Proceedings of the 2013 Joint JSTP/NTP Satellite Symposium

The first joint Japanese Society of Toxicologic Pathology (JSTP) and National Toxicology Program (NTP) Satellite Symposium, entitled “Pathology Potpourri,” was held on January 29^th at Okura Frontier Hotel in Tsukuba, Ibaraki, Japan, in advance of the JSTP’s 29^th Annual Meeting. The goal of this Symposium was to present current diagnostic pathology or nomenclature issues to the toxicologic pathology community. This article presents summaries of the speakers’ presentations, including diagnostic or nomenclature issues that were presented, select images that were used for audience voting or discussion, and the voting results. Some lesions and topics covered during the symposium include: treatment-related atypical hepatocellular foci of cellular alteration in B6C3F1 mice; purulent ventriculoencephalitis in a young BALB/c mouse; a subcutaneous malignant schwannoma in a RccHan:WIST rat; spontaneous nasal septum hyalinosis/eosinophilic substance in B6C3F1 mice; a rare pancreatic ductal cell adenoma in a young Lewis rat; eosinophilic crystalline pneumonia in a transgenic mouse model; hyaline glomerulopathy in two female ddY mice; treatment-related intrahepatic erythrocytes in B6C3F1 mice; treatment-related subendothelial hepatocytes in B6C3F1 mice; spontaneous thyroid follicular cell vacuolar degeneration in a cynomolgus monkey; congenital hepatic fibrosis in a 1-year-old cat; a spontaneous adenocarcinoma of the middle ear in a young Crl:CD(SD) rat; and finally a series of cases illustrating some differences between cholangiofibrosis and cholangiocarcinoma in Sprague Dawley and F344 rats.     

The quality and maturation of bitch oocytes recovered from ovaries by the slicing method

Oocytes were recovered from bitch ovaries at various stages of the estrous cycle by the slicing method. The proportion of Grade A oocytes (darkly pigmented and surrounded in part, or whole, by dense layers of cumulus cells) were counted. Only Grade A oocytes were cultured in TCM199 supplemented with 5% fetal calf serum for evaluation of meiotic competence. There were no significant differences in the total number of oocytes or the proportion of Grade A oocytes that were recovered from bitches at various stages of the estrous cycle. Only 11% of the oocytes reached metaphase II (MII) at 72 hr after initiation of maturation culture. However, the proportions of oocytes reaching MII did not increase with culturing for up to 120 hr.     

The role of CD4(+) or CD8(+) T cells in the protective immune response of BALB/c mice to Neospora caninum infection

The role of CD4(+) or CD8(+) T cells in the immune response of BALB/c mice against Neospora caninum infection was examined by using anti-CD4 and/or anti-CD8 monoclonal antibodies (mAbs). Mice were intraperitoneally inoculated with anti-CD4 and/or anti-CD8 mAbs before and after infection with N. caninum and observed for 30 days after infection. Most of the anti-CD4 mAb-treated mice and all of the anti-CD4 and anti-CD8 mAbs-treated mice died within 30 days post-infection (p.i.). In contrast, 100% of PBS-treated mice and 70% of anti-CD8 mAb-treated mice survived more than 30 days. When compared with phosphate-buffered saline (PBS)-treated mice, the weight of mice treated with mAbs tended to decrease. From these results CD4(+) T cells, but not CD8(+) T cells, have an important role for protection of mice against N. caninum infection. Serum antibody levels to N. caninum in infected-mice treated with anti-CD4 mAb or a mixture of anti-CD4 and anti-CD8 mAbs were lower than those in the infected mice treated with anti-CD8 mAb or PBS. The mice treated with anti-interferon-gamma (IFN-gamma) mAb produced high antibody levels to N. caninum, but all mice died within 18 days p.i. These results indicated that IFN-gamma is an important cytokine for protection against N. caninum infection at the early stage of infection. However, since CD4(+) T cells against N. caninum were essential to the production of specific antibody, these antibodies might have important roles in host protection at the later stage of infection.     

Localization of leptin and leptin receptor in the bovine adenohypophysis

The present study was carried out to detail the cellular localization of leptin (Lep) and the leptin receptor (LepR) in the bovine adenohypophysis. Lep immunoreactivity (Lep-ir) was found in about 30% of adenohypophysial cells in the gland. Immunochemistry of Lep and specific hormones using serial sections revealed that Lep-ir was present in 60.4% of somatotrophs, 15.9% of gonadotrophs, 6.5% of mammotrophs, 6.5% of thyrotrophs and 2.4% of corticotrophs. Both the common short isoform (OBRa) and the long isoform (OBRb) of LepR mRNA were expressed in the bovine adenohypophysis. LepR immunoreactivity (LepR-ir) was found in only 2.8% of the adenohypophysial cells and over 50% of LepR-ir cells were gonadotrophs, in which most of the cells were distributed in the zona tuberalis. The findings on Lep and LepR in the adenohypophysial cells indicate that Lep may regulate gonadotroph function through autocrine/paracrine pathway in the bovine adenohypophysis.     

Transglutaminase 2: a novel autoantigen in canine idiopathic central nervous system inflammatory diseases

Necrotizing meningoencephalitis (NME), necrotizing leukoencephalitis (NLE) and granulomatous meningoencephalomyelitis (GME) are common idiopathic inflammatory central nervous system (CNS) diseases with unknown etiology in dogs. We previously showed that IgG autoantibodies in the cerebrospinal fluid (CSF) of NME cases reacted to unknown brain proteins as well as to glial fibrillary acidic protein (GFAP). In the present report, we evaluated the autoantibodies against transglutaminase2 (TG2) in the canine CNS diseases. CSF samples obtained from dogs with NME (n=19), NLE (n=7), GME (n=11) and miscellaneous CNS diseases (n=12) were subjected. CSFs from 20 healthy dogs were used as controls. Indirect fluorescent antibody test on the canine cerebrum revealed astrocyte-binding IgG in the CSF of NME. After absorption of the CSF with bovine GFAP, the CSF still possessed the reactivity to astrocytes. Double-color staining showed clear colocalization of the autoantibodies and anti-human TG2 rabbit polyclonal IgG. An immunoblot assay against human recombinant TG2 revealed anti-TG2 IgG in the CSF from dogs with NME, NLE and GME. The CSF of canine idiopathic encephalitis cases, notably of NME, tended to show high ELISA OD values against human recombinant TG2 compared to healthy controls. The presence of anti-TG2 autoantibodies in the CSF may contribute to the elucidation of the etiology of canine NME, NLE and GME.     

Development and introduction of detection dogs in surveying for scats of small Indian mongoose as invasive alien species

The small Indian mongoose was introduced to the island of Okinawa in 1910 to control rats and snakes, however, it poses a threat to many endemic species in the Yambaru forest region in Okinawa Island. An important concern in eradication is that, as much as possible, capture should be carried out without affecting native species. To protect endemic species in the Yambaru region of Okinawa Island, we trained and used detection dogs to search for mongoose scats that indicated that mongoose were present in the area. We confirmed the ability of the dogs to detect scats in positive and negative control tests in the Yambaru forest region after basic, obedience, and mongoose-scat-detection specific training. In 30 trials, the dogs found 60 of 65 scats samples. For the trained alert, the dogs carried out 59 trained alerts for the 60 scats found. In the positive and negative control tests, the dogs showed no reaction to 129 of 130 negative controls. We then surveyed the forest in the Yambaru region for mongoose scats using the detection dogs. In this study, the detection dogs found 79 scats in a survey of a 69.0-km area. We divided the survey area into 43 grids and calculated the rate of detection in each grid. The rate of detection of scats was higher in the southern part of the Yambaru region. These result showed that our mongoose scat detection dogs can be useful tools to assist in determining the presence of mongoose in the Yambaru region of Okinawa Island.     

Proliferation capacity, neuronal differentiation potency and microstructures after the differentiation of canine bone marrow stromal cells into neurons

We examined the proliferation capacity and neuronal differentiation potency of canine bone marrow stromal cells (BMSCs). In addition, the microstructures of neuron-like cells after neuronal differentiation were observed under a scanning electron microscope. Canine BMSCs grew to confluency at 10.0 ± 2.5 days, and 3.8 ± 2.1 × 10(6) BMSCs were collected in one passage. Approximately 65% of canine BMSCs changed to neuron-like morphology after neuronal differentiation, and nearly all neuron-like cells stained positive against neuron-specific enolase. In addition, microstructures such as the cellular organelles, filaments and growth cones of these cells bore a close resemblance to those of the original mature neurons. These results suggested that canine BMSCs might be capable of differentiating into neurons.     

Effect of relationships among clinical mastitis incidence,reproductive performance,and culling rate on the lifetime of dairy cows at Hiroshima University Farm

Farm managers' decision to cull dairy cows is based on the cows' milk production, history of disorder(s), and reproductive performance, each of which affects dairy cows' lifetime (herd life and productive lifespan). We investigated the relationships among the incidence of clinical mastitis (CM), the reproductive performance, and the culling rate. We also assessed the effects of these relationships on the lifetimes of dairy cows, using the records made before and after the introduction of an automatic milking system (AMS) at Hiroshima University Farm. Milk yield, CM incidence density, and culling rate of dairy cows increased after the AMS introduction. The CM incidence was associated with an elongation of the calving interval in cows with the same parity. CM in the 1st parity might have caused the reductions of the cows' lifetime and their parity at culling. A higher age at first calving (AFC) was associated with an increase in culling rate but did not lead to a significant decrease in lifetime. Investigations of the factors mediating CM in the 1st parity or AFC with CM incidence or culling rate in the later stages might contribute to the control of lifetime of dairy cows.     

Comparison of N-terminal pro-atrial natriuretic peptide and three cardiac biomarkers for discriminatory ability of clinical stage in dogs with myxomatous mitral valve disease

Plasma N-terminal pro-atrial natriuretic peptide (NT-proANP) concentration increases with progression of myxomatous mitral valve disease (MMVD) in dogs. This multicentre, prospective study compared plasma NT-proANP, N-terminal pro-brain natriuretic peptide (NT-proBNP), ANP, and cardiac troponin I (cTnI) concentrations in dogs with MMVD for their characteristics and discriminatory ability to detect cardiac dilatation and congestive heart failure (CHF). Thirty-six healthy dogs and 69 dogs with MMVD were included. Clinical variables were obtained via physical examination, thoracic radiography, and echocardiography. The discriminatory ability of each cardiac biomarker (CB) to determine the presence or absence of cardiac dilatation (event 1) and CHF (event 2) was evaluated using the receiver operating characteristic curves. Plasma NT-proANP, NT-proBNP, and ANP concentrations showed a significant association with the left atrium/aorta ratio (P<0.01). The area under the curve of plasma NT-proANP and NT-proBNP concentrations were 0.72 and 0.75, respectively in event1 and 0.72 and 0.76, respectively in event2. Plasma NT-proANP and NT-proBNP concentrations showed sensitivity 80.0 and 80.0%; specificity 67.6 and 64.7% in event1 (cutoff value; 8,497.81 pg/ml and 1,453.00 pmol/l, respectively) and sensitivity 85.7 and 81.0%; specificity 60.4 and 64.6% in event2 (cutoff value; 8,684.33 pg/ml and 1,772.00 pmol/l, respectively). In dogs with MMVD, plasma NT-proANP, NT-proBNP, and ANP concentrations increase with left atrial enlargement. Particularly, plasma NT-proANP and NT-proBNP concentrations appeared to be equally useful in the discriminatory ability to detect cardiac dilatation and CHF.