Experimental Infection of Sheep and Goats with Caprine Arthritis-Encephalitis Virus

Sir, — The isolation of caprine arthritis-encephalitis virus (CAEV) from the carpal joint of a yearling goat with chronic interstitial pneumonia is reported in this issue of the Journal.⁽³⁾ We wish to report on the experimental infection of sheep and goats with this isolate of CAEV. Two 20-month-old Romney lambs (483, 485) and two 2-month-old feral goat kids (43, 45) were inoculated with CAEV suspension (10^4.0 median tissue culture infective doses [TCID₅₀ml]). Each animal was inoculated with 1.0 ml of virus suspension by each of the following routes: intravenous, intracerebral into the right cerebral hemisphere and intra-articularly into the right radio-carpal joint.⁽⁴⁾ An additional control lamb (490) and goat kid (44) were inoculated in identical fashion with cell culture fluid from uninfected goat synovial membrane cells.     

Seroreactivity of Peruvian sheep and goats to small ruminant lentivirus-ovine progressive pneumonia virus

Sera from 3,369 sheep and 1,394 goats in Peru were examined by agar-gel immunodiffusion for antibodies to ovine progressive pneumonia virus (OPPV). The point prevalence rates for antibodies to OPPV in sheep were 1.7% to 40.6% (mean, 19.02%) in the 7 flocks studied, whereas for goats, the point prevalence rates for antibodies that cross-reacted with OPPV in 12 herds were 0.0% to 45.1%. For sheep, a direct association between increasing age and increasing seroreactivity to OPPV was established, and there was evidence to indicate that lambs born to primiparous ewes and raised separated from all other sheep after they were weaned may have been less likely to become infected with OPPV than those lambs born to multiparous ewes and not separated from other sheep after they were weaned. For goats, antibodies to OPPV were detected in 7 of 12 herds studied, the highest infection rate being present within a herd in the Lima department (district).     

绵羊进行性肺炎病毒与山羊关节炎一脑炎病毒的血清学交叉反应的研究

本实验用琼脂凝胶免疫扩散试验（ＡＧＩＤＴ）和免疫印迹试验（ＩＢＡ）对实验感染绵羊进行性肺炎病毒（ＯＰＰＶ）的山羊血清与山羊关节炎—脑炎病毒（ＣＡＥＶ）抗原以及实验感染ＣＡＥＶ的绵羊血清与ＯＰＰＶ抗原的交叉反应进行了研究。４只接种ＯＰＰＶ的山羊中有一只山羊的血清可与ＣＡＥＶ琼扩抗原发生交叉反应,并在免疫印迹试验中可识别ＣＡＥＶ的ｇｐ４４、ｐ３５和ｐ２８。２只接种ＣＡＥＶ的绵羊中有一只绵羊的血清可与ＯＰＰＶ琼扩抗原发生交叉反应,并在免疫印迹试验中可识别ＯＰＰＶ的ｇｐ４４和ｐ２８。以上的交叉反应结果表明ＯＰＰＶ与ＣＡＥＶ的抗原之间具有密切的相关性,这对于ＯＰＰＶ通过山羊和ＣＡＥＶ通过绵羊的传代研究是非常重要的,并对将来的免疫预防策略具有重要的指导意义。     

Intrauterine transmission of ovine progressive pneumonia virus

Ovine fetuses, newborn lambs, and ovine colostrum were examined for ovine progressive pneumonia virus. The lambs and colostrum were also examined for specific antibody. Virus was isolated from 1 fetus, from 2 newborn lambs, and from most samples of colostrum. The fetus was about 100 days old and was carried by a seronegative ewe in contact with seropositive sheep. Both newborn lambs were carried by seropositive ewes. One lamb was dead at birth; the other lamb was normal and had not nursed. Antibody specific for the virus was present in the colostrum of 12 of 14 seropositive ewes and in the serum of 8 of 11 lambs that had nursed seropositive ewes, but not in the serum of lambs that had not nursed.     

Clinical and laboratorial findings in pregnant ewes and their progeny infected with Border disease virus (BDV-4 genotype)

To evaluate the pathogenicity of local isolates of ovine pestiviruses (BDV-4 genotype), 13 virus- and antibody-negative, artificially inseminated pregnant ewes were challenged on days 108 (5 ewes), 76 (5 ewes) and 55 of pregnancy (3 ewes) with 2 ml of ovine pestivirus containing 10⁶ TCID₅₀. Viraemia was detected by RT-PCR from 2 to 15 days pi in most ewes. No abortion due to the infection was observed but the number of stillbirths was high (32%), and bodyweight at lambing was significantly reduced compared to the experimental flock of origin used as control. Clinical symptoms in live lambs consisted on tremors, gait anomalies and inability to stand unaided. Skeletal abnormalities (brachygnathia, prognathia, arthrogryposis) were present in 44% of the lambs. Only 20% of the lambs were clinically normal. RT-PCR was a very sensitive technique compared to antigen ELISA in detecting viral presence in experimentally infected ewes and their progeny.     

Evidence of decreased concanavalin A induced suppressor cell activity in the peripheral blood and pulmonary lymph nodes of sheep with ovine progressive pneumonia

Concanavalin A (Con A) induced suppressor cell activity was evaluated in a group of ovine progressive pneumonia virus-antibody positive sheep (OPPV+). Decreased levels of suppressor activity were observed in the peripheral blood and regional pulmonary lymph nodes of animals with clinical and pathological evidence of ovine progressive pneumonia (OPP) when compared to animals with no lesions and animals with caseous lymphadenitis involving the lungs and pulmonary lymph nodes. Decreased levels of Con A stimulated lymphocyte proliferation was also observed in the peripheral blood and iliac lymph nodes of sheep with OPP. Sheep with OPP were found to be hypogammaglobulinemic. Sera from OPPV+ sheep had no effect on T and B cell mitogen stimulated responses of lymphocytes from sheep seronegative for antibodies to ovine progressive pneumonia virus (OPPV-) when compared to normal sheep serum.     

Experimental Dual Infection of Specific Pathogen‐Free Pigs with Porcine Reproductive and Respiratory Syndrome Virus and Pseudorabies Virus

Twenty 6‐week‐old specific pathogen‐free pigs were divided into four groups. On day 0 of the experiment, PRRSV–PRV (n = 6) and PRRSV (n = 4) groups were intranasally inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) (10^5.6 TCID₅₀). On day 7, the PRRSV–PRV and PRV (n = 6) groups were intranasally inoculated with pseudorabies virus (PRV) (10^3.6 TCID₅₀). Control pigs (n = 4) were kept as uninoculated negative controls. Half of the pigs in each group were euthanized and necropsied on day 14 or 21. Clinical signs such as depression and anorexia were observed in the PRRSV–PRV and PRV groups after inoculation with PRV. Although febrile response was observed after virus inoculations, the duration of that response was prolonged in the PRRSV–PRV group compared with the other groups. The lungs in the PRRSV–PRV group failed to collapse and were mottled or diffusely tan and red, whereas the lungs of the pigs in the other groups were grossly normal. Histopathologically, interstitial pneumonia was present in all PRRSV‐inoculated pigs, but the pneumonic lesions were more severe in the PRRSV–PRV group. Mean PRRSV titres of tonsil and lung in the PRRSV–PRV group were significantly (P < 0.05) higher than that in the PRRSV group on day 21. These results indicate that dual infection with PRRSV and PRV increased clinical signs and pneumonic lesions in pigs infected with both viruses, as compared to pigs infected with PRRSV or PRV only, at least in the present experimental conditions.     

山羊实验感染绵羊进行性肺炎病毒的抗体应答反应

用琼脂凝胶免疫扩散试验（ＡＧＩＤＴ）和免疫印迹试验（ＩＢＡ）对山羊实验感染绵羊进行性肺炎病毒（ＯＰＰＶ）的抗体应答反应进行了研究。结果,用ＡＧＩＤＴ和ＩＢＡ都可在接毒山羊的血清中检测到ＯＰＰＶ的抗体。ＡＧＩＤＴ最早于接毒后１５ｄ检测到抗体;ＩＢＡ最早于接毒后４ｄ检测到抗ＯＰＰＶ的ｇｐ４４和ｐ２８的抗体,以后又陆续检测到抗ｐ９４、ｐ１４和ｇｐ１２５的抗体。由此看出,ＩＢＡ比ＡＧＩＤＴ更为敏感。本研究结果表明,ＯＰ－ＰＶ可在山羊体内诱生较强的体液免疫应答反应,因此用ＯＰＰＶ通过山羊体传代的方法可能会得到具有良好抗原性的ＯＰＰＶ毒株。     

用绵羊进行性肺炎病毒实验感染山羊的研究

用绵羊进行肺炎病毒接种４只山羊３－４个月后，可观察到接毒山羊都出现了发育迟缓和消瘦现象，并有１只山羊山现了明显的临床病症。而未接毒的对照山羊则发育正常。接毒２８ｄ后，可以从接毒山羊的外周血单核细胞中分离到病毒。病理剖检发现４只接毒山羊中有１只山羊的多种器官出现了较为严重的病变，组织学检查则可看到典型的间质性肺炎、中度的脑炎和较为严重的脾炎的症变。以上的结果表明ＯＰＰＶ可以感染山羊，并对山羊有较强的     

Seroepidemiologic survey for antibodies to selected viruses in the respiratory tract of lambs

A serologic study was conducted to determine the prevalence of antibodies to, and infection rate of, Mastadenovirus ovi 5, M ovi 6, parainfluenza-3 (PI-3) virus, bovine herpesvirus-1 (BHV-1), respiratory syncytial virus (RSV), bovine viral diarrhea (BVD) virus, and ovine progressive pneumonia (OPP) virus in lambs at a ram lamb growth-rate test station. For 2 consecutive years, serum samples were prepared from blood collected from 1- to 2-month-old ram lambs as they entered the test station (1st sample) and again 2 months later (2nd sample). The 1st year, 59 producers submitted 237 lambs; the 2nd year, 65 producers submitted 253 lambs. Microtitration serum virus-neutralization tests were used to determine antibody titers for M ovi 5, M ovi 6, PI-3 virus, BHV-1, and BVD virus. Antibodies to RSV and OPP virus were determined, using indirect hemagglutination and agar-gel immunodiffusion, respectively. Based on results of the 1st blood samples collected, the mean prevalence for both years was as follows: 95% of the lambs were seropositive for M ovi 5; 87.2% for PI-3 virus; 84.5% for RSV; 41.7% for M ovi 6; 8.7% for BVD virus; 5.4% for BHV-1; and 3.3% for OPP virus. Based on the 2-year mean, M ovi 6 had the highest infection rate (207 of 484 [42.8%]) as determined by the number of lambs evaluated having a greater than or equal to 4-fold increase in serum antibody titer from the 1st to the 2nd sampling. Infection rates of the other viruses were: 31.0% for M ovi 5; 15.3% for PI-3 virus; 5.6% for RSV; 0.6% for BVD virus; and 0.4% for BHV-1. One lamb became seropositive for OPP virus the 2nd year.     

The length of BTV-8 viraemia in cattle according to infection doses and diagnostic techniques

Four groups of BTV free Frisian and cross bred calves were used to determine the length of viraemia following infection with different doses of BTV-8 Italian isolate. The first group of five animals was infected with 10 TCID₅₀ of BTV-8, the second group of four animals with 10³ TCID₅₀ and the third group, which also included four animals, was infected with 10⁶ TCID₅₀. A placebo containing uninfected tissue culture medium was given to the four animals of the fourth group. The viraemia was evaluated by real time RT-PCR and virus isolation. In all infected groups, virus isolation was able to detect infectious virus up to 39 days post infection (dpi) while RT-PCR was positive up to 151–157 dpi. Infectious dose did influence neither the length nor the pattern of BTV-8 viraemia and confirmed that real time RT-PCR remains positive although no circulating virus is detectable in the peripheral circulation.     

无血清全悬浮PK15细胞培养猪圆环病毒2型的研究

为改进猪圆环病毒2型的培养工艺,驯化了一株可无血清培养的全悬浮PK15细胞用于培养猪圆环病毒2型,并对病毒的敏感性、接毒时间、接毒量、收获方法进行了试验.结果表明,用该细胞培养猪圆环病毒2型,如果采用批次收获,接毒时细胞密度为0.5×106/mL,接毒量为0.1 MOI,接毒72 h后收毒,病毒滴度能达到106.4 T...     

Failure to Remove Bovine Diarrhea Virus (BVDV) from Bull Semen by Swim up and other Separatory Sperm Techniques Associated with in vitro Fertilization

Semen samples, collected from bulls pesistently infected with bovine viral diarrhea virus (BVDV) and containing BVDV (titer 10⁵ - 10⁶TCID₅₀/ml), were subjected to sperm separation procedures (washing, swim up, Percoll gradient, glass wool filtration, glass beads filtration) that are commonly used prior to in vitro fertilization (IVF) to determine if these procedures would yield spermatozoa free from BVDV. The final sperm pellets from frozen and fresh ejaculates were tested for the presence of BVDV by the immunoperoxidase technique; all tests were positive for BVDV in the range of 10³ - 10⁴TCID₅₀/ml). The study shows that when semen containing BVDV at the level of 10⁵ - 10⁶TCID₅₀/ml) is used for IVF, the virus is not completely removed by any of the simple physical methods commonly used to prepare sperm for IVF. Inhalt: Feldversuch das bovine Diarrhoe Virus (BVDV) aus Bullensperma durch Swim up oder andere Trennverfahren in Verbindung mit der In vitro Fertilisation zu entfernen Spermaproben, die von Bullen gewonnen wurden, welche dauerhaft mit dem Virus der bovinen Virus-Diarrhoe (BVDV) infiziert waren und einen Titer zwischen 10⁵ - 10⁶TCID₅₀/ml enthielten, wurden verschiedenen Trennverfahren unterzogen (Waschen, swim up, Percoll gradient, Glaswollenfiltration, Glaskugelfiltration). Diese Verfahren werden üblicherweise für die Vorbereitung zur In vitro Befruchtung verwendet, und es sollte geprüft werden, ob diese Verfahren auch das Sperma von dem BVD-Virus befreien können. Das endgültige Spermapellet von gefrorenen/aufgetauten und frischen Ejakulaten wurde in Gegenwart des BVD-Virus und mit Hilfe der immunoperoxidase Technik getestet. Alle Testergebnisse waren positiv für BVD-Virus in einem Bereich von 10³ - 10⁴ TCID₅₀/ml. Die Studie zeigt, daß Sperma, wenn es 10⁵-10⁶TCID₅₀/ml des Virus enhält, nicht vollständig mit den für die IVF üblichen, einfachen physikalischen Methoden von dem Virus befeit werden kann.     

Yeast-expressed classical swine fever virus glycoprotein E2 induces a protective immune response

Classical swine fever (CSF) is an economically important swine disease worldwide. The glycoprotein E2 of classical swine fever virus (CSFV) is a viral antigen that can induce a protective immune response against CSF. A recombinant E2 protein was constructed using the yeast Pichia pastoris expression system and evaluated for its vaccine efficacy. The yeast-expressed E2 (yE2) was shown to have N-linked glycosylation and to form homodimer molecules. Four 6-week-old specified-pathogen-free (SPF) piglets were intramuscularly immunized with yE2 twice at 3-week intervals. All yE2-vaccinated pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers ranging from 1:96 to 1:768. Neutralizing antibody titers at 10 weeks post booster vaccination ranged from 1:16 to 1:64. At this time, the pigs were subjected to challenge infection with a dose of 1 × 10⁵ TCID₅₀ (50% tissue culture infective dose) virulent CSFV strain. At 1 week post challenge infection, all of the yE2-immunized pigs were alive and without symptoms or signs of CSF. Neutralizing antibody titers at this time ranged from 1:4,800 to 1:12,800 and even to 1:51,200 one week later. In contrast, the control pigs continuously exhibited signs of CSF and had to be euthanized because of severe clinical symptoms at 6 days post challenge infection. All of the yE2-vaccinated pigs were E^rns antibody negative and had seroconverted against E^rns by post challenge day 11, suggesting that yE2 is a potential DIVA (differentiating infected from vaccinated animals) vaccine. The yeast-expressed E2 protein retains correct immunogenicity and is able to induce a protective immune response against CSFV infection.     

Variation in resistance to the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in Pietrain and Miniature pigs

Porcine reproductive and respiratory syndrome (PRRS) is one of the economically most important diseases of swine. Viraemia and the prolonged persistence of the virus are among the most critical factors. Virus replication and severity of disease vary with virus isolates, and there is rising evidence for a genetic component of the host susceptibility. Dissecting the genetic basis of resistance/susceptibility to PRRS virus (PRRSV) might lead to improved knowledge on the molecular mechanisms of PRRS and the establishment of genetic markers for future disease control. The aim of this study was to establish a porcine model with emphasized genetic differences in PRRSV susceptibility. Seven ‘Wiesenauer Miniature’ pigs (MI), a local German breed and eight commercial Pietrain (PI) pigs were challenged with 10⁵ TCID₅₀ of an attenuated PRRSV strain (Ingelvac^® PRRSV MLV). Clinical status, viraemia and seroconversion of the pigs were compared. No clinical signs were observed during the experiment. Viraemia peaked on day 6 p.i., with 100% of viraemic pigs in PI and on day 12 p.i with 87% of viraemic MI. Viraemia lasted for up to 35 days in MI and for at least 72 days in PI. This surprising result was confirmed by a second study with another four MI. MI and PI showed maximum virus titres of 10^2.5 TCID₅₀/ml of serum and 10^4.5 TCID₅₀/ml, respectively, indicating a virus replication in MI of approximately 3.3% that of PI over the complete period. MI were more efficient in antibody production. With such pronounced breed differences, the model is of high relevance for the genetic dissection of PRRS pathogenesis and susceptibility.     

Reduction of infectious bursal disease virus replication by shRNAs targeting the VP1 and VP2 genes driven by chicken U6 promoter

Infectious bursal disease virus (IBDV) causes a highly contagious and immunosuppressive disease in young chickens and results in considerable economic losses for the poultry industry. To suppress the replication of IBDV, two short hairpin RNAs (shRNAs) were designed for targeting the VP1 and VP2 genes of IBDV. Recombinant plasmids carrying each shRNA or two shRNAs were constructed based on vector pSilencer2.1-U6 in which the human U6 promoter was replaced with chicken U6 promoter. In chicken embryo fibroblasts, transfection with these shRNA plasmids 24 h before infection with IBDV B87 reduced 50% tissue culture infectious doses (TCID₅₀) from 10^8.75 TCID₅₀/0.1 mL to 10^3.75–10^1.0 TCID₅₀/0.1 mL. In 10-day old specific pathogen-free (SPF) chicken embryos, incubation with a mixture of IBDV B87 and a shRNA plasmid via the allantoic cavity resulted in 100% mortality and high IBDV virus titer in the control group but 25–0% mortality and near normal embryo development in the specific shRNA groups; additionally, IBDV VP1 and VP2 mRNA levels were reduced by 72–95% in the shRNA groups as compared with the control groups. When challenged with a virulent strain IBDV GX8/99, 14-day-old chickens pre-treated with the single shRNA plasmids or the dual shRNA plasmid showed approximately 70% or 90% survival at 5 days post-challenge while those pre-treated with control plasmid or saline had less than 5% survival. The current study suggests that two IBDV shRNAs expressed by a plasmid under chicken U6 promoter could effectively and synergistically reduce IBDV replication in vitro and in vivo.     

Individual animal risk factors for clinical mastitis in meat sheep in Norway

An m:n matched case–control study was conducted to identify risk factors for ovine clinical mastitis (CM). Data were from a national sheep registry and only ewes that lambed in the spring of 2004 were included. Eligible cases (n = 2857) and controls (n = 76,716) from 1056 flocks of meat sheep were matched on flock and conditional logistic regression was used for analysis of the data. CM risk was associated with age of the ewe and whether or not assistance at lambing was needed owing to dystocia; however, the effects of both these factors were modified by the number of lambs born. In ewes with 1 lamb, increasing age was associated with increased odds of CM (OR = 1.2 for each 1-year increase), while only a slight numerical increase in the odds was observed in ewes with >1 lamb. Dystocia was associated with increased odds of CM in ewes with 1 lamb (OR = 1.7) or 2 lambs (OR = 1.4), while no association was observed in ewes with >2 lambs. The odds of CM increased markedly with increasing number of lambs born to the ewe. For example, odds for 2-year-old ewes without dystocia were 6.7 times greater for those with >3 lambs than for those with 1 lamb. Compared with ewes of old Norwegian breeds, ewes of other breeds were more likely to experience CM (OR = 1.7). Ewes treated for CM at least once during the preceding 3 years had 4.0 times greater odds of CM compared with ewes without a CM history. It is likely that the effect estimates from this study, which are adjusted for breed and unaffected by inter-flock variations, are valid also for other meat sheep populations.     

一例猪伪狂犬病病毒的分离鉴定

从临床疑似猪伪狂犬病发病仔猪的脑组织等病料中,经PCR扩增出大小为217 bp的伪狂犬病病毒gp50的基因片段,结果证实为猪伪狂犬病病毒(porcine pseudorabies virus,PRV)感染。随后采用BHK-21细胞进行猪伪狂犬病病毒的分离培养,该分离株经细胞传代培养5代后,能够产生典型的细胞病变,经PCR鉴定为伪狂犬病病毒,其病毒感染力达10^8.68 TCID₅₀/0.1mL。最后用10^7.0 TCID₅₀/mL病毒培养物接种家兔,48 h后注射部位出现典型瘙痒、皮肤破损等症状,于72 h后全部死亡。结果表明,该伪狂犬病病毒分离株对易感动物具有高致病性,为进一步开展该病毒流行病学、致病机理、疫苗免疫及诊断研究奠定了基础。     

Effects on ovine fetuses of exposure to ovine progressive pneumonia virus

Fetuses of 20 pregnant ewes at 4 gestational periods (45, 55, 85, and 100 days) were inoculated with ovine progressive pneumonia virus. Fourteen of 16 fetuses exposed to virus before gestational day 80 were either resorbed or expelled, whereas 10 of 15 fetuses exposed to virus after day 80 were normal at birth. Three of the 9 expelled fetuses and 1 of 2 newborn lambs had accumulations of lymphoid cells in the lungs. Virus was readily isolated from the tissues of expelled fetuses and newborn lambs. Lambs did not have precipitating antibody to the virus at birth, but 3 to 5 lambs had specific antiviral antibody at 18 months of age.