Evaluation of two monoclonal antibodies for serotyping Haemophilus paragallinarum

Two monoclonal antibodies (MAbs) were evaluated for their ability to serotype 108 isolates of Haemophilus paragallinarum. One MAb (E5C12D10) was raised against a Page serovar A strain and the other (F2E6) against a Page serovar C strain. In both dot blot and hemagglutination-inhibition tests, MAb E5C12D10 recognized the type strains of Page serovar A and Kume serovars A-1, A-2, A-3, and A-4. MAb F2E6 recognized the type strains of Page serovar C and Kume serovars C-1, C-2, and C-3. Neither antibody recognized the type strains of Page serovar B or Kume serovars B-1 and C-4. When evaluated with 97 field isolates in a dot blot test, the MAbs serotyped 81 isolates, which was better than agglutinin typing by the Page scheme (69 isolates serotyped). The field isolates that did not react with the MAbs were either Page serovar B/Kume serovar B-1 (three isolates), Page serovar C/Kume serovar C-4 (12 isolates), or nontypable by either the Page or Kume scheme (one isolate).     

Protection conferred by bivalent and trivalent infectious coryza bacterins against prevalent serovars of Avibacterium (Haemophilus) paragallinarum in Mexico

The protection and level of hemagglutination-inhibition (HI) antibodies conferred in infectious coryza bivalent- and trivalent-immunized chickens against Avibacterium (Haemophilus) paragallinarum field isolates of the prevalent serovars in Mexico (A-1, A-2, B-1, and C-2) were investigated. The bivalent bacterin (A-1 and C-1) conferred significant protection and increased HI antibodies against isolates of serovars A-1, A-2, and C-2, but not against a serovar B-1 isolate. The trivalent bacterin (A-1, B-1, and C-2) conferred protection and increased HI antibodies against all four of the isolates. The results confirmed that in poultry areas where serovar B-1 is prevalent, the inclusion of this serovar in bacterins is needed to confer protection against infectious coryza caused by A. (H.) paragallinarum isolates of serovar B-1.     

The presence of nicotinamide adenine dinucleotide-independent Haemophilus paragallinarum in México

Two isolates of Haemophilus paragallinarum were obtained from a layer chicken in Mexico. The isolates were confirmed as H. paragallinarum by polymerase chain reaction and conventional biochemical identification. The isolates were nicotinamide adenine dinucleotide (NAD) independent-growing on blood agar without the need of a nurse colony as well as on a complex medium that lacked both NAD and chicken serum. Both isolates were pathogenic, causing the typical clinical signs of infectious coryza in susceptible chickens. One isolate was Page serovar B/Kume serovar B-1 and the other isolate was Page serovar C/Kume serovar C-2. The isolates were associated with a field outbreak that involved an egg drop of 20% over a 3-wk period and a doubling of weekly mortality (from 0.1% to 0.2%). This is the first report of NAD-independent H. paragallinarum outside South Africa and is the first time that NAD-independent H. paragallinarum of serovar B has been reported.     

An evaluation of the cross-protection afforded by inactivated infectious coryza vaccines

The cross-protection afforded by three inactivated infectious coryza vaccines was evaluated. Each vaccine contained one of the following strains of Haemophilus paragallinarum, HP31, HP60 and HP14. Strain HP31 belongs to Kume serovar C-2, strain HP60 belongs to Kume serovar C-4 while strain HP14 belongs to Kume serovar A-4. Four groups of twenty six-week-old specific-pathogen-free chickens were either given a single dose of an aluminium-hydroxide based vaccine (three groups) or left as unvaccinated controls. Three weeks after vaccination, all four groups were challenged with virulent H paragallinarum. One half of each group was challenged with the strain HP31 and the other half with strain HP60. The efficacy of the vaccines was assessed in terms of the prevention of the typical clinical signs and macroscopic lesions of infectious coryza as well as the prevention of any colonisation by the challenge organism. The serovar C-2 and serovar C-4 vaccines protected more than 50% of birds against either a serovar C-2 or C-4 challenge while the serovar A-4 vaccine protected less than 50% of birds.     

ERIC-PCR genotyping of emergent serovar C-1 isolates of Avibacterium paragallinarum from Mexico

Between 2008 and 2010, 14 isolates of Avibacterium paragallinarum were identified as serovar C-1 in Mexico. All isolates were obtained from commercial laying hens suffering infectious coryza despite a history of vaccination. The enterobacterial repetitive intergenic consensus-based PCR genotyping showed that all isolates had a common pattern. Until recently, serovars A-1, A-2, B-1, and C-2 were the serovars prevalent in Mexico. Serovar C-1 has been identified in Japan and recently in the Americas in Ecuador. Our current study suggests that Av. paragallinarum serovar C-1 is an emerging serovar in Mexico. Our results also indicate that the Mexican isolates of Av. paragallinarum serovar C-1 may have a clonal relationship. Knowledge of the genetic diversity of Av. paragallinarum may be of value in understanding vaccine performance and identifying the best combination to achieve broader protection.     

Typing of Haemophilus paragallinarum strains by using enterobacterial repetitive intergenic consensus-based polymerase chain reaction

The enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) technique was used for fingerprinting of reference strains and Mexican isolates of Haemophilus paragallinarum. A total of nine ERIC patterns were given by the nine serovar reference strains of this bacteria. Two Modesto (C-2) reference strains from different sources showed the same ERIC pattern. Seventeen ERIC patterns were obtained among 29 Mexican isolates included in the study, belonging to serovars prevalent in Mexico (A-1, A-2, B-1, and C-2). Obtained results indicate that the ERIC-PCR technique could be used as a molecular laboratory tool for subtyping of H. paragallinarum.     

Hemagglutinin serotyping of <Emphasis Type="Italic">Avibacterium paragallinarum</Emphasis> isolates from Ecuador

Avibacterium paragallinarum is the causative agent of infectious coryza, an acute respiratory disease of chickens. In this study, a total of 28 isolates of A. paragallinarum from Ecuador were serotyped by the hemagglutinin scheme which recognizes nine serovars. Out of 28 isolates, 17 isolates belonged to serovar A-3, and five isolates to each serovars B-1 and C-1, whereas one isolate was non-typeable. This is the first report of A. paragallinarum serovar A-3 outside Brazil and serovar C-1 outside Japan.     

马铃薯杂种优良株系遗传差异的AFLP分析

为明确从‘1867’ב陇薯7号’、‘MB09’ב陇薯7号’和‘MB09’ב陇薯6号’3个马铃薯杂交组合中选育出的17个杂种优良株系的遗传差异程度,用4个亲本材料作对照,利用AFLP分子标记技术对其进行了遗传差异分析。用筛选出的10对AFLP适宜引物进行PCR扩增共得到321个位点,多态性位点277个,多态性位点百分率87.29%。试验建立了各杂种优良株系的AFLP指纹图。21份马铃薯材料间的多态信息含量(PIC)平均为0.5617,Nei’s遗传多样性指数为0.3623,Shannon指数为0.5407。各材料间的平均遗传距离(GD)为0.5281,以GD值0.51为基准,21份材料分为4类:‘1867’、A-10、A-12、A-21和A-29为一类;‘陇薯6号’、‘陇薯7号’、A-14、A-23、A-26、B-13和B-14为一类;‘MB09’、B-1、B-2、B-7、B-15、B-20和C-22为一类;C-2和C-21为一类。该研究可为马铃薯杂交亲本的选择利用和杂种优良新品系的选育提供依据。     

Crossbreeding cattle in beef production programmes in Kenya

Data collected on a privately owned ranch located in the Machakos District of Kenya at approximately 2 degrees latitude south of the equator at an elevation varying from 1,675 to 2,000 m were analysed on five breed groups of cows: (1) purebred Boran, (2) 1/2 Charolais-1/2 Boran (1/2 C-1/2 B), (3) 3/4 Boran-1/4 Charolais (3/4 B-1/4 C), (4) 1/2 Ayrshire-1/2 Boran (1/2 A-1/2 B) and (5) 1/2 Santa Gertrudis-1/2 Boran (1/2 SG-1/2 B). The maternal traits evaluated included age at first calving, calving interval, calf weight at weaning and cow productivity index (calf weight weaned annually per cow calving). Mean cow productivity index for all cows was 192 kg; for purebred Boran, 174 kg; for 1/2 C-1/2 B, 200 kg; for 3/4 B-1/4 C, 191 kg; for 1/2 A-1/2 B, 210 kg; and for 1/2 SG-1/2 B, 185 kg. Cow breed groups 1/2 C-1/2 B, 3/4 B-1/4 C, 1/2 A-1/2 B and 1/2 SG-1/2 B exceeded (P less than 0.01) purebred Boran by 14.9, 9.8, 20.7 and 6.3%, respectively, in cow productivity index.     

Generation of monoclonal antibodies to porcine interleukin 6 (PIL-6) using the recombinant PIL-6 expressed in Escherichia coli

Porcine interleukin-6 (PIL-6) protein without signal peptide was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli. The fusion protein was expressed in an insoluble fraction, however, it was solubilized by refolding procedure using urea. From the solubilized protein, the recombinant PIL-6 (rPIL-6) was purified by a batch method using glutathione sepharose 4B and PreScission protease cleavage. By the B3B1 hybridoma cell proliferation assay, biological activity of the purified rPIL-6 was confirmed. Three monoclonal antibodies (MAbs) named 2B-1, 5A-8 and 4C-3 were generated by using the rPIL-6 as an immunogen. Immunoglobulin isotypes of the MAbs were IgG2a (4C-3) and IgG2b (2B-1 and 5A-8). For the epitope analysis, additive enzyme-linked immunosorbent assay and immunoblot analysis using deletion mutants of PIL-6 were performed. These experiments revealed that the two MAbs (2B-1 and 5A-8) recognize an overlapped epitope and the other (4C-3) recognizes a distinct epitope, and all epitopes reside in the region of aa26-64 of PIL-6.     

Evaluation of a panel of monoclonal antibodies in the subtyping of Haemophilus paragallinarum.

A panel of four monoclonal antibodies (MAbs) was evaluated, using a hemagglutination-inhibition test, for its ability to subtype 76 isolates of Haemophilus paragallinarum. The results of the MAb reactions were compared with the results of both the Page and Kume serotyping schemes (the serovars of the Page scheme correspond to the serogroups of the Kume scheme). One MAb (E5C12D10) was raised against a Page serovar A strain and the remaining MAbs (F2E6, D6D8D5, and B3E6F9) against a Page serovar C strain. Six different reaction patterns were found among the 76 isolates of H. paragallinarum. There was total correlation between the MAb reaction pattern and the Page scheme, and thus the Kume scheme, to the serogroup level. All 19 Page serovar A (= Kume serogroup A) strains reacted only with MAb E5C12D10, whereas all five Page serovar B (= Kume serogroup B) strains failed to react with any of the MAbs. All 52 remaining strains were Page serovar C (= Kume serogroup C), and all failed to react with MAb E5C12D10 but showed varying reaction patterns with the three other MAbs. Although the MAbs recognized four subdivisions within Kume serogroup C, these subdivisions differed from the four Kume C serovars. This panel of MAbs can be used to assign isolates of H. paragallinarum to either Page serovars or Kume serogroups. Although the subdivisions recognized by the MAbs within the Page serovar C strains do not correspond to the Kume serovars, they may be useful in epidemiological applications.     

Pathogenicity of various adenovirus serotypes in the presence of Escherichia coli in chickens

Ten strains of adenovirus representing 10 serotypes were administered intratracheally to 3-week-old specific-pathogen-free chickens, which also received 2.9 X 10(5) colony-forming units of a pathogenic Escherichia coli intranasally. One group was given only E. coli, and one was retained as an uninoculated control. Gross pathologic alterations post-mortem were minimal and limited to multiple scattered, pale areas in the lungs of an occasional chicken in various groups. Histopathologic changes in the lungs were those of multifocal, interstitial, and occasionally diffuse pneumonia. Moderate to marked interstitial pneumonia was incited by adenovirus strains 75-1A, B-3 A-2, C-2B, and X-11; Ind-C, Stein, Tipton, J-2, and T-8 caused similar but milder lesions. Strains 75-1A, A-2, C-2B, T-8, and X-11 incited moderate to marked multifocal pneumonia; Ind-C, Stein, Tipton, J-2, and B-3 caused mild multifocal pneumonia. In all groups, the pneumonic lesions were more severe 5 days postinoculation than 12 days postinoculation. Bronchiolitis and tracheitis lesions also varied in severity with serotype. A mild hepatitis was seen with serotypes T-8 and 75-1A. Neither the uninoculated control group nor the group inoculated with only E. coli exhibited gross or histopathologic alterations.     

Effects of differences in virulence of different serovars of Haemophilus paragallinarum on perceived vaccine efficacy

The virulence of four South African field isolates of NAD-dependent Haemophilus paragallinarum and two field isolates of NAD-independent H. paragallinarum has previously been tested in unvaccinated chickens. In this study, the disease profiles caused by the NAD-dependent isolates of H. paragallinarum in vaccinated chickens were studied. It was shown that the clinical signs induced in the vaccinated chickens were substantially less severe than were those in unvaccinated chickens, as was expected. However, due to the high virulence of the serovar C-3 isolates, clinical signs in the vaccinated chickens challenged with this isolate were still detected. These were as severe as those occurring in unvaccinated chickens challenged with serovar B-1 isolates. Although the clinical signs induced in unvaccinated birds challenged with serovar A-1 were more severe than those occurring when vaccinated birds were challenged with serovar C-3, the overall disease profiles were similar. Substantial clinical signs were recorded in vaccinated birds challenged with serovar C-3. This could be interpreted as vaccination failure if the disease profile obtained in unvaccinated birds is not considered. It was found that a high level of protection was provided by this vaccine against challenge by serovar C-3. The high virulence of this serovar resulted in the development of clinical signs in vaccinated birds. These findings could possibly explain the large number of so-called vaccination failures that are reported in South Africa.     

Isolation of serovar C-3 Haemophilus paragallinarum from Zimbabwe: A further indication of the need for the production of vaccines against infectious coryza containing local isolates of H. paragallinarum

Various isolates of Haemophilus paragallinarum, collected from a severe outbreak of infectious coryza in poultry from Zimbabwe, were serotyped and were found to belong to serovar C-3. Previously, isolates were serotyped using polyclonal antiserum produced against serogroup reference strains (0083 for serogroup A, 0222 for serogroup B and Modesto, or H-18 for serogroup C) of H. paragallinarum. In this case, polyclonal antiserum produced against these reference isolates were used, as well as polyclonal antiserum that has been raised specifically against the serovar C-3 isolate 46 C-3. When using the latter serum at a 1 in 50 dilution, no cross-reaction with other members of serogroup C were found. The severity of the disease outbreak in Zimbabwe, the vaccination history of the infected flocks on the sites and the isolation of the uniquely southern African serovar C-3, further highlights the need for vaccines composed of local isolates to control infectious coryza in regions where vaccination failures occur.     

配种前药物处理对母猪繁殖性能的影响

为了减少人工授精过程中人为操作引入的病原微生物,改善母猪断奶后不发情、延迟发情或发情后配种不受孕所导致的母猪繁殖性能降低的现象,在饲养管理良好、免疫程序健全的条件下,试验采用在母猪配种前对其进行药物处理。将试验母猪随机分为2组,A组为断奶经产母猪(n=65),B组为第2情期后的后备母猪(n=58)。将A组进一步分为A-Ⅰ组(n=36,投药组)和A-Ⅱ组(n=29,空白对照组),B组进一步分为B-Ⅰ组(n=34,投药组)和B-Ⅱ组(n=24,空白对照组)。统计发情率、受胎率、分娩率及产仔数等繁殖指标。结果表明:A-Ⅰ组每头母猪断奶10 d内发情率为100%,与A-Ⅱ组发情率(96.55%)相比差异不显著(P>0.05);A-Ⅰ组情期受胎率为91.67%,分娩率为86.11%,产仔数为11.32头,而A-Ⅱ组情期受胎率为85.71%,分娩率为78.57%,产仔数为9.95头,差异均显著(P<0.05);B组内,除健仔数外其余各指标差异均不显著(P>0.05)。说明在配种前给经产母猪投药,可以改善母猪繁殖力。     

塔克拉玛干沙漠生物结皮中几种藻类的系统发育分析

新疆塔克拉玛干沙漠中富含多种沙漠藻类,严酷的沙漠环境造就了这些沙漠微藻独特的生物学特性,但对于这些沙漠微藻的研究,相关报道较少。本研究以新疆塔克拉玛干沙漠沙样为素材,通过实验室培养,以有限稀释法和平板法进行分离,分离得到6株沙漠微藻。形态学观察后初步鉴定5株属于绿藻门(TLD2A1,TLD2B,TLD7A-2,TLD6B,TLD7B-5),1株为蓝藻门(TLD5A1)。分别比对分析了5株绿藻的18S rDNA和其中3株的5.8S rDNA-ITS序列,1株蓝藻TLD5A1的16S rDNA序列,并进行了系统发育分析。结果表明,TLD2A1和TLD6B与小球藻科物种亲缘关系很近,TLD2B和衣藻科物种亲缘关系很近。TLD7B-5形态呈新月形,分子鉴定与环藻科物种亲缘关系较近,TLD5A1与念珠藻科的物种聚为一支,同时与念珠藻属的椭孢念珠藻同源性高达97%。本研究确定了6株沙漠微藻的分类地位,为后续塔克拉玛干沙漠微藻的研究工作提供理论依据。     

紫花苜蓿粗灰分与矿质元素含量QTL定位分析

为初步鉴定与紫花苜蓿(Medicago sativa)粗灰分、钾、钙、镁、磷含量调控相关的数量性状基因座(Quantitative trait loci，QTL)和分子标记，本研究用低产早熟苜蓿和高产晚熟苜蓿杂交，构建了由392个个体组成的F₁群体，对这些性状进行3年表型数据测定，且基于前期已构建的高密度遗传连锁图谱开展QTL定位。结果表明：共检测到63个与粗灰分和4种矿质元素含量相关的QTL，分布于22个染色体上，单个QTL的贡献率为2.50%~29.85%；其中重复定位的QTL共8个(qCa-3C-1和qCa-3C-2，qCa-6B-1和qCa-6B-2，qCa-6D-1和qCa-6D-2，qAsh-8B-1和qAsh-8B-2)，共定位的QTL有6个(qP-2C和qK-2C，qP-3A和qMg-3A，qP-6D-3和qMg-6D-2)；经进一步验证，与这些QTL紧密连锁的标记可用于分子标记辅助选择育种。本研究为选育矿质营养更丰富的苜蓿新品种奠定了基础。     

饲喂燕麦-箭筈豌豆捆裹青贮对绵羊瘤胃pH与氮代谢参数的影响

选用6只装有永久瘤胃瘘管的羯羊作为试验动物,采用3×3拉丁方设计,研究了A(100%燕麦—箭筈豌豆捆裹青贮料,简称青贮料),B(80%青贮料+20%玉米)和C(63%青贮料+37%玉米)3种饲粮处理对绵羊瘤胃内环境的影响。结果表明,瘤胃液pH值为A>B>C,但A的瘤胃液总氮含量平均值显著低于处理C(P<0.05);除食前1 h处理间NH3-N浓度差异接近显著(P=0.085)外,其他时段A,B和C差异显著或不显著(P<0.05或P>0.05);瘤胃液中尿素氮仅B和C食后8 h的测值和平均值显著(P<0.01或P<0.05)高于A;C的瘤胃液蛋白氮含量平均值显著高于A(P<0.05),但3种处理差异不显著。因此,3种饲粮试羊的瘤胃液pH值和微生物蛋白合成状况间没有显著差异。     

Pandemic H1N1 influenza virus in Chilean commercial turkeys with genetic and serologic comparisons to U.S. H1N1 avian influenza vaccine isolates

Beginning in April 2009, a novel H1N1 influenza virus caused acute respiratory disease in humans, first in Mexico and then around the world. The resulting pandemic influenza A H1N1 2009 (pH1N1) virus was isolated in swine in Canada in June 2009 and later in breeder turkeys in Chile, Canada, and the United States. The pH1N1 virus consists of gene segments of avian, human, and swine influenza origin and has the potential for infection in poultry following exposure to infected humans or swine. We examined the clinical events following the initial outbreak of pH1N1 in turkeys and determined the relatedness of the hemagglutinin (HA) gene segments from the pH1N1 to two H1N1 avian influenza (AI) isolates used in commercial turkey inactivated vaccines. Overall, infection of turkey breeder hens with pH1N1 resulted in -50% reduction of egg production over 3-4 weeks. Genetic analysis indicated one H1N1 AI vaccine isolate (Alturkey/North Carolina/17026/1988) contained approximately 92% nucleotide sequence similarity to the pH1N1 virus (A/Mexico/4109/2009); whereas, a more recent AI vaccine isolate (A/ swine/North Carolina/00573/2005) contained 75.9% similarity. Comparison of amino acids found at antigenic sites of the HA protein indicated conserved epitopes at the Sa site; however, major differences were found at the Ca2 site between pH1N1 and A/ turkey/North Carolina/127026/1988. Hemagglutinin-inhibition (HI) tests were conducted with sera produced in vaccinated turkeys in North Carolina to determine if protection would be conferred using U.S. AI vaccine isolates. HI results indicate positive reactivity (HI titer > or = 5 log2) against the vaccine viruses over the course of study. However, limited cross-reactivity to the 2009 pH1N1 virus was observed, with positive titers in a limited number of birds (6 out of 20) beginning only after a third vaccination. Taken together, these results demonstrate that turkeys treated with these vaccines would likely not be protected against pH1N1 and current vaccines used in breeder turkeys in the United States against circulating H1N1 viruses should be updated to ensure adequate protection against field exposure.     

Virulence of the nine serovar reference strains of Haemophilus paragallinarum

The virulence of the reference strains of the nine currently recognized Kume serovars of Haemophilus paragallinarum was investigated. The capacity of the H. paragallinarum strains to cause the typical clinical signs of upper respiratory tract disease associated with infectious coryza in unvaccinated, nasal-challenged chickens was assessed. Differences in virulence were assessed by means of a standardized scoring system for clinical signs. All nine strains were pathogenic to chickens, producing typical clinical signs of infectious coryza. The highest clinical signs score was obtained for serovar C-1 (1.72), while the lowest clinical signs score was obtained for serovar C-4 (0.32). Our results indicate that virulence differences exist among the serovars of H. paragallinarum.