Immunization of chickens with temperature-sensitive mutants of Mycoplasma gallisepticum

Temperature-sensitive (TS) mutants of the S6 strain of Mycoplasma gallisepticum (MG) were used to immunize newly hatched chickens. Immunized chickens developed antibodies to the wild-type (WT) S6 strain as demonstrated by serologic tests. MG was recovered from nasal cavities but not from the lower respiratory tract of the immunized chicks. Three weeks after intranasal immunization, chickens were challenged via the air sacs with the virulent S6 strain. Immunized chickens were significantly better protected from development of air-sac lesions than were controls.     

Further studies on the immunization of chickens with temperature-sensitive Mycoplasma gallisepticum mutant

Newly hatched chickens were immunized with a temperature-sensitive (TS) Mycoplasma gallisepticum (MG) mutant (TS 100). Immunized chickens resisted challenge with the virulent S6 strain. The dose of TS MG needed for protection was less than 3.3 X 10(4) colony-forming units. After immunization with TS 100, chickens were subjected to a variety of virus infection and immunosuppressive treatments. Neonatal bursectomy or thymectomy, infectious bursal disease virus infection, and infectious bronchitis virus vaccination or a combination of infectious bronchitis virus and Newcastle disease virus vaccination did not contribute to the development of air-sac lesions in TS MG-immunized chickens. Infectious bronchitis virus vaccination enabled MG to migrate from the nasal cavity to the trachea but not to the air sacs. The TS MG vaccine appears to be a safe immunogen.     

Experimental infection of turkeys with Mycoplasma gallisepticum of low virulence, transmissibility, and immunogenicity.

Three-week-old turkeys were inoculated intranasally with approximately 10(6) colony-forming units (CFU) of putative variant Mycoplasma gallisepticum (MG) strains M876, M35, or the virulent S6 reference strain. Uninoculated turkeys in each group served as contact sentinels. The hemagglutination-inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA) were used to determine serologic responses. MG was isolated from 100% and 92% of S6- and M876-inoculated turkeys, respectively, on day 7 PI. However, culture-positive rates among M876-inoculated turkeys declined more rapidly, transmission to contact sentinels took longer and occurred at lower rates, and serologic responses measured by HI and ELISA were lower than in S6-infected turkeys. Testing sera from inoculated turkeys for antibodies to MG in homologous and heterologous ELISA systems indicated that strain M876 was significantly (P less than 0.05) less immunogenic than S6 (days 62 and 95 PI), and that the homologous ELISA was more sensitive (P less than 0.005). MG strain M35 failed to infect turkeys in three attempts, even though the inocula used were viable on culture media.     

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.     

Experimental infection of chickens and turkeys with Mycoplasma gallisepticum reference strain S6 and North Carolina field isolate RAPD type B

During an epidemic of mycoplasmosis in chicken and turkey flocks in North Carolina between 1999 and 2001, isolates of Mycoplasma gallisepticum (MG) from affected flocks were characterized by random amplification of polymorphic DNA (RAPD), and eight distinct RAPD types were identified. MG RAPD type B accounted for more than 90% of the isolates and was associated with moderate-to-severe clinical signs and mortality. The virulence of MG RAPD type B for chickens and turkeys was compared with sham-inoculated negative controls and MG S6 (a virulent strain)-inoculated positive controls. Clinical signs occurred in chickens and turkeys inoculated with either MG RAPD type B or MG S6. However, they were not as frequent or severe as those seen in naturally affected flocks, and there was no mortality in the experimental groups. Based on gross and microscopic findings, MG RAPD type B was equal to or more virulent than MG S6. All MG-inoculated birds were culture and PCR positive at 7 and 14 days postinoculation (PI). Among serological tests, the serum plate agglutination test was positive for the majority of chickens and turkeys (58%-100%) infected with either strain of MG at both 7 and 14 days PI. The hemagglutination inhibition test was negative for all birds at 7 days PI and positive for a few chickens (8%-17%) and several turkey sera (40%-60%) at 14 days PI. Only a single serum was positive by enzyme-linked immunosorbent assay (an MG S6-infected turkey) at 14 days PI.     

Evaluation of immune effects of fowlpox vaccine strains and field isolates

The immune effects of fowlpox virus (FPV) field isolates and vaccine strains were evaluated in chickens infected at the age of 1 day and 6 weeks. The field isolates and the obsolete vaccine strain (FPV S) contained integrated reticuloendotheliosis virus (REV) provirus, while the current vaccine strain (FPVST) carries only REV LTR sequences. An indirect antibody ELISA was used to measure the FPV-specific antibody response. The non-specific humoral response was evaluated by injection of two T-cell-dependent antigens, sheep red blood cells (SRBC) and bovine serum albumin (BSA). There was no significant difference in the antibody response to FPV between chickens infected with FPV various isolates and strains at either age. In contrast, antibody responses to both SRBC and BSA were significantly lower in 1-day-old chickens inoculated with field isolates and FPV S at 2-3 weeks post-inoculation. Furthermore, cell-mediated immune (CMI) responses measured by in vitro lymphocyte proliferation assay and in vivo using a PHA-P skin test were significantly depressed in chickens inoculated with field isolates and FPV S at the same periods. In addition, thymus and bursal weights were lower in infected chickens. These immunosuppressive effects were not observed in chickens inoculated with the current vaccine strain, FPVST, at any time. The results of this study suggest that virulent field isolates and FPV S have immunosuppressive effects when inoculated into young chickens, which appeared in the first 3 weeks post infection. REV integrated in the FPV field isolates and FPV S may have played a central role in the development of immunosuppression.     

Vaccination of turkeys against air-sac infection with a temperature-sensitive mutant of Mycoplasma gallisepticum

Turkeys were vaccinated with temperature-sensitive (TS) mutants of Mycoplasma gallisepticum (MG) to determine pathogenicity and immunogenicity. TS 37 was apathogenic yet immunogenic to turkeys, TS 100 was highly pathogenic, and TS 102 was slightly pathogenic and nonimmunogenic. Five or 7 weeks after intranasal vaccination of turkeys with the TS 37 mutant, a highly statistically significant resistance against intra-air-sac challenge with the S6 strain of MG was observed.     

Systemic and local immune responses of gnotobiotic calves to respiratory infection with Mycoplasma bovis

Gnotobiotic calves were inoculated by the intratracheal route with Mycoplasma bovis and the specific antibody response in sera and tracheo-bronchial washings examined by radioimmunoassay. In sera an IgM response which reached a peak two weeks post infection was followed by IgG1 and IgG2 antibody responses. Low levels of IgA antibody were detected in sera three and four weeks after infection. The predominant antibody in tracheo-bronchial washings 2 weeks after infection was IgA. Four weeks after infection IgG1 antibody predominated, but IgG2 and IgA antibodies were also present. Cells containing Ig were present in the cellular accumulations around the necrotic zones produced by M. bovis in the lung parenchyma two and four weeks after infection. IgG1 containing cells predominated in these cellular infiltrates. IgG2 producing cells were the next in frequency. It is concluded that the lung lesion caused by M. bovis is partly due to the host's immune response, presumably contributing to the control of the infection, and that the cells infiltrating the lung are a major source of the local and systemic IgG antibody that is detected after infection. IgA staining cells were observed in the submucosa of tissues from nasal cavity and trachea. These cells are probably the source of IgA in tracheo-bronchial washings and sera since IgA-producing cells were not a predominant component of the lesion in the lung parenchyma.     

Immunological differences between layer- and broiler-type chickens

In commercial poultry husbandry, alternatives for the use of antibiotics and vaccines are under investigation, which preferably have to be applicable for both layer- and broiler-type chickens. There are indications that the defense mechanisms vary between layer- and broiler-type chickens. Therefore, the difference in immune response between layer- and broiler-type chickens of the same age was investigated, using TNP-KLH (trinitrophenyl-conjugated keyhole limpet hemocyanin) as antigen without adjuvant. First different routes of immunization (intravenously, intramuscular, subcutaneous and ocular) were examined to find out which immunization route gives the highest antibody titers. The intravenous immunization route resulted in higher TNP-specific antibody responses than the other immunization routes tested and therefore this immunization route was used in both following experiments. In order to investigate the optimal dose of antigen needed for immunization, a dose-response curve in broiler- and layer-type chickens was completed. The humoral immune response was measured in serum by a TNP-specific ELISA and the in vitro cellular immune response by an antigen-specific lymphocyte proliferation assay.The antibody response of layer- and broiler-type chickens appeared to differ, not only in optimal dose and response, but also in kinetics of the response itself. Broiler chickens generated higher IgM anti-TNP titers whereas layer-type chickens generated higher IgG anti-TNP titers. This specific antibody response in broiler-type chickens did not last as long as in layer-type chickens. The TNP-specific cellular immune response was detectable in layer-type chickens, but not in broilers. Both types generate a non-specific cellular immune response, although this response in broilers is lower than in layer-type chickens.From these results, we conclude that broilers primarily respond to TNP-KLH with a high IgM antibody response whereas layer-type chickens respond with a high IgG response. In addition, the cellular response of layer-type chickens is much higher than the response of broilers. The results suggest that broilers are specialized in the production of a strong short-term humoral response and layer-type chickens in a long-term humoral response in combination with a strong cellular response, which is in conformity with their life expectancy.     

鸡毒支原体感染病理模型复制

以鸡毒支原体(Mycoplasma gallisepticum,MG)S6标准菌株不同浓度接种16日龄雏鸡,5d后用鸡新城疫疫苗(I系)诱导发病,对以鸡新城疫为诱导因子的鸡毒支原体野外环境病理模型复制进行研究。结果表明:鸡毒支原体S6攻毒菌液浓度为109CCU/mL,攻毒后第5天,用鸡新城疫疫苗(I系)喷雾,5羽份/只,进行激发,可成功复制出鸡毒支原体感染病理模型。     

Attenuation of virulence of Lawsonia intracellularis after in vitro passages and its effects on the experimental reproduction of porcine proliferative enteropathy

Non-pathogenic Lawsonia intracellularis variants have been obtained through multiple passages in cell culture but there is no information regarding the number of passages necessary to attenuate a pathogenic isolate. The present study evaluated the susceptibility of pigs to L. intracellularis after 10, 20 and 40 passages in vitro. Three groups (six animals/group) were inoculated with pure culture of L. intracellularis on passage 10, 20 or 40 and one group with placebo. The animals were monitored for clinical signs, fecal shedding and serological IgG response during 28 days post-inoculation. Gross and histologic lesions and the level of infection based on the amount of L. intracellularis-specific antigen in the intestinal mucosa identified by immunohistochemistry were evaluated in two animals from each group on days 14, 21 and 28. Animals inoculated with passages 10 and 20 demonstrated proliferative lesions typical of porcine proliferative enteropathy associated with the presence of Lawsonia-specific antigen in the intestinal mucosa. Passage 40-inoculated pigs did not show proliferative lesions or presence of Lawsonia antigen at any time point throughout the study. Similar patterns of the fecal shedding were observed in passage 10 and 20-infected pigs but those infected with passage 40 shed for a short period. Serological IgG responses in passage 10 and 20-inoculated pigs were detected from day 14 post-infection but not at all in passage 40-inoculated animals. These results demonstrate attenuation of the virulence properties of L. intracellularis between 20 and 40 cell passages in vitro. This information will be valuable for design of future experimental models and for studying the mechanisms involved in the attenuation of L. intracellularis virulence.     

Antibody response of calves after intranasal inoculation with parainfluenza-3 virus and resistance of inoculated calves to experimental homologous viral infection

The immunologic response of colostrum-deprived calves to parainfluenza-3 (PI-3) virus given by intranasal inoculation was studied. Inoculation of calves with 3.2 x 10(6) median cell culture infective doses (CCID50) of either virulent (SF-4) virus or a modified strain of PI-3 virus, or with 2.0 x 10(8) CCID50 of SF-4 virus, stimulated development of both serum antibody and nasal secretion (NS) antibody. However, NA antibody decreased in all calves between the 16th and 42nd postinoculation days and was present only at low or moderately low concentrations on the 126th day, when the immunity of the calves was challenged. Generally, calves that were inoculated with 3.2 x 10(6) CCID50 of SF-4 virus developed slightly higher concentrations of serum and NS antibodies than did calves inoculated with modified virus. Calves that were inoculated with 2.0 x 10(8) or 3.2 x 10(6) CCID 50 of SF-4 virus developed comparable concentrations of serum antibody, but large doses of SF-4 virus were less effective than smaller doses of the same virus in stimulating the development of NS antibody. Reinoculation of 3 calves with modified PI-3 virus resulted in a demonstrable increase in serum antibody in 2 calves and an increase and subsequent decrease in NS antibody in all calves. Challenge exposure of inoculated calves to aerosols of SF-4 virus failed to cause clinical signs of disease, and the challenge virus was not isolated from the nasal passages.     

Antibody response to experimental Salmonella typhimurium infection in chickens measured by ELISA

An indirect ELISA has been developed to detect Salmonella typhimurium antibodies in chicken sera, using whole bacterial cell protein, flagellar protein or lipopolysaccharide as antigens. In experimental infections high concentrations of S typhimurium-specific IgG persisted after the faecal excretion of S typhimurium had ceased, whereas the specific IgM response was transitory. Some uninfected chickens placed in contact with experimentally infected birds developed high IgG titres in the absence of detectable faecal excretion. Other S typhimurium strains, which varied in their invasive abilities, also induced high titres of IgG. The ELISA allowed chickens infected experimentally with S typhimurium to be differentiated from chickens infected with 10 other serotypes, including S enteritidis. The use of whole blood in place of serum in the ELISA reduced the titres slightly. The storage of serum dried on to filter paper strips for four weeks produced little change in ELISA antibody titre, and the treatment of such strips with phenol or chloroform vapour had little or no effect on the antibody titre.     

不同剂型新城疫-禽流感二联灭活疫苗免疫SPF鸡后抗体水平对比试验

不同剂型的佐剂对疫苗的免疫效果有较大的影响,本试验旨在对比不同剂型佐剂的新城疫-禽流感(简称"新-流")二联灭活疫苗的免疫效果,从而为生产上选择最佳剂型佐剂应用于新城疫-禽流流感二联灭活疫苗提供依据。分别将油包水型、水包油型、水包油包水型佐剂与新城疫及禽流感灭活抗原乳化成不同剂型的新城疫-禽流感(H9亚型,HP株)二联灭活疫苗,将这三种不同剂型的新城疫-禽流感(H9亚型,HP株)二联灭活疫苗分别免疫一组7日龄SPF鸡。每羽颈部皮下注射0.2 m L,同时设一组未免7日龄SPF鸡作为对照组,各免疫组与对照组SPF鸡于免疫组免后6、10、15、21、28、35 d进行采血,检测新城疫与禽流感抗体水平。结果表明:免疫油包水型疫苗组SPF鸡免疫后新城疫与禽流感抗体上升最快,在免后10、15、21、28、35 d的新城疫与禽流感抗体也最高,其次是免疫水包油包水型的,而免疫水包油型疫苗组的SPF鸡新城疫与禽流感抗体上升最慢,而且在免后10、15、21、28、35 d的新城疫与禽流感抗体也最低。此外,从各免疫组可以看出,在免后6 d时新城疫抗体已开始产生,在1log2以下,而禽流感抗体仍未产生。可见免疫新-流二联苗后,新城疫抗体比禽流感抗体更早产生,油包水型新-流二联苗的免后抗体高于水包油包水及水包油型,而且能持续刺激机体产生高水平抗体,更具优势。     

Development of nasal, fecal and serum isotype-specific antibodies in calves challenged with bovine coronavirus or rotavirus

Unsuckled specific pathogen free calves were inoculated at 3-4 weeks of age, either intranasally (IN) or orally (O) with bovine coronavirus or O plus IN (O/IN) or O with bovine rotavirus. Shedding of virus in nasal or fecal samples, and virus-infected nasal epithelial cells were detected using immunofluorescent staining (IF), ELISA or immune electron microscopy (IEM). Isotype-specific antibody titers in sera, nasal and fecal samples were determined by ELISA. Calves inoculated with coronavirus shed virus in feces and virus was detected in nasal epithelial cells. Nasal shedding persisted longer in IN-inoculated calves than in O-inoculated calves and longer than fecal shedding in both IN and O-inoculated calves. Diarrhea occurred in all calves, but there were no signs of respiratory disease. Calves inoculated with rotavirus had similar patterns of diarrhea and fecal shedding, but generally of shorter duration than in coronavirus-inoculated calves. No nasal shedding of rotavirus was detected. Peak IgM antibody responses, in most calves, were detected in fecal and nasal speciments at 7-10 days post-exposure (DPE), preceeding peak IgA responses which occurred at 10-14 DPE. The nasal antibody responses occurred in all virus-inoculated calves even in the absence of nasal shedding of virus in rotavirus-inoculated calves. Calves inoculated with coronavirus had higher titers of IgM and IgA antibodies in fecal and nasal samples than rotavirus-inoculated calves. In most inoculated calves, maximal titers of IgM or IgA antibodies correlated with the cessation of fecal or nasal virus shedding. A similar sequence of appearance of IgM and IgA antibodies occurred in serum, but IgA antibodies persisted for a shorter period than in fecal or nasal samples. Serum IgG1 antibody responses generally preceeded IgG2 responses and were predominant in most calves after 14-21 DPE.     

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。     

Humoral responses and salmonellosis protection in chickens given a vitamin-dependent Salmonella typhimurium mutant

Humoral responses in chickens inoculated with an aromatic vitamin dependent (Aro-) Salmonella typhimurium mutant (STM) were studied to ascertain the efficacy of the organism as a vaccine for salmonellosis and possibly as a delivery system for antigens from enteric pathogens of chickens. Serum antibody responses in chickens that were given oral or subcutaneous inoculations of the bacterium followed the classic order of antibody production, with IgM being detected first, followed by IgG and IgA. Antibody responses in the gut of orally inoculated chickens were restricted to IgG and IgA. Weight gain measured in chickens given high doses of STM (up to 5 x 10(9)) orally, revealed that the bacterium did not adversely affect the chickens; in fact, inoculated chickens had significantly higher body weights than controls at the same age. Salmonellosis protection of chickens by oral vaccination with STM was examined in a vaccination/challenge experiment. The experiment revealed that oral vaccination reduced excretion of a virulent S. typhimurium used as the challenge organism.     

Humoral and cellular immune responses of pigs inoculated with Mycoplasma hyopneumoniae

Cellular and humoral immune responses of pigs inoculated with Mycoplasma hyopneumoniae were investigated at postinoculation weeks (PIW) 2, 4, and 6. The response of blood lymphocytes (BL) and bronchial lymph node lymphocytes (LNL) to stimulation by M hyopneumoniae antigens was evaluated by a lymphocyte-stimulation test. Specific antibodies in serum and lung washing samples were assayed by ELISA. Immunoglobulin-positive cells in lungs and bronchial lymph nodes were identified by indirect fluorescent antibody test, using isotype-specific monoclonal antibodies. At PIW 0 to 6, BL from control and M hyopneumoniae-inoculated pigs were stimulated by M hyopneumoniae cells; however, BL from inoculated pigs generally had higher stimulation indices, especially at PIW 6. The response of LNL was influenced by previous exposure to M hyopneumoniae, as indicated by higher stimulation indices (P less than 0.01) of LNL from inoculated pigs killed at PIW 2 and 6. Specific ELISA antibodies to M hyopneumoniae in lung washings from inoculated pigs consisted mainly of IgG and IgA isotypes. Examination of lung sections by indirect immunofluorescence revealed that cells producing IgM and IgA were in controls as well as M hyopneumoniae-inoculated pigs, but IgG-positive cells were only in lungs of inoculated pigs. Resolution of pneumonia appeared to correlate with development of increased sensitization of BL, as well as development of marked increases in immunoglobulins, particularly IgG in lung washings at PIW 6.     

Avian eimeria: invasion in foreign host birds and generation of partial immunity against coccidiosis

Four species of avian Eimeria invaded the intestine of foreign host birds in the same areas in which they invaded the natural host. Repeated inoculation (immunization) of chickens with the turkey coccidian, Eimeria adenoeides, partially protected the chickens against a subsequent challenge with 5.8 x 10(4) E. tenella oocysts. At 6 days post-challenge, the weight gain and feed conversion efficiency of the immunized chickens was significantly better than those of the chickens that were not immunized with E. adenoeides. Lesion scores and cellular invasion by the sporozoites were significantly lower in the immunized birds than in the unimmunized group. Electrophoresis and Western blot analysis identified changes in the serum antibody profiles of the chickens that appeared to be associated with the immunization and challenge programs. An antibody or antibodies recognizing a 60,000-molecular-weight antigen of E. tenella sporozoites disappeared when chickens immunized with E. adenoeides were challenged with E. tenella; an antibody or antibodies recognizing a 23,000-molecular-weight sporozoite antigen appeared within 6 days of challenge. Reciprocal studies, in which turkeys were immunized with E. tenella and challenged with E. adenoeides, showed little evidence of protection.     

Experimental inoculation of heifers with bovine adenovirus type 3.

Nine 2-year-old heifers having BAd3-neutralizing antibody titers between 1:120 and 1:1080 were individually exposed intranasally to an aerosol of 10(8) pfu of wild type (wt) bovine adenovirus type 3 (BAd3). Four animals were kept as non-inoculated controls. The heifers were examined daily for rectal temperature, weight gain/loss, nasal and ocular discharges, and other clinical signs for 10 d post-inoculation. None of the animals showed any sign of clinical disease. Virus excretion was observed in one animal only on Day 3 post-inoculation. All BAd3-inoculated heifers demonstrated a significant (P < 0.005, paired t-test) rise in BAd3-specific serum IgG, IgG1, or IgG2 ELISA titers and virus-neutralizing antibody titers compared to the titers before inoculation. All virus-inoculated animals demonstrated increased levels of BAd3-specific IgA ELISA titers in nasal secretions. These results suggest that in the presence of circulating BAd3-neutralizing antibodies, intranasal inoculation of cattle with wt BAd3 would result in inapparent infection.