Aortic valve insufficiency caused by a hypoplastic aortic valve in a calf.

A 1-month-old crossbred calf was referred for examination due to marked systolic heart murmurs and poor growth. The heart murmur was most audible on the right side of the cranial thorax. Cardiomegaly was evident on chest radiography, and echocardiography demonstrated aortic regurgitation and decreased fractional shortening. Cardiomegaly, aortic root dilation and cardiac displacement were confirmed by computed tomography. At necropsy, the heart was enlarged, and all three aortic valve leaflets were irregularly shaped. In calves with chronic aortic insufficiency, remodeling displacement of the heart and aorta causes changes in the location and timing of heart murmurs. Therefore, aortic insufficiency cannot be ruled out when a systolic heart murmur can be observed in the right chest wall.

Cynomolgus macaque model of neuronal ceroid lipofuscinosis type 2 disease.

Neuronal ceroid lipofuscinoses (NCLs) are autosomal-recessive fatal neurodegenerative diseases that occur in children and young adults, with symptoms including ataxia, seizures and visual impairment. We report the discovery of cynomolgus macaques carrying the CLN2/TPP1 variant and our analysis of whether the macaques could be a new non-human primate model for NCL type 2 (CLN2) disease. Three cynomolgus macaques presented progressive neuronal clinical symptoms such as limb tremors and gait disturbance after about 2 years of age. Morphological analyses using brain MRI at the endpoint of approximately 3 years of age revealed marked cerebellar and cerebral atrophy of the gray matter, with sulcus dilation, gyrus thinning, and ventricular enlargement. Histopathological analyses of three affected macaques revealed severe neuronal loss and degeneration in the cerebellar and cerebral cortices, accompanied by glial activation and/or changes in axonal morphology. Neurons observed throughout the central nervous system contained autofluorescent cytoplasmic pigments, which were identified as ceroid-lipofuscin based on staining properties, and the cerebral cortex examined by transmission electron microscopy had curvilinear profiles, the typical ultrastructural pattern of CLN2. These findings are commonly observed in all forms of NCL. DNA sequencing analysis identified a homozygous single-base deletion (c.42delC) of the CLN2/TPP1 gene, resulting in a frameshifted premature stop codon. Immunohistochemical analysis showed that tissue from the affected macaques lacked a detectable signal against TPP1, the product of the CLN2/TPP1 gene. Analysis for transmission of the CLN2/TPP1 mutated gene revealed that 47 (49.5%) and 48 (50.5%) of the 95 individuals genotyped in the CLN2-affected macaque family were heterozygous carriers and homozygous wild-type individuals, respectively. Thus, we identified cynomolgus macaques as a non-human primate model of CLN2 disease. The CLN2 macaques reported here could become a useful resource for research and the development of drugs and methods for treating CLN2 disease, which involves severe symptoms in humans.

Aquatic environments change the cardiac morphology of dolphins.

Previous studies on dolphin electrocardiograms have shown that they are mainly composed of increased negative waves, similar to ungulates. The electrocardiogram waveform was determined by the distribution of the Purkinje fibers. Based on the waveform of the dolphin electrocardiogram, Hamlin predicted that the Purkinje fibers would be distributed within the ventricular muscle, as in ungulates. The purpose of this study was to confirm the histological distribution of Purkinje fibers in dolphins. In the present study, bottlenose dolphin hearts were observed both grossly and histologically, and the effects of Purkinje fiber distribution and cardiac morphology on electrocardiogram waveforms were examined. This study showed that the Purkinje fibers of dolphins run just below the endocardium, as in humans, dogs, and cats, whose electrocardiograms mainly show positive waves. When the cardiac morphology of dolphins was observed carefully, the right ventricle was found to be extremely dilated compared to that of terrestrial mammals. In human recreational divers, right ventricular dilatation is induced by diving. We hypothesized that the dolphin's heart is in a state similar to that of the right heart dilatation in terrestrial animals. The dolphin electrocardiogram waveform was considered to be due to right axis deviation. Based on the above, we concluded that the dolphin electrocardiogram waveform was due to its ability to live in water. We found that the dolphins are genetically related to ungulates, particularly the hippopotamus, but that their hearts have evolved differently.

The effect of different body positions on the cardiothoracic ratios obtained by chest radiography in Japanese macaques (Macaca fuscata) and rhesus macaques (Macaca mulatta).

BACKGROUND: Although some studies have reported cardiac diseases in macaques, an adequate screening method for cardiac enlargement has not yet been established. This study aimed to evaluate the positioning of macaques for radiographs and establish reference intervals for the cardiothoracic ratio (CTR). MATERIALS AND METHODS: We developed a device for chest radiography in the sitting position and performed chest radiography in 50 Japanese and 48 rhesus macaques to evaluate the CTR and chest cavity size. RESULTS: In Japanese and rhesus macaques, the thorax height was significantly larger, the heart width was significantly smaller, and the mean CTR was significantly smaller in the sitting position than in the prone position. The reference intervals for CTR in the sitting position were 51.6 ± 4.6% and 52.2 ± 5.1% in Japanese and rhesus macaques, respectively. CONCLUSION: Thoracic radiographic images obtained in a sitting position resulted in a smaller CTR and a larger thorax height, which could be useful for detecting pulmonary and cardiac abnormalities.

Activation of canine neutrophils by platelet-activating factor.

Neutrophils are essential for innate immunity as the first line of defence. Neutrophils act as phagocytic white blood cells to kill bacteria and other microorganisms. A strong respiratory burst of neutrophils, dependent on reactive oxygen species, is produced during phagocytosis. Platelet-activating factor (PAF) is a signalling molecule with several prominent roles in tissue injury, inflammation, and platelet aggregation. However, the detailed mechanisms and intracellular signalling pathways involved in PAF-mediated neutrophil activation remain unclear. Here, we investigated the effect of PAF on changes in calcium concentration ([Ca2+]i) and oxygen radical (O2-) generation in activating canine neutrophils. We further evaluated these effects of PAF with inhibition of G protein-coupled receptors using the specific inhibitor suramin. Blood samples were collected from a total of five dogs and neutrophils were isolated. PAF stimulation of canine neutrophils caused an increase in [Ca2+]i as well as the generation of O2-, and the PAF receptor was sensitive to suramin. The results suggested that PAF stimulation of canine neutrophils may cause Ca2+ influx from the endoplasmic reticulum into the cytoplasm (as the first wave) and then trigger store-operated Ca2+ entry (as the second wave), which is an important intracellular signal transduction pathway for neutrophil activation. Furthermore, O2- generation by PAF stimulation may depend on the intracellular signalling pathway, with increasing inositol trisphosphate levels and [Ca2+]i via G protein-coupled receptors. The finding that PAF-activating platelet aggregation is involved in canine neutrophil activation suggests a close relationship between haemostasis and neutrophil activation in dogs, offering new insight into the response to infection.

Prevalence and risk factors for feather-damaging behavior in psittacine birds: Analysis of a Japanese nationwide survey.

A case control study was conducted to estimate the prevalence of feather-damaging behavior and evaluate the correlation with risk factors among pet psittacine birds in Japan. Although feather-damaging behavior among pet parrots is frequently observed in Japan, its prevalence and potential risk factors have not been investigated. Therefore, we conducted an online questionnaire survey on parrot owners throughout Japan to examine regional differences in feather-damaging behavior and associated risk factors. In total, 2,331 valid responses were obtained. The prevalence of feather-damaging behavior was 11.7%, in general agreement with prior studies. The highest prevalence was among Cockatoos (Cacatua spp., etc.; 30.6%), followed by Lovebirds (Agapornis spp.; 24.5%) and African grey parrots (Psittacus erithacus; 23.7%). Multivariate logistic regression was carried out to calculate the adjusted odds ratio (ORadj) for potential risk factors and adjust the confounding of the variables. The odds of feather-damaging behavior were significantly higher for Conures (Aratinga spp., Pyrrhura spp., Thectocercus acuticaudatus, Cyanoliseus patagonus) (ORadj = 2.55, P = 0.005), Pacific parrotlets (Forpus coelestis) (ORadj = 3.96, P < 0.001), African grey parrots (ORadj = 6.74, P < 0.001), Lovebirds (ORadj = 6.79, P < 0.001) and Cockatoos (ORadj = 9.46, P < 0.001) than Budgerigars (Melopsittacus undulatus), and for young adults (ORadj = 1.81, P = 0.038) and adults (ORadj = 3.17, P < 0.001) than young birds, and for signs of separation anxiety (ORadj = 1.81, P < 0.001). Species, bird age and signs of separation anxiety were significantly higher risk factors for feather-damaging behavior than any other potential risk factors. Our findings, which include broad species diversity, are a good source of data for predicting risk factors for feather-damaging behavior and could be useful in preventing declines in welfare.

Usefulness of cardiac hormones for evaluating valvular disease in cynomolgus monkeys (Macaca fascicularis).

Nonhuman primates are commonly used as experimental animals due to their biological resemblance to humans. In patients with cardiac disease, the levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) tend to increase in response to cardiac damage, and they are thus used as indicators for the diagnosis of human heart failure. However, no reference values for ANP and BNP have been reported for heart disease in nonhuman primates. In this study, we recorded the age, sex, and body weight of 202 cynomolgus monkeys, and performed evaluations to assess the ANP and BNP levels, electrocardiography and echocardiography, and accordingly divided the monkeys into two groups: healthy monkeys and those with spontaneous cardiac disease. Statistical analysis was performed to determine the relationship of ANP and BNP with the factors of age, sex, and body weight. No significant relationship was found between the levels of ANP and BNP and the factors of age, sex, and body weight. However, both the ANP and BNP levels were significantly different between the healthy monkeys and monkeys with valvular disease. Similar to humans, the ANP and BNP levels tended to increase with the progression of cardiac disease in monkeys. Based on these results, we concluded that ANP and BNP are indicators of cardiac disease in nonhuman primates, and that this nonhuman primate cardiac disease model is applicable for cardiology research in humans.

Ultrasound-guided, Transabdominal, Intrauterine Artificial Insemination for Cynomolgus Macaques (Macaca fascicularis) Based on Estimated Timing of Ovulation.

Intrauterine sperm injection for artificial insemination is difficult in cynomolgus macaques (Macaca fascicularis) and rhesus macaques (M. mulatta) due to the complex structure of the cervical canal, which differs from that of humans. Despite the availability of several artificial insemination methods for macaques, pregnancy rates are inconsistent, and details regarding ovulation are unclear, thus warranting more effective methods. Therefore, we developed an effective, ultrasound-guided, transabdominal intrauterine artificial insemination method for cynomolgus macaques that involves timing sperm injection to coincide with the periovulation phase estimated according to rapid hormone measurement. We performed our intrauterine artificial insemination on 6 female macaques; 4 of the 5 animals that were predicted to have ovulated soon after insemination became pregnant, whereas the 1 macaque that was predicted not to have ovulated did not. Furthermore, we saw no evidence of injury, such as a conspicuous needle hole or bleeding on the surface of or inside the uterus, nor did our method result in any abnormalities in the mothers or their offspring. Thus, our ultrasound-guided, transabdominal, intrauterine artificial insemination method is rapid, safe, and effective in cynomolgus macaques.

Echocardiographic evaluation of cardiac function in cynomolgus monkeys over a wide age range.

Various cardiovascular diseases can be detected and diagnosed using echocardiography. The demand for cardiovascular system research using nonhuman primates is increasing, but echocardiographic references for nonhuman primates are limited. This report describes the first comparison of echocardiographic reference values in 247 normal cynomolgus monkeys (135 females, 112 males) over a wide age range. Echocardiography, electrocardiography, blood pressure and chest X-ray images were acquired under immobilization with intramuscular ketamine hydrochloride, then cardiac structure, function, and flow velocity were assessed. Cardiac hormone levels were also tested. We found that cardiac structures positively correlated with weight, that the size of these structures stabilized after reaching maturity and that cardiac output increased according to heart size. In contrast, fractional shortening of the left ventricle, ejection fraction and flow velocity showed no significant correlations with weight or age, and age and E wave correlated negatively. These findings appear sufficiently similar to those in humans to suggest that cynomolgus monkeys can serve as a suitable model of human cardiac disease. Our data should also prove useful for surveying cardiac dysfunction in monkeys.

Establishment of a new formula for QT interval correction using a large colony of cynomolgus monkeys.

The demand for monkeys for medical research is increasing, because their ionic mechanism of repolarization is similar to that of humans. The QT interval is the distance between the Q wave and T wave, but this interval is affected by heart rate. Therefore, QT correction methods are commonly used in clinical settings. However, an accurate correction formula for the QT interval in cynomolgus monkeys has not been reported. We assessed snapshot electrocardiograms (ECGs) of 353 ketamine-immobilized monkeys, including aged animals, and contrived a new formula for the corrected QT interval (QTc) as a marker of QT interval prolongation in cynomolgus monkeys. Values for QTc were calculated using the formula [QTc] = [QT] / [RR]n, along with several other formulas commonly used to calculate QTc. We found that the optimal exponent of the QT interval corrected for heart rate, n, was 0.576. The mean value of QTc in healthy monkeys determined using the new formula was 373 ± 31 mm, and there were no significant differences between the sexes. Other ECG parameters were not significantly different between the sexes and there were no age-related effects on QTc. Prolongation of QTc to over 405 ms, as calculated by the new formula, was observed in 50 monkeys with underlying diseases. Additionally, all monkeys with QTc above 440 ms by the new formula had some underlying disease. The results resemble those in humans, suggesting that the new QTc formula could be useful for diagnosis of QT interval prolongation in cynomolgus monkeys.

Utility of arterial blood gas, CBC, biochemistry and cardiac hormones as evaluation parameters of cardiovascular disease in nonhuman primates.

Cardiovascular disease (CVD) has a tremendous impact on the quality of life of humans. While experimental animals are valuable to medical research as models of human diseases, cardiac systems differ widely across various animal species. Thus, we examined a CVD model in cynomolgus monkeys. Laboratory primates are precious resources, making it imperative that symptoms of diseases and disorders are detected as early as possible. Thus, in this study we comprehensively examined important indicators of CVD in cynomolgus monkeys, including arterial blood gas, complete blood count (CBC), biochemistry and cardiac hormones. The control group included 20 healthy macaques showing non-abnormal findings in screening tests, whereas the CVD group included 20 macaques with valvular disease and cardiomyopathy. An increase of red blood cell distribution width was observed in the CBC, indicating chronic inflammation related to CVD. An increase of HCO3 was attributed to the correction of acidosis. Furthermore, development of the CVD model was supported by significant increases in natriuretic peptides. It is suggested that these results indicated a correlation between human CVD and the model in monkeys. Moreover, blood tests including arterial blood gas are non-invasive and can be performed more easily than other technical tests. CVD affected animals easily change their condition by anesthesia and surgical invasion. Pay attention to arterial blood gas and proper respond to their condition are important for research. This data may facilitate human research and aid in the management and veterinary care of nonhuman primates.

Superparamagnetic Iron Oxide-enhanced Magnetic Resonance Imaging of Spontaneous Hepatic Neoplasia in a Cynomolgus Macaque (Macaca fascicularis).

Although the number of reports describing tumors in aged NHP has increased, spontaneous neoplasias in NHP are extremely rare, with the notable exception of prosimians, in which spontaneous hepatic neoplasms arise. In addition to radiography and ultrasonography, superparamagnetic iron oxide (SPIO)-enhanced MRI tends to be applied in human practice to non-invasively locate, identify, and size liver tumors and to define the border between neoplastic and normal tissues. Here we report a 13-y-old female cynomolgus monkey with anorexia and serologically normal liver enzymes. After fluid therapy, the condition remained in remission for several months. Later, however, a palpable mass was assessed by using ultrasonography, radiology, and SPIO-MRI; T2-weighted images revealed a clear border between a hepatocellular carcinoma and normal liver tissue. Findings at necropsy supported the imaging data. Serologic assessment after euthanasia revealed a positive reaction to an abnormal form of prothrombin (PIVKA-II). We recommend SPIO-MRI as a practical and useful for diagnosing hepatocellular neoplasias in NHP. This study is the first to demonstrate the applicability of SPIO-MRI for the identification of hepatocellular carcinoma in NHP.

Establishment of reference values for complete blood count and blood gases in cynomolgus monkeys (Macaca fascicularis).

Cynomolgus monkeys are closely related to humans phylogenetically, and this has resulted in their widespread use as a preclinical model. Hematological data with regard to these monkeys are thus important. Although reference values for blood components and sex hormones have been established for cynomolgus monkeys, those for arterial blood gases have not. The arterial blood gases quickly reflect respiratory and circulatory dynamics, and are thus useful for animal management and safe general anesthesia and surgical operations. Furthermore, since O2 is transported by RBC, CBC and blood gases are closely related. The present study aimed to establish reference values for arterial blood gases and CBC in cynomolgus monkeys over a wide age range. Blood gases and CBC of arterial blood, collected from 41 female and 21 male anesthetized monkeys, were measured. Age correlated with RBC, HGB and HCT in the CBC. Values differed significantly between males and females in pCO2, CO2 concentration, MCV and MCH. The pH of blood was equivalent to that of humans and pCO2 was more stable, whereas MCV and MCH were lower than those in humans. Erythrocytes were smaller and less pigmented than in other Macaca species. Several relationships between gender and age, and blood gases and CBC were identified in cynomolgus monkeys. In conclusion, these reference values will be useful as markers for veterinary applications and in the care and maintenance of these animals.

The pectoral fin muscles of the coelacanth Latimeria chalumnae: Functional and evolutionary implications for the fin-to-limb transition and subsequent evolution of tetrapods.

To investigate the morphology and evolutionary origin of muscles in vertebrate limbs, we conducted anatomical dissections, computed tomography and kinematic analyses on the pectoral fin of the African coelacanth, Latimeria chalumnae. We discovered nine antagonistic pairs of pronators and supinators that are anatomically and functionally distinct from the abductor and adductor superficiales and profundi. In particular, the first pronator and supinator pair represents mono- and biarticular muscles; a portion of the muscle fibers is attached to ridges on the humerus and is separated into two monoarticular muscles, whereas, as a biarticular muscle, the main body is inserted into the radius by crossing two joints from the shoulder girdle. This pair, consisting of a pronator and supinator, constitutes a muscle arrangement equivalent to two human antagonistic pairs of monoarticular muscles and one antagonistic pair of biarticular muscles in the stylopod between the shoulder and elbow joints. Our recent kinesiological and biomechanical engineering studies on human limbs have demonstrated that two antagonistic pairs of monoarticular muscles and one antagonistic pair of biarticular muscles in the stylopod (1) coordinately control output force and force direction at the wrist and ankle and (2) achieve a contact task to carry out weight-bearing motion and maintain stable posture. Therefore, along with dissections of the pectoral fins in two lungfish species, Neoceratodus forsteri and Protopterus aethiopicus, we discuss the functional and evolutionary implications for the fin-to-limb transition and subsequent evolution of tetrapods. Anat Rec, 299:1203-1223, 2016. © 2016 Wiley Periodicals, Inc.

Tracking cells implanted into cynomolgus monkeys (Macaca fascicularis) using MRI.

Regenerative therapy with stem cell transplantation is used to treat various diseases such as coronary syndrome and Buerger's disease. For instance, stem-cell transplantation into the infarcted myocardium is an innovative and promising strategy for treating heart failure due to ischemic heart disease. Basic studies using small animals have shown that transplanted cells improve blood flow in the infarcted region. Magnetic resonance imaging (MRI) can noninvasively identify and track transplanted cells labeled with superparamagnetic iron oxide (SPIO). Although clinical regenerative therapies have been clinically applied to patients, the fate of implanted cells remains unknown. In addition, follow-up studies have shown that some adverse events can occur after recovery. Therefore, the present study evaluated the ability of MRI using a 3T scanner to track implanted peripheral blood mononuclear cells labeled with SPIO on days 0 and 7 after intramuscular (i.m.) and intravenous (i.v.) injection into a cynomolgus monkey. Labeled cells were visualized at the liver and triceps surae muscle on MR images using T1- and T2-weighted sequences and histologically localized by Prussian blue staining. The transplanted cells were tracked without abnormal clinical manifestations throughout this study. Hence, MRI of cynomolgus monkey transplanted SPIO-labeled cells is a safe and efficient method of tracking labeled cells that could help to determine the mechanisms involved in regenerative therapy.

Analysis of the breast milk of giant pandas (Ailuropoda melanoleuca) and the preparation of substitutes.

The first milk substitute for giant panda cubs was developed in 1988 based on limited data about giant panda breast milk and that of certain types of bear. Mixtures of other formulas have also been fed to cubs at some facilities. However, they are not of sufficient nutritional quality for promoting growth in panda cubs. Here, we report analysis of giant panda breast milk and propose new milk substitutes for cubs, which were developed based on the results of our analysis. The Chengdu Research Base of Giant Panda Breeding obtained breast milk samples from three giant pandas. Up to 30 ml of breast milk were collected from each mother by hand. Then, the milk samples were frozen and sent to Nihon University. The levels of protein, fat, carbohydrates, ash, moisture, vitamins, minerals, total amino acids, fatty acids, lactose and other carbohydrates in the milk were analyzed. The breast milk samples exhibited the following nutritional values: protein: 6.6-8.5%, fat: 6.9-16.4%, carbohydrates: 2.5-9.1%, ash: 0.9-1.0% and moisture: 67-83%. We designed two kinds of milk substitutes based on the data obtained and the nutritional requirements of dogs, cats and rodents. The nutritional composition of the milk substitutes for the first and second stages was as follows: protein: 38 and 26%, fat: 40 and 40%, carbohydrates: 13 and 25%, ash: 6 and 6% and moisture: 3 and 3%, respectively. In addition, the substitutes contained vitamins, minerals, taurine, docosahexaenoic acid, lactoferrin, nucleotides and other nutrients.

Effects of food enriched with egg yolk hydrolysate (bone peptide) on bone metabolism in orchidectomized dogs.

We examined the effects of chicken egg hydrolysate (also known as "bone peptide" or BP) on bone metabolism in 5- to 8-month-old orchidectomized dogs. The bone formation marker serum bone alkaline phosphatase (BAP) and the bone resorption marker urine deoxypyridinoline (DPD) were used as indicators to measure changes in bone metabolism. The following results were observed that Serum BAP was higher in dogs fed BP-enriched food throughout the clinical investigation. Serum BAP was statistically significantly higher in dogs fed BP-enriched food than in dogs fed non-BP-enriched food at 2 months after orchidectomy. This suggests that BP promoted bone formation immediately after orchidectomy.

Comparative investigation of body composition in male dogs using CT and body fat analysis software.

In small animal veterinary practices, body condition score (BCS) is generally used to diagnose obesity. However, BCS does not constitute objective data. In this study, we investigated the value of using human body fat analysis software for male dogs. We also compared changes in body fat after neutering. Changes in body fat at the time of neutering (age 1 year) and 1 year later were compared by performing CT scanning and using human body fat analysis software. We found that body fat increased in all the individuals tested. In terms of the site of fat accumulation, subcutaneous fat was more pronounced than visceral fat with a marked increase on the dorsal side of the abdomen rather than the thorax.

Standard electrocardiographic data of young Japanese monkeys (Macaca fusucata).

The electrocardiogram of nonhuman primates is similar to that of humans because of similar intrathoracic heart position and structure. Despite the frequent use of nonhuman primates in biologic studies, few electrocardiographic studies of Japanese monkeys (Macaca fusucata) have been reported, and no reference data are available for this species. We obtained limb-lead electrocardiograms from indoor-bred and housed ketamine-sedated Japanese macaques (48 male; 56 female; mean age, 44.3 mo; mean body weight, 4.84 kg) in the dorsal recumbency. The following quantitative data was obtained: heart rate, P wave amplitude and width, R wave amplitude, QRS duration, PR interval, QT interval, T wave height, and mean electrical axis. Corrected QT intervals were calculated by using the Bazett and Fridericia formulae. Measurements were evaluated according to sex and age. The duration of the QRS complex showed moderate correlation with age in male monkeys. All parameters, except heart rate, were similar to previous reports from Japanese, cynomolgus, and other macaques. P waves, R waves and mean electrical axis did not differ significantly between humans and Japanese macaques, but the wave amplitude in macaques was half that in humans. Our electrocardiographic measurements can serve as normal reference data for sedated, young Japanese monkeys.

Neuronal ceroid lipofuscinosis in Border Collie dogs in Japan: clinical and molecular epidemiological study (2000-2011).

Neuronal ceroid lipofuscinosis (NCL) is an inherited, neurodegenerative lysosomal disease that causes premature death. The present study describes the clinical and molecular epidemiologic findings of NCL in Border Collies in Japan for 12 years, between 2000 and 2011. The number of affected dogs was surveyed, and their clinical characteristics were analyzed. In 4 kennels with affected dogs, the dogs were genotyped. The genetic relationships of all affected dogs and carriers identified were analyzed. The survey revealed 27 affected dogs, but there was a decreasing trend at the end of the study period. The clinical characteristics of these affected dogs were updated in detail. The genotyping survey demonstrated a high mutant allele frequency in examined kennels (34.8%). The pedigree analysis demonstrated that all affected dogs and carriers in Japan are related to some presumptive carriers imported from Oceania and having a common ancestor. The current high prevalence in Japan might be due to an overuse of these carriers by breeders without any knowledge of the disease. For NCL control and prevention, it is necessary to examine all breeding dogs, especially in kennels with a high prevalence. Such endeavors will reduce NCL prevalence and may already be contributing to the recent decreasing trend in Japan.