Systemic and mucosal antibody responses to selected cell surface antigens of avian pathogenic Escherichia coli in experimentally infected chickens

The immune response to four cell surface antigens of avian pathogenic Escherichia coli (APEC) was investigated as the first step in identifying vaccine candidates. F1 pilus adhesin, P pilus adhesin, aerobactin receptor protein, and lipopolysaccharide (LPS) from an O78 E. coli (strain EC99) were used as antigens. The proteins were purified as 6xhistidine-tagged recombinant proteins and LPS was purified from a phenol/water extract. Groups of 12 broiler chickens were vaccinated intranasally with the EC99 strain and challenged with the same strain 10 days later via the intra-air sac route. The chickens that survived were euthanatized 10 days postchallenge. Scores were assigned to infected chickens on the basis of lesions and recovery of the challenge E. coli. The immunoglobulin (Ig) IgG, IgA, and IgM antibodies to the four antigens were measured in serum and air sac washings in an enzyme-linked immunosorbent assay. Among the chickens that were not vaccinated prior to challenge, two died and three of the survivors were ill, whereas, of the chickens that were vaccinated prior to challenge, one died and one of the survivors became ill. After the intranasal vaccination, high antibody activity against all four antigens was associated with each Ig isotype in serum and air sac washings. IgG was the predominant isotype of Ig in air sac washings as detected by radial immunodiffusion. Chickens that were not ill after challenge had greater IgG, IgA, and IgM antibody activity against all four antigens in serum and air sac washings than did sick chickens. Thus, all of the antigens tested appear to be suitable candidates for a vaccine to protect chickens from respiratory tract infections caused by APEC.     

Construction, characterization, and evaluation of the vaccine potential of three genetically defined mutants of avian pathogenic Escherichia coli

The delta galE, delta purA, and delta aroA derivatives of avian septicemic Escherichia coli EC99 strain (O78 serogroup) were constructed with a suicide vector containing the pir-dependent R6K replicon and the sacB gene of Bacillus subtilis. The resultant isogenic mutants were stable and lacked approximately 45%, 36%, and 52% of the genes for galE, purA, and aroA, respectively. The delta purA and delta aroA mutants did not grow on minimal medium, whereas the delta galE mutant grew on minimal medium but was sensitive to galactose-induced lysis. The reversion frequencies of all three mutants were <10(-12). The mutants were highly attenuated for virulence as determined by administration of approximately 10(7) colony-forming units of each mutant to 1-day-old chicks by the subcutaneous route. Chickens were vaccinated with the mutants by spray (droplet size approximately 20 microm) at 1 and 14 days of age to determine safety, immunogenicity, and efficacy. The mutants were found to be safe. Seven days after a second vaccination, immunoglobulin (Ig)Y antibodies to E. coli in serum and air sac washings were detected by enzyme-linked immunosorbent assay. In both serum and air sac washings, IgY antibodies were significantly higher in chickens vaccinated with the mutants as compared with phosphate-buffered saline-treated controls but were significantly lower compared with chickens that were vaccinated with the parent strain. In serum, but not in air sac washings, IgY antibodies were significantly lower in chickens vaccinated with the mutants compared with the parent strain. The vaccinated chickens were given infectious bronchitis virus intranasally at 17 days of age and were challenged with homologous (EC99 strain) or heterologous (O2 serogroup) E. coli 4 days later. Chickens that received wild-type EC99 strain or its mutant derivatives were protected from homologous but not from heterologous challenge. This study indicates that the delta galE, delta purA, and delta aroA mutants are safe and moderately immunogenic but the protection conferred by the mutants is serogroup specific.     

Immunogenicity of an Escherichia coli multivalent pilus vaccine in chickens

Immunogenicity of an oil-emulsified Escherichia coli multivalent pilus vaccine was evaluated in 4-week-old chickens. The vaccine contained 180 micrograms of pilus protein from each of serotypes O1 and O78 and 170 micrograms of pilus protein from serotype O2. Chickens were vaccinated twice subcutaneously at 4 and 6 weeks old and challenged via the posterior thoracic air sac with E. coli serotype O1, O2, or O78 2 weeks after the last vaccination. Unvaccinated challenged chickens suffered 8% to 26% mortality; no vaccinated chickens died. Vaccinated chickens had very mild gross lesions in the air sacs, livers, and pericardial sacs and eliminated E. coli more efficiently than the unvaccinated challenged chickens. The results showed that a multivalent pilus vaccine protects chickens against active respiratory infection.     

Recombinant Iss as a potential vaccine for avian colibacillosis

Avian pathogenic Escherichia coli (APEC) cause colibacillosis, a disease which is responsible for significant losses in poultry. Control of colibacillosis is problematic due to the restricted availability of relevant antimicrobial agents and to the frequent failure of vaccines to protect against the diverse range of APEC serogroups causing disease in birds. Previously, we reported that the increased serum survival gene (iss) is strongly associated with APEC strains, but not with fecal commensal E. coli in birds, making iss and the outer membrane protein it encodes (Iss) candidate targets for colibacillosis control procedures. Preliminary studies in birds showed that their immunization with Iss fusion proteins protected against challenge with two of the more-commonly occurring APEC serogroups (O2 and O78). Here, the potential of an Iss-based vaccine was further examined by assessing its effectiveness against an additional and widely occurring APEC serogroup (O1) and its ability to evoke both a serum and mucosal antibody response in immunized birds. In addition, tissues of selected birds were subjected to histopathologic examination in an effort to better characterize the protective response afforded by immunization with this vaccine. Iss fusion proteins were administered intramuscularly to four groups of 2-wk-old broiler chickens. At 2 wk postimmunization, chickens were challenged with APEC strains of the O1, O2, or O78 serogroups. One week after challenge, chickens were euthanatized, necropsied, any lesions consistent with colibacillosis were scored, and tissues from these birds were taken aseptically. Sera were collected pre-immunization, postimmunization, and post-challenge, and antibody titers to Iss were determined by enzyme-linked immunosorbent assay (ELISA). Also, air sac washings were collected to determine the mucosal antibody response to Iss by ELISA. During the observation period following challenge, 3/12 nonimmunized chickens, 1/12 chickens immunized with 10 microg of GST-Iss, and 1/12 chickens immunized with 50 microg of GST-Iss died when challenged with the O78 strain. No other deaths occurred. Immunized chickens produced a serum and mucosal antibody response to Iss and had significantly lower lesion scores than nonimmunized chickens following challenge, regardless of the challenge strain. This study expands on our previous report of the value of Iss as an immunoprotective antigen and demonstrates that immunization with Iss can provide significant protection of chickens against challenge with three different E. coli strains.     

鸡大肠杆菌Ⅰ型菌毛亚单位苗交叉保护的初步研究

含Ⅰ型菌毛的３ 个不同血清型（Ｏ１ 、Ｏ７８ 及Ｏ８８） 的菌株， 大容量培养后提取菌毛制备３ 种单价菌毛油乳苗。用１ 日龄雏鸡分别免疫３ 种单价菌毛油乳剂苗， 每雏免疫量为１２５ μｇ ， 隔离饲养至２ 周龄经气囊攻毒， 并评价疫苗的免疫原性。结果未免疫鸡出现８７－５ ％ ～１００ ％ 的死亡率， 免疫鸡用同源菌株攻毒后死亡率仅１２－５ ％ ， 用异源菌株攻毒出现３７－５％ ～６２－５ ％ 的死亡率。免疫鸡攻毒后未死亡者， 经扑杀观察， 可见在气囊、心包及肝脏的病变非常轻微， 且攻毒后比非免疫鸡能更有效地清除攻入气囊的大肠杆菌     

Evaluation of the non-temperature-sensitive field clonal isolates of the Mycoplasma synoviae vaccine strain MS-H

The live attenuated temperature-sensitive (ts+) Mycoplasma synovia (MS) strain, MS-H, is used as a vaccine in a number of countries to control virulent MS infection in commercial chicken flocks. Nine out of 50 isolates made from flocks vaccinated with MS-H were found to have lost the ts+ phenotype of the original vaccine strain. In order to examine the influence of the ts- phenotype on virulence of the isolates, four of the ts- isolates, the MS-H vaccine, and the vaccine parent strain 86079/7NS were administered by aerosol in conjunction with infectious bronchitis virus to 3-wk-old specific-pathogen-free chickens. The four ts- clones induced only minimal air sac lesions that were not different in severity from those caused by MS-H vaccine; however, the vaccine parent strain 86079/7NS caused air sac lesions that were significantly greater than those of MS-H and all ts- clones. The vaccine parent strain 86079/7NS and two of the ts- clones were recovered from the air sacs of the respectively infected chickens whereas the MS-H vaccine and two other ts- clones were not. Three of the ts- isolates caused increased tracheal mucosal thicknesses that were significantly greater than those from birds inoculated with MS-H, and one caused increased tracheal mucosal thicknesses that were significantly less than those from birds inoculated with 86079/7NS. In conclusion, unlike the MS-H vaccine, the MS-H ts- clones were associated with minor changes in tracheal mucosa; however, unlike the vaccine parent strain, they did not induce lesions in the air sacs. These results suggest that factors other than ts+ phenotype are involved in the attenuation of the MS-H vaccine.     

Effect of temperature-sensitive Mycoplasma gallisepticum vaccine preparations and routes of inoculation on resistance of white leghorns to challenge

One-week-old chickens were vaccinated with live or formalin-killed temperature-sensitive (TS) Mycoplasma gallisepticum (MG) either intranasally (IN) or subcutaneously (SQ). Live TS MG protected chickens against S6 strain challenge directly into the air sacs, regardless of route of vaccination. Killed MG, however, protected chickens only when administered SQ. Antibody to MG was detected in sera and in the tracheal and air-sac washings of only the chickens given live vaccine IN. The antibody present in tracheal and air-sac washings may be one of the mechanisms that play a role in resistance to MG challenge.     

Studies on the live-virus vaccine against infectious laryngotracheitis of chickens. II. Evaluation of the tissue-culture-modified strain C7 in laboratory and field trials

Laboratory and field tests were conducted to evaluate the immunogenicity of a live-virus vaccine against infectious laryngotracheitis (ILT) of chickens that was prepared using tissue-culture-modified strain C7. Eighty-three percent or more of chickens vaccinated by the ocular (OC) or intranasal (IN) route with 10(4.75) TCID50 of the attenuated strain C7 at 50 days of age were protected against challenge with a virulent strain of ILT virus without showing any clinical signs for 4 weeks post-vaccination (PV). When vaccine was administered by aerosol, however, only 65% of vaccinated chickens were protected against challenge. Fifty-seven percent of chickens vaccinated at 70 days of age maintained immunity for 6 months PV. Immune response in younger chickens was inferior to that in older ones. In the field trials, clinical observation revealed no adverse reactions caused by the vaccination, and 60% or more of broilers and 80% or more of layers vaccinated by the OC route were protected against challenge at 4 weeks PV. These results confirmed the safety and efficacy of the vaccine.     

Expression of Escherichia coli antigens in Salmonella typhimurium as a vaccine to prevent airsacculitis in chickens

Avian pathogenic Escherichia coli strains are associated with a variety of extraintestinal poultry diseases, including airsacculitis, colisepticemia, and cellulitis. A number of E. coli serotypes are associated with these diseases, although the most prevalent serotype is O78. Fimbrial proteins expressed by these strains appear to be important virulence factors, including type 1 fimbriae, P fimbriae, and curli. We have been working to develop an effective vaccine to protect chickens against these diseases. We have previously shown that an attenuated Salmonella typhimurium strain expressing O78 lipopolysaccharide provides protection against challenge with an O78 avian pathogenic E. coli strain. In this work, we have constructed an attenuated S. typhimurium that expresses both the O78 lipopolysaccharide and E. coli-derived type 1 fimbriae. In these studies, chickens were vaccinated at day of hatch and again at 2 wk of age. Birds were challenged at 4 wk of age. We found that the vaccine candidate provided significant protection against airsacculitis as compared to untreated controls or birds vaccinated with an attenuated S. typhimurium that did not express any E. coli antigens. In a separate experiment, challenged vaccinates showed significant weight gain compared to challenged nonvaccinates. We were not able to demonstrate protection against E. coli O1 or O2 serotype challenge, nor against challenge with wild-type S. typhimurium.     

鸡毒支原体SC克隆致弱株免疫原性测试——开放动物模型的建立与应用

对罗斯鸡和 Ｓ Ｐ Ｆ来航鸡用鸡毒支原体强毒攻击时,以鸡致病性大肠杆菌 Ｏ７ ８ 血清型菌株 １６ 小时培养物 ０２ｍ ｌ／羽皮下注射作人工诱导发病,试验鸡出现明显气囊病变,初步建立了以鸡致病性大肠杆菌为诱导因子的 Ｍ Ｇ 野外环境人工发病的动物模型。以此开放模型检测 Ｓ Ｐ Ｆ来航鸡以鸡毒支原体 Ｓ６ 克隆致弱株 Ｆ１５６ 代培养物点眼、滴鼻免疫后 ３０ 日、６０ 日龄的攻毒保护率,结果保护率分别达 ９０％ （１８／２０）、８４％ （４２／５０）,而对照组为 ３０％ （６／２０）、５０％ （１５／３０）,中期免疫效果证明,鸡毒支原体 Ｓ６ 克隆致弱株有良好的免疫保护作用。     

Immunogenicity of a temperature sensitive mutant Mycoplasma gaffisepticum vaccine

The immunogenicity of the ts-11 vaccine strain of Mycoplasma gallisepticum was assessed following eye drop or coarse aerosol administration in chickens of various ages. Protection was evalualted following intra-abdominal (IA) or fine droplet aerosol administration of virulent M. gallisepticum, usually the Ap3AS strain and was measured mainly by the scoring of gross air sac lesions or by egg production. Vaccination of chickens with ts-11 did not elicit a substantial serum antibody response as measured by rapid serum agglutination test, or ELISA. Protection was never demonstrated when no M. gallisepticum serum antibody response was detected in a vaccinated group of chickens. Failure to protect occurred usually, although not invariably, following aerosol administration of the vaccine. Vaccination by eye drop usually, although not invariably provided protection against challenge. In one experiment, chickens vaccinated by eye drop at 8-weeks were as susceptible as non vaccinated controls when challenged by IA inoculation at 13-weeks-of-age. Yet other birds from the same vaccinated group were resistant when challenged in an identical way at 23-weeks. No measurable increase in M. gallisepticum specific serum antibody concentrations occurred in the intervening period. Equally surprising was the response of another group of birds in the same experiment that had been vaccinated with a higher dose of ts-11. An antibody response was detected in this group, but they were susceptible to challenge at 23-weeks. Interestingly, a drop in egg production commenced 4 weeks after challenge, 2 weeks later than that observed in a non vaccinated group challenged at the same time.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunogenicity of Mycoplasma gallisepticum

The serological response and protective immunity elicited in the chicken by the pathogenic Ap3AS strain and the moderately pathogenic 80083 strain of Mycoplasma gallisepticum and variants of strain 80083 attenuated by repeated passage in mycoplasma broth were investigated. Strain 80083 elicited a substantial serum antibody response after administration either in drinking water or by conjunctival sac instillation to 7-week-old SPF chickens. No vaccinated chickens developed air sac lesions when challenged by intra-abdominal (IA) injection with the virulent Ap3AS strain. Chickens vaccinated with strain 80083M (50 broth passages) showed only a weak serological response but were substantially protected when challenged 4 weeks after vaccination. Chickens vaccinated with 80083H (100 broth passages) were serologically negative 4 weeks after vaccination and developed severe air sac lesions after challenge. Thirty-seven-week-old hens vaccinated 6 months previously with strain 80083 had high serum antibody levels and were completely protected against IA challenge with the homologous strain. However, 4/6 showed mild air sac lesions when challenged intra-abdominally with strain Ap3AS. Another group showed high M. gallisepticum serum antibody levels 6 months after vaccination with strain Ap3AS but 4/6 and 2/6 showed mild lesions after IA challenge with strains Ap3AS or 80083, respectively. Strains 80083 or 80083M were administered by conjunctival sac instillation to susceptible 11-week-old commercial pullets at the time of fowl pox vaccination. The concurrent use of both vaccines had no apparent adverse effect on the health of the chickens. Similar protection against IA challenge with strain Ap3AS was produced with the M. gallisepticum vaccines whether used alone or in combination with fowl pox.     

Nonspecific innate immunity against Escherichia coli infection in chickens induced by vaccine strains of Newcastle disease virus

The objective was to test the hypothesis that vaccine strains of Newcastle disease virus (NDV) induce nonspecific immunity against subsequent infection with Escherichia coli. White leghorn chickens at 5 wk of age were vaccinated with a NDV vaccine at various days before challenge exposure with O1:K1 strain of E. coli via an intra-air sac route. Immunity was determined on the basis of the viable number of E. coli in the spleen 24 hr after the infection. Roakin strain induced significant (P < 0.05) immunity against E. coli at 4, 6, and 8 days, and La Sota strain at 2, 4, and 8 days, postvaccination. Secondary NDV vaccination administered 14 days later failed to induce immunity against E. coli when chickens were infected 1 or 5 days after the vaccination. Significant (P < 0.05) suppression of this nonspecific immunity was observed in birds treated with corticosterone, 40 mg/kg in feed, given for three consecutive days immediately prior to the bacterial exposure but not in those treated prior to the period. The results indicate that innate immunity induced by the primary NDV vaccination may significantly suppress the multiplication of E. coli in chickens for a period of 2-8 days postvaccination. The NDV-induced immunity was inhibited by corticosterone, which is known to mediate physiological responses to stress.     

Virulence and transmissibility of Mycoplasma gallisepticum

The virulence of 4 low passage strains of Mycoplasma gallisepticum obtained from different sources within Australia was studied by experimental infection of chickens. Strain Ap3AS, originally isolated from the air sac of a broiler chicken, produced severe air sac lesions following injection into the abdominal air sacs of 2-week or 3-week-old chickens, and adult hens. Strain 80083 which was isolated from a clinically normal broiler breeder hen was also capable of producing gross air sac lesions following intra-abdominal (IA) injection, although it did so less consistently than strain Ap3AS. Strain 82078 isolated from a layer hen and strain QXO which was isolated from a turkey were also moderately pathogenic in terms of the incidence and severity of lesions elicited following IA injection. Strains Ap3AS and 80083 both caused a substantial loss of egg production over a 5 week period after IA infection of 27-week-old hens. Neither strain Ap3AS nor 80083 caused gross lesions or loss of egg production when administered alone into the upper respiratory tract. However, when inoculated into the conjunctival sac in combination with the Vic S strain of infectious bronchitis virus (IBV) strains Ap3AS and 80083 produced identical clinical signs of conjunctivitis. The mean numbers of M. gallisepticum in tracheal washings were significantly higher 2 weeks after infection in the group receiving strain 80083 in combination with IBV than in the group infected with strain Ap3AS and IBV (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

新城疫LaSota活疫苗对基因Ⅶ型分离株的免疫保护性试验

从山东省发病鸡群分离鉴定了一株新城疫病毒(NDV),命名为SDLY01。经蚀斑纯化后进行毒力测定和序列分析表明分离株SDLY01属于基因Ⅶ型NDV强毒。20只7日龄SPF鸡免疫新城疫活疫苗LaSot a后14 d分别用NDV标准强毒F48E8和分离株SDLY01攻毒,同时设同日龄SPF鸡为对照组,未免疫任何疫苗。攻毒后观察10 d,免疫组在攻毒后食欲、精神均正常;对照组在攻毒后2～4d发病死亡,并表现ND典型的临床症状和病理变化。攻毒后第3、5、7、9 d对免疫组试验鸡取喉头、泄殖腔棉拭进行病毒分离,F48E8攻毒组病毒分离均为NDV阴性,SDLYO1攻毒组第5 d病毒分离NDV阳性,第3、7和9d病毒分离阴性。本研究结果表明LaSot a活疫苗对F48E8和SDLY01均能提供100%免疫保护,但不能完全抑制基因Ⅶ NDV分离株在体内的复制和排毒。     

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.     

鸡大肠杆菌多价1型菌毛亚单位疫苗在鸡体内的免疫原性研究

用1日龄雏鸡免疫评价了鸡大肠杆菌多价1型菌毛油乳剂苗在鸡体内的免疫原性。用含1型菌毛的3个不同血清型（O1、O78及O88）菌株,大容量培养后提取菌毛制备多价菌毛油乳剂苗。接种多价菌毛油乳剂苗的鸡用同源菌株攻毒后出现10％～11．2％的死亡率,阳性对照组鸡攻毒后出现70％～80％的死亡率;用异源菌株（O36）攻毒后,免疫鸡出现33．3％的死亡率,而未免疫阳性对照组鸡死亡率达70％。免疫鸡在气囊,心包及肝脏的病变非常轻微,且攻毒后非免疫鸡能更有效地清除侵入气囊的大肠杆菌。     

Increased tracheal colonization in chickens without impairing pathogenic properties of avian pathogenic Escherichia coli MT78 with a fimH deletion

Several studies suggest that the expression of F1 fimbriae could be involved in the virulence of Escherichia coli for chickens. F1 fimbriae display multivalent properties such as adhesion to epithelia or interaction with the immune system that imply specific interactions between the adhesin FimH and different cell receptors. We constructed a delta fimH mutant of the avian pathogenic E. coli MT78 and evaluated its in vivo colonization and pathogenicity, as compared to that of the parent strain. The generated mutant PA68 was unable to adhere in vitro to chicken epithelial pharyngeal or tracheal cells; mutant bacteria were mostly afimbriated although a minority of them displayed altered piliation phenotypes. Two inoculation routes were used to compare the ability of MT78 and PA68 to colonize the respiratory tract and to induce colibacillosis in chickens. In the first model, 2-wk-old axenic chickens were inoculated intratracheally with one or both E. coli strains, after primary infection with infectious bronchitis virus. In the second model, 3-wk-old specific-pathogen-free chickens were inoculated via the caudal thoracic air sac. After intratracheal inoculation, the delta fimH mutant was found to be a better colonizer than MT78 in the trachea of inoculated chickens. Furthermore, when both strains were inoculated simultaneously, the delta fimH mutant constituted 98% of the bacterial population in the trachea at day 7 postinoculation. Irrespective to the inoculation route, MT78 and PA68 showed similar abilities to induce macroscopic lesions in chickens, to provoke bacteremia, and to colonize the internal organs. However, 4 days after intra-air sac inoculation, bacterial counts of the mutant were lower in the spleen and liver than those of MT78. Our results show that FimH is not required for colonization of the trachea of axenic chickens by E. coli and that it is not a major determinant of bacterial pathogenicity. On the contrary, the lack of expression of FimH seems to favor the in vivo colonization of the trachea of chickens by E. coli.     

Efficacy of soluble recombinant FliC protein from Salmonella enterica serovar enteritidis as a potential vaccine candidate against homologous challenge in chickens

FliC, the flagellin antigen of Salmonella Enteritidis, was tested as a vaccine candidate for protective effect against a homologous challenge in chickens. After immunization with recombinant FliC (rFliC) or administration of phosphate-buffered saline (PBS) at 56 days old, the chickens were challenged with 10(9) colony-forming units of Salmonella Enteritidis at 76 days old. The vaccinated birds showed significantly decreased bacterial counts in the liver and cecal contents compared to those administered PBS at 7 days postchallenge, but the protection was partial. The replication experiment also showed a similar result. In both experiments, vaccination induced an increased level of serum anti-rFliC IgG, which was also reactive to the native flagella. The intestinal IgA level was slightly higher in the vaccinated birds than in the control. However, neither the proliferative response nor interferon-gamma secretion of splenic cells upon stimulation with rFliC was induced. Therefore, the effect of rFliC as a vaccine is limited, and further improvement is needed.     

Improvements to the hemagglutination inhibition test for serological assessment of recombinant fowlpox-H5-avian-influenza vaccination in chickens and its use along with an agar gel immunodiffusion test for differentiating infected from noninfected vaccinated animals

In general, avian influenza (AI) vaccines protect chickens from morbidity and mortality and reduce, but do not completely prevent, replication of wild AI viruses in the respiratory and intestinal tracts of vaccinated chickens. Therefore, surveillance programs based on serological testing must be developed to differentiate vaccinated flocks infected with wild strains of AI virus from noninfected vaccinated flocks in order to evaluate the success of vaccination in a control program and allow continuation of national and international commerce of poultry and poultry products. In this study, chickens were immunized with a commercial recombinant fowlpox virus vaccine containing an H5 hemagglutinin gene from A/turkey/Ireland/83 (H5N8) avian influenza (AI) virus (rFP-H5) and evaluated for correlation of immunological response by hemagglutination inhibition (HI) or agar gel immunodiffusion (AGID) tests and determination of protection following challenge with a high pathogenicity AI (HPAI) virus. In two different trials, chickens immunized with the rFP-H5 vaccine did not develop AGID antibodies because the vaccine lacks AI nucleoprotein and matrix genes, but 0%-100% had HI antibodies, depending on the AI virus strain used in the HI test, the HI antigen inactivation procedure, and whether the birds had been preimmunized against fowlpox virus. The most consistent and highest HI titers were observed when using A/turkey/Ireland/83 (H5N8) HPAI virus strain as the beta-propiolactone (BPL)-inactivated HI test antigen, which matched the hemagglutinin gene insert in the rFP-H5 vaccine. In addition, higher HI titers were observed if ether or a combination of ether and BPL-inactivated virus was used in place of the BPL-inactivated virus. The rFP-H5 vaccinated chickens survived HPAI challenge and antibodies were detected by both AGID and HI tests. In conclusion, we demonstrated that the rFP-H5 vaccine allowed easy serological differentiation of infected from noninfected birds in vaccinated populations of chickens when using standard AGID and HI tests.