Long-term shedding from fully convalesced individuals indicates that Pacific herring are a reservoir for viral hemorrhagic septicemia virus.

Processes that allow viral hemorrhagic septicemia (VHS) virus to persist in the marine environment remain enigmatic, owing largely to the presence of covert and cryptic infections in marine fishes during typical sub-epizootic periods. As such, marine host reservoirs for VHS virus have not been fully demonstrated, nor have the mechanism(s) by which infected hosts contribute to virus perpetuation and transmission. Here, we demonstrate that after surviving VHS, convalesced Pacific herring continue to shed virus at a low rate for extended periods. Further, exposure of previously naïve conspecific sentinels to this shed virus can result in infections for at least 6 mo after cessation of overt disease. This transmission mechanism was not necessarily dependent on the magnitude of the disease outbreak, as prolonged transmission occurred from 2 groups of donor herring that experienced cumulative mortalities of 4 and 29%. The results further suggest that the virus persists in association with the gills of fully recovered individuals, and long-term viral shedding or shedding relapses are related to cooler or decreasing water temperatures. These results provide support for a new VHS virus perpetuation paradigm in the marine environment, whereby the virus can be maintained in convalesced survivors and trafficked from these carriers to sympatric susceptible individuals.

Intra-Annual Changes in Waterborne Nanophyetus salmincola.

An analysis of daily water samples collected from an index site on Big Soos Creek, Washington indicated intra-annual differences in the concentrations of waterborne Nanophyetus salmincola. Waterborne concentrations, quantified as gene copies/L, peaked during the fall (October-November 2016), decreased to very low concentrations over the winter (January-March 2017), and then increased in the spring and throughout the summer. High waterborne concentrations of N. salmincola DNA (2 × 106 gene copies/L) corresponded with live N. salmincola cercariae (mean = 3 cercariae/L) that were detected in companion water samples. Spikes in waterborne N. salmincola concentrations in October and November typically coincided with increases in streamflow; this combination resulted in elevated infection pressures during high water events in the fall. The peak in waterborne N. salmincola concentrations corresponded with an accompanying peak in tissue parasite density (metacercariae/posterior kidney) in Coho Salmon Oncorhynchus kisutch that were reared in the untreated water.

Molecular testing of adult Pacific salmon and trout (Oncorhynchus spp.) for several RNA viruses demonstrates widespread distribution of piscine orthoreovirus in Alaska and Washington.

This research was initiated in conjunction with a systematic, multiagency surveillance effort in the United States (U.S.) in response to reported findings of infectious salmon anaemia virus (ISAV) RNA in British Columbia, Canada. In the systematic surveillance study reported in a companion paper, tissues from various salmonids taken from Washington and Alaska were surveyed for ISAV RNA using the U.S.-approved diagnostic method, and samples were released for use in this present study only after testing negative. Here, we tested a subset of these samples for ISAV RNA with three additional published molecular assays, as well as for RNA from salmonid alphavirus (SAV), piscine myocarditis virus (PMCV) and piscine orthoreovirus (PRV). All samples (n = 2,252; 121 stock cohorts) tested negative for RNA from ISAV, PMCV, and SAV. In contrast, there were 25 stock cohorts from Washington and Alaska that had one or more individuals test positive for PRV RNA; prevalence within stocks varied and ranged from 2% to 73%. The overall prevalence of PRV RNA-positive individuals across the study was 3.4% (77 of 2,252 fish tested). Findings of PRV RNA were most common in coho (Oncorhynchus kisutch Walbaum) and Chinook (O. tshawytscha Walbaum) salmon.

A systematic surveillance programme for infectious salmon anaemia virus supports its absence in the Pacific Northwest of the United States.

In response to reported findings of infectious salmon anaemia virus (ISAV) in British Columbia (BC), Canada, in 2011, U.S. national, state and tribal fisheries managers and fish health specialists developed and implemented a collaborative ISAV surveillance plan for the Pacific Northwest region of the United States. Accordingly, over a 3-1/2-year period, 4,962 salmonids were sampled and successfully tested by real-time reverse-transcription PCR. The sample set included multiple tissues from free-ranging Pacific salmonids from coastal regions of Alaska and Washington and farmed Atlantic salmon (Salmo salar L.) from Washington, all representing fish exposed to marine environments. The survey design targeted physiologically compromised or moribund animals more vulnerable to infection as well as species considered susceptible to ISAV. Samples were handled with a documented chain of custody and testing protocols, and criteria for interpretation of test results were defined in advance. All 4,962 completed tests were negative for ISAV RNA. Results of this surveillance effort provide sound evidence to support the absence of ISAV in represented populations of free-ranging and marine-farmed salmonids on the northwest coast of the United States.

Atlantic salmon, Salmo salar L. are broadly susceptible to isolates representing the North American genogroups of infectious hematopoietic necrosis virus.

Beginning in 1992, three epidemic waves of infectious hematopoietic necrosis, often with high mortality, occurred in farmed Atlantic salmon Salmo salar L. on the west coast of North America. We compared the virulence of eleven strains of infectious hematopoietic necrosis virus (IHNV), representing the U, M and L genogroups, in experimental challenges of juvenile Atlantic salmon in freshwater. All strains caused mortality and there was wide variation within genogroups: cumulative mortality for five U-group strains ranged from 20 to 100%, four M-group strains ranged 30-63% and two L-group strains varied from 41 to 81%. Thus, unlike Pacific salmonids, there was no apparent correlation of virulence in a particular host species with virus genogroup. The mortality patterns indicated two different phenotypes in terms of kinetics of disease progression and final per cent mortality, with nine strains having moderate virulence and two strains (from the U and L genogroups) having high virulence. These phenotypes were investigated by histopathology and immunohistochemistry to describe the variation in the course of IHNV disease in Atlantic salmon. The results from this study demonstrate that IHNV may become a major threat to farmed Atlantic salmon in other regions of the world where the virus has been, or may be, introduced.

Effects of temperature on Renibacterium salmoninarum infection and transmission potential in Chinook salmon, Oncorhynchus tshawytscha (Walbaum).

Renibacterium salmoninarum is a significant pathogen of salmonids and the causative agent of bacterial kidney disease (BKD). Water temperature affects the replication rate of pathogens and the function of the fish immune system to influence the progression of disease. In addition, rapid shifts in temperature may serve as stressors that reduce host resistance. This study evaluated the effect of shifts in water temperature on established R. salmoninarum infections. We challenged Chinook salmon with R. salmoninarum at 12 °C for 2 weeks and then divided the fish into three temperature groups (8, 12 and 15 °C). Fish in the 8 °C group had significantly higher R. salmoninarum-specific mortality, kidney R. salmoninarum loads and bacterial shedding rates relative to the fish held at 12 or 15 °C. There was a trend towards suppressed bacterial load and shedding in the 15 °C group, but the results were not significant. Bacterial load was a significant predictor of shedding for the 8 and 12 °C groups but not for the 15 °C group. Overall, our results showed little effect of temperature stress on the progress of infection, but do support the conclusion that cooler water temperatures contribute to infection progression and increased transmission potential in Chinook salmon infected with R. salmoninarum.

Piscine reovirus, but not Jaundice Syndrome, was transmissible to Chinook Salmon, Oncorhynchus tshawytscha (Walbaum), Sockeye Salmon, Oncorhynchus nerka (Walbaum), and Atlantic Salmon, Salmo salar L.

A Jaundice Syndrome occurs sporadically among sea-pen-farmed Chinook Salmon in British Columbia, the westernmost province of Canada. Affected salmon are easily identified by a distinctive yellow discolouration of the abdominal and periorbital regions. Through traditional diagnostics, no bacterial or viral agents were cultured from tissues of jaundiced Chinook Salmon; however, piscine reovirus (PRV) was identified via RT-rPCR in all 10 affected fish sampled. By histopathology, Jaundice Syndrome is an acute to peracute systemic disease, and the time from first clinical signs to death is likely <48 h; renal tubular epithelial cell necrosis is the most consistent lesion. In an infectivity trial, Chinook Salmon, Sockeye Salmon and Atlantic Salmon, intraperitoneally inoculated with a PRV-positive organ homogenate from jaundiced Chinook Salmon, developed no gross or microscopic evidence of jaundice despite persistence of PRV for the 5-month holding period. The results from this study demonstrate that the Jaundice Syndrome was not transmissible by injection of material from infected fish and that PRV was not the sole aetiological factor for the condition. Additionally, these findings showed the Pacific coast strain of PRV, while transmissible, was of low pathogenicity for Atlantic Salmon, Chinook Salmon and Sockeye Salmon.

Bench-top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture.

No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.

Restricted growth of U-type infectious haematopoietic necrosis virus (IHNV) in rainbow trout cells may be linked to casein kinase II activity.

Previously, we demonstrated that a representative M genogroup type strain of infectious haematopoietic necrosis virus (IHNV) from rainbow trout grows well in rainbow trout-derived RTG-2 cells, but a U genogroup type strain from sockeye salmon has restricted growth, associated with reduced genome replication and mRNA transcription. Here, we analysed further the mechanisms for this growth restriction of U-type IHNV in RTG-2 cells, using strategies that assessed differences in viral genes, host immune regulation and phosphorylation. To determine whether the viral glycoprotein (G) or non-virion (NV) protein was responsible for the growth restriction, four recombinant IHNV viruses were generated in which the G gene of an infectious IHNV clone was replaced by the G gene of U- or M-type IHNV and the NV gene was replaced by NV of U- or M-type IHNV. There was no significant difference in the growth of these recombinants in RTG-2 cells, indicating that G and NV proteins are not major factors responsible for the differential growth of the U- and M-type strains. Poly I:C pretreatment of RTG-2 cells suppressed the growth of both U- and M-type IHNV, although the M virus continued to replicate at a reduced level. Both viruses induced type 1 interferon (IFN1) and the IFN1 stimulated gene Mx1, but the expression levels in M-infected cells were significantly higher than in U-infected cells and an inhibitor of the IFN1-inducible protein kinase PKR, 2-aminopurine (2-AP), did not affect the growth of U- or M-type IHNV in RTG-2 cells. These data did not indicate a role for the IFN1 system in the restricted growth of U-type IHNV in RTG-2 cells. Prediction of kinase-specific phosphorylation sites in the viral phosphoprotein (P) using the NetPhosK program revealed differences between U- and M-type P genes at five phosphorylation sites. Pretreatment of RTG-2 cells with a PKC inhibitor or a p38MAPK inhibitor did not affect the growth of the U- and M-type viruses. However, 100 µm of the casein kinase II (CKII) inhibitor, 5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole (DRB), reduced the titre of the U type 8.3-fold at 24 h post-infection. In contrast, 100 µm of the CKII inhibitor reduced the titre of the M type only 1.3-fold at 48 h post-infection. Our data suggest that the different growth of U- and M-type IHNV in RTG-2 cells may be linked to a differential requirement for cellular protein kinases such as CKII for their growth.

Erratum: Genomes reveal genetic diversity of Piscine orthoreovirus in farmed and free-ranging salmonids from Canada and USA.

[This corrects the article DOI: 10.1093/ve/veaa054.].

Sequence analysis of the internal transcribed spacer (ITS) region reveals a novel clade of Ichthyophonus sp. from rainbow trout.

The mesomycetozoean parasite Ichthyophonus hoferi is most commonly associated with marine fish hosts but also occurs in some components of the freshwater rainbow trout Oncorhynchus mykiss aquaculture industry in Idaho, USA. It is not certain how the parasite was introduced into rainbow trout culture, but it might have been associated with the historical practice of feeding raw, ground common carp Cyprinus carpio that were caught by commercial fisherman. Here, we report a major genetic division between west coast freshwater and marine isolates of Ichthyophonus hoferi. Sequence differences were not detected in 2 regions of the highly conserved small subunit (18S) rDNA gene; however, nucleotide variation was seen in internal transcribed spacer loci (ITS1 and ITS2), both within and among the isolates. Intra-isolate variation ranged from 2.4 to 7.6 nucleotides over a region consisting of approximately 740 bp. Majority consensus sequences from marine/anadromous hosts differed in only 0 to 3 nucleotides (99.6 to 100% nucleotide identity), while those derived from freshwater rainbow trout had no nucleotide substitutions relative to each other. However, the consensus sequences between isolates from freshwater rainbow trout and those from marine/anadromous hosts differed in 13 to 16 nucleotides (97.8 to 98.2% nucleotide identity).

Differential growth of U and M type infectious haematopoietic necrosis virus in a rainbow trout-derived cell line, RTG-2.

Infectious haematopoietic necrosis virus (IHNV) is one of the most important viral pathogens of salmonids. In rainbow trout, IHNV isolates in the M genogroup are highly pathogenic, while U genogroup isolates are significantly less pathogenic. We show here that, at a multiplicity of infection (MOI) of 1, a representative U type strain yielded 42-fold less infectious virus than an M type strain in the rainbow trout-derived RTG-2 cell line at 24 h post-infection (p.i.). However, at an MOI of 10, there was only fivefold difference in the yield of infectious virus between the U and M strains. Quantification of extracellular viral genomic RNA suggested that the number of virus particles released from cells infected with the U strain at a MOI of 1 was 47-fold lower than from M-infected cells, but U and M virions were equally infectious by particle to infectivity ratios. At an MOI of 1, U strain intracellular viral genome accumulation and transcription were 37- and 12-fold lower, respectively, than those of the M strain at 24 h p.i. Viral nucleocapsid (N) protein accumulation in U strain infections was fivefold lower than in M strain infections. These results suggest that the block in U type strain growth in RTG-2 cells was because of the effects of reduced genome replication and transcription. The reduced growth of the U strain does not seem to be caused by defective genes, because the U and M strains grew equally well in the permissive epithelioma papulosum cyprini cell line at an MOI of 1. This suggests that host-specific factors in RTG-2 cells control the growth of the IHNV U and M strains differently, leading to growth restriction of the U type virus during the RNA synthesis step.

Genomes reveal genetic diversity of Piscine orthoreovirus in farmed and free-ranging salmonids from Canada and USA.

Piscine orthoreovirus (PRV-1) is a segmented RNA virus, which is commonly found in salmonids in the Atlantic and Pacific Oceans. PRV-1 causes the heart and skeletal muscle inflammation disease in Atlantic salmon and is associated with several other disease conditions. Previous phylogenetic studies of genome segment 1 (S1) identified four main genogroups of PRV-1 (S1 genogroups I-IV). The goal of the present study was to use Bayesian phylogenetic inference to expand our understanding of the spatial, temporal, and host patterns of PRV-1 from the waters of the northeast Pacific. To that end, we determined the coding genome sequences of fourteen PRV-1 samples that were selected to improve our knowledge of genetic diversity across a broader temporal, geographic, and host range, including the first reported genome sequences from the northwest Atlantic (Eastern Canada). Nucleotide and amino acid sequences of the concatenated genomes and their individual segments revealed that established sequences from the northeast Pacific were monophyletic in all analyses. Bayesian inference phylogenetic trees of S1 sequences using BEAST and MrBayes also found that sequences from the northeast Pacific grouped separately from sequences from other areas. One PRV-1 sample (WCAN_BC17_AS_2017) from an escaped Atlantic salmon, collected in British Columbia but derived from Icelandic broodstock, grouped with other S1 sequences from Iceland. Our concatenated genome and S1 analysis demonstrated that PRV-1 from the northeast Pacific is genetically distinct but descended from PRV-1 from the North Atlantic. However, the analyses were inconclusive as to the timing and exact source of introduction into the northeast Pacific, either from eastern North America or from European waters of the North Atlantic. There was no evidence that PRV-1 was evolving differently between free-ranging Pacific Salmon and farmed Atlantic Salmon. The northeast Pacific PRV-1 sequences fall within genogroup II based on the classification of Garseth, Ekrem, and Biering (Garseth, A. H., Ekrem, T., and Biering, E. (2013) 'Phylogenetic Evidence of Long Distance Dispersal and Transmission of Piscine Reovirus (PRV) between Farmed and Wild Atlantic Salmon', PLoS One, 8: e82202.), which also includes North Atlantic sequences from Eastern Canada, Iceland, and Norway. The additional full-genome sequences herein strengthen our understanding of phylogeographical patterns related to the northeast Pacific, but a more balanced representation of full PRV-1 genomes from across its range, as well additional sequencing of archived samples, is still needed to better understand global relationships including potential transmission links among regions.

Development and validation of a quantitative PCR to detect Parvicapsula minibicornis and comparison to histologically ranked infection of juvenile Chinook salmon, Oncorhynchus tshawytscha (Walbaum), from the Klamath River, USA.

Parvicapsula minibicornis is a myxosporean parasite that is associated with disease in Pacific salmon during their freshwater life history phase. This study reports the development of a quantitative (real-time) polymerase chain reaction (QPCR) to detect P. minibicornis DNA. The QPCR assay targets the 18S ribosomal subunit gene. A plasmid DNA control was developed to calibrate cycle threshold (C(T)) score to plasmid molecular equivalent (PME) units, a measure of gene copy number. Assay validation revealed that the QPCR was sensitive and able to detect 50 ag of plasmid DNA, which was equivalent to 12.5 PME. The QPCR assay could detect single P. minibicornis actinospores well above assay sensitivity, indicating a single spore contains at least 100 times the 18S DNA copies required for detection. The QPCR assay was repeatable and highly specific; no detectable amplification was observed using DNA from related myxozoan parasites. The method was validated using kidney tissues from 218 juvenile Chinook salmon sampled during the emigration period of March to July 2005 from the Klamath River. The QPCR assay was compared with histological examination. The QPCR assay detected P. minibicornis infection in 88.1% of the fish sampled, while histological examination detected infection in 71.1% of the fish sampled. Good concordance was found between the methods as 80% of the samples were in agreement. The majority of the disconcordant fish were positive by QPCR, with low levels of P. minibicornis DNA, but negative by histology. The majority of the fish rated histologically as having subclinical or clinical infections had high QPCR levels. The results of this study demonstrate that QPCR is a sensitive quantitative tool for evaluating P. minibicornis infection in fish health monitoring studies.

Infectious haematopoietic necrosis virus genogroup-specific virulence mechanisms in sockeye salmon, Oncorhynchus nerka (Walbaum), from Redfish Lake, Idaho.

Characterization of infectious haematopoietic necrosis virus (IHNV) field isolates from North America has established three main genogroups (U, M and L) that differ in host-specific virulence. In sockeye salmon, Oncorhynchus nerka, the U genogroup is highly virulent, whereas the M genogroup is nearly non-pathogenic. In this study, we sought to characterize the virus-host dynamics that contribute to genogroup-specific virulence in a captive stock of sockeye salmon from Redfish Lake in Idaho. Juvenile sockeye salmon were challenged by immersion and injection with either a representative U or M viral strain and sampled periodically until 14 days post-infection (p.i.). Fish challenged with each strain had positive viral titre by day 3, regardless of challenge route, but the fish exposed to the M genogroup virus had significantly lower virus titres than fish exposed to the U genogroup virus. Gene expression analysis by quantitative reverse transcriptase PCR was used to simultaneously assess viral load and host interferon (IFN) response in the anterior kidney. Viral load was significantly higher in the U-challenged fish relative to M-challenged fish. Both viruses induced expression of the IFN-stimulated genes (ISGs), but expression was usually significantly lower in the M-challenged group, particularly at later time points (7 and 14 days p.i.). However, ISG expression was comparable with 3 days post-immersion challenge despite a significant difference in viral load. Our data indicated that the M genogroup virus entered the host, replicated and spread in the sockeye salmon tissues, but to a lesser extent than the U genogroup. Both virus types induced a host IFN response, but the high virulence strain (U) continued to replicate in the presence of this response, whereas the low virulence strain (M) was cleared below detectable levels. We hypothesize that high virulence is associated with early in vivo replication allowing the virus to achieve a threshold level, which the host innate immune system cannot control.

Differential survival of Ichthyophonus isolates indicates parasite adaptation to its host environment.

In vitro viability of Ichthyophonus spp. spores in seawater and freshwater corresponded with the water type of the host from which the spores were isolated. Among Ichthyophonus spp. spores from both marine and freshwater fish hosts (Pacific herring, Clupea pallasii, and rainbow trout, Oncorhynchus mykiss, respectively), viability was significantly greater (P < 0.05) after incubation in seawater than in freshwater at all time points from 1 to 60 min after immersion; however, magnitude of the spore tolerances to water type differed with host origin. Ichthyophonus sp. adaptation to its host environment was indicated by greater seawater tolerance of spores from the marine host and greater freshwater tolerance of spores from the freshwater host. Prolonged aqueous survival of Ichthyophonus spp. spores in the absence of a host provides insight into routes of transmission, particularly among planktivorous fishes, and should be considered when designing strategies to dispose of infected fish carcasses and tissues.

Characterization of Toll-like receptor 3 gene in rainbow trout (Oncorhynchus mykiss).

Antiviral immunity in fish is not well understood. In mammals, Toll-like receptor (TLR) 3 is involved in double-stranded RNA recognition and host immune response activation. Here, we report the first identification of a rainbow trout TLR3 ortholog (rtTLR3), its genomic structure, and mRNA regulation. Six exons and five introns were identified from bacterial artificial chromosome (BAC) and expressed sequence tag (EST) sequencing, and this genomic organization is similar to mammalian and fish TLR3 genes. The putative 913 amino acid protein has a Toll/interleukin (IL)-1R (TIR) domain, a transmembrane domain, and leucine-rich repeats. In healthy trout, rtTLR3 is highly expressed in the liver, pyloric ceca, intestine, spleen, and anterior and trunk kidney tissues. To investigate whether rtTLR3 is involved in antiviral immunity, transcriptional regulation in vivo was examined by quantitative real-time polymerase chain reaction (PCR) after poly inosinic:cytidylic (I:C) and infectious hematopoietic necrosis virus (IHNV) treatments. TLR3 mRNA expression peaked 1 day after poly (I:C) injection of live animals, while the peak of gene expression after live IHNV challenge was observed on day 3. In vitro stimulation of rainbow trout anterior kidney leukocytes with poly (I:C) also enhanced rtTLR3 expression. Up-regulation was specific to viral challenge as there was no significant up-regulation of rtTLR3 mRNA levels in the spleen and a modest down-regulation in the anterior kidney after bath challenge with a gram-negative bacterial trout pathogen, Yersinia ruckeri. The sequence conservation of trout TLR3 and mRNA regulation after poly (I:C) or RNA virus exposures strongly suggest a role for trout TLR3 in antiviral immunity.

Interdecadal heterogeneity in mitochondrial DNA of Atlantic haddock (Melanogrammus aeglefinus) from Georges Bank.

Atlantic haddock of Georges Bank are characterized by large fluctuations in population size and a recent collapse of the commercial fishery. DNA extracted from dried scales of Georges Bank haddock, archived by the U.S. National Marine Fisheries Service (NMFS), reveals significant heterogeneity in frequencies of four mitochondrial DNA control region haplotypes between 1975 and 1985 cohorts. Several processes may be responsible for this temporal variation, the most attractive hypothesis being that haddock from other geographic regions episodically contribute to the Georges Bank gene pool. Thus, the population of haddock spawning on Georges Bank may not be genetically discrete and, with respect to Atlantic haddock, Georges Bank may not be viewed as a closed system.

Fine mapping of Ath6, a quantitative trait locus for atherosclerosis in mice.

Ath6 is a novel quantitative trait locus associated with differences in susceptibility to atherosclerosis between C57BL/6J (B6) and C57BLKS/J (BKS) inbred mouse strains. Combining data from an intercross and a backcross (1593 meioses) between mice from B6 and BKS strains and from The Jackson Laboratory interspecific backcross panels, (C57BL/6J x Mus spretus) F1 x C57BL/6J and (C57BL/6J x SPRET/Ei) F1 x SPRET/Ei, we constructed a consensus genetic map and narrowed Ath6 to a 1.07 +/- 0.26 cM interval between the anonymous DNA marker D12Pgn4 and the gene Nmyc1. This region is near the proximal end of murine Chromosome (Chr) 12, which is homologous to the human chromosomal region 2p24-p25. Marker order in the Ath6 region was concordant among the two crosses and The Jackson Laboratory interspecific backcross panels. This high resolution map rules out candidate genes encoding apolipoprotein B, syndecan 1, and Adam17. The two Ath6 crosses have a combined potential resolution of 0.06 cM.