Determination of Feeder Cell‐Based Cellular Niches Supporting the Colonization and Maintenance of Spermatogonial Stem Cells from Prepubertal Domestic Cat Testes

Recently, isolation and in vitro culture of putative spermatogonial stem cells (SSCs) in the domestic cat have been conducted. However, the cellular niche conditions that facilitate the establishment and long‐term maintenance of feline SSCs (FSSCs) have not been described. Therefore, we investigated the type of feeder cells used to stimulate colony formation and growth of FSSCs among the various factors in the FSSC niche. Spermatogonial stem cells isolated from feline testes were cultured on mitotically inactivated testicular stromal cells (TSCs) derived from cats, dogs and mice, and mouse embryonic fibroblasts (MEFs). The formation and growth of colonies derived from SSCs cultured on each type of feeder cell were identified at passage 0, and the morphology, alkaline phosphatase (AP) activity and expression of SSC‐specific genes in surviving colonies were investigated at passage 4. Among these diverse feeder cells, TSCs from cat showed the greatest colony formation, growth and maintenance of FSSCs, and SSC colonies cultured by passage 4 showed a typical dome‐shaped morphology, AP activity and expression of SSC‐specific genes (NANOG, OCT4, SOX2 and CD9). Accordingly, these results demonstrate that feline TSCs could be used as feeder cells to support the establishment and maintenance of SSCs from domestic cats.     

Hemodynamic characteristics of vasopressin in dogs with severe hemorrhagic shock

The effect of vasopressin was compared with that of the established vasopressor epinephrine in experimentally induced hemorrhagic shock. After rapid crystalloid resuscitation in a ratio of three volumes of 0.9% saline to one volume of blood (3:1 crystalloid resuscitation), six dogs were given 0.4 IU/kg vasopressin and another six dogs were given 0.1 mg/kg epinephrine. Five dogs in the control group were given fluid resuscitation in the same manner as above without administration of any drugs. Administration of vasopressin increased diastolic arterial pressure (DAP) from 45.0 +/- 4.9 to 91.2 +/- 9.6 mmHg within 5 min, compared with epinephrine from 46 +/- 4.0 to 51.8 +/- 7.7, and control from 47.3 +/- 7.5 to 46.3 +/- 7.3. Systolic arterial pressure (SAP) did not increase significantly following vasopressin compared with epinephrine and control group. Results of DAP and systemic vascular resistance index (SVRI) suggested that vasopressin administration was vasoconstrictive after fluid resuscitation in decompensatory hemorrhagic shock in dogs, whereas epinephrine did not compared with control. In addition, epinephrine did not affect the cardiac index (CI) and SVRI, while a significant decrease in CI and increase in SVRI were observed in vasopressin group. The pressor effect of epinephrine in the vascular system was abrupt and only lasted a short period of time (within 5 min), while that of vasopressin was steady and lasted for more than 1 hr, especially regard to in DAP. When compared with epinephrine, vasopressin can be a more effective and safer choice in patients with severe hemorrhagic shock.     

Medial canthoplasty for epiphora in dogs: a retrospective study of 23 cases

The medical records of 23 dogs that underwent medial canthoplasty for treatment of epiphora were reviewed. The most prevalent breed encountered was the shih tzu. Other affected breeds included the Pekingese, Maltese, toy poodle, and pug. All dogs had epiphora associated with medial canthal trichiasis and/or entropion. Other ocular abnormalities included conjunctivitis, keratitis, pigmentary keratitis, corneal ulceration, globe prolapse, and nasal fold trichiasis. After medial canthoplasty, the epiphora resolved in all dogs.     

Disseminated transmissible venereal tumor in a dog.

Transmissible venereal tumor (TVT) is a well-documented transplantable tumor in dogs, with no breed or sex predilection and a low metastatic rate. In this report, a 2-year-old intact female Mastiff that had numerous, rapidly growing masses throughout the subcutis mainly at the dorsal body plane, the caudal half of the ventral abdomen, and around the vulva was euthanized due to poor prognosis. Neoplastic nodules similar to those seen in the subcutis were also noted in the lung, anterior mediastinum, liver, spleen, kidney, and superficial and deep lymph nodes in both abdominal and thoracic cavities. The neoplastic nodules from the subcutis as well as metastatic foci revealed similar cytologic and histologic features, which were consistent with canine TVT. By immunohistochemical staining, the neoplastic cells were positive for lysozyme and vimentin but were negative for cytokeratin, desmin, CD3, and CD79a. The diagnosis of the TVT was further supported by the identification and analysis of long interspersed nuclear elements (LINE) from paraffin-embedded tumor tissue. This case is a rare example of TVT with multiorgan metastasis. In this case, the polymerase chain reaction technique was useful in differential diagnosis of canine round cell tumors because this technique can be applied in retrospective as well as future study.     

Comparison of response to immunotherapy by intradermal skin test and antigen-specific IgE in canine atopy

The intradermal skin test (IDST) and serologic allergy test (SAT) has been developed for confirming a diagnosis of canine atopy and determining allergens for immunotherapy. To determine the prevalence of causative allergens for canine atopic dermatitis in Japan, IDST and SAT were performed with the CMG Immunodot strips on 95 atopic dogs using 9 allergens. In addition, we compared agreement rate, sensitivity and specificity between them (using IDST as the standard). The allergen most commonly positive in both tests was house dust mites (IDST: 69.5%, SAT: 48.4%). Moreover, Japanese cedar, mugwort and grass mix were detected as attendant causative allergens. Agreement rates between the two tests ranged from 67.4% to 96.8%; the overall mean agreement rate were 81%. SAT was shown to have sensitivity to IDST ranging from 16.7 to 68.2%. The specificities were very high for all allergens, on the order of 94.9-100% (median=98.7%). Finally, the efficacy of immunotherapy was evaluated on 27 atopic dogs based on IDST (15 dogs) and SAT (12 dogs) results. Overall, 60% (9/15) of the IDST group and 66.8% (8/12) of the SAT group experienced a 50% to 100% reduction in their symptomatology. No significant differences were found in response to immunotherapy during the follow-up period between allergen selection methods. These results indicate the value of serologic tests as an aid to identifying an allergen solution for immunotherapy.     

Development of a multiplex PCR assay to detect Edwardsiella tarda,Streptococcus parauberis,and Streptococcus iniae in olive flounder (Paralichthys olivaceus)

A multiplex PCR protocol was established to simultaneously detect major bacterial pathogens in olive flounder (Paralichthys olivaceus) including Edwardsiella (E.) tarda, Streptococcus (S.) parauberis, and S. iniae. The PCR assay was able to detect 0.01 ng of E. tarda, 0.1 ng of S. parauberis, and 1 ng of S. iniae genomic DNA. Furthermore, this technique was found to have high specificity when tested with related bacterial species. This method represents a cheaper, faster, and reliable alternative for identifying major bacterial pathogens in olive flounder, the most important farmed fish in Korea.     

Molecular cloning and expression analysis of pig CD81

CD81, also known as TAPA-1 (target of antiproliferative antibody 1), is a member of the tetraspanin family of proteins and a component of the B cell co-receptor complex. Several studies have shown that CD81 plays significant roles in a variety of immune responses, including activation of B cells and T cells. In this study, we cloned pig Cd81 cDNA using RT-PCR coupled with rapid amplification of cDNA ends (RACE)-PCR and determined the complete cDNA sequence of pig Cd81. Pig Cd81 cDNA contains an open reading frame (711 bp) encoding 236 amino acids. The identity of pig CD81 with those of human, cattle, rat, and mouse are 90.30%, 92.26%, 86.22%, and 86.22%, respectively. Alignment of the CD81 amino acid sequence with those of mammalian species showed that the large extracellular loop (LEL) is the most divergent, whereas other domains are largely conserved. Pig Cd81 mRNA was detected by RT-PCR in a broad range of tissues, including lymphoid tissues as well as nonlymphoid tissues, indicated variety of cellular functions of CD81 in most pig tissues. Flow cytometry analyses demonstrated that human CD81 antibody recognizes a pig CD81 on the cell surface. Further, immunohistochemistry analysis using human CD81 antibody on pig spleen was revealed that CD81 expression is widely diffused in spleen tissue. Future study will be focused on defining the functional role of CD81 during the course of pig infectious diseases.     

Differential effects of treadmill exercise on calretinin immunoreactivity in type 2 diabetic rats in early and chronic diabetic stages

In this study, we investigated the effects of treadmill exercise on calretinin (CR), a marker of early postmitotic neurons, immunoreactivity in the dentate gyrus (DG) of Zucker diabetic fatty (ZDF) rats, before or after diabetes onset, and Zucker lean control (ZLC) rats. For this study, 6-week-old ZLC and prediabetic ZDF rats, and 22-week-old ZLC and ZDF rats were exercised on the treadmill. Sedentary ZLC and ZDF rats of the same age were used as exercise experiment controls. The exercised prediabetic ZDF rats did not show diabetes onset, while the sedentary prediabetic ZDF rats showed significantly increased blood glucose levels. The exercised diabetic ZDF rats exhibited a decrease in their blood glucose levels compared to the sedentary diabetic ZDF rats, but the levels were still above 20 mmol/l. ZLC rats of both ages were in the normoglycemic range. CR immunoreactivity was detected throughout the DG, including the subgranular zone and the polymorphic layer. Diabetic rats exhibited a significant decrease in the number of CR-immunoreactive cells and fibers in the DG. Exercise in the prediabetic ZDF rats significantly increased the number of CR-immunoreactive cells and fibers in the subgranular zone of the DG. In the ZLC and ZDF rats of chronic diabetic phase, exercise increased CR-immunoreactive neurons in the hilar region. These results suggest that diabetes significantly reduces the number of postmitotic CR-immunoreactive neurons and the intensity of immunoreactivity and that exercise increases these CR-related parameters in a diabetic stage-dependent manner.