Equine Multinodular Pulmonary Fibrosis and Lymphoma in a Horse Associated with Equine Herpesvirus-5

A 9-year-old mare presented with a 2-week history of partial anorexia, multiple swellings in the area of the throatlatch, and purulent nasal discharge. On initial presentation, the horse had submandibular and retropharyngeal lymphadenopathy, mild ventral edema, and weight loss. Thoracic radiographs revealed a pulmonary interstitial pattern. Necropsy revealed enlarged lymph nodes throughout the body. The lung parenchyma contained multiple random, well-circumscribed nodules, which, on cut section, were pale, tan colored, and very firm with a distinct line of demarcation from the surrounding normal parenchyma. The subendocardium of the left ventricle, left atrium, and, multifocally, the right ventricle contained white, gritty areas of mineralization. There was marked subintimal mineralization of the aorta and pulmonary artery. Histopathology of the lymph nodes revealed effacement of the parenchyma by a neoplasm composed of large numbers of small mature lymphocytes, fewer large lymphocytes, and scattered moderate numbers of histiocytes. Immunohistochemistry tests for CD3, CD79a, and CD20, confirmed the lymphoma was T-cell-rich B-cell lymphoma. The lungs contained marked interstitial fibrosis with alveolar histiocytosis. Polymerase chain reaction test results of lymph node and lungs were positive for equine herpesvirus-5 (EHV-5), a gammaherpesvirus. Gammaherpesvirus infection has been associated with lymphoma and pulmonary fibrosis in other species. This report describes the association between EHV-5 and both pulmonary fibrosis and lymphoma.     

主要病毒性牛呼吸道疾病的研究进展

牛呼吸道疾病（bovine respiratory disease,BRD）是引起舍饲牛发病和死亡的主要原因,给北美和世界养牛业造成巨大的经济损失。BRD是由多种病毒、细菌与外界环境相互作用,如应激、环境因素与多种病毒、细菌和支原体等而引起的一种严重的呼吸系统疾病。作者就引起BRD的常见和严重的病原牛传染性鼻气管炎病毒（infectious bovine rhinotracheitis virus,IBRV）和牛呼吸道合胞体病毒（bovine respiratory syncytial virus,BRSV）的生物学特性、细胞感染和致病机制等进行简要概述,以期为该病的防制和研究提供参考。     

Antibody Responses Against Equine Influenza Virus Induced by Concurrent and by Consecutive Use of an Inactivated Equine Influenza Virus Vaccine and a Modified Live Equine Herpesvirus Type 1 Vaccine in Thoroughbred Racehorses

An inactivated equine influenza virus (EIV) vaccine and a live equine herpesvirus type 1 (EHV-1) vaccine are usually administered concurrently to Thoroughbred racehorses in Japan. The objective of this study was to evaluate whether concurrent administration of an inactivated EIV vaccine and a live EHV-1 vaccine in Thoroughbred racehorses influences the antibody response against EIV. We compared the antibody response against EIV in horses administered both vaccines on the same day (Group A; n = 27) and the response in horses administered an inactivated EIV vaccine first and then a live EHV-1 vaccine 1–2 weeks later (Group B; n = 20). In both groups, geometric mean hemagglutination inhibition (HI) titers against A/equine/Ibaraki/1/2007 and A/equine/Yokohama/aq13/2010 increased significantly after EIV vaccination. However, the percentage of horses that showed a twofold increase or greater in HI titers against A/equine/Yokohama/aq13/2010 was significantly higher in Group B (75%) than in Group A (37%; P = .02). These results suggest that the concurrent use of an inactivated EIV vaccine and a live EHV-1 vaccine reduced the immune response against EIV to some extent, and it would be better to use these vaccines consecutively, especially for naïve horses or horses whose vaccination history is incomplete.     

Equine Herpesvirus-I Infection in Horses: Recent Updates on its Pathogenicity,Vaccination, and Preventive Management Strategies

Equine herpesvirus-1 (EHV-1) is one of the most common and ubiquitous viral pathogens infecting equines, particularly horses worldwide. The EHV-1 is known to induce not only humoral but also cellular immune responses in horses. Respiratory distress, abortion in pregnant mares, neurological disorders, and neonatal foal deaths represent EHV-1 infection. Despite the limited success of inactivated, subunit, live, and DNA vaccines, over the past few decades, vaccination remains the prime preventive option to combat EHV-1 infection in horses. However, current vaccines lack the potentiality to protect the neurological form of infections in horses. There is desperate necessity to search effectual EHV-1 vaccines that may stimulate not only mucosal and systemic cellular immunity but also humoral immunity in the horses. This review highlights the state of knowledge regarding EHV-1 biology, EHV-1 pathogenesis, and disparate vaccines studied in the past to prevent EHV-1 infection. The review also underlines the best management strategies which certainly need to be adopted by veterinarians in order to avoid and prevent EHV-1 infection and outbreak in horses in the future.     

Comparison of the Head and Neck Positions in Ridden Horses Advertised in an Australian Horse Sales Magazine: 2005 Versus 2018

The impact of head and neck position (HNP) on horse welfare has received much attention in the scientific literature within the last two decades. Studies have identified physiological and behavioral signs of distress in horses ridden for prolonged time in an HNP with their noseline behind the vertical (BTV), which may compromise their welfare. The objective of this study was to compare potential differences of HNPs shown in horse sales photographs advertised in an Australian horse sales magazine (Horse Deals) from the years 2005 and 2018. In addition, factors potentially impacting HNPs, such as type of tack presented in (e.g., noseband type), riding discipline, and competition experience of the horse, were investigated. The sample population (n = 570) comprised horses ridden with headgear and bit in walk, trot, or canter/gallop, advertised in an Australian horse sales magazine. Issues from April 2005 and October/November 2018 were selected. Head and neck position was categorized as BTV, on the vertical (OV), slightly in front of the vertical (IFV), or extremely in front of the vertical (EIFV; any HNP >30° IFV). Data were analyzed using the chi-squared test and post hoc testing via a multiple regression approach through SPSS and test of proportions via the Z-score calculator for two independent population proportions. Analysis of combined data from years 2005 and 2018 showed 47.0% (n = 570) of the horse sample population were advertised with HNPs BTV. Behind-the-vertical HNP was observed as the predominant HNP (57.8%; n = 268) in the warmblood/eventers/show/performance (WESP) category (P < .0005). In 2005, 53.4% (n = 303) of the sample population were ridden BTV compared with 39.7% (n = 267) in 2018 (P < .001), 12.9% (n = 303) were OV in 2005 compared with 15.0% (n = 267) in 2018 (P > .05), and 10.9% (n = 303) were IFV in 2005 compared with 27.3% (n = 267) in 2018 (P < .0004). These results suggest a positive development with fewer vendors/riders selecting images where the horse’s nose was BTV. However, this may be explained by the larger proportion of horses advertised in the WESP category in 2005 (63.0%; n = 303) versus 2018 (28.5%; n = 267), and the WESP category predominantly comprised of dressage, jumper, and eventing horses. In addition, the reduction of HNPs BTV from 53.4% (n = 303) in 2005 to 39.7% (n = 267) in 2018 could be attributed to the observation that in 2018, a larger proportion of horses were listed in categories that do not require the horses to be worked with a flexed HNP referred to as “on-the-bit” (e.g., western, endurance, Australian sStock horses). The HNP BTV remains preferential by a substantial proportion of the horse-owning public when advertising horses for sale, particularly in disciplines where the horse is worked in a flexed HNP or “on-the-bit.”     

马流感病毒(A/equine/Qinghai/1/94)核蛋白基因的序列测定及同源性分析

根据已发表的马流感病毒核蛋白基因序列，设计并合成一对特异性引物，经反转录-聚合酶链反应（RT-PCR）成功扩增出了我国马流感病毒（A/equine/Qinghai/1/94核蛋白基因。将该片段连接到PGEM-T-EASY载体并转化DH5α，提取阳性菌落的质粒径EcRo1酶切的PCR鉴定其大小为1.5kb左右，对其测序并进行分析发现，与A/Equine/Kentucky/2/86,A/Equine/Miami/1/63等关系较近，同源率为93.3%--93.4%，而与我国马流感吉林A/Equine/Jilin/1/89株关系较远，同源率仅为84.6%。     

Detection and Management of an Outbreak of Equine Herpesvirus Type 1 Infection and Associated Neurological Disease in a Veterinary Teaching Hospital

Background: Because of the serious disease sequelae associated with equine herpesvirus type 1 (EHV‐1) infections, awareness and control measures used to control outbreaks are important issues for all horse populations. Objectives: Describe the occurrence and management of an outbreak of EHV‐1 infection at a veterinary hospital. Animals: Horses hospitalized at a referral veterinary hospital. Methods: A horse with myeloencephalopathy associated with EHV‐1 infection (EHM) was admitted for diagnostic evaluation and treatment under strict infection control procedures. We describe the occurrence and management of a nosocomial outbreak of EHV‐1 infections associated with admission of this patient. Results: Despite institution of rigorous biosecurity precautions at the time of admission of the index case, EHV‐1 infections spread to 6 other horses that were hospitalized at the James L. Voss Veterinary Teaching Hopsital, including 2 that served as sources of infection for horses on their home premises after discharge. Infection with EHV‐1 was confirmed by polymerase chain reaction (PCR) and by seroconversion documented by glycoprotein G ELISA. A voluntary quarantine was imposed and admissions were restricted to prevent additional horses from being exposed. Quarantine duration was abbreviated by serial testing of all horses with PCR. Conclusions and Clinical Importance: These findings illustrate the contagious disease risk that can accompany management of horses with EHM. Horses with active nasal EHV‐1 shedding should be isolated in an airspace that is separate from other horses by strictly enforced biosecurity and isolation procedures. Serial testing with PCR may be a useful adjunct to determine when the risk of transmission has been minimized.     

Detection of Foot and Mouth Disease and Porcine Reproductive and Respiratory Syndrome Viral Genes Using Microarray Chip

Two viral pathogens, namely, porcine reproductive and respiratory syndrome virus (PRRSV) and foot and mouth disease virus (FMDV), were selected as models for multiple pathogen detection in a cDNA microarray. Two signature regions selected from ORF2 (around 500 bp) and ORF5 (around 600 bp) of PRRVS (America serotype), and one signature region from structural genes VP1 (around 500 bp) of FMDV type O were designed and spotted on a nylon membrane. For PCR sensitivity study, the cloned FMDV–VP1 template could be diluted to near one copy and its PCR product was still detectable in gel electrophoresis. In the microarray detection, the labelling FMDV probes (3 mg/ml) could be diluted 320 times and still maintained a visible colour when hybridized with the chip. Using the mixing primers, the microarray chip demonstrated rapid and accurate detection of the specific genes. To our knowledge, this preliminary study is the first example reported applying the long signature sequences to the multiple pathogen detection in cDNA microarray.     

猪生殖——呼吸综合征病毒蛋白的结构与功能

猪生殖－－呼吸道综合征（ＰＲＲＳ）病毒是新发现的ＲＮＡ病毒，其基因组有８个开放阅读框架（ＯＲＦ），ＯＲＦ１编码病毒的ＲＮＡ聚合酶，ＯＲＦ２－７分别编码糖蛋白ＧＰ２－５、Ｍ蛋白和Ｎ蛋白。本文概括了近几年来国际上对其病毒蛋白的研究成果，对这些病毒蛋白的结构，功能及其免疫学地位进行了分析和讨论。     

Identification of equine herpesviruses 1 and 4 by polymerase chain reaction

OBJECTIVE: To develop and validate specific, sensitive and rapid (< 8 hour) diagnostic tests using polymerase chain reaction (PCR) for the diagnosis of abortion and respiratory disease caused by equine herpesvirus 1 (EHV1; equine abortion virus) and EHV4 (equine rhinopneumonitis virus). DESIGN: Primer sets based on nucleotide sequences encoding glycoprotein H (gH) of EHV1 and gB of EHV4 were designed and used in single round and second round (seminested) PCRs, and in a multiplex PCR for the diagnosis of EHV1 and EHV4 infections. METHODS: Oligonucleotide primers were designed for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The tests were applied to tissue samples from aborted equine foetuses and to nasopharyngeal swabs from horses with acute febrile respiratory disease. RESULTS: Individual single round and a second round (seminested) EHV1 and EHV4 PCRs were specific in that EHV1 primers amplified all (n = 30) EHV1 isolates and did not amplify EHV4. Similarly EHV4 primers amplified all (n = 6) EHV4 isolates and did not amplify EHV1. Both PCRs were sensitive in that the first round EHV1 PCR detected 1220 molecules of EHV1 plasmid DNA and the first round EHV4 PCR detected 7280 molecules of EHV4 plasmid DNA. The EHV1 second round PCR was 100 times more sensitive in that it detected 12 molecules of EHV1 DNA and the EHV4 second round PCR was 1000 times more sensitive in that it detected 8 molecules of EHV4 DNA. There was a high correlation between detection of EHV1 by virus isolation and PCR when tissue samples from 71 aborted foetuses were examined; all samples positive by virus isolation were positive by PCR. Similarly the EHV4 PCR was at least as sensitive as virus isolation when applied to nasaopharyngeal swabs from horses with respiratory disease in that all samples positive by virus isolation were also positive by PCR. CONCLUSION: Individual single round and second round (seminested) PCRs and a seminested multiplex PCR were developed that enabled reliable, rapid detection of EHV1 and EHV4 in aborted foetal tissues and nasopharyngeal swab samples.     

Surveillance for highly pathogenic avian influenza in migratory shorebirds at the terminus of the East Asian-Australasian Flyway

AIM: To determine if migratory birds arriving in New Zealand in the Southern Hemisphere spring of 2004 were infected with the highly pathogenic avian influenza (AI) virus, H5N1.

METHODS: Cloacal and faecal samples were collected from migratory red knots following their arrival in New Zealand in October 2004. Two species of resident sympatric birds, wrybill and mallard duck, were sampled prior to, and following, the arrival of migratory birds.

RESULTS: No AI viruses were isolated from migratory or resident shorebirds. Non-pathogenic AI viruses were isolated from six resident mallard ducks, comprising the endemic subtypes H4 (n=2), H7 (non-pathogenic), H10, and H11 (n=2).

CONCLUSIONS: Highly pathogenic AI H5N1 virus was not detected in migratory shorebirds or sympatric water birds in the Firth of Thames, New Zealand, in 2004-2005, despite the possible proximity of migratory birds to outbreaks of the disease in East Asia in 2004.     

牛磺脱氧胆酸钠和CpGDNA对灭活H9N2禽流感病毒鼻腔免疫鸭的呼吸道抗体分泌细胞的影响

运用牛磺脱氧胆酸钠和CpG DNA配合鸭源禽流感H9N2灭活病毒滴鼻免疫雏鸭,通过检测鸭呼吸道各组织中IgA和IgG抗体分泌细胞面积的变化对免疫效果进行评价.结果发现:应用牛磺脱氧胆酸钠和CpG DNA配合H9N2灭活病毒鼻腔免疫后3、5和7周,鼻腔、气管和气管叉组织中的IgA和IgG分泌细胞面积显著或极显著(P＜0.05或P＜0.01)高于单独H9N2灭活病毒免疫后的水平;应用CpG DNA配合H9N2灭活病毒鼻腔免疫后鼻腔和气管组织中IgA和IgG分泌细胞的面积有部分提高(P＜0.05);而单独运用H9N2灭活病毒鼻腔免疫后IgA和IgG分泌细胞面积同对照组相比没有显著差异.结果表明:在牛磺脱氧胆酸钠和CpG DNA的配合下,禽流感H9N2灭活病毒鼻腔免疫能够提高雏鸭呼吸道组织中IgA分泌细胞和IgG分泌细胞的面积,有效地增强呼吸道局部的免疫应答水平.     

Respiratory phagocytes are implicated in enhanced colibacillosis in chickens co-infected with influenza virus H9N2 and Escherichia coli

1. The aim of this study was to determine the most likely time interval after infection with influenza virus H9N2 for co-infection with Escherichia coli to cause colibacillosis, the importance of lung load of E. coli and the involvement of respiratory phagocytes.

2. Specific pathogen free chickens were inoculated intranasally with 10⁶EID₅₀ of influenza virus or uninfected. After specified time intervals, 10⁷ CFU E. coli or phosphate-buffered saline was inoculated. The presence of lesions, the number of respiratory phagocytes in the respiratory lavage fluid and the E. coli load in the lung were determined after different time intervals.

3. Compared with the number of lesions in chickens receiving only E. coli inoculation, the number lesions in co-infected chickens were increased at 0- and 3-d time intervals, but reduced in the groups at 6- and 9-d intervals between co-infection.

4. At 1–3 d after E. coli inoculation, the number of lesions chickens was correlated with the number of respiratory phagocytes harvested and related to the E. coli load in the lungs at 5 d.

5. These results suggest that the lesions caused by E. coli in chickens were increased within a 0–3 d interval following H9N2 virus inoculation and that this effect is related to the number of respiratory phagocytes.     

猪繁殖与呼吸综合征病毒、猪圆环病毒2型与副猪嗜血杆菌混合感染的诊断及病原分析

为确定河南开封某猪场发生猪呼吸系统疾病综合征（PRDC）的病原,本研究无菌采集病死保育猪肺脏、心脏和脾脏等组织样品,进行细菌学检验和药敏试验,通过PCR/RT-PCR检测样品中猪繁殖与呼吸综合征病毒（PRRSV）、猪圆环病毒2型（PCV2）、猪流感病毒（SIV）、猪伪狂犬病病毒（PRV）、猪瘟病毒（CSFV）和猪肺炎支原体（Mhp）等病原,并对核酸阳性病毒性病原的抗原结构基因进行测序和遗传演化分析。结果表明,通过细菌分离培养、形态观察、卫星现象观察和16S rRNA基因鉴定,从病死保育猪体内分离鉴定出1株副猪嗜血杆菌（Hps）,药敏实验表明该菌株对对氨苄西林、阿莫西林克拉维酸、头孢噻呋和四环素几种药物敏感。核酸检测PRRSV和PCV2核酸阳性,分别命名为PRRSV/HN-2019和PCV2/HN11-2019;进一步对PRRSV/HN-2019和PCV2/HN11-2019的结构基因分析发现,PRRSV/HN-2019与与NADC30分支的毒株亲缘关系较近,属于NADC30-like毒株;PCV2/HN11-2019与PCV-2d分支的毒株亲缘关系较近,属于PCV-2d分支。综上所述,本研究确定该猪场存在PRRSV、PCV2和Hps的混合感染,为该猪场下一步的PRDC有效防控提供了参考依据。     

Diagnosis and Pathogen Analysis of Mixed Infection of Porcine Reproductive and Respiratory Syndrome Virus,Porcine Circovirus Type 2 and Haemophilus parasuis

To identify the causative agent of porcine respiratory disease complex (PRDC) occurred at a pig farm in Kaifeng city,Henan province,we identified the potential causative bacteria of the morbid nursing piglets by bacterial test and drug sensitivity test of the clinical samples (lung,liver and spleen),and detected the common potential pathogens causing PRDC diseases by PCR and RT-PCR assays,including porcine reproductive and respiratory syndrome virus (PRRSV),porcine circovirus type 2 (PCV2),swine influenza virus (SIV),pseudorabies virus (PRV),classical swine fever virus (CSFV) and Mycoplasma hyopneumoniae (Mhp),and then sequcing and phylogenetic analysis of the structural gene of positive causative pathogens were carried out.The results showed that a Haemophilus parasuis (Hps) strain were isolated and identified by the observation of bacterial morphology and satellite phenomenon,and the sequence analysis of 16S rRNA gene.The drug sensitivity test showed that the Hps strain was sensitive to ampicillin,amoxicillin-clavulanic acid,ceftiofur and tetracycline.Meanwhile,PCR and RT-PCR assays indicated that all the samples were positive for PRRSV and PCV2,and named the involved strain as PRRSV/HN-2019 and PCV2/HN11-2019,respectively.Sequencing and phylogenetic analysis of the ORF5 gene of the newly identified PRRSV revealed that the PRRSV/HN-2019 strain was closely related to the NADC30-like strains and grouped into NADC30-like genotype clade.And cap gene of the newly identified PCV2 strain the PCV2/HN11-2019 strain was closely related to the PCV-2d strains and grouped into PCV-2d genotype clade.In conclusion,this study demonstrated that the morbid piglets were co-infected with PRRSV,PCV2 and Hps,which provided a basis for the development of effective control strategies in the pig farm.     

Phylogenetic and Molecular Characterization of the Equine Influenza Virus A (H3N8) Causing the 1997 and 2004 Outbreaks in Morocco

Reported here are the results of antigenic and genetic characterization of equine influenza strains causing local outbreaks reported in Morocco, respectively, in 1997 and 2004. The antigenic and genetic characterizations of the equine influenza virus H3N8 are reported here. The highest similarity between the HA1 nucleotide sequences of A/equine/Nador/1/1997 and those of A/equine/Rome/5/1991 and A/equine/Italy/1199/1992 demonstrate that A/equine/Nador/1/1997 belongs to the European lineage. On the other hand, A/equine/Essaouira/2/2004 and A/equine/Essaouira/3/2004 were classified in the predivergent lineage. The present work emphasizes the importance of a national influenza survey program, which requires a collaborative laboratory network to promote the collection and characterization (antigenic and genetic) of equine influenza viruses in real time.     

1株重组猪繁殖与呼吸综合征病毒的基因组特征

猪繁殖与呼吸综合征病毒（PRRSV）不断发生变异,其防控难度也逐渐增加。为了及时监测PRRSV在自然感染背景下的遗传进化特点,本研究从上海某发病猪场的临床样品中分离获得1株PRRSV毒株,命名为SHpd1/2018株,并对其生物学特性和基因组序列进行分析。结果表明,该毒株基因组全长为15 018 bp（不含poly A）,不能被针对HP-PRRSV强毒HuN4株的Nsp2的单抗所识别,但其增殖特性与强毒HuN4株相似。序列分析表明,SHpd1/2018株与HP-PRRSV强毒株的相似性最高（与HuN4相似性为94.3%）。重组分析结果显示,SHpd1/2018株是以HP-PRRSV-like为主要亲本毒株,NADC30-like为次要亲本毒株的重组病毒,两个重组断点nt 2002和nt 3205均位于Nsp2基因的高变区。研究证实SHpd1/2018株是1株发生变异重组的PRRSV分离株,该毒株是上海某猪场保育猪发病的主要原因。