Full‐length sequence of genotype 3 hepatitis E virus derived from a pig in Thailand

Hepatitis E virus (HEV) infection in pigs was investigated in two principal swine farming areas in Thailand. Anti‐HEV antibodies and HEV RNA in sera were examined in 258 pigs reared on five commercial farms from age 1 to 6.5 months and sows. Overall, 167 of 258 (64.7%) pigs were positive for anti‐HEV IgG, while 20 of 258 (7.75%) had detectable HEV RNA. Sequence analysis of 20 HEV isolates obtained from viremic pigs revealed that they were 92.3–100% identical to each other and had 82.2–88.2% nucleotide similarity to other reported genotype 3 isolates in 415 nucleotide sequences within ORF2 region. Further characterization by sequencing the complete genome of the Thai swine HEV isolate (named Thai‐swHEV07) and phylogenetic analysis showed that Thai‐swHEV07 segregated into a cluster consisting of swine isolates from Japan, Mongolia, and Kyrgyzstan within the HEV genotype 3. The Thai‐swHEV07 had a genomic length of 7,229 nt excluding the polyadenylated region at 3′ terminus of the genome. Comparison of Thai‐swHEV07 and 27 reported strains of genotype 3 revealed 80.4–85.9% nucleotide identity, with the highest identity of 85.9% to the novel swHEV strain from Mongolia. These findings suggest that genotype 3 HEV isolates are markedly heterogeneous. J. Med. Virol. 81:657–664, 2009 © 2009 Wiley‐Liss, Inc.     

Association between serum cortisol and progesterone concentrations and the infiltration of immune cells in the endometrium of gilts with vaginal discharge

The objective of the study was to investigate an association between serum cortisol and progesterone (P₄) concentrations and the distribution of immune cells in the endometrium of the gilts with vaginal discharge. Genital organs from 39 Landrace×Yorkshire crossbred gilts culled owing to vaginal discharge problem were collected from two commercial swine herds in Thailand. The estrous stage and gross pathology were examined. Blood samples were collected from the jugular vein prior to being slaughtered. Serum P₄ and cortisol were analyzed by means of enzyme immunoassay. The samples observed were in inactive (n = 4), follicular (n = 10), and luteal (n = 25) phases. They, afterwards, were processed in hematoxylin and eosin sections. The endometrium of the gilts was histologically divided into three layers, i.e., epithelial, subepithelial connective tissue, and glandular connective tissue layers. Immune cells, i.e., lymphocytes, neutrophils, eosinophils, macrophages, and plasma cells, in each layer were quantified under a light microscope (×400). The results revealed that mean serum cortisol was 430.6 ± 68.3 nmol/l. Serum P₄ varied by ovarian status. Serum P₄ of the gilts in the luteal phase was higher than those in the follicular phase (88.3 ± 7.7 versus 20.6 ± 6.2 nmol/l, P < 0.05). As for the endometrium condition, the gilts were classified into acute/subacute endometritis (n = 13), chronic endometritis (n = 9), and normal endometrium (n = 17). Neutrophils were the main local immune cells in the epithelial layer. Lymphocytes were the dominant population in the subepithelial and glandular connective tissue layers. Generally, the serum cortisol tended to negatively correlate with lymphocytes in the subepithelial connective tissue layer (r = −0.28, P = 0.081). In the gilts with acute/subacute endometritis, no correlation among serum cortisol, P₄, and immune cells was observed. In chronic endometritis gilts, only a negative correlation was remarked between P₄ and epithelial lymphocytes (r = −0.83, P = 0.010), epithelial neutrophils (r = −0.79, P = 0.019), and subepithelial neutrophils (r = −0.73, P = 0.025). In the gilts with normal endometrium, P₄ negatively correlated with subepithelial neutrophils (r = −0.55, P = 0.022) while positively correlated with subepithelial macrophages (r = 0.54, P = 0.024) and subepithelial eosinophils (r = 0.60, P = 0.011).     

The Sow Endometrium at Different Stages of the Oestrous Cycle: Immunohistochemical Study on the Distribution of SWC3‐Expressing Cells (Granulocytes,Monocytes and Macrophages)

Uterine samples from sows taken immediately after slaughter at late di‐oestrus, pro‐oestrus, oestrus, early di‐oestrus and di‐oestrus, were analysed by immunohistochemistry with an avidin–biotin–peroxidase method using a monoclonal antibody (anti‐SWC3) to granulocyte, monocyte and macrophage populations. The endometrium was then examined by light microscopy. In the surface and glandular epithelium, the largest numbers of SWC3‐expressing cells (P ≤ 0.01 and P ≤ 0.05) were found at oestrus, and at pro‐oestrus and oestrus, respectively. The numbers of SWC3‐expressing cells in the epithelium were positively correlated with the plasma levels of oestradiol‐17β. In the connective tissue of the subepithelial and glandular layers, no significant effect of the oestrous cycle stage was found on the number of SWC3‐expressing cells. The present study showed a variation in the distribution of SWC3‐expressing cells in the sow endometrium, especially in the surface and glandular epithelium, during different stages of the oestrous cycle.     

具有临床意义的药物相互作用

药物相互作用是药物不良反应问题中相当少的一部分,至少来自医院的监测报告是这样的。虽说在文献中已有大景有关药物相互作用的情报,但是如何科学地评价不良药物相互作用报告的有效性,尚无行之有效的办法。在药物文献中有许多矛盾着的、使人误解的报道和许多被曲解的数据,而且把我们引入歧途,以为在实验动物发生的相互作用也好象会在人类中出现。我们没有必要担心可能发生的没有临床关联的相互作用,但对具有临床意义的相互     

Endometritis in gilts: reproductive data, bacterial culture, histopathology, and infiltration of immune cells in the endometrium

The aim of the present study was to quantify the number of immune cells infiltrated in the endometrium of endometritis gilts. Based on gross morphology, a selected 28 genital organs of endometritis gilts were investigated. The gilts were classified according to the ovarian appearance into three groups, i.e. follicular, luteal, and ovarian quiescent phases. Historical data, reason for culling, histopathology, bacterial identification, and number and type of immune cells in different layers of the endometrium were examined. The gilts were culled at 336 ± 63 days of age at a body weight of 142 ± 20 kg. The culling reasons included abnormal vaginal discharge (n = 10), repeat breeding (n = 6), anestrus (n = 6), abortion (n = 4), and not pregnant (n = 2). Bacteria identified from pus exudates included Escherichia coli (33.3%), Staphylococcus sp. (17.5%), α-hemolytic Streptococcus sp. (14.3%), and β-hemolytic Streptococcus sp. (9.5%). Neutrophils were the most common immune cells in the epithelial and subepithelial tissue layers of the endometrium, while lymphocytes were the most common immune cells in the glandular layer. Neutrophils in the epithelial and subepithelial layers of the endometrium in the luteal phase were lower than in the follicular and ovarian quiescent phase. During the acute stage, neutrophils were the most common immune cells in the endometrium, while during the chronic stages, lymphocytes, plasma cells, and eosinophils were the dominant immune cells. In conclusion, the number and type of immune cells in the endometrium of the endometritis gilts varied according to both the reproductive cycles and the stage of endometritis. Neutrophils, lymphocytes, plasma cells, and eosinophils indicate stages and the severity of endometritis.     

富含DHA的鸡蛋黄和L-半胱氨酸盐酸盐添加物对冷冻猪精液质量的影响

本文旨在研究富含DHA的鸡蛋黄和L-半胱氨酸添加物对冷冻猪精液质量的影响。从5只皮特兰猪种获得15个射精样本，根据冷冻添加物的组成将其分为4组：正常鸡蛋黄组（组1），富含DHA的鸡蛋黄组（组2），添加了5mmol／L半胱氨酸添加物的正常鸡蛋黄（组3），添加了5mmol／L的半胱氨酸添加物的富含DHA的鸡蛋黄（组4）。精液冷冻保存过后在50℃下解冻12秒。之后检测精子直线前进运动，精子生存力，顶体完整性，解冻精液中精子质膜的功能完整性。只添加IL-半胱氨酸（组3）可以提高精子直线前进运动（P〈0．05），与富含DHA的鸡蛋黄一起添加（组4）既能增加直线前进运动（P〈0．05）又能改善顶体完整性（P〈0．01）。富含DHA的鸡蛋黄单独添加对解冻后精液的质量没有任何作用（P〉0．05）。总之，抗氧化的三．半胱氨酸单独或者与富含DHA的鸡蛋黄一起作用可以明显改善解冻后精液质量，尤其是直线前进运动力和顶体完整性。     

Influence of Post‐ovulatory Insemination on Sperm Distribution,Pregnancy and the Infiltration by Cells of the Immune System,and the Distribution of CD2, CD4, CD8 and MHC Class II Expressing Cells in the Sow Endometrium

This study investigates the distribution of leucocytes, CD2+, CD4+, CD8+ lymphocyte subpopulations and MHC class II expressing cells in the sow endometrium following post‐ovulatory insemination in relation to clinical findings and pregnancy outcome. Crossbred multiparous sows were inseminated once either at 15–20 h after ovulation [experiment 1, slaughtered at 20–25 h (5–6 h after artificial insemination (AI), group 1‐A, n = 4), at 70 h after ovulation (group 1‐B, n = 4), on day 11 (group 1‐C, n = 4, first day of standing oestrus = day 1) or on day 19 (group 1‐D, n = 4)] or 30 h after ovulation [experiment 2, slaughtered at 5–6 h after AI (group 2‐A, n = 4) or on day 19 (group 2‐D, n = 3)]. The uterine horns were flushed to control for the presence of spermatozoa and neutrophils and/or for recovery of oocytes and/or embryos. Mesometrial uterine samples were plastic embedded and stained. Cryofixed uterine samples were analysed by immunohistochemistry using mAbs to lymphocyte subpopulations and MHC class II molecules. Light microscopy was used to examine surface (SE) and glandular epithelia (GE), and connective tissue layers, both subepithelially (SL) and glandular (GL). In experiment 1, group 1‐A, only one sow had spermatozoa in the utero‐tubal junction (UTJ). Marked/moderated numbers of neutrophils and spermatozoa were observed in the flushings of two sows. In group 1‐B, altogether 23 of 48 oocytes were cleaved. Day 11 (1‐C), embryos with small diameter were observed. Day 19 (1‐D), no embryos were found but small pieces of foetal membrane were observed in one of the sows. In group 1‐A, large numbers of neutrophils were found within the SE and SL but with high individual variation. For T lymphocyte subpopulations, in the SE, most CD2+ cells were found in group 1‐A. For both SE and GE in all groups, the number of CD8+ cells was significantly larger than that of CD4+ cells. In experiment 2, group 2‐A, no sow had spermatozoa in the UTJ or in the uterine flushings. At day 19, no sow was pregnant. In group 2‐A, large numbers of neutrophils were found within the SE and SL but with high individual variation. At day 19, high E₂ levels showed a hormonal prooestrous stage but the endometrial neutrophil infiltration normally expected at pro‐oestrus was absent. In conclusion, post‐ovulatory insemination (about 18 h after ovulation) resulted in impaired spermatozoa transport within the uterus and embryonic degeneration. In sows post‐ovulatory inseminated at a later stage (30 h after ovulation), no sow was pregnant. In both experiments, disturbed immune cell patterns were observed in some individuals.     

In vivo evaluation of coumarin and nicotine as probe drugs to predict the metabolic capacity of CYP2A6 due to genetic polymorphism in Thais

The association between the distribution characteristics of CYP2A6 catalytic activities toward nicotine and coumarin, and the frequency distribution of CYP2A6 variant alleles reported was estimated in 120 healthy Thais. The distributions of the subjects as classified by the amounts of 7-hydroxycoumarin (7-OHC) excreted in the urine and by cotinine/nicotine ratio in the plasma were clearly bimodal. However, the numbers of apparently poor metabolizers for coumarin and nicotine were different. The inter-individual variability in the in vivo dispositions of coumarin and nicotine closely related to the CYP2A6 genetic polymorphism. There was a close correlation between the rate of 7-OHC excretion in the urine and cotinine/nicotine ratio in the plasma among subjects (R=0.92, p<0.001). The frequency of CYP2A6 allele found in the present study was: CYP2A6*1A=32% (95% CI, 22.1-39.4%), CYP2A6*1B=27% (95% CI, 19.4-33.5%), CYP2A6*9=20% (95% CI, 17.6-23.3%), CYP2A6*4=14% (95% CI, 9.6-17.8%), CYP2A6*7=5% (95% CI, 3.7-9.4%), CYP2A6*10=2% (95% CI, 0.8-5.1%). Subjects having CYP2A6*1A/*1B were found to have a higher rate of 7-OHC excretion, as well as a higher cotinine/nicotine ratio in the plasma compared with those of the other genotypes. In contrast, subjects with CYP2A6*4/*7 and CYP2A6*7/*7 almost lacked any cotinine formation, whereas urinary 7-OHC was still detectable. CYP2A6*9 allele clearly resulted in reduced enzyme activities. Despite the absence of the homozygote for CYP2A6*10 allele, the presence of CYP2A6*10 allele significantly decreased the enzyme activities. The results of the present study demonstrate that in vivo phenotyping of CYP2A6 using nicotine and coumarin are not metabolically equivalent. Nicotine is a better probe according to its specificity, while coumarin is still valuable to be used for a routine CYP2A6 phenotyping since the test employs a non-invasive method.     

Supplemental effect of varying L-cysteine concentrations on the quality of cryopreserved boar semen

Cryopreservation is associated with the production of reactive oxygen species, which leads to lipid peroxidation of the sperm membrane and consequently a reduction in sperm motility and decreased fertility potential. The aim of this study was to determine the optimal concentration of L-cysteine needed for cryopreservation of boar semen. Twelve boars provided semen of proven motility and morphology for this study. The semen was divided into four portions in which the lactose-egg yolk （LEY） extender used to resuspend the centrifuged sperm pellet was supplemented with various concentrations of L-cysteine to reach 0 mmol L^-1 （group Ⅰ, control）, 5 mmol L^-1 （group Ⅱ）, 10 mmol L^-1 （group Ⅲ） and 15 mmol L^-1 （group Ⅳ）. Semen suspensions were loaded in straws （0.5 mL） and placed in a controlled-rate freezer. After cryopreservation, frozen semen samples were thawed and investigated for progressive motility, viability using SYBR-14/EthD-1 staining and acrosome integrity using FITC-PNA/EthD-1 staining. There was a significantly higher （P 〈 0.01） percentage of progressive motility, viability and acrosomal integrity in two L-cysteine-supplemented groups （group Ⅱ and group Ⅲ） compared with the control. There was a biphasic effect of L-cysteine, with the highest percentage of progressive motility, viability and acrosomal integrity in group Ⅲ. In conclusion, 5 or 10 mmol L^-1 was the optimum concentration of L-cysteine to be added to the LEY extender for improving the quality of frozen-thawed boar semen.