Deep Learning for Face Detection and Pain Assessment in Japanese macaques (Macaca fuscata).

Facial expressions have increasingly been used to assess emotional states in mammals. The recognition of pain in research animals is essential for their well-being and leads to more reliable research outcomes. Automating this process could contribute to early pain diagnosis and treatment. Artificial neural networks have become a popular option for image classification tasks in recent years due to the development of deep learning. In this study, we investigated the ability of a deep learning model to detect pain in Japanese macaques based on their facial expression. Thirty to 60 min of video footage from Japanese macaques undergoing laparotomy was used in the study. Macaques were recorded undisturbed in their cages before surgery (No Pain) and one day after the surgery before scheduled analgesia (Pain). Videos were processed for facial detection and image extraction with the algorithms RetinaFace (adding a bounding box around the face for image extraction) or Mask R-CNN (contouring the face for extraction). ResNet50 used 75% of the images to train systems; the other 25% were used for testing. Test accuracy varied from 48 to 54% after box extraction. The low accuracy of classification after box extraction was likely due to the incorporation of features that were not relevant for pain (for example, background, illumination, skin color, or objects in the enclosure). However, using contour extraction, preprocessing the images, and fine-tuning, the network resulted in 64% appropriate generalization. These results suggest that Mask R-CNN can be used for facial feature extractions and that the performance of the classifying model is relatively accurate for nonannotated single-frame images.

Investigating subtle changes in facial expression to assess acute pain in Japanese macaques.

Changes in facial expression provide cues for assessing emotional states in mammals and may provide non-verbal signals of pain. This study uses geometric morphometrics (GMM) to explore the facial shape variation in female Japanese macaques who underwent experimental laparotomy. Face image samples were collected from video footage of fourteen macaques before surgery and 1, 3, and 7 days after the procedure. Image samples in the pre-surgical condition were considered pain-free, and facial expressions emerging after surgery were investigated as potential indicators of pain. Landmarks for shape analysis were selected based on the underlying facial musculature and their corresponding facial action units and then annotated in 324 pre-surgical and 750 post-surgical images. The expression of pain is likely to vary between individuals. Tightly closed eyelids or squeezed eyes and lip tension were the most commonly observed facial changes on day 1 after surgery (p < 0.01974). A good overall inter-rater reliability [ICC = 0.99 (95% CI 0.75-1.0)] was observed with the method. The study emphasizes the importance of individualized assessment and provides a better understanding of facial cues to pain for captive macaque care.

Identification and Structure of an MHC Class I-Encoded Protein with the Potential to Present N-Myristoylated 4-mer Peptides to T Cells.

Similar to host proteins, N-myristoylation occurs for viral proteins to dictate their pathological function. However, this lipid-modifying reaction creates a novel class of "lipopeptide" Ags targeted by host CTLs. The primate MHC class I-encoded protein, Mamu-B*098, was previously shown to bind N-myristoylated 5-mer peptides. Nevertheless, T cells exist that recognize even shorter lipopeptides, and much remains to be elucidated concerning the molecular mechanisms of lipopeptide presentation. We, in this study, demonstrate that the MHC class I allele, Mamu-B*05104, binds the N-myristoylated 4-mer peptide (C14-Gly-Gly-Ala-Ile) derived from the viral Nef protein for its presentation to CTLs. A phylogenetic tree analysis indicates that these classical MHC class I alleles are not closely associated; however, the high-resolution x-ray crystallographic analyses indicate that both molecules share lipid-binding structures defined by the exceptionally large, hydrophobic B pocket to accommodate the acylated glycine (G1) as an anchor. The C-terminal isoleucine (I4) of C14-Gly-Gly-Ala-Ile anchors at the F pocket, which is distinct from that of Mamu-B*098 and is virtually identical to that of the peptide-presenting MHC class I molecule, HLA-B51. The two central amino acid residues (G2 and A3) are only exposed externally for recognition by T cells, and the methyl side chain on A3 constitutes a major T cell epitope, underscoring that the epitopic diversity is highly limited for lipopeptides as compared with that for MHC class I-presented long peptides. These structural features suggest that lipopeptide-presenting MHC class I alleles comprise a distinct MHC class I subset that mediates an alternative pathway for CTL activation.

Experimental infection of Japanese macaques with simian retrovirus 5.

Recently, a large number of Japanese macaques (Macaca fuscata) died of an unknown hemorrhagic syndrome at Kyoto University Primate Research Institute (KUPRI) and an external breeding facility for National Institute for Physiological Sciences (NIPS). We previously reported that the hemorrhagic syndrome of Japanese macaques at KUPRI was caused by infection with simian retrovirus 4 (SRV-4); however, the cause of similar diseases that occurred at the external breeding facility for NIPS was still unknown. In this study, we isolated SRV-5 from Japanese macaques exhibiting thrombocytopenia and then constructed an infectious molecular clone of the SRV-5 isolate. When the SRV-5 isolate was inoculated into two Japanese macaques, severe thrombocytopenia was induced in one of two macaques within 22 days after inoculation. Similarly, the clone-derived virus was inoculated into the other two Japanese macaques, and one of two macaques developed severe thrombocytopenia within 22 days. On the other hand, the remaining two of four macaques survived as asymptomatic carriers even after administering an immunosuppressive agent, dexamethasone. As determined by real-time PCR, SRV-5 infected a variety of tissues in Japanese macaques, especially in digestive and lymph organs. We also identified the SRV-5 receptor as ASCT2, a neutral amino acid transporter in Japanese macaques. Taken together, we conclude that the causative agent of hemorrhagic syndrome occurred at the external breeding facility for NIPS was SRV-5.

Hepatic neuroendocrine carcinoma in a Japanese macaque (Macaca fuscata).

Primary neuroendocrine neoplasm of the liver is extremely rare in both humans and non-human primates. The present report describes the clinical and pathological findings of an aged Japanese macaque (Macaca fuscata) with hepatic neuroendocrine carcinoma. To our knowledge, this is the first report of hepatic neuroendocrine neoplasm in macaques.

Hepatocellular carcinoma with intracranial metastasis in a Japanese macaque (Macaca fuscata).

BACKGROUND: A 23-year-old male Japanese macaque (Macaca fuscata) showed left ptosis, which progressed to exophthalmos. METHODS: The macaque underwent a clinical examination, CT and MRI, and was euthanized. Necropsy and histopathological examination were performed after euthanasia. RESULTS: The CT revealed and MRI confirmed an intracranial mass at the skull base with orbital extension. At necropsy, there were a large hepatic mass and an intracranial mass compressing the left temporal lobe of the brain. Histopathological and immunohistological examinations revealed that the masses were hepatocellular carcinoma (HCC) and a metastatic lesion. In both the primary and metastatic lesions, neoplastic hepatocytes were arranged mainly in a trabecular pattern. Immunohistochemically, the tumor cells were positive for cytokeratin (AE1/AE3 and CAM5.2) and hepatocyte paraffin 1 and negative for cytokeratin 7 and 20 and vimentin. CONCLUSION: To our knowledge, this is the first case report of HCC with intracranial metastasis in a macaque.

Epidemiological Surveillance of Lymphocryptovirus Infection in Wild Bonobos.

Lymphocryptovirus (LCV) is one of the major gena in the herpesvirus family and is widely disseminated among primates. LCVs of human and rhesus macaques are shown to be causative agents of a number of malignant diseases including lymphoma and carcinoma. Bonobos (Pan paniscus) are highly endangered and the least studied species of the great apes. Considering the potential pathogenicity of the LCV that might threaten the fate of wild bonobos, population-based epidemiological information in terms of LCV prevalence in different location of Bonobo's habitats will help propose improved conservation strategies for the bonobos. However, such data are not available yet because it is very difficult to collect blood samples in the wild and thus virtually impossible to conduct sero-epidemiological study on the wild ape. In order to overcome this issue, we focused on evaluating anti-LCV IgA in the feces of bonobos, which are available in a non-invasive manner. Preliminary study showed that anti-LCV IgA but not IgG was efficiently and reproducibly detected in the feces of captive chimpanzees. It is noteworthy that the fecal IgA-positive individuals were seropositive for both anti-LCV IgG and IgA and that the IgA antibodies in both sera and feces were also detectable by Western blotting assay. These results indicate that the detection of fecal anti-LCV IgA is likely a reliable and feasible for epidemiological surveillance of LCV prevalence in the great apes. We then applied this method and found that 31% of wild bonobos tested were positive for anti-LCV IgA antibody in the feces. Notably, the positivity rates varied extensively among their sampled populations. In conclusion, our results in this study demonstrate that LCV is highly disseminated among wild bonobos while the prevalence is remarkably diverse in their population-dependent manner.

Emergence of infectious malignant thrombocytopenia in Japanese macaques (Macaca fuscata) by SRV-4 after transmission to a novel host.

We discovered a lethal hemorrhagic syndrome arising from severe thrombocytopenia in Japanese macaques kept at the Primate Research Institute, Kyoto University. Extensive investigation identified that simian retrovirus type 4 (SRV-4) was the causative agent of the disease. SRV-4 had previously been isolated only from cynomolgus macaques in which it is usually asymptomatic. We consider that the SRV-4 crossed the so-called species barrier between cynomolgus and Japanese macaques, leading to extremely severe acute symptoms in the latter. Infectious agents that cross the species barrier occasionally amplify in virulence, which is not observed in the original hosts. In such cases, the new hosts are usually distantly related to the original hosts. However, Japanese macaques are closely related to cynomolgus macaques, and can even hybridize when given the opportunity. This lethal outbreak of a novel pathogen in Japanese macaques highlights the need to modify our expectations about virulence with regards crossing species barriers.

Intracranial arachnoid cysts in a chimpanzee (Pan troglodytes).

An intracranial arachnoid cyst was detected in a 32-year-old, 44.6-kg, female chimpanzee at the Primate Research Institute, Kyoto University. Magnetic resonance imaging (MRI) and computed tomography (CT) were performed and the cognitive studies in which she participated were reviewed. MRI revealed that the cyst was present in the chimpanzee's right occipital convexity, and was located in close proximity to the posterior horn of the right lateral ventricle without ventriculomegaly. CT confirmed the presence of the cyst and no apparent signs indicating previous skull fractures were found. The thickness of the mandible was asymmetrical, whereas the temporomandibular joints and dentition were symmetrical. She showed no abnormalities in various cognitive studies since she was 3 years old, except a different behavioural pattern during a recent study, indicating a possible visual field defect. Detailed cognitive studies, long-term observation of her physical condition and follow-up MRI will be continued.

Malignant NK/T-cell lymphoma associated with simian Epstein-Barr virus infection in a Japanese macaque (Macaca fuscata).

A case of spontaneous malignant lymphoma in a Japanese macaque (Macaca fuscata) was pathologically, etiologically and virologically studied. Nasal cavity was involved in the neoplastic lesions in addition to lymphoid and visceral tissues. Histopathological analyses revealed the presence of neoplastic cells classified into histiocytic Hodgkin-like cells and Reed-Sternberg-like cells. Histiocytic Hodgkin-like cells were CD16+ and CD20+, and the CD16+ cells were also positive for simian Epstein-Barr virus (sEBV)-encoded early RNA transcripts. RS-like cells were negative for CD3, CD16 and CD20. Antibodies to early antigen of sEBV were detected, while antibodies to simian T-cell leukemia virus-1 were negative. The case may correspond to EBV-associated nasal type NK/T-cell lymphoma in humans rather than Hodgkin lymphoma.

Changes in testicular and nipple volume related to age and seasonality in Japanese macaques (Macaca fuscata), especially in the pre- and post-pubertal periods.

We investigated, longitudinally and cross-sectionally, age and seasonal change in both the testis and nipple volume of Japanese macaques (Macaca fuscata) in relation to concentration profiles of gonadal steroids: testosterone (T) in males and progesterone (P) in females. Testicular volume (TV) and nipple volume (NV) showed rapid growth at puberty, 4.5 and 3.5 years of age in males and females, respectively, but in both sexes there were precocious individuals. The testis as a whole matures at about 10 years of age. TV change is closely related to T concentration profile. The pattern of TV change is composed of maturation and seasonal effects, with individual variation evident mainly in the latter. Some individuals show a simple pattern consisting of one peak in the breeding season (from summer to winter) and one trough in the non-breeding season. Other individuals exhibit a more complicated pattern composed of two or more peaks and troughs before and during the breeding season. The nipple matures at about 7 years but it is difficult to determine the exact maturational age as there are many confounding factors relating to NV. NV shows seasonal fluctuations similar to that of TV. Many animals have periods of substantial growth whereas others do not. The NV in adults from 10 to 25 years does not appear to change much with age, but animals older than 25 years of age have significantly smaller nipples. Seasonal fluctuation in NV mirrors that of the P level. Considered to be controlled by estrogen and P, the NV is a good indicator of the physiological status of reproduction, with its peak about 2 weeks earlier than that of P, that is, at the mid-follicular phase. NV and P level show a similar pattern in pregnancy; from conception, indicated by a P peak, NV and P concentration first decrease, then they increase until peri-parturition and slowly decrease again until the next breeding season.

Long-term treatment effects of Pueraria mirifica phytoestrogens on parathyroid hormone and calcium levels in aged menopausal cynomolgus monkeys.

To determine the effect of Pueraria mirifica (PM) on serum parathyroid hormone (PTH) and calcium levels on aged menopausal monkeys (Macaca fascicularis), subjects were treated with 10, 100, or 1,000 mg/day of PM. Blood samples were collected every 5 days for 30, 90, and 60 days during pre-treatment, treatment, and post-treatment periods, respectively. Sera were assayed for PTH, estradiol, and calcium levels. PM-1,000 had the strongest effect on the decrease in PTH (0.001

In vitro aging of macaque adherent cells: similar pattern of cellular aging between human and macaque.

To explore new models for human cellular aging as well as to evaluate aging of the macaques, profiles of cellular aging in macaques were studied. Adherent cells were obtained from five Japanese macaques (Macaca fuscata), 14 long-tailed macaques (Macaca fascicularis), two bonnet monkeys (Macaca radiata) and a rhesus monkey (Macaca mulatta). A total of 35 cultures were performed and cell morphology, doubling time, telomere length and telomerase activity were studied. They were classified into three groups; group I: cell strains with a definite replicative life-span (-41 PDLs) (presence of M1), group II: cell strains with a limited extension of replicative life-span (79-106 PDLs) with p53 mutation(s) (presence of M2), and group III: a cell strain with an indefinite replicative life-span (>150 PDLs) with characteristics of transformation. Except for the last group, telomerase activity was not observed. Macaque cells demonstrated three chronological patterns comprising both human and rodent patterns, however, presence of the two limits of proliferation in vitro grants macaque cells to be more appropriate than rodents in both studying human aging and oncogenesis.