Antimicrobial resistance in Escherichia coli isolates from Japanese raccoon dogs (Nyctereutes viverrinus) in Kanagawa Prefecture, Japan: Emergence of extended-spectrum cephalosporin-resistant human-related clones.

Introduction. Wild animals are one of the putative reservoirs of antimicrobial-resistant bacteria, but the significance of raccoon dogs remains to be investigated.Hypothesis. Raccoon dogs can be a reservoir of antimicrobial-resistant bacteria.Aim. This study aimed to explore the prevalence of antimicrobial resistance, mainly extended-spectrum cephalosporins resistance, in Escherichia coli isolates from faeces of 80 Japanese raccoon dogs in Kanagawa Prefecture, Japan.Methodology. All of the 80 faecal samples were streaked onto deoxycholate-hydrogen sulfate-lactose (DHL) and cefotaxime (CTX)-supplemented DHL (DHL-CTX) agars. Susceptibilities to ten antimicrobials were determined using the agar dilution method. Additionally, extended-spectrum ß-lactamases (ESBLs) and AmpC-type ß-lactamases (ABLs) were identified in addition to sequence types (STs), in ESC-resistant isolates by a polymerase chain reaction and sequencing.Results. Out of all the samples, 75 (93.8 %) and 20 (25.0 %) E. coli isolates were isolated by DHL and DHL-CTX agars, respectively. Significantly higher resistance rates to most of the drugs were found in DHL-CTX-derived isolates than DHL-derived isolates (P<0.01). Genetic analysis identified CTX-M-14 (n=6), CTX-M-2 (n=2), CTX-M-1 (n=1) and CTX-M-55 (n=1) as ESBLs, and CMY-2 (n=8) and DHA-1 (n=1) as ABLs in 20 DHL-CTX-derived isolates. Most of the detected STs were related to Japanese humans (i.e. ST10, ST58, ST69, ST131, ST357, ST648 and ST4038). Notably, this is the first report on ST69, ST131, ST155 and ST648, which are well-known international high-risk clones in Japanese raccoon dogs.Conclusion. Our findings underscore the need to understand the significance of raccoon dogs as an antimicrobial-resistant bacteria reservoir using one health approach.

Development of a rapid scabies immunodiagnostic assay based on transcriptomic analysis of Sarcoptes scabiei var. nyctereutis.

Scabies is a highly contagious skin disease caused by the mite Sarcoptes scabiei that affects many mammals. However, the sensitivity of traditional tests for scabies diagnosis in humans is less than 50%. To simplify the diagnosis of scabies, methods that are simple, sensitive, specific, and cost-effective are required. We developed an immunodiagnostic test based on S. scabiei var. nyctereutis RNA-seq data collected from Japanese raccoon dogs with sarcoptic mange. Three candidate antigens-a highly expressed hypothetical protein "QR98_0091190," another mite allergen known as "SMIPP-Cc," and an abundant "vitellogenin-like protein"-were evaluated by western-blot analysis. A lateral flow immunoassay, using specific antibodies against the vitellogenin-like protein, successfully detected scabies in the skin flakes of S. scabiei-infected raccoon dogs. This assay can potentially diagnose scabies more accurately in wildlife, as well as in humans.

Captive environmental change and induction of natural breeding and egg-laying in ploughshare tortoises (Astrochelys yniphora).

The ploughshare tortoise (Astrochelys yniphora) is an endangered species. It is important to increase the number of tortoises in the wild and captivity; however, breeding in captivity, especially outside of the natural habitat area, has been unsuccessful worldwide. As a result of an emergency rescue due to a violation of the Act of Conservation of Endangered Species of Wild Fauna and Flora, Nogeyama Zoological Gardens (Yokohama, Japan) has housed ploughshare tortoises, and a natural breeding trial started in 2011. In this case, the captive environment in Nogeyama, especially room temperature and humidity, imitated the environment in the natural habitat of northwest Madagascar. The maximum temperature during the day was 30.8-32.5°C throughout the year, but the minimum temperature was 24°C during December and January, representing the rainy season, and 17°C during July, representing the dry season. The humidity was adjusted and the room was not humidified between November and February (dry season) but was humidified by sprinkling water once every few days in March and daily between April and October (rainy season). Natural breeding and egg-laying were observed 1 year after the captive environmental change. Three neonates were hatched from nine eggs in 3 years after the beginning of the breeding program. This study showed that for natural breeding of the ploughshare tortoises, it is important to adjust the temperature and humidity of the captive environment to imitate the temperature and humidity of the natural habitat.

Multilobular tumor of bone on the forehead of a guinea pig.

Multilobular tumor of bone (MLTB) is an infrequent, slow-growing, bone neoplasm formed predominantly on the head. These tumors can behave as malignant neoplasms clinically and pathologically and can metastasize occasionally. No cases of MLTB in rodents have been reported, to our knowledge. We describe a novel case of an MLTB in a guinea pig. An adult guinea pig had an exophytic mass fixed on the frontal bone, maxilla, and nasal bone. On radiography, the mass had a spherical contour and variable density and was formed on the surface of the cranial bones. The mass was excised surgically. The cut surface was light-yellow to milky-white and had a granular texture with fine fibrous septa. Histologically, the neoplasm had a multilobular pattern, which consisted of many islands of bone and/or cartilage matrix surrounded by small cells and separated by fibrous septa, which closely resembles the equivalent neoplasm in dogs.

Emotion estimation using a wearable heart rate monitoring device in Asian elephants (Elephas maximus) during veterinary clinical procedures.

Fatal accidents in captive elephants occasionally occur because humans are unable to gauge elephants' emotions solely by their behavior. The intellectual capacity of elephants makes them capable of understanding circumstantial changes and associated emotions, allowing them to react accordingly. Physiological markers, such as heart rate variability, may be effective in determining an elephant's emotional state. In this study, a wearable heart rate monitor was used to determine the emotional state of a female Indian captive elephant (Elephas maximus indicus). The average heart rate was higher when the elephant underwent painful treatment than when it underwent non-painful treatment. In addition, the heart rate increased both before and after the treatment, which included radiography and blood collection.

Possible transmission of Sarcoptes scabiei between herbivorous Japanese serows and omnivorous Caniformia in Japan: a cryptic transmission and persistence?

BACKGROUND: Two transmission patterns of Sarcoptes scabiei in host mammal communities have been reported based on microsatellite-level genetic studies in the last two decades. While one involves restrictions among different host taxa, the other is associated with predator-prey interactions between different host taxa. In contrast to these observations, the present study reports a possible irregular case of transmission of S. scabiei between herbivorous Japanese serow and omnivorous Caniformia mammals in Japan, though under very weak predator-prey relationships. METHODS: DNA from 93 Sarcoptes mites isolated from omnivorous Caniformia (such as the domestic dog, raccoon dog, raccoon and Japanese marten), omnivorous Cetartiodactyla (wild boar) and herbivorous Cetartiodactyla (Japanese serow) in Japan were analyzed by amplifying nine microsatellite markers. Principal components analyses (PCA), Bayesian clustering analyses using STRUCTURE software, and phylogenetic analyses by constructing a NeighborNet network were applied to determine the genetic relationships among mites associated with host populations. RESULTS: In all the analyses, the genetic differentiation of Sarcoptes mites from wild boars and Japanese serows was observed. Conversely, considerably close genetic relationships were detected between Caniformia-derived and Japanese serow-derived mites. Because the predator-prey interactions between the omnivorous Caniformia and herbivorous Japanese serow are quite limited and epidemiological history shows at least a 10-year lag between the emergence of sarcoptic mange in Japanese serow and that in Caniformia, the transmission of S. scabiei from Caniformia to Japanese serow is highly suspected. CONCLUSIONS: The close genetic relationships among mites beyond Host-taxon relationships and without obvious predator-prey interactions in Caniformia and Japanese serow deviate from previously reported S. scabiei transmission patterns. This type of cryptic relationship of S. scabiei populations may exist in local mammalian communities worldwide and become a risk factor for the conservation of the remnant and fragmented populations of wild mammals.

The complete mitochondrial genome of Sarcoptes scabiei var. nyctereutis from the Japanese raccoon dog: Prediction and detection of two transfer RNAs (tRNA-A and tRNA-Y).

Sarcoptes scabiei (Acari: Sarcoptidae) causes a common contagious skin disease that affects many mammals. Here, the complete mitochondrial genome of a mite, S. scabiei var. nyctereutis, from Japanese wild raccoon dogs was analyzed. The 13,837bp circular genome contained 13 protein-coding genes, two rRNA genes, and 22 tRNA genes. For the first time, two tRNAs (alanine and tyrosine), that were thought to be absent in scabies mites from other animals, were predicted to have short, non-cloverleaf structures by in silico annotation and detected by RT-PCR, sequencing, and northern analysis. The mitochondrial genome structure of S. scabiei is similar to that of Psoroptes cuniculi and Dermatophagoides farinae. While small and unusual tRNA genes seem to be common among acariform mites, further experimental evidence for their presence is needed. Furthermore, through an analysis of the cox1 gene, we have provided new evidence to confirm the transmission of this mite between different animal hosts.

Novel treatment for chronic pododermatitis in an Indian elephant (Elephas maximus indicus) with Mohs' paste.

Asian and African elephants are frequently afflicted by foot disorders that can be very challenging to manage even with aggressive therapy. Such conditions may have indirect life-threatening effects. Mohs' paste (zinc chloride based escharotic agent) was used to treat a female Indian elephant (Elephas maximus indicus) aged 39 years with foot disorder at Kanazawa Zoological Gardens. Degenerated hyperplastic tissue was observed inside the hoofs of digits 2 and 5. Mohs' paste was applied on the lesions, which coagulated the hyperplastic tissue and restrained its proliferation. Subsequently, the hyperplastic tissue could be trimmed with little pain, and the disorder became manageable. Mohs' paste treatment was effective and is expected to be an alternative treatment for hoof disorder.

Evaluation of somatotype in the reticulated giraffe (Giraffa camelopardalis reticulata) using three-dimensional laser measurement.

The giraffe (Giraffa camelopardalis) is a difficult animal to keep in captivity as it has high mortality due to nutrition-related disorders, perhaps because the giraffe's condition is difficult to evaluate. Image analysis techniques have recently become popular and may be useful for evaluating the giraffe's somatotype. The present study aimed to evaluate the giraffe somatotype using a three-dimensional laser measurement device, and to examine the usefulness of this method. First, ten zoo staff members visually evaluated the somatotypes of three giraffes housed at Kanazawa Zoological Gardens, Japan. Next, three-dimensional point cloud datasets were obtained from these giraffes using the device. The point cloud datasets indicated that the cross-sectional area and width of the largest giraffe's body were large in the abdominal region in the transverse sections. However, by visual examination, the ten zoo staff members deemed a different giraffe to be the largest. These results indicated that the three-dimensional laser measurement device could be used to evaluate giraffe somatotype in detail, and that this method may be an alternative to visual evaluation.

Molar eruption and identification of the eastern grey kangaroo (Macropus giganteus) at different ages.

To treat dental disorders and estimate age, it is important that veterinarians understand teeth eruption sequence for the animal in question. Few dental images of the eastern grey kangaroo have been published. In the present study, radiographic imagings of 29 kangaroos, ranging in age from 12 months to 10 years 9 months, was used to surveil the replacement of premolars and the eruption of molars. These images revealed eruption patterns in five stages, while the second and third deciduous premolars were shed non-systematically. Furthermore, the third premolars and fourth molars erupted in the mandible earlier than in the maxilla, which may contribute to the frequency of mandibular dental disorders.

Factors affecting the occurrence of enteritis in 10- to 12-month-old eastern grey kangaroos (Macropus giganteus).

Eastern grey kangaroos (Macropus giganteus) younger than 19 months at Kanazawa Zoological Gardens, Japan, frequently died of enteritis. The main cause of death was coccidiosis. This study aimed to reveal retrospectively the clinical features of enteritis and factors contributing to its high occurrence. In addition, haematological and serum biochemical parameters in kangaroos younger than 24 months were analyzed. The findings suggested that enteritis occurrence was higher in 10- to 12-month-old kangaroos than other ages and during seasons with high temperature and humidity than during seasons with low temperature and humidity. The haematological and serum biochemical analyses showed decreases in glucose, total cholesterol, calcium, and triglyceride levels at approximately 10 months of age. Joeys emerge from their mother's pouch at 8 months of age and permanently leave it at approximately 10 months of age. Subsequently, the joeys are exposed to the external environment and infectious agents. Although the joeys continue suckling until 13-19 months of age, the joeys start to eat the similar feed to the adult and their nutritional status may change at approximately 10 months of age. Seasonal influences, species behavior, and shifts in dietary composition are proposed to be contributory to the increased occurrence of enteritis between 10 and 12 months of age.

New techniques to collect live Sarcoptes scabiei and evaluation of methods as alternative diagnostics for infection.

Sarcoptes scabiei is a widespread, highly contagious skin disease that affects many mammals including humans. The biological characteristics of S. scabiei remain unclear. Therefore, the ability to collect adequate amount of mites for studies is required to advance our understanding of the parasite. The present study aimed to find a method to collect an adequate amount of live S. scabiei mites within a short time frame. The cornified layer and fur from an infected raccoon dog were inserted into a 50-ml catheter tip-type syringe. A 1.5-ml microtube was attached at the tip of the syringe to collect the mites, which crawled out from the cornified layer and fur. Four conditions were examined, and the following condition was determined to be the best: the syringe and microtube were shaded by aluminum foil, and the microtube was heated using a pet heater (36 °C). In addition, the effectiveness of this method as an alternative method to diagnose S. scabiei infections in animal was evaluated. S. scabiei live mites were not detected in the raccoon dog samples 24 h after the administration of medication (ivermectin or selamectin). The present study revealed that this technique was useful to collect adequate amounts of live mites, and the mites prefer a heated environment and actively move when using the shaded conditions. In addition, this technique was effective as an alternative diagnostic technique to detect live mites on an animal body.

Supplemental feeding of captive neonatal koalas (Phascolarctos cinereus).

Koalas (Phascolarctos cinereus) are cautious animals, making supplemental feeding of neonates challenging because of disturbances to the normal routine. However, supplemental feeding is beneficial in improving juvenile nutrition using less formula than required for hand-rearing, and allowing maternal bonding to continue through suckling. In this study, two neonatal koalas, delivered by the same mother in 2 years, exhibited insufficient growth post-emergence from the pouch; supplemental feeding was therefore initiated. The amount of formula fed was determined according to the product instructions, and offspring weight was monitored. Slower than normal growth was not initially noticed in the first offspring. This caused delayed commencement of supplemental feeding. An attempt was made to counteract this by providing more formula for a longer period; however, this meant No. 1 was unable to eat enough eucalyptus when weaning. Supplemental feeding was started earlier for the second offspring than for the first, and was terminated at weaning; this juvenile showed a healthy body weight increase. Furthermore, it was able to eat eucalyptus leaves at an earlier stage than No. 1. Although No. 1 showed delayed growth, both koalas matured and are still living. This study showed that supplemental feeding is useful for koalas, if the mother will accept human intervention. The key factors for successful supplemental feeding of koalas identified by comparing the two feeding systems observed in this study are that: (1) it should be initiated as soon as insufficient growth is identified; and (2) it should be terminated before weaning age. Zoo Biol. 36:62-65, 2017. © 2016 Wiley Periodicals, Inc.

Hematological and pathological features of massive hepatic necrosis in two radiated tortoises (Astrochelys radiata).

Two radiated tortoises (Astrochelys radiata) exhibited anorexia and hypokinesia. In both cases, hematological and serum biochemical examinations revealed high alkaline phosphatase levels, moderately high aspartate aminotransferase levels and white blood cell counts approximately within the normal range. Despite being treated, the tortoises died 9 and 43 days after the first clinical examination. Gross pathological examinations revealed that the livers of both animals were extremely swollen and contained pale yellow necrotic tissue. Histopathological assessment revealed that the livers contained a massive area of hepatic necrosis surrounded by migration of macrophages and multinucleated giant cells. In one of the cases, severe fibrosis was observed. The present study provides reference information for similar cases in the future.

Extraction of an incisor embedded within the nasal cavity in two guinea pigs.

Oral examination of two guinea pigs revealed that the unilateral incisor was absent. On radiographic examination, the incisor was identified within the nasal cavity in both patients. Under anesthesia in both patients, the skin was incised from the nostril to 1.5 cm proximal, and the premaxilla and part of the maxilla were exposed. The bone was removed using a surgical drill, and the incisor was exposed in the nasal cavity. The root was grasped with forceps and carefully extracted as it was degraded and very fragile. Diagnosis was easy using oral and radiographic examination. In guinea pig patients where an incisor is absent on oral examination, this condition should be considered.

FOLLICULAR CELL CARCINOMA OF THE THYROID GLAND IN THREE CAPTIVE AGED RACCOON DOGS (NYCTEREUTES PROCYONOIDES).

The clinical and histologic features of thyroid carcinoma in raccoon dogs have not been previously reported. Three of four raccoon dogs (Nyctereutes procyonoides) over 8 yr of age at the Nogeyama Zoological Gardens developed thyroid follicular cell carcinomas that were detected at necropsy. The affected raccoon dogs were rescued from the wild and were housed at the Nogeyama Zoological Gardens for 8 yr 8 mo, 8 yr 10 mo, and 10 yr 3 mo, respectively. Although all of them appeared lethargic and developed partial alopecia or desquamation of their skin, they did not display any other specific clinical signs associated with a thyroid lesion. Serum thyroid hormone values were examined in two of the affected raccoon dogs and the average and standard deviation values (free-thyroxin [FT4]: 0.078 ± 0.077 pM/L and 0.062 ± 0.0039 pM/L; free-triiodothyronine [FT3]: 3.261 ± 0.765 pM/L and 3.407 ± 0.919 pM/L) were lower than the reference range (FT4: 0.141 ± 0.117 pM/L; FT3: 5.139 ± 2.412 pM/L) derived from a clinically normal raccoon dog. On necropsy, the thyroid lobes were markedly enlarged bilaterally. Histopathologically, the neoplastic cells in the thyroid gland appeared round or oval and columnar or cuboidal with minimal heteromorphism. Moreover, mostly small (but occasionally large) follicles were identified, and the neoplastic cells had infiltrated into the surrounding capsule and blood vessels. The histopathologic features of the thyroid tumors in the raccoon dogs revealed that the tumors were derived from follicular cells.

A new voluntary blood collection method for the Andean bear (Tremarctos ornatus) and Asiatic black bear (Ursus thibetanus).

Various training methods have been developed for animal husbandry and health care in zoos and one of these trainings is blood collection. One training method, recently widely used for blood collection in Ursidae, requires setting up a sleeve outside the cage and gives access to limited blood collection sites. A new voluntary blood collection method without a sleeve was applied to the Andean bear (Tremarctos ornatus) and Asiatic black bear (Ursus thibetanus) with access to various veins at the same time. The present study evaluated the effectiveness of this new method and suggests improvements. Two Andean and two Asiatic black bears in Yokohama and Nogeyama Zoological Gardens, respectively, were trained to hold a bamboo pipe outside their cages. We could, thereby, simultaneously access superficial dorsal veins, the dorsal venous network of the hand, the cephalic vein from the carpal joint, and an area approximately 10 cm proximal to the carpal joint. This allowed us to evaluate which vein was most suitable for blood collection. We found that the cephalic vein, approximately 10 cm proximal to the carpal joint, was the most suitable for blood collection. This new method requires little or no modification of zoo facilities and provides a useful alternative method for blood collection. It could be adapted for use in other clinical examinations such as ultrasound examination.

Effective treatment for improving the survival rate of raccoon dogs infected with Sarcoptes scabiei.

Sarcoptes scabiei is one of the important external parasites. Although ivermectin is the recommended treatment, many raccoon dogs (Nyctereutes procyonoides) that were rescued and brought to the Kanazawa Zoological Gardens (Yokohama, Japan) have died because of S. scabiei, even after receiving single ivermectin treatment. Therefore, supportive treatment should be required. The present study revealed the number of animals that survived was greater after the administration of ivermectin along with an antibiotic for all raccoon dogs, as well as following the administration of fluid therapy to the debilitated raccoon dogs infected with S. scabiei, immediately after the rescue. During the initial period, treatment to improve the general clinical condition was required prior to deworming treatment for S. scabiei.

Insufficient colostrum ingestion is a risk factor for polyarthritis and/or phlegmon in hand-reared reticulated giraffes (Giraffa camelopardalis reticulata): 7 cases (2003-2012).

Seven reticulated giraffes were hand-reared at Nogeyama Zoological Gardens, because the dam had agalactia. Six of the 7 calves exhibited polyarthritis and/or phlegmon in the lower legs. However, the cause of the disorder was unclear. The present study reviewed the clinical records of the 7 giraffes, including the type and amount of colostrum ingested during the first 72 hr. The disorder involved the fetlocks and carpal and tarsal joints in 6 of the 7 calves within an average of 8 days of birth. The average amount of fed bovine or powdered colostrum was 0-2.4 l in the first 24 hr and 2.0-6.2 l during the first 72 hr. Insufficient colostrum quantity might be a factor in polyarthritis and/or phlegmon.

Perivertebral B-cell lymphoma in a Queensland koala (Phascolarctos cinereus adustus) with paralytic symptoms in the hind limbs.

A male Queensland koala (Phascolarctos cinereus adustus) at Kanazawa Zoological Gardens (Kanagawa, Japan) exhibited paralytic symptoms in the hind limbs. Computed tomography and magnetic resonance imaging revealed a mass on the left ventral side of the 11th to 13th thoracic vertebrae, and the presence of myelitis or edema in the spinal cord. The koala was under anesthesia during the examination and suddenly developed ventricular fibrillation and died. Necropsy revealed a firm flat ovoid hemorrhagic mass on the vertebrae. Following a microscopic examination including immunohistochemistry, the perivertebral mass was diagnosed as B cell lymphoma. Therefore, neoplastic cell infiltration into the spinal cord may cause paralytic symptoms in the hind limbs.