Kidney cell culture Macaca fascicularis as a candidate for vaccine development and in vitro model.

BACKGROUND: Cell culture is the proliferation of a cell population in vitro by isolating from the original tissue or growing from existing ones. One essential source is the monkey kidney cell cultures which have an essential role in biomedical study. This is due to the significant homology between the human and macaque genomes making these useful for cultivating human viruses, especially enteroviruses, and growing vaccines. METHODS: This study developed cell cultures derived from the kidney of Macaca fascicularis (Mf) and validated its gene expression. RESULTS: The primary cultures were successfully subcultured up to six passages, grew as monolayers, and exhibited epithelial-like morphology. The cultured cells remained heterogeneous in phenotype and they expressed CD155 and CD46 as viral receptors, cell morphology (CD24, endosialin, and vWF), proliferation, also apoptosis markers (Ki67 and p53). CONCLUSIONS: These results indicated that the cell cultures can be used as in vitro model cells for vaccine development and bioactive compound.

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.

Increased expression of GAPDH in cynomolgus monkeys with spontaneous cognitive decline and amyloidopathy reminiscent of an Alzheimer's-type disease is reflected in the circulation.

Previous studies of aging cynomolgus monkeys from our group identified spontaneous age-associated cognitive declines associated with biomarkers and brain lesions reminiscent of Alzheimer's Disease (AD), in a proportion of aged monkeys. However, the molecular mechanisms that underlie the spontaneous amyloid disorders and cognitive declines observed in these affected monkeys have yet to be investigated in detail. Using reverse transcriptase quantitative real time PCR techniques, normalized to the ACTB housekeeping gene, we analyzed the expression patterns of a number of genes which have been implicated in amyloid and tau abnormalities, in well-characterized aged cynomolgus monkeys with cognitive decline. A significantly increased expression of the genes coding for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was found in aged-cognitive decline monkeys compared to age-matched healthy controls. GAPDH has been implicated in several neurodegenerative diseases and interacts with beta amyloid precursor proteins. These findings provide support for the utilization of cynomolgus macaques in translational preclinical research as valid spontaneous models in experimental investigations of the relationships among aging, cognitive decline, and the neuropathy of AD.

Non-invasive surveillance for Plasmodium in reservoir macaque species.

BACKGROUND: Primates are important reservoirs for human diseases, but their infection status and disease dynamics are difficult to track in the wild. Within the last decade, a macaque malaria, Plasmodium knowlesi, has caused disease in hundreds of humans in Southeast Asia. In order to track cases and understand zoonotic risk, it is imperative to be able to quantify infection status in reservoir macaque species. In this study, protocols for the collection of non-invasive samples and isolation of malaria parasites from naturally infected macaques are optimized. METHODS: Paired faecal and blood samples from 60 Macaca fascicularis and four Macaca nemestrina were collected. All animals came from Sumatra or Java and were housed in semi-captive breeding colonies around West Java. DNA was extracted from samples using a modified protocol. Nested polymerase chain reactions (PCR) were run to detect Plasmodium using primers targeting mitochondrial DNA. Sensitivity of screening faecal samples for Plasmodium was compared to other studies using Kruskal Wallis tests and logistic regression models. RESULTS: The best primer set was 96.7 % (95 % confidence intervals (CI): 83.3-99.4 %) sensitive for detecting Plasmodium in faecal samples of naturally infected macaques (n = 30). This is the first study to produce definitive estimates of Plasmodium sensitivity and specificity in faecal samples from naturally infected hosts. The sensitivity was significantly higher than some other studies involving wild primates. CONCLUSIONS: Faecal samples can be used for detection of malaria infection in field surveys of macaques, even when there are no parasites visible in thin blood smears. Repeating samples from individuals will improve inferences of the epidemiology of malaria in wild primates.

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.

Identification and molecular characterization of simian endogenous retrovirus in Macaca fascicularis and Macaca nemestrina from captive breeding facilities in Bogor, Indonesia.

Background and Aim: Endogenous retroviruses (ERVs) found in all vertebrates, including non-human primates (NHPs), are known to be genetically inherited. Thus, recent studies have explored ERVs for human immunodeficiency virus vaccine development using human ERV (HERV) due to the hypervariability of exogenous retroviruses which cause conventional vaccines to be ineffective. HERV was also found to be able to induce an immune response in cancer patients. This study aimed to identify and molecularly characterize ERVs from Indonesian NHPs: Macaca fascicularis and Macaca nemestrina. Then, we described the phylogenetic relationship of these isolates with those of the simian ERVs (SERVs) characterized in other species and countries. Materials and Methods: First, 5 mL of whole blood samples was taken from 131 long-tailed macaques and 58 pig-tailed macaques in captive breeding facilities at Bogor, Indonesia, for DNA extraction. Next, the DNA samples were screened using the SYBR Green real-time polymerase chain reaction (PCR) technique with specific primers for env (simian retroviruses [SRV]1-5 7585U19 and SRV1-5 7695L21). Positive SERV results were those with cycle threshold (CT) values < 24 (CT < 24) and melting temperature (TM) ranges of 80°C-82°C. Then, whole-genome nucleotide sequences from two pig-tailed macaques samples detected as positive SERV were generated using a nucleic acid sequencing technique which utilized the walking primer method. Subsequently, the sequences were analyzed using bioinformatics programs, such as 4Peaks, Clustal Omega, and BLAST (NCBI). Subsequently, a phylogenetic tree was constructed using the neighbor-joining method in MEGA X. Results: SYBR Green real-time PCR amplification results indicated that SERV (Mn B1 and Mn B140910)-positive samples had CT values of 22.37-22.54 and TM of 82°C. Moreover, whole-genome sequences resulted in 7991 nucleotide sequences, comprising long terminal repeat, gag, pro, pol, and env genes identical between the sequenced samples. Furthermore, the phylogenetic tree results indicated that both samples from M. nemestrina had 99%-100% nucleotide identities to the Mn 92227 sample identified at the National Primate Center University of Washington (NaPRC UW) which was imported from Indonesia in 1998, confirmed as a novel SERV strain. The phylogenetic tree results also indicated that although SERV whole-genome nucleotide and env amino acid sequences were clustered with SRV-2 (identity values of 82% and 79%, respectively), they had a 99%-100% nucleotide identity to Mn 92227. Meanwhile, the gag, pro, and pol amino acids were clustered with SRV-1, SRV-3, SRV-4, SRV-5, SRV-8, and SERV/1997, with 82% and 88% identity values. Conclusion: Based on the SYBR Green real-time PCR profiles generated, similarities with Mn 92227 were observed. Subsequent phylogenetic analysis confirmed that both samples (Mn B1 and Mn B140919) from pig-tailed macaques in the country of origin were novel SERV strains at NaPRC UW. Therefore, it could be used in biomedical research on ERVs.

Spontaneous expression of the gene of KI67 and P53 in cynomolgus monkeys infected with papillomavirus.

Background and Aim: Cynomolgus monkeys (Macaca fascicularis) develop spontaneous infection of Papillomavirus (PV); thus, potentially beneficial for modeling human PV (HPV) infection study. Contrary to human origin, infection in cynomolgus monkeys does not always show evident clinical symptoms of cervical cancer. The absence of cervical cancer clinical symptoms leads us to investigate the molecular mechanism of the HPV infection in cynomolgus monkeys. This study aimed to investigate the messenger ribonucleic acid (mRNA) expression levels of KI67 and P53 genes, majorly known as biomarker oncogenesis of PV infection. Materials and Methods: The polymerase chain reaction (PCR) technique was used with MY11/MY09 primer to screen PV in cynomolgus monkey, further grouped as positive-PV and negative-PV infection groups. Real-time quantitative PCR was also applied to quantify the mRNA expression levels of KI67 and P53 genes in animals. Results: Increased expression of mRNA level of KI67 genes was significantly higher in Positive- PV group than negative-PV group. In contrast, the P53 mRNA expression level increased markedly higher in the negative-PV group than in the positive-PV group. Conclusion: Our study describes the potential of cynomolgus monkeys as a spontaneous oncogenesis model of PV infection-type. However, we used a limited number of cancer genetic markers. So, further study of other genetic markers is required to prove that cervical cancer could be developed naturally in cynomolgus monkeys.

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.

Screening and confirmatory testing of MHC class I alleles in pig-tailed macaques.

Pig-tailed macaques (Macaca nemestrina) are a commonly studied primate model of human AIDS. The Mane-A1*084:01 MHC class I allele (previously named Mane-A*10) is important for the control of SIV infection by CD8+ T cells in this model. Validated methods to detect this allele in large numbers of macaques are lacking. We studied this MHC allele using sequence-specific PCRs in 217 pig-tailed macaques and identified 75 (35%) positive animals. We then performed massively parallel pyrosequencing with a universal 568-bp MHC class I cDNA-PCR amplicon for 50 of these 75 macaques. All 50 animals expressed Mane-A1*084:01 or closely related variants of the Mane-A1*084 lineage. Mane-A1*084 transcripts accounted for an average of 20.9% of all class I sequences identified per animal. SIV infection of a subset of these macaques resulted in the induction of SIV-specific CD8+ T cell responses detected by Mane-A1*084:01 tetramers. An average of 19 distinct class I transcripts were identified per animal by pyrosequencing. This analysis revealed 89 new Mane class I sequences as well as 32 previously described sequences that were extended with the longer amplicons employed in the current study. In addition, multiple Mane class I haplotypes that had been inferred previously based on shared transcript profiles between unrelated animals were confirmed for a subset of animals where pedigree information was available. We conclude that sequence-specific PCR is useful to screen pig-tailed macaques for Mane-A1*084:01, although pyrosequencing permits a much broader identification of the repertoire of MHC class I sequences and haplotypes expressed by individual animals.

Construction of A Preliminary Three-Dimensional Structure Simian betaretrovirus Serotype-2 (SRV-2) Reverse Transcriptase Isolated from Indonesian Cynomolgus Monkey.

Simian betaretrovirus serotype-2 (SRV-2) is an important pathogenic agent in Asian macaques. It is a potential confounding variable in biomedical research. SRV-2 also provides a valuable viral model compared to other retroviruses which can be used for understanding many aspects of retroviral-host interactions and immunosuppression, infection mechanism, retroviral structure, antiretroviral and vaccine development. In this study, we isolated the gene encoding reverse transcriptase enzyme (RT) of SRV-2 that infected Indonesian cynomolgus monkey (Mf ET1006) and predicted the three dimensional structure model using the iterative threading assembly refinement (I-TASSER) computational programme. This SRV-2 RT Mf ET1006 consisted of 547 amino acids at nucleotide position 3284-4925 of whole genome SRV-2. The polymerase active site located in the finger/palm subdomain characterised by three conserved catalytic aspartates (Asp90, Asp165, Asp166), and has a highly conserved YMDD motif as Tyr163, Met164, Asp165 and Asp166. We estimated that this SRV-2 RT Mf ET1006 structure has the accuracy of template modelling score (TM-score 0.90 ± 0.06) and root mean square deviation (RMSD) 4.7 ± 3.1Å, indicating that this model can be trusted and the accuracy can be seen from the appearance of protein folding in tertiary structure. The superpositionings between SRV-2 RT Mf ET1006 and Human Immunodeficiency Virus-1 (HIV-1) RT were performed to predict the structural in details and to optimise the best fits for illustrations. This SRV-2 RT Mf ET1006 structure model has the highest homology to HIV-1 RT (2B6A.pdb) with estimated accuracy at TM-score 0.911, RMSD 1.85 Å, and coverage of 0.953. This preliminary study of SRV-2 RT Mf ET1006 structure modelling is intriguing and provide some information to explore the molecular characteristic and biochemical mechanism of this enzyme.

Characterization of Burkholderia pseudomallei from spontaneous melioidosis in a Bornean orangutan.

BACKGROUND AND AIM: Melioidosis is a potentially fatal disease affecting humans and a wide range of animal species; it is often underdiagnosed and underreported in veterinary medicine in Indonesia. This study aimed to characterize morphological and molecular features of Burkholderia pseudomallei, the causative agent of melioidosis which caused the death of a Bornean orangutan. MATERIALS AND METHODS: Pulmonary abscess samples were cultured on several types of media, including Ashdown agar, Ashdown broth, and MacConkey agar. Type three secretion system orf 2 real-time polymerase chain reaction (PCR) and latex agglutination tests were performed to identify the bacteria. Morphological characteristics were compared to all previously published morphotypes. Subsequently, the bacteria were characterized by multilocus sequence typing (MLST) and Yersinia-like flagellum/Burkholderia thailandensis-like flagellum and chemotaxis PCR. The results of the genotyping were afterward compared to all genotypes from Southeast Asia. RESULTS: Multiple morphotypes of B. pseudomallei were perceived during the growth on Ashdown agar. Furthermore, it was identified by MLST that the Type I and Type II morphotypes observed in this study were clones of a single ST, ST54, which is predominantly found in humans and the environment in Malaysia and Thailand, although a very limited number of reports was published in association with animals. Moreover, the E-BURST analysis showed that the ST is grouped together with isolates from Southeast Asian countries, including Malaysia, Thailand, Singapore, and Cambodia. ST54 was predicted to be the founding genotype of several STs from those regions. CONCLUSION: B. pseudomallei ST54 that caused the death of a Bornean orangutan has a distant genetic relationship with other STs which were previously reported in Indonesia, implying a vast genetic diversity in Indonesia that has not been discovered yet.