modification-treatment-methods-for-wasting-marmoset-syndrome-with

Modification of the Treatment Methods for Wasting Marmoset Syndrome with Tranexamic Acid and Supportive Measures

Wasting marmoset syndrome (WMS), a serious disease in captive common marmoset (Callithrix jacchus) colonies, is associated with a high mortality rate. The ...

RIKEN Center for Brain Science

Chronic diarrhea in laboratory-bred marmosets poses a serious health problem during experiments. Despite a growing demand for laboratory-bred experimental marmosets, the mechanisms underlying the development of diarrhea and measures for its treatment and prevention remain unclear. To explore the factors affecting development of chronic diarrhea in laboratory-bred marmosets, the gut microbiota composition (GMC) of 58 laboratory-bred marmosets, including 19 animals with chronic diarrhea, was analyzed using terminal restriction fragment length polymorphism. We found that the GMCs in these animals cluster into two groups that differ significantly in rate of chronic diarrhea (56.5% in one group, Cluster 1, and 17.1% in Cluster 2). Additionally, a higher α-diversity and a lower proportion of Bifidobacterium spp. according to quantitative PCR was found the animals in the Cluster 1 than in those in Cluster 2. Taken together, our findings indicate that there is a relationship between GMC and development of chronic diarrhea in laboratory-bred marmosets. This is the first study to highlight the potential of assessing GMC in relation to development of chronic diarrhea in laboratory-bred marmosets.

Comparison of gut microbiota composition between laboratory-bred marmosets (Callithrix jacchus) with chronic diarrhea and healthy animals using terminal restriction fragment length polymorphism analysis.

Although wasting marmoset syndrome (WMS) is one of the biggest problems facing captive marmoset colonies, the mechanisms underlying its pathogenesis remain unclear. In our clinical experience, it is difficult to cure WMS-affected marmosets with severe hypoalbuminemia. Thus, the mechanisms underlying hypoalbuminemia in WMS must be understood. In the present study, we investigated whether intestinal protein loss, a known reason for hypoalbuminemia, occurs in this disease. Fecal α1-proteinase inhibitor (α1-PI, also known as α1-antitrypsin) has been used to diagnose intestinal protein loss in other species. To develop an assay system for this protein, marmoset α1-PI was purified from plasma and antibodies against it were developed using the purified protein. Using the antibodies, a sandwich enzyme-linked immunosorbent assay (ELISA) to measure marmoset α1-PI was developed, and its detection sensitivity for fecal samples was ∼20-fold higher than that of a commercial kit for human α1-PI. From this ELISA, the reference intervals for serum and feces of healthy marmosets were 0.87-1.85 mg/ml and 0.53-395.58 μg/g, respectively. The average concentrations of α1-PI in serum and feces of seven WMS-affected marmosets were 1.17 mg/ml and 1357.58 μg/g, respectively. Although there were no significant differences in the serum concentrations between healthy and WMS-affected marmosets, the fecal concentrations were significantly higher in WMS-affected marmosets than in healthy individuals, suggesting that intestinal protein loss occurs in WMS. Intestinal protein loss of WMS-affected marmosets was significantly attenuated with treatment, suggesting that it is one of the mechanisms involved in the hypoalbuminemia observed in WMS.

Measurement of the α1-proteinase inhibitor (α1-antitrypsin) of common marmoset and intestinal protein loss in wasting syndrome.

Germ-free (GF) mice are useful models for the examination of host-microbe interactions in health and disease. We recently reported on the maintenance of individual GF mice for more than 1 year in a sealed positive-pressure cage. However, no useful system exists to automatically record basic behavioral patterns, such as activity and the intake of water and food, under GF status. In this study, we examined basic behavior by combining the sealed positive-pressure cage with a behavioral monitoring system and observed the gross morphology of GF mice at 4 weeks and 8 months of age. GF mice exhibited cecal enlargement and had lower body and adipose tissue weights compared with age-matched specific pathogen-free (SPF) mice. Although both strains had similar circadian rhythms, GF mice exhibited decreased activity compared with age-matched SPF mice. GF mice also exhibited increased levels of water intake compared with age-matched SPF mice. Although GF mice demonstrated decreased food intake levels at the age of 4 weeks, they exhibited increased food intake levels compared with age-matched SPF mice at the age of 8 months. The present research indicates that automated measurement systems that record the basic behaviors of GF mice for long periods are useful for the acceleration of the study of metabolic functions and host-microbe interactions.

New system to examine the activity and water and food intake of germ-free mice in a sealed positive-pressure cage.

Treatment of epilepsy remains difficult because patients suffer from pharmacoresistant forms of the disease and drug side-effects. Thus, there is an urgent need to identify not only new antiepileptic drug candidates but also novel epileptic animal models. Here, we characterize seizures induced with kainic acid (KA) in the common marmoset (Callithrix jacchus). Adult marmosets received 0.1, 1, or 10 mg/kg of KA subcutaneously. All animals exhibited early convulsive behavior (seizure scores of I and II on the Racine scale). Seizure scores were low at lower KA doses, but the highest dose of KA tested triggered generalized seizures (scores IV and V on the Racine scale). We next performed preliminary evaluation of the efficacy of the antiepileptic drug diazepam. This drug at 1 mg/kg (delivered subcutaneously) prevented 10 mg/kg KA-induced stage V seizures. KA administration to marmosets reliably triggers generalized seizures; therefore, the marmoset is a useful animal model in which to analyze the seizures of a nonhuman primate brain and to develop new treatments for epilepsy.

Kainic acid-induced seizures in the common marmoset.

Desbuquois dysplasia (DD) type 1 is a rare skeletal dysplasia characterized by a short stature, round face, progressive scoliosis, and joint laxity. The causative gene has been identified as calcium-activated nucleotidase 1 (CANT1), which encodes a nucleotidase that preferentially hydrolyzes UDP to UMP and phosphate. In this study, we generated Cant1 KO mice using CRISPR/Cas9-mediated genome editing. All F0 mice possessing frameshift deletions at both Cant1 alleles exhibited a dwarf phenotype. Germline transmission of the edited allele was confirmed in an F0 heterozygous mouse, and KO mice were generated by crossing of the heterozygous breeding pairs. Cant1 KO mice exhibited skeletal defects, including short stature, thoracic kyphosis, and delta phalanx, all of which are observed in DD type 1 patients. The glycosaminoglycan (GAG) content and extracellular matrix space were reduced in the growth plate cartilage of mutants, and proliferating chondrocytes lost their typical flat shape and became round. Chondrocyte differentiation, especially terminal differentiation to hypertrophic chondrocytes, was impaired in Cant1 KO mice. These findings indicate that CANT1 is involved in the synthesis of GAG and regulation of chondrocyte differentiation in the cartilage and contribute to a better understanding of the pathogenesis of DD type 1.

CANT1 deficiency in a mouse model of Desbuquois dysplasia impairs glycosaminoglycan synthesis and chondrocyte differentiation in growth plate cartilage.

Fecal microbiota in seven different monogastric animal species, elephant, horse, human, marmoset, mouse, pig and, rat were compared using the same analytical protocol of 16S rRNA metagenome. Fecal microbiota in herbivores showed higher alpha diversity than omnivores except for pigs. Additionally, principal coordinate analysis based on weighted UniFrac distance demonstrated that herbivores and pigs clustered together, whereas other animal species were separately aggregated. In view of butyrate- and lactate-producing bacteria, predominant genera were different depending on animal species. For example, the abundance of Faecalibacterium, a known butyrate producer, was 8.02% ± 3.22% in human while it was less than 1% in other animal species. Additionally, Bifidobacterium was a predominant lactate producer in human and marmoset, while it was rarely detected in other omnivores. The abundance of lactate-producing bacteria in herbivores was notably lower than omnivores. On the other hand, herbivores as well as pig possess Fibrobacter, a cellulolytic bacterium. This study demonstrated that fecal microbiota in herbivorous animals is similar, sharing some common features such as higher alpha diversity and higher abundance of cellulolytic bacterium. On the other hand, omnivorous animals seem to possess unique fecal microbiota. It is of interest that pigs, although omnivore, have fecal microbiota showing some common features with herbivores.

Comparison of the fecal microbiota of two monogastric herbivorous and five omnivorous mammals.

Wasting marmoset syndrome (WMS) is a serious disease in captive common marmoset (Callithrix jacchus) colonies. Because of the high mortality rates, elucidation of the underlying mechanisms is essential. In this study, we compared the histopathology, the number of each epithelial cell in the jejunum and colon, and the expression patterns of some molecular markers between healthy and WMS-affected marmosets. Atrophy of villi in the jejunum and mononuclear cell infiltration in the lamina propria were observed in the intestinal tract of WMS-affected marmosets. Although the numbers of transient amplifying cells and tuft cells were increased, the number of goblet cells was obviously decreased in the jejunum and colon of WMS-affected marmosets compared to healthy marmosets. In addition, the number of enterocytes in the jejunum was decreased in WMS animals. There was no apparent difference in the numbers of stem cells, enteroendocrine cells, or Paneth cells. The expression of β-catenin and Tcf7l2 was increased in WMS, and the co-existence of β-catenin and Tcf7l2/Cyclin D1 was observed around the crypts in WMS-affected marmosets. These findings suggest that cell proliferation continues, but cell differentiation is halted in the intestinal tract due to the enhanced β-catenin/Tcf7l2/Cyclin D1signaling pathway in WMS, which results in malfunction of the villus and mucosa.

Reduced differentiation of intestinal epithelial cells in wasting marmoset syndrome.

Laboratory animals are used for scientific research in various fields. In recent years, there has been a concern that the gut microbiota may differ among laboratory animals, which may yield different results in different laboratories where in-vivo experiments are performed. Our knowledge of the gut microbiota of laboratory-reared common marmosets (Callithrix jacchus) is limited; thus, in this study, we analyzed the daily changes in fecal microbiome composition, individual variations, and effects of the birth facility in healthy female laboratory-reared marmosets, supplied by three vendors. We showed that the marmoset fecal microbiome varied among animals from the same vendor and among animals from different vendors (birth facility), with daily changes of approximately 37%. The fecal microbiome per vendor is characterized by alpha diversity and specific bacteria, with Bifidobacterium for vendor A, Phascolarctobacterium for vendor B, and Megamonas for vendor C. Furthermore, we found that plasma progesterone concentrations and estrous cycles were not correlated with daily fecal microbiome changes. In contrast, animals with an anovulatory cycle lacked Megamonas and Desulfovibrio bacteria compared to normal estrous females. This study suggests that the source of the animal, such as breeding and housing facilities, is important for in-vivo experiments on the marmoset gut microbiota.

Individual variations and effects of birth facilities on the fecal microbiome of laboratory-bred marmosets (Callithrix jacchus) assessed by a longitudinal study.

Article	Year
Comparison of gut microbiota composition between laboratory-bred marmosets (Callithrix jacchus) with chronic diarrhea and healthy animals using terminal restriction fragment length polymorph... Microbiology and immunology\| PubMed ID: 30350387	2018
Measurement of the α1-proteinase inhibitor (α1-antitrypsin) of common marmoset and intestinal protein loss in wasting syndrome. Bioscience reports\| PubMed ID: 31243120	2019
New system to examine the activity and water and food intake of germ-free mice in a sealed positive-pressure cage. Heliyon\| PubMed ID: 31463382	2019
Kainic acid-induced seizures in the common marmoset. Biochemical and biophysical research communications\| PubMed ID: 32115153	2020
CANT1 deficiency in a mouse model of Desbuquois dysplasia impairs glycosaminoglycan synthesis and chondrocyte differentiation in growth plate cartilage. FEBS open bio\| PubMed ID: 32277574	2020
Comparison of the fecal microbiota of two monogastric herbivorous and five omnivorous mammals. Animal science journal = Nihon chikusan Gakkaiho\| PubMed ID: 32285557	2020
Reduced differentiation of intestinal epithelial cells in wasting marmoset syndrome. The Journal of veterinary medical science\| PubMed ID: 33731497	2021
Individual variations and effects of birth facilities on the fecal microbiome of laboratory-bred marmosets (Callithrix jacchus) assessed by a longitudinal study. PloS one\| PubMed ID: 36040908	2022

Kimie Niimi

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