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Short Communications |
1 Veterinary Sciences Division, Agri-Food and Biosciences Institute, Stoney
Road, Stormont, Belfast BT4 3SD
2 Veterinary Sciences Division, Agri-Food and Biosciences Institute, 43 Beltany
Road, Omagh BT78 5NF
Correspondence: E-mail for correspondence: david.graham{at}afbini.gov.uk
BOVINE viral diarrhoea virus (BVDV) is an economically significant pathogen in many countries, including the UK and Ireland (Bennett and others 1999, Saatkamp and others 2008). Overall, losses attributable to fetal damage are recognised to make a significant contribution to economic losses (Houe 1999, Gunn and others 2004). BVDV in aborted bovine fetuses is likely to be underdetected for two reasons. First, the actual abortion may be undetected or appropriate samples may not be submitted for laboratory investigation (Carpenter and others 2006). Secondly, even when samples are submitted, the cause of the abortion is often not identified (Murray 1990, Jamaluddin and others 1996). In the case of BVDV, the possibility of false-negative results due to factors including autolysis and cell culture cytotoxicity is well recognised, and the need for improved diagnostic tools has been highlighted (Hyndman and others 1998, Sandvik and others 2008). This short communication reports an investigation into the performance of a range of diagnostic tests for detecting fetal infection with BVDV.
Between December 2005 and August 2006, samples were collected from 140 aborted and stillborn bovine fetuses submitted for postmortem examination. Samples comprised fetal fluid (typically heart blood), spleen, ileum and lung, and an ear tissue plug with two full skin thicknesses. Spleen, ileum and lung were processed for immunofluorescent antibody testing (IFAT) as they were collected. Otherwise, samples were stored at -20°C or below before testing. Samples were also collected for histological and bacteriological examination as part of the routine processing of these submissions, and the crown-rump length was recorded as an indicator of fetal age. The fetal fluid and ear skin samples were tested for BVDV antigen using a commercial ELISA (HerdChek BVDV Antigen/Serum Plus Test Kit; IDEXX). The fetal fluid samples were also tested for BVDV-specific antibodies using a blocking ELISA kit (BVD/BD p80 Blocking One-Step; LSI). IFAT was performed using a commercial polyclonal BVDV antiserum (VMRD). The spleen, ileum and lung samples from each fetus were pooled, and a 10 per cent w/v suspension was prepared and processed for virus isolation according to standard procedures (Graham and others 1998). Each pool received two passages on fetal bovine lung cells followed by IFAT for BVDV. Viral RNA was extracted from a 200 µl volume of each fetal fluid using the QIAamp Virus BioRobot MDX kit on a BioRobot Universal system (Qiagen). Real-time RT-PCR was subsequently carried out using the Quantitect SYBR Green RT-PCR kit (Qiagen) with the primer pair F2/PESTR (Letellier and Kerkhofs 2003). Briefly, 25 µl of the reaction mixture, containing 2·5 µl of extracted RNA and 300 pmol of each primer, was subjected to the following thermal conditions: 50°C for 30 minutes, 95°C for 15 minutes, then 40 cycles of 95°C for 15 seconds and 55°C for 30 seconds, followed by 72°C for 30 seconds. Thereafter, melt analysis was performed, with samples that gave both a typical amplification curve and a melt temperature between 83°C and 86°C being considered positive. All kits were used according to the manufacturers' instructions. For a minority of fetuses, not all tests were performed, due primarily to insufficient sample being available.
Based on the calculation reported by Rexroad and others (1974), the fetuses examined ranged from 122 to 302 days of gestation (median 210 days). One or more tests were positive from a total of 25 (17·9 per cent) fetuses (Table 1). Virus isolation gave positive results in three of 134 (2·2 per cent) fetuses, and IFAT gave a positive result in one of 134 (0·7 per cent) fetuses. Specific fluorescence was detected in all three tissues (spleen, ileum and lung) of the IFAT-positive fetus, which was also culture positive. In contrast, 20 of 140 (14·3 per cent) fetal fluids and 16 of 129 (12·4 per cent) ear skin samples were positive by antigen ELISA. The overall agreement between antigen ELISA results for fetal fluids and ear skin was 95·3 per cent. The real-time RT-PCR was positive in 18 of 138 (13 per cent) fetal fluids, with positive cycle threshold values ranging from 17·9 to 26·9 (mean 23·5). The agreement between RT-PCR results and antigen ELISA results on fetal fluids was 97·1 per cent. Three fetal fluid samples positive by ELISA were negative by RT-PCR. This could have been due to inhibitors in the samples, although this cannot be stated conclusively. Alternatively, it could reflect a greater resistance of viral proteins relative to RNA to degradation in the fetal environment. All three of the culture-positive fetuses were also positive by antigen ELISA on fetal fluid and by real-time RT-PCR, with the ear skin sample from two of these also positive by ELISA. Antibodies were detected in two of 140 (1·4 per cent) samples. No other tests for BVDV on these two fetuses were positive. A possible alternative cause of abortion was diagnosed in only six of the 25 fetuses testing positive for BVDV.
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Overall, the results show that use of the traditional diagnostic methods of virus isolation and IFAT produced a significant number of false-negative results, with consequent underestimation of the incidence of BVDV in aborted and stillborn calves. In contrast, both real-time PCR and antigen ELISA were found to be superior, with good overall correlation between these tests. This finding is consistent with that of Jamaluddin and others (1996) and Hyndman and others (1998). In the latter study, BVDV was isolated from only 1 per cent of aborted bovine fetuses compared with 8 per cent found positive by a conventional nested RT-PCR. In that study, a large number of fetal fluid samples were toxic in cell culture. Although this was not the case in the present study, a similar low rate of isolation was observed. The real-time RT-PCR used in the present study offers a number of advantages over the nested assay described by Hyndman and others (1998). These include speed and the lack of handling, both between assay steps and after amplification, with a consequent reduction in the likelihood of cross-contamination between samples.
While antigen capture ELISA, using both blood samples and, more recently, ear tissue, has been used widely for the detection of BVDV in postnatal cattle (Cornish and others 2005), it is not used extensively for this purpose in fetuses. Ear skin was included in this study because of the possibility that viral antigens in this tissue might be less susceptible to autolysis and degradation. However, the results did not support this supposition.
It was not possible to state whether fetuses testing positive for viral antigen or RNA were persistently infected. Fredriksen and others (1999) showed that antigen may be detected by immunostaining in multiple organs of persistently infected fetuses. The fact that only one fetus was positive by IFAT may suggest that the majority of infections detected in this study were transient, and therefore associated with lower viral loads. On the other hand, the optical density values detected by antigen ELISA were largely typical of those in persistently infected cattle (D. A. Graham, unpublished data).
The use of serology to detect fetal infection with BVDV has been reported previously (Murray 1990, Hyndman and others 1998), with fetuses considered capable of mounting a humoral immune response from approximately 120 days of gestation (Schultz 1973, Ohmann and others 1982). An isotype-independent blocking ELISA capable of detecting both specific IgG and IgM was selected for use in this study. Despite this, only two fetuses (1·4 per cent) were found to be seropositive, with no RT-PCR- or antigen ELISA-positive results from these animals. In contrast, Hyndman and others (1999) found 5 per cent of fetuses to be seropositive, with all of these also being positive by RT-PCR, and Murray (1990) found 9·6 per cent of fetuses (the majority of which were stillbirths) to be seropositive. These differences in seroprevalence may reflect differences in the test methods, the fetal ages or the true prevalence in the study populations.
The results of this study highlight both the need to use optimal diagnostic methods and the significant cost to the cattle industry in Northern Ireland due to abortion associated with BVDV. While direct or indirect evidence of exposure does not confirm BVDV as the cause of these abortions, the failure to find any other potential causes in 19 of the 25 positive fetuses increases the likelihood of a causal relationship. The level of infection in fetuses in this study is consistent with the known relatively high prevalence of BVDV in cattle herds in Northern Ireland (Graham and others 2001). This contrasts with the situation in several other European countries, particularly in Scandinavia, that have achieved, or are pursuing, systematic control and eradication of BVDV based on the identification and removal of persistently infected cattle (Lindberg and others 2008).
Antigen-detecting ELISAs or real-time RT-PCR for BVDV were found to be superior to IFAT, virus isolation or serology for detection of fetal exposure. It is recommended that these tools be incorporated into diagnostic protocols for investigating abortions. The evidence of exposure detected in 17·9 per cent of fetuses further highlights the need for herd owners and veterinarians to adopt a systematic approach to BVDV control.
Acknowledgements
This work was funded by the Department of Agriculture and Rural Development, Northern Ireland. The authors acknowledge the assistance of the staff at the Veterinary Services Division, Omagh, with the postmortem collection of the samples and additional diagnostic testing, and that of Ms Julie Forster with the RNA extractions.
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