Sensitivity assessment of diphacinone by pharmacokinetic analysis in invasive black rats in the Bonin (Ogasawara) Archipelago, Japan.

The Bonin Archipelago is a United Nations Educational, Scientific and Cultural Organization's World Natural Heritage Site in Japan with a unique ecosystem; however, the invasive rodents preying on endemic species have been a significant concern. The anticoagulant rodenticide, diphacinone, sprayed by the Ministry of the Environment, has succeeded; however, its repeated use leads to rodenticide resistance. This study evaluated the sensitivity by in vivo pharmacokinetics/pharmacodynamics (PK/PD) analysis and physiologically-based pharmacokinetic modeling to diphacinone in black rats (Rattus rattus) captured on the Bonin Archipelago in February 2022. The Bonin rats exhibited prolonged coagulation time after diphacinone administration. They recovered earlier than susceptible black rats, indicating that Bonin rats were less susceptible, though there were no genetic mutations in Vkorc1, the target enzyme of diphacinone. After the administration of diphacinone, hepatic expression levels of Fsp1, identified as the vitamin K reductase, was decreased, however, the Bonin rats exhibited the most minor suppression. The PK analysis showed that the excretion capacity of the Bonin rats was lower than that of the resistant black rats. In the PBPK modeling, the resistant black rats showed higher clearance than the Bonin and susceptible black rats due to high hepatic metabolic capacity. The Bonin rats demonstrated slow absorption and relatively low clearance. This study highlighted the reduced rodenticide-sensitive tendency of wild black rats in the Bonin Archipelago at an in vivo phenotype level. At the same time, they do not have known rodenticide resistance mechanisms, such as hepatic metabolic enhancement or Vkorc1 mutations. It is crucial to monitor the biological levels to evaluate rodenticide sensitivity accurately.

Streptobacillus notomytis Bacteremia after Exposure to Rat Feces.

To determine the source of Streptobacillus notomytis bacteremia in a woman in Japan with signs of rat-bite fever, we examined rat feces from her home. After culture and PCR failed to identify the causative organism in the feces, next-generation sequencing detected Streptobacillus spp., illustrating this procedure's value for identifying causative environmental organisms.

The VKORC1 ER-luminal loop mutation (Leu76Pro) leads to a significant resistance to warfarin in black rats (Rattus rattus).

Well-known 4-hydroxycoumarin derivatives, such as warfarin, act as inhibitors of the vitamin K epoxide reductase (VKOR) and are used as anticoagulants. Mutations of the VKOR enzyme can lead to resistance to those compounds. This has been a problem in using them as medicine or rodenticide. Most of these mutations lie in the vicinity of potential warfarin-binding sites within the ER-luminal loop structure (Lys30, Phe55) and the transmembrane helix (Tyr138). However, a VKOR mutation found in Tokyo in warfarin-resistant rats does not follow that pattern (Leu76Pro), and its effect on VKOR function and structure remains unclear. We conducted both in vitro kinetic analyses and in silico docking studies to characterize the VKOR mutant. On the one hand, resistant rats (R-rats) showed a 37.5-fold increased IC50 value to warfarin when compared to susceptible rats (S-rats); on the other hand, R-rats showed a 16.5-fold lower basal VKOR activity (Vmax/Km). Docking calculations exhibited that the mutated VKOR of R-rats has a decreased affinity for warfarin. Molecular dynamics simulations further revealed that VKOR-associated warfarin was more exposed to solvents in R-rats and key interactions between Lys30, Phe55, and warfarin were less favored. This study concludes that a single mutation of VKOR at position 76 leads to a significant resistance to warfarin by modifying the types and numbers of intermolecular interactions between the two.

Investigation of hepatic warfarin metabolism activity in rodenticide-resistant black rats (Rattus rattus) in Tokyo by in situ liver perfusion.

Anti-blood coagulation rodenticides, such as warfarin, have been used all over the world. They inhibit vitamin K epoxide reductase (VKOR), which is necessary for producing several blood clotting factors. This inhibition by rodenticides results in lethal hemorrhage in rodents. However, heavy usage of these agents has led to the appearance of rodenticide-resistant rats. There are two major mechanisms underlying this resistance, i.e., mutation of the target enzyme of warfarin, VKOR, and enhanced metabolism of warfarin. However, there have been few studies regarding the hepatic metabolism of warfarin, which should be related to resistance. To investigate warfarin metabolism in resistant rats, in situ liver perfusion of warfarin was performed with resistant black rats (Rattus rattus) from Tokyo, Japan. Liver perfusion is an in situ methodology that can reveal hepatic function specifically with natural composition of the liver. The results indicated enhanced hepatic warfarin hydroxylation activity compared with sensitive black rats. On the other hand, in an in vitro microsomal warfarin metabolism assay to investigate kinetic parameters of cytochrome P450, which plays a major role in warfarin hydroxylation, the Vmax of resistant rats was slightly but significantly higher compared to the results obtained in the in situ study. These results indicated that another factor like electron donators may also contribute to the enhanced metabolism in addition to high expression of cytochrome P450.

Streptobacillus ratti sp. nov., isolated from a black rat (Rattus rattus).

An indole-, oxidase- and catalase-negative, non-motile bacterium, strain OGS16T, was isolated from an oral swab of a feral black rat (Rattus rattus) in 2007 in Japan. It stained Gram-negative and had pleomorphic, rod-shaped, non-spore-forming cells. Based on 16S rRNA gene sequence analyses, strain OGS16T was assigned to the genus Streptobacillus, with 16S rRNA gene sequence similarities of 99.3, 99.0, 98.6 and 95.5% to the type strains of Streptobacillus moniliformis, Streptobacillus notomytis, Streptobacillus felis and Streptobacillus hongkongensis, respectively. Strain OGS16T could also be differentiated clearly from other species of the genus Streptobacillus by rpoB, groEL and recA nucleotide and deduced amino acid sequence analysis. DNA-DNA relatedness as obtained by average nucleotide identity was 89.10% between strain OGS16T and Streptobacillus moniliformis DSM 12112T. Chemotaxonomic and physiological data for strain OGS16T were congruent with results for other closely related members of the family Leptotrichiaceae, represented by highly similar enzyme profiles and fatty acid patterns. MALDI-TOF MS analysis also proved suitable in discriminating strain OGS16T unequivocally from all currently described taxa of the genus Streptobacillus. On the basis of these data, we propose the novel species Streptobacillus ratti sp. nov., with the type strain OGS16T (=JCM 31098T=DSM 101843T). The G+C content of the DNA of the type strain is 25.9 mol% and the genome size is 1.50 Mbp.

Novel revelation of warfarin resistant mechanism in roof rats (Rattus rattus) using pharmacokinetic/pharmacodynamic analysis.

Roof rats (Rattus rattus) live mainly in human habitats. Heavy use of rodenticides, such as warfarin, has led to the development of drug resistance, making pest control difficult. There have been many reports regarding mutations of vitamin K epoxide reductase (VKOR), the target enzyme of warfarin, in resistant rats. However, it has been suggested there are other mechanisms of warfarin resistance. To confirm these possibilities, closed colonies of warfarin-susceptible roof rats (S) and resistant rats from Tokyo (R) were established, and the pharmacokinetics/pharmacodynamics of warfarin in rats from both colonies was investigated. R rats had low levels of warfarin in serum and high clearance activity. These rats can rapidly metabolize warfarin by hydroxylation. The levels of accumulation in the organs were lower than those of S rats. R rats administered warfarin showed high expression levels of CYP2B, 2C, and 3A, which play roles in warfarin hydroxylation, and may explain the high clearance ability of R rats. The mechanism of warfarin resistance in roof rats from Tokyo involved not only mutation of VKOR but also high clearance ability due to high levels of CYP2B, 2C and 3A expression possibly induced by warfarin.

Coexistence of roof rats and carnivores in barns on a livestock farm in Japan.

Brown rats (Rattus norvegicus), roof rats (Rattus rattus), and house mice (Mus musculus) are considered to be important pests on livestock farms. Although the diel activity patterns of rodents are key to their control, information on this aspect of their ecology is limited. Furthermore, the effect of carnivores on rodent activity patterns as well as the carnivore species present on livestock farms is unclear. Here, we set camera traps in an open-type cow barn and in an enclosed pig barn on the same livestock farm in Japan from August through October 2021. The only rodents observed in both barns were roof rats, and the carnivore species observed were dogs (Canis familiaris), cats (Felis catus), and Japanese weasels (Mustela itatsi). Roof rats showed different patterns of activity and behavior between the barns. However, because the pattern in both barns was nocturnal, the activity patterns of roof rats and carnivores showed a moderate to high degree of overlap. Therefore, roof rats did not appear to shift their activity patterns to avoid nocturnal carnivores. Taken together, the present study provides valuable information for rodent control on livestock farms in Japan.

Approach/Avoidance Behavior to Novel Objects is Correlated with the Serotonergic and Dopaminergic Systems in the Brown Rat (Rattus norvegicus).

The brown rat (Rattus norvegicus) is known to show three types of behavioral responses to novel objects. Whereas some rats are indifferent to novel objects, neophobic and neophilic rats show avoidance and approach behavior, respectively. Here, we compared the dopaminergic, serotonergic, and noradrenergic systems immunohistochemically among these rats. Trapped wild rats and laboratory rats were first individually exposed to the novel objects in their home cage. Wild rats were divided into neophobic and indifferent rats depending on their behavioral responses. Similarly, laboratory rats were divided into neophilic and indifferent rats. Consistent with the behavioral differences, in the paraventricular nucleus of the hypothalamus, Fos expression in corticotropin-releasing hormone-containing neurons was higher in the neophobic rats than in the indifferent rats. In the anterior basal amygdala, the neophobic rats showed higher Fos expression than the indifferent rats. In the posterior basal amygdala, the neophobic and neophilic rats showed lower and higher Fos expressions than the indifferent rats, respectively. When we compared the neuromodulatory systems, in the dorsal raphe, the number of serotonergic neurons and Fos expression in serotonergic neurons increased linearly from neophobic to indifferent to neophilic rats. In the ventral tegmental area, Fos expression in dopaminergic neurons was higher in the neophilic rats than in the indifferent rats. These results demonstrate that approach/avoidance behavior to novel objects is correlated with the serotonergic and dopaminergic systems in the brown rat. We propose that the serotonergic system suppresses avoidance behavior while the dopaminergic system enhances approach behavior to novel objects.

Pesticide Contamination Levels in the Stomach Contents of Wild Raccoons (Procyon lotor) and Masked Palm Civets (Paguma larvata) in Japan.

Pesticides, which are vital for agriculture, pose a significant threat to wildlife in transformed Japanese landscapes. Despite global reports of pesticide poisoning in animals, limited studies have examined current wildlife exposure in croplands or metropolitan areas in the region. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS), our study aimed to assess the contamination status of 368 commonly used pesticides. The stomach contents of raccoons living in croplands contained 13 pesticides, including six herbicides and 11 fungicides. Neonicotinoid insecticides, some fungicides, and previously banned insecticides (benzene hexachloride and dichlofenthion) were most frequently detected and found at the highest concentrations, suggesting direct soil-plant transfer and direct consumption by crop-eating species. In masked palm civets living in metropolitan areas, four insecticides and six fungicides were detected, indicating urban wildlife exposure from raided dustbins, urban gardens, and lumber from houses. Although the maximum measured concentrations of all pesticides were lower than the acceptable daily intake for humans, it remains unclear whether these concentrations may have toxic or adverse health effects on the species evaluated in these transformed landscapes. Our study is the first to examine recent pesticide exposures in wild mammals in Japan. Application of the method we developed will lay the foundation for the examination of pesticides in other wildlife species to assist conservation management efforts in the region. Environ Toxicol Chem 2024;43:943-951. © 2024 SETAC.

Surveillance of SARS-CoV-2 Infection in Rodent Populations in Tokyo, Japan.

The maintenance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among wildlife populations poses a potential risk for the emergence of novel variants. Therefore, monitoring SARS-CoV-2 infection among animals is crucial. As urban rodents live in close proximity to human habitats, there is concern that they may be a potential source of zoonoses. To examine the prevalence of SARS-CoV-2 in rodent populations, we analyzed 128 serum samples and 129 oral swabs collected from 128 brown rats (Rattus norvegicus) and 2 black rats (Rattus rattus) captured for pest control purposes in Tokyo, Japan, between May and December 2023. A virus-neutralizing test using the Omicron variant revealed no evidence of SARS-CoV-2 infection in these populations. Real-time RT-PCR from oral swabs did not detect any SARS-CoV-2 RNA-positive rats. These results indicate the low probability of SARS-CoV-2 circulation among rat populations in Tokyo.

An appeasing pheromone ameliorates fear responses in the brown rat (Rattus norvegicus).

The brown rat (Rattus norvegicus) is one of the major animals both in the laboratory and in urban centers. Brown rats communicate various types of information using pheromones, the chemicals that mediate intra-species communication in minute amounts. Therefore, analyses of pheromones would further our understanding of the mode of life of rats. We show that a minute amount of 2-methylbutyric acid (2-MB) released from the neck region can ameliorate fear responses both in laboratory rats and in wild brown rats. Based on these findings, we conclude that 2-MB is an appeasing pheromone in the brown rat. A better understanding of rats themselves would allow us to perform more effective ecologically based research on social skills and pest management campaigns with low animal welfare impacts, which might contribute to furthering the advancement of science and improving public health.

A new loop-mediated isothermal amplification method for rapid, simple, and sensitive detection of Leptospira spp. in urine.

We developed a new loop-mediated isothermal amplification (LAMP) method to detect rrs, a 16S rRNA gene of pathogenic Leptospira spp. in urine. The method enables detection of two leptospiral cells per reaction mixture following boiling of urine specimens. The sensitivity of this method is higher than that of culture or of flaB nested PCR.

Social distancing measures differentially affected rats in North America and Tokyo.

Because rats are commensal organisms that depend on human activities for food, shifts in human behavior will have pronounced effects on local rat populations. In the spring of 2020, social distancing measures were implemented globally to curtail the spread of SARS-CoV-2. This presented a unique opportunity to obtain information regarding the immediate effects of shifts in human behavior on rat populations in a variety of countries. In response to increased sightings of rats in the USA that were reported in American media, we analyzed the changes in the number of public service calls in Tokyo, Japan. We found that the number of calls increased after the implementation of social distancing measures, suggesting that rat sightings had also increased in Tokyo. We then surveyed the changes in the business activities of pest management professionals in the USA, Canada, and Tokyo. We found that the activities were increased in 50 to 60% of the respondents from the USA and Canada. In contrast, 60 to 70% of the respondents from Tokyo answered that their activities were not changed. These results implied that, following the implementation of social distancing measures, rat infestations increased in North America, but not in Tokyo. The survey also suggested that roof rats were considered to be the predominant rodent species in Tokyo. This may account for the limited infestations in Tokyo because roof rats are more sedentary than brown rats. Taken together, our findings suggest that social distancing measures differentially affected rat populations in North America and Tokyo. Supplementary Information: The online version contains supplementary material available at 10.1007/s10340-021-01405-z.

Origin of agouti-melanistic polymorphism in Wild Black Rats (Rattus rattus) inferred from Mc1r gene sequences.

We examined nucleotide changes that underlie coat color variation in Black Rats (the Rattus rattus species complex), which show polymorphism in dorsal fur color, including either grayish brown (agouti) or black (melanistic) forms. We examined the full coding sequence of a gene known to produce melanism in other vertebrates-melanocortin-1-receptor gene Mc1r (954 bp) -using samples of both R. rattus (with 2n = 38) and its close relative Asian Black Rat (R. tanezumi; 2n = 42). We used 61 specimens from Japan with karyotype-known individuals and four samples from Pakistan. We found 11 allele sequences and constructed a network tree that shows two distinct clusters, with allelic segregation according to karyotype and by inference, representing the two species. We found that a nucleotide substitution from G to A at site 280, producing an amino acid change from glutamic acid to lysine, was associated with the dominant trait of the melanistic form of the coat color in R. rattus. Notably, the derived SNP 280A was found in a single allele, with the ancestral SNP 280G present in seven alleles. By contrast, all three alleles for R. tanezumi retain the ancestral SNP 280G. These results suggest a possible recent origin of melanism in R. rattus.

Existence of wild brown rats (Rattus norvegicus) that are indifferent to novel objects.

Exposure to novel objects typically evokes avoidance behavior in wild animals, which is called neophobia. We previously found that wild brown rats (Rattus norvegicus) that were trapped in a park in downtown Tokyo, Japan, exhibited neophobia. We also found that this behavior was accompanied by the activation of the basolateral complex of the amygdala (BLA). Previous studies have suggested that genetic factors are the primary determinants of neophobia. Since rats in cities form populations with distinct genetic characteristics, it is reasonable to assume that wild rats caught at different locations in urban centers will exhibit different levels of neophobia. Here we assessed the intensity of neophobia in wild rats trapped at a wholesale market in Tokyo. Although we performed exactly the same experiment in which neophobia was observed in wild rats trapped at the park, the presence of novel objects did not affect the behaviors of wild rats trapped at the market. Conversely, laboratory rats showed approach and exploratory behaviors as seen in the previous study, suggesting that the experiment was performed appropriately. Compared to the laboratory rats, the lack of behavioral changes in the wild rats was accompanied by fewer Fos immunoreactive cells in the BLA. In addition, the numbers of Fos immunoreactive cells in the bed nucleus of the stria terminalis and ventromedial hypothalamus were similar between the two types of rats. The results demonstrated the existence of wild rats that were indifferent to novel objects.

Common substitution mutation F348Y of acetylcholinesterase gene contributes to organophosphate and carbamate resistance in Cimex lectularius and C. hemipterus.

Bed bug control highly depends on insecticides with a limited number of modes of action, especially since the global prevalence of pyrethroid resistance. De facto insecticide options against bed bugs in Japan are acetylcholinesterase inhibitors (AChEis) that consist of organophosphates and carbamates. However, the status of AChEi resistance and the mechanisms involved have not been ascertained. An amino acid substitution mutation, F348Y (or F331Y in standard numbering), occurring at an acyl-binding site of the paralogous AChE gene (p-Ace), was identified among AChEi-resistant colonies of both common and tropical bed bugs (Cimex lectularius and C. hemipterus, respectively). This mutation was genetically associated with propoxur and fenitrothion resistance in F348Y-segregating colonies of C. hemipterus. Inhibition of heterologously expressed C. lectularius p-Ace with insecticides revealed that the sensitivities of F348Y-carrying AChE decreased by orders of 10- to more than 100-fold for diazoxon, carbaryl, fenitroxon, paraoxon, chlorpyrifos-methyl, malaoxon, azamethiphos, methyl-paraoxon, and propoxur. In contrast, the mutant AChE showed a slightly decreased degree of sensitivity for dichlorvos and almost unchanged sensitivity for metoxadiazone. Further studies are needed to ascertain whether the practical efficacies of dichlorvos and metoxadiazone are ensured against F348Y-carrying bed bugs and whether other resistance mechanisms are involved.

Human leptospirosis cases and the prevalence of rats harbouring Leptospira interrogans in urban areas of Tokyo, Japan.

Thirteen patients with leptospirosis were identified, as confirmed by laboratory analysis during the last 5 years in our laboratory, who came from urban areas of Tokyo, Japan. All of the patients came into contact with rats before the onset of illness. Seventeen per cent of Norway rats captured in the inner cities of Tokyo carried leptospires in their kidneys. Most of these rat isolates were Leptospira interrogans serovar Copenhageni/Icterohaemorrhagiae. Antibodies against these serovars and their DNA were detected in the patients. This suggests that rats are important reservoirs of leptospirosis, and that rat-borne leptospires occur in urban areas of Tokyo.

Novel objects elicit greater activation in the basolateral complex of the amygdala of wild rats compared with laboratory rats.

Wild animals tend to avoid novel objects that do not elicit clear avoidance behaviors in domesticated animals. We previously found that the basolateral complex of the amygdala (BLA) and dorsal bed nucleus of the stria terminalis (dBNST) were larger in trapped wild rats compared with laboratory rats. Based on these findings, we hypothesized that the BLA and/or dBNST would be differentially activated when wild and laboratory rats showed different avoidance behaviors towards novel objects. In this study, we placed novel objects at one end of the home cage. We measured the time spent in that half of the cage and expressed the data as a percentage of the time spent in that region with no object placement. We found that this percentage was lower in the wild rats compared with the laboratory rats. These behavioral differences were accompanied by increased Fos expression in the BLA, but not in the dBNST, of the wild rats. These results suggest that wild rats show greater BLA activation compared with laboratory rats in response to novel objects. We also found increased Fos expression in the paraventricular nucleus of the hypothalamus, ventral BNST, and ventromedial hypothalamus, but not in the central amygdala of wild rats. Taken together, our data represent new information regarding differences in behavioral and neural responses towards novel objects in wild vs. laboratory rats.

PCR-based identification of Culex pipiens complex collected in Japan.

The Culex pipiens complex consists of vector mosquitoes that transmit important human pathogens. In this study we established a simplified method to distinguish three members of the Cx. pipiens complex, Cx. p. pallens Coquillet, Cx. p. form molestus Forskal, and Cx. quinquefasciatus Say, collected in Japan. Sequence analysis of the Drosophila Ace-orthologous acetylcholinesterase (Ace) gene (668 to 680 bp) revealed that a single polymorphic region characterizes each species. Based on this region, specific primers that distinguish Cx. p. form molestus (ACEpip2) and Cx. p. pallens (ACEpall2) were newly designed. Polymerase chain reactions were performed with the genomic DNA of Culex mosquitoes as the template, and these primers clearly distinguished two Culex spp. The accuracy of the designed primers was evaluated with 38 colonies of mosquito samples collected from 9 prefectures of Japan. The testing revealed that the distribution of anautogenous Cx. p. pipiens has not been confirmed in Japan. It also revealed that the male of Cx. p. pallens possesses an Ace gene haplotype that is highly similar to the sequence of Cx. quinquefasciatus. This improved method allows the evaluation of vector competence of Cx. p.molestus, which is the suspected vector of West Nile virus.

Structural differences in the brain between wild and laboratory rats (Rattus norvegicus): Potential contribution to wariness.

Wild animals typically exhibit defensive behaviors in response to a wider range and/or a weaker intensity of stimuli compared with domestic animals. However, little is known about the neural mechanisms underlying "wariness" in wild animals. Wild rats are one of the most accessible wild animals for experimental research. Laboratory rats are a domesticated form of wild rat, belonging to the same species, and are therefore considered suitable control animals for wild rats. Based on these factors, we analyzed structural differences in the brain between wild and laboratory rats to elucidate the neural mechanisms underlying wariness. We examined wild rats trapped in Tokyo, and weight-matched laboratory rats. We then prepared brain sections and compared the basolateral complex of the amygdala (BLA), the bed nucleus of the stria terminalis (BNST), the main olfactory bulb and the accessory olfactory bulb. The results revealed that wild rats exhibited larger BLA, BNST and caudal part of the accessory olfactory bulb compared with laboratory rats. These results suggest that the BLA, BNST, and vomeronasal system potentially contribute to wariness in wild rats.