Avian influenza virus subtypes inside and outside the live bird markets, 1993-2000: a spatial and temporal relationship

Between 1993 and 2000, gallinaceous birds, waterfowl, and environmental specimens from the live bird markets (LBMs) of the northeastern United States and non-LBM premises were tested for the presence of avian influenza virus (AIV), pathogenic properties of AIV subtypes, especially of hemagglutinin (H) subtypes H5 and H7, and a possible association between LBM and non-LBM infections. Ten H subtypes of AIV were isolated from the LBM specimens: H1, H2, H3, H4, H5, H6, H7, H9, H10, and H11. During this period, the 10 subtypes also were isolated from birds in non-LBM premises. In the LBMs, subtypes H2, H3, H4, H6, H7, and H11 were present for 5-8 yr despite efforts to clean and disinfect the premises. The H5 or H7 subtypes present during the same year in both LBMs and non-LBMs within a state or in contiguous states were (subtype/year): H5N2/1993, 1999, and H7N2/1994-99. The AIV subtypes including the H5 and H7 that were evaluated for pathogenicity in chickens were low pathogenic. The deduced amino acid sequence at the H cleavage site of H5 and H7 subtypes was consistent with those of low pathogenic AIV. Although the H5N2 and H7N2 subtypes remained low pathogenic, they did undergo mutations and acquired an additional basic amino acid at the H cleavage site; however, the minimum number of basic amino acids in correct sequence (B-X-B-R, where B = basic amino acid, X = need not be basic amino acid, and R = arginine) required for high pathogenicity was lacking. A low pathogenic H5 or H7 subtype may become highly pathogenic by acquiring additional basic amino acids at the H cleavage site. The LBMs have been and will likely continue to be a source of AIV for commercial poultry.     

Development of vaccine strains of H5 and H7 influenza viruses

To establish vaccine strains of H5 and H7 influenza viruses, A/duck/Hokkaido/Vac-1/04 (H5N1) [Vac-1/04 (H5N1)], A/duck/Hokkaido/Vac-3/07 (H5N1) [Vac-3/07 (H5N1)], and A/duck/Hokkaido/ Vac-2/04 (H7N7) [Vac-2/04 (H7N7)] were generated from non-pathogenic avian influenza viruses isolated from migratory ducks. Vac-1/04 (H5N1) and Vac-3/07 (H5N1) were generated by genetic reassortment between H5N2 or H5N3 virus as an HA gene provider and H7N1 or H6N1 viruses as an NA gene provider. Vac-2/04 (H7N7) was a genetic reassortant obtained using H7N7 and H9 N2 viruses to give high growth character of the H9N2 virus in chicken embryonated eggs. The results of sequence analyses and experimental infections revealed that these H5N1 and H7N7 reassortant viruses were non-pathogenic in chickens and embryos, and had good growth potential in embryonated eggs. These viruses should be useful to develop vaccines against H5 and H7 highly pathogenic avian influenza viruses.     

Performance of Hereford and two-breed rotational crosses of Hereford with Angus and Simmental cattle: I. Calf production through weaning

Cow size, reproductive traits and calf performance through weaning were evaluated in a range environment for Simmental (S) x Hereford (H) and Angus (A) x H crosses in two-breed rotations and straightbred H. Data were grouped into seven dam breed categories: straightbred Hereford (H), crossbred F1 S x H cows (SH), S x H cows of low percentage H (SHS), S x H cows of high percentage H (HSH), F1 A x H cows (AH), A x H cows of low percentage H (AHA) and A x H cows of high percentage H (HAH). Straightbred H, SH, AH, SHS and AHA cows were mated to H bulls, HSH cows were mated to S bulls and HAH cows were mated to A bulls. Cows in the SHS and AHA groups ranged from 1/4 to 3/8 H and their calves from 5/8 to 11/16 H. Cows within the HSH and HAH groups ranged from 5/8 to 3/4 H and their calves from 5/16 to 3/8 H. Cow age ranged from 3 to 10 yr. Simmental-cross cows were heavier and taller and produced heavier calves at birth and weaning than A-cross. Pregnancy rate, calf birth date and percentage of difficult births did not vary significantly among dam breed groups. Within the A x H and S x H rotations, dam breed group rankings for calf birth weight were inverse to rankings for proportion of H in the breed makeup of the calf.(ABSTRACT TRUNCATED AT 250 WORDS)     

长顺绿壳蛋鸡SLC8A1基因多态性及其对蛋壳品质的影响

【目的】探究溶质载体家族8成员A1（solute carrier family 8 member A1，SLC8A1）基因多态性对长顺绿壳蛋鸡蛋壳品质的影响，为今后改善蛋壳品质提供参考。【方法】根据GenBank数据库中鸡SLC8A1基因序列（登录号：NC_052534.1），使用Primer Premier 3.0软件设计引物进行PCR扩增，采用直接测序法筛选SLC8A1基因SNP位点，并利用SPSS 22.0软件检测SNP位点不同基因型蛋壳指标的差异。【结果】在SLC8A1基因外显子11上共发现3个新的SNPs：g.16085681 T>C、g.16085766 C>T和g.16085781 T>C，共存在3种单倍型：H1（TCT）、H2（CTC）、H3（TTC），以及6种双倍型：H1H1（TTCCTT）、H1H2（TCTCTC）、H1H3（TTTCTC）、H2H2（CCTTCC）、H2H3（CTTTCC）、H3H3（TTTTCC）。χ²检验结果显示，3个SNPs位点均处于Hardy-Weinberg平衡状态（P>0.05），均表现为中度多态。关联性分析结果显示，g.16085681 T>C位点TC基因型个体蛋壳强度显著高于TT基因型（P<0.05），g.16085681 T>C位点和单倍型H2（C_16085681T_16085766C_16085781）对SLC8A1基因mRNA二级结构产生明显的影响；g.16085766 C>T位点CC基因型个体蛋形指数显著高于TT基因型（P<0.05）；双倍型H1H1、H1H3个体蛋形指数显著高于H2H3，H2H3个体蛋壳强度和蛋壳厚度均显著高于H1H3，H1H2、H2H2个体蛋重均显著高于H3H3（P<0.05）。【结论】g.16085681 T>C位点可作为影响蛋壳强度的标记位点，g.16085766 C>T位点可作为影响蛋形指数的标记位点，双倍型H2H3可能是蛋壳强度和蛋壳厚度的有利双倍型。     

Immune responses and protective efficacy of a recombinant swinepox virus co-expressing HA1 genes of H3N2 and H1N1 swine influenza virus in mice and pigs

The recombinant swine poxvirus rSPV/H3-2A-H1 co-expressing HA1 genes of H3N2 and H1N1 subtype SIV has been constructed and identified. Inoculations of rSPV/H3-2A-H1 yielded ELISA and neutralization antibodies against SIV H1N1 and H3N2, and elicited potent H1N1 and H3N2 SIV-specific INF-γ response from T-lymphocytes in mice and pigs in this study. Complete protection against SIV H1N1 or H3N2 challenge in pigs was observed.     

关岭牛TBC1D7基因多态性及其与生长性状的关联分析

为探究TBC1蛋白家族第7成员（TBC1 domain family member 7，TBC1D7）基因单核苷酸多态性（SNP）对关岭牛生长性状的影响，试验选取116头贵州关岭牛，测定其8项生长性状指标，提取血液基因组DNA，并利用DNA混池扩增TBC1D7基因全部外显子，通过直接测序法筛查SNP。利用不同独立样本DNA扩增SNP位点对应外显子并测序，以验证位点并进行基因分型。利用SPSS 19.0软件单因素方差分析TBC1D7基因不同基因型和关岭牛生长性状之间的相关性。结果显示，在TBC1D7基因第5、6和8外显子上共发现5个SNPs位点：g.12972 T>C、g.12984 A>G、g.20538 C>T、g.20577 T>C和g.24807 G>A，均存在3种基因型；χ²检验结果显示，5个SNPs位点均显著偏离Hardy-Weinberg平衡状态（P<0.05）；群体遗传参数分析发现，5个SNPs位点杂合度较高，多态信息含量均表现为中度多态（0.25<PIC<0.5）。关联性分析结果发现，关岭牛TBC1D7基因g.24807 G>A位点GA基因型个体体斜长、胸围和后臀围均显著高于AA基因型个体（P<0.05）。在本试验群体中，5个SNPs存在6种单倍型（H1~H6）和9种双倍型（H1H1、H2H2、H2H3、H2H6、H3H3、H3H4、H3H5、H3H6和H5H5），其中H3和H2为优势单倍型，H3H3、H2H2和H2H3为优势双倍型；双倍型关联分析结果显示，H3H6和H5H5个体体斜长和后臀围相比其他双倍型个体占有明显优势，可能是有利基因型。结果表明，TBC1D7基因g.24807 G>A位点为影响关岭牛生长发育的重要多态位点，本研究为筛选有助于关岭牛生长发育的分子标记提供了理论参考。     

禾本科8种牧草DNA条形码通用序列筛选

DNA条形码技术是利用DNA保守片段对物种进行快速准确鉴定的新兴技术。本研究根据GenBank中禾本科牧草matK和rbcL基因的核苷酸序列,设计4对通用引物,建立并优化了针对禾本科7个主要牧草属8种牧草11个样品[高丹草(Sorghum bicolor×S.sudanense)、玉米(Zea mays)、针茅(Stipa capillata)、"贝克"多年生黑麦草(Lolium perenne‘Plxie)、"凯帝莎"多年生黑麦草(L.perenne‘Caddieshack’)、甘肃羊茅(Festuca kansuensis)、"百琪"紫羊茅(F.rubra‘Bargena’)、"梦神"紫羊茅(F.rubra‘Maxima’)、"百胜"草地早熟禾(Poa pratensis‘Barvictor’)、"钻石"草地早熟禾(P.pratensis‘Diamond’)和芨芨草(Achnatherum splendens)]的目的片段的扩增条件。对扩增产物进行测序和分析,经分别比对,筛选出8种牧草的4个标记位点5’端和3’端保守序列。对各标记位点保守区内的核苷酸进行单核苷酸多态性(SNPs)的单倍型分析。结果表明,matK1、matK2、matK3分别有6个单倍型(H1~A、H1~B、H1~C、H1~D、H1~E和H1~F)、7个单倍型(H2~A、H2~B、H2~C、H2~D、H2~E、H2~F和H2~G)和3个单倍型(H3~A、H3~B和H3~C),rbcL基因有5个单倍型(H4~A、H4~B、H4~C、H4~D和H4~E)。根据matK(matK1、matK2、matK3)和rbcL基因筛选的4个标记位点为8种牧草建立了相对应的特异DNA识别码。本研究可为混合禾本科牧草饲料中的高粱属、玉蜀黍属、芨芨草属、针茅属、黑麦草属、羊茅属和早熟禾属的8种牧草准确识别提供分子水平上的科学依据。     

Cytochrome oxidase I gene sequence of Hypoderma sinense infecting yaks in the Qinghai-Tibet high plateau of China

The previous scanning electron microscopy study of the Hypoderma species suggested that Hypoderma sinense Pleske (H. sinense) was different from Hypoderma bovis (H. bovis) and Hypoderma lineatum (H. lineatum). In this study, the mitochondrial cytochrome oxidase I (COI) gene sequence of H. sinense was compared with those of the other two species. The H. sinense sequence showed only 88.3% homology to H. bovis and 88.5% to H. lineatum. The results of the sequencing analysis confirm that H. sinense is a different species from H. bovis and H. lineatum.     

猪流感与公共卫生

猪流感(Swine influenza,SI)是目前危害全世界养猪业的重要呼吸道传染病之一.导致猪发病的致病毒株主要有H1N1、H1N2、H3N1、H3N2、H2N3、H5N1和H9N2等亚型流感病毒,特别是从猪体分离H5N1和H9N2亚型流感病毒对禽流感的控制及人类公共卫生有重要意义.针对目前流行的甲型H1N1疫情,对猪流感病毒的分子生物学、临床症状、病理变化及公共卫生意义等方面进行了综述,以期对其有一个较为全面的了解.     

H1、H3亚型猪流感病毒双重实时荧光定量RT-PCR检测方法的建立及应用

为建立一种快速、准确地检测H1和H3亚型猪流感病毒（swine influence virus,SIV）的方法,根据H1和H3亚型SIV血凝素（hemagglutinin,HA）基因保守序列,分别设计2对特异性引物和2条TaqMan探针,建立双重实时荧光定量RT-PCR方法。结果显示,该方法敏感性高,可检测到最低拷贝数为10²拷贝/μL;重复性良好,重复孔Ct值的变异系数均在5%以下;特异性好,除H1和H3亚型SIV外,H4、H5、H7、H9亚型SIV、猪瘟病毒、猪繁殖与呼吸综合征病毒、口蹄疫病毒、伪狂犬病病毒检测均为阴性;在田间样品中成功检出1株H1和1株H3亚型SIV,检出率为1.16%。该方法准确、快速、灵敏、特异,可为H1、H3亚型SIV的快速鉴别检测及流行病学调查提供有效的技术手段。     

应用多重引物RT-PCR快速诊断禽流感亚型

将本实验室分离保存、已经过PCR扩增、核酸测序鉴定的三种不同亚型禽流感病毒(AIVH5N1、H6N2、H9N2)分别接种鸡胚,收集尿囊液,分离提取AIV总RNA。参考H1￣H15亚型禽流感病毒的核蛋白(NP)基因序列以及H5、H6、H9亚型AIV血凝素(HA)基因序列,设计四对引物:NP1,NP2;H5P1,H5P2;H6P1,H6P2;H9P1,H9P2,混合为PCR扩增多重引物MP1,下游引物NP2、H5P2、H6P2,、H9P2混合为逆转录反应多重引物MP2。AIV总RNA在相同反应条件下经MP2逆转录,用MP1进行PCR扩增,AIVH5亚型材料得到205bp,563bp两条扩增带,AIVH6亚型材料得到205bp,233bp两条扩增带,AIVH9亚型材料得到205bp,690bp两条扩增带,都与实验设计相符合,阴性材料没有扩增带。该方法成功实现四对引物进行RT-PCR反应同时检测出AIV,准确诊断H5、H6、H9三种禽流感亚型,检测时间短,特异性强,能及时检测出AIV亚型。     

Development and Application of Duplex Real-time RT-PCR Assay for Detection of H1 and H3 Subtype Swine Influenza Viruses

To establish a rapid, accurate method to diagnose and detect H1 and H3 subtype swine influenza viruses (SIV) at the same time, the specific primers and TaqMan probes were designed according to the conserved region of the HA gene of H1 and H3 subtype SIV. A duplex Real-time RT-PCR assay was developed for detection of H1 and H3 subtype SIV. The results showed that the Real-time RT-PCR could detect 10² copies/μL of H1 and H3 subtype SIV, the sensibility was well. Coefficient of variation of Ct value between repeating groups were all below 5%, the repeatability was favorable. The results were negative for the detection of H4, H5, H7, H9 subtypes SIV, classical swine fever virus, porcine reproductive and respiratory syndrome virus, foot and disease virus and pseudorabies virus, the specificity was fine. One sample was H1 subtype SIV, and one sample was H3 subtype SIV, by the established assay, the positive rate was 1.16%. The method was highly accurate, rapid, sensitive and specific, and could provide a method for rapid detection and epidemiological investigation of H1 and H3 subtype SIV.     

修剪频率和留茬高度对草地早熟禾与多年生黑麦草混播草坪质量的影响

研究了不同修剪频率（1次/周、1次/2周、1次/3周3个处理水平）和留茬高度3、5、7、9cm处理组合对草地早熟禾与多年生黑麦草混播草坪质量的影响。结果表明：随修剪频率降低、留茬高度的增加草坪草密度减小,叶片宽度增加,质地变劣,叶片颜色加深,生长速度先增大后减小,地上植物量和地下植物量均呈先增大后减小的趋势。草坪综合质量评价显示,H5F1处理草坪综合质量最优,H5F2处理次之,H3F2处理最差,12种处理草坪的综合质量从优到劣依次为：H5F1〉H5F2〉H7F2〉H7F3〉H7F1〉H5F3〉H9F1〉H9F2〉H9F3〉H3F3〉H3F1〉H3F2。     

Study on association of single nucleotide polymorphism of CAPN1 gene with muscle fibre and carcass traits in quality chicken populations

This study was aimed at investigating the effect of the calpain 1 (CAPN1) gene on carcass and meat quality traits in eight meat-type chicken populations, including five pure lines (developed from Chinese local breeds) and three cross-breeds. Primer pairs for the Coding Sequence (CDS) region in CAPN1 were designed from the chicken genomic sequence database. Polymorphisms were detected by polymerase chain reaction (PCR)-Single Strand Conformation Polymorphism (SSCP) and DNA sequencing. Three single nucleotide polymorphisms (SNP; C2546T, G3535A and C7198A) were detected among individuals in each population. The associations of their haplotypes (H1 = CGA, H2 = CGC, H3 = CAA, H4 = CAC, H5 = TGA and H7 = TAA) with chicken breast muscle fibre and carcass traits were analysed. Results showed that the haplotypes were associated with live weight (LW), carcass weight (CW), breast muscle weight (BMW) and leg muscle weight (LMW) (p < 0.05), and were also related to eviscerated percentage (%EP) and breast muscle fibre density (p < 0.01). H1H3 haplotype was dominant for LW, CW and BMW; H1H5 haplotype was dominant for EP; H3H4 haplotype was dominant for LMW and H1H1 haplotype was dominant for BFD. It was concluded that the CAPN1 gene may be a major gene affecting meat quality traits of chicken or it is linked with the major gene. H1H3, H1H5 and H3H4 were the most advantageous haplotypes for carcass traits whereas H1H1 was the positive haplotype for breast muscle fibre trait.     

Adjuvanticity and inflammatory response following administration of water-in-oil emulsions prepared with saturated hydrocarbons in chickens

Water-in-oil emulsions containing bovine serum albumin (BSA) as a model antigen were prepared using aliphatic saturated hydrocarbons with carbon number from 12 to 18, and were tested in chickens. Straight-chain hydrocarbons induced higher antibody titers against BSA after administration than branched-chain hydrocarbons. n-C16H34 and n-C18H38 maintained high antibody titers even at 32 weeks after administration, compared with n-C12H26, n-C14H30 and n-C15H32. n-C12H26 and n-C14H30 raised concentrations of sialic acid and creatine kinase in plasma, both of which are important markers of inflammatory responses, immediately after administration. n-C16H34 and n-C18H38 did not raise the values of these markers. These results indicated that n-C16H34 and n-C18H38 induced elevated and sustained immune responses without severe adverse reactions in chickens.     

Serologic surveillance of swine H1 and H3 and avian H5 and H9 influenza A virus infections in swine population in Korea

Influenza A is a respiratory disease common in the swine industry. Three subtypes, H1N1, H1N2 and H3N2 influenza A viruses, are currently co-circulating in swine populations in Korea. An outbreak of the highly pathogenic avian influenza H5N1 virus occurred in domestic bird farms in Korea during the winter season of 2003. Pigs can serve as hosts for avian influenza viruses, enabling passage of the virus to other mammals and recombination of mammalian and avian influenza viruses, which are more readily transmissible to humans. This study reports the current seroprevalence of swine H1 and H3 influenza in swine populations in Korea by hemagglutination inhibition (HI) assay. We also investigated whether avian H5 and H9 influenza transmission occurred in pigs from Korea using both the HI and neutralization (NT) tests. 51.2% (380/742) of serum samples tested were positive against the swine H1 virus and 43.7% (324/742) were positive against the swine H3 virus by HI assay. The incidence of seropositivity against both the swine H1 virus and the swine H3 virus was 25.3% (188/742). On the other hand, none of the samples tested showed seropositivity against either the avian H5 virus or the avian H9 virus by the HI and NT tests. Therefore, we report the high current seroprevalence and co-infectivity of swine H1 and H3 influenza viruses in swine populations and the lack of seroepidemiological evidence of avian H5 and H9 influenza transmission to Korean pigs.     

An enzyme-linked immunosorbent assay for detection of avian influenza virus subtypes H5 and H7 antibodies

Background

Avian influenza virus (AIV) subtypes H5 and H7 attracts particular attention because of the risk of their potential pathogenicity in poultry. The haemagglutination inhibition (HI) test is widely used as subtype specific test for serological diagnostics despite the laborious nature of this method. However, enzyme-linked immunosorbent assays (ELISAs) are being explored as an alternative test method.H5 and H7 specific monoclonal antibodies were experimentally raised and used in the development of inhibition ELISAs for detection of serological response specifically directed against AIV subtypes H5 and H7. The ELISAs were evaluated with polyclonal chicken anti-AIV antibodies against AIV subtypes: H1N2, H5N2, H5N7, H7N1, H7N7, H9N9, H10N4 and H16N3.

Results

Both the H5 and H7 ELISA proved to have a high sensitivity and specificity and the ELISAs detected H5 and H7 antibodies earlier during experimental infection than the HI test did. The reproducibility of the ELISA’s performed at different times was high with Pearson correlation coefficients of 0.96-0.98.

Conclusions

The ELISAs are a potential alternative to the HI test for screening of large amounts of avian sera, although only experimental sera were tested in this study.     

猪白细胞抗原与产仔性能的关系

采用微量淋巴细胞毒试验对９０头经产母猪（二花脸６５头，大约克猪２５头）进行了血清学分型，计算了单倍型在两品种中的分布频率，运用单因子和双因子最小二乘分析法分析了ＳＬＡ单倍型与产活仔数之间的相关性。结果发现：（１）不同单倍型对猪产活仔数影响有明显的差异，Ｈ６、Ｈ１０有明显的正效应，Ｈ２、Ｈ１２、Ｈ３１有负效应。（２）单倍型对产活仔数影响在品种间有差异，在太湖猪中Ｈ６、Ｈ７、Ｈ１０和Ｈ２３几种单倍型频     

Heat tolerance of Boran and Tuli crossbred steers.

Experiments were conducted to evaluate the heat tolerance of the following breeds: Hereford (H), Brahman (B), H x B, H x Boran (H x Bo), and H x Tuli (H x T). Heat tolerance was evaluated in a climatically controlled room (Exp. 1) and under summer environmental conditions (Exp. 2) by comparing rectal temperatures (RT), respiration rates (RR), and sweating rates (SW). In Exp. 1, under extremely hot conditions (mean temperature-humidity index [THI] > 90), purebred B had significantly (P < .05) lower RT and RR than other genotypes, which may be indicative of greater surface area per mass to dissipate heat and a lower metabolic rate than other genotypes. Boran and Tuli crosses had RT (39.5 degrees C) that were intermediate to those of B (39.0 degrees C) and H x B (40.0 degrees C). The H genotype had the greatest RT at 40.3 degrees C. Among the breeds, trends in RR were similar to RR observed at THI < 77; B had the lowest RR, and H x B were intermediate. However, in these extreme conditions, RR did not differ among the purebred H and the Boran and Tuli crossbred steers, but H x B steers had lower RR than the other H crossbred steers. Sweating rates were significantly greater for the Bos indicus x Bos taurus crosses (H x B and H x Bo) than for the purebred genotypes (H and B) and the Bos taurus cross (H x T). In Exp. 2, mean RT for B, H x B, H x Bo, and H x T were very similar to those recorded under the moderate heat stress conditions found in Exp. 1. There were no differences in RT among B, H x Bo, and H x T genotypes. The RR increased over time for H only, and RR for other genotypes tended to be elevated only slightly over time. Among genotypes, SW was significantly greater for the H x Bo steers. The ability of the Bos indicus crosses to dissipate heat through enhanced SW and associated evaporative cooling was evident. However, the heat-tolerant nature of the Bos taurus cross (H x T) was not evident through enhanced RR or SW in either experiment. Compared with other genotypes, the lower RR of B steers was clearly evident and is assumed to be due to greater surface area and other skin characteristics that allow them to dissipate heat to maintain lower RT. These data suggest that the H x Bo and H x T are similar to H x B and intermediate to H and B genotypes in maintaining homeostasis when exposed to a high heat load.     

商品代AA肉鸡接种禽流感疫苗的免疫效力观察

分别应用不同禽流感疫苗接种不同批次的商品代AA肉鸡,通过血凝抑制试验测定免疫后不同时期的抗体水平,确定各种疫苗对商品代AA肉鸡的免疫效果。结果表明,在免疫20天后,禽流感（H5＋H9）二价灭活疫苗（H5N1 Re-1＋H9N2 Re-2株）刺激肉鸡群产生的HI抗体均高于禽流感灭活疫苗（H5亚型,N28株）、禽流感（H5N2＋H9N2）二价灭活疫苗（N28株＋SD696株）和禽流感-新城疫重组二联活疫苗（rL-H5株）,与后三种疫苗接种商品代AA肉鸡的免疫抗体相比差异显著（P〈0.05）。