Detection of Borrelia burgdorferi Sensu Lato and Relapsing Fever Borrelia in Feeding Ixodes Ticks and Rodents in Sarawak, Malaysia: New Geographical Records of Borrelia yangtzensis and Borrelia miyamotoi

Members of the Borrelia burgdorferi sensu lato (Bbsl) complex are etiological agents of Lyme disease (LD), and Borrelia miyamotoi is one of the relapsing fever Borrelia (RFB). Despite the serological evidence of LD in Malaysia, there has been no report from Sarawak, Malaysian Borneo. Thus, this study aimed to detect and characterize Borrelia in rodents and Ixodes ticks from primary forests and an oil palm (OP) plantation in Sarawak. Borrelia yangtzensis (a member of the Bbsl complex) was detected in 43.8% (14/32) of Ixodes granulatus; most of the positive ticks were from the OP plantation (13/14). Out of 56 rodents, B. yangtzensis was detected in four Rattus spp. from the OP plantation and B. miyamotoi was detected in one rodent, Sundamys muelleri, from the primary forest. Further, the positive samples of B. yangtzensis were randomly selected for multilocus sequence analysis (MLSA). The MLSA results of successfully amplified tick samples revealed a clustering with the sequences isolated from Japan and China. This study is the first evidence of B. miyamotoi, a known human pathogen in Malaysia, and B. yangtzensis, which is circulating in ticks and rodents in Sarawak, Malaysian Borneo, and presenting a new geographical record of the Borrelia spp

The dynamics of the microbiome in Ixodidae are shaped by tick ontogeny and pathogens in Sarawak, Malaysian Borneo

Tick-borne diseases have recently been considered a potential emerging public health threat in Malaysia; however, fundamental studies into tick-borne pathogens and microbiome appear limited. In this study, six tick species (Ixodes granulatus, Haemaphysalis hystricis, Haemaphysalis shimoga, Dermacentor compactus, Dermacentor steini and Dermacentor atrosignatus) collected from two primary forests and an oil palm plantation in Sarawak, Malaysian Borneo, were used for microbiome analysis targeting bacterial 16S rDNA using next-generation sequencing (NGS). In addition, bacterial species were further characterized in conventional PCRs to identify potential pathogens. Sequences generated from NGS were first filtered with the Decontam package in R before subsequent microbial diversity analyses. Alpha and beta analyses revealed that the genus Dermacentor had the highest microbial diversity, and H. shimoga significantly differed in microbial composition from other tick species. Alpha and beta diversities were also significantly different between developmental stages of H. shimoga. Furthermore, we observed that some bacterial groups were significantly more abundant in certain tick species and developmental stages of H. shimoga. We tested the relative abundances using pairwise linear discriminant analysis effect size (LEfSe), which also revealed significant microbial composition differences between Borrelia -positive and Borrelia -negative I. granulatus ticks. Finally, pathogenic and potentially pathogenic bacteria circulating in different tick species, such as Rickettsia heilongjiangensis , Ehrlichia sp., Anaplasma sp. and Bartonella spp. were characterized by PCR and sequencing. Moreover, Coxiella and Francisella -like potential symbionts were identified from H. shimoga and D. steini, respectively. More studies are required to unravel the factors associated with the variations observed in this study

Changes in blood glucose and insulin responses to intravenous glucose tolerance tests and blood biochemical values in adult female Japanese black bears (Ursus thibetanus japonicus)

The metabolic mechanisms to circannual changes in body mass of bears have yet to be elucidated. We hypothesized that the Japanese black bear (Ursus thibetanus japonicus) has a metabolic mechanism that efficiently converts carbohydrates into body fat by altering insulin sensitivity during the hyperphagic stage before hibernation. To test this hypothesis, we investigated the changes in blood biochemical values and glucose and insulin responses to intravenous glucose tolerance tests (IVGTT) during the active season (August, early and late November). Four, adult, female bears (5-17 years old) were anesthetized with 6 mg/kg TZ (tiletamine HCl and zolazepam HCl) in combination with 0.1 mg/kg acepromazine maleate. The bears were injected intravenously with glucose (0.5 g/kg of body mass), and blood samples were obtained before, at, and intermittently after glucose injection. The basal triglycerides concentration decreased significantly with increase in body mass from August to November. Basal levels of plasma glucose and serum insulin concentrations were not significantly different among groups. The results of IVGTT demonstrated the increased peripheral insulin sensitivity and glucose tolerance in early November. In contrast, peripheral insulin resistance was indicated by the exaggerated insulin response in late November. Our findings suggest that bears shift their glucose and lipid metabolism from the stage of normal activity to the hyperphagic stage in which they show lipogenicpredominant metabolism and accelerate glucose uptake by increasing the peripheral insulin sensitivity

Skeletal muscles of hibernating black bears show minimal atrophy and phenotype shifting despite prolonged physical inactivity and starvation.

Hibernating mammals experience prolonged periods of torpor and starvation during winter for up to 5-7 months. Though physical inactivity and malnutrition generally lead to profound loss of muscle mass and metabolic dysfunction in humans, hibernating bears show limited muscle atrophy and can successfully maintain locomotive function. These physiological features in bears allow us to hypothesize that hibernating bears uniquely alter the regulation of protein and energy metabolism in skeletal muscle which then contributes to "muscle atrophy resistance" against continued physical inactivity. In this study, alteration of signaling pathways governing protein and energy metabolisms was examined in skeletal muscle of the Japanese black bear (Ursus thibetanus japonicus). Sartorius muscle samples were collected from bear legs during late November (pre-hibernation) and early April (post-hibernation). Protein degradation pathways, through a ubiquitin-proteasome system (as assessed by increased expression of murf1 mRNA) and an autophagy-dependent system (as assessed by increased expression of atg7, beclin1, and map1lc3 mRNAs), were significantly activated in skeletal muscle following hibernation. In contrast, as indicated by a significant increase in S6K1 phosphorylation, an activation state of mTOR (mammalian/mechanistic target of rapamycin), which functions as a central regulator of protein synthesis, increased in post-hibernation samples. Gene expression of myostatin, a negative regulator of skeletal muscle mass, was significantly decreased post-hibernation. We also confirmed that the phenotype shifted toward slow-oxidative muscle and mitochondrial biogenesis. These observations suggest that protein and energy metabolism may be altered in skeletal muscle of hibernating bears, which then may contribute to limited loss of muscle mass and efficient energy utilization

Supplementing cultured human myotubes with hibernating bear serum results in increased protein content by modulating Akt/FOXO3a signaling

Hibernating bears remain in their dens for 5-7 months during winter and survive without eating or drinking while staying inactive. However, they maintain their physical functions with minimal skeletal muscle atrophy and metabolic dysfunction. In bears, resistance to skeletal muscle atrophy during hibernation is likely mediated by seasonally altered systemic factors that are independent of neuromuscular activity. To determine whether there are components in bear serum that regulate protein and energy metabolism, differentiated human skeletal muscle cells were treated with bear serum (5% in DMEM/Ham's F-12, 24 h) collected during active summer (July) and hibernating winter (February) periods. The serum samples were collected from the same individual bears (Ursus thibetanus japonicus, n = 7 in each season). Total protein content in cultured skeletal muscle cells was significantly increased following a 24 h treatment with hibernating bear serum. Although the protein synthesis rate was not altered, the expression of MuRF1 protein, a muscle-specific E3 ubiquitin ligase was significantly decreased along with a concomitant activation of Akt/FOXO3a signaling. Increased levels of insulin-like growth factor-1 (IGF-1) were also observed in hibernating bear serum. These observations suggest that protein metabolism in cultured human myotubes may be altered when incubated with hibernating bear serum, with a significant increase in serum IGF-1 and diminished MuRF1 expression, a potential target of Akt/FOXO3a signaling. A protein sparing phenotype in cultured muscle cells by treatment with hibernating bear serum holds potential for the development of methods to prevent human muscle atrophy and related disorders

Evaluation of body condition using body mass and chest girth in brown bears of Hokkaido, Japan (Ursus arctos yesoensis)

Chest girth and body mass of 3,576 brown bears (Ursus arctos yesoensis) harvested for conflict management in Hokkaido, Japan during 1991-2012 were used to establish methods to assess body condition and to compare the body condition of bears by sex, month, year, and reproductive status. The body mass was estimated based on the chest girth in cases with no measurements of the bear body mass. Using the measured and estimated body mass, a growth curve by age was demonstrated to ascertain the mean asymptotical body mass (245 kg for males, 114 kg for females) and ages at 95% asymptotic body mass (14.2 years for males, 7.1 years for females). The body condition value of bears was evaluated as body mass difference (kg) between the individual body mass and the standard body mass as estimated from the growth curve. Body condition value changed seasonally with a low in summer and the highest in the autumnal hyperphagic period. Female body condition value was higher than the males during September. Fluctuation in annual body condition value was found for females; however, there was no difference between solitary adult females and females with offspring (cubs, yearlings, or offspring of unknown age). No significant fluctuation was found for males. Our body condition evaluation method using chest girth and body mass of brown bears is useful to elucidate different trends across sex, year, and season

Borrelia miyamotoi Infections among Wild Rodents Show Age and Month Independence and Correlation with Ixodes persulcatus Larval Attachment in Hokkaido, Japan

To clarify how Borrelia miyamotoi is maintained in the environment in Hokkaido, we examined Ixodes persulcatus for its prevalence among wild rodents and its tick vector by detecting a portion of the borrelial flaB gene in rodent urinary bladder and blood samples, and from whole ticks. We compared B. miyamotoi infection rates to Borrelia garinii and Borrelia afzelii, which are human Lyme disease pathogens also carried by wild rodents, and which are transmitted by the same vector tick. Whereas B. garinii and B. afzelii showed age dependence of infection rates among wild rodents (18.4% and 9.9% among adults and 6.0% and 3.4% among sub-adults, respectively) when looking at urinary bladder samples, B. miyamotoi infection rates were not age dependent for either blood (4.2% among adults, and 7.9% among sub-adults) or urinary bladder samples (1.0% among adults, and 1.7% among sub-adults). Moreover, while B. garinii and B. afzelii infection rates showed increases across months (June, July [p < 0.05] and August [p < 0.01] had higher rates than in May for adult rodents with B. garinii, and July and August had higher rates than in May [p < 0.01] for adult rodents with B. afzelii), B. miyamotoi infection rates did not show significant month dependence. These differences in month and age dependence led us to suspect that B. miyamotoi may not develop persistent infections in wild rodents, as B. garinii and B. afzelii are thought to. Furthermore, we examined the extent of rodent exposure to I. persulcatus nymphs and larvae throughout most of the tick's active season (May through September), and determined that B. miyamotoi infection rates in sub-adult rodents were correlated with larval burden (p < 0.01), suggesting that larvae may be very important in transmission of B. miyamotoi to wild rodents