Purification and characterization of two anionic trypsins from the hepatopancreas of carp

SUMMARY: Two trypsins, designated as trypsin A and trypsin B, have been purified from the hepatopancreas of carp. The purification procedures consisted of ammonium sulfate fractionation, and chromatographies on DEAE-Sephacel, Ultrogel AcA54 and Q-Sepharose. Trypsin A was purified to homogeneity with the molecular mass of approximately 28 kDa, while trypsin B gave two close bands of 28.5 kDa and 28 kDa on sodium dodecylsulfate polyacrylamide gel electrophoresis both under reducing and non-reducing conditions. On native-PAGE, both trypsin A and trypsin B showed a single band. Trypsin A and trypsin B revealed optimum temperature of 40°C and 45°C, respectively, and shared the same optimum pH 9.0 using Boc-Phe-Ser-Arg-MCA as substrate. Both enzymes were effectively inhibited by trypsin inhibitors and their susceptibilities were similar. The NH₂-terminal amino acid sequences of trypsin A and trypsin B were determined to 37th and 40th amino acid residue, respectively. Their sequences were very homologous, but not identical to that of a trypsin-type serine proteinase from carp muscle and these of other trypsins. Immunoblotting test using the antibody raised against trypsin A cross-reacted with trypsin B positively.     

Analysis of genomic mutation and immunohistochemistry of platelet‐derived growth factor receptors in canine vascular tumours

We examined whether mutation of the platelet‐derived growth factor receptor protein tyrosine kinase (PDGFR)‐α and PDGFR‐β genes contributes to their overexpression in canine vascular tumours. Genomic sequences of trans‐ or juxtamembrane regions of PDGFR‐α and PDGFR‐β were analysed with immunohistochemical staining and polymerase chain reaction‐direct sequencing using DNA from paraffin‐embedded neoplastic tissues of 27 hemangiosarcomas (HSAs) and 20 hemangiomas (HAs). Immunohistochemically, 75% of the HA cases were positive for PDGFR‐α and almost most of the HA cases were negative for PDGFR‐β. Of the HSA cases, 55.6% were negative for PDGFR‐α and 63% were strongly positive for PDGFR‐β. Among the HA cases, 1 missense mutation was detected in PDGFR‐α exon 18 and 1 in PDGFR‐β exon 17. Two HSA cases had missense mutations in exon 14 and 1 in exon 17 of PDGFR‐β. Thus, genomic mutation of trans‐ or juxtamembrane regions of PDGFRs was not the main mechanism driving the activation of receptors in HSA and HA.     

Seroprevalence and risk factors for Neospora caninum in sheep in the state Minas Gerais, southeastern Brazil

The aim of this study was to determine the prevalence and risk factors associated with infection due to Neospora caninum in serum samples from 488 sheep originating from 63 farms in 63 municipalities distributed across eight of the twelve mesoregions of the state Minas Gerais, southeastern Brazil. For detection of N. caninum the sheep serum samples were subjected to the indirect fluorescence antibody test (IFAT ≥ 50). To identify the risk factors associated with infection due to N. caninum a questionnaire was filled out for each herd by interviewing, the individual responsible for the herd, demanding information on the general characteristics of the property. Sixty-four sheep sera (13.1%; 95% CI=10.3-16.4) presented IgG-specific anti-N. caninum antibodies with the following titers: 50 (49; 76.6%), 100 (7; 10.9%), 200 (4; 6.2%), 400 (3; 4.7%) and 800 (1; 1.6%). The prevalence of infected sheep per mesoregion ranged from 0 to 28.1%. Out of the 63 farms sampled, 31 (49.2%; 95% CI=36.4-62.1) presented at least one seropositive sheep. No significant association was found between the presence of anti-N. caninum antibodies and the risk factors evaluated on the farms, except for the mesoregion variable (p=0.004; OR=0.429; CI95%=0.182-1.008). These results indicate that there is a need for additional research to define the epidemiological importance of this parasite as a cause of reproductive problems in sheep herds in Minas Gerais.     

Tumour necrosis factor‐related apoptosis‐inducing ligand induces apoptosis in canine hemangiosarcoma cells in vitro

Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is an apoptosis‐inducing cytokine that shows potential therapeutic value for human neoplasms, and is effective in some canine tumours; however, its potential for killing canine hemangiosarcoma (HSA) cells is unknown. Thus, we evaluated the proapoptotic effect of TRAIL in nine canine HSA cell lines. Cells (JuA1, JuB2, JuB2‐1, JuB4, Re11, Re12, Re21, Ud2 and Ud6) were cultured with three recombinant human TRAILs (rhTRAILs): TRAIL‐TEC derived from Escherichia coli, TRAIL‐TL derived from mammalian cells and isoleucine zipper recombinant human TRAIL (izTRAIL) containing an isoleucine‐zippered structure that facilitates trimerization. TRAIL‐TEC did not decrease the cell viability in any of the cell lines tested, whereas the other two rhTRAILs effectively decreased the viability of all cell lines as assessed by the WST‐1 assay. In canine HSA cells, izTRAIL induced apoptosis more effectively than TRAIL‐TL. In JuB4, Re12, and Ud6 cells, izTRAIL increased the activation of caspase‐3 and caspase‐8 and caused poly (ADP‐ribose) polymerase degradation. Moreover, izTRAIL treatment increased the proportion of Annexin V+/ Propidium iodide (PI)? apoptotic cells and nuclear fragmentation in izTRAIL‐sensitive cells. These results show that rhTRAIL can induce apoptosis in canine HSA cells, but the sensitivity of TRAIL was different depending on the cell lines. Therefore, TRAIL could be an effective therapeutic agent against canine HSA, but the specific mechanism of resistance should be determined to clarify under what conditions this treatment would be most effective.     

A case of feline sporotrichosis

We excised surgically a feline granulomatous lesion and performed histopathological, mycological and molecular examinations. As a result, it was diagnosed as sporotrichosis, which was the second recorded case of a cat so afflicted in Japan. After the operation, we recognized another nodule on the lymph node. Histopathological examination was therefore performed, but no fungi were detected. To prevent recurrence, the cat was administered a antimycotic drug, itraconazole. As a result, no recurrence was found. Excision of the lesion is the treatment of choice for feline sporotrichosis.     

Genetic polymorphism as a background of animal behavior

Various studies have shown the associations between differences in human behavioral traits and genetic polymorphism of neurotransmitter-related proteins such as receptors, transporters and monoamine oxidase. To clarify the genetic background of animal behavior, corresponding regions in animals have been analyzed. The study has been especially focused on primates, as the evolutionally closest animal to humans, and on dogs, as the socially closest animal to humans. In primates, polymorphisms were discovered between or within species, and the functional effects on neural transmission were found to be different by alleles. Even in apes, the closest species to humans, function was different from that in humans. In dogs, allele distributions of several genes were different among breeds showing different behavioral traits, and genes associated with individual differences in aggressiveness and aptitude of working dogs were surveyed. The survey of behavior-related genes has also been carried out in other mammals such as horses and cetaceans. Genes controlling various behaviors in birds have also been reported. The marker genes for behavior will provide useful information for human evolution, welfare of zoo animals and effective selection of working dogs and industry animals.     

Dirofilaria immitis infection of a snow leopard (Uncia uncia) in a Japanese zoo with mitochondrial DNA analysis

Three dog heartworms (Dirofilaria immitis) were detected in the lumen of the right cardiac ventriculus and of the pulmonary artery of a captive female snow leopard (Uncia uncia) that died of pancreatic carcinoma at a zoo in Japan. Neither clinical respiratory nor circulatory symptoms caused by the heartworm infection were observed. The filarial worms were identified as D. immitis from the morphologic characteristics of the esophagus, the presence of faint longitudinal ridges on the cuticular surface, the situation of vulva posterior to the esophagus, and the measurements of the body. The heartworms from the snow leopard were identical to that of D. immitis from dogs in the sequence of the cytochrome oxidase I region in the mitochondrial DNA. This host record is the first of D. immitis in U. uncia.     

Artificial insemination with frozen epididymal sperm in cats

Artificial insemination with frozen cauda epididymal sperm was performed in cats. Sperm were transmigrated from the epididymides in 10 male cats. The mean sperm motility and viability were 67% and 82.5%, respectively, and 11.6 x 10(7) sperm were recovered. The mean sperm motility after thawing was 24.0%. Eleven female cats received unilateral intrauterine insemination of 5 x 10(7) sperm, and the conception rate was 27.3% (3/11). This was the first case of conception obtained with frozen epididymal sperm in cats.