禽大肠埃希氏菌融合菌株疫苗的研制及应用

对从陕西省主要养鸡地区病死鸡分离的禽致病性大肠埃希氏菌进行Omp分型，用不同Omp型的菌株构建成融合菌株。用融合菌株制作铝胶灭活疫苗，与单价疫苗和两亲本菌株双价疫苗的免疫保护效果进行比较。结果表明，融合菌株疫苗可对多种O血清型致病性大肠埃希氏菌的攻毒试验产生坚强的保护，保护率高达93％～100％，而O血清型单价疫苗的保护率仅67％～80％，双价疫苗的保护率仅80％。在生产中对蛋用鸡的应用结果表明，融合菌株疫苗可明显降低鸡的死淘率。     

安宁河流域鸭瘟、鸭霍乱综合防制研究

采用Ｃａｒｔｅｒ荚膜群鉴定法，并参考Ｈｅｄｄｌｅｓｔｏｎ热稳定抗原鉴定法，对从安宁河流域分离的３７株禽源多杀性巴氏杆菌和中监所提供的Ｃ４８－１株菌进行了血清学鉴定。３７株菌中，鸭源３２株，鹅源４株，鸡源１株。鉴定结果表明，有３７株为荚膜Ａ群菌，占９７．４％，其中Ａ∶５型３４株，占８９．５％，Ａ∶２型３株，占７．９％；１株为荚膜Ｂ群菌，占２．６％。少部分菌株具有明显的交叉反应     

禽霍乱基因缺陷菌株双型联合免疫的研究

将禽源多杀性巴氏杆菌Ｃ４８－１株（血清型为５∶Ａ）用化学诱变剂亚硝基胍（ＮＴＧ）处理，从３０００多个克隆中筛选出９株链霉素依赖性基因缺陷型菌株（Ｃｓｔｄ株），经２０代连续回复突变测定，除１株外，其余８株表型稳定。将８株Ｃｓｔｄ株用小鼠做毒力比较试验，其中５株加注链霉素表现毒力，不加注链霉素则不表现毒力；用小鼠做免疫力测定，其中Ｃｓｔｄ－１和Ｃｓｔｄ－７能保护１０个最小致死量（ＭＬＤ）同型强毒菌的攻击。将Ｃｓｔｄ－１株与另一禽源多杀性巴氏杆菌Ｐ１０５９株（血清型为８∶Ａ）的链霉素依赖型菌株Ｐｓｔｄ－６混合，用小鼠做双型联合免疫试验，结果小鼠能抵抗Ｃ４８－１和Ｐ１０５９两株强毒菌混合培养物１０个ＭＬＤ的攻击，保护率达１００％。     

辽宁省禽大肠杆菌血清型分布与大肠杆菌病防治措施研究

以病原菌分离、血清型鉴定及本动物致病性试验对流行于辽宁省致病性禽大肠埃希氏菌的血清型分布、致病性以及用蜂胶苗防治该病进行了研究。2001至2005年从14市的病死禽和死胚中分离出大肠杆菌226株,定型105株,其中078型占29．52％（31／105）;0109型占10．48％（11／105）。结果证明078、0109血清型是流行于本省致病性禽大肠埃希氏菌的绝对优势血清型。致病性试验研究证明,0109型菌对鸡的致病性强于01、02、078居第一位。用2001年分离的优势菌株的11个血清型大肠杆菌蜂胶苗和大肠杆菌新城疫二联蜂胶苗对鸡免疫,从2005年分离的优势菌株中选078、0109、01、0142、093型菌分别以致死量进行攻毒,保护率均达100％。免疫鸡与发病鸡同居感染试验,100％健康存活。     

兔源巴氏杆菌弱毒833菌苗对鸡的安全和免疫试验

鸡皮下注射兔源巴氏杆菌弱毒833菌苗150亿活菌,对鸡是安全的,注射10万个活菌可以保护80％以上,安全量与免疫量相差15万倍以上,远远超过其他禽霍乱弱毒菌苗。因此,它是一株颇有希望的禽霍乱菌苗。     

B型副鸡禽杆菌田间分离株的毒力及免疫原性研究

为探索分离自安徽省某大型白羽鸡场的1株B型副鸡禽杆菌的毒力及免疫原性,本试验测定了该菌对5～6胚龄SPF鸡胚及35～40日龄SPF鸡的毒力,并用该菌株制备免疫原,免疫35～40日龄SPF鸡,用攻毒的方法测定菌株的免疫原性,同时对本菌株制备的抗原及目前国内、国外市场上销售的鸡传染性鼻炎A型、B型、C型三价或二价灭活疫苗的免疫效力进行比较。毒力试验结果显示,本菌株对SPF鸡胚的最小致死剂量为110 CFU,对SPF鸡的最小发病剂量为5.5×10~3 CFU。免疫原性试验结果显示,本菌株制备的抗原免疫鸡能有效抵抗本菌株的攻击,说明本菌株的免疫原性良好,而市场上销售的鸡传染性鼻炎三价/二价灭活疫苗免疫鸡均无法抵抗本菌株的攻击,说明市场上销售的疫苗对本菌株的保护效果较差,B型菌疫苗间的交叉保护效果不理想。本试验为B型鸡传染性鼻炎疫苗的研究提供参考和物质基础。     

饲用复合微生物添加剂的研究

采用以多个乳酸菌为主，加入芽胞菌及枯草杆菌，研制而成的复合微生物添加剂。其制剂性能稳定，含菌量为１０×１０＾８个菌／ｇ，菌粉用塑料袋包装，常温下保８个月活菌数未下降，对动物安全，经对小鼠试验结果ＬＤ５０为１０＾１１个菌／ｋｇ，以含６×１０＾８个菌／ｋｇ，０．６×１０＾８个菌／ｋｇ饲料， 饲养试验取得较满意的效果。生长育肥猪全期试验结果：增竽率提高２０．９％，饲料利用率提高２０．６７％。肉用仔鸡试验     

猪丹毒QL251弱毒株安全性及免疫原性试验

猪丹毒ＱＬ２５１弱毒株是我所保存的猪丹毒强毒菌种Ｃ４３００９的变异株，对其进行了毒力和免疫原性的测定，该菌株毒力原来是３－５个活菌可致死小白鼠，现在对小白鼠皮下注射７．４×１０~８活菌，小白鼠全部健活，对敏感猪皮下注射９．４×１０~（１０）活菌均健活且无任何反应；小白鼠免疫９－１２个活菌７２％保护，对猪免疫２９０－５９０×１０~４活菌１００％保护。     

禽多杀巴氏杆菌交叉保护因子的研究

禽多杀巴氏杆菌交叉保护因子存在于鸡活体培养物中，用鸡活体培养禽多杀巴氏杆菌Ｃ４８－１强毒株（Ｉ型），菌血症时收获细菌制成死菌苗，免疫鸡对异型菌株Ｐ１０５９（３型）攻击产生交叉保护，而４１℃肉汤培养时Ｃ４８－１菌不具备这种交叉保护，活体培养的细菌冻融后对异型菌株保护率达１００％，经溶菌酶等处理溶解后的溶解物上清及沉淀对异型菌株产生６６、７％和８０％的交叉保护，但其沉淀经蛋白酶处理后则失去了交叉保护能     

6株嗜水气单胞菌的毒力因子及其对小鼠的致死性

根据嗜水气单胞菌毒素、蛋白酶及Ｓ蛋白３种毒力因子的检测结果,从６６株嗜水气单胞菌中选出３种毒力因子分布情况各异的有代表性的６个菌株。分别用含菌量均为１０８ＣＦＵ／ｍＬ的６株嗜水气单胞菌肉汤培养物注射小鼠,结果,毒素、蛋白酶及Ｓ蛋白均有的菌株以及具有前两者而无Ｓ蛋白的菌株对小鼠的致死率均达１００％;仅有蛋白酶的菌株对小鼠致死率达６０％;而只有Ｓ蛋白的,或既有毒素又有Ｓ蛋白的,或３种毒力因子均无的菌株对小鼠无致死性。     

天津地区猪链球菌3型菌株的致病性及耐药特性研究

试验旨在对分离自天津地区发病猪场的7株猪链球菌3型菌株（Streptococcus suis type 3,SS 3）进行致病性和耐药特性研究。应用剂量为1.0×10⁷、1.0×10⁸和1.0×10⁹ CFU/只的细菌对小鼠进行致病力研究,用PCR方法检测7株SS 3型菌株的毒力基因mrp、ef、sly、gdh、gapdh、fbps和orf2,并对这7株SS 3型菌株进行药物敏感试验。致病力试验结果显示,接种剂量为1.0×10⁹和1.0×10⁸ CFU/只时分别有6和3株SS 3型菌株可使小鼠100.0%（5/5）发病死亡,接种剂量1.0×10⁷ CFU时仍有1株SS 3型菌株可使80.0%（4/5）小鼠发病死亡,这7株SS 3型菌株对小鼠的致病力依次为R15056 > R15042=S15030 > Y12024=Y09011 > Y13125 > Y13164。毒力基因检测结果表明,共有3种毒力基因型,R12024、Y13164、R15042和S15030株为gdh、gapdh、fbps和orf2毒力基因阳性,Y13125和R15056株为sly、gdh、gapdh、fbps和orf2毒力基因阳性,Y09011株为gdh、fbps和orf2毒力基因阳性。药物敏感性试验结果显示,7株SS 3型对头孢喹肟相对最敏感,其次为阿莫西林、环丙沙星和磺胺间甲氧嘧啶钠,但对强力霉素100.0%耐药,且所有菌株均呈现3重以上的耐药性。本研究为进一步开展天津地区SS 3型流行特点及致病机理研究奠定了基础,可为天津地区SS 3型菌株的综合防控提供理论指导。     

Duration of strain 2308 infection and immunogenicity of Brucella abortus lipopolysaccharide in five strains of mice

A study was conducted to compare immunogenicity of a Brucella abortus lipopolysaccharide (LPS) and the duration of infection in 5 strains of mice. Mice of strains CBA/NJ, BALB/c, CD-1, C3H/HeN, and C3H/HeJ were allotted into 2 large groups (vaccinated with proteinase K-treated LPS or nonvaccinated) and 6 subgroups based on the intervals between challenge exposure to B abortus strain 2308 and the week the response data were obtained. Criteria used in comparing responses between the various strains of mice as well as between vaccinated and nonvaccinated mice were splenomegaly, colony-forming units (CFU) from spleens, and antibody titers. Responses were evaluated at 1, 2, 3, 5, 8, and 12 weeks after challenge exposure. Results indicated that all strains of mice became infected and maintained infection throughout the 12-week period, the percentages of mice infected were significantly (P less than 0.05) less in vaccinated mice for the first 5 weeks after challenge exposure, and there were no direct correlations between increased immunoglobulins (IgM and IgG titers) and reduction in CFU. Vaccinated mice of strains BALB/c, CD-1, C3H/HeN, and C3H/HeJ had increased titers when challenge exposed and also had significantly (P less than 0.05) smaller spleens and lower CFU. Vaccinated CBA/NJ mice did not have marked antibody titers. The overall results indicated that vaccination with LPS offers some initial protection against B abortus strain 2308 infection, but this protection disappears gradually and in various degrees in the 5 strains of mice studied.     

Safety and efficacy of two live Pasteurella multocida aro-A mutant vaccines in chickens

Two auxotrophic aro-A mutants of Pasteurella multocida designated PMP1 (serotype 1) and PMP3 (serotype 3) were tested as vaccine candidates to protect chickens against fowl cholera. A reliable intratracheal challenge method was established that resulted in > or = 75% mortality in both specific-pathogen-free chickens and commercial broiler breeders 24 hr after challenge. Dose protection studies indicated that at least 10(6) colony-forming units (CFU) of PMP1 and 10(8) CFU of PMP3 were required to provide complete protection against challenge in all birds. Although high doses of 10(9) CFU of the vaccine strains produced some endotoxinlike reactions, lower but protective dose levels produced no clinical sign or lesion in any chicken. Both vaccine strains provided cross-protection with a heterologous challenge strain PM206 (serotype 4). Future studies will examine the duration of protective immunity induced by the two vaccine candidates, PMP1 and PMP3, and cross-protection against other serovars.     

猪饲料添加剂复合菌种的筛选和特性研究

根据微生态理论,文章选用嗜酸乳杆菌、纳豆芽孢杆菌、酿酒酵母作为制备复合微生物饲料添加剂的试验菌株。经一系列实验,结果表明:所选3种试验菌均能耐受0.3%的猪胆盐,对pH值为3.0的酸性环境也有较强的抵抗力,活菌数均在106CFU/ml以上;3种试验菌对人工胃肠液均有理想的耐受能力,能顺利通过胃、十二指肠到达小肠发挥作用,并能抑制猪体常见致病菌,是制备复合微生物饲料添加剂的优良菌种。     

The relationship between the numbers of Salmonella Enteritidis, Salmonella Heidelberg, or Salmonella Hadar colonizing reproductive tissues of experimentally infected laying hens and deposition inside eggs

Contamination of eggs by Salmonella Enteritidis has been a prominent cause of human illness for several decades and is the focus of a recently implemented national regulatory plan for egg-producing flocks in the United States. Salmonella Heidelberg has also been identified as an egg-transmitted pathogen. The deposition of Salmonella strains inside eggs is a consequence of reproductive tract colonization in infected laying hens, but prior research has not determined the relationship between the numbers of Salmonella that colonize reproductive organs and the associated frequency of egg contamination. In the present study, groups of laying hens in two trials were experimentally infected with large oral doses of strains of Salmonella Enteritidis (phage type 13a), Salmonella Heidelberg, or Salmonella Hadar. Reproductive tissues of selected hens were cultured to detect and enumerate Salmonella at 5 days postinoculation, and the interior contents of eggs laid between 6 and 25 days postinoculation were tested for contamination. Significantly more internally contaminated eggs were laid by hens infected with Salmonella Enteritidis (3.58%) than with strains of either Salmonella Heidelberg (0.47%) or Salmonella Hadar (0%). However, no significant differences were observed between Salmonella strains in either isolation frequency or the number of colony-forming units (CFU) isolated from ovaries or oviducts. Salmonella isolation frequencies ranged from 20.8% to 41.7% for ovaries and from 8.3% to 33.3% for oviducts. Mean Salmonella colonization levels ranged from 0.10 to 0.51 log CFU/g for ovaries and from 0.25 to 0.46 log CFU/g for oviducts. Although parallel rank-orders were observed for Salmonella enumeration (in both ovaries and oviducts) and egg contamination frequency, a statistically significant relationship could not be established between these two parameters of infection.     

猪链球菌3型疫苗候选菌株筛选及3＋9型二价灭活疫苗对小鼠免疫效果评估

【目的】 筛选猪链球菌血清3型疫苗候选菌株,制备猪链球菌3＋9型二价灭活疫苗并评估其在BALB/c小鼠上的免疫保护效果。【方法】 从发病猪病料中分离猪链球菌血清3型菌株,通过蜡螟幼虫和BALB/c小鼠模型筛选出强毒株作为疫苗候选菌株,并对候选菌株进行生物学特性研究。将筛选得到的3型疫苗候选菌株和前期筛选的9型疫苗候选菌株灭活浓缩,使其抗原浓度为2×10¹⁰ CFU/mL,无菌检测后将浓缩抗原液按1：1配比混合,再混合抗原与Summit Poly Solution佐剂按4：1比例混合,制备猪链球菌3＋9型二价灭活疫苗,疫苗中猪链球菌血清3和9型含菌量均为2×10⁹ CFU/mL。将制备的疫苗免疫6周龄BALB/c小鼠,首免后第14天进行二免,二免后第14天进行攻毒。同时设立商品化疫苗免疫组和阴性对照组,评估疫苗的安全性和免疫保护效果。【结果】 PCR鉴定结果显示,23株临床分离株均为血清3型猪链球菌,依次命名为KQ3-1~KQ3-23。分别通过蜡螟和小鼠进行初筛和复筛,筛选到强毒株KQ3-1。毒力基因检测显示,该菌株基因型为gapdh^＋/sly^＋/fbps^-/orf2^-/mrp^-/89K^-/gdh^＋/epf^-;生长曲线显示,该菌株在37 ℃培养8~10 h时生长达到对数生长后期;BALB/c小鼠致病性结果显示,腹腔接种12 h内可引起小鼠精神萎靡、扎堆、毛发耸立、运动迟缓、死亡等临床症状,该菌株的LD₅₀为5.2×10⁷ CFU/只。制备的疫苗免疫小鼠后,小鼠精神状况、采食等均正常,疫苗注射部位无肿胀、硬块等不良反应,无死亡发生,表明该疫苗具有良好的安全性。免疫后进行猪链球菌血清2型菌株ZYS、猪链球菌血清3型菌株KQ3-1和猪链球菌血清9型菌株YT攻毒,对照组死亡率分别为90%、100%和100%,猪链球菌3＋9型二价灭活疫苗免疫组保护率分别为30%、80%和70%,商品化疫苗组的保护效果分别为70%、0和10%。【结论】 本研究研制的疫苗能对猪链球菌血清3和9型强毒株提供良好的免疫保护,该疫苗具备疫苗市场开发的潜在价值。     

Experimental infection of specific pathogen free (SPF) cats with two different strains of bartonella henselae type I: a comparative study

Domestic cats are the reservoir of Bartonella henselae, the main causative agent of cat scratch disease. We compared B. henselae type I infection characteristics in 6 SPF cats infected with a feline strain (4.8 x 10(7) colony-forming units (CFU)/mL) and in 6 SPF cats infected with the reference Houston I strain (6.6 x 10(6) CFU/mL to 9.6 x 10(7) /mL). All the cats inoculated with the feline strain, but none of the cats inoculated with B. henselae Houston I, developed a fever within 2-12 days (mean: 5.8 days) post inoculation (PI), which lasted for 1-2 weeks. However, all 12 cats became bacteremic. The duration of bacteremia was significantly longer in the cats inoculated with the feline strain (mean: 237 days) than in the cats inoculated with Houston I strain (mean: 60 days) (p < 0.01). Five (83%) cats inoculated with the feline strain and none of the six cats inoculated with B. henselae Houston I had relapsing bacteremia (p = 0.02). IgG antibodies were detected by IFA within 1-2 weeks for both strains, but peaked later (week 10 versus week 3 PI) for the feline strain. By ELISA, using antigens of each B. henselae strain, all 12 cats developed Bartonella specific IgM and IgG antibodies, but the cats infected with B. henselae Houston I antigen yielded significantly lower optical density values (p < 0.05). By SDS-PAGE, PFGE and Western blotting, protein profile differences (84 to 89% homology) were observed between the two strains. If a feline vaccine is to be developed in order to prevent human infection, the choice of the vaccine strain will be critical, since major differences were identified even between strains belonging to the same sero/genotype.     

Development of a PCR Assay Based on a Single–Base Pair Substitution for the Detection of Aeromonas caviae by Targeting the gyrB Gene

Aeromonas caviae is a bacterial pathogen that causes various infectious diseases in both humans and animals. To facilitate its detection, we developed species-specific primer sets targeting polymorphisms in the gyrB gene for use in a PCR assay. The technique was able to detect 100% (29/29) of the A. caviae strains tested using either of two sets of primers (designated ACF1-ACR and ACF3-ACR), which produced 293-bp and 206-bp amplicons, respectively. Another set of primers (designated ACF2-ACR) yielded a 237-bp amplicon and exhibited 90% (26/29) positive results with respect to A. caviae. None of the primer sets exhibited cross-reactivity with 12 non–A. caviae isolates and 52 other non-Aeromonas bacteria. The detection limit using the ACF2-ACR and ACF3-ACR primer sets in pure culture was 1.6?×?10³ CFU/mL, or 6 CFU per reaction, whereas that of the ACF1-ACR primer set was 1.6?×?10⁴ CFU/mL, or 60 CFU per reaction. In the case of spiked Nile Tilapia Oreochromis niloticus, the sensitivity of all primer sets without enrichment was 1.8?×?10⁴ CFU/g, or 30 CFU per reaction. Primer set ACF3-ACR was the best for a PCR assay targeting the gyrB gene, and the PCR technique developed was rapid, specific, and sensitive for the identification of A. caviae.

Received December 2, 2014; accepted April 9, 2015     

Campylobacter jejuni contamination of eggs

Contamination of commercial table eggs with a fecal suspension containing 4.4 X 10(6) CFU/g Campylobacter jejuni resulted in shell penetration in 3/70 eggs and recovery of the organism from homogenized egg contents in 1/70 eggs. Viability of C. jejuni on the shell surface was retained for only 16 hours, attributed to desiccation of the fecal suspension. A field survey of three commercial laying farms and their associated egg-packing plants showed that hens demonstrated to be fecal shedders of C. jejuni (12% to 62% incidence) did not produce infected eggs. The organism could not be detected in the environment of the packing plant, including grading machinery and effluent.     

Hormonal profiles of late gestation ewes following intra-uterine inoculation with and without lux-modified Escherichia coli

The objectives of these investigations were to develop an ovine model for Escherichia coli (E. coli)-induced preterm delivery, and monitor ewe hormonal response. EXP 1: Ewes (105 +/- 13 days of gestation) were allotted to the following intra-uterine inoculations: Saline-(CON; n=5); 1 x 10(6) CFU/ml (Low Treatment, LT; n=6); or 1 x 10(7) CFU/ml (High Treatment, HT; n=6) E. coli. Twenty-four h after inoculation, the HT ewes had increased (P<0.05) cortisol compared to LT and CON ewes, and HT and LT ewes had increased (P<0.05) progesterone compared to CON ewes. Preterm delivery was 33% for LT ewes and 0% for HT and CON ewes. EXP 2: Ewes (124 +/- 18 days of gestation) were allotted to the following intra-uterine inoculations using lux-modified E. coli: Trial-1: Luria Broth (LB; CT1; n=5); 4.0 x 10(6) CFU (n=5), 20.0 x 10(6) CFU (n=5); and Trial-2: LB (CT2; n=5), 1.2 x 10(6) CFU (n=5), and 5.6 x 10(6) CFU (n=5) E. coli-lux. Preterm delivery occurred between 48 and 120 h post-inoculation in 60, 25, 60 and 75% of ewes infected with 1.2, 4.0, 5.6, and 20 x 10(6) CFU, respectively. Serum cortisol and progesterone did not differ (P>0.05) between CT1 or CT2 and inoculated ewes. In summary, 25 to 75% of ewes inoculated preterm delivered. However, variable results in cortisol and progesterone profiles between Control and inoculated ewes were observed between the two studies.