禽腺病毒QU分离株的致病性及细胞适应性研究

QU分离株是一株类似产蛋下降综合征病毒,属于鸭腺病毒1型病毒。通过人工感染和细胞增殖试验,结果显示QU分离株接种无特定病原雏鸡未出现临床病症及生长发育障碍,不致死鸡胚,对鸭胚的致死率明显比引起产蛋下降的HS株低。QU株在鸡胚肝细胞、鸭胚成纤维细胞及鸡胚成纤维细胞上生长良好,产生典型细胞病变,且在鸡胚肝细胞上的增殖滴度最高,但不适应鸡胚肾细胞。这些数据说明QU株系对鸡具有低毒力的腺病毒,有可能用作禽用基因疫苗或基因治疗的候选病毒载体。     

Evaluation of a modified-live virus vaccine administered in ovo to protect chickens against Newcastle disease.

The B1 strain of Newcastle disease virus (NDV-B1), which is nonpathogenic for newly hatched chickens, killed embryos when it was used to inoculate chicken eggs at embryonation day 18. Treatment of NDV-B1 with an alkylating agent, ethylmethane sulfonate (EMS) markedly reduced the pathogenicity of the virus for 18-day-old chicken embryos. Eggs inoculated with the modified virus (NDV-B1-EMS) hatched, and the virus was isolated from lungs and spleen of 1-day-old chickens. The hatched chickens developed antibody to NDV and were protected against challenge exposure (at 4 weeks of age) with a highly virulent GB-Texas strain of NDV. Presence of maternal antibody to NDV in embryonating eggs did not influence the protective ability of NDV-B1-EMS, which also induced protective immunity when administered to 4-week-old chickens. The 50% protective dose of NDV-B1-EMS in maternal antibody-negative and -positive embryos was calculated to be 10.77 and 17.70 embryo 50% lethal doses, respectively. Results of the study indicated that NDV-B1-EMS may be used as an embryo vaccine to protect chickens against Newcastle disease.     

Pathogenicity of Mycoplasma synoviae in chicken embryos.

Pathogenicity of Mycoplasma synoviae (MS) was examined in specific-pathogen-free (SPF) white leghorn chicken embryos. Six isolates of MS were inoculated into 7-day embryos via the yolk sac. Isolates were evaluated for gross and microscopic lesions through 19 days' incubation and for embryo lethality through 20 days' incubation. Isolates in decreasing order of lethality, from lowest to highest 50% embryo lethal dose, were WVU 1853, K1968, K1858, FMT, 92D8034, and F10-2AS. Embryo lethality was consistent with lesion incidence and severity. Embryo lethality did not correlate with previous results regarding pathogenicity of these same six isolates in SPF broiler chickens.     

Pathogenicity of embryo-adapted serotype 2 OH strain of infectious bursal disease virus in chickens and turkeys

The pathogenicity of serotype 2 OH strain of infectious bursal disease virus (IBDV) to specific-pathogen-free (SPF) chicken embryos and 2-wk-old SPF chickens and turkey poults was investigated. The virus was pathogenic for chicken embryos after five passages as evidenced by pathologic changes in inoculated embryos. The embryo-adapted virus was not pathogenic for 2-wk-old SPF chickens and turkey poults as indicated by lack of clinical signs, gross or microscopic lesions in the bursa of Fabricius of inoculated birds. Bursa-to-body-weight ratios of the inoculated chickens and turkey poults were not significantly different from those of uninoculated controls. Virus-neutralizing antibodies to serotype 2 IBDV were detected in inoculated chickens and turkeys. Results of this study indicated that the embryo-adapted serotype 2 OH IBDV isolate that is pathogenic for chicken embryos is infectious but not pathogenic in chickens and turkeys.     

In situ production of interferon in tissues of chickens exposed as embryos to turkey herpesvirus and Marek's disease virus

Chicken eggs at embryonation day (ED) 18 or newly hatched chicks were inoculated with turkey herpesvirus (HVT), Marek's disease virus (MDV), or virus-free diluent and, at intervals after inoculation, tissue homogenates of virus-exposed and virus-free chickens or chicken embryos were examined for interferon (IFN) activity. Homogenates of lung, thymus and spleen specimens from chickens given HVT at ED 18 had IFN activity. Activity of IFN in the lungs was studied further. Homogenates of lung specimens from chickens exposed to HVT at hatching also had IFN activity, although the concentration of IFN was lower than that in chickens given HVT at ED 18. The pathogenic isolates of MDV (JM-MDV), but not the attenuated (Md11/75C-MDV) or nonpathogenic (SB1-MDV) isolates, inoculated at ED 18 also induced high lung IFN activity. Exposure to a combination of HVT and SB1-MDV induced IFN activity comparable with that in chickens given HVT alone. The IFN activity in homogenates of lung specimens from virus-exposed chickens was species specific and heat and pH stable, but was destroyed by trypsin treatment. Occasionally, low IFN activity also was detected in homogenates of tissue specimens from virus-free chickens or chicken embryos. This IFN activity could have been produced constitutively or may have been induced by substances (inducers) in the environment.     

Effect of Mycoplasma gallinarum on embryonated village chicken eggs

The effect of Mycoplasma gallinarum isolated from village chickens on embryonated village chicken eggs was investigated. Seven-, 10−, 12− and 18-day-old embryos were inoculated with medium containing 10⁶ colony forming units of M gallinarum or with uninfected medium and incubated at 37°C until they hatched. There was no significant difference between the infected embryos which failed to hatch and those which hatched or between them and the group inoculated with medium only. A much higher percentage of the eggs inoculated as 18-day-old embryos hatched than of those inoculated at seven, 10 and 12 days old. The hatchability of the uninoculated village chicken eggs used in the four trials ranged from 60 to 100 per cent. Mycoplasmas were isolated from the oesophagus, trachea and yolk of the infected chicks and from the dead-in-shell embryos.     

改良背主动脉注射法对鸡胚发育与转基因表达效率的影响

背主动脉注射是生产转基因鸡的经典方法,该方法不需换壳培养,但壳内注射技术难度大,并且无法实时观察后期胚胎发育情况.本实验对背主动脉注射法进行了改进,将增强型绿色荧光蛋白(EGFP)腺相关病毒(Adeno-associated Virus,AAV)壳外注射至150枚HH 14～16期(Hamburger-Hamilton...     

The influence of methimazole on the thyrotrophic and peripheral activity of thyrotrophin and thyrotrophin-releasing hormone in the chick embryo and growing chicken

Plasma concentrations of thyroxine (T₄) and triiodothyronine (T₃) were profoundly depressed both in chick embryos and growing chickens after methimazole (MMI) treatment. There was no response of T₄ and T₃ levels to TRH or TSH injections in the MMI group, either in embryos or growing chickens.

Peroxidase activity measured in the thyroid gland was significantly higher in embryos and growing chickens treated with MMI. However, neither TRH nor TSH affected this activity 2 hr after injection in either control or the MMI-treated group.

Hepatic 5′-monodeiodinase activity was significantly stimulated in the MMI-treated groups of embryos and growing chickens but only when additional sulphydryl groups (DTT) were provided. In embryos, monodeiodination activity 2 hr after TSH injection was not significantly different from control values for either DTT-stimulated or unstimulated conditions within the control and MMI-infused groups. However, in both control and MMI-treated embryos monodeiodination activity significantly increased 2 hr after TRH injection. In the growing chickens, monodeiodination activity 2 hr after TRH or TSH injection was not significantly different from control values in either stimulated or unstimulated conditions of each group.     

禽脑脊髓炎的调查及病毒分离

对陕西省宝鸡、渭南等地的24个鸡群进行了禽脑脊髓炎（AE）的调查,所调查鸡群都有产蛋下降史或产蛋正在下降,下降幅度平均为17.2%。用琼扩试验（AGP）检测所采集的256份血清,AE阳性率平均高达85.6%。有10个鸡群阳性率达100%。跟踪调查2个种鸡群,发现孵出的雏鸡从3日龄开始发病,而且有AE的症状,采发病雏鸡的脑组织,接种6龄鸡胚分离病毒,连传3代,琼扩检测分离毒,呈AE抗原阳性,人工感染1日龄雏鸡复制出与原发病鸡群相同的疾例,表明分离毒是AE病毒。     

Detection of chicken anemia virus DNA in embryonal tissues and eggshell membranes

Chicken infectious anemia virus (CIAV) is a ubiquitous and highly resistant virus of chickens that causes anemia and death in chicks less than 3 wk of age and immunosuppression in chickens older than 3 wk of age. The production of specific-pathogen-free eggs free of CIAV is essential for research and vaccine production. Currently, flocks are screened for CIAV by antibody tests to ensure freedom from CIAV infection. Recent evidence, however, indicates that chickens may carry and vertically transmit CIAV DNA independently of their antibody status. In this study, we tested embryos and eggshell membrane residues by nested polymerase chain reaction (PCR) as a sensitive method of detecting CIAV DNA. CIAV DNA could be detected in the blastodisks and semen obtained from antibody-positive and -negative chickens. Examination of different tissues between 18 and 20 days of incubation indicated that many but not all organs of individual embryos were positive. The lymphoid organs and gonads had the highest incidence of CIAV DNA, which was significantly different (P < 0.05) from the incidence in the liver. Eggshell membrane samples from embryos or newly hatched chicks were an excellent noninvasive source for the detection of CIAV DNA, identifying significantly more positive embryos than did pooled lymphoid organs. The use of dexamethasone injections as a method to improve the detection of carrier birds did not result in an increase of vertical transmission or cause seroconversion in the treated hens. A combination of testing eggshell membrane residues at hatch and periodic testing of blood DNA by nested PCR can be used to identify chickens carrying CIAV DNA and may be used to eradicate carrier birds.     

Embryo vaccination against Marek's disease with serotypes 1, 2 and 3 vaccines administered singly or in combination

Marek's disease virus (MDV) vaccines of serotypes 1 and 2 administered in 18-day-old embryonated eggs induced better protection against post-hatch challenge at 3 days with virulent MDV than vaccines given at hatch. Embryonal vaccination with a polyvalent vaccine containing equal quantities of serotypes 1 and 2 of MDV and serotype 3 virus (turkey herpesvirus, HVT) was also significantly more effective than post-hatch vaccination. These and earlier results indicate that protective efficacy of single or combined Marek's disease vaccine serotypes against post-hatch challenge at 3 days can be substantially improved if the vaccines are injected into 18-day embryos rather than at hatch. Injection of vaccines of serotypes 1 or 2 into embryonated eggs or hatched chicks did not cause detectable gross or microscopic lesions in chickens. Vaccine viruses of serotypes 1 and 2 could be isolated from spleen cells of chickens 1 week post-vaccination, and the titer of recoverable viruses was higher in chickens that received the vaccines at the 18th day of embryonation than in chickens vaccinated at hatch. Although embryo vaccination with HVT usually provided better protection than post-hatch vaccination against early post-hatch challenge with variant pathotypes of MDV, the protection was poor regardless of vaccination protocol. If challenge with variant pathotypes of MDV was delayed until embryonally or post-hatch HVT-vaccinated chickens were 21 days of age, protection of chickens by HVT was not enhanced. Thus, resistance induced by embryonal vaccination with HVT was qualitatively similar to that induced by post-hatch vaccination with this virus.     

绵羊肺炎支原体人工感染SPF鸡胚的研究

为探索SPF鸡胚作为绵羊肺炎支原体实验室感染模型的可行性,本试验用不同浓度绵羊肺炎支原体（10⁸、10⁹、10¹⁰ ccu/mL）经由卵黄囊和尿囊腔两个部位接种7日龄SPF鸡胚,通过统计鸡胚死亡情况和不同鸡胚组织样品中绵羊肺炎支原体检测阳性率（PCR检测和支原体分离鉴定）,确定绵羊肺炎支原体鸡胚感染方式、感染剂量和最佳分离部位,再用3株不同来源的绵羊肺炎支原体分离株感染鸡胚,观察其对鸡胚的致病力。结果表明,绵羊肺炎支原体感染鸡胚最佳接种途径为卵黄囊接种,感染剂量为10⁹ ccu/mL、0.2 mL/只,最佳分离部位为卵黄液。3株支原体均能感染和致死鸡胚,并均能从卵黄液中分离到绵羊肺炎支原体,但对鸡胚的致病性存在一定差异：FL3株致鸡胚死亡率为45%,略高于MoGH3-3株（40%）,二者均高于A3株（25%）,但差异均不显著（P>0.05）;FL3株鸡胚检测阳率为100%,高于MoGH3-3株（85%）及A3株（90%）,但差异也不显著（P>0.05）。本试验初步确定了绵羊肺炎支原体可感染和致死SPF鸡胚,不同分离株对SPF鸡胚致病力有差异,表明SPF鸡胚可作为下一步建立绵羊肺炎支原体实验室感染模型的候选,为绵羊肺炎支原体致病性研究和疫苗研制奠定基础。     

Experimental Infection of Mycoplasma ovipneumoniae in SPF Chicken Embryos

In order to evaluate the SPF chicken embryos as a candidate of the experimental model of Mycoplasma ovipneumoniae (Mo) infection,the 7-day-old SPF chicken embryos were infected with different concentrations of Mo(10⁸,10⁹ and 10¹⁰ ccu/mL) by injection of vitelline fluid and allantoic cavity.The mortality and Mo detection rate (PCR detection and isolation) of infected chicken embryos were employed to evaluate the optimal inoculation route,dose and sampling site.Three different isolates of Mo were submitted to injection of chicken embryos for pathogenicity comparison.The results showed that the optimal inoculation route and dose were vitelline fluid injection with 0.2 mL (10⁹ ccu/mL) Mo per embryo and the sampling site for detection was also vitelline fluid.Three Mo isolates revealed good infectivity and lethality to the chicken embryo.The chicken embryos mortality caused by FL3 strain (45%) and MoGH3-3 (40%) were both higher than that by A3 strain (25%),but with no significance (P>0.05).The detection rate of Mo from the chicken embryos in FL3 group (100%) was higher than that of MoGH3-3 strain(85%) and that of A3 strain (90%),but with no significance(P>0.05).This experiment preliminarily proved that the SPF chicken embryos could be infected and partially killed by Mo,and the virulence of different strains of Mo to the chicken embryos was different.The results provided the data for further establishment of the chicken embryo infection model of Mo,which would benefit the research on Mo pathogenicity and vaccine development.     

新城疫病毒山东强毒株的分离鉴定

从山东流行烈性传染病的鸡群中分离出7株具有血凝活性的病毒，该7株病毒的血凝活性均能被新城疫La Sota株标准阳性血清所抑制，但病毒不能被中和，仍能致死鸡胚。经分离鉴定，分离毒均为新成疫病毒株。通过鸡胚半致死量（ELD50）、最小致死量致鸡胚的平均时间（MDT）、1日龄雏鸡脑内致死指数（ICPI）、6周龄雏鸡静脉致死指数（IVPI）、血凝解脱及血凝素稳定性等试验表明：7株分离病毒均为新城疫病毒强毒性，其毒力与标准强毒株F48E9株相似。     

Comparison of in ovo and post-hatch vaccination with particular reference to infectious bursal disease. A review

In ovo vaccination is an alternative approach to post-hatch vaccination of chickens, particularly in broilers. Vaccination at embryonation day 18 helps to 'close the window' of susceptibility i.e. the time between vaccination and early exposure to infectious agents compared with post-hatch vaccination. Attempts on embryonal vaccination as a mode of vaccine delivery were approached from the observation that chickens already develop certain immunologic functions before hatching. The immune system in birds begins to develop early during embryogenesis and various immune reactions have been induced in the late stage chicken embryos. Compared with post-hatch vaccination, in ovo vaccination stimulates both the innate and adaptive immune responses with the advantage that because of the prenatal immunization, in ovo vaccinated chicks have developed an appreciable degree of protection by the time of hatch. Effects of maternal antibodies on vaccines to be used for in ovo vaccination can be prevented by developing vaccines that are insensitive to maternal antibodies. It has been described that vaccination of chicken embryos at embryonation day 18 did not significantly affect the immune competence of hatched chickens. The apparent absence of tolerance in chicks hatched from embryos exposed to an antigen at the late stage of embryonation implies the feasibility of in ovo vaccination. Investigations on in ovo vaccination to produce safe and efficient vaccines are still in progress. Currently a large number of vaccines are under investigation for viral, bacterial and protozoal diseases.     

Detection of the EGFP sequence in breast muscle of 3‐year‐old chicken after transfection using sonoporation

In our continuing effort to generate transgenic chickens, sonoporation was chosen to insert an exogenous gene into the chicken genome. An EGFP expression vector (pCAG‐EGFPac) and microbubbles were injected into the central disc of stage‐X blastoderm or the germinal crescent of stage‐4 embryos, followed by ultrasonic vibration. Nineteen chicks out of 108 treated embryos hatched, six females and six males out of these 19 chicks grew to sexual maturity and two females and three males lived for 3 years. Genomic DNA from 17 out of 35 gonads from embryos and chicks that died before sexual maturity was EGFP‐positive by PCR. No EGFP sequence was detected in the genomic DNA of 322 embryos from six sexually mature females and the semen from four sexually mature males by PCR. When genomic DNA was obtained from various tissues of five 3‐year‐old chickens, the EGFP sequence was amplified from the genomic DNA of the breast muscle of a female (No. 85). The above sequence was subjected to DNA sequencing and verified to be the EGFP sequence. These results showed that sonoporation is an effective tool for the transduction of exogenous genes into chicken embryos for the generation of transgenic chickens.     

Characterization of a velogenic Newcastle disease virus isolated from broilers in Saudi Arabia

A highly virulent Newcastle disease virus (SA84) was isolated from a large broiler operation in Saudi Arabia. The mean death time of chicken embryos given the minimum lethal dose, the pathogenicity of the isolate for 8-week-old chickens, the plaque characteristics, and the intracerebral pathogenicity index indicated that the isolate is of the viscerotropic velogenic pathotype.     

Clinical effects, virological and serological responses in chickens following in-ovo inoculation of reticuloendotheliosis virus

Six-day-old embryonated specific pathogen free chicken eggs were inoculated with reticuloendotheliosis virus (REV) into the yolk sac and were incubated until they hatched. The hatchability of eggs inoculated with REV was significantly less (P less than 0.025) than that of media-inoculated controls. Although there were no significant differences in the body weights of these chickens at hatching, there were differences (P less than 0.001) at 6, 25 and 51 days of age between the infected and control chickens. Six of 10 chickens hatched from eggs inoculated with REV had feathering defects at 6 days of age. All chickens hatched from infected eggs had cell-free viraemia and antigenaemia, but not precipitating antibodies. Some of these chickens had very low neutralising antibody titres (less than 45) when examined at 25 and 37 days of age, as did all 10 chickens at 51 days of age. A low rate of horizontal transmission was indicated by the detection of antibodies at 37 and 51 days of age in chickens running in contact with the chickens hatched from eggs inoculated with REV.     

Immunohistochemical and polymerase chain reaction studies in Neospora caninum experimentally infected broiler chicken embryonated eggs

The diagnostic characteristics of immunohistochemistry (IHC) and polymerase chain reaction (PCR) methods were studied in the tissues of broiler chicken embryos experimentally infected by Neospora caninum. An infection with N. caninum NC-1 isolate was conducted in 70 broiler chicken embryonated eggs randomly divided into seven equal groups. After 8 days of incubation, six groups were inoculated with 10, 10(2), 10(3), 10(4), 10(5), and 10(6) doses of tachyzoites/embryonated egg. The 7th group was considered as control. The mortality rate and pathological changes of the dead embryos and hatched chickens up to 60 days old were noticed. Consecutive sections to those used for histopathological examination including the liver, heart, brain, and chorioalantoic (CA) membrane were subjected to IHC. The intensity and distribution of the immunostaining was graded as highly to mildly positive. For PCR procedure, DNA was extracted from 50mg of the tissues and primer pair Np21/Np6 was used for amplification of the Nc-5 gene. The results of the immunosignaling ranged from variable degrees of mild to moderate staining as dark-brown to brown and coarsely to finely granular, mostly within the cytoplasm of infected cells such as the endothelial cells of blood vessels. The parasite aggregation was more predominant in the heart than other tissues. Immunoreactivity for N. caninum antigen was multifocally moderate positive in the heart, liver and CA of the 10(3) dose, and also heart, liver, brain and CA of the 10(4) dose. IHC showed mildly positive in the liver and heart of the chicken embryos infected with 10 and 10(2) tachyzoites, as well. The results of the PCR confirmed the existence of the parasite in all of the examined tissues from the 10(3) and 10(4) doses. In conclusion, the results indicate a good agreement between IHC and PCR in diagnosis of neospora antigen in the infected tissues.     

interactions between escherichia coli and Newcastle disease virus in chickens

We investigated the interaction between Newcastle disease virus (NDV) and Escherichia coli in cell cultures, embryonated eggs, and 8-wk-old chickens. We measured the interactions on the basis of bacterial adherence and NDV hemagglutination titer in chickens, chicken embryos, and chicken embryo cell culture. Depending on the inoculation order of E. coli, a significant alteration of the growth of NDV was observed in both chickens and chicken embryos. When certain strains of E. coli were given before NDV exposure, the virus titers were lowered. In chickens, the mean virus titer was significantly (P < 0.05) lowered in the crop, the proventriculus, the gizzard, and the jejunum. However, there were no significant differences (P < 0.05) between the two groups for NDV titers in the duodenum, ileum, and cecum. In chicken embryos, when E. coli serotypes O78 and O119:B14 were inoculated before NDV exposure, the mean NDV titers were significantly (P < 0.5) lowered. However, there were no significant differences (P < 0.05) in NDV titer between the two groups when E. coli serotypes O78:K80:NM and O1ab:K NM were inoculated 24 hr before NDV exposure. When NDV was given prior to E. coli exposure, NDV titer was higher in both chickens and chicken embryos. In chickens, when NDV was given 48 hr before E. coli inoculation, NDV was detected in the proventriculus, gizzard, jejunum, ileum, and cecum, whereas no virus was detected in the control groups (NDV only). In the crop, NDV was detected at a significantly (P < 0.05) higher titer in the E. coli-inoculated group when compared with the control group that received NDV alone. In chicken embryos, virus titer was significantly (P < 0.05) higher when NDV was given 24 hr before E. coli inoculation for all three NDV strains used (Ulster and V4 strains). Adherence of E. coli to chicken embryo kidney (CEK) cells was significantly higher (P < 0.05) when the CEK cells were infected first with NDV and then by E. coli. The mean bacterial count per microscopic field in NDV-uninfected monolayers was eight compared with 112 for the NDV-infected monolayers. In approximately 10% of the fields in NDV-infected monolayers, the bacteria were too numerous to count.