are closely related to morbidity in infants and children less than 2 years in the developing world^{147, 148}. E. coli is also one of the most common bacterial pathogens causing diarrhea in HIV-infected immunocompromised humans in whom both ETEC and EPEC are important opportunistic diarrheagenic pathogens¹⁴⁹. EPEC strains have also been identified in SIV-infected rhesus¹⁵⁰ and cynomolgus monkeys used in preclinical safety studies¹⁵¹.

Clinical and anatomical pathologic features. EPEC-induced diarrhea associated histologic changes are observed in the large or small intestine or both. To identify the characteristic histologic lesions, several sections must be evaluated^{148, 150, 151}. In the small intestine of infected cynomolgus monkeys, the villi exhibit clubbing and shortening with vacuolation in the subepithelial lamina propria^{150, 151}. In the colons of infected marmosets and rhesus monkeys, bacteria can be detected on the surface of the affected epithelium along with decreased crypt size or onset of reactive crypt hyperplasia, epithelial vacuolation, and neutrophilic inflammatory infiltration^{148, 150}. EPEC may be observed in lesions on the surfaces of villi in toluidine blue-stained semithin sections subjected to electron microscopy examination. The tight attachment of bacteria to the cell surface causes epithelial cell injury, which is characterized by degeneration and loss of cilia in the brush border¹⁵¹.

Diagnosis. As several E. coli can cause diarrhea, fecal culture and serotype identification are the traditional methods to obtain a definitive diagnosis. Commercial PCR diagnostic kits are also available.

Parasitic Infections

Pulmonary acariasis (Pneumonyssus simicola)

Overview. Pulmonary acariasis (Pneumonyssus simicola) is parasitic infections observed in NHPs, including cynomolgus monkeys¹⁵². The prevalence of pulmonary acariasis in a colony increases with the age of the monkeys^{153, 154}. Recently, mite bodies were not so commonly observed in the lung sections in toxicological studies. In combination with other evidence of acariasis, including granulomatous inflammation with or without bronchiolar dilatation, dark pigments phagocytosed by macrophages around the lesions are indicators of acariasis even if the mite is not observed in the lung section^{128, 152}.

Clinical and anatomical pathologic features. When infested with a small number of pulmonary achalasia, animals do not show clinical signs. In heavily infested monkeys, scattered small (1–5 mm) yellow or pale green nodules can be observed grossly in the lung^{155, 156}. The definitive histopathologic finding is the presence of mite bodies in a section. Mites are surrounded by a thin wall of connective tissue, forming cysts or bullae. Around the cysts, granulomatous tissue with inflammatory cells, including pigmented or unpigmented macrophages, lymphocytes, neutrophils, and eosinophils, is observed. Bronchitis or peribronchiolitis, which is sometimes accompanied by bronchiolar dilatation, is evident^{128, 154, 155, 156}. Ivermectin treatment effectively eliminates mites but cyst-like structures with granulomatous inflammation may persist^{153, 154}.

Diagnosis: Infested monkeys usually do not exhibit clinical signs^{154, 155}. Pneumothorax due to cyst or bulla rupture can occur in severely infested macaques. Clinical diagnosis of acariasis using X-ray is difficult in monkeys^{153, 155}. Tracheobronchial lavage examination and computed tomography are potential tools for detecting the presence of Pneumonyssus spp. or multiple bullae in the lung¹⁵³.

Helminths, including nematodes, cestodes, and trematodes

Overview. Helminths include nematodes and platyhelminths (cestodes and trematodes). Wild-caught macaque monkeys can be infested with a large number of a wide variety of helminths¹⁵⁵. Of these, Strongyloides fuelleborni, Trichuris trichiura, and Oesophagostomum spp. are intestinal nematodes that have been detected with high incidence in macaque monkeys^{36, 152, 157, 158, 159, 160, 161} and are recommended to be monitored^{36, 160}. Although cestodes and trematodes have also been reported to infest macaque monkeys¹⁵⁵, nematodes are frequently reported and are the focus of this section. In NHPs maintained in internal housing, the incidence of nematode infestations is low. Anthelmintics, including ivermectin and moxidectin, effectively decrease the egg counts per gram of feces in the laboratory^{160, 161, 162}. However, the complete elimination of nematodes from the intestine is difficult, requiring sequential or combined anthelmintics^{138, 160, 161}.

Clinical and anatomical pathologic features. Severe infestations of nematodes that cause clinical signs in macaque monkeys are rare³⁶. Pathologists may encounter helminths in specimens during histopathologic examination in studies and investigations (Fig. 7a and 7b).

Fig. 7.

Helminths (Nematode). (a) A cross and longitudinal section of nematode in the esophageal mucosa. (b) A cross-section of nematode in the cecal mucosa. Hematoxylin and eosin (H&E) staining.

Oesophagostomum spp. form small (approximately 8 mm in diameter) dark nodules in the large intestine that are can be observed via gross examination¹⁵². Lesions can be distributed from the submucosa to the serosal surface and are sometimes detected in the mesentery or intestinal wall^{152, 155}. The histopathologic characteristics of the nodules included multiple cross-sections of the nematode. The inflammatory cells are predominantly neutrophils and macrophages with occasional sometimes foreign-body giant cells. The nodules may be encapsulated by fibrous tissue, and forming granulomatous lesions^{152, 155}. Granulomatous lesions with mineralization in the center can be observed. These are old lesions that suggest evidence of previous infestation of helminths¹⁵⁵.

Strongyloides spp. are usually observed in the mucosa of the small intestine and occasionally in the large intestine with inflammation in the lamina propria^{152, 155}. In the case of severe autoinfection, the lymphatics of the intestine and lung may be obstructed by the larvae¹⁵⁵.

Trichuris trichiura on the mucosal surface of the large intestine does not cause clinical signs. Histologically, cross- (or longitudinal) sections of T. trichiura can be observed on the surface of the mucosa or embedded in the mucosal folds without any reaction or inflammation.

Diagnosis. To determine whether animals were infested with intestinal helminths, microscopic examination of fecal samples is performed to detect parasite eggs^{36, 157, 160, 162}. Pooled fecal samples can also be used for the diagnosis³⁶. The number of eggs per gram (EPG) of feces indicates the severity of infestation^{160, 162, 163}. Molecular techniques have also been used to identify infectious species^{164, 165}.

Protozoa: general overview

In macaque monkeys, protozoa are more common than nematodes. Infections with some species can be opportunistically activated in immunosuppressed macaques^{138, 160}. Protozoal parasites that may be encountered during the histopathologic examination of laboratory macaque monkeys include flagellates (Giardia spp., Trypanosoma spp., and Trichomonas spp.), amoebae (Entamoeba spp.), coccidia (Cryptosporidium spp., Plasmodium spp., Hepatocystis spp., Toxoplasma gondii, Sarcocystis spp., and Babesia spp.), and ciliates (Balantidium spp.)¹⁵⁵. In macaques housed in laboratory settings, infections may have occurred and carried in from outdoor breeding colonies.

Gastrointestinal protozoa

Overview. Protozoa infecting the gastrointestinal tract of macaque monkeys include Entamoeba spp., Giardia spp., Balantidium spp., and Cryptosporidium spp. Of these, Balantidium spp. are frequently observed in the lumen and on the surface of mucosa of the large intestine during routine histopathologic examinations. The prevalence of Entamoeba spp. in macaques in East Asia is high although their virulence is low. Meanwhile, the prevalence of the virulent Entamoeba histolytica is low^{166, 167, 168}. Cryptosporidium and Giardia spp. are uncommon in normal healthy macaques^{155, 160, 169}. However, gastrointestinal infection with Cryptosporidium sp. has been reported in juvenile and immunocompromised macaques^{169, 170}.

Clinical and anatomical pathologic features.

Entamoeba: The pathogenicity of Entamoeba depends on the strain and the host species, nutritional status, environmental factors, and bacterial flora¹⁷¹. Entamoeba spp. infection becomes pathogenic when protozoa invade the mucosa, leading to amebic dysentery^{172, 173}. In clinically apparent cases, anorexia, vomiting, severe diarrhea, and ulcerative hemorrhagic colitis with trophozoites in ulcerated lesions, as well as liver abscesses, are reported under immunosuppressive conditions^{155, 166, 171}. Trophozoites may not be visible in H&E-stained sections but are stained bright red in periodic acid Schiff (PAS)-stained sections¹⁵⁵.

Cryptosporidium: Cryptosporidium spp. are detected on the epithelial surfaces of gastric pits or intestinal crypts^{169, 170} and can be activated under immunosuppressive conditions. In the stomachs of monkeys with the proliferation of Cryptosporidium spp., small (1–4 μm) and round protozoa (stained blue with Giemsa staining¹⁷⁰) have been detected on the surface of the affected mucosa. Additionally, associated reactive mucosal hyperplasia with increased mucus secretion has also been reported (Fig. 8). Cryptosporidium spp. infection in the small intestine of juvenile macaques (rhesus and cynomolgus) can result in life-threatening diarrhea with histological lesions, including epithelial vacuolation, necrosis, and villus atrophy.

Fig. 8.

Cryptosporidium sp. Numerous cocci were found in the fundic pits of the stomach of cynomolgus monkeys with hypertrophy/hyperplasia of superficial mucus cells. Hematoxylin and eosin (H&E) staining.

Balantidium coli: In most cases, B. coli infection in macaques is asymptomatic¹⁷⁴. B. coli, which can be identified in the intestinal lumen and mucosal folds (Fig. 9), can become pathogenic in severely immunocompromised animals, causing severe diarrhea with the multiplication of protozoa and their invasion into the mucosa, and mucosal erosions or ulcerations¹⁷⁵.

Fig. 9.

Balantidium coli.Balantidium coli on the mucosa of the cecum. Hematoxylin and eosin (H&E) staining.

Giardia spp.: Although the prevalence of intestinal Giardia spp. in rhesus macaques is high¹⁷⁶, limited information is available on its pathologic features. This may be because similar to humans, infection by a small number of parasites does not induce clinical signs in monkeys. However, in humans, symptomatic diarrhea with gas formation has been reported¹⁷⁷.

Diagnosis. In addition to the identification of characteristic protozoa in intestinal sections and fecal examination, which is one of the most common tools to screen for intestinal protozoal infestation¹⁶⁶, other laboratory methods, including biochemical analysis of isoenzymes of Entamoeba spp., ELISA, or PCR evaluation should be employed to identify the species and virulence of infectious protozoa to detect virulent E. histolytica^{168, 178, 179, 180}.

Other protozoa: Trypanosoma spp. (flagellates)

Overview. The prevalence of Trypanosoma cruzi in Central and South America is higher than that in other parts of the world because of the dissemination of the insect vector kissing-bug^{181, 182}. The prevalence of T. cruzi in NHPs ranges from 2% to 10% in facilities in the southern USA¹⁸¹.

Clinical and anatomical pathologic features. Infected animals may be asymptomatic. Trypanosoma spp. induce histopathologic lesions, including lymphocytic myocarditis and gastritis, with increased levels of proinflammatory cytokines in the blood. These characteristics are similar to those of Chagas disease in humans. The myocardium, muscle fibers, or esophagus may also be affected and may accompanied by arrythmia or esophageal dilatation^{181, 182, 183, 184, 185, 186}.

Diagnosis. In addition to clinical and histopathologic characteristics, examination of Giemsa-stained thin or thick blood smears can be used to detect the active phase of Trypanosoma spp. PCR can also be used to detect parasite-specific DNA in the blood or tissues¹⁸⁷.

Other protozoa: Toxoplasma gondii (coccidia)

Overview. Although Toxoplasma gondii is widely distributed in wild and domestic animals^{188, 189}, its seroprevalence in breeding colonies of rhesus and cynomolgus monkeys is low^{190, 191}.

Clinical and anatomical pathologic features. Toxoplasmosis in macaques is less prevalent and there is little information on its clinical manifestations, perhaps because it is asymptomatic as in humans. Some studies have reported pathologic lesions associated with natural T. gondii infection in macaques. In humans and other animals, Toxoplasma-induced lesions include cellular necrosis and inflammation associated with invasion by tachyzoites, as well as the formation of bradyzoite cysts in various organs and tissues, including the blood vessels, lung, liver, and brain^{188, 189}.

Diagnosis. Serological examination can be used to identify characteristic cysts and tachyzoites in the affected tissues^{191, 192}. PCR can be performed to detect Toxoplasma DNA in the blood, tissues, and cerebrospinal fluid^{193, 194, 195}.

Other protozoa: Sarcocystis spp. (coccidia)

Overview. The prevalence of Sarcocystis infection is high in monkeys caught from the wild and low in captive-born monkeys¹⁹⁶.

Clinical and anatomical pathologic features. In one reported case of a rhesus monkey with clinically apparent sarcocystosis, the animal became moribund with anorexia and systemic edema. In this animal, coalescing myocardial edema and necrosis of the myocardium with infiltration of macrophages and lymphocytes, as well as Sarcocystis spp. infestation in endothelial cells, were observed¹⁹⁷. Cysts of Sarcocystis are incidentally observed in skeletal muscle in the absence of any associated degenerative or inflammatory lesions (Fig. 10).

Fig. 10.

Sarcocystis spp. Sarcocystis in the skeletal muscle without any associated degenerative or inflammatory changes.

Diagnosis. In addition to histopathologic analysis, PCR can be used¹⁹⁷.

Other protozoa: Babesia spp., including Entopolypoides macaci (coccidia)

Overview. Babesia, which is an intra-erythrocytic parasite transmitted by ticks in rhesus and cynomolgus monkeys, can cause opportunistic infections in immunocompromised monkeys¹⁹⁸. Although the tick, vector of Babesia spp., is not observed in the laboratory setting, latent infection can be caused by exposure to ticks outside breeding colonies. Babesiosis is recognized worldwide as a zoonotic disease and has been sporadically reported in mammals, including humans and NHPs^{199, 200}.

Clinical and anatomical pathologic features. Babesia infections are generally latent and not clinically apparent. Recurrence can occur when animals are immunocompromised or have undergone splenectomy^{198, 201, 202}. Anemia, parasites in red blood cells, and splenomegaly are observed in animals with clinically apparent babesiosis^{198, 202}. In contrast to the malaria-causing Plasmodium, this organism does not produce pigments (hemozoin) in the affected red blood cells²⁰².

Diagnosis. The presence of parasites without hemozoin pigments is confirmed in red blood cells in Giemsa-stained thin blood smears. PCR detection of Babesia-specific DNA is used to differentiate Babesia from Plasmodium infection.

Other protozoa: Plasmodium spp. (coccidia)

Overview. Plasmodium spp. cause malaria in various animals. Plasmodium cynomolgi, P. semiovale, and P. fieldi have been identified in macaques. Infections are usually latent and take the form of hepatocellular hypnozoites that can lead to relapse. Although infection rates are high in macaque monkeys^{203, 204}, clinical manifestations of malaria are rare in healthy macaques. Relapse can be induced in laboratory macaques under immune suppressive conditions due to viral infection, treatment with immunosuppressive chemicals or biologics, splenectomy, or increased turnover of red blood cells^{59, 203, 204, 205}.

Clinical and anatomical pathologic features. In the case of clinically evident malaria that we experienced in a 13-week toxicity study, severe anemia with a marked decrease in bodyweight (by −15% compared with controls), spontaneous activity, erythrocyte count (by 0.89 × 10⁶/µL), and hematocrit values (by 9.0%) was observed. In this animal, cyclic decreases in erythroid parameters were observed. The presence of hemoparasites morphologically consistent with Plasmodium spp. in red blood cells was confirmed by the examination of thin blood smears (Fig. 11a–11c). At necropsy, massive enlargement and dark discoloration of the spleen (Fig. 11d) and liver were observed. Histopathologic evaluation revealed depositions of black-brown hemozoin pigment in macrophages in the spleen and Kupffer cells in the liver (Fig. 11e and 11f) along with Berlin-blue-positive hemosiderin. Hemozoin pigments are birefringent under polarized light and are negative for iron staining (Berlin blue)²⁰⁷. These changes are accompanied by lymphoid depletion in the spleen²⁰⁴.

Fig. 11.

Plasmodium spp. In thin blood smears, various stages of parasites in red blood cells were observed: (a) schizont, (b) trophozoite, and (c) ring form. The spleen was enlarged (d) with the expansion of the red pulp and lymphoid depletion (bar=3 mm) and (e) several brown-black pigments in the macrophages in the red pulp. (f) Pigments were observed in the livers of the Kupffer cells. (a, b, and c) Wright’s staining and (d, e, and f) hematoxylin and eosin (H&E) staining. Photographs are from a previous study from the author of this manuscript (E. Ohta).

Diagnosis. To detect low-level parasitemia, Giemsa-stained thick blood smears have been reported to be better than tin blood smear examinations in NHPs. Other diagnostic methods, including PCR, can be used although their efficacy has not been thoroughly established in NHPs^{203, 204, 206, 207}.

Fungi

Many fungal infections are regarded as opportunistic and may not have clinical or histopathologic manifestations unless the immune function of the host is compromised by the disease or experimental treatment. In addition to Candida albicans and Pneumocystis spp. described in this section, Histoplasma capsulatum, Cryptococcus neoformans, and Aspergillus fumigatus have been identified as rare opportunistic agents in monkeys^{22, 208}.

Candida albicans

Overview. Candidiasis is the most common opportunistic yeast infection worldwide. Infection can be superficial or deep/systemic and can affect the skin, mucosa of the mouth and alimentary tract, genital or urinary tract, or respiratory system^{208, 209, 210, 211}. In immunocompromised humans, endocarditis, meningitis, and Candida sepsis have been reported as forms of deep systemic candidiasis²¹¹.

Clinical and anatomical pathologic features. In the upper alimentary tracts of macaques affected with symptomatic candidiasis, clinical signs related to ulceration of mucosa thought to be observed. The ulcers can be observed as macroscopic whitish streaks or plaques on the mucosa of the tongue, oral cavity, and esophagus. A yellowish pseudomembrane is also observed in the intestine. Histologically, clusters of fungus (3–5-μm pseudohyphae and blastospores that are PAS-positive and Gomori methenamine silver [GMS]-positive) can be observed on the mucosal epithelium. If the fungus invades the tissue, ulceration with inflammatory cell infiltration can occur²⁰⁸.

Diagnosis. In addition to the characteristic gross and histological findings in the presence of PAS-positive or GMS-positive yeasts in the affected organs, fungal culture, PCR, and serum antibodies against Candida or its glycoproteins can be used to diagnose candidiasis^{208, 212, 213}.

Pneumocystis spp.

Overview. Pneumocystis is a yeast-like fungus that is predominantly observed in the lung tissues. Strains of Pneumocystis spp. have a high host specificity^{214, 215}. Only the genomes of human P. jirovecii, rat P. carinii, and mouse P. murina have been identified^{215, 216}. Additionally, the genome of P. macacae in macaque monkeys^{215, 217} has been proposed. Similar to their pathologic effects in humans, Pneumocystis spp. are opportunistic fungal organisms that cause Pneumocystis pneumonia in macaque monkeys. Clinical manifestations can be observed in immunologically compromised animals^{22, 218}.

Clinical and anatomical pathologic features. In lungs with symptomatic pneumonia, the alveolar septa are thickened with infiltration of lymphocytes and neutrophils. In alveolar spaces filled with eosinophilic foamy material, the presence of Pneumocystis spp. can be confirmed using GMS staining and IHC for Pneumocystis antigen^{219, 220, 221}. A nodular type of Pneumocystis pneumonia has been reported in monkeys with AIDS. This pneumonia is characterized by numerous grossly apparent large nodules. Histological analysis revealed foamy material, central necrosis, and necrotizing vasculitis in the nodules²²⁰.

Diagnosis. Infection can be diagnosed based on the typical histopathologic features, as well as based on IHC detection of Pneumocystis-specific antigens and PCR detection of DNA^{220, 221}.

Discussion and Conclusion

This review presents a summary of the clinical and anatomical manifestations, pathologic features, and diagnosis of representative infectious diseases, including opportunistic infections, in NHPs, especially macaque monkeys. The macaque monkeys that were the focus of this review were predominantly the cynomolgus macaques (Macaca fascicularis) and rhesus macaques (Macaca mulatta), which are the most common NHP models in biomedical research, including preclinical toxicity studies²²². Several efforts have been undertaken to screen out representative infectious agents via periodic examination during breeding and quarantine at export or import. However, latent or incidental background infections can be observed. In particular, the potential recurrence of latent infection should be considered under experimental conditions in which the immune system can be compromised. Recurrence of latent infection can be severe in immunocompromised animals with systemic or local manifestations and can mask or complicate the real pathologic features induced by the test agent. In such case the interpretation of the study results is complicated and challenging. For example, changes in erythroid parameters in a toxicity study described in the Plasmodium spp. section can be misunderstood as being directly caused by the test article because the article was not an intended immune modulator. In this case, a thin blood smear examination was decided to conduct because careful monitoring of hematological parameters revealed cyclic changes in red blood cells. The smear examination revealed parasites in the red blood cells and thus the changes in the animal were diagnosed to be related to the relapse of latent malaria in the animal and not directly related to the test article. In particular, when the intended pharmacological effects of test articles are not related to the immune system, the changes related to incidental infection or relapse of latent spontaneous infection are misinterpreted as direct effects of test articles.

In addition to the effects of test article on the immune system, background or incidental infection with immunosuppressive viruses (e.g. measles transmitted from humans to monkeys), malnutrition, and stress due to experimental procedures, changes in animal husbandry conditions (including changes in the animal room), or shipping can cause recurrence. To accurately interpret changes associated with the test article or experiment, toxicologic pathologists must understand the prevalence of these infections, their basic pathologic features, and the current diagnostic methods. When unexpected findings are observed and infectious diseases are suspected in studies, pathologists should not only collect information on the origin of the monkeys, the quarantine methods, and the results of routine testing for infectious agents but also on the time-course changes in clinical findings, the health status of the handlers, and any hints of accidents or procedures that could have stressed the monkeys. If an intervention has been performed on an animal diagnosed with an infection, pathologists should also be aware of the type of intervention (e.g. allowing dosing holidays for animals or drugs that were used). This information will help pathologists to distinguish both infectious disease-related and test-item-related lesions and precisely interpret the results of experiments or studies. If the pathogen is identified, the pathologist and onsite veterinarian should check whether the laws of each country require notification of the infectious disease and take appropriate action.

Disclosure of Potential Conflicts of Interest

The author declares no potential conflicts of interest regarding the research, authorship, or publication of this article.