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.     

乳胶凝集试验与血清中和试验检测猪伪狂犬病血清抗体的 …

应用血清中和试验（ＳＮＴ）和伪狂犬病乳胶凝集试验（ＬＡＴ）诊断试剂盒对两种伪狂犬病是性血清、伪狂犬病病毒（ＰＲＶ）高兔血甭及６０份被检猪血清进行了ＰＲＶ抗体效价测定和相关性分析,两种方法测得的抗体效价之间呈强相关性（ｒ＝０．９６）,且ＬＡＴ效价比ＳＮＴ一般高出一个滴度;能干为自３５个猪场的４１４份猪血清进行了ＰＲＶ抗体检测,并与ＳＮＴ检测结果进行了对比,结果在ＳＮＴ检测为阳笥的１７１份血清中,ＬＡ     

A study of the ability of a TK-negative and gI/gE-negative pseudorabies virus (PRV) mutant inoculated by different routes to protect pigs against PRV infection

The capacity of a TK-negative (TK-) and gI/gE-negative (gI/gE-) pseudorabies virus (PRV) mutant to protect pigs against Aujeszky's disease carried out by experimental infection with a virulent PRV strain, was tested. There were three groups, each of four susceptible pigs which were inoculated twice by two different schedules. Group 1 received the modified virus by the intradermal (first inoculation)-intramuscular (second inoculation) routes; group 2 was treated by the intranasal (first inoculation)-intramuscular (second inoculation) routes. The third group was left untreated as the control. All of the pigs were challenged intranasally with a virulent PRV strain and they were subsequently injected with dexamethasone. Two pigs in each group were necropsied on days 5 and 15 after dexamethasone inoculation. The challenge exposure resulted in mild clinical signs, increase in growth and a shorter period of virus shedding in vaccinated pigs, whereas the control group showed severe signs of Aujeszky's disease. No difference in the titre of the virulent virus which was excreted by pigs of all three groups, was observed and all animals seroconverted. Both the mutant strain and the wild-type virus established a latent infection although only the latter was reactivated and shed. Slight lesions were observed in target tissues of the vaccinated animals and no significant differences were detected between the two inoculation schedules.     

间接ELISA检测猪伪狂犬病血清抗体

用猪肾传代细胞IBRS－2增殖猪伪狂犬病病毒（PRV）鄂A株，病毒培养上清液经硫酸铵沉淀、聚乙二醇（Mr20000）浓缩后作为包被抗原。用纯化的猪血清IgG免疫家兔，HRP村记撮的兔抗猪IgG，制备出高效价的酶标抗体，酶标抗体工作浓度为1：50000；经各种条件的选择，建立了检测猪伪狂犬病血清抗体的间接ELISA。所建立的间接ELISA抗原包被浓度为39.2mg/L，血清最佳稀释度为1：20，与猪细小病毒、猪、O型口蹄疫、猪衣原体标准阳性血清呈阴性反应，与标准阴性血清和临床未感染PRV的猪血清呈阴性反应；与猪伪狂犬病标准阳性血清、免疫猪血清和临床发病猪血清呈明显的阳性反应；与美国进口的PRV抗体检测ELISA诊断试剂盒检测结果比较，45份猪血清的阴、阳性检出符合率均为100％。表明建立的间接ELISA具有敏感性高、特异性强、重复性好的优点，可用于猪伪狂犬病血清抗体的定性和定量检测。     

A Cross‐Sectional Study of Hepatitis E Virus Infection in Healthy People Directly Exposed and Unexposed to Pigs in a Rural Community in Northern Thailand

A cross‐sectional study of the association between occupational pig exposure and hepatitis E virus (HEV) infection in adult pig farmers and the general population who were not directly exposed to pigs was conducted in Nan Province, Thailand, from November 2010 to April 2011. All participants were interviewed to provide information on their job history, eating habits and other potential confounders. The prevalence of anti‐HEV immunoglobulin G antibodies (IgG) among 513 subjects was 23.0%. Hand washing with water and soap was associated with a lower seroprevalence of HEV infection, whereas living in an area with frequent flooding (OR 1.64, 95% CI: 1.00–2.68) and consuming internal pig organs more than twice per week (OR 3.23, 95%CI: 1.15–9.01) were both associated with a higher seroprevalence of anti‐HEV IgG. There was no association between HEV seroprevalence and frequent, direct occupational pig contact.     

Pathogenesis and Subsequent Cross‐Protection of Influenza Virus Infection in Pigs Sustained by an H1N2 Strain

The H1N1, H3N2 and, more recently, H1N2 subtypes of influenza A virus are presently co‐circulating in swine herds in several countries. The objectives of this study were to investigate the pathogenesis of Sw/Italy/1521/98 (H1N2) influenza virus, isolated from respiratory tissues of pigs from herds in Northern Italy, and to evaluate its potential cross‐protection against the Sw/Fin/2899/82 (H1N1) strain. In the pathogenesis test, eight pigs were intranasally infected with H1N2 virus; at pre‐determined intervals, these animals were killed and necropsied, along with eight uninfected animals. In the cross‐protection test, sixteen pigs were infected by intranasal (i.n.) and intratracheal (i.t.) routes with either H1N2 or H1N1 virus. Twenty days later, all pigs were challenged (by the same route), with either the homologous H1N2 or heterologous H1N1 virus strains. Control group was inoculated with culture medium alone. On post‐challenge days (PCD) 1 and 3, two pigs from each infected group, along with one control pig, were killed. Clinical, virological, serological and histopathological investigations were performed in both the pathogenicity and cross‐protection tests. In the pathogenicity test, mild clinical signs were observed in two pigs during 3 and 4 days, respectively. Virus was isolated from two pigs over 6 days and from lung samples of pigs killed on post‐infection days 2 and 4. Seroconversion was detected in the two infected animals killed 15 days after infection. In the cross‐protection study, mild clinical respiratory signs were detected in all pigs infected with either the H1N2 or H1N1 virus. The virus was isolated from nasal swabs of almost all pigs till 6 days. After the challenge infection, the pigs remained clinically healthy and virus isolation from the nasal secretions or lung samples was sporadic. Antibody titres in H1N1 or H1N2 infected groups were similar, whereas the H1N2 sub‐type induced less protection against re‐infection by homologous and heterologous virus than H1N1 sub‐type. The controls had no signs of the disease. In the H1N2 infected pigs, a reduced number of goblet cells in nasal and tracheal mucosa and small foci of lymphomononuclear cell infiltrates in the submucosa were detected. Furthermore, the goblet cell reduction was related to the time of infection. Diffuse mild interstitial pneumonia was also recorded in pigs infected with the H1N2 virus and challenged with either H1N1or H1N2 pigs. These studies showed the moderate virulence of the H1N2 virus and a partial cross‐protection against heterologous infection.     

First Evidence of Porcine Circovirus Type 2 (PCV‐2) Infection of Pigs in the Czech Republic by Semi‐Nested PCR

Three oligonucleotide primers for semi‐nested polymerase chain reaction (PCR) were designed according to already published sequences of porcine circovirus types 1 (PCV‐1) and 2 (PCV‐2) isolates. These primers were used to detect PCV‐2 DNA. A positive amplification reaction was visualized from a DNA suspension containing as few as 10 copies of virus DNA. In total, 77 samples of inguinal lymph nodes and nasal swabs from pigs in the Czech Republic were used to detect the virus. Thirty‐seven of them were positive for PCV‐2 DNA. In order to confirm specificity of the PCR reaction, seven DNA fragments were sequenced. Czech PCV sequences were found to have a 92–97% homology with other known PCV‐2 strains and only 80–83% homology with PCV‐1 strains.     

Simultaneous Infection of Pigs and People with Triple‐Reassortant Swine Influenza Virus H1N1 at a U.S. County Fair

Influenza‐like illness was noted in people and pigs in attendance at an Ohio county fair in August 2007. The morbidity rate in swine approached 100% within 1–2 days of initial clinical signs being recognized, and approximately two dozen people developed influenza‐like illness. Triple‐reassortant swine H1N1 influenza viruses were identified in both pigs and people at the fair. The identified viruses (A/Sw/OH/511445/2007, A/Ohio/01/2007, and A/Ohio/02/2007) were similar to H1N1 swine influenza viruses currently found in the U.S. swine population. This case illustrates the possibility of transmission of swine influenza in settings where there is close human/swine interaction.     

Serological Survey of Bluetongue Virus Serotype‐8 Infection in South American Camelids in Switzerland (2007–2008)

Background: Outbreak of bluetongue virus serotype‐8 (BTV‐8) infection in domestic ruminants in Northern Europe. Objective: To investigate the South American camelids' (SAC) susceptibility to BTV‐8 infection, their role in the epidemiology of the disease, and the use of currently available serological screening tests in SAC in an endemic region. Animals: Three hundred and fifty‐four unvaccinated and 27 vaccinated SAC (170 llamas, 201 alpacas), ranging in age from 1 month to 17 years between June and August 2008. The SAC originated from 44 herds throughout the country, representing 10% of the Swiss SAC population. Methods: Prospective, observational study of a convenience sample of SAC. Serum samples were analyzed with 2 serological screening tests. When results diverged, a 3rd ELISA was carried out for confirmation (ID Screen Bluetongue Competition ELISA kit). Results: All sera from the 354 unvaccinated animals were negative in the endemic region. Reliable seroconversion was observed after administration of 2 doses of vaccine. Conclusions and Clinical Importance: This study suggests a low susceptibility of SAC to BTV‐8 despite the presence of the virus in the cattle and small ruminant population, indicating that SAC do not play a major role in the epidemiology of BTV‐8. Furthermore, these results indicate that commercially available serological tests for BTV‐8 can be used in SAC.     

Efficacy of Tilmicosin Phosphate (Pulmotil® Premix) in Feed for the Treatment of a Clinical Outbreak of Actinobacilluspleuropneumoniae Infection in Growing–Finishing Pigs

A double‐blind randomized clinical trial was carried out to investigate the efficacy of tilmicosin (Pulmotil^® premix) for the treatment of a clinical outbreak of Actinobacillus pleuropneumoniae infection in growing–finishing pigs. The effects of tilmicosin administration in the feed at 400 mg/kg and an injection therapy of clinically diseased pigs with long‐acting oxytetracycline (Terramycine^® LA) at 20 mg/kg bodyweight were compared. Both groups, totalling 147 pigs, were compared during a medication period of 15 days and a post‐medication period of 11 days by means of different clinical and performance parameters. During the medication period, the tilmicosin group showed a significant advantage with respect to the number of new disease cases (P < 0.01), and a non‐significant advantage regarding the number of removed pigs (P=0.16), the number of sick pigs that recovered (P=0.27) and the time to recovery (P=0.42). During the post‐medication period, the pigs of the tilmicosin group showed numerical non‐significant benefits (P > 0.05) with respect to the clinical parameters. During the overall study period (26 days), the average daily gain and the feed conversion ratio were both significantly (P < 0.01) better in pigs from the tilmicosin group compared with pigs from the oxytetracycline group. This study demonstrated that in‐feed medication of tilmicosin at a dosage of 400 mg/kg is efficacious for the treatment of a clinical respiratory disease outbreak of A. pleuropneumoniae infection in growing–finishing pigs. Compared with oxytetracycline injection of clinically diseased pigs, the tilmicosin treatment is particularly beneficial in the prevention of new disease cases while increasing or maintaining the performance of the pigs.     

ELISA and Direct Immunofluorescence Test to Detect Equine Arteritis Virus (EAV) using a Monoclonal Antibody Directed to the EAV‐N Protein

A monoclonal antibody (mAb) directed against the equine arteritis virus (EAV) nucleocapsid (N) protein was used for indirect enzyme‐linked immunosorbent assays (ELISAs) using viral antigen from different sources. The same mAb was labelled with fluorescein isothiocyanate for direct immunofluorescence tests (DIFTs). The N‐specific mAb appeared to be suitable for the detection in both ELISA and DIFT of different EAV strains and field isolates from semen and tissue samples after passage in lines of RK‐13, Vero and fetal equine kidney cells. The ELISA described is an easy and fast method which can be used in most cases to replace the microneutralization test to prove the EAV specificity of the cytopathic effect of cell cultures. The DIFT, however, is more sensitive than both the ELISA and the microneutralization test because EAV antigen can be detected even in cell cultures without or with very weak cytopathic effect.     

Avian Influenza A(H5N1) Virus Outbreak Investigation: Application of the FAO‐OIE‐WHO Four‐way Linking Framework in Indonesia

WHO, FAO and OIE developed a ‘four‐way linking’ framework to enhance the cross‐sectoral sharing of epidemiological and virological information in responding to zoonotic disease outbreaks. In Indonesia, outbreak response challenges include completeness of data shared between human and animal health authorities. The four‐way linking framework (human health laboratory/epidemiology and animal health laboratory/epidemiology) was applied in the investigation of the 193rd human case of avian influenza A(H5N1) virus infection. As recommended by the framework, outbreak investigation and risk assessment findings were shared. On 18 June 2013, a hospital in West Java Province reported a suspect H5N1 case in a 2‐year‐old male. The case was laboratory‐confirmed that evening, and the information was immediately shared with the Ministry of Agriculture. The human health epidemiology/laboratory team investigated the outbreak and conducted an initial risk assessment on 19 June. The likelihood of secondary cases was deemed low as none of the case contacts were sick. By 3 July, no secondary cases associated with the outbreak were identified. The animal health epidemiology/laboratory investigation was conducted on 19–25 June and found that a live bird market visited by the case was positive for H5N1 virus. Once both human and market virus isolates were sequenced, a second risk assessment was conducted jointly by the human health and animal health epidemiology/laboratory teams. This assessment concluded that the likelihood of additional human cases associated with this outbreak was low but that future sporadic human infections could not be ruled out because of challenges in controlling H5N1 virus contamination in markets. Findings from the outbreak investigation and risk assessments were shared with stakeholders at both Ministries. The four‐way linking framework clarified the type of data to be shared. Both human health and animal health teams made ample data available, and there was cooperation to achieve risk assessment objectives.     

Immunization of Pups with Maternally Derived Antibodies to Canine Parvovirus (CPV) Using a Modified‐Live Variant(CPV‐2b)

The results of vaccination trials carried out on pups with maternally derived antibodies (MDA) to canine parvovirus (CPV), using a modified‐live CPV‐2b variant vaccine (29‐97/40 strain), are reported. The vaccine was able to overcome the obstacle of MDA, and to elicit protective immunity in 100% of the pups whose antibody titres were 1:10–1:40, 83% of the pups with titres of 1:80, 57% of the pups with titres of 1:160, and even in 60% of the pups with antibody titres of 1:320.     

Risk of Transmission of Bovine Herpesvirus‐1 (BHV‐1) by Infected Semen to Embryo Recipients and Offspring

Infection with bovine herpesvirus‐1 (BHV‐1), also called infectious bovine rhinotracheitis/infectious pustular vulvovaginitis virus, is associated with a variety of respiratory, neurological and infertility health problems causing worldwide economic losses and trading restrictions to the livestock industry. Although there is a considerable amount of information about the risk of BHV‐1 transmission through contaminated semen used for artificial insemination, there is no available evidence to indicate whether the resulting embryos, when used for embryo transfer (ET), can lead to the transmission of BHV‐1 to recipients and offspring. For this study, cryopreserved bull semen contaminated with BHV‐1 was used for artificial insemination (AI) of seronegative, superovulated heifers (N = 43). Embryos were collected post‐mortem at 7 days post‐insemination and were washed according to the International Embryo Transfer Society (IETS) guidelines. BHV‐1 was detected in all samples of follicular fluid, oviductal epithelial cells, endometrium and corpora lutea tissues and a proportion of unwashed (52 of 120, 43%) and washed oocytes and embryos (7 of 113, 6%) collected from embryo donors. Of the 396 collected, unfertilized oocytes and embryos, only 29 (7%) were of ET quality. Most of the embryos and oocytes were degenerated (N = 224, 57%) or unfertilized (N = 143, 36%). The 13 heifers, which each received a single morula‐stage washed embryo, maintained seronegative status, but only two (15%) became pregnant and delivered BHV‐1‐free calves. In conclusion, results suggest that embryos fertilized with BHV‐1‐contaminated semen may not result in disease transmission to embryo recipients or their offspring when embryos are processed according to IETS and the World Organization for Animal Health (OIE) guidelines. However, due to the transmission of BHV‐1 via AI to embryo donors and the apparent adverse effect of BHV‐1 on the quality of the embryos, it is unlikely that the procedure can be justified for a commercial application.     

Quality and Developmental Ability of Dromedary (Camelus dromedarius) Embryos Obtained by IVM/IVF, In Vivo Matured/IVF or In Vivo Matured/Fertilized Oocytes

The effect of source of cumulus-oocytes-complexes (COCs), maturation and fertilization conditions on developmental competence of dromedary embryos was examined. Thirty-six adult females were superovulated with equine Chorionic Gonadotropin (eCG) injection (3500 IU, IM) and divided in three groups of 12 females each. Group 1 provided 138 COC's collected from follicles >or= 5 mm 10 days after stimulation prior hCG treatment and matured in vitro for 30 h. Group 2 provided 120 in vivo matured oocytes which were aspirated from their follicles 20 h after hCG (3000 IU, IV) given on day 10 follow eCG injection. Group 3 provided 65 in vivo matured/fertilized oocytes. Females in Group 3 received hCG on day 10 following eCG treatment and then were mated 24 h later. Fertilized oocytes were collected from the oviducts of females 48-h post-mating. Quality of the oocytes was assessed after in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of COCs. All cultures were performed in three replicates (n = 3) at 38.5 degrees C, under 5% CO(2) and high humidity (>95%). Only COCs with cumulus and homogenous (dark) cytoplasm were used. Nuclear maturation rate for Groups 1 and 2 was determined by epifluorescence microscopy in a sample of COCs (n = 30) denuded, fixed and stained with Hoechst 33342. To study the viability of obtained embryos, hatched blastocysts from each group were transferred to recipients followed by pregnancy diagnosis using ultrasonography at 15, 60 and 90 days. The percentage of COCs reaching metaphase II (MII) after 30 h of maturation was slightly but not significantly higher for in vivo matured oocytes (28/30; 93%) than those in vitro matured (25/30; 84%). The total rate of cleavage (2 cells to blastocyst stage) was not different for the three groups. However, significantly (p < 0.05) more blastocyst and hatched blastocysts were obtained from in vivo matured and in vivo fertilized oocytes (Group 3; 52% and 73%) than from in vitro fertilized oocytes whether they were matured in vitro (Group 1; 35% and 32%) or in vivo (Group 2; 32% and 45%). Pregnancy rates were not significantly different amongst all groups for the three first months following embryo transfer. All pregnancies were lost after day 90 follow transfer except for in vivo matured and in vivo matured/fertilized groups. Only in vivo matured/in vitro fertilized and in vivo matured/fertilized produced embryos continued normal development until term and resulted in the birth of normal and healthy live calves. Six claves (29%; 6/21) were born from Group 3 and one (8%; 1/13) calf was born from Group 2. This study shows that the IVC system used is able to support camel embryo development. However, developmental competence and viability of dromedary embryos may be directly related to the intrinsic quality (cytoplasmic maturation) of oocytes.     

猪流感病毒A/swine/Guangdong/2/2012(H1N1)毒株攻毒BALB/c小鼠细胞因子动态变化的研究

为了测定H1N1亚型猪流感病毒(swine influenza virus,SIV)对小鼠的致病性,本试验对A/swine/Guangdong/2/2012(H1N1)株SIV HA基因进行克隆及遗传分析,并将SIV尿囊液经鼻腔感染6周龄BALB/c小鼠,观察感染后小鼠的一般状况、器官系数和组织病理学变化,在病毒感染后第1、3和7天使用荧光定量PCR测定小鼠肺脏、脾脏、脑组织中7种细胞因子mRNA的表达量,研究其对小鼠的致病特性。结果显示,该病毒属于经典SIV,病毒经鼻腔感染后可引起小鼠活动减少、采食量降低,但无咳嗽和死亡;病理组织学变化为肺间隔较正常组织明显增厚,毛细血管明显扩张充血,周围肺泡腔呈代偿性肺气肿;小鼠肺脏、脾脏组织样本中IFN-α、IFN-β、IP-10、IL-1β、TNF-α、IRF-3和IL-10 mRNA含量在感染后第3天均显著升高(P<0.05),而脑组织样本中IL-1β和IL-10在小鼠攻毒后第3和7天均显著上调(P<0.05)。