The cell-free antigen (CFA) obtained from the culture supernatant of Pasteurella multocida (P. multocida) and the toxin (PMT) purified from CFA were inactivated and mixed with oil adjuvant to prepare a trial vaccine. Both of the mice immunized with CFA and PMT toxoid vaccine were noticeably protected against intratracheal challenge with toxigenic strains of P. multocida. Nevertheless, the protective indices of the mice immunized with CFA vaccine indicate that it is more protective and clears away the bacteria more promptly than in the mice immunized with PMT vaccine. The results suggested that CFA would possibly be good as an effective antigen to toxigenic strains of P. multocida infection.
Lactoferrin purified from canine seminal plasma by a three-step chromatography procedure had a molecular mass of 75.2 kDa and cross-reacted with antiserum to equine seminal plasma lactoferrin. Seminal plasma lactoferrin concentrations were determined by a competitive enzyme-linked immunosorbent assay (ELISA) by using rabbit anti-equine lactoferrin antibody and alkaline phosphatase-labeled goat anti-rabbit IgG antibody in 14 normal dogs and found to range from 12 to 197 μg/ml, with a mean value of 77 ± 59 μg/ml (the mean ± SD). Seminal plasma transferrin concentrations were determined by a sandwich ELISA with goat antibody to canine serum transferrin and alkaline phosphatase-conjugated goat anti-canine transferrin antibody and found to range from 0.32 to 12.6 μg/m l, with a mean value of 2.44 ± 3.25 μg/m l. The lactoferrin concentration significantly correlated with the sperm concentration (r=0.7025, P<0.01), but there was no significant correlation between the seminal plasma transferrin concentration and sperm density. These results indicate that seminal plasma lactoferrin, but not transferrin, reflects gonadal function.
The aim of this study was to assess the practicality of the metabolic profile test (MPT) for feeding evaluation in dairy cattle. Stepwise regression analysis was used to evaluate the relationships of MPT to feeding and milk production of 4,679 cows in 343 commercial dairy herds. Significant explanatory variables were determined by forward set-up selection, among the deviated values from the reference values of 10 blood metabolites and body condition score, to predict dependent variables, i.e., milk production and the rate of feeding to nutrient requirements, in each or all lactation stages and the dry period. The milk production model of the all-lactation stage showed the greatest goodness-of-fit (adjusted R2=0.214, p<0.0001) with high positive regression coefficients for serum cholesterol, magnesium, urea nitrogen and albumin, and negative for glucose and calcium. In the feeding models, goodness-of-fit of crude protein was relatively high (R2=0.072, p<0.0001) with a positive relationship to blood urea nitrogen. Although the other feeding models were low in goodness-of-fit, several significant explanatory variables to feeding were found. All feeding models in the late lactation stage and the dry period, in which the feeding was stable, had greater goodness-of-fit than those in the early lactation stage in which milk production varied. It was concluded that the values which deviated from the reference values for the MPT components could assess milk production and feeding, and the MPT is a practical tool for auxiliary feeding evaluation.
A buffalo disease, called "Degnala", causing lameness, edema, gangrenous ulceration of hooves or tail, emaciation, recumbency and eventual death, occurs in Eastern Nepal. Clinical examinations manifested lice eggs on hairs, bradycardia, hypothermia, dehydration, exanthema and icterus. Hematologically, increase of band neutrophil, giant platelet, hypoalbuminemia and hyperglobulinemia were characteristics. Microscopically, dark blue tiny particles were seen on red blood cell (RBC) after Giemsa staining. Administration of tetracycline at an early stage of the disease was effective.
The functions of polymorphonuclear cells (PMN) are the important non-specific defense mechanisms in the immune system. Especially marine mammals are protected by these mechanisms from the aquatic environment with a large variety of microorganisms. Therefore, we examined the PMN functions of bottlenose dolphins in order to obtain the normal ranges and to standardize the techniques. PMNs were isolated by using lymphocyte isolate solution whose density was 1.077; superoxide production was assessed by nitroblue tetrazolium reduction test (NBT) and phagocytosis was tested by using polystyrene latex beads. We showed that the optimal incubation time was 30 min in NBT assay and 12 hr in phagocytosis assay for dolphin PMNs.
The normal concentrations of salivary secretory IgA (sIgA) were examined, and the response of sIgA to acute stress was evaluated in dogs. Ten clinically healthy beagle dogs familiarized with the method of saliva sampling were used. During the non-stress period, saliva samples were collected between 0800 hr and 1700 hr at 1-hr intervals for 7 consecutive days and analyzed for sIgA concentration. After a 1-day control period, a noise stressor was presented for 15 min between 0845 hr and 0900 hr on 2 consecutive days. Saliva was collected at pre-stress, immediately after, 30 min after and 60 min after the stress. The average sIgA concentration over the 2-day period was compared with the control value. Environmental stimuli were restricted. During the non-stress period, significant variations were observed during the diurnal pattern, in which sIgA increased in the morning and then decreased; and the day-to-day variations were significant except at 0800 hr and 0900 hr. During the stress experiments, the sIgA concentration decreased significantly, immediately after and 30 min after the noise stress, and then increased to the same level as the control value by 60 min after the stress. When estimating the effectiveness of salivary sIgA as a marker of stress in dogs, the appropriate time for saliva sampling appears to be in the morning. Salivary sIgA was deemed potentially useful as a marker of stress in dogs.
Dendritic cells (DCs) are the most potent antigen-presenting cells that are expected to be therapeutic agents for tumor immunotherapy. In this study, we generated DCs of sufficient number for DC-based immunotherapy from peripheral blood mononuclear cells (PBMC) in dogs. PBMC were cultured in the presence of phytohemagglutinin (PHA). On day 6, large adherent cells with dendrite-like projections were seen, and the number of these large cells with projections increased on day 8. These cells were positive for esterase staining. They expressed MHC class II, CD11b, CD8 and weakly CD4 on their surface. They tended to make contact with lymphocytes under culture conditions. We obtained about 2-5 × 106 of DCs from 10 ml of peripheral blood. These DCs phagocytosed HEK-293 cells by overnight co-culturing. These cells generated from PBMC are possible canine DCs and are applicable to clinical trials of DC-based whole tumor cell immunotherapy in dogs.
To examine whether an angiotensin converting enzyme (ACE) inhibitor, benazepril, can be transformed to the active metabolite, benazeprilat, by severely injured liver of dogs with ascitic heartworm disease, benazepril hydrochloride was administered orally to dogs once daily for 7 consecutive days at a dose rate of 0.29 mg/kg to 0.63 mg/kg of body weight, and plasma benazepril and benazeprilat concentrations were determined on the 1st and 7th administration days. In 7 dogs with ascitic pulmonary heartworm disease, plasma benazeprilat concentrations tended to be higher than in 7 control dogs both on the 1st and 7th administration days. The peak concentration and area under the concentration-time curve tended to be greater in dogs of the ascites group than in control dogs, but the statistics could not detect significant differences in the time to peak concentration and t1/2 between the control and ascites groups. Plasma ACE activities decreased after administration of benazepril. In dogs with ascitic heartworm disease, benazepril was readily transformed to benazeprilat by the liver, and was effective for suppression of plasma ACE activity.
Two dogs with severe exercise intolerance (Cases 1 and 2) and another dog with cardiac dilation (Case 3) were referred to the Nihon University Animal Medical Center (ANMEC). Case 1 was diagnosed as pericardial effusion (PE), Case 2 as pericardial hemorrhage, and Case 3 as pericardiophrenic hernia. When these causative disorders were removed, the heart expanded, and clinical symptoms markedly improved in these three dogs. In particular, the cardiac chamber diameters and left ventricle fractional shortening (LVFS) was normalized in all 3 dogs postoperatively. There is only one case report that compares before effusion extractions in the pericardial sac with the after echocardiography findings. In this paper, echocardiography was conducted on the three endocardial disease cases, comparing before removing these causative disorders with the findings after echocardiography.
A 14-year-old female Yorkshire terrier was presented with the complaint of cardiac murmur and convulsive seizure. Thickened mitral valve, left atrial enlargement, excess motions of the left ventricular (LV) free wall and the ventricular septum, and tricuspid, mitral and aortic valve regurgitations were recognized on echocardiography. Follow-up echocardiography revealed the progression of concentric LV hypertrophy and LV outflow obstruction. Clinical symptoms associated with cardiac failure did not develop during the observation period. The pathological examination of the heart revealed that the dog had the morphological hallmarks of hypertrophic cardiomyopathy: massive ventricular hypertrophy, disorganization of cardiac muscle cells, interstitial myocardial fibrosis, and abnormal intramural coronary arteries.
Western blotting was performed to analyze Neospora caninum tachyzoite antigens recognized by mouse IgG at different stages of infection including recrudescence. At the early stage of infection, a 36-38 kDa antigen was clearly recognized by the mouse antisera. After day 48 postinoculation, the signal of the 36-38 kDa antigen gradually weakened. Meanwhile, a 43 kDa antigen was intensely and continuously recognized from 48 to 125 days postinoculation. This 43 kDa antigen was clearly detectable with the antisera from the mice under immunosuppression. Sera from naturally infected cattle strongly reacted with the 43 kDa antigen. Therefore, the 43 kDa antigen may be useful for immunological reactions to detect infected animals except in the early stage of the infection.
Larvae of the raccoon roundworm, Baylisascaris procyonis (B. procyonis) are a known cause of cerebrospinal larva migrans in animals and humans. The present paper described details of the central nervous lesion in the rabbits (Oryctolagus cuniculus) affected with B. procyonis larva migrans in Japan. Clinically affected animals showed neurological signs including circling, torticollis, tremor of head, or ataxic gait. The most characteristic pathological alterations were large malacic lesions associated with an activated astroglial proliferation which was seen at the corpus medullare in the cerebellum including the cerebellar peduncle. Moreover, focal malacic lesions with perivascular cuffing and infiltration by lymphocytes and heterophiles were scattered everywhere throughout the brain. In these lesions or normal-appearing areas away from obvious lesions, ascarid larvae, about a maximum 65-75 μm in diameter, were recognized. Other prominent features were minute lesions (we call them migration tract-like lesions) composed of lymphocytes, hemosiderin-laden macrophages and reactive astrocytes scattering throughout the cerebrum. In this study, we demonstrated ascarid larvae in only eight out of 23 animals diagnosed as B. procyonis larva migrans. Since it is not always possible to detect the larvae, the possibility of B. procyonis larva migrans must be given serious consideration to the characteristic lesions described above.
A new canine cell line, named CCT, was established from the cutaneous malignant histiocytosis in a 4-year-old male Borzoi. CCT proliferated with loose adherence and doubling time was approximately 30 hr. When co-cultured with latex beads, CCT phagocytized beads vigorously. Lysozyme and vimentin were positive by immunostaining, and non-specific esterase and acid phosphatase were positive by cytochemical staining. These features indicated the cells had a histiocytic nature. Furthermore, by subcutaneous injection to nude mice CCT could successfully form tumors with the morphological and immunohistochemical features similar to the original tumor.
Equine protozoal myeloencephalitis developed in a three-year-old male Thoroughbred racehorse imported from the United States. The animal showed astasia five days after the onset of ataxia. Histopathologically, focal nonpurulent myelitis accompanied by hemorrhage and perivascular infiltration was observed in the fourth and fifth cervical spinal cord. Immunohistochemically, shizonts were occasionally observed and were positive for anti-Sarcocystis neurona (S. neurona) antiserum. S. neurona-specific antibodies were detected in the serum and cerebrospinal fluid by Western blot. This is the first equine protozoal myeloencephalitis case in Japan.
Oxytocin receptor (OTR) mRNA levels increase dramatically near term and is potently stimulated by estrogen because increased OTR mRNA levels result from estrogen treatment in ovariectomized rat uterus. In this study, OTR, estrogen receptor (ER) α and ERβ mRNA levels in the rat uterus during the estrous cycle were examined by quantitative RT-PCR. OTR mRNA levels during the estrous cycle began to increase on diestrus (P<0.05, vs value on estrus), reached maximal increase both in the morning (1000-1130 hr) and afternoon (1600-1630 hr) on proestrus (P<0.01, vs metestrus, diestrus and estrus) and then declined on estrus. In contrast ER α mRNA levels began to decrease on diestrus, reached statistical significance both in the morning and the afternoon on proestrus (P<0.01, vs metestrus, diestrus and estrus) and returned to the value of metestrus on estrus. ERβ mRNA levels were low in the morning and the afternoon on proestrus (P<0.01, vs metestrus and estrus) and also returned to metestrus values on estrus. Treatments with estrogen for 3 days significantly decreased both ERα and ERβ mRNA levels. It can be concluded from these results that during the estrous cycle, OTR mRNA levels in rat uterus predominantly increase at proestrus with a decrease in ERα and ERβ mRNA levels, which is probably due to the increased estrogen levels in circulation before ovulation.
To clarify the morphological and immunohistochemical characteristics in mares with granulosa theca cell tumor (GTCT), the localization of inhibin subunits (α, βA, βB) and aromatase in the granulosa cell layers and theca layers in the ovarian follicles were determined by immunohistochemical staining. The follicles were obtained from the ovaries of 6 mares with GTCT and 4 normal mares as controls. Immunohistochemically, inhibin α-subunit was localized in the granulosa cells of all follicles showing different sizes in all GTCT cases and βA- subunit was localized in two GTCT cases in all sized follicles. But inhibin βB- subunit and aromatase were not localized in GTCT cases. On the other hand, inhibin α-, βA-, and βB-subunits and aromatase were localized in the large and medium sized follicles, but inhibin βA- and βB-subunits and aromatase were not stained in the small sized follicles in normal cases. These findings suggest that some mares with GTCT can secrete dimeric inhibin (inhibin A), but all GTCT cases cannot secrete inhibin B. By the results of aromatase staining it is clear that testosterone is not converted into estradiol due to the lack of aromatase in the GTCT follicles.
A 21 year old thoroughbred mare with granulosa theca cell tumor (GTCT) in the right side and atrophic contralateral ovary was investigated in this study. After arrival at our laboratory on 10th December 1999, the clinical diagnosis of GTCT was examined by rectal palpation and ultrasonographic image of ovaries. Plasma from peripheral blood was collected in the breeding and non-breeding seasons for hormonal analysis. The results showed that the contralateral ovary regained normal activity without any treatment of the GTCT affected ovary and contained follicles showing different sizes 19 months later. However, the affected right ovary, which became smaller after 4 months, was totally inactive without any follicle. The observations clearly demonstrate that without any treatment of the GTCT affected ovary, a mare can return to her normal estrous cycle within a certain period in some GTCT cases.
The ELISA we developed was able to determine the antigen content and was suitable for a potency test, and we described a relative potency assay method which determines the potency of test vaccines by comparing the ELISA value of a test vaccine to that of a reference vaccine. In the present study, we standardized the reference vaccine used for determining the potencies of test vaccines, and established a potency test by ELISA. We evaluated the proposed reference vaccine by the neutralizing antibody responses in dogs after vaccination, by the challenge protection test in guinea pigs (GP potency test), which is the earlier official potency test used in Japan, and by the NIH potency test, which is widely used throughout the world. The results showed that a 4-fold dilution of the proposed reference vaccine induced sufficient immunity in dogs. A 3-fold dilution of the proposed reference vaccine passed the GP potency test. The international units (IU) calibrated by the NIH potency test were 3.7 IU/dose. From the results and the WHO recommendation that veterinary rabies vaccines should have a potency of at least 1.0 IU/dose, we determined to dilute the proposed reference vaccine by 3 fold and regarded it as the reference vaccine. Finally, we confirmed that there is a good agreement between the results of the potency test by ELISA and the results of the GP potency test. The establishment of the potency test by ELISA has made it possible to monitor the potency in the production process and has contributed to the stable production of the vaccine.
A multiplex seminested polymerase chain reaction (PCR) was developed for the simultaneous detection and differentiation among porcine circovirus 1 (PCV1), PCV2, and porcine parvovirus (PPV) from boar semen. Primers of PCV1, PCV2 and PPV were specific and did not react with other viruses respectively. Twenty (20.4%) and 42 (42.9%) out of 98 whole semen samples were found to be positive for PCV and PPV using multiplex conventional and seminested PCR, respectively. When the separated fractions of PCV or PPV-contaminated semen were analyzed using multiplex seminested PCR, PCV and PPV DNA were found to be present mainly in the seminal fluid and nonsperm cell fractions. This multiplex seminested PCR assay was sensitive, rapid and a good alternative method for the detection and differentiation of these viruses in boar semen.