Further Development of an Equine Cell Line that can be Propagated over 100 Times

Cell lines originating from horses are necessary for isolation and propagation of equine herpesviruses (EHV). Although we established an equine-derived cell line, FHK-Tcl3, propagation ceased after fewer than 40 passages. In this study, FHK-Tcl3 cell propagation continued beyond 40 passages, achieving over 100 passages. FHK-Tcl3 cells were then cloned by limiting dilution at the 100th passage. Cloned cells were termed FHK-Tcl3.1. FHK-Tcl3.1 cells grew well and were propagated every 3 to 4 days by splitting 1:5. In addition, EHV-1, -2 and -4 showed a clear cytopathic effect (CPE) in FHK-Tcl3.1 cells, and this CPE was very similar to those seen in parental FHK-Tcl3 and primary fetal horse kidney cells. FHK-Tcl3.1 cells continue to propagate and the current passage record is over 100 times after cloning. Therefore, this cell appears to have been immortalized. FHK-Tcl3.1 cells have potential for growth and diagnosis of various equine viruses, including equine herpesviruses.     

Chronic Peripheral Administration of Kappa-Opioid Receptor Antagonist Advances Puberty Onset Associated with Acceleration of Pulsatile Luteinizing Hormone Secretion in Female Rats

Puberty in mammals is timed by an increase in gonadotropin-releasing hormone (GnRH) secretion. Previous studies have shown involvement of the two neuropeptides, kisspeptin and neurokinin B (NKB), in controlling puberty onset. Little is known about the role of the other key neuropeptide, dynorphin, in controlling puberty onset, although these three neuropeptides colocalize in the arcuate kisspeptin neurons. The arcuate kisspeptin neuron, which is also referred to as the KNDy neuron, has recently been considered to play a role as an intrinsic source of the GnRH pulse generator. The present study aimed to determine if attenuation of inhibitory dynorphin-kappa-opioid receptor (KOR) signaling triggers the initiation of puberty in normal developing female rats. The present study also determined if stimulatory NKB-neurokinin 3 receptor (NK3R) signaling advances puberty onset. Female Wistar-Imamichi rats were weaned and intraperitoneally implanted with osmotic minipumps filled with nor-binaltorphimine (nor-BNI), a KOR antagonist, or senktide, a NK3R agonist, at 20 days of age. Fourteen days of intraperitoneal infusion of nor-BNI or senktide advanced puberty onset, manifested as vaginal opening and the first vaginal estrus in female rats. Frequent blood sampling showed that nor-BNI significantly increased luteinizing hormone (LH) pulse frequency at 29 days of age compared with vehicle-treated controls. Senktide tended to increase this frequency, but its effect was not statistically significant. The present results suggest that the inhibitory input of dynorphin-KOR signaling plays a role in the prepubertal restraint of GnRH/LH secretion in normal developing female rats and that attenuation of dynorphin-KOR signaling and increase in NKB-NK3R signaling trigger the onset of puberty in female rats.     

Prevalence of serum antibodies to Toxoplasma gondii in free-ranging cats on Tokunoshima Island,Japan

The prevalence of Toxoplasma gondii infection in free-ranging cats on Tokunoshima Island was assessed by testing 125 serum samples using anti-T. gondii IgG indirect enzyme-linked immunosorbent assay. The overall seropositivity rate was 47.2% (59/125). Seropositivity rates in cats with body weight >2.0 kg (57.4%) were significantly higher than in those with body weight ≤2.0 kg (12.5%, P<0.01). Analysis of the number of seropositive cats by settlement revealed the presence of possibly-infected cats in 17 of 23 settlements, indicating the widespread prevalence of T. gondii on the island. This is the first study to show the seroprevalence of T. gondii in free-ranging cats on Tokunoshima Island. The information revealed in this paper will help to prevent the transmission of T. gondii among cats and also in both wild and domestic animals and humans on the island.     

Mitochondrial diversity of native pigs in the mainland South and South-east Asian countries and its relationships between local wild boars

In this study, we analyzed DNA sequence of mitochondrial DNA (mtDNA) control regions on the 130 native domestic pigs and eight wild boars in the mainland South and South-east Asian countries including Bhutan, Cambodia, Laos, Myanmar, and Vietnam. Forty-four haplotypes were found in the 138 individuals, 41 were in the domestic and four were in wild boars. Only one haplotype was shared by domestic and wild population in Bhutan. In other cases, mtDNA of wild boars did not show close affinity to that of the domestic pigs in the same location, indicating that the native domestic pigs in these countries did not originate in the present habitat. Phylogenetic analyzes of mtDNA haplotypes recapitulated several major clusters identified in other studies, but 11 haplotypes were grouped in a new cluster we named MTSEA. In most cases, more than one lineage group were present in a sampling station, indicating that the present indigenous domestic pigs may have multiple origins. The MTSEA haplotypes were present in relatively high frequencies in domestic pigs in the mountainous area of mainland South-east Asia (Cambodia and Laos), with a few found in Myanmar and Bhutan. The distributions of MTSEA haplotypes are in great conformity with the distribution of present-day Mon-Khmer language and indicated the existence of yet another independent domestication. The D2 haplotypes that distribute high frequency (almost 100%) throughout the Chinese breeds were dominant in Bhutan, Myanmar, and Vietnam. These results suggest an existence of human-mediated dispersal of domestic pigs from north to the south during the historical expansion of Sino-Tibetan and Tai peoples. The D3 haplotypes previously reported in north India were found in sympatric domestic and wild pigs in Bhutan. The D3 haplotype is an important proof of independent domestication event and/or great gene flow between wild and domestic pigs in the foot of Himalaya.     

Equine Endometrial Gland Density and Endometrial Thickness Vary among Sampling Sites in Thoroughbred Mares

The secretions of the equine endometrial glands are essential for the survival, growth, and development of the conceptus in early pregnancy, and endometrial gland density is directly related to successful pregnancy outcome. Endometrial biopsy is routinely used to assess the reproductive potential of broodmares. Some previous studies have shown that equine endometrial glands are uniformly distributed throughout the uterus; however, other work has shown variation of the endometrial architecture between biopsy sites, suggesting that a single biopsy is not representative of the entire endometrium. The aims of this study were to assess and compare the endometrial gland density and thickness at four sampling sites in the uterus (the central segment of each uterine horn, the uterine horn-body junction, and the caudal portion of the uterine body). Endometrial samples from five nulliparous Thoroughbred mares in diestrus were obtained at necropsy and used for subsequent histomorphometric analysis. The caudal uterine body had a significantly lower endometrial gland density and endometrial thickness than the other sites. This may result in nutrient deprivation and reduced survival of embryos or fetuses in this region of the uterus. The endometrial gland density and endometrial thickness did not significantly differ between the other regions sampled, indicating that they are similarly suitable for embryonic implantation and fetal development. Our results suggest that the endometrial structure of the caudal uterine body of the mare is not representative of the endometrial morphology at other sites. Thus, the caudal uterine body is not a suitable site for routine endometrial biopsy.     

Urinary albumin and transferrin as early diagnostic markers of chronic kidney disease

Feline renal diseases are increasingly noted in veterinary practice. It is important to diagnose and identify the pathological basis of renal dysfunction accurately at an early stage, but there are only a few reports on this area in clinical veterinary medicine. We investigated the efficacy of measurement of urinary albumin (u-Alb) and urinary transferrin (u-Tf) for early diagnosis using 5-µl urine samples collected noninvasively by catheterization from normal (IRIS stage I) cats and cats with stage I chronic kidney disease (CKD). The u-Alb levels in normal and stage I CKD cats were 6.0 ± 4.5 and 11.2 ± 8.4 mg/dl, respectively, and the u-Tf levels were 0.09 ± 0.42 and 0.52 ± 0.79 mg/dl, respectively. Based on ROC curve analysis, the sensitivity and specificity of u-Alb and u-Tf were higher than those of the currently used biomarker, the plasma creatinine level. The sensitivity of u-Alb was higher than that of u-Tf, whereas the specificity of u-Tf was higher than that of u-Alb. The validity of the threshold albumin level (20 mg/dl) was confirmed by measurements using SDS-PAGE. Since leakage of u-Tf in urine precedes leakage of u-Alb, inclusion of u-Tf in biochemistry tests may be appropriate for IRIS staging as a diagnostic marker of early diagnosis of renal disorder in cats.     

ErbB2 copy number gain is associated with adverse outcome in canine mammary carcinoma

Copy number gain (CNG) and/or protein overexpression of ErbB2 have been observed in human breast cancer patients and are associated with poor prognosis. Similarly, ErbB2 overexpression has also been observed in canine mammary carcinoma; however, data on ErbB2 copy number is limited. The purposes of this study were to evaluate ErbB2 copy number in dogs with mammary carcinoma and to investigate associations of ErbB2 CNG with ErbB2 expression, histological and clinical characteristics, and survival. DNA samples were isolated from 59 formalin-fixed paraffin-embedded canine mammary gland tissues (34 carcinoma, 14 adenoma, and 11 normal). Using a digital PCR assay, the ErbB2 copy number in these samples was determined as compared to a reference gene on canine chromosome 8. ErbB2 CNG was detected in 14/34 (41%) carcinomas and 2/14 (14%) adenomas. ErbB2 overexpression was observed in 3/34 (9%) carcinomas but not in adenomas. Neither ErbB2 CNG nor ErbB2 overexpression were detected in the normal controls. There was no significant association of the ErbB2 CNG with histological and clinical characteristics such as age, neutered status, histological grade, tumor size, lymph node involvement, distant metastasis, and clinical stage in the dogs with mammary carcinoma. The presence of ErbB2 CNG, but not ErbB2 overexpression, was significantly related to the shorter overall survival. These findings suggest that ErbB2 CNG is a prognostic factor in dogs with mammary carcinoma.     

不同种植密度下温室间作玉米的水分利用情况与其营养元素吸收特征的关系

Dent corn, as a catch crop used for salt removal, was cultivated at different densities, i.e., 7.3 （low density）, 59.7 （normal density）, and 119.5 plants m-2 （high density）, during a 50 d fallow period after cultivation of a commercial crop in a greenhouse, to analyze the characteristics of nutrient salt （N, K, Mg, and Ca） uptake by roots and to study the effect of plant density on the characteristics associated with crop water use. Leaf area index for the high and normal density treatments reached extremely high values of 24.3 and 14.9, respectively. These values induced higher transpiration rates that were estimated using the Penman-Monteith model with the incorporation of specific parameters for crop and greenhouse conditions. The total N, K, Mg, and Ca contents in the crop canopy at harvest were 26.8, 13.0, 1.0, and 1.7 g m-S, respectively, under the high density treatment. The dynamics of salt uptake rates for high, normal, and low density treatments were evaluated by assessing weekly changes in salt content, and were subsequently compared against the transpiration rate. A positive linear relationship was obtained between these 2 parameters for all 3 density treatments and all tested salts. Hence, higher transpiration rates caused higher salt uptake rates through water absorption. On the other hand, salt uptake efficiency per unit water use by cultivation was lower in the low density treatment. Therefore, management procedures with dense planting that induce higher transpiration rates and lower evaporation rate are extremely important for the effective cultivation of corn catch crops.