中国传染性法氏囊病病毒野毒株致病性的流行病学研究
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摘要
传染性法氏囊病(IBD)是一种危害幼龄鸡的一种急性、高度接触传染性疫病，其病原传染性法氏囊病病毒(IBDV)是一种双股RNA病毒。为了弄清我国各地流行的IBDV毒力程度，在3—7周龄SPF鸡进行人工感染的致病性试验，以此比较研究了1997—2001年期间从我国不同地区分离到的31个IBDV野毒株。结果表明，这些野毒株的毒力差异甚大。致病性最强的GX8／99可在易感年龄的SPF鸡引起70-92％的死亡率，是典型的超强毒。而人工接种YN2／99株的SPF鸡的死亡率几乎为零。其余毒株对SPF鸡的致死率则介于6—77％之间，但是所有毒株都能造成法氏囊和胸腺的显著萎缩。对具有不同致死率毒株的比较表明，近几年中，我国多数IBDV流行毒株的毒力都已远远超过标准毒IBDV，而接近超强毒。选择致病性最强的来自广西的GX8／99株，在对法氏囊悬液中病毒用鸡胚半数致死量(ELD_(50))定量测定后，在不同年龄的SPF鸡和商品代蛋用型坞多次重复地比较研究了该毒株的致病性。随鸡的日龄和接种剂量的不同，其致死率也有很大差异。对28-30日龄SPF鸡，病毒接种量为200个ELD_(50)时，感染后7日内死亡率最高可达60-90.5％(18／30和19／21)，感染量为20个ELD_(50)时死亡率亦可达53-92％(8／15和23／25)。以500个ELD_(50)感染28-104日龄期间SPF鸡，死亡率均在55.1％-67.2％。直至113-120日龄时，感染后仍有10-15％致死率。但在128日龄接种时，既不引起死亡也不表现任何症状，但都产生了抗IBDV抗体。人工种发病死亡鸡法氏囊出血程度随感染量和年龄而异。50日龄鸡接种2000个ELD_(50)后死亡的鸡，100％(12／12)的法氏囊严重出血，而在40日龄感染200个ELD_(50)后死亡鸡中，仅17％(3／18)发生出血。在2、3、4周龄带有母源抗
山东农业大学硕士学位论义（2003）
体的商品代蛋鸡，u 2000个ELD50病毒接种后只引起 10o（2／20）、35O
（7／20）和35o（7／20）的死亡率，但在5周龄时，随着母源抗体的消失，
仅接触感染的致死率就可达 61．3％（98／1 60）。而在 4和 5周龄商品代蛋
鸡人工接种200个ELD50病毒后，死亡率分别是sl．6－94．3％（62／76－33／35）
和 93．9－94O（31／33－47／50）。通过在 1900多羽鸡的试验表明，GX－8／99株
确实是一个超强毒IBDV毒株，表现为高死亡率（最高可达94％），易感
年龄延长至4月龄，中枢性免疫器官法氏囊出血严重和胸腺明显萎缩。此
外，GX－8／99株在 SPF鸡胚上增殖 2代的鸡胚毒（GX－8／99／HZ），每 0．lml
含 ELD。。可达 10b，对鸡胚的致死率大大提高，但对 SPF鸡的致病力较
囊毒显著降低。
为研究病毒的核酸分子结构与其致病性的关系，本研究选取了在致死
率上不同的4个IBDV野毒株，比较了它们的VPZ基因高变区共494个
碱基序列。结果表明，4个毒株对SPF鸡的致病性存在较大差异（G－94％
之间），但VPZ基因高变区的变异较小。SD－l／97、SD－3／98、JS－30／99相
互之间及其与＿IBDV参考株HK46间均具有相当高的同源性，结果似乎
说明IBDV毒株的致病性与VPZ基因高变区变异的关系不大。
在SPF鸡分别比较研究了两个中等毒力IBDV疫苗接种后对法氏囊和
胸腺的影响，以及对超强毒GX－8／99攻毒后的免疫保护力。结果表明，
用于试验的两个中等毒力疫苗虽然在SPF鸡可引起法氏囊和胸腺不同程
度的萎缩，但确实能够 100％保护己经免疫的 SPF鸡免于该超强毒 IBDV
人工感染造成的死亡。而且，还能减轻该株病毒感染后诱发的法氏囊的炎
症、出血和变性。这表明，在不受母源抗体干扰条件下，中等毒力的IBDV
弱毒疫苗仍能诱发对IBDV超强毒株GX－8／99的有效的免疫保护作用。
通过对 24日龄 SPF鸡人工感染传染性贫血病病毒（CAV）、36日龄
一3
我国传染。性法氏囊病病毒野每株致病性的流行病学研究
SPF鸡人工感染网状内皮增生病病毒（REV）、52日龄SPF鸡分别人工感
染马立克氏病病毒（MDV）、CAV、REV、MDV＋REV＋CAV后不同天数，
再用传染性法氏囊病病毒（IBDV）SD－3／98株做人工攻毒。结果表明，
不同日龄的 SPF鸡在感染 MDV、CAV、REV、MDV＋REV＋CAV后对 IBDV
强毒攻毒抵抗力下降。主要表现为在其它毒株共感染的作用下胸腺和法氏
囊的萎缩程度更为严重，而引起死亡率的差异不甚明显。
这只是对几种病毒共感染对IBDV致病性的影响进行了初步探讨。关
于 IR龄肉鸡感染其它免疫抑制病毒如 REV。J－ALV、REV＋J－ALV与
IBDV共感染的试验正在进行之中。并且有待于在不同年龄不同间隔做更
加深入的比较研究。
Pathogenicity of 31 field strains of infections bursal disease viruses(IBDV) isolated during 1997-2001 in China was compared in SPF chickens at different ages. The results indicated that the mortality induced by different strains varied very much. The mortality was also influenced by the age of chickens when infected. Chickens at 4 weeks of age were the most sensitive. Among 31 strains, GX-8/99 was the most virulent, which could cause as high mortality as 70%-94% in the relatively wide ranges of ages. In contract to GX-8/99, another strain YN2/99 caused no mortality. The left strains induced mortality in the ranges of 6%-77%. However, all strains could cause severe atrophy of the Bursa and thymuses, no matter they caused high or low mortality. The results also indicated that the virulence of the most IBDV field collected in recent years was between the typical very virulent and standard virulent strains.
A very virulent strain GX8/99 of IBDV was studied for its mortality in SPF chickens and commercial egg-type chickens at different age inoculated with bursal suspension of 20-2000 ELD50 in repeated experiments. It demonstrated that GX8/99 could induce high mortality between 55%-92% at the age of 4-13 weeks. It still caused some mortality of 10%-15% at the age of 120 days, but did not induce any mortality after 128 days of age. In commercial egg-type chickens of 4-5 weeks, GX8/99 of 2000 or 200 ELD50 caused mortality between 35%-94% from frock to frock. In 2 week-old commercial egg-type chickens with certain some level of maternal antibody to IBDV, the mortality was only 10%. On the other side, the replication ability of GX8/99 in embryoo increased when it was passed in embryoo for 2 passages. Its ELD50 would be 10 95/0.1ml. But the Pathogenicity in chickens is becoming lower.
From field strains of IBDV isolated in China during 1997-1999 were compared for their pathogenity and sequences in high viriable (hv) region of VP2 genes. It was indicated that GX8/99 was a real very vilulant IBDV strain
(vvIBDV), pathogenicity of other 3 strains was not as high as the typical wIBDV but still muck higher than reguler standard vIBDV strains. The hv region of VP2 of the 4 strains have a very high homology with wIBDV reference strain HK46, 96.8-99.5% at DNA level and 96.6%-100% at amino acid level, but a lower homology with vaccine strain D78, as only 91.7%-93.6% or 91.8-93.2% at DNA level or amino acid level respectively. It seems like that there was some relationship between hv region and puthogenicity. Relatively, the new wIBDV strains GX8/99 had less homology to wIBDV reference strain HK46 and other 3 field strains as 96.8%-97.2% at DNA or aa levels, than the homology among HK46 and 3 strains, strains GX8/99 more than 98.4%-98.6% at DNA or aa levels. There was a 100% homology which was among SD-3/98, JS30/99 and HK46 for the hv region of VP2. It was demoastrated that the new wIBDV strains GX8/99 had changed in both pathogenicity and VP2 gene.
To understand of the current commercial vaccine could also protect chickens from wIBDV challenges, two vaccines A and B were tested and compared for their protective immune efficacy in SPF chickens against wIBDV strain GX8/99. The results indicated that both commercial vaccines protected mothernal antibody-free chickens very effectively against GX8/99 challenge. But both vaccine theselves also incuce atrophy of the Bursa and thymuses.
Many reports has been demonstrated that the sub-clinic infections of immunosuppressive viruses in chickens are very common. And immunosuppression and other pathogenic changes could become very clear and severe only when dual or multiple viral infections and their interactions happened. In this study work, we tested the roles of multiple
immunosuppressive viral infection(as MDV. REV and CAV) in pathogenesis
in SPF chickens. But it was only a primary tries, the results were not conclusive.
山东农业大学硕士学位论义（2003）
体的商品代蛋鸡，u 2000个ELD50病毒接种后只引起 10o（2／20）、35O
（7／20）和35o（7／20）的死亡率，但在5周龄时，随着母源抗体的消失，
仅接触感染的致死率就可达 61．3％（98／1 60）。而在 4和 5周龄商品代蛋
鸡人工接种200个ELD50病毒后，死亡率分别是sl．6－94．3％（62／76－33／35）
和 93．9－94O（31／33－47／50）。通过在 1900多羽鸡的试验表明，GX－8／99株
确实是一个超强毒IBDV毒株，表现为高死亡率（最高可达94％），易感
年龄延长至4月龄，中枢性免疫器官法氏囊出血严重和胸腺明显萎缩。此
外，GX－8／99株在 SPF鸡胚上增殖 2代的鸡胚毒（GX－8／99／HZ），每 0．lml
含 ELD。。可达 10b，对鸡胚的致死率大大提高，但对 SPF鸡的致病力较
囊毒显著降低。
为研究病毒的核酸分子结构与其致病性的关系，本研究选取了在致死
率上不同的4个IBDV野毒株，比较了它们的VPZ基因高变区共494个
碱基序列。结果表明，4个毒株对SPF鸡的致病性存在较大差异（G－94％
之间），但VPZ基因高变区的变异较小。SD－l／97、SD－3／98、JS－30／99相
互之间及其与＿IBDV参考株HK46间均具有相当高的同源性，结果似乎
说明IBDV毒株的致病性与VPZ基因高变区变异的关系不大。
在SPF鸡分别比较研究了两个中等毒力IBDV疫苗接种后对法氏囊和
胸腺的影响，以及对超强毒GX－8／99攻毒后的免疫保护力。结果表明，
用于试验的两个中等毒力疫苗虽然在SPF鸡可引起法氏囊和胸腺不同程
度的萎缩，但确实能够 100％保护己经免疫的 SPF鸡免于该超强毒 IBDV
人工感染造成的死亡。而且，还能减轻该株病毒感染后诱发的法氏囊的炎
症、出血和变性。这表明，在不受母源抗体干扰条件下，中等毒力的IBDV
弱毒疫苗仍能诱发对IBDV超强毒株GX－8／99的有效的免疫保护作用。
通过对 24日龄 SPF鸡人工感染传染性贫血病病毒（CAV）、36日龄
一3
我国传染。性法氏囊病病毒野每株致病性的流行病学研究
SPF鸡人工感染网状内皮增生病病毒（REV）、52日龄SPF鸡分别人工感
染马立克氏病病毒（MDV）、CAV、REV、MDV＋REV＋CAV后不同天数，
再用传染性法氏囊病病毒（IBDV）SD－3／98株做人工攻毒。结果表明，
不同日龄的 SPF鸡在感染 MDV、CAV、REV、MDV＋REV＋CAV后对 IBDV
强毒攻毒抵抗力下降。主要表现为在其它毒株共感染的作用下胸腺和法氏
囊的萎缩程度更为严重，而引起死亡率的差异不甚明显。
这只是对几种病毒共感染对IBDV致病性的影响进行了初步探讨。关
于 IR龄肉鸡感染其它免疫抑制病毒如 REV。J－ALV、REV＋J－ALV与
IBDV共感染的试验正在进行之中。并且有待于在不同年龄不同间隔做更
加深入的比较研究。
Pathogenicity of 31 field strains of infections bursal disease viruses(IBDV) isolated during 1997-2001 in China was compared in SPF chickens at different ages. The results indicated that the mortality induced by different strains varied very much. The mortality was also influenced by the age of chickens when infected. Chickens at 4 weeks of age were the most sensitive. Among 31 strains, GX-8/99 was the most virulent, which could cause as high mortality as 70%-94% in the relatively wide ranges of ages. In contract to GX-8/99, another strain YN2/99 caused no mortality. The left strains induced mortality in the ranges of 6%-77%. However, all strains could cause severe atrophy of the Bursa and thymuses, no matter they caused high or low mortality. The results also indicated that the virulence of the most IBDV field collected in recent years was between the typical very virulent and standard virulent strains.
A very virulent strain GX8/99 of IBDV was studied for its mortality in SPF chickens and commercial egg-type chickens at different age inoculated with bursal suspension of 20-2000 ELD50 in repeated experiments. It demonstrated that GX8/99 could induce high mortality between 55%-92% at the age of 4-13 weeks. It still caused some mortality of 10%-15% at the age of 120 days, but did not induce any mortality after 128 days of age. In commercial egg-type chickens of 4-5 weeks, GX8/99 of 2000 or 200 ELD50 caused mortality between 35%-94% from frock to frock. In 2 week-old commercial egg-type chickens with certain some level of maternal antibody to IBDV, the mortality was only 10%. On the other side, the replication ability of GX8/99 in embryoo increased when it was passed in embryoo for 2 passages. Its ELD50 would be 10 95/0.1ml. But the Pathogenicity in chickens is becoming lower.
From field strains of IBDV isolated in China during 1997-1999 were compared for their pathogenity and sequences in high viriable (hv) region of VP2 genes. It was indicated that GX8/99 was a real very vilulant IBDV strain
(vvIBDV), pathogenicity of other 3 strains was not as high as the typical wIBDV but still muck higher than reguler standard vIBDV strains. The hv region of VP2 of the 4 strains have a very high homology with wIBDV reference strain HK46, 96.8-99.5% at DNA level and 96.6%-100% at amino acid level, but a lower homology with vaccine strain D78, as only 91.7%-93.6% or 91.8-93.2% at DNA level or amino acid level respectively. It seems like that there was some relationship between hv region and puthogenicity. Relatively, the new wIBDV strains GX8/99 had less homology to wIBDV reference strain HK46 and other 3 field strains as 96.8%-97.2% at DNA or aa levels, than the homology among HK46 and 3 strains, strains GX8/99 more than 98.4%-98.6% at DNA or aa levels. There was a 100% homology which was among SD-3/98, JS30/99 and HK46 for the hv region of VP2. It was demoastrated that the new wIBDV strains GX8/99 had changed in both pathogenicity and VP2 gene.
To understand of the current commercial vaccine could also protect chickens from wIBDV challenges, two vaccines A and B were tested and compared for their protective immune efficacy in SPF chickens against wIBDV strain GX8/99. The results indicated that both commercial vaccines protected mothernal antibody-free chickens very effectively against GX8/99 challenge. But both vaccine theselves also incuce atrophy of the Bursa and thymuses.
Many reports has been demonstrated that the sub-clinic infections of immunosuppressive viruses in chickens are very common. And immunosuppression and other pathogenic changes could become very clear and severe only when dual or multiple viral infections and their interactions happened. In this study work, we tested the roles of multiple
immunosuppressive viral infection(as MDV. REV and CAV) in pathogenesis
in SPF chickens. But it was only a primary tries, the results were not conclusive.
引文
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50. Kibenge, F.S. and V.Dhama. Evidence that virion-associated VP1 of avibimaviruses contains viral RNA sequences. Arch Virol. 1997.142:1227-1236.
51. Kibenge, F. S. and B. Qian. Sequence conservation in the RNA polymerase gene of infectious bursal disease viruses. Arch Virol. 1994. 134:441-449.
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