BCSFA responds to Kibenge et al study results

CAMPBELL RIVER, B.C. – The BC Salmon Farmers Association has spoken with a number of scientists who all have serious questions about the recent study results published by Kibenge et al in Virology Journal.

"For over four years Ms. Morton has been reporting positive PCR tests (PCR is a laboratory method used for making a very large number of copies of short sections of DNA from a very small sample of genetic material) for Infectious Salmon Anemia Virus (ISAV) from B.C. samples, none of these results have been confirmed by the Canadian Food and Inspection agency (CFIA) using World Organization for Animal Health (OIE) standards, said Jeremy Dunn, Executive Director, BC Salmon Farmers Association. “We have great concerns about the methodology, and the ethics of the researchers involved, given their history of reporting false positives with respect to ISA. None of the results reported in this paper have been confirmed by an outside laboratory.”

ISA has never been detected in fish on the West Coast of North America. This has been confirmed through thousands of tests by CFIA, as well as thousands of tests by authorities in Washington State, Oregon, and Alaska. Farm-raised salmon in B.C. are healthy and have never shown signs of sickness from ISA. This report claims to find an ISA sequence, but the researchers admit they were unable to verify it using necessary, globally standard follow-up tests. Their study also confirms they found no evidence of the ISA disease in B.C. fish.

The laboratory that conducted the tests in this paper was audited by the OIE in 2012: “Conclusions of the audit were unfavourable and showed that a series of weaknesses in the system have a direct impact on the quality of diagnoses conducted by the OIE Reference Laboratory at AVC.”
Source: http://www.oie.int/for-the-media/press-releases/detail/article/information-on-oie-reference-laboratories/

Sample contamination is particularly a problem for laboratories that do research and diagnostics on tested organisms. Spillover of nucleic acid from research samples can easily contaminate diagnostic samples. This is one of the many things identified by the 2012 OIE audit of Dr. Kibenge’s laboratory (e.g., on page 3, “Dr Kibenge has five rooms at his disposal for the activities undertaken as the OIE-RL for ISA. The same rooms are in use for research and other diagnostic activities.” Then, on p. 5, “The panel has serious concerns regarding the current setup in the OIE-RL. The primary concerns were the cramped, untidy conditions of the laboratories, particularly the general laboratory [room 329(S)] where both sample preparation and post-PCR analysis were performed in close proximity to each other. … The panel believes that there is a serious risk that the integrity of the test samples will be compromised. ” Source: http://www.oie.int/fileadmin/Home/eng/Internationa_Standard_Setting/docs/pdf/Aquatic_Commissio n/Evaluation_OIE_Ref_Lab_ISA_website.pdf

One of the scientists the Association spoke with, Dr. Gary Marty, Senior Fish Pathologist with Ministry of Agriculture, has provided a review of the paper.

Gary D. Marty, D.V.M., Ph.D., Diplomate, A.C.V.P. Critique

Paper: Kibenge, M.J.T., T. Iwamoto, Y. Wang, A. Morton, R. Routledge, and F.S.B.
Kibenge. 2016. Discovery of variant infectious salmon anaemia virus (ISAV) of European genotype in British Columbia, Canada. Virology Journal 201613:3. DOI: 10.1186/s12985-015-0459-1.

The primary change that this new paper brings to B.C. is that we now have unconfirmed PCR test results for ISAV that are published in the peer-reviewed scientific literature. For the past 4+ years, positive PCR test results for ISAV from B.C. samples have been reported, primarily by Alexandra Morton and Kristi Miller. None of these results have been confirmed by CFIA using OIE standards, and none of these results have been confirmed by other standards of diagnostic medicine. The recent paper (Kibenge et al. 2016) does not change this status.

I am confident that these unconfirmed test results (Kibenge et al. 2016) are not a threat to wild or farmed salmon. My confidence is based on results from extensive monitoring over the past decade for ISA and other diseases in B.C. farm salmon. This study is led by government and farm veterinary scientists, and it is consistent with type of further study recommended in the paper. The unconfirmed PCR test results reported by Kibenge et al. (2016) will not change our approach to rigorous science-based sampling and testing hundreds of farm salmon every year for diseases, including ISA.

Kibenge et al. (2016) is based solely on PCR results alone; therefore, their results remain unconfirmed. Because the chance for false positive PCR test results is high in regions of the world like B.C. that are free of reportable diseases like ISA, OIE standards for disease confirmation never rely on PCR results alone. Nucleic acid sequencing, as done by Kibenge et al. (2016) is helpful to rule out nonspecific amplification as a source of false positive tests, but sequencing does not rule out the possibility of sample contamination.

Lack of confirmation of results by an outside laboratory decreases my confidence in the results. Kibenge et al. (2016) did not provide information that their results were tested or confirmed by any outside laboratory.

Lack of repeatability of the results within Dr. Kibenge’s laboratory decreases my confidence in their results. Kibenge et al. (2016) did provide information that their own results were not consistently repeatable. All of their samples (n = 1,106) were tested by RT-PCR; all samples with positive results with at least one replicate (n = 20) and a subset with negative RT-PCR results were also tested by conventional PCR. Based on data in Supplementary Table S2, of the 10 samples that were positive by RT-PCR with both replicates, only one sample was also positive by both conventional assays (= 10% of the 10 RT-PCR positives, and 1/1,106 samples tested = 0.09%). From supplementary Table S2, only 9% (6/65) of samples that tested positive by conventional PCR were also positive by RT-PCR, even though the samples tested by conventional PCR were biased towards fish that were already positive by RT-PCR. Only 2% (1/50) of the samples positive by segment 8 conventional PCR were also positive by RT-PCR.

The reported mutation in the sequence that is used in a common RT-PCR test for detecting the ISA virus would not eliminate our ability to detect ISAV if it was present in our samples. The paper reports 10 positive RT-PCR tests from 1,1,06 samples tested using the same PCR primers and probe that we have used since October 2014. If the virus were truly present in the samples that we were testing, we would be detecting it at least as often as Dr. Kibenge’s laboratory; the fact that we are not detecting ISAV in our samples decreases my confidence in the results reported in Kibenge et al. (2016).

As part of the BC Fish Health Audit and Surveillance Program, the BC Animal Health Centre tests moribund and recently dead fish from salmon farms. Since October 2014, samples from 930 fish were tested, and all results were negative—no virus. Before October 2014, we used different PCR tests for ISAV that did not rely on detecting the reported mutation in the probe sequence; from 2003 through the third quarter of 2014 (n = 6,980), the test results were the same: all negative—no virus. Our veterinary virologist, Dr. Tomy Joseph, assures me that a single mutation in the probe sequence would not significantly affect our ability to detect levels of virus that might be causing disease.

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Gary D. Marty, D.V.M., Ph.D., Diplomate, A.C.V.P.
Senior Fish Pathologist
Animal Health Centre
Ministry of Agriculture

About the BC Salmon Farmers Association:

B.C. salmon farmers grow 58% of all salmon raised in Canada and account for 60% of the total landed value of seafood in British Columbia, generating more than $1.14-billion towards the provincial economy. The BCSFA represents the province’s vibrant and diversified salmon aquaculture sector, inclusive of companies raising salmon, as well as the businesses that proudly provide services and supplies.

For more information visit BCSalmonFarmers.ca

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