Experimental respiratory Marburg virus haemorrhagic fever infection in the common marmoset (Callithrix jacchus)

Marburg virus causes a highly infectious and lethal haemorrhagic fever in primates and may be exploited as a potential biothreat pathogen. To combat the infection and threat of Marburg haemorrhagic fever, there is a need to develop and license appropriate medical countermeasures. To determine whether the common marmoset (Callithrix jacchus) would be an appropriate model to assess therapies against Marburg haemorrhagic fever, initial susceptibility, lethality and pathogenesis studies were performed. Low doses of virus, between 4 and 28 TCID₅₀, were sufficient to cause a lethal, reproducible infection. Animals became febrile between days 5 and 6, maintaining a high fever before succumbing to disease between 8 and 11 days postchallenge. Typical signs of Marburg virus infection were observed including haemorrhaging and a transient rash. In pathogenesis studies, virus was isolated from the animals’ lungs from day 3 postchallenge and from the liver, spleen and blood from day 5 postchallenge. Early signs of histopathology were apparent in the kidney and liver from day 3. The most striking features were observed in animals exhibiting severe clinical signs, which included high viral titres in all organs, with the highest levels in the blood, increased levels in liver function enzymes and blood clotting times, decreased levels in platelets, multifocal moderate‐to‐severe hepatitis and perivascular oedema.     

Establishment of lethal inhalational infection with Francisella tularensis (tularaemia) in the common marmoset (Callithrix jacchus)

Susceptibility and lethality studies of inhalational tularaemia were undertaken using the common marmoset ( Callithrix jacchus ) to determine its suitability as a non-human primate model. Pairs of marmosets were exposed to varying challenge doses of Francisella tularensis by the airborne route and monitored for up to 14 days postchallenge (p.c.). Lethal infection was achieved following a retained dose of less than 10 bacterial colony-forming units (CFU). However, precise LD₅₀ determination was not possible. The model was characterized using a target challenge dose of approximately 100 CFU. Increased core body temperature was the first indicator of disease, at approximately 2.5 days p.c. Overt clinical signs were first observed 12–18 h after the temperature increase. Significantly decreased activity was observed after approximately 3 days. All animals succumbed to infection between 4.5 and 7 days p.c. At postmortem examination, gross pathology was evident in the liver, spleen and lungs of all animals and high bacterial numbers were detected in all the organs assessed. Bacteraemia was demonstrated in all animals postmortem. Histopathological observations included severe suppurative bronchopneumonia, severe multifocal pyogranulomatous hepatitis, splenitis and lymphadenitis. Tularaemia disease progression in the common marmoset therefore appears to be consistent with the disease seen in humans and other animal models. The common marmoset may therefore be considered a suitable model for further studies of inhalational tularaemia.     

Development of an acute model of inhalational melioidosis in the common marmoset (Callithrix jacchus)

Studies of inhalational melioidosis were undertaken in the common marmoset (Callithrix jacchus). Following exposure to an inhaled challenge with aerosolized Burkholderia pseudomallei, lethal infection was observed in marmosets challenged with doses below 10 cfu; a precise LD(50) determination was not possible. The model was further characterized using a target challenge dose of approximately 10(2) cfu. A separate pathogenesis time-course experiment was also conducted. All animals succumbed, between 27 and 78 h postchallenge. The challenge dose received and the time to the humane endpoint (1 °C below normal body temperature postfever) were correlated. The first indicator of disease was an increased core body temperature (T(c) ), at 22 h postchallenge. This coincided with bacteraemia and bacterial dissemination. Overt clinical signs were first observed 3-5 h later. A sharp decrease (typically within 3-6 h) in the T(c) was observed prior to humanely culling the animals in the lethality study. Pathology was noted in the lung, liver and spleen. Disease progression in the common marmoset appears to be consistent with human infection in terms of bacterial spread, pathology and physiology. The common marmoset can therefore be considered a suitable animal model for further studies of inhalational melioidosis.     

Induction of endometriosis in the marmoset monkey (Callithrix jacchus)

Endometriosis is an estrogen-dependent gynaecological disease associated with pain and infertility, which occurs in humans and menstruating primates. In this study, the marmoset monkey (Callithrix jacchus), which is a non-menstruating primate with high circulating estrogen levels, was used to test firstly the hypothesis that endometriosis is based on uterine shedding into the peritoneal cavity, secondly to study the pathogenesis of endometriosis due to its estrogenic situation. Female marmoset monkeys (n = 29) were exposed to two different experimental procedures (non-invasive versus invasive) for intrapelvic placement of endometrial cells by uterine flushing over an experimental period of 2-3 years. First endometriotic foci were detected by colour Doppler ultrasound at the bladder, the uterus and the ovaries at the earliest after 4 months of either treatments. However, invasive induction was more effective in terms of the time-course of induction and the number of resulting endometriotic foci. The analysis of the endometriotic foci by histology, immunohistochemistry and molecular techniques allowed a division into two distinct groups: an initial developing stage occurred, which under further treatment led to the second stage of established endometriosis. Both procedures showed a treatment-dependent increase of vascular supply to the endometriotic foci over the experimental period. The invasive method induced the final established stage of endometriosis more rapidly, with the expression of steroid receptors, aromatase, 17betaHSD1 and CD10. Altogether, 72% of the treated marmoset monkeys developed endometriosis under our endometrial reflux protocols. Our data support the theory that endometriosis can be induced artificially in a non-menstruating primate (C. jacchus) by endometrial shedding into the peritoneal cavity. Because the marmoset is a primate with very high peripheral estrogen levels, this offers an interesting model for studying the pathogenesis of this estrogen-dependent disease, as well as for therapeutic impacts on enzymes involved in steroid metabolism.     

Discrete retinal input to the parabrachial complex of a new-world primate, the common marmoset (Callithrix jacchus)

Traditional retinal projections target three functionally complementary systems in the brain of mammals: the primary visual system, the visuomotor integration systems and the circadian timing system. In recent years, studies in several animals have been conducted to investigate the retinal projections to these three systems, despite some evidence of additional targets. The aim of this study was to disclose a previously unknown connection between the retina and the parabrachial complex of the common marmoset, by means of the intraocular injection of cholera toxin subunit b. A few labeled retinal fibers/terminals that are detected in the medial parabrachial portion of the marmoset brain show clear varicosities, suggesting terminal fields. Although the possible role of these projections remains unknown, they may provide a modulation of the cholinergic parabrachial neurons which project to the thalamic dorsal lateral geniculate nucleus.     

Preparation of acute living hippocampal slice from common marmoset (Callithrix jacchus) for synaptic function analysis

We described the preparation of acute living slices from the hippocampus of the neonatal common marmoset (Callithrix jacchus). Slices from a temporal lobe section were prepared quickly using a rotary slicer. By this method, we successfully recorded field potentials, namely, pre-synaptic fiber volley and field excitatory post-synaptic potentials, from the hippocampal CA1 region with conventional electrophysiological techniques, and analyzed the indicators of synaptic function such as input-output curve. This study thus presents an efficient preparation method for acute living hippocampal slice from which synaptic function of the hippocampus in non-human primate can be analyzed.     

The effect of stress on semen reduction in the marmoset monkey (Callithrix jacchus)

This study compared a number of semen parameters of two separategroups of the common marmoset monkey (Callithrix jacchus) inorder to determine the effect of a continuous potentially stressfulsituation on these parameters, and thus on the monkey's reproductiveability. The semen from 16 adult male marmoset monkeys was collectedand analysed to compare semen parameters between a ’normal‘(control) group (n = 9) and a ’blood withdrawn‘(’stress‘) group (n = 7). The semen parameter valuesobserved in the control were: pH 7.51 ± 0.22, volume40.2 ± 27.2 µ1, concentration 27.3 ± l4.8xl04/ul,motility 47.4 ± 15.9%, grade of velocity 3.5 ±1.2, and normal morphological forms 51.8 ± 13.7%. The’blood withdrawn‘ group of marmoset monkeys showedsignificantly lower semen volume and sperm concentration thanthe ’normal‘ group. In addition, total count ofspermatozoa, normal spermatozoa, motile spermatozoa and normalmotile spermatozoa per ejaculate per monkey was significantlyreduced in the ‘blood withdrawn’ group. The semenof these monkeys also revealed a significantly higher percentageof abnormally-shaped sperm heads than the normal group, andcases of impotence and sham ejaculation were recorded. Our studyrevealed that the continuous withdrawal of a small volume ofblood from a group of marmoset monkeys appeared to be stressfulto these monkeys and as a result, influenced their semen parameters,possibly making them less fertile. In addition, electroejaculationwas found to be possibly harmful to the monkey's reproductiveability.     

The facial artery of the common marmoset (Callithrix jacchus)

The facial artery and its ramifications in 7 adult common marmosets (Callithrix jacchus) were studied by the plastic injection method. The findings obtained are discussed in comparison with those for other primates. In the submandibular region, the facial artery arose from the external carotid artery at the height of the atlas via the linguofacial trunk on 10 of the total of 14 sides examined and independently on the other 4 sides. This common trunk always gave rise to the superior thyroid artery. The facial artery passed anterolaterally between the styloglossus muscle and the intermediate tendon of the digastricus muscle, giving off the styloglossal and the submandibular glandular branches, and anteroinferiorly medial to the pterygoideus medialis muscle. In a position anterior to this muscle, the submental artery and masseteric branch were derived. The submental artery gave off the medial pterygoid, the digastric, the cutaneous, the sublingual glandular and the mylohyoid branches, and then continued up to the median line, where it terminated to supply the genioglossus muscle. In the facial region, the facial artery passed anterosuperiorly along the anterior margin of the masseter muscle on 12 sides and away from it forwards on 2 sides, giving off the premasseteric branch in one of these 2 sides. It gave rise to the cutaneous, the buccal and the buccinator branches, the inferior labial artery and the communicating branch with the zygomatic artery. It terminated to divide into the superior labial and the naris lateral arteries, although the latter was lacking on 4 sides. The inferior labial artery gave off the mandibular marginal, the inferior labial marginal and the inferior labial glandular branches and terminated to anastomose with the mental artery. The superior labial artery divided into the superficial and deep branches, each of which continued as a nasal septal branch. The facial artery of the common marmoset usually ascended along the anterior margin of the masseter muscle and did not reach the medial angle of the eye.     

Localization and synthesis of zona pellucida proteins in the marmoset monkey (Callithrix jacchus) ovary

In most species, the zona pellucida (ZP), an extracellular matrix surrounding the mammalian oocyte, is composed of three glycoproteins: ZPA, ZPB and ZPC. Based mainly on results with mice, the site of zona pellucida biosynthesis has been suggested to be exclusively in the oocyte cytoplasm. However, evidence is accumulating that among various species cumulus/granulosa cells may be involved. Because knowledge of ZP biosynthesis in primates is lacking, we used the common marmoset (Callithrix jacchus) to acquire information about the localization and the site of synthesis of ZP proteins in this species. Using antibodies against synthetic ZPA and ZPC peptides, immunoreactivity was found in the marmoset ZP and in surrounding cumulus cells. Interestingly, the amounts of ZPA and ZPC proteins expressed appeared to differ depending on the stage of folliculogenesis. RT-PCR analysis of mRNA from marmoset oocytes and from oocyte-free follicle cells revealed expression of ZPA, ZPB and ZPC in oocytes and in follicle cells of different stages of marmoset monkey folliculogenesis. Our data suggest that the biosynthesis of marmoset ZPA, ZPB and ZPC proteins takes place both in oocytes and in follicle cells of different follicle stages, although the abundance of ZP glycoproteins may differ depending on the individual ZP protein.     

Platelet measurements in the common marmoset, Callithrix jacchus

Platelet counts and mean platelet volumes were determined for 39 male and 32 female adult marmoset whole blood samples and these results are compared with previously published data. There is no evidence of a difference between sexes but there is some evidence of a relationship between platelet count and mean platelet volume in the Callithrix jacchus population studied.     

Comparative experimental subcutaneous glanders and melioidosis in the common marmoset (Callithrix jacchus)

Glanders and melioidosis are caused by two distinct Burkholderia species and have generally been considered to have similar disease progression. While both of these pathogens are HHS/CDC Tier 1 agents, natural infection with both these pathogens is primarily through skin inoculation. The common marmoset (Callithrix jacchus) was used to compare disease following experimental subcutaneous challenge. Acute, lethal disease was observed in marmosets following challenge with between 26 and 1.2 × 10⁸ cfu Burkholderia pseudomallei within 22–85 h. The reproducibility and progression of the disease were assessed following a challenge of 1 × 10² cfu of B. pseudomallei. Melioidosis was characterised by high levels of bacteraemia, focal microgranuloma progressing to non‐necrotic multifocal solid lesions in the livers and spleens and multi‐organ failure. Lethal disease was observed in 93% of animals challenged with Burkholderia mallei, occurring between 5 and 10.6 days. Following challenge with 1 × 10² cfu of B. mallei, glanders was characterised with lymphatic spread of the bacteria and non‐necrotic, multifocal solid lesions progressing to a multifocal lesion with severe necrosis and pneumonia. The experimental results confirmed that the disease pathology and presentation is strikingly different between the two pathogens. The marmoset provides a model of the human syndrome for both diseases facilitating the development of medical countermeasures.     

Background pathology of the common marmoset (Callithrix jacchus) in toxicological studies.

Two hundred common marmosets (Callithrix jacchus) from control groups (100 males, 100 females) of toxicological studies were examined histopathologically to evaluate the spectrum of spontaneous lesions in this species. Frequent findings were extramedullary hematopoiesis affecting adrenal glands, liver, kidney and spleen, cystic Brunner's glands in the duodenum, acute or subacute inflammation in the large intestine and gall bladder, renal mineralization and pigmentation, hepatic Ito-cell vacuolation, microgranuloma and glycogen storage, pituitary cysts, C-cell hyperplasia and lymphocytic foci of the thyroid gland, luteal hyperplasia of the ovary and inflammatory cell foci in various organs.     

Early implantation stages in the marmoset monkey (Callithrix jacchus)

The morphology of the initial stages of implantation in the marmoset monkey (Callithrix jacchus) was studied by obtaining embryos and associated endometrium at timed intervals after ovulation. Estrus cycles were detected by measuring daily levels of plasma progesterone. Following a short follicular phase, circulating levels of progesterone above 20 ng/ml were taken as representing day 1 after ovulation. On this basis, single, twin, and triplet embryos were recovered from six perfused-fixed females on days 13, 16, 19, 23, and 29 after ovulation and prepared in resin for light microscopy. Early implantation stages, 13 and 16 days after ovulation, were characterized by the intrusion of syncytial trophoblast between epithelial cells of the endometrium with minimal cellular damage. Some hyperplasia of epithelium at the margin of the implantation site was evident. The consolidation of the initial attachment was achieved by an increase in syncytial trophoblast underlying the inner cell mass of the embryo which rapidly surrounded and breached maternal capillaries. Although initially separate, the chorions of twin or triplet embyros started to fuse by day 19 after ovulation. This process was complete by day 29 such that embryos shared a common uterine exocoelom surrounded by continuous trophoblast. It was concluded that implantation in the marmoset monkey commenced on days 11–12.5 after ovulation and involved an intrusive mechanism. Although trophoblast penetration of endometrium was superficial, maternal capillaries were tapped at an early stage of implantation. The fusion of chorions of twins and triplets first occurred around day 19 after ovulation.     

Novel monoclonal antibodies recognizing different subsets of lymphocytes from the common marmoset (Callithrix jacchus)

Callithrix jacchus, the common marmoset, is a small new world primate that is considered effective as an experimental animal model for various human diseases. In this study, we generated monoclonal antibodies (mAbs) against common marmoset lymphocytes for immunological studies on the common marmoset. We established five hybridoma clones, 6C9, 10D7, 6F10, 7A4 and 5A1, producing anti-marmoset mAbs against cell surface antigens on marmoset T and/or B lymphocytes. We confirmed that 6C9 and 10D7 antibodies recognized CD45 antigen, and 6F10 antibody recognized CD8 antigen by flow cytometry using marmoset cDNA transfectants. We also tested them for application of immunoprecipitation, Western blot analysis and immunohistochemistry. We found that immunohistochemistry using marmoset spleen sections could be applied with all established mAbs but immunoprecipitation and the Western blot analysis could be applied with 6F10 and 10D7 antibodies but not with the other three mAbs. These results show that these monoclonal antibodies are useful for advancing immunological research on the common marmoset.     

Establishment of novel embryonic stem cell lines derived from the common marmoset (Callithrix jacchus)

The successful establishment of human embryonic stem cell (hESC) lines has inaugurated a new era in regenerative medicine by facilitating the transplantation of differentiated ESCs to specific organs. However, problems with the safety and efficacy of hESC therapy in vivo remain to be resolved. Preclinical studies using animal model systems, including nonhuman primates, are essential to evaluate the safety and efficacy of hESC therapies. Previously, we demonstrated that common marmosets are suitable laboratory animal models for preclinical studies of hematopoietic stem cell therapies. As this animal model is also applicable to preclinical trials of ESC therapies, we have established novel common marmoset ESC (CMESC) lines. To obtain marmoset embryos, we developed a new embryo collection system, in which blastocysts can be obtained every 3 weeks from each marmoset pair. The inner cell mass was isolated by immunosurgery and plated on a mouse embryonic feeder layer. Some of the CMESC lines were cultured continuously for more than 1 year. These CMESC lines showed alkaline phosphatase activity and expressed stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, and TRA-1-81. On the other hand, SSEA-1 was not detected. Furthermore, our novel CMESCs are pluripotent, as evidenced by in vivo teratoma formation in immunodeficient mice and in vitro differentiation experiments. Our established CMESC lines and the common marmoset provide an excellent experimental model system for understanding differentiation mechanisms, as well as the development of regenerative therapies using hESCs.     

Central olfactory connections in the microsmatic marmoset monkey (Callithrix jacchus)

The mammalian primary olfactory system consists of a set of different telencephalic structures, including paleo-, archi-, periarchi- and mesocortical components. We present the first characterisation of the normal and connectional anatomy of the primary olfactory cortex of the common marmoset, a microsmatic simian species increasingly used in primate research. The centrifugal and centripetal bulbar projections were determined by injections of the anterograde and retrograde tracer wheat germ agglutinin-conjugated horseradish peroxidase and fluorescent dyes into the ipsilateral main olfactory bulb. The efferent projections of the marmoset bulb are organised entirely ipsilaterally and are established via a rudimentary medial olfactory tract and the dominant lateral olfactory tract. Target areas are the anterior olfactory nucleus, the entire prepiriform cortex, ventral tenia tecta, periamygdaloid cortex and the rostral part of the entorhinal cortex. The bulbar axons predominantly terminate in the outer part of layer I. The anterior olfactory nucleus receives a weak additional input within layer II and III, which is not found in macrosmatic rodents. Further anterograde labelling was found in the endopiriform nucleus deep under the prepiriform cortex and within an anterolateral strip of the olfactory tubercle. However, control injections into the olfactory tubercle suggest that the marmoset olfactory tubercle receives a bisynaptic olfactory input only. Retrograde labelling after bulb injections revealed that, except for the olfactory tubercle, all primary olfactory cortices contributed to an ipsilateral bulbopetal feedback projection. Like in rodents, the only bulbopetal projection organised bilaterally in the marmoset is maintained by the anterior olfactory nucleus. With few exceptions, the projections of the marmoset olfactory brain are organised similarly to that of the macaque monkey or those of macrosmatic species.     

Gene conversion of the major histocompatibility complex class I Caja‐G in common marmosets (Callithrix jacchus)

Currently, the amount of sequenced and classified MHC class I genes of the common marmoset is limited, in spite of the wide use of this species as an animal model for biomedical research. In this study, 480 clones of MHC class I G locus (Caja‐G) cDNA sequences were obtained from 21 common marmosets. Up to 10 different alleles were detected in each common marmoset, leading to the assumption that the Caja‐G loci duplicated in the marmoset genome. In the investigated population, four alleles occurred more often, giving evidence for higher immunological advantage of these alleles. In contrast to the human non‐classical MHC class I genes, Caja‐G shows high rates of polymorphism at the relevant peptide‐binding sites, despite its phylogenetic relationship to the non‐classical HLA‐G. Our results provide information for better understanding of the immunological properties of the common marmoset and confirm the theory of a gene conversion of the Caja‐G due to its detected plasticity and the absence of any known HLA‐A equivalent.