Some infectious and parasitic diseases in Oklahoma raptors.

Blood films and sera samples from wild Oklahoma raptors (Strigiformes--36 birds, 3 species; Falconiformes--50 birds, 7 species) were examined for hematozoa and tested for serologic antibody response to Newcastle Disease Virus (NDV), encephalitis (EEE and WEE), ornithosis, and influenza. Twenty-nine of 36 (80.5) Strigiformes and 24 of 50 (48.0%) Falconiformes showed the presence of one or more hematozoa. Serologic testing revealed the serum of one adult male red-tailed hawk positive for antibody to NDV and one additional adult male red-tailed hawk positive for antibody to type-A influenza.     

Recombinant Newcastle Disease Virus as a Vaccine Vector

A complete cDNA clone of the Newcastle disease virus (NDV) vaccine strain Hitchner B1 was constructed, and infectious recombinant virus expressing an influenza virus hemagglutinin was generated by reverse genetics. The rescued virus induces a strong humoral antibody response against influenza virus and provides complete protection against a lethal dose of influenza virus challenge in mice, demonstrating the potential of recombinant NDV as a vaccine vector.     

禽呼肠孤病毒感染对鸡法氏囊及免疫应答的影响

研究LY株禽呼肠孤病毒(ARV)感染1日龄SPF鸡后对法氏囊发育影响,对传染性法氏囊病毒(IBDV)、禽流感病毒(AIV)、新城疫病毒(NDV)疫苗免疫诱发的抗体的影响,及对强毒株IBDV致病作用的影响。结果表明,LY株ARV感染1日龄SPF鸡可引起法氏囊萎缩和部分淋巴细胞减少,但对增重及AIV和NDV疫苗免疫后抗体滴度却没有显著影响。ARV感染可降低弱毒IBDV疫苗免疫后的抗体反应,但对随后IBDV强毒株攻毒的抵抗力却与对照鸡无显著差异。经IBDV弱毒疫苗免疫后,再接种强毒株IBDV,不会引起死亡,但却仍能显著抑制对AIV、NDV疫苗免疫后的抗体滴度。然而,对于1~7日龄经ARV感染的鸡,IBDV强毒的这种免疫抑制作用又显著低于未经ARV感染的对照鸡。     

深圳市龙岗区流感监测及健康人群流感抗体水平分析

对深圳市龙岗区近几年流感病原学及人群流感抗体水平的分析,为流感防控提供科学依据。通过流感监测系统对流感进行病原学和血清学监测的结果显示,2007年流感病毒分离率为1.08%(3/277),2009年分离率为6.68%(56/838),两年流感病毒分离率差别具有统计学意义(χ2=13.03,P=0.000)。2007年健康人群血清样本中H1N1、H3N2、BY、BV流感抗体阳性率分别为60.5%、82.1%、59.3%、50.2%,2010年健康人群血清样本中H1N1、H3N2、BY、BV、甲型H1N1流感抗体阳性率分别为36.2%、37.6%、40.2%、21.9%、37.6%,2007年和2009年各亚型流感抗体阳性率具有统计学意义(P<0.05),两年中均表现H3N2亚型抗体阳性率最高,BV抗体阳性率最低。2007年和2010年H1N1、H3N2、BY、BV抗体阳性率均高于2010年,差别均具有统计学意义(P<0.01)。因此应加强流感病毒抗原变异株和人群流感水平的监测,对5岁以下及20岁以上的易感人群加强流感疫苗的接种,预防流感大流行。     

网状内皮增生病病毒感染SPF鸡对疫苗免疫反应的抑制作用

我国鸡群中网状内皮增生病病毒(REV)感染已相当普遍,但对其造成的实际危害却不太清楚。本研究结果表明,1日龄SPF鸡感染REV会显著抑制对新城疫病毒(NDV)和禽流感病毒(AIV,H5和H9)疫苗的免疫反应。1周龄用相应灭活疫苗免疫后3周、4周和5周,REV感染组对不同病毒疫苗免疫后的HI效价显著低于对照组。高剂量REV感染组的抑制作用大于低剂量感染组,但统计学差异不显著。REV感染可造成中枢免疫器官萎缩,REV感染组的胸腺、法氏囊与体重比显著低于对照组。本研究证明了,REV早期感染会干扰鸡群对NDV、AIV的免疫效果,特别是会严重干扰对AIV疫苗的免疫效果。     

新城疫病毒单克隆抗体的特性及应用

建立了8个分泌抗新城疫病毒(NDV)特异性单克隆抗体(McAb)的杂交瘤细胞株,根据它们的免疫生物学特性可以分为三种类型:(1)具有FA和ELISA特性(FN1、FN4、FN29、FN30、FN35、FNl22);(2)具有FA、ELISA和HI特性(FN7);(3)具有ELISA、HI特性和中和能力(FN106),根据FN30和FN106的ELISA试验,可将11个NDV毒株分为二种不同的抗原群,应用FN4-FITC,FN7-FITC和FN29-HRP试剂,对人工感染NDV和野外送检病例检测结果表明,单抗试剂的DFA阳性率(92.3％)高于病毒分离阳性率(87.2％),两种方法的符合率89.7％,这些单抗试剂用于临床诊断敏感性和特异性高,且方法快速、简便。     

Protective Efficacy of Newcastle Disease Virus Expressing Soluble Trimeric Hemagglutinin against Highly Pathogenic H5N1 Influenza in Chickens and Mice

Background

Highly pathogenic avian influenza virus (HPAIV) causes a highly contagious often fatal disease in poultry, resulting in significant economic losses in the poultry industry. HPAIV H5N1 also poses a major public health threat as it can be transmitted directly from infected poultry to humans. One effective way to combat avian influenza with pandemic potential is through the vaccination of poultry. Several live vaccines based on attenuated Newcastle disease virus (NDV) that express influenza hemagglutinin (HA) have been developed to protect chickens or mammalian species against HPAIV. However, the zoonotic potential of NDV raises safety concerns regarding the use of live NDV recombinants, as the incorporation of a heterologous attachment protein may result in the generation of NDV with altered tropism and/or pathogenicity.

Methodology/Principal Findings

In the present study we generated recombinant NDVs expressing either full length, membrane-anchored HA of the H5 subtype (NDV-H5) or a soluble trimeric form thereof (NDV-sH5³). A single intramuscular immunization with NDV-sH5³ or NDV-H5 fully protected chickens against disease after a lethal challenge with H5N1 and reduced levels of virus shedding in tracheal and cloacal swabs. NDV-sH5³ was less protective than NDV-H5 (50% vs 80% protection) when administered via the respiratory tract. The NDV-sH5³ was ineffective in mice, regardless of whether administered oculonasally or intramuscularly. In this species, NDV-H5 induced protective immunity against HPAIV H5N1, but only after oculonasal administration, despite the poor H5-specific serum antibody response it elicited.

Conclusions/Significance

Although NDV expressing membrane anchored H5 in general provided better protection than its counterpart expressing soluble H5, chickens could be fully protected against a lethal challenge with H5N1 by using the latter NDV vector. This study thus provides proof of concept for the use of recombinant vector vaccines expressing a soluble form of a heterologous viral membrane protein. Such vectors may be advantageous as they preclude the incorporation of heterologous membrane proteins into the viral vector particles.     

Further observations on the blood parasites of birds in Uganda

Birds from three National Parks (Bwindi Impenetrable, Kibale, and Queen Elizabeth) in western Uganda were surveyed during the dry season in July 2003 and investigated for hematozoa by microscopic examination of stained blood films. Of 307 birds examined, representing 68 species of 15 families and four orders, 61.9% were found to be infected with blood parasites. Species of Haemoproteus (15.3% prevalence), Plasmodium (20.5%), Leucocytozoon (40.1%), Trypanosoma (11.4%), Hepatozoon (2.6%), Atoxoplasma (0.3%), and microfilariae (3.9%) were recorded. Except for Haemoproteus spp. infections, the overall prevalence of hematozoa belonging to all genera was significantly higher in this study than was previously reported in Uganda. Thirty-six species of birds were examined for blood parasites for the first time and 112 new host-parasite associations were identified. Eighty-one were at the generic and 31 at the specific level of the hematozoa. Hepatozoon and Atoxoplasma spp. were detected for the first time in Uganda.     

表达H5亚型AIV血凝素的重组NDV构建及免疫原性

利用反向遗传技术获得表达H5亚型禽流感病毒(AIV)血凝素(HA)的新城疫病毒(NDV)。克隆NDV clone 30的全长基因,通过在NDV的融合蛋白基因和血凝素-神经氨酸酶(HN)基因之间插入编码高致病性AIV分离株A/chicken/italy/8/98(H5N2)的血凝素基因开放阅读框从而获得两株重组新城疫病毒NDVH5和NDVH5m。NDVH5感染的细胞可以检测到两种HA转录产物。对于重组病毒NDVH5m,NDV位于HA ORF的转录终止信号序列被沉默突变消除,产生2.7个全长HA转录产物的折叠,从而使修饰过的HA得到稳定地高表达。1日龄小鸡的脑内接种证实了两种重组病毒均无致病性。鸡群在NDVH5m诱导产生的NDV和H5亚型AIV HA特异性抗体的免疫力下能够免于致死剂量的NDV与高致病性AIV的感染。血清学研究结果表明NDVH5m免疫鸡群产生的抗体可结合NP蛋白抗体的检测从而用于区分免疫和感染AIV的动物。因此,NDVH5m重组病毒可作为抗NDV和AIV的"二联疫苗",也可成为控制AJ的标记疫苗。     

Newcastle disease virus-based live attenuated vaccine completely protects chickens and mice from lethal challenge of homologous and heterologous H5N1 avian influenza viruses

H5N1 highly pathogenic avian influenza virus (HPAIV) has continued to spread and poses a significant threat to both animal and human health. Current influenza vaccine strategies have limitations that prevent their effective use for widespread inoculation of animals in the field. Vaccine strains of Newcastle disease virus (NDV), however, have been used successfully to easily vaccinate large numbers of animals. In this study, we used reverse genetics to construct a NDV that expressed an H5 subtype avian influenza virus (AIV) hemagglutinin (HA). Both a wild-type and a mutated HA open reading frame (ORF) from the HPAIV wild bird isolate, A/Bar-headed goose/Qinghai/3/2005 (H5N1), were inserted into the intergenic region between the P and M genes of the LaSota NDV vaccine strain. The recombinant viruses stably expressing the wild-type and mutant HA genes were found to be innocuous after intracerebral inoculation of 1-day-old chickens. A single dose of the recombinant viruses in chickens induced both NDV- and AIV H5-specific antibodies and completely protected chickens from challenge with a lethal dose of both velogenic NDV and homologous and heterologous H5N1 HPAIV. In addition, BALB/c mice immunized with the recombinant NDV-based vaccine produced H5 AIV-specific antibodies and were completely protected from homologous and heterologous lethal virus challenge. Our results indicate that recombinant NDV is suitable as a bivalent live attenuated vaccine against both NDV and AIV infection in poultry. The recombinant NDV vaccine may also have potential use in high-risk human individuals to control the pandemic spread of lethal avian influenza.     

Hematozoa of hatch-year common mergansers from Michigan

Fifty-five hatch-year common mergansers (Mergus merganser) were sampled for hematozoa from Douglas Lake (Michigan, USA) on 17 July 1995. Forty-one (75%) were infected with hematozoa. Haemoproteus greineri and Leucocytozoon simondi were common, infecting 28 (51%) and 26 (47%) common mergansers, respectively. Plasmodium circumflexum infected two (4%) birds. The common merganser is a new host record for H. greineri and P. circumflexum. Intensity data indicate possible negative interspecific interaction between H. greineri and L. simondi.     

Subtyping of influenza virus A hemagglutinin with a hybridization microarray

An oligonucleotide microarray for influenza A hemagglutinin subtyping was presented. The number of probes for the determination of each subtype of hemagglutinin (H1-H13, H15, H16, pandemic flu H1N1) varied from 13 to 28. When testing the microarray using 40 type-A influenza virus isolates, the hemagglutinin subtypes were unambiguously determined for 36 specimens.     

Hematozoa from Southern African vultures, with a description of Haemoproteus janovyi sp. n.

This study represents the first major survey of avian hematozoa from southern Africa and the only one dealing with blood parasites of vultures. Blood smears from 506 Rhodesian, Botswanan and South African vultures (Hooded, White-headed, Lappetfaced, Cape Griffon and Whitebacked Vultures) were examined for hematozoa. Haemoproteus janovyi sp. n. was observed in 35.2% of the vultures, Leucocytozoon toddi in 0.8%, Plasmodium fallax in 0.6%, Atoxoplasma sp. in 1.4% and microfilariae in 0.2%. Hematozoan prevalence increased with age of the vultures. Only 2 of 133 nestlings sampled during the dry season had patent parasitemias (L. toddi). Haemoproteid prevalence in immature vultures was depressed during the dry season, whereas it was stable throughout the year in adults. The only species which nests on cliffs (the Cape Griffon Vulture) did not harbor hematozoa whereas the other species which nest and roost in trees were infected with at least one hematozoan species.     

Persistence of avian influenza viruses in lake sediment, duck feces, and duck meat

The persistence of 3 low-pathogenicity avian influenza viruses (LPAIV) (H4N6, H5N1, and H6N8) and one human influenza virus (H1N1) as well as Newcastle disease virus (NDV) and enteric cytopathogenic bovine orphan (ECBO) virus was investigated in lake sediment, duck feces, and duck meat at 30, 20, 10, and 0°C using a germ carrier technique. Virus-loaded germ carriers were incubated in each substrate, and residual infectivity of the eluted virus was quantified on cell culture after regular intervals for a maximum of 24 weeks. Data were analyzed by a linear regression model to calculate T(90) values (time required for 90% loss of virus infectivity) and estimated persistence of the viruses. In general, the persistence of all of the viruses was highest in lake sediment, followed by feces, and was the lowest in duck meat at all temperatures. For the avian influenza virus subtypes, T(90) values in sediment ranged from 5 to 11, 13 to 18, 43 to 54, and 66 to 394 days at 30, 20, 10, and 0°C, respectively, which were 2 to 5 times higher than the T(90) values of the viruses in the feces and meat. Although the individual viruses vary in tenacity, the survival time of influenza viruses was shorter than that of NDV and ECBO virus in all substrates. The results of this study suggest that lake sediment may act as a long-term source of influenza viruses in the aquatic habitat, while the viruses may remain infectious for extended periods of time in duck feces and meat at low temperatures, allowing persistence of the viruses in the environment over winter.     

Sensitive enzyme immunoassay for the rapid diagnosis of influenza a virus infections in clinical specimens

Samples of nasopharyngeal secretion (NPS) from 100 infants and small children admitted for acute respiratory disease during the period from January to March 1989 were examined for the presence of influenza A virus. All samples were tested by enzyme immunoassay (EIA), fluorescent antibody (FA) technique and by isolation in cell culture 3–6 h after they were obtained from the patients. Of 24 influenza strains found by isolation, 21 were detected by EIA and 19 were FA⁺. In comparison with virus isolation, EIA gave the following values: sensitivity 88 %, specificity 100 %, positive prognostic value (PPV) 100 %, and negative prognostic value (NPV) 96 %. A rabbit anti-influenza-A serum (A-13) was used as catching antibody and a monoclonal anti-influenza-A pool against NP protein was used as detector antibody in EIA. A-13 gave bands corresponding to influenza A core proteins (NP and M1) in Western blot (WB) studies when different H3N2 strains were employed as antigens. A-13 gave only a band corresponding to the NP protein when H1N1 strains were examined by WB. The detection level by EIA for both H3N2 and H1N1 strains precipitated by polyethylene glycol from tissue culture maintenance medium was 1–2 ng.     

禽流感和新城疫病毒二重荧光定量RT-PCR检测方法的建立

目的：建立二重荧光定量RT-PCR方法,用于禽流感病毒（AIV）和新城疫病毒（NDV）的检测。方法：根据AIV和NDV的基因保守序列,设计了AIV和NDV的2对特异性引物和2条用不同荧光基团标记的TaqMan探针;对反应条件和试剂浓度进行优化,建立了能够同时检测AIV和NDV的Z-重荧光定量RT-PCR方法。结果：所建方法特异性好,对AIV和NDV的检测敏感性均达到2000个模板拷贝数,比常规RT-PCR敏感性高100倍;抗干扰能力强,对AIV和NDV不同模板浓度进行组合,仍可有效地同时检测2种病毒。对保存的AIV或NDV鸡胚尿囊液及临床病料进行二重荧光定量RT-PCR检测,结果尿囊液检测的拷贝数均达到10^10/μL以上,临床病料的拷贝数为2．13x10^8-6．52x10^4／μL。结论：建立了用于检测AIV和NDV的二重荧光定量RT-PCR法,该方法特异、敏感、快速、可定量,对AIV和NDV的防制有重要意义。