Immunohistochemical characterization of integrin alfa 6 in uterus and cervix of the cynomolgus monkeys (Macaca fascicularis) with MfPV3 infection.

BACKGROUND: Cervical cancer is an abnormal growth of cervical tissue epithelial cells due to persistent human papilloma virus (HPV) infection. Cynomolgus monkeys (Macaca fascicularis) can be naturally and spontaneously infected with M. fascicularis Papillomavirus Type 3 (MfPV3), a virus that is phylogenetically closely related to human oncogenic HPV (HPV-16 and HPV-34), and therefore a potentially beneficial for modeling HPV disease. This study aims to evaluate the expression of the integrin alpha 6 (ITGα6) receptor in cynomolgus monkeys spontaneously infected with MfPV3, which this receptor also found in human infected with HPV. METHODS: The study was done on archived Formalin-fixed Paraffin-Embedded (FFPE) samples of uterine and cervix tissue of cynomolgus monkeys. Immunohistochemistry was also performed to quantify the expression levels of ITGα6. RESULTS: The results showed 80% of the samples positive Cervical Intraepithelial Neoplasia (CIN) and increased expression of ITGα6 significantly in Positive-MfPV3 group than negative-MfPV3 group. CONCLUSIONS: This indicated the potential of cynomolgus monkeys as a spontaneous oncogenesis model of PV infection type.

Klebsiella pneumoniae infection in cynomolgus monkeys at primate research center facility in Indonesia.

BACKGROUND: Klebsiella pneumoniae infection in nonhuman primates has been widely reported and causes significant morbidity and mortality. Animal deaths occur routinely at the Primate Research Center of IPB University. The results of necropsy and culture suggested a K. pneumoniae infection. METHODS: A mass health assessment of Cynomolgus monkeys (n = 429) was carried out by physical examination and molecular targeting K. pneumoniae (n = 96), family of Coronaviridae (n = 148) and Paramyxoviridae (n = 148). RESULTS: A total of 49.18% of the animals had clinical symptoms of respiratory disorders, abscesses, trauma, and others. PCR results indicated that 28.57% were positive for K. pneumoniae with 35.71% mortality, while all samples were negative for both virus families. CONCLUSIONS: There have been outbreaks caused by K. pneumoniae and/or K. pneumoniae subsp. pneumoniae. This disease is chronic, infects all of the buildings, and no tendency for disease transmission according to gender and age class.

Age-related cognitive impairment is associated with low serum concentrations of testosterone and CSF levels of amyloid beta 42 in male cynomolgus monkeys (Macaca fascicularis).

Aged memory-impaired cynomolgus monkeys had significantly lower levels of cerebrospinal amyloid (Aß42 ) and serum testosterone compared with young animals and non-memory-impaired controls. Our findings confirm similar findings in the human and substantiate the usefulness of the cynomolgus monkey as a spontaneous model for aging-associated senile dementia of the Alzheimer type.

Molecular detection and identification of Plasmodium spp. isolated from captive-bred cynomolgus monkeys in Bogor, Indonesia.

Background and Aim: Asian macaques are natural hosts of several Plasmodium species. Some monkey malaria parasites may infect humans and cause zoonotic infections. This study was conducted to estimate the prevalence of monkey malaria parasites in Bogor, Indonesia, based on molecular detection and identification, particularly in cynomolgus monkeys, which have a wide geographic distribution and share extensive habitats with humans. These data are needed to evaluate the status of simian malaria among macaques in Bogor and to study the potential risks to human health. These updated data will provide sufficient information for implementing malaria control strategies in the future and for developing a potential malaria vaccine using monkeys as an animal model. Materials and Methods: Blood samples of 274 cynomolgus monkeys (Macaca fascicularis) were collected and identified using microscopy. DNA was extracted from positive blood samples and analyzed using polymerase chain reaction (PCR) to amplify the small subunit ribosomal RNA (SSU rRNA) target gene using consensus primers for Plasmodium species. The PCR-positive samples were then nucleotide-sequenced using commercial sequencing services, analyzed using the BioEdit program, and aligned using Basic Local Alignment Search Tool from the National Center for Biotechnology Information. Phylogenetic trees were constructed using MEGA 11.0 and the neighbor-joining (NJ) method to determine the kinship of Plasmodium. Bootstrapping was performed using 500 replicates to assess the robustness of tree topologies. Results: Thirty-eight of the 274 microscopically positive samples for Plasmodium spp. were also positive using PCR, resulting in a 1640 bp amplicon. Further, analysis using nucleotide sequencing confirmed that these positive samples were Plasmodium inui with more than 99% nucleotide identity compared to GenBank sequences. Phylogenetic tree analysis of the SSU rRNA partial gene showed that all our isolates clustered and were closely related to a P. inui strain isolated from cynomolgus macaques in South China in 2011. Conclusion: P. inui is the predominant malaria parasite in cynomolgus monkeys from Bogor.

Comparison of diurnal biting activity, life table, and demographic attributes of Aedes albopictus (Asian tiger mosquito) from different urbanized settings in West Java, Indonesia.

The Asian tiger mosquito, Aedes albopictus has well-adaptive behavior to environmental changes, including human urbanization, and has an essential role as the main vector of important pathogenic arboviruses. This study aims to analyze the biology and life table of the Ae. albopictus populations collected from urban and peri-urban areas of Bogor, West Java, Indonesia under laboratory conditions. Mosquito eggs collection was carried out in urban and peri-urban areas using ovitraps. The observation of the life table experiment that followed the development of Ae. albopictus started from the emergence of the first individual to the last surviving individual. Several biological parameters comparing Ae. albopictus from two collection sites based on life table analysis were shown to be significantly different. Biting activity of all mosquitoes from urban and peri-urban areas showed a clear bimodal activity with morning peak at 09:00-10.00 and evening peak at 16:00-17:00. Ae. albopictus from the urban area have higher fecundity, considerably longer lifespan, more gonotrophic cycles, and a higher net reproduction rate (R0) than Ae. albopictus from the peri-urban area. These findings will provide valuable information about the well-adapted Ae. albopictus in urban areas and assist in providing basic reproductive data to improve vector control and current surveillance strategies especially in the study area.

Novel polymorphisms of dopa decarboxylase gene and their association with lamb quality traits in Indonesian sheep.

OBJECTIVE: This study aimed to investigate the polymorphisms of the dopa decarboxylase (DDC) gene and association analysis with lamb quality and expression quantification of the DDC gene in phenotypically divergent Indonesian sheep. METHODS: The totals of 189 rams with an average body weight of 24.12 kg at 10 to 12 months were used to identify DDC gene polymorphism using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Among 189 rams, several rams representing various sheep genotypes were used for an association study between genotypes and phenotypic traits with proc general linear model (GLM) analysis. In addition, the gene expression analysis of the DDC mRNA in the phenotypically divergent sheep population was analyzed using quantitative reverse-transcription PCR. RESULTS: The DDC gene (g. 5377439 G>A) showed polymorphisms that indicated three genotypes: AA, AG, and GG. The DDC gene polymorphism was significantly associated (p≤0.05) with carcass characteristics including carcass percentage, carcass length, hot and cold carcass; physical properties of lamb quality including pH value; retail cut carcass; fatty acid composition such as fat content, pentadecanoic acid (C15:0), tricosylic acid (C23:0), lignoceric acid (C24:0), oleic acid (C18:1n9c), elaidic acid (C18:1n9t), nervonic acid (C24:1), linoleic acid (C18:2n6c), arachidonic acid (C20:4n6), cervonic acid (C22:6n3); and mineral content including potassium (K). The GG genotype of the DDC gene had the best association with lamb quality traits. The DDC gene expression analysis mRNA showed no significant difference (p≥0.05) between lamb quality traits. CONCLUSION: The DDC gene could be used as a potential candidate gene to improve lamb quality.

Diversity of mosquito species and potential arbovirus transmission in long-tailed macaque (Macaca fascicularis) breeding facilities.

Background and Aim: Mosquito-borne viral infections are diseases that reduce human and animal health levels. Their transmission involves wildlife animals as reservoirs and amplifying hosts, including long-tailed macaques (Macaca fascicularis), and potentially transmits to humans and vice versa. This study aimed to determine the species diversity, richness, and biting activity of mosquitoes in a long-tailed macaque breeding area facility and discover the presence of Flavivirus and Alphavirus as the two main arboviruses reported to infect macaques. Materials and Methods: Human landing catch, light trap, and sweep net methods were used for mosquito collection around long-tailed macaques cages at parallel times for 12 h (18:00-06:00) for 12 nights. Mosquito species were identified to the species level based on the morphological identification key for Indonesian mosquitoes. Mosquito diversity was analyzed by several diversity indices. Mosquitoes caught using the human landing catch method were pooled based on mosquito species for viral ribonucleic acid extraction. Reverse transcription-polymerase chain reaction (RT-PCR) detected the non-structural protein 5 of the Flavivirus region and the non-structural protein 4 of the Alphavirus region. This study used the man-hour density and man-biting rate formulas for mosquito density. Results: Ten mosquito species were collected, namely, Aedes albopictus, Anopheles aconitus, Anopheles minimus, Anopheles vagus, Armigeres foliatus, Armigeres subalbatus, Culex gelidus, Culex hutchinsoni, Culex tritaeniorhynchus, and Culex quinquefasciatus. The number of mosquitoes caught using the light trap method had the highest abundance. In contrast, the number of mosquito species caught using the sweep net method had lower diversity than the other two methods. Seven mosquito species were obtained using the human landing catch method. The mosquito species with the highest density was Cx. quinquefasciatus within the observed densest period from 20:00 to 21:00. Negative results were obtained from RT-PCR testing on five species detected using universal Flavivirus and Alphavirus primers. Conclusion: The occurrence of mosquitoes in long-tailed macaque breeding facilities can be a source of transmission of zoonotic vector-borne diseases between animals and humans and vice versa.

Determination of thick and thin fibres distribution in Sunda porcupine dorsal skin (Hystrix javanica) using Picrosirius red staining.

The complexity of the Sunda porcupine skin has become an important topic due to the unique characteristics of its quill follicles. The structure and chemical composition of the skin has affected many physiological and other conditions. Generally, quills are larger, stronger and stiffer than hair; therefore, the skin structure needs to adapt to support their physiology. The strength of the skin is determined by its collagen composition and arrangement; therefore, this study aims to analyse the composition and distribution of thick and thin fibres based on the specific characteristics of Sunda porcupine skin under polarized light using picrosirius red staining. The skin samples used were from the thoracodorsal and lumbosacral regions of eight Sunda porcupine adults. The histological staining was carried out using the picrosirius red method, while the samples were observed under a polarized light microscope and analysed with software. The results showed that the skin is composed of 36%-65% thick fibres, 20%-35% thin fibres and small amounts of other types with the lumbosacral region having higher compositions of thick and thin fibres than those in the thoracodorsal region. Furthermore, the thoracodorsal and lumbosacral regions have the highest composition of thick fibre in the deeper dermis and quill follicle, respectively. These demonstrated that the complexity of the skin structure of Sunda porcupine due to its quill derivates correlated with its collagen composition and distribution.

Molecular diagnosis with the corresponding clinical symptoms of canine distemper virus infection in javan leopard (Panthera pardus ssp. melas).

Diseases are one of the possible threats to the conservation of wild cat populations. One disease that has been reported to infect and cause death, including in various wildlife, is the canine distemper virus (CDV). Here, we report the first case of CDV in an adolescent melanistic Javan female leopard in Indonesia. We combined the clinical report with the Reverse Transcription PCR (RT-PCR) analyses on the faecal and brain samples of partial nucleoprotein (CDV-N) and hemagglutinin (CDV-H) genes. We also performed analyses of urine, haematology, and blood chemistry. The CDV-H nucleotide sequence confirmed the CDV infection in the female leopard and was clustered to the CDV's Asia 1 genotype. This finding opens an investigating window to analyse the pathogen transmission between animals in wildlife, particularly to support the management of conservation in natural habitats in Indonesia.

Relative white blood cell counts, heterophil-to-lymphocyte ratio, and discovery of blood parasites in wild dugong (Dugong dugon) at Lingayan Island, Toli-toli, Indonesia.

AIM: This study was conducted to investigate the relative white blood cell (WBC) counts and the heterophil-to-lymphocyte (H/L) ratio and to analyze the presence of blood parasites in wild dugongs at Lingayan Island. It is expected that the results of this study could provide additional knowledge about the physiological conditions of wild dugongs in their natural habitats, which can serve as basic data in dugong conservation efforts, especially in Indonesia. MATERIALS AND METHODS: A wild dugong was captured around Lingayan Island. Blood samples were collected from the saphenous vein, and blood smears were prepared immediately. The blood smears were examined for leukocyte identification, calculation of relative WBC counts, and presence of blood parasites. The H/L ratio was calculated based on the obtained relative WBC counts. RESULTS: The relative WBC counts included heterophils 19.4%, lymphocytes 76.4%, and monocytes 3.6%, and the H/L ratio was 0.25. Intraerythrocytic parasites were identified and suspected to be Anaplasma and Babesia. CONCLUSION: This study reports leukocyte values from free-ranging dugongs captured in Lingayan Island, Indonesia. Based on the H/L ratio, the dugong examined, in this study, did not experience chronic stress. However, the discovery of blood parasites could be one of the threatening factors for the dugong population.

Localization of uncoupling protein 1 (UCP-1) in the sebaceous gland of the dorsal region in the Sunda porcupine (Hystrix javanica).

Uncoupling protein 1 (UCP-1) was believed to be an exclusive protein found in the brown adipose tissue of small rodents and humans; however, recent studies show that the expression of UCP-1 protein has been found in the sebaceous glands of the mouse tail and human skin. There are a few reports about the presence of UCP-1 in the sebaceous glands of other rodents, such as the Sunda porcupine (Hystrix javanica), a wild spiny rodent commonly found in Indonesia with a large sebaceous gland. The aim of this study was to identify the presence of UCP-1 in the sebaceous glands on the skin of the Sunda porcupine. The skin from three regions (thoracodorsal, lumbosacral and apex caudal) of eight adult Sunda porcupines was used to detect UCP-1-immunopositive cells through immunohistochemistry. All three regions were found immunopositive to anti-UCP-1 antibody in the sebaceous gland of quill and hair follicles, and the epidermal layer in quill and hair follicles with various intensities. The result of immunohistochemistry revealed that the thoracodorsal and apex caudal region was the most intense immunoreaction followed by the lumbosacral region. These findings proved that the presence of UCP-1 was also identified in the sebaceous glands of other rodent (Hystrix javanica) and regions of the body, which has not been reported previously.

Morphological evaluation of polysaccharide content and collagen composition during cutaneous wound healing in the Sunda porcupine (Hystrix javanica).

Wound healing in the Sunda porcupine is believed to occur quickly, although the wound is large and severe. Wound enclosure involves many processes to restore the lost or damaged skin structure where conjugated polysaccharide-protein and collagen, as the main components deposited in wound tissue to restore it. The aim of this study was to evaluate alteration of polysaccharide contents and collagen in untreated full-thickness wound healing in the thoracodorsal and lumbosacral regions in the Sunda porcupines. Histological analysis was performed by periodic acid Schiff, alcian blue pH 2.5, picrosirius red staining method and Low Vacuum Scanning Electron Microscope (LV-SEM) imaging to obtain the fundamental data of healing process. Wound healing began with re-epithelization followed by progressive wound contraction with 4 overlapping stages in about 30-50 days until the wound closed (21-30 days in thoracodorsal and 30-50 days in lumbosacral). Neutral polysaccharide was more widely distributed compared to the acid polysaccharide in almost all stages of wound healing. The ratio of collagen I to III appeared to be higher in the thoracodorsal region than the lumbosacral region during healing process. LV-SEM imaging showed changes in connective tissue structure in the wound border and granulation tissue which appeared abundant and mixed of thin and thick fiber. In conclusion, cutaneous full thickness wound healing in the Sunda porcupine occurred faster in the thoracodorsal region, which might be correlated to the role of neutral polysaccharide and a high ratio of collagen I to III.

Amyloid Beta1-42 and the Phoshorylated Tau Threonine 231 in Brains of Aged Cynomolgus Monkeys (Macaca fascicularis).

Pathological hallmarks indicative of Alzheimer's disease (AD), which are the plaques of amyloid beta1-42 and neurofibrillary tangles, were found in brain of aged cynomolgus monkey. The aim of this study was to investigate if aged monkeys exhibiting spatial memory impairment and levels of biomarkers indicative of AD, had brain lesions similar to human patients suffering from senile dementia. Generating immunohistochemistry technique to biomarkers of amyloid beta1-42 and the phosphorylated tau 231, our study assessed the amyloidopathy, such as indicative to the senile plaques and cerebral amyloid angiopathy, and the tauopathy, to possible neurofibrillary tangles. Six aged monkeys were selected based on their spatial memory performance and profile of biomarkers of AD, divided equally to affected aged subject - with Memory-affected and low amyloid level, and aged with higher performance in memory and amyloid, as the age-matched subjects. Using immunohistochemistry, plaques of amyloid beta1-42 were observed in two out of three brains of aged subjects with memory impairment and biomarkers indicative of AD. The cerebral amyloid angiopathy was observed in both aged monkey groups, and unlike in the human, the amyloids were found to deposit in the small veins and capillaries. In one of the affected individuals, phosphorylated tau was positively stained intracellularly of the neurons, indicating a possibility of an early stage of the formation of tangles. These findings add to the body of evidence of the utility of the aged cynomolgus monkeys as a spontaneous model for Alzheimer-related disease.