How is ISAv detected?
There are 3 common tests (“assays”) for ISAv:
- The classic test is cell culture; salmon cells are grown in the laboratory and tissues from infected fish are ground up and added to the cell culture. If pathogenic (disease causing) viruses are present, they will infect and kill the cells, resulting in “plaques” or cytopathic effects (CPE)- areas where the cells have died. This indicates that a virus is present. Once CPE are detected (under a microscope), other assays are used to figure out what type of virus is infecting the cells. Cell culture can yield results slowly and, if the virus present is not pathogenic or the cell type used is not “permissive” (does not support viral infection by that particular strain of virus), no CPE will be observed (a “false negative” result).
- A second test uses antibodies to detect viral particles (for ISAv, the test is called “indirect fluorescent antibody test,” IFAT). IFAT tests have problems with low sensitivity and/or reacting to other things besides ISAv (a “false positive”) – sometimes the antibodies are not specific for ISAv alone, creating problems. For these reasons IFAT is not widely used to detect ISAv.
- The third and most recent assay type developed relies on molecular biology: the polymerase chain reaction (PCR) test. PCR assays detect DNA (because the ISAv genome is encoded as RNA, the first step is to convert this to DNA; this additional step, called “reverse transcription”, means the assay has the acronym RT-PCR). PCR tests are extremely sensitive and, in theory, do not generate false positive results. Standard PCR tests yield a positive or negative result; either the virus is present or it is not. Another type of PCR, “quantitative” PCR (qPCR, or qRT-PCR), yields an estimate of the number of viral genomes present in the sample. If the virus is present in large quantities, this assay detects it in fewer steps – so a low number indicates a highly infected sample. A problem with all PCR assays is contamination because the tests are very sensitive. Because of this drawback, samples positive by PCR are typically tested multiple times, and the product should also be sequenced to make sure that the “target” (viral) DNA was present. Often, cell culture is also used to confirm a PCR positive sample, but studies published in 2001, 2004 and 2009 show that there are virulent strains of ISAv that do not grow in culture, indicating that this method doesn’t always work for ISAv.
What is the best assay to detect ISAv?
The best type of assay, in terms of speed and sensitivity, is the RT-PCR assay. However, there is a great deal of confusion about which specific PCR assay is best – ISAv has 8 genome segments, and various researchers have developed different RT-PCR assays, some even for the same segment of the genome. Until very recently it was commonly accepted that RT-PCRs aimed at segment 8 would detect all strains of ISAv, but some of the recently detected Fraser River fish samples were positive only by segment 7 RT-PCR. The selection of the correct RT-PCR assay is critical; if the wrong test is run, ISAv will not be detected even if it is present in great abundance – a false negative result. PCR results should be confirmed by another assay and/or by sequencing the PCR product to make sure it matches the ISAv sequence.
When was ISAv first detected in the Pacific Northwest?
The recent detections, in juvenile sockeye from the West Coast of Vancouver Island, B.C., were reported on October 15, 2011. Within 10 days, the virus had also been detected in adult Chinook, coho and chum salmon from the Fraser River. Both of these results were reported by the laboratory of Dr. Fred Kibenge, the OIE (World Organization for Animal Health) reference lab for ISAv in the western hemisphere. Shortly thereafter, these results were confirmed by Dr. Are Nylund, the head of the OIE reference lab for ISAv in the eastern hemisphere (Norway). The result was a weak positive that was not reproducible in subsequent tests, and only the segment 7 RT-PCR was positive. The Canadian government then took the samples from the Kibenge lab and reran them in an attempt to confirm these results, and could not do so. However, they specifically cited that the samples were in poor condition, which may have affected their results, and they have not yet explained which tests they ran; if they chose the wrong assay, they would not have detected the virus even if it were present.
On November 28, 2011, a manuscript dating from 2004 on ISAv detection in the Pacific Northwest was leaked to the press. The work was performed by Molly Kibenge (at the time a postdoctoral student at the Pacific Biological Station in Nanaimo) but was never submitted for publication because of the objections of her supervisor, who works for the Department of Fisheries and Oceans, Canada (DFO). The paper describes the detection of ISAv in Pacific salmon species (as well as some farmed Atlantic salmon) by RT-PCR from the Bering Sea south to the Fraser River and the west coast of Vancouver Island. The infected fish were caught at sea and did not have overt signs of infection or disease (“asymptomatic”), suggesting a less virulent strain of virus was present. Kibenge used RT-PCR assays aimed at both segment 7 and 8 of the ISAv genome and also sequenced the gene segments that were amplified to confirm ISAv. Importantly, she also sequenced her positive controls (which are run to make sure that the assay is working correctly), which had different sequences than the virus that was detected in the salmon sampled. This makes the work compelling, even though she could not get the virus to infect tissue culture cells. Although this manuscript was not entirely complete, it should have provoked enough interest to be shared with the broader community and pursued further.
On December 15, 2011, during the reconvened Cohen Commission hearings, Dr. Kristi Miller, head of molecular genetics at the federal Pacific Biological Station in Nanaimo, provided testimony that suggested ISAv has been present in BC salmon as far back as 1986
Canadian Food Inspection Agency and other agencies have done sampling/testing to try to find ISAv. Why haven’t they been able to find a case? Are they doing different tests than the laboratory of Dr. Fred Kibenge at the Atlantic Veterinary College? Why are there conflicting laboratory results?
At present the B.C. government/Canadian Food Inspection Agency (CFIA)/Department of Fisheries and Oceans (DFO) have not released the methods that they are employing to look for ISAv in the samples that were reported positive. Without this information we cannot interpret their results. Insistence on finding the virus in culture is not scientifically valid; we have known since 2001 that there are strains of ISAv that do not grow well or at all in tissue culture, including some virulent strains (in fact, this finding was first reported in the scientific literature in 1999, and has since been confirmed several times). If the Canadian agencies are using RT-PCR to test for ISAv (see “How is ISAv detected?”), we need to know which specific assay is being used and what gene segment it targets. If they are not using a segment 7 assay for detection, or the primers used by M. Kibenge in the unpublished 2004 manuscript, they may be testing samples that contain ISAv and they would not detect it (false negatives). [see also “What is the best assay to detect ISAv?”]
What are the OIE regulations for reporting ISAv?
7.2. Definition of confirmed case
The following criteria in i) should be met for confirmation of ISA. The criteria given in ii) and iii) should be met for the confirmation of ISAv infection.
i) Mortality, clinical signs and pathological changes consistent with ISA (Section 4.2), and detection of ISAv in tissue preparations by means of specific antibodies against ISAv (IFAT on tissue imprints [Section 184.108.40.206.2] or fixed sections as described in Section 220.127.116.11.3) in addition to either:
a) isolation and identification of ISAv in cell culture from at least one sample from any fish on the farm, as described in Section 18.104.22.168.1
b) detection of ISAv by RT-PCR by the methods described in Section 22.214.171.124.3;
ii) Isolation and identification of ISAv in cell culture from at least two independent samples (targeted or routine) from any fish on the farm tested on separate occasions as described in Section 126.96.36.199.1;
iii) Isolation and identification of ISAv in cell culture from at least one sample from any fish on the farm with corroborating evidence of ISAv in tissue preparations using either RT-PCR (Section 188.8.131.52.3) or IFAT (Sections 184.108.40.206.2 and 220.127.116.11.3).