Isolation of Bacteria from Freeze-Dried Samples and the Functional Characterization of Species-Specific Lactic Acid Bacteria with a Comparison of Wild and Captive Proboscis Monkeys.

Previously, we isolated a novel lactic acid bacteria species (Lactobacillus nasalidis) from the fresh forestomach contents of a captive proboscis monkey (Nasalis larvatus) in a Japanese zoo. In this study, we isolated two strains of L. nasalidis from the freeze-dried forestomach contents of a wild proboscis monkey inhabiting a riverine forest in Malaysia. The samples had been stored for more than six years. Phenotypic analysis showed that strains isolated from the wild individual had more diverse sugar utilization and lower salt tolerance than strains previously isolated from the captive counterpart. These phenotypic differences are most likely induced by feeding conditions; wild individuals consume a wide variety of natural food, unlike their zoo-raised counterparts that consume formula feed with sodium sufficiency. Since 16s rRNA sequences of L. nasalidis were detected in the previously created 16S rRNA libraries of wild, provisioned, and captive proboscis monkeys in Malaysia and Japan, L. nasalidis may be an essential bacterium of the foregut microbial community of the proboscis monkey. The currently established method for the isolation of gut bacteria from freeze-dried samples under storage will be applicable to many already-stored precious samples.

Modifying the diets of captive proboscis monkeys in a temperate zoo to reduce weight loss and renal disease.

In animal husbandry, diets should help in maintaining a healthy body condition, support reproduction, and promote species-specific longevity. It is recommended to feed folivorous primates kept in zoos a high-fiber diet, i.e., leaves, although satisfying such a requirement is challenging in temperate regions because it is difficult to obtain fresh leaves, especially in autumn and winter. As equally important for their appropriate treatment, it is valuable to provide details of clinical reports of medical problems and pathological findings, although such clinical reports are rather limited. Therefore, in foregut-fermenting proboscis monkeys (Nasalis larvatus), we (1) described the individual clinical reports of renal disease and weight loss at the Yokohama Zoological Gardens in Japan, (2) determined the nutritional profile of the diets supplied to these animals because other potential triggers for their renal disease and weight loss could be excluded, (3) modified the diet regimen to minimize weight loss and the development of hypercalcemia and hypophosphatemia, and (4) assessed the effects of such dietary modification by comparing the body weight and the Ca and P concentrations and the Ca/P ratios in the blood before and after diet modification with a comparison of these measurements between zoo and free-ranging individuals. Based on the nutritional profile of the diets, we concluded that the reported cases of renal failure might be caused by consumption of leaves with a Ca/P ratio far above the appropriate level in autumn and winter. Additionally, the dietary modification of minerals and metabolizable energy achieved certain beneficial effects on zoo-kept proboscis monkeys.

Successful Laparoscopic Oviductal Artificial Insemination in the Endangered Tsushima Leopard Cat (Prionailurus bengalensis euptilurus).

The Tsushima leopard cat (Prionailurus bengalensis euptilurus) is an endangered wild felid that lives solely on Tsushima Island, Nagasaki, Japan. Japan's Ministry of the Environment is working with the Japanese Association of Zoo and Aquariums for ex situ conservation of this species. However, considering genetic diversity, it is difficult to conduct captive breeding programs by natural breeding alone; hence, assisted reproductive technologies are required. This study aimed to breed Tsushima leopard cats, which otherwise cannot be paired, using laparoscopic oviductal artificial insemination (AI). Female Tsushima leopard cats (female 1, aged 7 years; female 2, aged 6 years) were treated with 200 IU equine chorionic gonadotropin, followed by administration of 1000 IU porcine luteinizing hormone (pLH) after 96 h to induce ovulation. Laparoscopic AI was performed 32 h post-pLH administration. Females 1 and 2 were inseminated in the oviduct with 2.4 × 106 and 3.3 × 106 motile spermatozoa, respectively, collected from two males. Pregnancy was confirmed by radiography 45 and 51 days post-AI in females 1 and 2, respectively; one fetus was found in female 2. Moreover, female 2 had spontaneous delivery of a female kitten 66 days post-AI. This is the first successful case of AI in a Tsushima leopard cat.

Lactobacillus nasalidis sp. nov., isolated from the forestomach of a captive proboscis monkey (Nasalis larvatus).

Three strains (YZ01T, YZ02 and YZ03) of Gram-stain-positive, facultatively anaerobic rods were isolated from the forestomach contents collected from a captive male proboscis monkey (Nasalis larvatus) at Yokohama Zoo in Japan. Phylogenetic analysis of the 16S rRNA gene sequences revealed that these strains belonged to the genus Lactobacillus. Based on the sequence similarity of the 16S rRNA gene, Lactobacillus delbrueckii subsp. indicus JCM 15610T was the closest phylogenetic neighbour to YZ01T. Sequence analyses of two partial concatenated housekeeping genes, the RNA polymerase alpha subunit (rpoA) and phenylalanyl-tRNA synthase alpha subunit (pheS) also indicated that the novel strains belonged to the genus Lactobacillus. The average nucleotide identity and digital DNA-DNA hybridization (dDDH) between L. delbrueckii subsp. indicus and YZ01T were 85.9 and 31.4 %, respectively. The phylogenetic tree based on the whole genomic data of strains YZ01T, YZ02 and YZ03 suggested that these three strains formed a single monophyletic cluster in the genus Lactobacillus, indicating that it belonged to a new species. The DNA G+C content of strain YZ01T was 51.6 mol%. The major fatty acids were C16 : 0 and C18 : 1 ω9c. Therefore, based on phylogenetic, phenotypic and physiological evidence, strains YZ01T, YZ02 and YZ03 represent a novel species of the genus Lactobacillus, for which the name Lactobacillus nasalidis sp. nov. is proposed with the type strain YZ01T (=JCM 33769T=DSM 110539T).

A cluster epidemic of influenza A(H1N1)pdm09 virus infection in four captive cheetahs (Acinonyx jubatus).

In January 2019, four cheetahs (Acinonyx jubatus) kept at a Japanese zoo intermittently showed respiratory signs following the incidence of seasonal influenza in animal caregivers. Respiratory materials (saliva, sputum and food tray swabs) were non-invasively collected from the four cheetahs. Although we were unable to isolate the virus, the NP gene of influenza A virus was detected in three of the cheetahs but not in the fourth cheetah that had nearly recovered. From a food tray swab which tested weakly positive by a commercial influenza detection kit, we were able to obtain the whole-genome sequence of the influenza A virus. Analysis of the genome, A/cheetah/Kanagawa/2/2019(H1N1), revealed that the virus was closely related to influenza A(H1N1)pdm09 viruses isolated from humans in Japan in the 2018-2019 winter. Production of haemagglutinin inhibition (HI) antibodies (64-128 HI) against an A(H1N1)pdm09 virus in plasma samples confirmed infection of all four cheetahs. The animals continued to produce antibodies for at least 314 days after disease onset. These findings strongly suggest that reverse zoonotic transmission of A(H1N1)pdm09 virus occurred from human to cheetah and subsequently from cheetah to cheetah in the zoo. We also show that specimens can be safely and non-invasively collected from non-domesticated animals and used to investigate respiratory infectious diseases.

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.

Analysis of glycosuria in okapi (Okapia johnstoni): examination of urinary N-acetyl-ß-D-glucosaminidase.

We analyzed the urinary excretion of glucose and N-acetyl-ß-D-glucosaminidase (NAG) in six okapis (Okapia johnstoni) in captivity to investigate the cause of their urinary sugar excretion. The urinary glucose-positive okapi had significantly higher urinary NAG indices than the urinary glucose-negative okapi. There was also a positive correlation between urinary glucose levels and urinary NAG indices. These results suggest that the proximal tubular function of the glycosuric okapi may have been obstructed, which impaired glucose reabsorption.

Effect of multiple dosing of ketoconazole on pharmacokinetics of midazolam, a cytochrome P-450 3A substrate in beagle dogs.

To evaluate effects of multiple dosing of ketoconazole (KTZ) on hepatic CYP3A, the pharmacokinetics of intravenous midazolam (MDZ, 0.5 mg/kg) before and during multiple dosing of KTZ were investigated in beagle dogs. KTZ tablets were given orally to dogs (n = 4) for 30 days (200 mg b.i.d.). With coadministration of KTZ, t(1/2beta) of MDZ were significantly increased both on day 1 (2-fold) and on day 30 (3-fold). Total body clearance (CL(tot)) of MDZ declined gradually during the first 5 days after the start of KTZ treatment, and thereafter CL(tot) appeared to reach a plateau phase (one-fourth), depending on plasma KTZ concentrations. The effects of KTZ on the biotransformation of MDZ were also investigated using dog liver microsomes (n = 5). The K(i) values of KTZ for MDZ 1'-hydroxylation and 4-hydroxylation were 0.0237 and 0.111 microM, respectively, indicating that KTZ extensively inhibits hepatic CYP3A activity in dogs. CL(tot) values estimated from in vitro K(i) values corrected by unbound fraction of KTZ and unbound concentrations of the drug in plasma were consistent with in vivo CL(tot) of MDZ. The results in this study suggest that KTZ treatment is necessary until plasma concentrations of the drug reach a steady state to evaluate the effect of multiple dosing of the drug on hepatic CYP3A in vivo. In addition, it is suggested that K(i) values corrected by unbound fraction of KTZ and unbound concentrations of the drug in plasma enable precise in vitro-in vivo scaling.