WAVLD Symposium Handbook_V4.indd - csiro

World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007
EVALUATION OF ELISAS TO DETECT ANTIBODIES TO BLUETONGUE VIRUS IN
INDIVIDUAL AND TANK MILK SAMPLES.
G Delbridge* 1 , RA Hawkes 1 , A Jugow 1 , B Hoffmann 2 and PD Kirkland 1
1 Virology Laboratory, Elizabeth Macarthur Agricultural Institute, NSW DPI, Menangle, NSW Australia
2 Friederick Loeffler Institute, Insel Reims, Germany
Introduction
Milk samples have been used for the identification of individual animals that have been infected with viruses,
either by the detection of antibodies or direct agent detection. Further, testing of tank (bulk) milk samples has
been used as a very efficient and cost effective method for the identification of infected herds and may be
used to define the geographical distribution of an agent. This study describes the evaluation of ELISAs to
detect antibodies to bluetongue virus (BTV) in both individual and tank milk samples and the subsequent use
to map geographical areas where there has been BTV infection.
Material & methods
BTV group reactive ELISAs using monoclonal antibodies (mAbs) in either competitive or blocking formats
have been used for the detection of antibodies to viruses from the BTV serogroup for many years 1 . In this
project, a range of antigens derived from different BTV serotypes and different monoclonal antibodies were
compared to establish a combination that would provide an assay with optimal sensitivity and specificity. A
limited number of reagent combinations were later compared by testing a panel of serum samples from cattle
and sheep infected with one of the 23 serotypes of BTV. Several thousand samples from naturally infected
cattle, sheep and goats from Australia, China, Italy and Germany and sheep and cattle sera from a BTV free
country were tested to confirm the sensitivity and specificity of the preferred reagent combination. An indirect
ELISA was also developed using cell culture derived antigen and a peroxidase conjugated antibovine IgG.
Matching individual serum and milk samples from naturally infected cattle were tested in both the blocking
and indirect ELISAs. Positive milk samples were titrated to determine the limits of detection and as a means
of estimating herd prevalence. The performance of the blocking ELISA was also compared with all of the
commercial ELISA kits available. Finally, tank milk samples from dairy herds in NSW were tested to evaluate
the use of these assays to map the distribution of BTV.
Results
A blocking assay employing antigen purified BTV23 infected cell cultures was found to provide optimal
sensitivity and specificity and was shown to reliably detect antibodies to each serotype of BTV, both soon
after the onset of infection and also after several years in the absence of re-infection with another serotype.
This blocking ELISA gave superior results in correctly identifying naturally infected cattle, sheep and goats
from Australia, China, Italy and Germany to any of the commercially available assays and also had higher
analytical sensitivity. There was a very high level of agreement between the results for matching serum and
milk samples from individual animals. The indirect ELISA also had good diagnostic performance and was
able to reliably identify BTV infected cattle with any of the serotypes. Both assays could detect antibodies in
tank milk samples from herds where there was a moderate to low prevalence of infection, usually giving
positive results with a prevalence of 5-10%. There was a high specificity when testing either individual or
tank milk samples.
Discussion & conclusions
The results of this study show that these assays are suitable for the detection of BTV antibodies in both milk
and serum samples from individual animals and can detect antibodies to any of the 23 serotypes of BTV. In
regions where there are dairy cattle, the capacity to be able to detect antibodies in tank milk samples
provides a useful tool to define areas in which there has been BTV transmission and to identify BTV free
areas. These should be valuable assays for surveillance in regions where the distribution of BTV is
expanding. Both assays provide advantages. A blocking assay can be used to test specimens from any
animal species while the indirect ELISA can be used in combination with the blocking ELISA when testing
cattle as a confirmatory assay. Further, because of the inclusion of a control antigen, the indirect assay can
be used to clarify the BTV status of suspicious reactors in the blocking ELISA.
References
1. Jeggo, M., Wright, P., Anderson, J., Eaton, B., Afshar, A., Pearson, J., Kirkland, P., and Ozawa, Y. 1992.
Standardization of the competitive ELISA test and reagents for the diagnosis of Bluetongue. In: Bluetongue,
African Horse Sickness, and Related Orbiviruses, Eds Walton, T.E. and Osborne, B.I., CRC, Boca Raton, pp
547-560.
Mon 12 November
Mon 12 November
World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007
MOLECULAR EPIDEMIOLOGY OF BLUETONGUE VIRUS TYPE 8 FROM THE NETHERLANDS 2006
S. Maan*, N.S. Maan, S.J. Anthony, K.E. Darpel, A.E. Shaw, C.A. Batten, H. Attoui & P.P.C. Mertens
Arbovirology Dept., Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey, GU24 0NF (UK)
Introduction: Bluetongue virus (BTV) (genus Orbivirus, family Reoviridae) is an ‘arbovirus’ with a ten
segmented dsRNA genome that is transmitted between its ruminant hosts by Culicoides biting midges. Since
1998, eight strains of six BTV serotypes (types-1, 2, 4, 8, 9 and 16) have invaded Europe, collectively
representing the largest outbreak of the disease on record, with the deaths of > 2 million animals. Since
1998, the virus has spread further west and north into Europe, initially reflecting changes in the distribution of
Culicoides imicola, the major vector species for BTV in southern Europe and the influence of climate change
[9]. In the summer of 2006, BTV-8 caused a major outbreak of disease in northern Europe, which was
transmitted by novel vector species (C. obsoletus and C. pulicaris groups) [3, 5, 8]. The virus arrived in the
UK (for the first time ever) in September 2007, infecting a Highland cow at a rare-breeds farm in Great
Blakenham, Near Ipswich, in the East Anglian region of England.
BTV serotype is controlled primarily by the outer coat protein VP2 (encoded by genome segment 2 (Seg-2).
Sequencing studies of Seg-2 from different BTV strains, were used to create a sequence database for
molecular epidemiology studies [4, 5, 6, 7, 8], which showed that most serotypes can be divided in eastern
and western strains (topotypes). Live attenuated ‘vaccine’ strains of BTV-2, 4, 9 (western topotype) and
BTV-16 (eastern topotype) were used in attempts to minimise the circulation of BTV in Europe. The South
African ‘Group B’ vaccine strains (types 3, 8, 9, 10 and 11) were also used briefly, in Bulgaria. The release of
these ‘attenuated’ strains, has added genetic diversity to the pool of field strains circulating in southern
Europe, generating an unprecedented mix of eastern and western viruses, leading to reassortment [2].
RT-PCR assays were developed for identification of the 24 BTV serotypes [7, 8] and used to identify the
strains circulating in Europe, including the most recent isolates from the UK (UKG2007/01). The entire
genome (segments 1-10) of BTV-8 from the Netherlands 2006 (NET2006/04) was sequenced and compared
to other European field and vaccine strains, to help determine the origin of the outbreak. This is the first
report of the full genome sequence of the BTV-8 strain from northern Europe.
Material & methods: RNA was extracted from cell-free supernatants, or EDTA treated blood samples, using
the QIAamp Viral RNA Mini Kit (QIAGEN), for serogroup or serotype specific RT-PCR assays [1, 7]. RNA for
synthesis of full length cDNAs and sequencing was purified from cell cultures using Trizol® (Invitrogen), [5,
6]. Sequences were aligned and subjected to phylogenetic analysis.
Results: Phylogenetic analyses of nucleotide (nt) sequence data for all ten genome segments of
NET2006/04 showed up to 98% identity with other viruses derived from a western origin (from the reference
collection at IAH Pirbright), although the most closely related strain varied for each segment. Seg-2 from
NET2006/04 showed >93% sequence identity with the reference strain of BTV-8 (identifying its serotype)
and was most closely related (> 97%) to BTV-8, from Nigeria 1982 (NIG1982/07) showing that it originated in
sub-Saharan Africa. None of the genome segments of NET2006/04 was identical to corresponding segments
of other field or vaccine strains, indicating that it had not exchanged genome segments (reassorted) with
other European viruses. The most recent BTV-8 strain from the UK (UKG2007/01) clusters with other BTV-
8s from northern Europe (~99% nt identity in Seg-2, with strains from Belgium or the Netherlands,
2006/2007), confirming that the UK strain was derived from the northern European outbreak.
Discussions & conclusions: The BTV-8 strain from northern European (2006) was not derived either
directly or by reassortment, from field or vaccine strains already present in the Mediterranean region, but
represents a new introduction to the region. Although its route of entry to northern Europe has not been
established, it originated in Sub-Saharan Africa and is distinct from the BTV-8 vaccine strain. The most
recent strain of BTV-8 from the UK (UKG2007/01) is derived from the outbreak in Belgium or the Netherlands
(2006-2007).
References
1. Anthony S, Jones H, Darpel KE, Elliott H, Maan S, Samuel A, Mellor PS & Mertens PPC (2007). J Virol Methods 141, 188-
197.
2. Batten CA, Shaw AE, Maan S, Maan NS and Mertens PPC (2007). J Gen Virol (Submitted).
3. Darpel KE, Batten CA, Veronesi E, and 11 other authors (2007). Vet Rec 161, 253-261.
4. IAH reference collection: http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/BTV-isolates.htm
5. Maan S, Maan NS, Samuel AR, Rao S, Attoui H, Mertens PPC. (2007a). J Gen Virol 88, 621-630.
6. Maan S, Rao S, Maan NS, Anthony SJ, Attoui H, Samuel AR & Mertens PPC (2007b). J Virol Methods 143,132-139.
7. Mertens PPC Maan NS, Prasad G, Samuel AR, Shaw AE, Potgieter AC, Anthony SJ & Maan S (2007a). J Gen Virol 88,
2811–2823.
8. Mertens PPC, Maan S, Maan NS, Bankowska K, Swain A, Batten C, Carpenter S, Gloster J, Mellor PS & Oura C (2007b).
Promed report Archive Number 20070926.3196, 26-SEP-2007.
9. Purse BV, Mellor PS, Rogers DJ, Samuel AR, Mertens PPC & Baylis M (2005). Nat Rev Microbiol 3, 171–181.