Diagnostic specificity of an equine influenza blocking ELISA estimated from New South Wales field data from the Australian epidemic in 2007

This observational study was undertaken in order to evaluate the diagnostic specificity of the blocking enzyme-linked immunosorbent assay (bELISA) for serum antibodies to influenza A virus nucleoprotein during the equine influenza (EI) outbreak response in New South Wales, Australia, in 2007. Using data collected during the outbreak response, bELISA testing data were collated for assumed uninfected horses from areas where EI infection was never recorded. Diagnostic specificity of the bELISA used during the EI response was high, but varied significantly between some regions, although the reason(s) for this variation could not be determined.     

Use of a blocking ELISA for antibodies to equine influenza virus as a test to distinguish between naturally infected and vaccinated horses: proof of concept studies

An important consideration in the selection of a vaccine during the Australian equine influenza (EI) outbreak in 2007 was the ability to differentiate between infected and vaccinated animals (DIVA). A blocking enzyme-linked immunosorbent assay (bELISA) targeted for the nucleoprotein of influenza A viruses was developed to differentiate between naturally infected horses and horses vaccinated with the ProteqFlu? vaccine, which only induces a response to viral haemagglutinin. This bELISA assay met the DIVA requirements and was used extensively during the EI control and eradication programs and 'proof of freedom' testing.     

Multiple diagnostic tests to identify cattle with Bovine viral diarrhea virus and duration of positive test results in persistently infected cattle

Several tests for Bovine viral diarrhea virus (BVDV) were applied to samples collected monthly from December 20, 2005, through November 27, 2006 (day 0 to day 342) from 12 persistently infected (PI) cattle with BVDV subtypes found in US cattle: BVDV-1a, BVDV-1b, and BVDV-2a. The samples included clotted blood for serum, nasal swabs, and fresh and formalin-fixed ear notches. The tests were as follows: titration of infectious virus in serum and nasal swabs; antigen-capture (AC) enzyme-linked immunosorbent assay (ELISA), or ACE, on serum, nasal swabs, and fresh ear notches; gel-based polymerase chain reaction (PCR) testing of serum, nasal swabs, and fresh ear notches; immunohistochemical (IHC) testing of formalin-fixed ear notches; and serologic testing for BVDV antibodies in serum. Of the 12 animals starting the study, 3 died with mucosal disease. The ACE and IHC tests on ear notches had positive results throughout the study, as did the ACE and PCR tests on serum. There was detectable virus in nasal swabs from all the cattle throughout the study except for a few samples that were toxic to cell cultures. The serum had a virus titer ≥ log₁₀ 1.60 in all samples from all the cattle except for 3 collections from 1 animal. Although there were several equivocal results, the PCR test most often had positive results. The BVDV antibodies were due to vaccination or exposure to heterologous strains and did not appear to interfere with any BVDV test. These findings illustrate that PI cattle may be identified by several tests, but differentiation of PI cattle from cattle with acute BVDV infection requires additional testing, especially of blood samples and nasal swabs positive on initial testing. Also, calves PI with BVDV are continual shedders of infectious virus, as shown by the infectivity of nasal swabs over the 11-mo study.     

Detection of equine herpesvirus-1 in nasal swabs of horses by quantitative real-time PCR

Background: Early identification of inhalation-transmitted equine herpesvirus type 1 (EHV-1) infections has been facilitated by the availability of a number of real-time quantitative PCR (qPCR) tests. A direct comparison between nasal swab qPCR and traditional virus isolation (VI) requires a method for normalizing the qPCR samples and controlling for PCR inhibitors present in some clinical samples.
Objectives: To quantify EHV-1 shedding in viral swabs using an internal control and to compare fast qPCR to VI for the detection of EHV-1 in nasal swabs from horses.
Animals: Fifteen horses experimentally infected with EHV-1.
Methods: Experimental study : Nasal swab samples were collected daily after experimental infection for up to 21 days. VI was performed by conventional methods. The DNA was prepared for qPCR with the addition of a known quantity DNA of Marek's disease virus as an internal control. qPCR was performed.
Results: The qPCR method detected virus up to day 21 after challenge, whereas VI detected virus only to day 5. The median Kaplan-Meier estimates for EHV-1 detection were 12 days for qPCR and 2 days for VI ( P < .0001). When compared with VI, the sensitivity and specificity of qPCR were 97 (95% CI: 86–100) and 27% (95% CI: 20–35).
Conclusions and Clinical Importance: We conclude that fast qPCR of nasal swab samples should be chosen for diagnosis and monitoring of herpesvirus-induced disease in horses. Recommended reference ranges of C _T values are provided as well as justification of a minimum 10-day quarantine period.     

Evaluation of an immunofluorescent test for the rapid diagnosis of field infections of infectious bovine rhinotracheitis

An indirect immunofluorescent test for the rapid detection of infectious bovine rhinotracheitis (IBR) virus in smears of nasal and ocular secretions from infected cattle, was compared with conventional virus isolation procedures using 200 swabs from 107 field outbreaks of suspected IBR. Virus was isolated from 38 per cent of the swabs and the indirect immunofluorescent test detected virus in 14.5 per cent of the positive swabs. Examination of samples from more than one animal increased the confirmation rates of infection during outbreaks to 39 per cent by virus isolation and 21.5 per cent by the immunofluorescent test. Ocular swabs were better than nasal swabs for confirming infection both by virus isolation and immunofluorescence, and agreement between the two tests increased with the number of samples collected during an outbreak.     

Experimental inoculation study indicates swine as a potential host for Hendra virus

Hendra virus (HeV) is a zoonotic virus from the family Paramyxoviridae causing fatal disease in humans and horses. Five-week-old Landrace pigs and 5-month-old Gottingen minipigs were inoculated with approximately 10⁷ plaque forming units per animal. In addition to fever and depression exhibited in all infected pigs, one of the two Landrace pigs developed respiratory signs at 5 days post-inoculation (dpi) and one of the Gottingen minipigs developed respiratory signs at 5 dpi and mild neurological signs at 7 dpi. Virus was detected in all infected pigs at 2–5 dpi from oral, nasal, and rectal swabs and at 3–5 dpi from ocular swabs by real-time RT-PCR targeting the HeV M gene. Virus titers in nasal swab samples were as high as 10^4.6 TCID₅₀/mL. The viral RNA was mainly distributed in tissues from respiratory and lymphoid systems at an early stage of infection and the presence of virus was confirmed by virus isolation. Pathological changes and immunohistochemical staining for viral antigen were consistent with the tissue distribution of the virus. This new finding indicates that pigs are susceptible to HeV infections and could potentially play a role as an intermediate host in transmission to humans.     

Diagnosis of equine infectious anaemia during the 2006 outbreak in Ireland

In 2006 there was an outbreak of equine infectious anaemia (EIA) in Ireland. This paper describes the use of the diagnosis of clinical and subclinical cases of the disease. In acute cases the ELISAs and the immunoblot were more sensitive than the AGID. In one mare, fluctuating antibody levels were observed in all the serological assays before it seroconverted by AGID. Viral RNA and DNA were detected by RT-PCR and PCR in all the tissues from the infected animals examined postmortem. The PCR detected viral DNA in plasma regardless of the stage of the disease. In contrast, the RT-PCR detected RNA in only 52 per cent of the seropositive animals tested and appeared to be most sensitive for the detection of virus early in infection. Both PCR and RT-PCR demonstrated potential to detect acutely infected horses earlier than some of the official tests. The serological data suggest that the usual incubation/seroconversion period for this strain of the virus was approximately 37 days but may be more than 60 days in a few cases.     

Viruses associated with outbreaks of equine respiratory disease in New Zealand

AIM: To identify viruses associated with respiratory disease in young horses in New Zealand.

METHODS: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3).

RESULTS: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV. Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV-2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR)=2.67, 95% CI=1.59-4.47, p=0.017].

CONCLUSIONS: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV-2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.     

Viruses associated with outbreaks of equine respiratory disease in New Zealand

AIM: To identify viruses associated with respiratory disease in young horses in New Zealand. METHODS: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3). RESULTS: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV. Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV-2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR)=2.67, 95% CI=1.59-4.47, p=0.017]. CONCLUSIONS: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV-2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.     

2007年华北地区H3N8亚型马流感病毒的分离与鉴定

2007年10月,华北地区某赛马场的马同时发生了以发烧、流水样鼻汁或脓性分泌物、咳嗽等临床症状为主的疾病,疑似马流行性感冒。采集患病赛马的鼻腔分泌物,发病期和发病后14d血清,经鸡胚接种法分离病毒,并用鸡红细胞血凝抑制试验(HI)、神经氨酸酶抑制试验(NI)、病毒回归试验、血清学检测和基因序列分析对分离的病毒进行了系统鉴定。结果表明分离的毒株(A/equine/Huabei/1/2007(H3N8)为马源H3N8亚型马流感病毒,基因型属于美洲分支。我们通过动物回归感染试验建立起分离毒株的实验感染模型。     

Detection of antibodies to equine arteritis virus by a monoclonal antibody-based blocking ELISA.

A potent ELISA antigen was prepared from equine arteritis virus (EAV) by differential centrifugation of EAV-infected cell culture fluid, followed by solubilization of the preparation by Triton X-100 treatment. Using this antigen and a mouse monoclonal antibody against the G(L) protein of EAV, a reliable blocking ELISA (bELISA) was developed for the detection of EAV antibodies in equine sera. The bELISA was evaluated using a total of 837 test serum samples. The relative sensitivity (n = 320) of the bELISA compared to the serum neutralization (SN) test was 99.4%. The bELISA appears to be a highly specific test, the specificity of which did not appear to be adversely affected by previous exposure of horses to non-EAV-containing biologicals. Of 119 serum samples, 21 from horses without any history of exposure to EAV and 98 from racetrack Thoroughbreds, 118 were negative in the SN test and bELISA. One sample was SN-negative but suspicious with the bELISA. Based on testing 465 SN-negative field samples and 52 SN-negative samples from experimental horses, and excluding any sera giving a suspicious reaction, the relative specificity of the bELISA was 97.7%. Samples should be examined undiluted and diluted 1/10 in the bELISA because the testing of sera of high neutralizing antibody titer may be affected by a prozone-like phenomenon. The bELISA is a more rapid and cost-efficient test than the SN test for the detection of EAV antibodies in equine sera.     

Neurological disease associated with EHV-1-infection in a riding school: clinical and virological characteristics

An outbreak of neurological disease caused by EHV-1 infection is described with emphasis on diagnosis and prognosis for recumbent horses. In April 1995, an outbreak of the neurological form of Equine herpesvirus type 1 (EHV-1) occurred in a well-managed riding school with 41 horses: 34 horses showed a temperature spike and 20 some degree of neurological signs, of which 10 were nursed intensively in the indoor arena of the riding school for 3 to 20 days, 8 having to be maintained in slings for 2-18 days, while 9 needed bladder catheterisation b.i.d. for 2-16 days. Within the first 3 days, one horse was subjected to euthanasia and another horse died. Postmortem examination revealed a mild vasculitis with perivascular mononuclear cuffing and axonal degeneration in the central nervous system. Clinical diagnosis was confirmed by serology and virology: 28 horses seroconverted in one or more tests during the outbreak, whereas 12 had already high CF and SN titres in the first sample, suggestive of recent infection. Virus was isolated from nasal swabs of 4 horses, and identified as EHV-1 with type-specific monoclonal antibodies. Restriction enzyme analysis revealed that the EHV-1 strains from this outbreak belonged to genome type EHV-1.IP. The electropherotypes were identical to those from another, epidemiologically unrelated, outbreak of neurological disease 2 months earlier. The timing of the temperature spikes and seroconversions indicated that the infection was probably introduced by a horse purchased 3 weeks before neurological signs occurred. At follow-up one year later, the 10 horses that showed mild neurological signs had recovered completely. Of the 8 horses that survived intensive care, 3 had returned to around their former performance level (2 of which had been in slings), while the other 5 had become pasture-sound. At follow-up 4 years later, all pasture-sound horses had been subjected to euthanasia because of persistent mild ataxia and incontinence. In conclusion, the prognosis for recumbent horses due to EHV-1 infection is grave. For virological diagnosis, extensive and strategic sampling of febrile in-contact horses is required, and the EHV-1-specific glycoprotein G (gG) ELISA is a valuable tool for specific serological diagnosis of EHV-1 infection causing neurological disease.     

Encephalitis caused by louping ill virus in a group of horses in Ireland.

An outbreak of LI infection in a group of free range horses is described. Three of 4 horses displayed signs of CNS disturbance and 2 of these died after illnesses ranging from 2-12 days duration. In both cases a variable degree of viral polioencephalomyelitis was observed. A virus antigenically indistinguishable from a reference strain of LI virus was isolated from the brain and cervical cord of a 3 y.o. draft mare. Serum samples obtained from 3 of the horses contained HI, CF, precipitating and neutralising antibodies to LI virus, with a rise in antibody titre being demonstrated in 2 animals.     

Bubaline alphaherpesvirus 1 induces a latent/reactivable infection in goats

Latent infection is a common mechanism used by several alphaherpesviruses to persist in their host but it is not clear whether this mechanism is also triggered in heterologous infections. Cross-species infections have been documented repeatedly for alphaherpesviruses of ruminants, a group of closely related viruses. Herewith we report latent infection with bubaline alphaherpesvirus 1 (BuHV-1) in experimentally infected goats and subsequent virus reactivation after treatment with dexamethasone (DMS) at 10 months after infection. After DMS treatment, the virus was isolated in one such animal in the nasal swabs from day 3 to 9 post treatment and in the ocular swabs at day 6. The goat was euthanized 48 days after DMS treatment and viral DNA was detected by PCR in the trigeminal ganglia and in two cervical ganglia. Additionally, BuHV-1 DNA was detected by PCR in the trigeminal ganglia of the other 3 goats.     

Significant features of the epidemiology of equine influenza in Queensland, Australia, 2007

An outbreak of equine influenza (EI) caused by influenza A H3N8 subtype virus occurred in the Australian states of Queensland and New South Wales in August 2007. Infection in the Australian horse population was associated with the introduction of infection by horses from overseas. The first case of EI in Queensland was detected on 25 August 2007 at an equestrian sporting event. Infection subsequently spread locally and to other clusters through horse movements prior to the implementation of an official standstill. There were five main clusters of infected properties during this outbreak and several outliers, which were investigated to find the potential mechanism of disease spread. To contain the outbreak, Queensland was divided into infection status zones, with different movement controls applied to each zone. Vaccination was implemented strategically in infected areas and within horse subpopulations. Control and eventual eradication of EI from Queensland was achieved through a combination of quarantine, biosecurity measures, movement control, rapid diagnostic testing and vaccination.     

Nasal Shedding of Equid Herpesvirus Type 1 and Type 4 in Hospitalized,Febrile Horses

The objective of this study was to determine the prevalence of shedding of equid herpesvirus 1 (EHV-1) or EHV-4 in nasal swab samples from any febrile, hospitalized horses during a 1-year period. It was hypothesized that some fevers in horses are associated with viral replication following recrudescence of latent virus or following a horizontal viral infection prior to or during admission to a referral hospital. During the observational period, nasal swab samples were collected from 64 febrile and 10 nonfebrile hospitalized horses. Routine DNA extraction was performed, and a validated quantitative polymerase chain reaction (qPCR) assay was used to detect and quantify genomic EHV-1 and -4 DNA. Genomic DNA of EHV-4 was detected in the nasal swab specimen of 1 of 64 febrile horses. EHV-1 DNA was not detected in any of the febrile horses. Samples from all nonfebrile horses were negative for both viruses. Considering the known association between fever and shedding of EHV-1 and EHV-4, we anticipated finding a higher percentage of PCR-positive samples from febrile patients. Fevers detected were likely a result of active disease processes for which the horses were hospitalized; concurrent other diseases appeared not to affect viral recrudescence. Further studies are warranted to examine frequency and factors of EHV latency and reactivation.