An enzyme-linked immunosorbent assay for detection of antibodies against Escherichia coli: association between indirect hemagglutination test and survival

An enzyme-linked immunosorbent assay (ELISA) was modified for detection of antibodies against the two main pathogenic serotypes of Escherichia coli: serotypes O78:K80 and O2:K1. The ELISA was a more sensitive and repeatable test than the indirect hemagglutination test (IHT), which is a common method for detecting antibodies against E. coli. Cross-reactivity between the two strains was measured by reacting antisera of each serotype against homologous and heterologous antigens. The results suggest that aside from similar determinants expressed by the two serotypes, serotype O2:K1 expresses more strain-specific determinants than does O78:K80. Comparison of mean antibody titers of immunized chicks by IHT and ELISA along the primary response revealed that during the first 15 days after immunization with inactivated E. coli, the titers in both tests were parallel. After 15 days post-immunization, antibody titers measured by IHT decreased rapidly, whereas titers measured by ELISA decreased only slightly. In addition, a higher correlation was found between titers detected by ELISA and survival through challenge with E. coli than between titers detected with IHT and survival through challenge. The results suggest that the ELISA is a better test for detection of antibody in flocks suspected of being infected with E. coli.     

鸡新城疫－传染性支气管炎－传染性法氏囊病三联灭活疫苗对不同日龄雏鸡的免疫效力研究

为评估鸡新城疫（ND）-传染性支气管炎（IB）-传染性法氏囊病（IBD）三联灭活疫苗对不同日龄和不同水平母源抗体雏鸡的免疫效力和持续期,本试验用该疫苗免疫7、14、21日龄SPF雏鸡和有母源抗体的普通雏鸡,免疫后采血测定ND血凝抑制抗体（HI Ab）、IB血凝抑制抗体（HI Ab）及IBD中和抗体（NA）,并用传染性法氏囊病病毒（IBDV）强毒攻击。结果显示,7日龄SPF雏鸡免疫后21 d ND HI抗体、IB HI抗体及IBD中和抗体效价分别为7.9log2、6.9log2和14.1log2,SPF鸡日龄越大,抗体水平越高;28日龄SPF鸡免疫后3个月,0.3 mL免疫剂量组试验鸡ND HI、IB HI及IBD中和抗体效价分别达6.5log2、6.1log2和13.6log2,IBDV攻毒保护率均为100%（10/10）;不同日龄普通雏鸡免疫效果与SPF鸡试验一致,抗体水平随鸡日龄增大而升高,IBD攻毒保护率也都达到100%（10/10）。试验结果证实,鸡新城疫-传染性支气管炎-传染性法氏囊病三联灭活疫苗可使7、14及21日龄SPF雏鸡和普通雏鸡产生良好的免疫力,对雏鸡的免疫期至少为3个月。     

Maternal antibody to infectious bronchitis virus: its role in protection against infection and development of active immunity to vaccine

Chicks hatched with high levels of maternal antibody had excellent protection (>95%) against infectious bronchitis virus (IBV) challenge at 1 day of age, but not at 7 days (<30%). This protection significantly (P<0.05) correlated with levels of local respiratory antibody and not with serum antibody.A high percentage of both maternal antibody-positive (Mab+) and maternal antibody-negative (Mab-) chicks failed to produce IBV antibody when vaccinated at 1 day of age by the intraocular route. In addition, Mab+ chickens had a weaker virus-neutralizing antibody response to a second IBV vaccination compared to Mab- birds (P<0.05). Mab+ chicks experienced a more rapid decline (P<0.01) in maternal antibody after 1-day-of-age vaccination compared to their unvaccinated counterparts.A monoclonal antibody-based blocking ELISA that measured antibody levels specific to S1 glycoprotein of IBV correlated well with virus-neutralizing antibody titers.     

新城疫La Sota疫苗对山东分离株的免疫保护试验

用标准疫苗株La Sota活疫苗在隔离器中接种SPF鸡,进行免疫保护试验.免疫后,每7 d采血监测NDV抗体,免疫后2周,利用经过鉴定的新城疫病毒(NDV)潍坊毒SGM01、昌乐毒SCL03、东营毒HY、日照毒SRZ03、莘县毒SSX03和标准强度F48E9分别进行攻毒试验,同时设SPF鸡对照.每天观察,及时剖检发病鸡,检查鸡群病变,确定疫苗的保护性.结果表明,La Sota疫苗能对除SGM01和HY以外的病毒攻击的SPF鸡提供较好的保护.     

Efficacy of mannanoligosaccharides and humate on immune response to Avian Influenza (H9) disease vaccination in broiler chickens

The effect of mannanoligosaccharides (MOS) and Humate (HU) in broiler diets on antibody titers against Avian Influenza virus (AIV) was evaluated. A completely randomized experimental design was used, and chicks were divided into 8 treatment groups, with 4 replicates per treatment and 10 chicks per replicate. Treatments were: 1) negative control group (CTL−), neither vaccinated against AIV nor given additives; 2) positive control (CTL+) or broilers were vaccinated against AIV + 0 additives; 3,4 and 5) CTL+ plus diet supplemented with 0.1, 0.2 and 0.3% HU, respectively; 6,7 and 8) CTL+ plus diet supplemented with 0.1, 0.2 and 0.3% MOS, respectively. For antibody analyses, blood samples were weekly collected by wing veins and the titers of antibody against AIV were measured by haemagglutination-inhibition test (HI). Compared to the positive control, the antibody titers against AIV were determined significantly lower in negative control group from 28 to 42 days of age. The inclusion of MOS resulted in increased antibody titers against AIV in the fourth, fifth and sixth weeks of age. MOS was effective in stimulating the humoral immune responses against AIV vaccine viruses. This study demonstrated an increase in the antibody titers in broilers fed diets containing 0.3% HU. In general, results of this study demonstrated that MOS proved to be much more effective on antibody production against AIV in broiler chicks than HU. Immune function could be modified with dietary HU and MOS supplementation.     

鸡新城疫、传染性法氏囊病二联活疫苗和单苗研究

用实验室研制的鸡新城疫(La Sota株),鸡传染性法氏囊病(NF8株)单苗和二联活疫苗分别免疫7日龄SPF鸡以及有母源抗体的雏鸡研究其免疫效力。结果表明:用二联活疫苗免疫SPF雏鸡后,NF8株与La Sota株间不产生明显的相互干扰作用,攻毒保护率与各自的单苗相比无明显差异。用二联活疫苗免疫有母源抗体的雏鸡后,只进行一次免疫,新城疫免疫效果较好,但法氏囊免疫效果稍差,不能完全保护;一免后10天再进行第二次加强免疫,其抗体水平、攻毒后的保护率与各自的单苗相比无明显差异,具有完全保护效力。     

Resistance of broiler chickens to Escherichia coli respiratory tract infection induced by passively transferred egg-yolk antibodies

Egg-yolk antibodies induced by immunizing hens with selected Escherichia coli antigens were evaluated for their ability to protect broiler chickens against respiratory/septicemic disease caused by avian pathogenic E. coli (APEC). Seven groups of broiler breeder hens were vaccinated three times, 1 week apart with live E. coli, killed E. coli, E. coli antigens [lipopolysaccharide (LPS), type 1 pilus adhesin (FimH), P pilus adhesin (PapG), aerobactin outer membrane receptor (IutA)] or phosphate buffered saline (PBS). An O78 APEC strain was used for preparation of all the antigens. Egg yolk immunoglobulins (IgY) were purified from eggs of each group and antibody activity in serum and purified IgY was determined by enzyme-linked immunosorbent assay (ELISA). IgY (100mg) was injected intramuscularly into 11-day-old broiler chickens, which were challenged 3 days later with homologous (O78) or heterologous (O1 or O2) E. coli by the intra-air sac route. Mortality was recorded and surviving chickens were euthanized 1 week after the challenge and examined for macroscopic lesions. Passive antibodies against all antigens except FimH were protective (90-100%) against the homologous challenge, but only anti-PapG and anti-IutA were effective against heterologous challenge. Anti-PapG IgY provided the greatest protection against the three serogroups of E. coli used for challenge. Hence vaccination of broiler breeders to induce anti-PapG and anti-IutA antibodies may provide passive protection of progeny chicks against respiratory/septicemic disease caused by APEC.     

Construction and evaluation of a delta cya delta crp Salmonella typhimurium strain expressing avian pathogenic Escherichia coli O78 LPS as a vaccine to prevent airsacculitis in chickens.

Avian pathogenic strains of Escherichia coli cause a number of extraintestinal diseases in poultry, including airsacculitis and colisepticemia. Expression of O78 lipopolysaccharide (LPS) is frequently associated with pathogenic isolates. Salmonella, a common poultry contaminant, is a major public health concern. The purpose of this work was to develop an E. coli vaccine for poultry with the use of an attenuated Salmonella typhimurium carrier that would benefit both the bird and the consumer. Orally administered attenuated S. typhimurium delta cya delta crp strains have been shown to provide excellent protection against wild-type Salmonella challenge in chickens. This work describes the construction of a delta cya delta crp derivative of an avian pathogenic S. typhimurium that expresses both the homologous group B determinants (O1,4,5,12) and the heterologous E. coli O78 LPS O antigens. This was accomplished by inserting the E. coli rfb region, which encodes the genes required for O78 expression, into the chromosomal cya gene of S. typhimurium, creating a defined deletion/insertion mutation. A delta crp mutation was introduced in a subsequent step. Expression of both O antigens was stable in vitro and in vivo. Vaccination of white leghorn chicks at day of hatch and 14 days with the recombinant vaccine strain induced serum immune responses against both S. typhimurium and E. coli LPS and protected the birds against subsequent challenge with an avian pathogenic E. coli O78 strain. Introduction of a mutation in rfc, which encodes the O antigen polymerase, reduced the chain length of the S. typhimurium LPS without affecting the expression of O78. The rfc mutation further enhanced the ability of the vaccine strain to protect chickens against E. coli challenge.     

Immunogenicity and efficacy of a bivalent vaccine against infectious bronchitis virus

Infectious bronchitis (IB) is a highly contagious viral disease and is responsible for considerable economic losses in the poultry industry, worldwide. To mitigate the IB-associated losses, multiple vaccines are being applied in the sector with variable successes and thus necessitating the development of a potent vaccine to protect against the IB in the poultry. In the present study, we investigated a bivalent live attenuated vaccine consisting of IB virus (IBV) strain H120 (GI-1 lineage) and D274 (GI-12 lineage) to evaluate its protection against heterologous variant of IBV (GI-23 lineage) in chicken. Protection efficacy was evaluated based on the serology, clinical signs, survival rates, tracheal and kidney histopathology and the viral shedding. Results demonstrated that administering live H120 and D274 (named here Classivar®) vaccine in one day-old and 14 days-old provided 100 % protection. We observed a significant increase in the mean antibody titers, reduced virus shedding, and ameliorated histopathology lesions compared to routinely used vaccination regimes. These results revealed that usage of different IBV vaccines combination can successfully ameliorate the clinical outcome and pathology in vaccinated chicks especially after booster vaccination regime using Classivar®. In conclusions, our data indicate that Classivar® vaccine is safe in chicks and may serve as an effective vaccine against the threat posed by commonly circulating IBV strains in the poultry industry.     

鸡新城疫病毒分离株与La Sota株灭活疫苗效力比较试验

用NDV分离株及La Sota株为抗源液，经福尔马林灭活后，与油佐剂混合，分别制成分离株灭活苗、La Sota株灭活苗及分离株与La Sota株二价灭活苗。将这三种灭活疫苗分别免疫SPF鸡后，均获得100%抵抗NDV分离株及F48株强毒攻击的保护力；而用这3种灭活苗与La Sota活苗单独或联合使用，免疫带有ND母源抗体的普通鸡后，3种灭活苗的免疫效力不同，分离株灭活苗与价灭活苗对NDV分离株攻击的免疫保护效力明显优于La Sota灭活苗；灭活苗与活苗同时使用，其免疫效力明显优于单独使用灭活苗或活苗。     

Effect of maternal antibody on timing of initial vaccination of young white leghorn chickens against infectious bursal disease virus

Maternal antibody titers in white leghorn chicks against infectious bursal disease virus (IBDV) were measured by a computer-assisted, single-serum-dilution, indirect kinetic-based enzyme-linked immunosorbent assay (KELISA) and by a virus-neutralization (VN) test in order to predict the timing of initial vaccination. Day-old white leghorns were from unvaccinated pullets or from pullets vaccinated either four times or twice with IBDV commercial vaccines. The chicks were immunized once via the drinking water with a commercial "intermediate" live IBDV vaccine at 1, 15, or 28 days of age. Effective initial immunization was confirmed by an increase in antibody to IBDV (serologic conversion) that occurred when maternal antibody decreased to 8 and 9 on a log2 scale. This concentration of antibody was detected between 24 and 28 days of age. The computer-assisted IBDV-KELISA increased the sample processing speed for detecting IBDV antibody, and it was as sensitive as the VN test for predicting the timing of initial IBDV vaccination.     

牛病毒性腹泻病毒弱毒活疫苗免疫持续期的研究

为检测牛病毒性腹泻病毒(BVDV)弱毒活疫苗在免疫牛体内抗体产生及其消长规律,评价弱毒疫苗的保护效力,并确定免疫持续期,本试验对免疫试验牛每头颈部肌肉接种BVDV SM株弱毒疫苗104.5TCID50,监测血清抗体效价,进行免疫持续期的确定。在疫苗免疫后的6个月、9个月和12个月,分别抽取5头免疫组和5头对照组牛,采用BVDV-JL强毒株进行攻毒试验,每头牛攻毒剂量为6×107.0TCID50/mL。结果显示疫苗免疫后12个月时血清中和抗体效价仍维持在1∶1048以上。攻毒结果显示,在3个不同时间点进行强毒攻击后,免疫组所有动物白细胞数量都没有下降也没有分离到病毒,而对照组动物白细胞数下降均超过30%,6个月和9个月时动物血清中均能分离到病毒,而12个月对照组动物由于年龄大,没有分离到病毒,因此暂定此疫苗的免疫持续期为9个月。     

禽大肠杆菌病免疫保护机理的研究

以禽病原性大肠杆菌O18、O78分离株制成超声波裂解铝佐剂灭活苗免疫14日龄鸡，以相同或不同外膜蛋白型（Outer membrane protein pattern,OMP型）的O18、O78分离株攻毒。结果表明：O78血清相同和不同OMP型分离株间能获得最大保护；O18血清型相同OMP型分离株间获得最大保护，而不同OMP型分离株间不能保护；上述两个血清型的分离株间不论OMP型是否相同，均缺乏保护。以间接ELISA试验、间接血凝试验分别测定了试验鸡临攻毒前针对大肠杆菌OMPs和脂多糖(Lipopolysaccharide,LPS）的抗体。结果表明：免疫组鸡血清上述两种抗体明显高于攻毒对照组；在免疫组，存活鸡临攻毒前血清中上述两种抗体滴度恒高于死亡鸡，但除3个组外，多数组差异不显著。攻毒对照组这一关系不稳定。结果说明：禽大肠杆菌疫苗的免疫保护，主要与O血清型有关，部分与OMP型有关，如O18分离株，免疫保护性抗原含OMPs,LPS等多个抗原表位。     

牛病毒性腹泻弱毒活疫苗免疫持续期的研究

为检测牛病毒性腹泻病毒(BVDV)弱毒活疫苗在免疫牛体内抗体产生及其消长规律,评价弱毒疫苗的保护效力,并确定免疫持续期,本试验对免疫试验牛每头颈部肌肉接种BVDV SM株弱毒疫苗10^4.5TCID₅₀/头,监测血清抗体效价,进行免疫持续期的确定。在疫苗免疫后的6、9和12个月分别抽取5头免疫组和5头对照组牛采用BVDV-JL强毒株进行攻毒试验,每头牛攻毒剂量为6×10^7.0 TCID₅₀/mL。结果显示疫苗免疫后12个月时血清中和抗体效价仍维持在1∶1048以上,攻毒结果显示3个时间点强毒攻击后,免疫组所有动物白细胞数量都没有下降也没有分离到病毒,而对照组动物白细胞数下降均超过30%,6和9个月动物均分离到病毒,而12个月对照组动物由于年龄大,没有分离到病毒,因此暂定此疫苗的免疫持续期为9个月。     

Antibody isotype response in adult cattle vaccinated with Brucella abortus S19

In a chronological study of sera collected from eight adult cattle vaccinated with 3 X 10(-10) cfu of Brucella abortus S19, antibody of each of the four major isotypes was measured by indirect enzyme immunoassay (ELISA) and by direct and modified complement fixation tests (CFT). Six of the cattle gave antibody responses to the vaccine strain that commenced between days 5 and 8 for all the isotypes in the ELISA, peaked by 1 to 4 months and then declined to low levels by 10 months. Direct CFT and modified CFT titers were measurable by 7 or 8 days post-vaccination, and peaked by 1 month; direct CFT titers disappeared by 5 months while the modified CFT titers lingered for 10 months. Two animals gave cyclical direct CFT and modified CFT antibody responses, a cyclical IgG1 response, a low IgG2 and an elevated IgA response. The amplitude of the cycles was uniform over three cycles while the wavelength increased with time. A year post-vaccination, B. abortus S19 was isolated twice from milk from one of the animals (no attempt was made to culture B. abortus from the other). Sera from B. abortus naturally infected cattle were analysed for comparison.     

Monitoring the Immune Status of Broilers Against Reoviruses Using Challenge and Serologic Data

Reoviruses are an important cause of suboptimum performance in commercial broilers worldwide. Integrators use the enzyme-linked immunosorbent assay against the S1133 antigen for monitoring serum of breeders for indicating pullet vaccine success. However, without correlating serology to reovirus challenge, it is difficult to determine whether titers reflect protective immunity. We developed a broiler challenge test against 2 common reovirus isolates (2408 and S1133) to evaluate the efficacy of reovirus pullet vaccine programs. Two reovirus serologic and challenge studies were undertaken using chicks from broiler integrators from the southeastern United States. Breeder flocks, from which the chicks were obtained, received at least 1 live and 2 inactivated reovirus vaccines during their pullet phase. One-day-old progeny were collected from 6 breeder flocks. At 1 d of age, 20 chicks from each broiler flock were bled, and serum was analyzed for antibodies. At 3 to 4 d of age, 20 progeny per flock were challenged with the 2408 reovirus by intratracheal route. At 10 to 14 d of age, another 20 birds per flock were challenged with the S1133 reovirus by footpad. Twenty birds per flock were used as nonchallenged controls. At 3 wk of age, all birds were killed and weighed. Percentage of protection was calculated for each flock based on the absence of gross lesions. Flocks with at least 50% protection were considered well protected. Most flocks were well protected against both viruses. The percentage of protection correlated with day-old enzyme-linked immunosorbent assay titers. Chicks from younger hens had higher titers and the best protection against challenge. Producers, whose hen flocks were monitored herein, were doing a good job of immunizing pullets against reovirus. They are now using reovirus progeny challenge studies along with breeder antibody titers to determine vaccination success of their pullets.     

Protection against enteric colibacillosis in pigs suckling orally vaccinated dams: evidence for pili as protective antigens

Pregnant gilts were vaccinated orally with Escherichia coli that produced pilus antigens K99 or 987P. The vaccines were live or dead enterotoxigenic E coli (ETEC) or a liver rough non-ETEC strain which has little ability to colonize pig intestine. Pigs born to the gilts were challenge exposed orally with K99+ or 987P+ ETEC, which did not produce heat-labile enterotoxin or flagella and which produced somatic and capsular antigens different from those of the vaccine strains. Control gilts had low titers of serum and colostral antibodies against pilus antigens, and their suckling pigs frequently had fatal diarrhea after challenge exposure. Serum antibody titers against pilus antigens of the vaccine strains increased in the gilts after vaccination with liver ETEC, and the colostral antibody titers of these gilts were higher than those of controls. Pigs suckling such vaccinated gilts were more resistant than controls to challenge strains were of different pilus types, and it could not be attributed to enterotoxin neutralization by colostrum. In contrast to the live ETEC vaccines given to the pregnant gilts, the liver rough non-ETEC and dead ETEC vaccines stimulated little or no production of antibody against pilu, and the pigs born of these vaccinated gilts remained highly susceptible to challenge exposure. The results support the hypothesis that pilu can be protective antigens in oral ETEC vaccines. It was indicated that in the system reported, protection depended on living bacteria for the production of pilus antigens in vivo or for the transport of pilus antigens across intestinal epithelium.     

Safety,immunogenicity, and efficacy of two Escherichia coli cya crp mutants as vaccines for broilers

Attenuated derivatives (delta cya delta crp mutants) of an O2 and an O78 avian septicemic Escherichia coli strain were used to immunize broiler chickens by spray to determine the safety, immunogenicity, and efficacy of the derivatives in single- and double-dose regimens. In the safety and immunogenicity studies, groups of 10 chickens were vaccinated by spray (droplet size approximately 20 microm) with the parent E. coli, the mutant organisms, or phosphate-buffered saline (PBS) at 14 days of age and euthanatised 21 days later. There was no deaths or gross pathologic finding in any of the chickens immunized with the vaccine strains. Compared with the levels in chickens exposed to PBS, there were significantly higher levels of immunoglobulin (Ig) G antibody in serum and air sac washings and of IgA antibody in air sac washings in response to the virulent parent strains than to the vaccine strains. In efficacy studies, chickens were immunized with the O2 or the O78 vaccine strain or PBS at day 14 and with the O2 vaccine strain or PBS at days 10 and 14 and challenged with the parent strain 10 days after the last vaccination. There was no significant difference in local IgA and IgG and serum IgG responses between vaccinated and control groups. Chickens vaccinated with the O2 strain, but not the O78 strain, had significantly lower air sac lesion scores compared with those of the unvaccinated groups in both single- and double-dose regimens. We conclude that the mutant O2 strain provided moderate protection against airsacculitis.     

Increased humoral antibody response of foot-and-mouth disease virus vaccine in growing pigs pre-treated with poly-γ-glutamic acid

This study was conducted to determine if humoral antibody response of foot-and-mouth disease (FMD) vaccine improved in 8-week-old growing pigs born to well-vaccinated sows pre-treated with 60 mg of poly-γ-glutamic acid (γ-PGA) three days before vaccination. Antibody against FMD virus serotype O was measured 0, 2, 4 and 6 weeks post-vaccination, using a PrioCHECK FMDV type O ELISA kit. The results showed that positive antibody reactions against FMDV serotype O antigen among a component of the vaccine significantly increased in response to pre-injection with γ-PGA.     

新城疫分离株疫苗免疫保护试验

用经过鉴定的新城疫病毒分离株按国家兽医生物制品质量标准制备油乳剂灭活疫苗，与标准的La Sota疫苗分别免疫带有新城疫母源抗体的白来航鸡，免疫后每7d采血监测新城疫抗体，并于免疫后21，28，35d用鸡新城疫分离毒和F48E9分别进行人工感染试验。结果显示，分离株灭活苗对鸡新城疫分离株的免疫保护效力优于La Sota灭活苗。