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.

Fatty acid composition profiling in the dorsal skin of Sunda porcupine (Hystrix javanica).

The fatty acid composition in the skin of Sunda porcupine (Hystrix javanica) is an interesting topic due to the special features of quills, especially in the dorsal region. Therefore, this study aims to analyze the composition of fatty acids in the dorsal region of Sunda porcupine skin. It was conducted using skin samples of the thoracodorsal and lumbosacral regions taken by biopsies and from frozen specimens. The skin lipid was extracted and then derivatized into fatty acid methyl ester before analyzing with gas chromatography mass spectrometry. The results showed that the skin is composed of up to 25 fatty acids ranging from C12 to C25 with various types but only 16 were found in both regions and sexes. Fatty acids with an antibacterial effect were found abundantly, such as oleic, palmitic, stearic, and linoleic acids. The total abundance in the thoracodorsal region was higher than lumbosacral, while the composition in male was higher than in female. Based on the results, the fatty acid composition in the dorsal skin region of Sunda porcupine consists of at least 16 types ranging from C12-C25. Additionally, the region and sex were observed to contribute significantly to the variation in skin fatty acid composition.

Comparison of Nutritional and Meat Quality Characteristics between Two Primal Cuts from Aceh Cattle in Aceh Province, Indonesia.

The Aceh cattle are local Indonesian beef cattle that are farmed in Aceh Province. This type of cattle is one of the sources of meat for the Aceh people. This study aims to analyze the quality of two primal cuts (longissimus lumborum and semitendinosus muscle) from Aceh cattle based on the muscle microstructure characteristics and MSTN gene expression. This study used a sample of longissimus lumborum and semitendinosus muscles from 18 adult male Aceh cattle with the age of 2-2.5 years and a BCS of 3.24. Muscle samples were obtained shortly after the cattle were slaughtered in slaughterhouses in Banda Aceh and Aceh Besar districts. Muscle microstructure analysis was performed using the HE, Masson's trichrome, and immunohistochemistry staining methods, while the MSTN gene expression analysis was performed using the qPCR method. The analysis of the physical quality of meat includes pH, meat color, fat color, cooking loss, water holding capacity, and WBSF value. The results showed that the area of LL muscle fibers was smaller than that of ST with relatively the same diameter. Both muscles were dominated by fast fibers with a percentage of 82.37% (LL muscle) and 91.80% (ST muscle). The area and composition of the type of muscle fibers are the main factors that influence the tenderness of Aceh beef. A higher distribution of collagen was found in ST muscles than in LL muscles. MSTN gene expression in both muscle types was relatively the same. Aceh cattle have large muscle fibers and are dominated by fast fibers with a high percentage, resulting in a low level of the tenderness of Aceh beef. However, the level of tenderness of Aceh beef is still in accordance with the cooking preparation of original and favorite cuisine of Aceh people.

Viral-derived DNA invasion and individual variation in an Indonesian population of large flying fox Pteropus vampyrus.

Here, we performed next-generation sequencing (NGS) on six large flying foxes (Pteropus vampyrus) collected in Indonesia. Seventy-five virus species in the liver tissue of each specimen were listed. Viral homologous sequences in the bat genome were identified from the listed viruses. This finding provides collateral evidence of viral endogenization into the host genome. We found that two of the six specimens bore partial sequences that were homologous to the plant pathogens Geminiviridae and Luteoviridae. These sequences were absent in the P. vampyrus chromosomal sequences. Hence, plant viral homologous sequences were localized to the hepatocytes as extrachromosomal DNA fragments. Therefore, this suggests that the bat is a potential carrier or vector of plant viruses. The present investigation on wild animals offered novel perspectives on viral invasion, variation, and host interaction.

Radiographic anatomy of the heart of fruit bats.

As the only mammal that can fly, bats have organ systems with a unique morphophysiology. One of the highlights is the heart and blood circulation system, which must be able to meet the needs of blood and oxygen supply when flying. This study examined the radiography of the normal condition of the heart organ in 3 species of fruit bats, namely Cynopterus titthaecheilus, Cynopterus brachyotis and Rousettus leschenaultii using radiological silhouette analysis and clock analogy. The results showed that the heart positions of the three bat species tend to be tilted to the left with the apex moving away from the midsagittal plane. Analysis of intercostal space (ICS) value and vertebral heart score (VHS), and evaluation of radiographic features showed R. leschenaultii has a relatively larger heart size than the other two species. All three bat species have a higher VHS than mammals in general. Radiographic images obtained, and interpretation results show the position, size and normal heart parts of the three bat species. They will be useful in diagnostic efforts related to heart problems in these three species.

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.

Sonoanatomy of female reproductive organ of Sunda porcupine (Hystrix javanica).

The conventional anatomical study of specimens requires cutting processes which destruct the limited specimens. A non-destructive method, namely an ultrasonography, can be used to assess the anatomical organ information of those specimens. The aim of this research is to analyse the macroanatomy of the female reproductive organ in the Sunda porcupine (Hystrix javanica), using ultrasonographical imaging. In this study, four formaldehyde-fixed reproductive organ specimens of the Sunda porcupine were used. A 10-12 MHz linear ultrasound transducer was utilized to provide an imaging format of both longitudinal and transversal views. Photographic images were then used as comparison with a sonographic image. The results show that the ultrasound image of the Sunda porcupine reproductive organ soft tissue was hypoechoic, the lumen and antrum follicles were anechoic, while atretic follicles and the mons pubis were hyperechoic. Generally, the size of the organ was not significantly different between photographical and ultrasonographical imaging (p > .05). In conclusion, ultrasound images can be utilized for anatomical studies of the Sunda porcupine reproductive organs without destructing the specimen.

Mosquito-borne viruses, insect-specific flaviviruses (family Flaviviridae, genus Flavivirus), Banna virus (family Reoviridae, genus Seadornavirus), Bogor virus (unassigned member of family Permutotetraviridae), and alphamesoniviruses 2 and 3 (family Mesoniviridae, genus Alphamesonivirus) isolated from Indonesian mosquitoes.

Mosquitoes transmit many kinds of arboviruses (arthropod-borne viruses), and numerous arboviral diseases have become serious problems in Indonesia. In this study, we conducted surveillance of mosquito-borne viruses at several sites in Indonesia during 2016-2018 for risk assessment of arbovirus infection and analysis of virus biodiversity in mosquito populations. We collected 10,015 mosquitoes comprising at least 11 species from 4 genera. Major collected mosquito species were Culex quinquefasciatus, Aedes albopictus, Culex tritaeniorhynchus, Aedes aegypti, and Armigeres subalbatus. The collected mosquitoes were divided into 285 pools and used for virus isolation using two mammalian cell lines, Vero and BHK-21, and one mosquito cell line, C6/36. Seventy-two pools showed clear cytopathic effects only in C6/36 cells. Using RT-PCR and next-generation sequencing approaches, these isolates were identified as insect flaviviruses (family Flaviviridae, genus Flavivirus), Banna virus (family Reoviridae, genus Seadornavirus), new permutotetravirus (designed as Bogor virus) (family Permutotetraviridae, genus Alphapermutotetravirus), and alphamesoniviruses 2 and 3 (family Mesoniviridae, genus Alphamesonivirus). We believed that this large surveillance of mosquitoes and mosquito-borne viruses provides basic information for the prevention and control of emerging and re-emerging arboviral diseases.

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.

Characteristics of testicular cell development of 5-day-old mice in culture in vitro.

The crude testicular cells (CTCs) contain many cell types, such as Sertoli cells, leydig cells, spermatogonial stem cells (SSCs), spermatocytes, and other somatic testicular cells, that secrete various growth factors needed in spermatogenesis. The objective of this study was to characterize development of 5-day-old mice testicular cells cultured. Crude testicular cells prepared from the testes of 5-day-old male mice were cultured in Dulbecco's Modified Eagle Medium and incubated at 37°C in a 5% CO2 atmosphere for 6 days. The results demonstrated that the testicular cells developed rapidly with a population doubling time (PDT) of 0.63 days and more than 90% of cells were viable after being cultured for 3 days. The number of Sertoli-like cells increased significantly over days 1, 3, and 6 to 22.1%, 34.6%, and 50.1%, respectively. A significant increase was also observed in fibroblast-like cells (15.5% on day 1 to 28.8% on day 3 and to 26.6% on day 6). In contrast, the number of spermatogonia-like cells decreased significantly (54.3%, 30.4%, and 18.7%, on days 1, 3, and 6, respectively). These data indicated that the developmental pattern of the testicular cell in this study might be affected by the niche provided by the cultured testicular cells.

Distribution of Japanese Encephalitis Virus, Japan and Southeast Asia, 2016-2018.

During 2016-2018, we conducted surveillance for Japanese encephalitis virus (JEV) in mosquitoes and pigs in Japan, Thailand, the Philippines, and Indonesia. Phylogenetic analyses demonstrated that our isolates (genotypes Ia, Ib, III, IV) were related to JEV isolates obtained from the same regions many years ago. Indigenous JEV strains persist in Asia.

Extracellular matrix composition of different spleen compartments of fruit bats.

The distribution and composition of extracellular matrix (ECM) of the spleen in two species of fruit bats, namely Cynopterus titthaecheilus and Rousettus leschenaultii, were examined by histochemistry and immunohistochemistry. Reticular fibres accompanied by laminin were identified to make up the splenic stromal network. Types I and III collagen were identified in various spleen compartments with varying intensities. Thin and short elastin fibres were scattered in several parts of the spleen. Visualization of the ECM of the spleen can better demonstrate spleen compartmentalization. The alleged vascular space structure in the fruit bats spleen was the sinus structure that was strengthened by the presence of reticular fibres that limit the sinus basement membrane. The present study identified periellipsoidal lymphoid sheath (PELS)-like structure in fruit bats spleen that had never been identified in mammals before. In addition to describing the structure, this study highlighted the variations in ECM composition of the spleen between species that can provide new insight into the phylogenetic study of spleen morphology.

Detection and isolation of tick-borne bacteria (Anaplasma spp., Rickettsia spp., and Borrelia spp.) in Amblyomma varanense ticks on lizard (Varanus salvator).

Ticks are one of the arthropods that play an important role in the transmission of numerous pathogens to livestock and humans. We investigated the presence of tick-borne bacteria in 23 Amblyomma varanense that fed on a water monitor (Varanus salvator) in Indonesia. Anaplasmataceae and borreliae were detected by PCR in 17.4% and 95.7% of ticks, respectively. "Candidatus Rickettsia sepangensis", spotted fever group of Rickettsia, was detected in 21.7% of ticks. The water monitor is a common reptile that is widely encountered in city areas in Asian countries. Our results suggested that Am. varanense on water monitor in Indonesia harbored several kinds of bacteria.

The spleen morphophysiology of fruit bats.

Spleen is one of the important lymphoid organs with wide variations of morphological and physiological functions according to species. Morphology and function of the spleen in bats, which are hosts to several viral strains without exhibiting clinical symptoms, remain to be fully elucidated. This study aims to examine the spleen morphology of fruit bats associated with their physiological functions. Spleen histological observations were performed in three fruit bats species: Cynopterus titthaecheilus (n = 9), Rousettus leschenaultii (n = 3) and Pteropus vampyrus (n = 3). The spleens of these fruit bats were surrounded by a thin capsule. Red pulp consisted of splenic cord and wide vascular space filled with blood. Ellipsoids in all three studied species were found numerously and adjacent to one another forming macrophages aggregates. White pulp consisted of periarteriolar lymphoid sheaths (PALS), lymphoid follicles and marginal zone. The lymphoid follicle contained a germinal centre and a tingible body macrophage that might reflect an active immune system. The marginal zone was prominent and well developed. This study reports some differences in spleen structure of fruit bats compared to other bat species previously reported and discusses possible physiological implications of the spleen based on its morphology.

Isolation of Pteropine orthoreovirus from Pteropus vampyrus in Garut, Indonesia.

Flying foxes belonging to the genus Pteropus are known to be reservoirs of zoonotic viruses. In this study, we describe the isolation of Pteropine orthoreovirus (PRV) from rectal swab samples of Pteropus vampyrus in Indonesia. PRV is an emerging zoonotic respiratory virus that can be transmitted from bats to humans. Rectal swabs (n = 91) were screened by PCR for PRV and 10 (11%) were positive. Phylogenetic analysis based on nucleotide sequences indicated that the S2, S3, S4, M3, L2, and L3 segments of one isolate (Garut-69) were closely related to previously isolated strains in Indonesia. The remaining gene segments showed both similarity and genetic divergence with other PRV strains, suggesting that re-assortment events had occurred. This is the first report of PRV infection to P. vampyrus in West Java, Indonesia.

The dorsal skin structure contributes to the surface bacteria populations of Sunda Porcupine (Hystrix javanica).

Skin becomes the largest organ in the body and protects its own inner layer. The structure and chemical composition of the skin contribute to skin condition and affect the habitat of certain bacteria. The Sunda Porcupine is one of endemic animals of Indonesia which possesses quill as the main derivate of its skin and as a defence tool against predators. The present study used nine adults (five females and four males) of Sunda Porcupine and aimed to observe the correlation of skin structure with bacterial population at the surface level. The skin was wavy due to the protrusion of quill follicle orifices on the skin surface and formed clusters. The skin of Sunda Porcupine was also wrinkled and had a lot of flakiness. Histologically, the skin was composed of epidermis, dermis, hypodermis and subcutaneous muscle. The quill follicles and their properties were the dominant structure component of the skin. No sweat gland was observed in the skin of the Sunda Porcupine, and sebaceous gland was found only around quill and hair follicles. The bacterias identified in the skin were Staphylococcus aureus, S. epidermidis, Micrococcus sp. and Salmonella sp. When compared, the bacterial population was higher in the lumbosacral region than in the thoracodorsal region, but the difference was not significant. The density of quill clusters was negatively correlated to the bacterial population. It was suggested the structure of the skin has contribution to bacterial population in dorsal trunk of the Sunda Porcupine.

Characterization of Seminiferous Epithelium Stages in the Wild Javan Muntjac (Muntiacus muntjak muntjak) Using the Tubular Morphology Method.

Stages of the seminiferous epithelium of the testis of the wild Javan muntjac (Muntiacus muntjak muntjak) in hard antler period were characterized based on the tubular morphology method. The number and the relative frequencies of seminiferous epithelium stages and the morphometry of germinal cell nuclei were identified microscopically. We identified eight stages of seminiferous epithelium in testicular tissue of the Javan muntjac and found that the relative frequencies of stages I to VIII were 14.87, 15.12, 17.75, 6.87, 7.37, 12.37, 13, and 12.62%, respectively. The diameter of the nuclei of germinal cells varied in each stage of seminiferous epithelium. Diplotene-stage primary spermatocytes had prominent and large nuclei ~8.97 ± 1.0 µm in stages III and IV. Pachytene primary spermatocytes appeared in most stages, except stage IV, whereas leptotene- and diplotene-stage primary spermatocytes were found in stages I and II, and III and IV, respectively. Round spermatids were observed in stages IV to VIII and in stage I but were absent in stages II and III, while elongated spermatids were observed in all stages except stage I. Our findings show that the stages of seminiferous epithelium in the Javan muntjac are similar to those found in neotropical cervids, small ruminants, and other domestic animals.

Nighttime behavioral study of flying foxes on the southern coast of West Java, Indonesia.

Flying foxes are important in the maintenance of forests and diversity. However, knowledge of their behavioral ecology, especially of movement and foraging patterns, which are essential for conservation and management of their populations, are not well known. Therefore, movement behavior of two individuals of Pteropus vampyrus were examined using an Argos telemetry system, and foraging pattern of Pteropus spp. was directly observed, at West Java province, Indonesia in October 2017. The maximum distance between the location at which bats were released and their furthest roost, recorded via satellite telemetry, was approximately 100 km. This reflects the long-distance flight ability of P. vampyrus. Daytime roosting sites and nighttime foraging places consisted of several types of habitats, such as intact forests, agricultural lands, and residential areas. This evidence indicated that there was habitat overlap between humans and bats in West Java province. According to direct observation of the behaviors of flying foxes at two locations within residential areas, various activities such as wing spreading, excretion, fighting, aggressive calls, movement, hanging relax, and hanging alert were found. The number of bat-visits to the trees varied among night hours, and had a positive correlation with the number of fruit dropping. The data obtained in this study have improved our understanding of nighttime behavior and habitat utilization of P. vampyrus, that can be used to support landscape management, species conservation, and disease prevention in regions of Southeast Asia.

Serological and molecular prevalence of equine piroplasmosis in Western Java, Indonesia.

Equine piroplasmosis is an economically significant disease caused by Theileria equi and Babesia caballi, which are tick-borne hemoprotozoan parasites. Infections with these parasite species had never been reported in horses in Indonesia. The aim of the present study was to investigate the prevalence of T. equi and B. caballi in horses reared in parts of Western Java, Indonesia. Blood samples were collected randomly from 235 horses in four different districts (Bandung, Depok, Tangerang, and Bogor) in Western Java, Indonesia. Thin blood smears prepared from the sampled animals were stained by Giemsa and observed under a light microscope. Serum samples prepared from blood were screened by enzyme-linked immunosorbent assays (ELISAs) based on recombinant forms of EMA-2 and BC48 antigens to determine the seroprevalence of T. equi and B. caballi, respectively. DNA samples extracted from the same blood samples were screened by EMA-2 and BC48 gene-based nested polymerase chain reaction (nPCR) assays for T. equi and B. caballi infections, respectively. Of 235 surveyed animals, five (2.1%) and 15 (6.4%) were seropositive for T. equi and B. caballi, respectively, whereas one and four horses were nPCR-positive for T. equi and B. caballi, respectively. All of the surveyed animals were negative for T. equi and B. caballi by microscopy. The T. equi EMA-2 and B. caballi BC48 gene fragments amplified by the nPCR assays were cloned, sequenced, and subjected to bioinformatic and phylogenetic analyses. The T. equi EMA-2 gene sequence from an Indonesian horse was identical to sequences from Florida and Washington strains and clustered together with these sequences in phylogeny. On the other hand, four Indonesian BC48 gene sequences shared 99.8-100% identity scores. This present study is the first to report T. equi and B. caballi in horses in Indonesia. Our findings highlight the need for monitoring horses in Indonesia for clinical piroplasmosis caused by T. equi and B. caballi.

Potential risk of viral transmission from flying foxes to domestic animals and humans on the southern coast of West Java, Indonesia.

Flying foxes have been considered to be involved in the transmission of serious infectious diseases to humans. Using questionnaires, we aimed to determine the direct and/or indirect contacts of flying foxes in an Indonesian nature conservation area with domestic animals and humans living in the surrounding area. We surveyed 150 residents of 10 villages in West Java. Villages were classified into 3 groups: inside and/or within 1 km from the outer border of the conservation area and 1-5 km or 5-10 km away from the reserve's outer border. Data were collected by direct interview using a structured questionnaire consisting of the respondent characteristics (age, sex and occupation); histories of contacts between flying foxes and humans, dogs and other domestic animals; and knowledge about infectious diseases, mainly rabies, in flying foxes. We found that flying foxes from the nature conservation area often enter residential areas at night to look for food, especially during the fruit season. In these residential areas, flying foxes had direct contacts with humans and a few contacts with domestic animals, especially dogs. People who encounter flying foxes seldom used personal protective equipment, such as leather gloves, goggles and caps. The residents living around the conservation area mostly had poor knowledge about flying foxes and disease transmission. This situation shows that the population in this region is at a quite high risk for contracting infectious diseases from flying foxes.