Clostridium ventriculi in a cynomolgus monkey with acute gastric dilatation and rupture: A case report.

Acute gastric dilatation (AGD) is one of the most prevalent and life-threatening diseases in nonhuman primates worldwide. However, the etiology of this syndrome has not been determined. Recently, sudden death occurred in a 7-year-old female cynomolgus monkey with a history of fecal microbiota transplantation using diarrheic stools. The monkey had undergone surgery previously. On necropsy, gastric dilatation and rupture demonstrated a tetrad arrangement on histopathologic examination. On 16S rRNA sequencing, a high population of Clostridium ventriculi was identified in the duodenum adjacent to stomach but not in the colon. This paper is the first report of Clostridium ventriculi infection in a cynomolgus macaque with acute gastric dilatation and rupture.

Germinal Center-Induced Immunity Is Correlated With Protection Against SARS-CoV-2 Reinfection But Not Lung Damage.

Germinal centers (GCs) elicit protective humoral immunity through a combination of antibody-secreting cells and memory B cells, following pathogen invasion or vaccination. However, the possibility of a GC response inducing protective immunity against reinfection following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains unknown. We found GC activity was consistent with seroconversion observed in recovered macaques and humans. Rechallenge with a different clade of virus resulted in significant reduction in replicating virus titers in respiratory tracts in macaques with high GC activity. However, diffuse alveolar damage and increased fibrotic tissue were observed in lungs of reinfected macaques. Our study highlights the importance of GCs developed during natural SARS-CoV-2 infection in managing viral loads in subsequent infections. However, their ability to alleviate lung damage remains to be determined. These results may improve understanding of SARS-CoV-2-induced immune responses, resulting in better coronavirus disease 2019 (COVID-19) diagnosis, treatment, and vaccine development.

Host- and Species-Dependent Quasispecies Divergence of Severe Acute Respiratory Syndrome Coronavirus-2 in Non-human Primate Models.

Recently, newly emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been continuously reported worldwide. However, the precise evaluation of SARS-CoV-2 microevolution in host is very limited because the exact genetic information of infected virus could not be acquired in human researches. In this report, we performed deep sequencing for seed virus and SARS-CoV-2 isolated in eight cynomolgus and rhesus macaques at 3 days postinoculation and evaluated single-nucleotide polymorphisms (SNPs) in SARS-CoV-2 by variant analysis. A total of 69 single-nucleotide variants (SNVs) were present in the 5'-untranslated region (UTR), 3'-UTR, ORF1ab, S, ORF3a, ORF8, and N genes of the seed virus passaged in VERO cells. Between those present on the seed virus and those on each SARS-CoV-2 isolated from the lungs of the macaques, a total of 29 variants was identified in 4 coding proteins (ORF1ab, S, ORF3a, and N) and non-coding regions (5'- and 3'-UTR). Variant number was significantly different according to individuals and ranged from 2 to 11. Moreover, the average major frequency variation was identified in six sites between the cynomolgus monkeys and rhesus macaques. As with diverse SNPs in SARS-CoV-2, the values of viral titers in lungs were significantly different according to individuals and species. Our study first revealed that the genomes of SARS-CoV-2 differ according to individuals and species despite infection of the identical virus in non-human primates (NHPs). These results are important for the interpretation of longitudinal studies evaluating the evolution of the SARS-CoV-2 in human beings and development of new diagnostics, vaccine, and therapeutics targeting SARS-CoV-2.

Effect of Human or Mouse IL-7 on the Homeostasis of Porcine T Lymphocytes.

Due to the inconsistent fluctuation of blood supply for transfusion, much attention has been paid to the development of artificial blood using other animals. Although mini-pigs are candidate animals, contamination of mini-pig T cells in artificial blood may cause a major safety concern. Therefore, it is important to analyze the cross-reactivity of IL-7, the major survival factor for T lymphocytes, between human, mouse, and mini-pig. Thus, we compared the protein sequences of IL-7 and found that porcine IL-7 was evolutionarily different from human IL-7. We also observed that when porcine T cells were cultured with either human or mouse IL-7, these cells did not increase the survival or proliferation compared to negative controls. These results suggest that porcine T cells do not recognize human or mouse IL-7 as their survival factor.

Induction of autophagy protects against extreme hypoxia-induced damage in porcine embryo.

In the mammalian female reproductive tract, physiological oxygen tension is lower than that of the atmosphere. Therefore, to mimic in vivo conditions during in vitro culture (IVC) of mammalian early embryos, 5% oxygen has been extensively used instead of 20%. However, the potential effect of hypoxia on the yield of early embryos with high developmental competence remains unknown or controversial, especially in pigs. In the present study, we examined the effects of low oxygen tension under different oxygen tension levels on early developmental competence of parthenogenetically activated (PA) and in vitro-fertilized (IVF) porcine embryos. Unlike the 5% and 20% oxygen groups, exposure of PA embryos to 1% oxygen tension, especially in early-phase IVC (0-2 days), greatly decreased several developmental competence parameters including blastocyst formation rate, blastocyst size, total cell number, inner cell mass (ICM) to trophectoderm (TE) ratio, and cellular survival rate. In contrast, 1% oxygen tension did not affect developmental parameters during the middle (2-4 days) and late phases (4-6 days) of IVC. Interestingly, induction of autophagy by rapamycin treatment markedly restored the developmental parameters of PA and IVF embryos cultured with 1% oxygen tension during early-phase IVC, to meet the levels of the other groups. Together, these results suggest that the early development of porcine embryos depends on crosstalk between oxygen tension and autophagy. Future studies of this relationship should explore the developmental events governing early embryonic development to produce embryos with high developmental competence in vitro.

Impaired Hand Dexterity Function in a Non-human Primate Model with Chronic Parkinson's Disease.

Symptoms of Parkinson's disease (PD) caused by loss of dopaminergic neurons are accompanied by movement disorders, including tremors, rigidity, bradykinesia, and akinesia. Non-human primate (NHP) models with PD play an essential role in the analysis of PD pathophysiology and behavior symptoms. As impairments of hand dexterity function can affect activities of daily living in patients with PD, research on hand dexterity function in NHP models with chronic PD is essential. Traditional rating scales previously used in the evaluation of animal spontaneous behavior were insufficient due to factors related to subjectivity and passivity. Thus, experimentally designed applications for an appropriate apparatus are necessary. In this study, we aimed to longitudinally assess hand dexterity function using hand dexterity task (HDT) in NHP-PD models. To validate this assessment, we analyzed the alteration in Parkinsonian tremor signs and the functionality of presynaptic dopaminergic neuron using positron emission tomography imaging of dopamine transporters in these models. In addition, a significant inverse correlation between HDT and DAT level was identified, but no local bias was found. The correlation with intention tremor signs was lower than the resting tremor. In conclusion, the evaluation of HDT may reflect behavioral symptoms of NHP-PD models. Furthermore, HDT was effectively used to experimentally distinguish intention tremors from other tremors.

Establishment and Characterization of Immortalized Miniature Pig Pancreatic Cell Lines Expressing Oncogenic K-RasG12D.

In recent decades, many studies on the treatment and prevention of pancreatic cancer have been conducted. However, pancreatic cancer remains incurable, with a high mortality rate. Although mouse models have been widely used for preclinical pancreatic cancer research, these models have many differences from humans. Therefore, large animals may be more useful for the investigation of pancreatic cancer. Pigs have recently emerged as a new model of pancreatic cancer due to their similarities to humans, but no pig pancreatic cancer cell lines have been established for use in drug screening or analysis of tumor biology. Here, we established and characterized an immortalized miniature pig pancreatic cell line derived from primary pancreatic cells and pancreatic cancer-like cells expressing K-rasG12D regulated by the human PTF1A promoter. Using this immortalized cell line, we analyzed the gene expression and phenotypes associated with cancer cell characteristics. Notably, we found that acinar-to-ductal transition was caused by K-rasG12D in the cell line constructed from acinar cells. This may constitute a good research model for the analysis of acinar-to-ductal metaplasia in human pancreatic cancer.

Transient Lymphopenia and Interstitial Pneumonia With Endotheliitis in SARS-CoV-2-Infected Macaques.

Using a reliable primate model is critical for developing therapeutic advances to treat humans infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Here, we exposed macaques to high titers of SARS-CoV-2 via combined transmission routes. We observed acute interstitial pneumonia with endotheliitis in the lungs of all infected macaques. All macaques had a significant loss of total lymphocytes during infection, which were restored over time. These data show that SARS-CoV-2 causes a coronavirus disease 2019 (COVID-19)-like disease in macaques. This new model could investigate the interaction between SARS-CoV-2 and the immune system to test therapeutic strategies.

Prevalence and characterization of Clostridium perfringens isolated from feces of captive cynomolgus monkeys (Macaca fascicularis).

Clostridium perfringens is ubiquitous in the environment and the gastrointestinal tract of warm-blooded animals. While part of the gut microbiome, abnormal growth of C. perfringens causes histotoxic, neurologic, and enteric diseases in a variety of animal species, including humans, due to the production of toxins. There is extremely limited information on C. perfringens infection in non-human primates. Presently, 10 strains were successfully isolated from 126 monkeys and confirmed by molecular and biochemical analyses. All isolates were genotype A based on molecular analysis. Alpha toxin was identified in all isolates. Beta 2 toxin was detected in only three isolates. No other toxins, including enterotoxin, beta, iota, epsilon, and net B toxin, were identified in any isolate. All isolates were highly susceptible to ß-lactam antibiotics. Double hemolysis and lecithinase activity were commonly observed in all strains. Biofilm formation, which can increase antibiotic resistance, was identified in 90% of the isolates. The data are the first report the prevalence and characteristics of C. perfringens isolated from captive cynomolgus monkeys.

Chronic Infiltration of T Lymphocytes into the Brain in a Non-human Primate Model of Parkinson's Disease.

Study of interactions between the nervous system and immunity offers insights into the pathogenesis of Parkinson's disease (PD) and potential therapeutic strategies for neurodegenerative diseases. Studies on rodents have revealed regulatory mechanisms of microglial activation and T lymphocyte recruitment in PD. However, the mechanisms underlying chronic T lymphocyte infiltration into the brain after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injection into a non-human primate (NHP) model of PD remain unknown. This study aimed to investigate changes in serum RANTES (regulated on activation, normal T cell expression and secretion) and analyze the chronic infiltration of T lymphocytes into the brain and microglia activation in NHPs at 48 weeks post-MPTP administration. We found selective and local chronic infiltration of CD4+ and CD8+ T lymphocytes, loss of dopaminergic neurons, dopamine transporter expression, chronic normalization of RANTES in the peripheral blood, and altered microglial morphology at 48 weeks after MPTP injection. This study confirms the involvement of CD4+ and CD8+ T lymphocyte infiltration in MPTP-induced NHP models of PD. Additionally, we corroborated previous findings regarding the mechanisms of T lymphocyte-induced neurodegeneration. The findings of chronic infiltration of T lymphocytes in our NHP model of PD provide novel insights into PD pathogenesis and the development of preventive and therapeutic agents.

Idiopathic chronic diarrhea associated with dysbiosis in a captive cynomolgus macaque (Macaca fascicularis).

Chronic inflammatory enteric diseases occur commonly in humans and animals, especially in captive bred macaques. However, information about the etiology of idiopathic chronic inflammatory diarrhea in cynomolgus monkeys is limited. In this paper, we reported the unusual case of idiopathic chronic diarrhea in a captive cynomolgus monkey based on microbial, imaging, and microbiome examinations.

A pilot study on assessment of locomotor behavior using a video tracking system in minipigs.

Pigs are often selected for large animal models including for neuroscience and behavioral research, because their anatomy and biochemistry are similar to those of humans. However, behavioral assessments, in combination with objective long-term monitoring, is difficult. In this study, we introduced an automated video tracking system which was previously used in rodent studies, for use with pig models. Locomotor behaviors (total distance, number of zone transitions, and velocity) were evaluated and their changes were validated by different 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration methods and dosing regimens. Three minipigs (23-29 kg) received subcutaneous or intravenous MPTP, either 1 or 3 times per week. Immediately after MPTP injection, the minipigs remained in a corner and exhibited reduced trajectory. In addition, the total distance travelled, number of zone transitions, and velocity were greatly reduced at every MPTP administration in all the minipigs, accompanying to increased resting time. However, the MPTP-induced symptoms were reversed when MPTP administration was terminated. In conclusion, this automated video-tracking system was able to monitor long-term locomotor activity and differentiate detailed alterations in large animals. It has the advantages of being easy to use, higher resolution, less effort, and more delicate tracking. Additionally, as our method can be applied to the animals' home pen, no habituation is needed.

Transient meiotic arrest maintained by DON (6-diazo-5-oxo-l-norleucine) enhances nuclear/cytoplasmic maturation of porcine oocytes.

The developmental competence of in vitro-matured oocytes is still lower than that of the in vivo-matured oocytes due to precocious meiotic resumption and inappropriate cytoplasmic maturation. Although numerous efforts have been attempted to accomplish better in vitro maturation (IVM) condition, only limited progress has been achieved. Thus, a current study was conducted to examine the effects of 6-diazo-5-oxo-l-norleucine (DON, an inhibitor of hyaluronan synthesis) during the first half period of IVM on nuclear/cytoplasmic maturation of porcine oocytes and subsequent embryonic development. Based on the observation of the nucleus pattern, metaphase II (MII) oocyte production rate in 1 µM DON group was significantly higher than other groups at 44 h of IVM. The 1 µM of DON was suggested to be optimal for porcine IVM and was therefore used for further investigation. Meiotic arrest effect of DON was maximal at 6 h of IVM, which was supported by the maintenance of significantly higher intra-oocyte cAMP level. In addition, increased pERK1/2 levels and clear rearrangement of cortical granules in membrane of MII oocytes matured with DON provided the evidence for balanced meiosis progression between nuclear and cytoplasmic maturation. Subsequently, DON significantly improved blastocyst formation rate, total cell numbers, and cellular survival in blastocysts after parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. Altogether, our results showed for the first time that 1 µM DON can be used to increase the yield of developmentally competent MII oocytes by synchronizing nuclear/cytoplasmic maturation, and it subsequently improves embryo developmental competence.

Increased CD68/TGFß Co-expressing Microglia/ Macrophages after Transient Middle Cerebral Artery Occlusion in Rhesus Monkeys.

The function of microglia/macrophages after ischemic stroke is poorly understood. This study examines the role of microglia/macrophages in the focal infarct area after transient middle cerebral artery occlusion (MCAO) in rhesus monkeys. We measured infarct volume and neurological function by magnetic resonance imaging (MRI) and non-human primate stroke scale (NHPSS), respectively, to assess temporal changes following MCAO. Activated phagocytic microglia/macrophages were examined by immunohistochemistry in post-mortem brains (n=6 MCAO, n=2 controls) at 3 and 24 hours (acute stage), 2 and 4 weeks (subacute stage), and 4, and 20 months (chronic stage) following MCAO. We found that the infarct volume progressively decreased between 1 and 4 weeks following MCAO, in parallel with the neurological recovery. Greater presence of cluster of differentiation 68 (CD68)-expressing microglia/macrophages was detected in the infarct lesion in the subacute and chronic stage, compared to the acute stage. Surprisingly, 98~99% of transforming growth factor beta (TGFß) was found co-localized with CD68-expressing cells. CD68-expressing microglia/macrophages, rather than CD206+ cells, may exert anti-inflammatory effects by secreting TGFß after the subacute stage of ischemic stroke. CD68+ microglia/macrophages can therefore be used as a potential therapeutic target.

Abnormal Mitochondria in a Non-human Primate Model of MPTP-induced Parkinson's Disease: Drp1 and CDK5/p25 Signaling.

Mitochondria continuously fuse and divide to maintain homeostasis. An impairment in the balance between the fusion and fission processes can trigger mitochondrial dysfunction. Accumulating evidence suggests that mitochondrial dysfunction is related to neurodegenerative diseases such as Parkinson's disease (PD), with excessive mitochondrial fission in dopaminergic neurons being one of the pathological mechanisms of PD. Here, we investigated the balance between mitochondrial fusion and fission in the substantia nigra of a non-human primate model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD. We found that MPTP induced shorter and abnormally distributed mitochondria. This phenomenon was accompanied by the activation of dynamin-related protein 1 (Drp1), a mitochondrial fission protein, through increased phosphorylation at S616. Thereafter, we assessed for activation of the components of the cyclin-dependent kinase 5 (CDK5) and extracellular signal-regulated kinase (ERK) signaling cascades, which are known regulators of Drp1(S616) phosphorylation. MPTP induced an increase in p25 and p35, which are required for CDK5 activation. Together, these findings suggest that the phosphorylation of Drp1(S616) by CDK5 is involved in mitochondrial fission in the substantia nigra of a non-human primate model of MPTP-induced PD.

Evaluation of fecal microbiomes associated with obesity in captive cynomolgus monkeys (Macaca fascicularis).

Microorganisms play important roles in obesity; however, the role of the gut microbiomes in obesity is controversial because of the inconsistent findings. This study investigated the gut microbiome communities in obese and lean groups of captive healthy cynomolgus monkeys reared under strict identical environmental conditions, including their diet. No significant differences in the relative abundance of Firmicutes, Bacteroidetes and Prevotella were observed between the obese and lean groups, but a significant difference in Spirochetes (p < 0.05) was noted. Microbial diversity and richness were similar, but highly variable results in microbial composition, diversity, and richness were observed in individuals, irrespective of their state of obesity. Distinct clustering between the groups was not observed by principal coordinate analysis using an unweighted pair group method. Higher sharedness values (95.81% ± 2.28% at the genus level, and 79.54% ± 5.88% at the species level) were identified among individual monkeys. This paper reports the association between the gut microbiome and obesity in captive non-human primate models reared under controlled environments. The relative proportion of Firmicutes and Bacteroidetes as well as the microbial diversity known to affect obesity were similar in the obese and lean groups of monkeys reared under identical conditions. Therefore, obesity-associated microbial changes reported previously appear to be associated directly with environmental factors, particularly diet, rather than obesity.

Reference values of hematological and biochemical parameters in young-adult cynomolgus monkey (Macaca fascicularis) and rhesus monkey (Macaca mulatta) anesthetized with ketamine hydrochloride.

Nonhuman primate models are valuable in biomedical research. However, reference data for clinical pathology parameters in cynomolgus and rhesus monkeys are limited. In the present study, we established hematologic and biochemical reference intervals for healthy cynomolgus and rhesus monkeys anesthetized with ketamine hydrochloride. A total of 142 cynomolgus monkeys (28 males and 114 females) and 42 rhesus monkeys (22 males and 20 females) were selected and analyzed in order to examine reference intervals of 20 hematological and 16 biochemical parameters. The effects of sex were also investigated. Reference intervals for hematological and biochemical parameters were separately established by species (cynomolgus and rhesus) and sex (male and female). No sex-related differences were determined in erythrocyte-related parameters for cynomolgus and rhesus monkey housed in indoor laboratory conditions. Alkaline phosphatase and gamma glutamyltransferase were significantly lower in females than males in both cynomolgus and rhesus monkeys aged 48-96 months. The reference values for hematological and biochemical parameters established herein might provide valuable information for researchers using cynomolgus and rhesus monkeys in experimental conditions for biomedical studies.

A non-human primate model for stable chronic Parkinson's disease induced by MPTP administration based on individual behavioral quantification.

BACKGROUND: The guidelines for applying individual adjustments to macaques according to the severity of behavioral symptoms during 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment were provided to reproduce stable chronic Parkinsonism in a recent study (Potts et al., 2014). But, since there are insufficient guidelines regarding objective severity criteria of individual symptoms for adjustments of MPTP treatment, it is difficult to develop MPTP-induced chronic non-human primate (NHP) models with stable symptoms. NEW METHOD: The individual adjustments of MPTP administration based on results of automatic quantification of global activity (GA) using a video-based tracking system were applied to develop MPTP-PD model. Low-dose (0.2 mg/kg) intramuscular injection was repeated continuously until GA was lower than 8% of baseline Parkinsonian behavior scores. The positron emission tomography imaging were used to follow the longitudinal course of Parkinson's disease (PD). RESULTS: Significant reductions in GA and dopamine transporter activity, along with significant increases in Parkinsonian behavior scores were found from 4 to 48 weeks following the first administration. GA was correlated with the Parkinsonian behavior score. The dopamine transporter activity was correlated with GA and the Parkinsonian behavior score. However, it was not correlated with the total dose of MPTP. Damage of dopaminergic neuronal systems in the basal ganglia was confirmed by immunohistochemistry and Western blot. COMPARISON WITH EXISTING METHOD: This study reinforces previous guidelines regarding production of NHP models with stable Parkinsonian symptoms. CONCLUSIONS: This novel strategy of MPTP administration based on global activity evaluations provides an important conceptual advance for the development of chronic NHP Parkinsonian models.

In vivo study of paraspinal muscle weakness using botulinum toxin in one primate model.

BACKGROUND: It has been generally speculated that paraspinal muscle weakness is related to the spinal degeneration including intervertebral disc failure. The purpose of this study was to investigate the effects of paraspinal muscle weakness induced by the botulinum toxin type-A on the lumbar spine and behavior pattern in an in-vivo primate model which has an upright locomotion similar to that of humans. METHODS: Botox injections into paraspinal muscle of one cynomolgus monkey were conducted biweekly up to 19 weeks at L2-L3, L3-L4 and L4-L5. MRIs were performed for measurement of muscle cross-sectional areas and behavioral data were collected using a high-resolution portable digital video camera. FINDINGS: The cross-sectional areas of the paraspinal muscles at L2-L3, L3-L4 and L4-L5 decreased by 8%, 12% and 8% at 21 weeks after the Botox injection, respectively. Intervertebral disc thickness at L2-L3, L3-L4 and L4-L5 decreased by 6%, 8% and 5% at 21 weeks after initial Botox injection, respectively. After the Botox injections, locomotion and movement activity of the monkey was decreased. The duration of sitting increased from 21% to a maximum of 97% at 9 weeks after the Botox injection, while stance time decreased from 9% to a minimum of 1% at 11 weeks post Botox injection. INTERPRETATION: The findings of this study revealed that paraspinal muscle atrophy affects intervertebral disc morphology and locomotion activity of a primate and may lead to an onset of intervertebral disc degeneration.