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Bovine isoleucyl-tRNA synthetase (IARS) disorder, a major cause of weak calf syndrome, is caused by a homozygous missense (c.235G>C) mutation in the bovine IARS gene of Japanese Black (JB) cattle, which was identified in 2013. However, the extent to which the carrier rate has changed at Kagoshima prefecture, Japan, and whether the carrier status is associated with any clinical or reproductive problems, have yet to be ascertained. In this study, using a real-time polymerase chain reaction-based genotyping assay, we determined the carrier rate in a regional JB cow population at Kagoshima prefecture. Comparative analyses were performed on the metabolic profile test (MPT) results and reproductive performance data obtained for heterozygous carrier and homozygous wild-type cows. In 2009 and 2018, DNA samples were collected from 130 and 462 clinically healthy JB cows, respectively, in Kagoshima prefecture. MPT results and reproductive performance data were evaluated for 62 cows, comprising four heterozygous carriers and 58 wild-type cows. Genotyping revealed that the carrier rate was 6.9% in 2009 and 1.5% in 2018, the difference of which was statistically significant (P<0.005). There were no statistically significant differences between the carrier and wild-type cows with respect to either MPT results or reproductive performance, indicating that the carrier cows have necessary IARS activity to maintain minimal health and reproductive potential.

This study investigated the effect of heat-killed Lactobacillus sakei HS-1 (HK-LS HS-1) on the health and fecal bacteriological change of suckling Japanese Black calves as a supplement in milk replacers. Twelve calves were separated from dams to calf-hatch after calving for milk replacers feeding. They were randomly assigned to an HK-LS HS-1 supplement or a control without HK-LS HS-1 group in milk replacers. HK-LS HS-1 was administered from separation day to 3 weeks. Blood and fecal samples were examined. Two calves with a haptoglobin concentration of >500 µg/ml on day 0 were excluded from the experiment, and 10 calves were finally included. Glucose and vitamin A levels on day 7 were significantly higher (P<0.05) in the supplement group than in the control group. No significant differences were observed in haptoglobin or serum amyloid A between the groups. The number of Escherichia coli in feces was lower in the control group than in the supplement group on day 21 (P=0.06). No difference was observed in the number of bifidobacteria, but that of lactic acid bacteria was significantly higher (P<0.05) in the supplement group on day 21. The number of medications administered was significantly lower (P<0.05) in the supplement group (5.2 ± 3.9) than in the control group (10.6 ± 5.9) during the experimental period. The results indicated that HK-LS HS-1 is potentially beneficial for improving intestinal microbes and reducing the number of medical treatments.

The objective of the present study was to evaluate the cross-protective immunity between type 1 and type 2 porcine reproductive and respiratory syndrome virus (PRRSV) isolates in growing pigs. Japanese type 1 PRRSV, first isolated from a pig with respiratory disorders in a farm in 2009, exhibits unique genetic characteristics. The pathogenicity of a Japanese standard strain of type 2 PRRSV, EDRD1, in pigs immunized by the type 1 PRRSV isolate, Jpn EU 4-37 was determined by evaluating clinical signs, viremia, antibody response, and pathological lesions. Similarly, we evaluated the pathogenicity of Jpn EU 4-37 in pigs immunized by EDRD1 and compared the cross-protective immunity between these isolates. The EDRD1 challenge after Jpn EU 4-37 inoculation reduced viral clearance and shedding in pigs, compared to those treated with the EDRD1 single infection. On the other hand, the pathogenicity of Jpn EU 4-37 after EDRD1 infection did not differ significantly compared to non-immunized pigs treated with Jpn EU 4-37. Therefore, exposure to Jpn EU 4-37 could not induce enough immunity to reduce the viremia against subsequent infection by type 2 PRRSV. However, the immunity induced by Jpn EU 4-37 infection may play a role in reducing viremia caused by type 2 PRRSV. Moreover, the immunity induced by the EDRD1 and other genetically related viruses, which are broadly distributed in Japan, may not contribute to cross-protection against Jpn EU 4-37 as an emerging virus.

The objective of this study was to evaluate the breeding performance of Indonesian beef cattle (Ongole cross) as recipients for embryo transfer using Limousin embryos. As a result, the pregnancy rate was 35% (7 out of 20 cows). There was a significant difference (P<0.01) in the serum progesterone concentration between the nonpregnant and pregnant cows at the time of the embryo transfer (day 7 after the estrus). The pregnancy rate in the Indonesian beef cows was low, which may be due to their insufficient genetic quality and/or low physical conditions caused by the poor management, like a low-nutrition diet. The low progesterone concentration in the nonpregnant cows on day 7 may be associated with the failure of embryo implantation.

The present study aimed to determine possible risk factors affecting reproductive performance, especially time taken to establish pregnancy in dairy cows, using Cox's proportional hazard model. The data were collected from 154 Holstein Friesian cows (199 lactations). Cows diagnosed with a vaginal discharge score (VDS) of one or calving abnormality showed significantly delayed pregnancy: hazard ratio (HR)=0.654 (95% confidence interval [CI]: 0.436-0.983; P=0.041) and HR=0.457 (95% CI: 0.270-0.774; P=0.004), respectively. Our study suggested that the occurrence of VDS of one or calving abnormality might be possible risk factors increasing the number of days open and affecting reproductive performance in dairy cows.

In this study, the prevalence, effectiveness of diagnosis, and treatment based on vaginal discharge score (VDS) of clinical endometritis in cattle were evaluated. To detect clinical endometritis and classify its severity, vaginoscopy was performed during 21 to 60 days postpartum in 164 Holstein cows consisting of 229 lactations. Groups were defined using the 4-point VDS scale. Study groups included the following: non-endometritis (VDS=0; no/clear mucus; NEM group; n=168); mild endometritis, no treatment (VDS=1; mucus containing flecks of white/off-white pus; NTR group; n=30); and severe endometritis, treated with PGF2α (VDS≥2; discharge containing <50% pus; and VDS=3; discharge containing >50% pus, and fluid or uterine horn asymmetry; TEM group; n=31). Cows treated with PGF2α that did not recover (VDS≥1, n=5) received intrauterine procaine penicillin and streptomycin. Prevalence of clinical endometritis (VDS≥1) was 26.6%. The NTR group required significantly more artificial inseminations per pregnancy than NEM and TEM groups (2.8 ± 1.8 vs 2.0 ± 1.3, 1.9 ± 0.8, P<0.05). In survival analysis, the proportion of non-pregnant cows was higher in the NTR group compared to the NEM (P=0.012) and TEM (P=0.076) groups. In the TEM group, calving to first artificial insemination interval tended to be higher in cows treated 41 to 60 days postpartum than cows treated 29 to 40 days postpartum (97.2 ± 27.1 vs 74.4 ± 19.7, P=0.084). Our study suggests that cows with VDS=1 may require treatment to recover fertility. Diagnosis and treatment of clinical endometritis based on a VDS grading system may improve dairy herd reproductive performance.

One Holstein cow housed with 21 other cows exhibited clinical signs of pyrexia, anorexia and diarrhea along with severe hemoglobinuria. Hematological and biochemical analyses conducted before and after antibiotic therapy indicated severe hemolytic anemia and disruption of hepatic function. A general improvement in conditions was observed after an 11-day program of treatment comprising a regular dose of antibiotics and prescribed supportive therapies. A tentative diagnosis of bacillary hemoglobinuria was made based on the clinical and clinico-pathologic features on day 7. A molecular diagnosis was made by a PCR amplification of the flagellin gene of Clostridium haemolyticum using DNA extracted from the whole blood. The cow was diagnosed with the first recorded occurrence of bacillary hemoglobinuria of Holstein cattle in Japan.

The changes triggered by sperm-induced activation of oocytes, which are required for normal oocyte development, can be mediated by other agents, thereby inducing the parthenogenesis. In this study, we exposed porcine oocytes to 1 mM Ca-EDTA, a metal-ion chelator, at various intervals during 48 hr of in vitro maturation to determine the optimum period of Ca-EDTA treatment for parthenogenetic activation. When the oocytes were cultured with or without Ca-EDTA from 36 hr (post-12), 24 hr (post-24), 12 hr (post-36) and 0 hr (post-48) after the start of maturation culture, the blastocyst formation rates were significantly higher (P<0.05) in the post-24, post-36 and post-48 groups (3.3%, 4.0% and 2.6%, respectively) than those in the control group without treatment (0%). Furthermore, when the oocytes were cultured with Ca-EDTA for 0 hr (control), 12 hr (pre-12), 24 hr (pre-24), 36 hr (pre-36) and 48 hr (pre-48) from the start of maturation culture, the oocytes formed blastocysts only in the pre-36 and pre-48 groups (0.4% or 0.8%, respectively). Pronuclei (<66.7%) were observed only when the periods of Ca-EDTA treatment were more than 12 hr during maturation culture. In the control group, no pronuclei were detected. Our findings demonstrate that porcine immature oocytes can be parthenogenetically activated by Ca-EDTA treatment for at least 24 hr to 36 hr during maturation culture, leading to pronucleus formation followed by the formation of blastocysts.

To clarify the pathogenicity of Japanese type 1 porcine reproductive and respiratory syndrome virus (PRRSV) isolate in experimentally infected pigs, we evaluated clinical signs and monitored viremia for 21 days post-inoculation (dpi). Lungs were mottled, tanned and reddish in appearance; had lesions predominantly in the cranial, middle and accessory lobes; and failed to collapse at 10 dpi. Although microscopic lesions of lungs were reproduced using the Japanese emerging type 1 PRRSV isolate under experimental conditions, no significant differences were noted between the challenge and control groups regarding mean rectal temperature and daily weight gain. These results provide useful insights into the limited pathogenicity of single infection with the Japanese type 1 PRRSV isolate in piglets, which differ from findings in reported field cases.

Eosinophilic enteritis (EOE) is a type of inflammatory bowel disease and is characterized clinically by chronic obstinate diarrhea. Three Japanese Black (JB) fattening cattle (2 males and 1 female) on different cattle farms presented with chronic episodic diarrhea without fever or dehydration. Soft reddish spherical carneous tissues (1-3 cm) were occasionally excreted within the diarrheic feces. Administration of antibiotics, antidiarrheal drugs and vermicides had no therapeutic effect, but dexamethasone improved the fecal characteristics. The symptoms persisted until the animals were slaughtered at 27-30 months of age. Histopathological examination of the intestines revealed marked eosinophilic infiltration in the lamina propria and submucosa. From these findings, we diagnosed these cattle as the first cases of EOE in JB cattle.

The emergence in Japan of field isolates of type 1 porcine reproductive and respiratory syndrome virus (PRRSV) suggests problems with control. We therefore developed a one-step real-time reverse transcription polymerase chain reaction (qRT-PCR) with improved sensitivity that detects as little as 1 × 10(-2) TCID50/ml of viral RNA. We tested serum samples collected in January and September 2008, October 2009 and January 2011 from a farm with an outbreak and found infected pigs between January and September 2008, but not in January 2011. Further, between 2008 and 2011, we did not detect infection in pigs at 8 nearby farms or in 2,052 serum samples collected from pigs from 74 farms in 12 prefectures. This assay should help prevent future outbreaks.

We developed a concise and broadly applicable method for accurate genomic sequencing of North American genotype (NA-type) porcine reproductive and respiratory syndrome viruses (PRRSVs) that overcomes high genetic variability of the viruses. The method, designated "combination of consensus oligonucleotide reverse transcription and multiple displacement amplification" (CORT-MDA), involves reverse-transcription of viral RNA followed by shotgun sequencing after amplification using only 11 degenerate oligonucleotide primers; these primers were designed against consensus regions within the open reading frames of the 124 NA-type PRRSV strains with reported full-length genomic sequences. Sequencing of the 192 shotgun clones generated per virus showed 80% to 94% coverage on the reported PRRSV genomic sequence, such that only 2 or 3 unread regions had to be resequenced after PCR amplification using custom primers. Direct sequencing of RT-PCR products confirmed absolute consistency between sequences determined by the CORT-MDA method and those from RT-PCR. These results suggest that our method is applicable to diverse NA-type viruses.

The study aims were (1) to confirm the effects of nutritional improvement in prepartal and postpartal periods, monitored using the serum metabolic profile test (MPT) and reproductive performance, and (2) to clarify regional characteristics of the MPT results within our jurisdiction by using our MPT database. Experiment 1: Among 42 breeding cattle herds in our jurisdiction mainly fed home-pasture roughage, 3 experimental herds showing subnormal blood urea nitrogen (BUN) levels were selected and compared with 1 representative excellent herd. Dietary remedial measures were implemented from feed analysis in each herd. BUN concentration in all 3 herds increased significantly, and open days postpartum in 2 of the herds were significantly reduced, compared with values before dietary supplementation. Experiment 2: Thirty-seven herds within our jurisdiction were grouped into 3 categories (Area 1, 2 and 3) by location and soil condition of the herd pastureland. The MPT and reproductive performance in cows whose blood samples were collected at both prepartum (60-20 days before calving) and postpartum (30-90 days after calving) were compared among the 3 areas. Significant regional differences were found in prepartal albumin, total cholesterol, BUN, and glucose and postpartal BUN, glucose and open days (P<0.05). Overall, the MPT (especially BUN) might be useful for determining the metabolic nutritional status of breeding cattle herds, particularly those fed home-pasture roughage. Additionally, poor/unsatisfactory reproductive performance of beef breeding cattle herds probably reflects inadequate nutritional content of the diet, possibly arising from regional pastureland differences.

This study was carried out to investigate the presence of Torque teno sus virus types 1 (TTSuV1) and 2 (TTSuV2) in a longitudinally (14 to 150 days of age) collected paired pooled sera (pSE) and peripheral blood mononuclear cells (pPBMCs) using nested polymerase chain reaction. The detection rate of TTSuV1 in pSE increased from 14 to 90 days of age, but a progressive decline was observed from 120 to 150 days of age, while in pPBMC, a high value was maintained till the end of growing-finishing period. On the contrary, except in PBMCs at 30 days of age, high detection rates of TTSuV2 were found in both pSE and pPBMCs in all sampling ages. The detection rate of TTSuVs between pSE and pPBMCs was positively correlated at all sampling ages except for TTSuV1 at 150 days of age. This is the first study showing the presence of TTSuVs in PBMCs from pigs and describing the in vivo infection dynamics of TTSuV in paired sera and PBMCs during the entire growing and finishing periods of pigs reared in conventional farms.

This study was performed to clarify the sow-to-fetus transmission pathway of Torque teno sus virus (TTSuV) types 1 (TTSuV1) and 2 (TTSuV2). For this purpose, detection of TTSuV1 and TTSuV2 (TTSuVs) in sera of 6 sows (Sows 1-6) at parturition and in sera of their newborn piglets immediately after birth without suckling colostrum was performed by nested polymerase chain reaction (nPCR). These sows were bred using semen that had tested negative for TTSuVs. In a TTSuV1- and TTSuV2-positive sow (Sow 1), TTSuV1 and TTSuV2 were detected in 4 and 5 of 12 newborn littermates, respectively. In a TTSuV1-positive sow (Sow 2), TTSuV1 was detected in 1 of 8 newborn littermates. In 4 TTSuV1- and TTSuV2-negative sows (Sows 3-6), TTSuV1 was detected in 6 out of the 25 newborn piglets of 3 sows (Sows 3-5), while TTSuVs were not detected in all 13 piglets of 1 sow (Sow 6). In addition, to investigate the possibility of a sow-to-piglet transmission pathway of TTSuV via colostrum, TTSuV1 and TTSuV2 in sera of 12 newborn piglets from Sows 1-3 were examined by nPCR. Immediately after birth without suckling colostrum, TTSuV1 and TTSuV2 were not detected in 10 and 8 of 12 newborn piglets, respectively; however, at 24 hr after suckling colostrum, TTSuV1 was detected in 6 piglets, while TTSuV2 was not detected in any piglets. These results confirmed the existence of a sow-to-fetus transmission pathway of TTSuV during normal pregnancy and suggested a possibility of sow-to-piglet transmission of TTSuV via colostrum.

We investigated the seroprevalence rate of Torque teno sus virus types 1 (TTSuV1) and 2 (TTSuV2) in the sera of 38 post weaning multisystemic wasting syndrome (PMWS)-suspected pigs and 43 porcine circovirus type 2 (PCV2)-vaccinated normal pigs on 3 commercial pig farms in southern Japan by using nested polymerase chain reaction. High seroprevalence rate of TTSuVs was observed in both PMWS-suspected pigs (100%) and PCV2-vaccinated normal pigs (90.7%). The seroprevalence rate of TTSuV2 was significantly higher in the PMWS-suspected pigs than in PCV2-vaccinated normal pigs (97.4% versus 81.4%, P<0.05), whereas no such difference was observed for TTSuV1 between the 2 groups of pigs. In both pig groups, the seroprevalence rate of TTSuV2 was significantly higher than that of TTSuV1 (P<0.01-0.05). These results show that TTSuVs are highly seroprevalent in both PMWS-suspected pigs and PCV2-vaccinated normal pigs with TTSuV2 being more seroprevalent in the former than in the latter.

We found a case of salivary mucocele that originated in the minor salivary gland (palatine gland) of the soft palate in a dog. At first admission, the soft palate swelled remarkably. Computed tomography (CT) revealed cystic radiolucency inside a large quantity of liquid in the soft palate, and most of the airway was occupied. Marsupialization was performed, but since a recurrence was observed one month later, the salivary mucocele was removed. There has been no report of salivary mucocele arising from the minor salivary gland of the soft palate in dogs. To our knowledge, this case is the first. Complete removal, including minor salivary glands surrounding the lesion, is necessary for treatment of salivary mucocele in dogs.

In order to compare the stimulation ability of insulin secretion, we determined changes in plasma glucose and insulin concentrations after intravenous administration of various amino acids and essential fatty acids in clinically healthy adult cats. Plasma glucose concentrations were within the normal ranges after injection of amino acids and fatty acids. Plasma insulin concentrations increased rapidly 2 to 4 min after injection of arginine, then decreased to the basal levels at 20 min in all five cats. Insulin peak responses were significantly greater in arginine injections than in normal saline (P<0.01). Areas under the curve (AUC) of plasma insulin concentrations from 0 to 10 min after injection of arginine were significantly larger than after injection of normal saline (P<0.01) and glucose (P<0.05). Increases in AUC of plasma insulin concentration from 0 to 60 min were observed after injection of arginine, leucine, alanine, and fat emulsion. Arginine had a strong insulinotropic effect, and leucine, alanine, and fatty acids had weak ones. Besides, valine, methionine, taurine and glutamine had no stimulant activity of insulin. Given the risk of glucose toxication and required time for testing, the intravenous arginine tolerance test may be useful for estimation of insulin responses in cats.

For 6 years, 5 Japanese Black cows of the same herd showed anorexia, depression, and dehydration with no feces in the rectum. Biomedical examination of 3 animals showed severe hypokalemia and hypochloremia. Although the first 3 animals died or were slaughtered (causes unknown), necropsy results showed that the cow in case 4 had intestinal obstruction due to phytobezoar derived from napier grass, fed mainly to the cattle as roughage. Therefore, farmers were recommended to avoid the hard root-stem portion of napier grass as roughage. Consequently, less phytobezoar was recovered from the fifth cow, and no similar clinical case of intestinal obstruction was observed thereafter. This is the first report on intestinal obstruction caused by phytobezoars derived from napier grass.

Three Japanese Black cows housed with 6 other cows exhibited main clinical symptoms of severe hemoglobinuria. Hematological analyses conducted after antibiotic therapy demonstrated severe anemia, and biochemical analyses indicated both severe hemolysis and disruption of hepatic function. One of the three cows died. Based on the above analyses and observation of typical clinical symptoms, a speculative diagnosis of bacillary hemoglobinuria was made, and immediate high-dose antibiotic treatment improved the general conditions of the surviving animals. Blood samples from the other 2 cows were collected sequentially after antibacterial therapy. Clostridium haemolyticum was detected by a nested polymerase chain reaction analysis of the blood samples. The cows were diagnosed with the second recorded occurrence of bacillary hemoglobinuria in Japan.

A total of 2,703 pig sera from 171 farms in six regions in Japan were screened for virus-neutralizing (VN) antibody against transmissible gastroenteritis virus (TGEV). Although none of the farms had clinical signs of transmissible gastroenteritis (TGE) or vaccination against TGEV, VN antibody was detected in 14.4% of sera at 30 farms (17.5%) across all six regions. On testing of 263 VN antibody-positive sera from 27 farms with a commercial blocking ELISA to distinguish TGEV and porcine respiratory coronavirus (PRCV) antibodies, 78.3% were positive for PRCV antibody only, while 12.5% were positive for TGEV antibody only or both TGEV and PRCV antibodies. Seven of the eight TGEV antibody-positive farms were also positive for PRCV antibody. Five months after the antibody examination, a TGE outbreak occurred at one of these seven farms. These results suggest that most of the detected VN antibodies were to PRCV, and that TGEV infection might be present at some PRCV-positive farms in Japan.

In order to investigate the origin of tetracycline resistance in Erysipelothrix rhusiopathiae, conjugative transpositions of Tn916 were tested. The frequency of transfer between strains of E. rhusiopathiae was about 10-fold higher than that between Enterococcus faecalis and E. rhusiopathiae. In addition, detection of a Tn916-like transposon was performed by PCR assay and DNA sequencing in E. rhusiopathiae field isolates. Of 49 tetracycline-resistant isolates, 38 (77.6 %) carried a Tn916-like transposon, while 11 (22.4 %) carried tet(M) only. These results suggested that Tn916-like transposon may be widely present in the E. rhsuiopathiae field isolates resistant to tetracycline.

Four pasture-fed Japanese Black cows showed the main clinical symptoms of severe hemoglobinuria at different periods between 2003 and 2007. Hematological analyses at the first consultation revealed severe anemia, and biochemical analyses indicated both severe hemolysis and disruption of hepatic function. Although the first 2 patients died, the hemoglobinuria and general condition of the remaining 2 cows, who were immediately initiated on large doses of antibiotics, improved within 3 days. Clostridium haemolyticum was detected by polymerase chain reaction (PCR) analysis of the blood sample of 1 of the infected cows. Anti-fascioliasis medicine is administered, and since then, no case of hemoglobinuria has been observed. The cows were diagnosed with bacillary hemoglobinuria, and they represent the first few cases in Japan.

We investigated 66 Erysipelothrix rhusiopathiae strains isolated from pigs affected with swine erysipelas in Japan from 1994 to 2001 for serotype, pathogenicity towards mice, protection in vaccinated mice and antimicrobial susceptibility. Most of the isolates (84.8%) were serotype 1 or 2. For the first time, strains belonging to serotype 21 were isolated from cases of septicemia. Fifty isolates (75.8%) were highly virulent, 12 isolates (18.2%) were weakly virulent and 4 isolates were avirulent strains. All the mice vaccinated with the Koganei 65-0.15 vaccine strain survived challenge exposure with 50 highly virulent isolates. Six isolates (9.1%) grew on TPB-T80 agar containing 0.02% of acriflavine, and this was identical to the growth of the vaccine strain. Forty-seven isolates (71.2%) were resistant to oxytetracycline. The number of strains resistant to oxytetracycline among field isolates increased rapidly each year. Tylosin-resistant strains were also isolated (6.1%). These results suggest that certain characteristics, particularly antimicrobial susceptibility of E. rhusiopathiae isolates, change yearly in the field. Therefore, further investigation of the characteristics of E. rhusiopathiae field isolates is necessary.

The efficacy of a gamma-aminobutyric acid (GABA) preparation in improving general health condition and growth rate was evaluated in suckling Japanese Black beef calves housed together and fed using an automatic milk feeder. The GABA preparation was administered from 4 to 10 d postpartum until weaning. A significant difference in mean body weight was observed at 3 and 8 weeks after introduction of the GABA preparation. The calves in the GABA group tended to require less medication than those in the control group (3.0 d/calf for BABA; 7.6 d/calf for the control; P=0.06). The health statuses of the 2 groups may be reflected in their blood parameters. GABA may possibly have etiotropic effects in group-housed calves fed using an automatic milk feeder.

The objective of this field study was to determine the therapeutic efficacy of mammary irrigation for the treatment of dairy cattle diagnosed with acute coliform mastitis caused by gram-negative bacteria. Additionally, the effects of different mammary irrigation regimen fluids such as ozone water and normal saline were compared. Dairy cattle clinically diagnosed with acute coliform mastitis (n = 57) were enrolled in the study, randomly assigned to 1 of 3 groups, and received the following treatments: systemic antibiotic administration (SAA group; n = 40), mammary irrigation regimen (MIR group; n = 10), and both treatments (MIX group; n=7). Significant antipyretic effects, as assessed by rectal temperature measurement, were observed in the MIX and MIR groups. Although 2 irrigating fluids were used, namely, ozone water and normal saline, no significant difference was observed between the 2 groups. Fourteen days after the onset of the treatments, the milk yield recovery rate in MIR group tended to be higher (p = 0.06) than that in the SAA group. Additionally, after 30 days of treatment, the MIR group cows demonstrated significantly higher successful recovery rates (p<0.05) than the SAA group cows. These results indicate that mammary irrigation with normal saline is an effective treatment for acute coliform mastitis in dairy cattle.

Several species of birds in Minami Daito Island, an oceanic island located in the far south from the main islands of Japan, were found to be infected with avian Plasmodium. However, no vector species of the avian malaria in this island have been revealed yet. To speculate potential vectors, we collected mosquitoes there and investigated using a PCR procedure whether the mosquitoes harbor avian malaria or not. Totally 1,264 mosquitoes including 9 species were collected during March 2006 to February 2007. The mosquitoes collected were stored every species, sampled date and location for DNA extraction. Fifteen out of 399 DNA samples showed positive for the partial mtDNA cytb gene of avian Plasmodium. Estimated minimum infection rate among collected mosquitoes was 1.2% in this study. Four species of mosquitoes; Aedes albopictus, Culex quinquefasciatus, Lutzia fuscanus and Mansonia sp. had avian Plasmodium gene sequences. Detected DNA sequences from A. albopictus and L. fuscanus were identical to an avian Plasmodium lineage detected in bull-headed shrike (Lanius bucephalus) captured in the island. Different sequences were detected from C. quinquefasciatus, which were corresponding to an avian Plasmodium from a sparrow (Passer montanus) and Plasmodium gallinaceum. Our results suggest that A. albopictus, Lutzia fuscanus, C. quinquefasciatus, and Mansonia sp. could be potential vectors of avian malaria in Minami Daito Island. This study was the first report of molecular detection of avian Plasmodium from mosquitoes in Japan.

Haemosporidian parasites infection among wild birds inhabiting Minami-daito Island was studied. Blood films from 183 birds representing 4 species of 4 families were examined microscopically. Avian haemosporidian parasites were detected in 3 species with an overall prevalence of 59.6%. None of the 30 Daito scops owls (Otus scops interpositus) examined were infected. Either Haemoproteus sp. or Plasmodium sp. infection was found in 14 of 31 (45.2%) Borodino islands white-eyes (Zosterops japonicus daitoensis). Plasmodium spp. were found in 94 of 102 (92.2%) bull-headed shrikes (Lanius bucephalus) and 1 of 20 (5%) tree sparrows (Passer montanus).

Antibiotic therapy administered for treatment of gram-negative bacterial infections can exert a negative effect in the form of endotoxin shock. Therefore, a sterilization method that releases little endotoxin is required. This in vitro study aimed to compare the amounts of endotoxin released from Escherichia coli exposed to antibiotics (aminobenzylpenicillin, kanamycin, oxytetracycline, sulfadimethoxine and enrofloxacin) or ozone. The bacterial growth and amount of endotoxin released were measured at specific time points. Compared with antibiotic treatment, ozone sterilization induced release of smaller amounts of endotoxin from the bacteria. Moreover, no major differences were observed when ozone was used against the purified endotoxin. We conclude that compared with antibiotic treatment, ozone sterilization may release smaller amounts of endotoxin.

An 11-month-old Japanese Black steer with chronic bloat underwent clinical and histological analyses. During the observation period, it showed normal appetite and fecal volume but persistent chronic bloat symptoms. Compared to controls, the steer's feces contained undigested large straws. Necropsy revealed normal rumen, reticulum, and abomasum but a small omasum. The rumen, reticulum, and abomasum mucosa was normal, with well-developed ruminal papillae. However, severe hypoplasia of the omasal laminae was observed along with hypoplasia reticular groove and ruminoreticular fold. The contents of the reticulum, omasum, and abomasums comprised undigested large sized hay particles. The omasum papillae showed no pathological abnormalities. This is a rare case of a steer with chronic bloat probably caused by severe hypoplasia of the omasal laminae.

To examine the roles of a short form of p53 in the regulation of apoptosis in chicken lymphoblastoid tumor cell lines derived from Marek's disease (MD) and avian leukosis (AL), the expressions of the p53 proteins were analyzed in these cell lines in which apoptosis was chemically induced. In MSB1-O derived from MD, the expression of a 40 kDa protein of p53 was decreased and that of a 32 kDa protein, a short form of p53, was increased during apoptosis induced by actinomycin D. In 1104B1 derived from AL, the expressions of 42 and 32 kDa of p53 were increased during the apoptosis. The short form of p53 was undetectable in these cell lines when apoptosis was blocked by the pretreatment with endonuclease inhibitor, Zn2+, protease inhibitors, TPCK and TLCK, or caspase inhibitor, Z-VAD-FMK. In the transcriptional level, the expressions of bcl-2 and IAP were decreased in these cell lines during actinomycin D-induced apoptosis, but no change was detected in the expression level of p53. These results suggest that, in these chicken tumors, the short form of p53 could play a role in the initiation of apoptosis induced by the chemotherapeutic compound, and that the regulation of its expression may be important for the maintenance of transformation status.

Microdialysis System (MDS) is a novel technique used for investigation of molecule secretion between different cell populations. Local hormonal secretion at follicular wall has been still unclear. This MDS study was used to determine progesterone (P4), androstenedione (A4), estradiol-17beta (E2) and Prostaglandin F2alpha (PGF2alpha) release in mare pre-ovulatory follicles. Follicles larger than 30 mm were isolated from the ovary and follicular fluid aspirated for hormone assay. Follicular fluid collected from small, middle and large follicles were analyzed by EIA. The concentrations of P4 and PGF2alpha were similar among the different sizes of follicles. The release of A4 was observed in middle and large follicles. E2 concentration was observed in middle follicles and was higher in large follicles compared with middle follicles. Follicular wall was cut and incubated for MDS and when LH was infused, there was an increase in P4 and A4 release. PGF2alpha release was considerably high after LH infusion compared to the control group. Infusion of PGF2alpha increased P4 and A4 release but there was no change in E2 release. This results suggest that in pre-ovulatory follicles, LH stimulates theca interna cells and also PGF2alpha seemed to have a mediator role to induce steroid hormone production and luteinization of follicular cells. The nature of the mechanisms involved in selection of large follicles is still a perplexing research problem in reproduction.

A total of 90 strains of Staphylococcus intermedius isolated from dogs were examined for antimicrobial susceptibility. There were no significant differences in the distribution patterns of MICs between strains from 1982 to 1985 and those from 1999, and between strains from healthy dogs and those from diseased dogs. All of the strains were susceptible to ABPC, DMPPC, CEX, TDM, ERFX, BFLX, and FF at concentrations of 0.05 to 6.25 microg/ml. The MICs of OTC, KM, EM, AIV-TS, and LCM were distributed in a broad range of 0.1 to >100 microg/ml, indicating the existence of resistant as well as susceptible populations of S. intermedius. Thirty-three strains (36.7%) were resistant to one or more anitmicrobial agents such as OTC (n=32), KM (n=9), EM (n=7), AIV-TS (n=7), and LCM (n=7).

Serotype 1 strains of Marek's disease virus (MDV1), except attenuated vaccine strains, are known to cause lymphomas in visceral organs of infected chickens. To know additional genetic differences between oncogenic and nononcogenic MDV1, polymerase chain reaction (PCR) was performed to amplify the meq gene of the viral genome. In addition to the 1,062-bp band including the native meq open reading frame (ORF), a 1.2-kb band was amplified from the DNA sample prepared from chick embryo fibroblast infected with an attenuated strain, CVI988, but not with oncogenic strains. Sequence analysis of the 1.2-kb band showed that a 178-bp sequence was inserted to the meq ORF of CVI988. This ORF could encode for the Meq protein with a different transactivator domain. Southern blot analysis also confirmed the insertion of the 178-bp sequence in the meq ORF of CVI988. This insertion of 178-bp sequence may explain the reason why CVI988 is not oncogenic.

To determine whether there is any abnormalities of the p53 gene in chicken lymphoblastoid tumor cell lines derived from Marek's disease (MD), lymphoid leukosis, reticuloendotheliosis, and field tumors, some portions of p53 cDNA corresponding to core and C-terminal domains (nucleotide positions 277-1104 in the p53 open reading frame (ORF)) were sequenced. Several mutations were identified in both cell lines and field tumors. However, none of these mutations is localized at the "hot spot", which has been reported as the site for transformation-activating mutations. Moreover, partial cDNA clones with a 122-bp deletion in the p53 ORF were identified in two cell lines, MSB1 and MTB1 derived from MD tumors. Southern blot analysis showed that no deletion occurred in the genome of p53 in MSB1, indicating that deletion occurred at the transcriptional level. This deletion could cause a frame shift of the encoding p53 protein, possibly resulting in the generation of a functionally different p53 protein. However, we confirmed that p53 mRNA without deletion is also present in each of these cell lines. These mutations of the p53 gene and deletion in the p53 transcript may be ones of molecular changes specific to the transformation induced by MD virus.

The rate of DNA synthesis in the inner cell mass (ICM) of frozen-thawed bovine embryos was examined. Bovine blastocysts derived from in vitro matured/in vitro fertilized oocytes were frozen with 1.8 M ethylene glycol (EG), 1.6 M propylene glycol (PG) and 1.4 M glycerol plus 0.25 M sucrose (GL). Viable embryos, after thawing and culture beyond the blastocysts stage, were examined by immunocytochemical staining for detection of DNA synthesis by ICM cells. The numbers of bromodeoxyuridine-immunoreactive ICM cells of frozen-thawed embryos prepared with EG (10.7) and GL (11.5) were significantly lower than those of unfrozen embryos (17.3). The results suggest that the rates of proliferation of ICM cells of frozen-thawed bovine embryos tend to be lower than those of unfrozen embryos irrespective of the cryoprotectant used.

Two strains (Strains 39 & B) of Haemophilus paragallinarum were isolated and identified as serotype C by dot-blotting analysis in Taiwan in 1994. To identify their virulence and pathogenicity in Taiwanese local chickens, local chickens were inoculated via nostril with the isolates, and examined for 14 days. Clinical signs of swollen face, and nasal discharge were first seen at 24 hr after inoculation, and the infectivity of Strains 39 and B at 14 days after inoculation among the inoculated chickens was 50.0-71.4% and 55.6-77.8%, respectively. From the results, we concluded that the two bacterial isolates were pathogenic to local chickens with a maximum morbidity of 77.8%.

Serovars of 1,046 Erysipelothrix strains isolated from diseased pigs during a period from 1983 to 1993 in Japan were determined by an agar gel double-diffusion precipitation system using typing sera representing all the known serovars, 1 through 23 and type N, of 2 species of Erysipelothrix. A total of 943 strains could be serotyped within the serovars. The remaining 103 strains could not be determined for their serovars and were classified as untypable. Of the 938 (99.5%) strains serologically identified as Erysipelothrix rhusiopathiae, 423 (40.4%) belonged to serovar 1a, 359 (34.3%) to serovar 2, 65 (6.2%) to serovar 1b, 35 (3.3%) to serovar 6, 19 (1.8%) to serovar 5, 11 (1.1%) to serovar 21, and the remaining 26 to serovars 4, 8, 11, 12, 15, 17, or 19. Only 5 (0.5%) strains isolated from the cases of erysipelas belonged to Erysipelothrix tonsillarum and represented serovars 7 or 23.

In 1989 and 1990, we isolated five strains of Haemophilus paragallinarum in the Philippines. Serological studies were done on three of these isolates. The results of haemagglutination, reactability to monoclonal antibodies and cross immunization with standard strains showed that they belonged to serotypes A, B and C respectively. This is the first report on the isolation of H. paragallinarum and on a related serological study in the Philippines.

An epizootic of foal diarrhea due to serotype 3 rotavirus (RV) was observed in 89 of 168 cases (53%) during the period from March to July in 1987. A total of 51 strains of RV were isolated from the 62 diarrheal feces examined, and one isolate (CH-3) showed a unique electropherotype of viral RNA which differed from the others that widely prevailed on this farm. No positive reaction was observed between strain CH-3 and each of the antisera against serotypes 1 to 12 of human and animal RV in neutralization tests. However, dsRNAs of the CH-3 virus were hybridized with a probe prepared from a strain of equine serotype 3 RV.

The genomic DNA of Haemophilus paragallinarum (Hpg) serotype A strain 221 was cloned into vector plasmid pBR322. The recombinant plasmids were introduced into Escherichia coli (E. coli) strain C600. Subsequently, a total of 277 transformants were obtained. One, designated strain no. 6, expressed hemagglutination activity against chicken erythrocytes. Strain no. 6 contained the recombinant plasmid pNV102, and DNA of about 2.57 kb was inserted into pNV102. When strain no. 6 was cured of pNV102, the strain lost hemagglutination activity. When the cured strain was retransformed with pNV102, hemagglutination activity was restored. E. coli strain no. 6 reacted with monoclonal antibody specific to the hemagglutinin of Hpg serotype A in a dot-blotting analysis. Chickens immunized with the inactivated strain no. 6 produced the hemagglutination inhibition (HI) antibody, and chickens possessing the HI antibody showed protection against challenge exposure by Hpg strain 221.

A survey of infectious coryza of chickens was performed in West Jawa of Indonesia between 1987 to 1988 by the detection of hemagglutination-inhibition (HI) antibody against Haemophilus paragallinarum (Hpg) in non-vaccinated healthy laying (12 farms, 196 chickens) and native (8 farms, 197 chickens) chickens. HI antibodies against Hpg were detected not only in the native chickens but also in the laying chickens, regardless of the district, and were observed in 70% (14/20) of farms and 19% (73/393) of chickens. HI antibodies against serotype A were detected from 11 farms (55%) and 11% (45/393) in chickens. Those against serotype C were detected from 5 farms (25%) and 8% (30/393) in chickens. Three Hpg strains were isolated from different chickens affected with infectious coryza. Two of them were identified as type A and the other as type C by the rapid plate agglutination test. These results demonstrated that the outbreaks of infectious coryza caused by serotype A and C strains had occurred in Indonesia.