An extensive breakout of equine influenza occurred for the first time in Japan, involving the Tokyo, Nakayama, and Fukushima Race Courses and the Equestrian Park, Tokyo, belonging to the Japan Racing Association, in 1971. In the Equestrian Park, 168 (98.3%) horses of 171 in total were infected with equine influenza within only 6 days, or from December 15 to 20, 1971. Hematological examination was carried out on the infected horses in the Equestrian Park. Ten horses were selected at random and examined for the erythrocyte and leukocyte counts and blood pictures on the 2nd, 4th, 7th, 11th and 22nd days of sickness. All of them were proved to have a high hemagglutination inhibitor in the serum. Equine influenza viruses were isolated from two of them. Consequently, erythrocytes and leukocytes showed a tendency to decrease a little. In the blood picture, a very remarkable increase in the segment form of neutrophil leukocytes was recognized on the 2nd day of sickness, although no increase was seen in the rod form of neutrophil leukocytes, myelocytes, or metamyelocytes. An obvious increase in monocytes was found on the 4th day of sickness. On the 7th day of sickness, these hematological changes caused by the disease disappeared and normal conditions were regained. Therefore, the hematological changes by the disease could be found within a very short period, unless the disease was associated with any secondary infection. It was considered that a hematological finding characteristic of the disease at the beginning would be a very remarkable increase in the segment form of neutrophil leukocytes, in spite of a slight decrease in leukocyte count. Such finding would be a characteristic of the disease, as compared with any other equine viral disease (equine infectious anemia, Japanese B encephalitis, equine rhinopneumonitis, etc.). The disease shows a violent spread, so it should be quickly diagnosed on early time of the outbreak. Therefore the characteristic findings mentioned above would play an auxiliary role in the diagnosis of the early stage of the disease, if they are used at the same time with the results of clinical observation.

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An extensive outbreak of equine influenza occurred in Japan for the first time. It spread over many racecourses, farms and riding clubs in the eastern part of the country. Then, it involved the concentration center of imported horses which had passed quarantine in December, 1971, to be distributed to four places. The hemagglutination-inhibition (HI) test was performed on sera collected from horses in six places involved in the outbreak. Influenza A/equi 1/Prague /'56, Influenza A/equi 2/Miami/'63, and other viruses were isolated from the nasal secretions of infected horses in the allantoic fluid of incubated eggs and used as antigens for the HI test. The test was carried out by the standard tray method. Sera were inactivated at 60°C for 30 minutes after treatment with RDE in the case of the Prague strain. Sera were only inactivated at 60°C for 30 minutes in the case of the Miami and the isolated strain. In addition 5% suspension of chicken erythrocytes was used. The results obtained are as follows. 1) All the horses of riding club A into which some imported horses had been introduced were infected within 3 days. Their sera were proved to have HI titers ranging from 1:4 to 1:64 against the Miami strain and from 1:16 to 1:128< against the isolated strain between 10 and 16 days after the outbreak of the infection. 2) In the Equestrian Park, Tokyo, 168 horses (98.3%) out of 171 in total were infected within 6 days. Influenza viruses were isolated from among the infected horses. The sera of these horses showed an increase in HI antibody titer against the Miami and the isolated virus strain. The antibody titer maintained its peak for 40-50 days and then decreased. The sera of these horses, however, possessed no HI antibody against the prague strain. 3) In the Tokyo Race Course, 957 horses (99.4%) of 963 in total and in the Nakayama Race Course, 674 horses (93.3%) of 721 in total were infected. The sera of the infected horses of both racecourses exhibited an increase in HI titers against the Miami and the isolated strain. 4) In the Fukushima Race Course, 42 horses (39.6%) of 106 in total were considered to be infected, although the method of diagnosis used was not so reliable. The sera of the infected horses showed such increase in HI titer as similar to that mentioned above. 5) In the Shiroi Branch of the Nakayama Race Course, 120 horses of 209 in total were administered with killed vaccine (E1 150 CCA and E2 250 CCA) prepared by the Nippon Institute for Biological Science, Tokyo, but 135 horses out of these were infected with equine influenza. In the Shiroi Branch, many of the horses which had been free from the disease and administered with no vaccine were recognized to show lower HI titers than horses under similar conditions in the Tokyo and Nakayama Race Courses and the Equestrian Park. In the Shiroi Branch, horses which had been free from the disease and administered with vaccine were proved to show HI titers equal to or higher than those of horses recovered from natural infection. There was a large difference in HI titer between the horses under such conditions and horses free from the disease administered with no vaccine in any other racecourse. It was considered that the horses which had been free from the disease and had shown low HI titers might not have obviously been infected or not have been infected at all, and that the horses which had been free from the disease and had shown high HI titers might have been infected with equine influenza. 6) It was supposed that the present outbreak of equine influenza might have been caused by influenza A/equi 2 virus, but not by influenza A/equi 1 virus.

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An acute “coughing disease” broke out among racehorses and riding ones in Japan in December, 1971. In this outbreak, affected horses showed the same symptoms, including fever at 39-41°C, dry cough characterized by stregth, and the secretion of nasal mucus . The disease was suspected to be equine influenza from clinical symptoms. The direct fluorescent antibody technique was applied to some horses involved. The results obtained are summarized as follows. 1.The nasal secretion collected from affected horses was positive for direct fluorescent antibody. It was found that this epizootic had been caused by equine influenza which had never occurred in Japan. 2.Of 29 samples collected from 5 epizootic regions, 18 samples (62.1 %) were proved to be positive. 3.The direct fluorescent antibody technique was considered to be extremely effective for the early diagnosis of equine influenza and for the prevention of the disease. 4.The positive cells were composed mainly of round epithelial cells containing fluorescenn in the cytoplasm . There were 4-8 positive cells perten thousand normal epithelial cells in the direct nasal smear.

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An acute "coughing disease" broke ont in the greater part of the racehorses and riding ones belonging to the Equestrian Park, the Fukushima, Tokyo, and Nakayama Race Courses in December, 1971. The epzootic seemed to have been spread quickly by imported horses which might have been infected with the "coughing disease". In this outbreak infected horses showed the same clinical symptoms, such as fever at 39-41°C, dry cough characterized by strength. Equine influenza viruses were studied to be isolated from 20 of the samples collected from the masopharngeal secretion of febrile horses, inoculated into the amnion of 8 day chick embryos and cnltivated at 35°C for 72 hours. They have been adapted to grow in the chorio allantois of chick embryos. The hemagglutination-inhibition (HI) and neutralization tests were performed with chicken and ferret antisera against A/Equi 1/Prague/56, A/Equi 2/Miami/63, and the isolated (Tokyo/ 71) viruses. The intact viruses were used as antigens. Furthermore, when the antibody response of convalescent horses was compared between Equi 2 and the isolated virus, it was found that the antigenicity of the isolated virus was about 8-16 times higher than that of Equi 2/Miami/63 virus. On the other hand, there was not so marked a difference in HI titer between the antigens of Equi 2/Miami/63 and the isolated viruses for chicken and ferret antisera against the Equi 2/Miami/63 strain. In this result 9 strains of equine influenza virus were isolated. It is for the first time in Japan that equine influenza virus was isolated. It was confirmed that the isolated viruses were similar to the Equi 2 type, although they were distingushed from the Equi 1 type.

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As part of basic studies on the clinical application of phonocardiograms to the racehorse, an experiment was carried out with 32 clinically healthy horses of a light breed. Phonocardiograms were recorded from the aortic, pulmonary, and mitral areas in horses standing in the stall or in the stocks without administration of sedatives or anesthetics. A Fukuda Denshi RS 200S thermal stylus type direct recording phonocardiograph was used in combination with a moving coil contact type microphone. Two filters, L and M₁, were applied. Filter L had a cut-off frequency of 50 hertz and a slope of 18 decibels per octave, and filter M₁, a cut-off frequency of 80 hertz and a slope of 24 decibels per octave. The paper speed adopted was 50 mm or 100 mm per second. 1.The configuration of the first heart sound was either a diamond shape or decrescendo at the rate of nearly half and half, while more than two-thirds of that of the second heart sound was decrescendo. 2.In more than half of the horses studied, the intensity of the first heart sound was higher than that of the second heart sound in the aortic area, but that of the second heart sound was higher in the pulmonary area . In more than two-thirds of the horses studied, the intensity of the first heart sound was higher in the mitral area. 3.The duration of the first heart sound was 0.10 to 0.23 second and that of the second heart sound 0.07 to 0.14 second. 4.The S₁-S₂ interval was 0.42 to 0.57 second and the Q-S₁ interval 0.03 to 0.10 second.

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In research of serum lactate dehydrogenase (S-LDH) isoenzyme for clinical application to racehorses, the first attempt was made to clarify the normal distribution of LDH Isoenzyme in the organs of 1 healthy horse and in the sera of 19 healthy horses by means of YOSHIDA's detecting method of LDH Isoenzyme. Besides, studies were made on changes in S-LDH isoenzyme in 2 horses intoxicated with carbon tetrachloride, 3 horses experimentally infected with equine infectious anemia (EIA), and a horse infected with EIA (chronic type) in the field. Discussion was made especially on the relationship between the appearance of S-LDH₅ isoenzyme and the hepatic dysfunction . The following results were obtained. 1)In the normal equine organs, LDH Isoenzyme consisted of 5 fractions. Of these fractions, LDH₁ occupied a high percentage in each tissue of erythrocyte, heart muscle, cerebellum, pancreas, and kidney, LDH₂ in salivary gland, LDH₃ in adrenal gland, spleen, lymph node, and cerebrum, and LDH₄ in thyroid gland, lung, and skeletal muscle . LDH₅ was contained exclusively in liver, skeletal muscle, and leukocyte. The lactate dehydrogenase isoenzyme contained in the tissues of the equine digestive tract was chiefly composed of LDH₃ and LDH₄. Part of the stomach (pars pylolica and pars fundica) had a high percentage of LDH₁ and LDH₂. 2)Of the fractions of S-LDH isoenzyme contained in the sera of 19 healthy horses, LDH₃ showed the highest percentage, 42.1±4.3 %, and wasfollowed by LDH₂ (34.0±4.0%), LDH₁ (19.4±4.8%), and LDH₄ (4 .2±2.0 %). Fraction LDH₅ could not be found in the serum of any horse. It was better to preserve normal equine serum in the deep-freezer than in the refrigerator. 3) The fraction of S-LDH isoenzyme were located among the serum protein fraction by the aid of relative mobility. Fraction LDH₁ was detected in the post-position of albumin, LDH₂ in the position of α₂-globulin, LDH₃ in the position of β₂-globulin (the position of application), LDH₄ in the middle position of β2 and γ-globulin, and LDH₅ in the fore-end of γ-globulin. 4)It was presumed that LDH₅ might appear during one or two days in horses intoxicated with carbon tetrachloride. When horses were administered with 0.4 ml/kg of carbon tetrachloride 9 times at a week's intervals, the relative percentage of LDH₅ and the activity of S-GOT showed a tendency to decrease after every administration. 5)In horses infected with EIA, the total activity of S-LDH increased a little late in the febrile stage. LDH₃, LDH₄, and LDH₂ increased in value in the order listed in this stage. LDH₄ showed the most characteristic increase. In horses inoculated with EIA virus, there was a tendency for LDH₅ to appear frequently in an early febrile stage (during a period from the 15th to 35th day after inoculation). In this stage, a high ratio of LDH₄ could be found. In the field case classified as chronic type, these tendencies were not so outstanding as in the inoculated cases. On the basis of these findings, it is concluded that the confirmation of LDH₅, which cannot be found in normal equine serum, may be a valuable index for the early diagnosis of hepatocellular damage in racehorses.

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Observation was made on the blinking action of horses at each time of rest, epirenamine injection, and additional stimulus of picking. The change of heart rate was used as index of excitement at that time. The results obtained are as follows. 1.Heart rate : It increased markedly by the injection of epirenamine. In some cases, it increased at the time of additional stimulus of picking. 2.Frequency of blinking : There were differences in frequency of blinking among animals at rest. This frequency increased by the injection of epire namine, but decreased markedly by the addition of stimulus of picking. The interval of blinking time was still irregular in both cases. 3.Blinking time : It was generally 0.38 second in the horses studied. The movement of opening the eyelids became slower than that of closing them. 4.Length of the eyelids : By the injection of epirenamine the eyelids increased in length markedly after they became narrow temporarily. This is contrary to the fact that the eyelids increase in length in man at psychic excitement. 5.The flutter : A phenomenon considered to be peculiar to horses, was recognized at a higher frequency than true blinking, though at irregular intervals.

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Conventionally, identification of horses has been conducted by various methods. The proposed method is basically an application of the freezing technique to horses. Marks were made by instruments in solid carbon dioxide (dry ice) and 95% ethanol. These were made with pressure on the handle of the applicator instrument. Freeze-brand were applied at 10 to 60 seconds. White-haired marks appeared most consistently when application time was between 20 and 30 seconds. At about 3 months, the hair will grow in white and number or mark will be more discernible.

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Spectrofluorophotometric determination of quinidine sulfate in horse plasma was established, using deproteinization with 20 % metaphosphoric acid. The plasma levels of quinidine sulfate after a single oral administration with 40 or 60 mg/kg were determined. A peak value of 10 pg/ml was found 3 hours after administration with 60 mg/kg. The substance was detectable even 30 hours after administration. A single oral dose of 40 mg/kg showed no significant side effects, but one of 60 mg/kg induced colic or uterine pain in one horse as side effects.

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In light horses fed a complete pelleted ration or a conventional ration, respiratory gases were analyzed in the state of resting. The effects of diet and season on gas metabolism were studied by means of the method of Douglas-mask. The results obtained are summarized as follows. 1.Even when a different diet was given in winter, there were no significant differences in respiratory quotient (RQ), expired volume of air, consumed oxygen, and produced carbon dioxide, which were 0.86-0.90, 40-50 1 /mm., 1.5-1.6 1/min., and 1.3-1.4 1/min., respectively, in this season. 2.RQ was lower in summer than in winter in light horses fed a pelleted ration. O₂ consumption increased to de significantly high in summer. 3.Basal metabolic energy calculated was Q=107.4 kcal/W^0.75kg in winter and Q=160.7 kcal/W^0.75kg in summer.

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The influence of infusion with epinephrine on expired gases was examined in horses fed a complete pelleted ration. The horses were in the state of excitement, similar to that during light exercise, after administration with epinephrine at the rate of 1μg /kg. The results obtained are summarized as follows. 1. Respiratory volume and 0₂ consumption kept increasing for about 10 minutes after infusion with epinephrine. 2. C0₂ production increased remarkably after administration and decreased slowly after a certain period of excitement. So that the respiratory quotient (RQ) was elevated from 0.86 at rest to 0.92-0.95 after infusion and remained at this level up to 60 minutes after infusion. Since blood glucose kept increasing for 60 minutes after a certain period of decline, the promotion of carbohydrate metabolism after infusion with epinephrine was presumed to continue considerably long. 3. To express the correlation between respiratory volume and 0₂ consumption after the infusion, the following reduction formula was obtained : Y=29. 16 X-0. 22, where Y is respiratory volume and X, consumed oxygen (1/mm.). nThe presence of a closely proportional interrelationship was shown by this formula.

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Little is known concerning the regulation or tolerance of carbohydrate metabolism in the horse, in spite of its importance as racehorse. In order to approach this problem, the glucose tolerance test was carried out in horses to clarify the glucose clearance and insulin response. The results indicated that our general concept on glucose metabolism in the horse was wrong. That is, the plasma glucose was removed approximately with first order reaction, but its clearance rate was relatively lower than in any other species. On the other hand, there were two types of insulin response. In one type, the appearance of a peak was much delayed. Naturally, this resulted in the reduction of carbohydrate metabolism. In other type, the insulin response was quick, and two peaks of insulin response were observed. However, it is not clear yet whether these insulin responses are playing a primary role in their relatively lower carbohydrate metabolism at rest in the horse.

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Ten cases of hemolytic icterus of foals were studied clinically and hematologically. The results obtained are summarized as follows. 1. The mode of occurrence of the disease in question and the clinical signs of diseased foals coincided with those of the hemolytic icterus of foals reported already. 2. Anemia, high icterus index, appearance of erythrophages and sideroleu kocytes, and reduced red blood cell fragility were characteristic findings in the blood. 3. The red blood cells of diseased foals were hemolyzed in the hemolytic test. They were positive for the direct Coombs' test. 4. Erythrophages and slight or almost perfect destruction of red blood cells were seen by electron microscopy.

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