Establishment of a potency test by ELISA for a rabies vaccine for animal use in Japan

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.     

Evaluation of the drug sensitivity and expression of 16 drug resistance-related genes in canine histiocytic sarcoma cell lines

Canine histiocytic sarcoma (HS) is an aggressive tumor type originating from histiocytic cell lineages. This disease is characterized by poor response to chemotherapy and short survival time. Therefore, it is of critical importance to identify and develop effective antitumor drugs against HS. The objectives of this study were to examine the drug sensitivities of 10 antitumor drugs. Using a real-time RT-PCR system, the mRNA expression levels of 16 genes related to drug resistance in 4 canine HS cell lines established from dogs with disseminated HS were determined and compared to 2 canine lymphoma cell lines (B-cell and T-cell). These 4 canine HS cell lines showed sensitivities toward microtubule inhibitors (vincristine, vinblastine and paclitaxel), comparable to those in the canine B-cell lymphoma cell line. Moreover, it was shown that P-gp in the HS cell lines used in this study did not have enough function to efflux its substrate. Sensitivities to melphalan, nimustine, methotrexate, cytarabine, doxorubicin and etoposide were lower in the 4 HS cell lines than in the 2 canine lymphoma cell lines. The data obtained in this study using cultured cell lines could prove helpful in the developing of advanced and effective chemotherapies for treating dogs that are suffering from HS.     

Distribution of a fish pathogen Listonella anguillarum in the Japanese flounder Paralichthys olivaceus hatchery

The present study was undertaken to investigate the distribution of Listonella anguillarum in a Japanese flounder (Paralichthys olivaceus) hatchery. A total of 2704 isolates were obtained from the developing fish, live diets and artificial feeds of Japanese flounder and their rearing water, 439 of which were identified as L. anguillarum by the combining incubation on thiosulfate-citrate-bile salt-sucrose (TCBS) agar at 35 °C overnight with polymerase chain reaction (PCR) detection for the VAH1 hemolysin gene. L. anguillarum was detected in all seven rotifer samples, with densities of 2.5 × 10³ to 4.6 × 10⁶ colony forming units (CFU) g^− 1. Both the analyzed samples of Nannochloropsis oculata contained this bacterium at densities of 1.6 × 10⁴ to 1.4 × 10⁵ CFU g^− 1. L. anguillarum was detected in only one of four samples of Artemia nauplii with a density of 4.8 × 10⁵ CFU g^− 1 (35%) and it was not detected in the two analyzed artificial feed samples. L. anguillarum was detected in 11 of 18 specimens of larval and juvenile Japanese flounder at densities of 5.0 × 10¹ to 7.4 × 10⁵ CFU g^− 1, while it was not detected in the two analyzed egg specimens of Japanese flounder. These results indicate that L. anguillarum associated with the developing Japanese flounder is likely derived from rearing water and live diets such as rotifers. Further, it is strongly suggested that L. anguillarum is a transient bacterium of the intestinal microflora for the Japanese flounder but is a permanently indigenous one for the Japanese flounder hatcheries.     

Mortierella wolfii keratomycosis in a horse

Purpose To describe a case of superficial keratomycosis caused by Mortierella wolfii (M. wolfii) in a horse. Methods A thoroughbred filly was presented with painful right eye of 2 days’ duration. A superficial corneal ulcer was observed ventrally together with multifocal punctuate opacities axially. Samples were collected by swabbing and scraping the ulcerated lesion and submitted for microbiologic and cytologic examination. Results Microscopic evaluation of debrided corneal tissue revealed the presence of nonseptate fungal hyphae, and culture of a corneal swab yielded fungal growth. Medical treatment with topical antifungal, antibiotic and autogenous serum and systemic anti‐inflammatory resolved the problem within 2 weeks. Conclusions Cytologic evaluation of a corneal scraping was useful to make a clinical diagnosis of keratomycosis. Based on the mycological characteristics, the fungus isolated from the corneal lesion was identified as M. wolfii. To the authors’ knowledge, this is the first case report of equine keratomycosis associated with this fungus, although the organism is known to infect various organs of cattle.     

Tracheal Endoscopic and Cytological Findings and Blood Examination Results in Thoroughbred Racehorses Suspected to have Lower Respiratory Tract Disease

Cytology of tracheal aspirates, tracheal endoscopic and blood tests were carried out to 86 Thoroughbred racehorses presenting coughs or poor performance which were suspected to have lower respiratory tract disease (LRTD) to assess the conditions of the disorders. Racehorses were classified into coughing (66 horses) and non-coughing (20 horses) groups based on clinical symptoms. Nine Thoroughbred racehorses without respiratory abnormality were used as controls. Assessment of grades of airway mucus, cytology of tracheal aspirates and serum amyloid A (SAA), fibrinogen (Fbg) and pulmonary surfactant protein D (SP-D) measurements were performed. Relationships between age, gender and racing careers were also investigated to understand the characteristics of LRTD in racehorses. Mean age was significantly higher in non-coughing group compared to coughing group. Existence of racing career and number of starts were significantly greater in non-coughing group compared to coughing group. On the other hand, grades of airway mucus were significantly higher in coughing group compared to control group. Percentages of neutrophils in tracheal aspirates were significantly higher in coughing group compared to non-coughing and control groups. SAA, Fbg and SP-D were higher in coughing group compared to non-coughing and control groups indicating that condition of coughing group is in the acute phase. Positive rate of inflammatory airway disease was significantly higher in coughing and non-coughing groups compared to control group. It was concluded that carrying out comprehensive evaluation including investigation on SAA, Fbg and SP-D analysis with airway assessment to Thoroughbred racehorses which were suspected to have LRTD are useful procedure to understand the pathological condition which aid to initiate appropriate treatment, prognosis judgment or to advise trainers to consider altering training regimen.     

Clinical evaluation of growth hormone secretion in cattle using insulin tolerance test

Growth hormone secretion was evaluated in cattle. Clinically healthy bovine growth hormone (bGH) concentrations were 10.7 +/- 1.6 ng/ml in Holstein and 7.8 +/- 3.9 ng/ml in Japanese black cattle. The bGH concentration alternated at three-hour intervals, and tended to be higher at midnight and lower in the morning and before feeding. Insulin tolerance test (ITT) at an insulin dosage of 0.25 U/kg showed a significant increase of bGH concentration to 331 +/- 153% at 60 to 90 min after injection. In ITT applied to five under-growth calves of Japanese black cattle, the basal bGH concentrations were lower and peak values after insulin injection were shown to be significantly low. The ITT is useful for the clinical examination of bGH secretion in cattle.     

Isolation and pathogenic characterization of an OB1 variant of Babesia rodhaini which has a glycophorin A-independent pathway to murine red blood cells

Recent studies using several Babesia spp. have demonstrated that these species commonly recognize host sialic acids of red blood cells (RBCs) for their invasion. Glycophorin A (GPA), which is a major carrier of the sialic acids on RBCs, is a possible invasive receptor for Babesia parasites. In the present study, a variant of Babesia rodhaini was successfully isolated from a GPA homozygous knockout (GPA^−/−) mouse infected with an Australian strain of B. rodhaini which had originally been unable to replicate in GPA^−/− mice. The isolated parasite (designated as an OB1 variant) caused lethal infection to wild-type mice, as in the case of the parent Australian strain. However, although the growth of the OB1 variant in GPA^−/− mice was comparable with that in wild-type mice at 1–4 days after infection, the growth was significantly inhibited from day 5 onward, leading to the eventual survival of the GPA^−/− mice. Resistance of GPA^−/− mice against OB1 infection was lost by splenectomy, although the cytokine responses to the infection in the sera of GPA^−/− mice were similar to those of wild-type mice. The autoantibody levels to GPA-defective RBCs in the sera of GPA^−/− mice were depressed at a lower level at 0–2 days after infection than those of wild-type mice, while the levels of GPA^−/− mice progressively increased and reached comparable levels to those of wild-type mice at day 3 or later. These results indicate that the isolated OB1 variant has a GPA-independent invasion pathway into murine RBCs and suggest that the resistance of GPA^−/− mice against infection with the OB1 variant may be attributed to the effective clearance of the parasitized RBCs lacking GPA in the spleen, possibly mediated by preferential autoantibody binding to the RBC membrane.