Effect of selected water temperatures used in Mycoplasma gallisepticum vaccine reconstitution on titer at selected time intervals

Numerous methods are currently used throughout the poultry industry for the administration of vaccines. Each utilizes water for vaccine reconstitution and/or administration, including two of the three commercially available live Mycoplasma gallisepticum (MG) vaccines. Selected water temperatures were used to reconstitute and/or dilute the three commercially available live MG vaccines. Water temperatures included 4 C, 22 C (room temperature), and 32 C, and titer (color change units) was recorded at four time intervals, at point of reconstitution (time 0), 15, 30, and 60 min postreconstitution of the vaccines (time periods 15, 30, and 60, respectively). Results for F strain MG (FMG) vaccine showed significant decreases in titer from time 0 to time 15 for the 22 C and 32 C water temperatures but no significant decrease for any time period for FMG reconstituted with 4 C water. For 6/85 strain MG no significant difference in titer was noted for any of four time periods within any of the three water temperatures. For ts-11 strain MG a significant decrease was observed in titer at each of the four postdilution time periods when diluted with 32 C water. There was no significant decrease in titer at any time period for ts-11 MG vaccine when diluted with either 4 C or 22 C water.     

Stabilization of Live Mycoplasma gallisepticum Vaccines During Vaccination with Second-Generation Spray-Vac Vaccine Stabilizer

Dilution and application of live Mycoplasma gallisepticum vaccines without the use of vaccine-stabilizing compounds may lead to significant loss of vaccine viability and loss of vaccine efficacy. Vaccine viability may decrease because of osmotic lysis of the mycoplasma as well as the presence of free chlorine or other detrimental chemicals in the water. Second-generation Spray-Vac vaccine stabilizer was developed and shown to maintain live Mycoplasma gallisepticum vaccine viability during exposure to free chlorine while protecting from the other factors that appear to decrease vaccine survival in solution. Increased vaccine survival in solution should lead to increased survival of the vaccine during vaccination. Field trial results demonstrate that second-generation Spray-Vac vaccine stabilizer yields excellent results without the need for distilled water or other vaccine-stabilizing compounds.     

抗菌药对马立克氏病活疫苗毒性的作用

用不同剂量的青霉素钾、硫酸链霉素、硫酸卡那霉素、硫酸庆大霉素和恩诺沙星等５种常见抗菌药加入ＨＶＴ冻干菌、ＣＶＩ９８８细胞结合苗和Ｚ４＋ＦＣ１２６双价活疫苗中,作用１ｈ后,分别取样计数,研究其对马立克氏病（ＭＤ）疫苗的毒性作用。结果表明：５种抗菌药加入ＭＤ疫苗稀释液后,ｐＨ值变化较大,ＭＤ疫苗空斑数显著减少,以至使ＭＤ疫苗失效。     

Determination of optimal conditions for thawing and diluting cell-bound CVI 988 Marek's disease vaccine and stability of the diluted vaccine

The cell-associated vaccine strain CVI 988, which is the active component of several commercial Marek's disease vaccines, normally is frozen and stored in liquid nitrogen. In order to ascertain good efficacy of the vaccine, it is crucial that the right procedures are followed for thawing and diluting of the virus. In the study presented here, ampoules containing the frozen product were taken from storage in liquid nitrogen and were thawed in a water bath at 27 C, which is similar to a lukewarm bath, and in a water bath at 37 C, with and without agitation. The effect of the thawing procedure on the live virus titer of the vaccine was investigated. Samples of thawed vaccine were diluted in diluent with different temperatures, and live virus titers were determined directly after dilution and after incubation of the diluted vaccine at different temperatures. The results show that directly after thawing in the water baths at 27 C and 37 C, with or without agitation, the live virus titers for CVI 988 were all in the same range. After incubation of the thawed virus at both temperatures for 15 min, the live virus titers were still in the same range. Decreases in live virus titers were observed after incubation for 4 hr. Live virus titration of the vaccine in diluent in different temperatures revealed that the highest titers were found with diluent at a temperature of 30 C to 37 C and the lowest titers in diluent at a temperature of 5 C. In addition, a combination product containing cell-associated CVI 988 and cell-associated herpesvirus of turkeys (HVT) was tested. For this combination product, the titers for HVT also were highest in diluent with a high temperature (i.e., 37 C), whereas the titers for CVI were highest in diluent at a temperature of 22 C. Both strains had relatively low titers in diluent at 5 C. After incubation of the diluted vaccine at the various temperatures for several hours, again, live virus titrations were done. Live virus titers were most stable with diluent at temperatures of 22 C. Vaccine virus diluted in diluent at 37 C could be stabilized by placing the diluted vaccine at 5 C directly after diluting. After evaluation of these data, the following is recommended. For thawing of the vaccine, a water bath at approximately 27 C, which is similar to a lukewarm bath, is preferred. For diluting the vaccine, diluent should be used at a temperature of 22 C or higher. If diluted in diluent at temperatures higher than 22 C, the diluted vaccine should be stored under cooling in order to avoid titer losses.     

The efficacy of three commercial Mycoplasma gallisepticum vaccines in laying hens

The efficacy of three commercial Mycoplasma gallisepticum (MG) immunizing agents-a bacterin, a recombinant fowlpox-MG vaccine, and a live F-strain vaccine-was compared in specific-pathogen-free hens in egg production. Three groups of 25 chickens were vaccinated with one of the vaccines at 10 wk of age and 25 birds were not vaccinated. At 25 wk of age (and approximately 50% egg production), 20 birds from each of the three vaccinated groups and 15 nonvaccinated controls were challenged with virulent R-strain via aerosol; the birds were necropsied and evaluated at 10 days post-challenge. The MG bacterin and live F-strain vaccinations were both protective and resulted in significant differences in air sac lesions, tracheal lesions, and ovarian regression compared to the nonvaccinated controls and the recombinant fowlpox-MG vaccine (P < or = 0.05). The evaluation of ovarian regression is a useful method of testing the efficacy of MG vaccines in laying hens.     

Eye surface area and dosage rates for spray vaccination

Spray application of Mycoplasma gallisepticum (MG) vaccines is a labor- and time-saving means of mass vaccination of layer chickens. Recent assessment of spray characteristics of nozzles commonly used to apply MG vaccine in layer chicken operations has shown that the amount of respirable droplets (< 5 microm) is negligible. Topical application of vaccine onto the eye surface has been suggested as a route of vaccination, but no estimates of vaccine load delivered via spray application were found in the literature. Estimates of eye surface area were developed using digital imaging; 24 layer pullets were used for analysis, and the mean eye surface area, corrected for corneal curvature, was found to be 0.609 cm2. This surface area was then used to estimate vaccine load for commercially available live MG vaccine sprayed through popular nozzles. Less than 3000 colony-forming units can be expected for direct deposition onto the surface of an eye.     

Vaccination of day-old broiler chicks against Newcastle disease and infectious bursal disease using commercial live and/or inactivated vaccines

Day-old broilers were administered live and/or inactivated vaccines to assess vaccine efficacy against challenge with Newcastle disease (ND) and infectious bursal disease (IBD). Chicks were from commercial breeder pullets vaccinated against ND and IBD using several live vaccine primers followed by an inactivated ND-IBD vaccine at 18 weeks. The most efficacious initial ND-IBD vaccination program was live ND virus by eye drop and live IBD vaccine injected subcutaneously (SQ) followed 2 hours later with inactivated ND-IBD vaccine SQ. The next two most efficacious programs were live vaccine alone and the inactivated vaccine only. Inactivated vaccine given SQ had no adverse effect on live IBD vaccine given 2 hours earlier in a similar site. Administration of inactivated vaccine by vent was not as efficacious as administration SQ. A booster of a second live ND-IBD vaccine drinking water at 18 days significantly increased levels of circulating antibody, regardless of the initial vaccination program.     

4株鸡IBD活疫苗对鸡ND免疫抗体的影响

４株不同毒力鸡传染性法氏囊病（ＩＢＤ）活疫苗,分别接种３０ｄＳＰＦ公维,４ｄ后再接种鸡新城（ＮＤ）Ｌａｓｏｔａ系活疫苗,同时每组捕杀５只,观察法氏囊损伤情况,另４组用同１天的ＳＰＦ公无接种ＮＤ－Ｌａｓｏｔｑ系疫苗４天后再接种不同毒力的ＩＢＤ疫苗。试验结果表明,４株ＩＢＤ活疫苗对雏鸡法氏囊不同程度损伤,并且对ＮＤ免疫抗体产生有明显抑制作用。     

Use of mgc2-polymerase chain reaction-restriction fragment length polymorphism for rapid differentiation between field isolates and vaccine strains of Mycoplasma gallisepticum in Israel

Increasing use of Mycoplasma gallisepticum (MG) live vaccines has led to a need for a rapid test for differentiation of MG field strains from the live vaccine strains ts-11 and 6/85. We examined the differentiating potential of diagnostic polymerase chain reaction (PCR) primers targeted to the gene mgc2, encoding a cytadherence-related surface protein uniquely present in MG. The mgc2-PCR diagnostic primers are specific for MG in tests of all avian mycoplasmas or bacteria present in the chicken trachea and are sensitive enough to readily detect MG in tracheal swabs from field outbreaks. Differentiation of vaccine strain ts-11 was based on identification of restriction enzyme sites in the 300-base-pair (bp) mgc2-PCR amplicon present in ts-11 and missing in MG isolates from field outbreaks in Israel. Restriction sites for the enzymes HaeII and SfaN1 were identified in the amplified region in strain ts-11 and were not found in 28 field isolates of MG, comprising a representative cross section of all the MG isolates from the period 1997-2003. In practice, differential diagnosis of MG is achieved within 1 day of submission of tracheal swab samples by mgc2-PCR amplification and restriction of the amplicon with HaeII, giving a 270-bp fragment for ts-11 or no restriction for other MG strains tested. Application of the mgc2-PCR-restriction fragment length polymorphism (mgc2-PCR-RFLP) assay enabled differential diagnosis of both components of a mixture of ts-11 and non-ts-11 DNA, detecting the field strain in the presence of a large excess of ts-11. The test was successfully applied in vivo for monitoring vaccinates in a ts-11 vaccine trial. In principle, the test may also be used to identify the 6/85 vaccine strain, which yields a 237-bp product, readily differentiated from the approximately 300-bp PCR product of all other strains tested. Further testing of field isolates will be necessary to determine the applicability of this test in the United States and other countries.     

Antibody responses in ostriches (Struthio camelus) vaccinated with commercial live and killed Newcastle disease vaccines

Three ostriches (Struthio camelus) were immunized with commercially available live and killed Newcastle disease (ND) vaccines for chickens and the antibody responses to the ND vaccines were evaluated by a virus-neutralization (VN) test. Primary vaccination with the live vaccine, B1, by eye drop was followed with two shots of alum-precipitated killed vaccine via subcutaneous injection in the neck. As a final booster, another live vaccine, Clone 30, was used by eye drop. A VN antibody titer, more than 1:10 was observed for 6 months. This is the first report on the use of a live vaccine by eye drop as a booster in ostriches as well as evaluating responses to ND vaccines using the VN test in this avian species.     

Differentiation of mycoplasma gallisepticum strains using molecular methods

Increasing use of Mycoplasma gallisepticum (MG) live vaccines has led to a need for the differentiation of MG strains. The MG strains MK-7, MS-16, S6, FS-9 and R strains and the MG live vaccine strain F were compared by random amplification of polymorphic DNA (RAPD) in this study. Using RAPD, different patterns were found among the MG strains. In addition to this, we examined the differentiating potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) primers targeted at the crmA, crmB, crmC, gapA, mgc2 and pvpA genes encoding cytadherence-related surface proteins. These proteins may take part in the pathogenesis of MG-induced disease. Differentiation of strain F is based on the identification of restriction enzyme sites in the PCR amplicons. Using HphI enzyme, crmC PCR amplicons produced different RFLP patterns. Digestion of amplicons of gapA-specific PCR with MboI enzyme also produced distinct patterns. Differences were observed among strains R and F by digestion of mgc2 PCR amplicons with HaelIl and VspI enzymes and digestion of pvpA PCR amplicons with AccI, PvulI and ScrFI endonucleases. This method can be used for the rapid differentiation of vaccine strain from wild strains. Differentiation of MG strains is a great advantage for diagnosticians or practitioners and it is useful for epidemiological studies.     

几种法氏囊活疫苗对鸡法氏囊损伤及免疫抑制作用的比较

探讨了鸡传染性法氏囊病（ＩＢＤ）活疫苗对鸡新城疫活苗免疫的影响。将四种ＩＢＤ活苗分别接种３０日龄ＳＰＦ公雏,４天后再免疫鸡新城疫ＬａＳｏｔａ系活疫苗,同时法氏囊损伤情况。试验结果表明,所选ＩＢＤ活疫苗对雏鸡法氏囊均有不同程度地损伤,并且诱导产生ＮＤ－ＨＩ抗体的时间被推迟。     

A chronicle of serologic response in commercial layer chickens to vaccination with commercial F strain Mycoplasma gallisepticum vaccine

Vaccination of multi-age layer operations, wherein one million plus commercial layer chickens are housed, has been spurious until the development of a self-propelled, constant-speed spray vaccinator. Still, even with its use, live Mycoplasma gallisepticum (MG) vaccinations have been questionable in terms of seroconversion. Using the vaccinator as a research tool over the past 5 yr, factors have been elucidated which impact seroconversion to one live MG vaccine in particular, the F strain of MG (FMG). These factors include the type of nozzle used to spray the vaccine, the temperature of the water used to rehydrate and administer the vaccine, and the pH and osmolarity of the fluid used to apply the vaccine. In the present study, one farm was monitored for its seroconversion rates over 4 1/2 yr, during which time the FMG vaccination protocol was amended as factors were identified that enhanced seroconversion rates. The results of this study showed that implementation and inclusion of the optimized factors into the vaccination protocol for FMG enhanced seroconversion rates because they went from an initial 50%-55% positive seroconversion rate to a consistent 100% positive seroconversion rate over the 56-mo study period.     

Will the effectiveness of the immunization of chicks with live Salmonella vaccines be affected by maternal antibodies?

In the progeny of breeder birds which had been vaccinated with live Salmonella Typhimurium and inactivated Salmonella Enteritidis vaccines, the caecal and systemic colonisation by a live Salmonella Enteritidis and a live Salmonella Typhimurium vaccine was studied. The efficacy of the oral immunisation of chicks from vaccinated and non-vaccinated breeders with a live Salmonella Enteritidis vaccine on day 1 of age was studied by an experimental challenge with Salmonella Enteritidis on day 30 of age. Antibody production of isotypes IgG, IgA and IgM was determined in sera and jejunum of the birds. Vaccination of parent birds resulted in an increase of the antibody concentration in sera and jejunum of the chicks. Own antibody production after administration of the live Salmonella vaccine to the day-old chicks was not detected until day 21 of life. Compared to controls, the number of vaccine organisms in the caeca of the progeny of vaccinated breeder birds was reduced by 0.5-1.5 log10 units. The reduction of the Salmonella Enteritidis vaccine was more pronounced than that of the Salmonella Typhimurium vaccine. However, the reduced colonisation by the live Salmonella vaccine strain did not impair the efficacy of the immunisation of the chicks. To ensure efficacy of the active oral immunisation of chicks from vaccinated parent birds with attenuated live Salmonella vaccines also in case where amounts of maternally transferred antibodies are even higher, it should be guaranteed that chicks take in via drinking water the recommended dose of the vaccine strain. In this connection, factors like the low intake of drinking water by very young chicks, the concentration of the vaccine organisms in the water and the survival of the vaccine should also be considered.     

三个厂家猪瘟活疫苗免疫效果比较试验

在同一条件下对3个厂家生产的猪瘟细胞源活疫苗进行了免疫效果评价试验,并与猪瘟传代细胞苗免疫效果进行比较。结果发现3个厂家生产的猪瘟细胞源活疫苗存在一定差异,2个厂家的免疫效果较好,1个厂家的免疫效果较差。猪瘟传代细胞苗免疫效果优于猪瘟细胞源活疫苗,猪瘟传代细胞苗免疫1次,抗体合格率高,持续时间长,猪瘟细胞源活疫苗要免疫两次才能获得比较好的效果。同时,我们发现高致病性猪蓝耳病活疫苗（JXA1-R株）对猪瘟抗体产生有一定影响。     

禽多杀性巴氏杆菌外膜蛋白和脂多糖的免疫保护效果

为确定禽多杀性巴氏杆菌(P.multocida)的保护性抗原外膜蛋白(OMPs)和脂多糖(LPS)在抵抗感染中的作用,本研究提取了禽P.multocida CVCC 474的OMPs和LPS成分,将该两种成分分别与弗氏佐剂混合制备免疫原,进行动物免疫。小鼠分为4组:OMPs组、LPS组、禽P.multocida弱毒活疫苗组和PBS对照组,每组16只。各组均免疫3次,每次间隔两周。间接ELISA检测免疫后小鼠血清特异性抗体水平,MTT法检测小鼠脾淋巴细胞增殖情况。以禽P.multocida强毒株CVCC 474进行攻毒,计算小鼠死亡数及保护率。实验结果表明,动物免疫后,OMPs组与LPS组免疫小鼠特异性血清抗体水平持续升高,与弱毒活疫苗组水平相近,与PBS组相比差异极显著(p<0.01)。脾淋巴细胞增殖试验表明,OMPs组、LPS组和弱毒活疫苗组的SI值极显著高于PBS对照组(p<0.01),但3个免疫组之间则无明显差异(p>0.05)。攻毒保护试验结果显示,OMPs免疫组的保护率与弱毒活疫苗相当,为8/10,高于LPS免疫组的保护率(7/10),表明OMPs作为研制禽P.multocida亚单位疫苗具有良好的...     

斑点杂交法在鸡毒支原体污染检测中的应用

试验根据GenBank中登录的鸡毒支原体16S rRNA基因序列,用DNAStar和Primer 5.0软件自行设计探针,并进行地高辛标记。采用带正电的尼龙膜对提取的鸡毒支原体DNA进行斑点杂交反应,建立最佳的反应条件,并采用建立的条件对市场上随机购买的鸡新城疫活疫苗进行检测。结果显示,运用斑点杂交反应可以从疫苗样品中检测到支原体的污染,检出率为23.3%(7/30)。表明该方法快速、特异性、敏感性高,对生物制品中支原体污染的快速检测具有较高的应用价值。     

A study on latency in calves by five vaccines against bovine herpesvirus-1 infection

Four bovine herpesvirus-1 (BHV-1) commercial vaccines, three of which (vaccines B, D, E) were modified live vaccines (MLV) and one (vaccine A) identified as a live strain of BHV-1 gE negative, were used for vaccination of calves, using three calves for each vaccine. Three months after vaccination calves were subjected to dexamethasone (DMS) treatment following which virus was recovered from calves inoculated with vaccine B and from those given vaccine D. No virus reactivation was obtained in calves, which received vaccines A or E. The DNA extracted from the two reactivated viruses was subjected to restriction endonuclease analysis. The restriction pattern of the isolate obtained from calves vaccinated with vaccine D differs significantly from that of the original vaccine, whereas the reactivated virus from calves given vaccine B conserved the general pattern of the original vaccine strain. For each reactivated virus in this experiment (B and D) as well as for the isolate obtained from calves vaccinated with a further MLV (vaccine C) in a previous trial, three calves were inoculated. No clinical signs of disease were detected in any of the inoculated calves during the observation period. When the nine calves were exposed 40 days later to challenge infection with virulent BHV-1, they remained healthy and no virus was isolated from their nasal swabbings. These results indicate that some BHV-1 vaccines considered in the project can establish latency in the vaccinated calves, however, the latency does not appear to interfere with the original properties of the vaccines in terms of safety and efficacy.     

Fingerprinting of Mycoplasma gallisepticum strains isolated from multiple-age layers vaccinated with live F strain

Mycoplasma gallisepticum (MG) isolates were obtained from three multiple-age commercial layer farms on which live F strain vaccine had been administered to each replacement flock for at least 2 years. All such isolates had restriction endonuclease DNA and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein patterns characteristic of F strain. These cultures also hybridized in dot blot assays with both the MG strain-specific and species-specific DNA probes. In contrast, the original MG isolate that came from one of the farms before vaccination began clearly was not F strain. These results suggest that continuous use of live F strain vaccine in each replacement pullet flock on multiple-age commercial layer sites will result in displacement of the original field strain of MG with the vaccine strain.     

Efficacy of live virus vaccines against infectious laryngotracheitis assessed by polymerase chain reaction-restriction fragment length polymorphism

The efficacy of four different commercial live vaccines (vaccines A, B, C, and D) against the infectious laryngotracheitis virus (ILTV) was assessed in specific-pathogen-free (SPF) chickens. SPF chickens were vaccinated intraocularly at 6 wk old with ILTV live vaccines and were challenged intratracheally with the N91B01 strain of virulent Korean ILTV 2 wk after vaccination. The immunity against ILTV live vaccines was assessed by the incidence of latent infection by the challenge virus in the chickens' tracheas and trigeminal ganglia, the reisolation rate of the challenge virus, and the clinical signs in the chickens challenged with the N91B01 strain of ILTV. The latent infection in chickens was assessed by nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Our data showed that the clinical signs and challenge virus isolation were negative in all chickens receiving four difference commercial ILTV live vaccines. The viral DNA of the vaccine strain, but not that of the challenge virus, was detected in chickens vaccinated with vaccine A by nested PCR-RFLP. The viral DNAs of both the vaccine and challenge strains were detected from chickens vaccinated with vaccines B, C, and D. This study showed that only vaccine A can protect chickens from latent infection with the field virulent ILTV. We speculate that the efficacy of infectious laryngotracheitis live vaccines to protect chickens from latent infection with virulent ILTVs can be assessed by nested PCR-RFLP analysis.