Elevated Serum IgG4 Levels in Diagnosis, Treatment Response, Organ Involvement, and Relapse in a Prospective IgG4-Related Disease UK Cohort.

OBJECTIVES: Elevated serum immunoglobulin G4 (IgG4) levels have been associated with autoimmune pancreatitis and IgG4-related disease (IgG4-RD) for over a decade. However, an elevated serum IgG4 is not specific for the disease. There have been inconsistent reports of its use in diagnosis, as a marker of disease relapse, and its relationship to organ involvement in retrospective cohorts. The aims of this study were to ascertain conditions that are associated with an elevated serum IgG4 and to investigate the role of IgG4 in diagnosis, relapse, and organ involvement in a prospective cohort of patients with IgG4-RD. METHODS: We evaluated serum IgG4 measurements in the Oxford Immunology Laboratory over 6 years. Patients in whom serum IgG4 was requested to differentiate IgG4-RD from other diseases were recruited into a longitudinal follow-up study to determine final diagnosis. In a prospective cohort of IgG4-RD patients, organ involvement, response to therapy, and disease relapse were determined. RESULTS: Two thousand and sixty-seven samples from 1,510 patients had serum IgG4 measured. Of these, IgG4 was elevated (≥1.4 g l(-1)) in 243 (16.1%) patients. The main indication (85.6%) was to distinguish between IgG4-RD and non-IgG4-RD conditions. Only 5.1% of patients who had serum IgG4 measured for this purpose had a final diagnosis of IgG4-RD. Of those with an elevated serum IgG4, 22.4% met IgG4-RD diagnostic criteria. Serum IgG4 was elevated in 48 (82.8%) of IgG4-RD patients. An IgG4 cutoff of 1.4 g l(-1) gave a sensitivity of 82.8% and specificity of 84.7% to diagnose IgG4-RD. Increasing this to 2.8 g l(-1) increased specificity to 96.2% and negative predictive value to 97.7%, with a lower sensitivity of 56.9% and positive predictive value of 44.5%. Serum IgG4 levels fell with corticosteroid therapy, but this was not disease-specific. A serum IgG4 of ≥2.8 g l(-1) at diagnosis was associated with multi-organ involvement and risk of relapse. CONCLUSIONS: Serum IgG4 levels are elevated in multiple non-IgG4-RD inflammatory and malignant conditions, with less than one-quarter of those with an elevated IgG4 meeting IgG4-RD diagnostic criteria. A serum IgG4 of ≥2.8 g l(-1) is useful in distinguishing between IgG4-RD and non-IgG4-RD diagnoses, predicting multiple-organ involvement and risk of relapse in IgG4-RD.

Tylosis with oesophageal cancer: Diagnosis, management and molecular mechanisms.

Tylosis (hyperkeratosis palmaris et plantaris) is characterised by focal thickening of the skin of the hands and feet and is associated with a very high lifetime risk of developing squamous cell carcinoma of the oesophagus. This risk has been calculated to be 95% at the age of 65 in one large family, however the frequency of the disorder in the general population is not known and is likely to be less than one in 1,000,000. Oesophageal lesions appear as small (2-5 mm), white, polyploid lesions dotted throughout the oesophagus and oral leukokeratosis has also been described. Although symptoms of oesophageal cancer can include dysphagia, odynophagia, anorexia and weight loss, there may be an absence of symptoms in early disease, highlighting the importance of endoscopic surveillance in these patients. Oesophageal cancer associated with tylosis usually presents in middle to late life (from mid-fifties onwards) and shows no earlier development than the sporadic form of the disease. Tylosis with oesophageal cancer is inherited as an autosomal dominant trait with complete penetrance of the cutaneous features, usually by 7 to 8 years of age but can present as late as puberty. Mutations in RHBDF2 located on 17q25.1 have recently been found to be causative. A diagnosis of tylosis with oesophageal cancer is made on the basis of a positive family history, characteristic clinical features, including cutaneous and oesophageal lesions, and genetic analysis for mutations in RHBDF2. The key management goal is surveillance for early detection and treatment of oesophageal dysplasia. Surveillance includes annual gastroscopy with biopsy of any suspicious lesion together with quadratic biopsies from the upper, middle and lower oesophagus. This is coupled with dietary and lifestyle modification advice and symptom education. Symptomatic management of the palmoplantar keratoderma includes regular application of emollients, specialist footwear and early treatment of fissures and super-added infection, particularly tinea pedis. More specific treatment for the thick skin is available in the form of oral retinoids, which are very effective but commonly produce side effects, including nasal excoriation and bleeding, hypercholesterolaemia, and abnormal liver function tests. Genetic counselling can be offered to patients and family members once a family history has been established. The prognosis of tylosis with oesophageal cancer is difficult to determine due to the limited number of affected individuals. In the last 40 years of surveillance, five out of six cases of squamous oesophageal cancer in the Liverpool family were detected endoscopically and were surgically removed. Four of five patients had stage 1 disease at presentation and remain alive and well more than 8 years later. This suggests that the presence of a screening program improves prognosis for these patients.

RHBDF2 mutations are associated with tylosis, a familial esophageal cancer syndrome.

Tylosis esophageal cancer (TOC) is an autosomal-dominant syndrome characterized by palmoplantar keratoderma, oral precursor lesions, and a high lifetime risk of esophageal cancer. We have previously localized the TOC locus to a small genomic interval within chromosomal region 17q25. Using a targeted capture array and next-generation sequencing, we have now identified missense mutations (c.557T>C [p.Ile186Thr] and c.566C>T [p.Pro189Leu] in RHBDF2, which encodes the inactive rhomboid protease RHBDF2 (also known as iRhom2), as the underlying cause of TOC. We show that the distribution of RHBDF2 in tylotic skin is altered in comparison with that in normal skin, and immortalized tylotic keratinocytes have decreased levels of total epidermal growth factor receptor (EGFR) and display an increased proliferative and migratory potential relative to normal cells, even when normal cells are stimulated with exogenous epidermal growth factor. It would thus appear that EGFR signaling is dysregulated in tylotic cells. Furthermore, we also show an altered localization of RHBDF2 in both tylotic and sporadic squamous esophageal tumors. The elucidation of a role of RHBDF2 in growth-factor signaling in esophageal cancer will help to determine whether targeting this pathway in chemotherapy for this and other squamous cell carcinomas will be effective.

A randomized placebo-controlled prevention trial of aspirin and/or resistant starch in young people with familial adenomatous polyposis.

Evidence supporting aspirin and resistant starch (RS) for colorectal cancer prevention comes from epidemiologic and laboratory studies (aspirin and RS) and randomized controlled clinical trials (aspirin). Familial adenomatous polyposis (FAP) strikes young people and, untreated, confers virtually a 100% risk of colorectal cancer and early death. We conducted an international, multicenter, randomized, placebo-controlled trial of aspirin (600 mg/d) and/or RS (30 g/d) for from 1 to 12 years to prevent disease progression in FAP patients from 10 to 21 years of age. In a 2 × 2 factorial design, patients were randomly assigned to the following four study arms: aspirin plus RS placebo; RS plus aspirin placebo; aspirin plus RS; RS placebo plus aspirin placebo; they were followed with standard annual clinical examinations including endoscopy. The primary endpoint was polyp number in the rectum and sigmoid colon (at the end of intervention), and the major secondary endpoint was size of the largest polyp. A total of 206 randomized FAP patients commenced intervention, of whom 133 had at least one follow-up endoscopy and were therefore included in the primary analysis. Neither intervention significantly reduced polyp count in the rectum and sigmoid colon: aspirin relative risk = 0.77 (95% CI, 0.54-1.10; versus nonaspirin arms); RS relative risk = 1.05 (95% CI, 0.73-1.49; versus non-RS arms). There was a trend toward a smaller size of largest polyp in patients treated with aspirin versus nonaspirin--mean 3.8 mm versus 5.5 mm for patients treated 1 or more years (adjusted P = 0.09) and mean 3.0 mm versus 6.0 mm for patients treated more than 1 year (P = 0.02); there were similar weaker trends with RS versus non-RS. Exploratory translational endpoints included crypt length (which was significantly shorter in normal-appearing mucosa in the RS group over time) and laboratory measures of proliferation (including Ki67). This clinical trial is the largest ever conducted in the setting of FAP and found a trend of reduced polyp load (number and size) with 600 mg of aspirin daily. RS had no clinical effect on adenomas.

Biloma: an unusual complication in a patient with pancreatic cancer.

The term biloma describes an encapsulated collection of bile within the abdomen, usually secondary to bile duct disruption. The commonest causes reported in the literature are iatrogenic (secondary to hepatobiliary surgery), trauma or complications due to choledocholithiasis. A few cases have been reported as complications of cholangiocarcinoma or acute cholecystitis. We report the case of a 64-year-old man initially diagnosed with a non-obstructive malignancy of the pancreas, who developed a spontaneous intrahepatic biloma 8 mo later. This was identified following a 1-wk history of fever, rigors and icterus. The biloma was identified on computed tomography and subsequently drained under ultrasound guidance. Forty-eight hours later, a stent was inserted endoscopically into his common bile duct and he made an uneventful in-hospital recovery. We believe this is the first documented case of spontaneous intrahepatic biloma to occur secondary to pancreatic malignancy.

Survival and replication of Piscirickettsia salmonis in rainbow trout head kidney macrophages.

Piscirickettsia salmonis is pathogenic for a variety of cultured marine fish species worldwide. The organism has been observed within host macrophages in natural disease outbreaks among coho salmon and European sea bass. In vitro studies, incorporating transmission electron microscopy (TEM) and ferritin loading of lysosomes, have confirmed that P. salmonis is capable of surviving and replicating in rainbow trout macrophages. Certain features of this intracellular survival underline its difference to other intracellular pathogens and suggest that a novel combination of defence mechanisms may be involved. Escape into the macrophage cytoplasm is not used as a means to avoid phago-lysosomal fusion and the organism remains at least partly enclosed within a vacuole membrane. While the piscirickettsial vacuole is often incomplete, survival and replication appear to require occupation of a complete, tightly-apposed, vacuolar membrane which does not fuse with lysosomes. Unlike some mammalian rickettsiae, actin-based motility (ABM) is not used as a means of intercellular spread. It is postulated that the presence of numerous small vesicles within vacuoles, and at gaps in the vacuolar membrane, may result from the blebbing of the piscirickettsial outer membrane seen early in the infection.

Infectious pancreatic necrosis virus suppresses type I interferon signalling in rainbow trout gonad cell line but not in Atlantic salmon macrophages.

RTG-P1 cells are a rainbow trout fibroblastic cell line permanently transfected with the luciferase gene under the control of the Mx promoter. On exposure to interferon (IFN) or IFN inducing agents, the cells produce luciferase. IPNV did not induce luciferase production up to 24h post-infection but did not suppress constitutive luciferase production. Furthermore, IPNV suppressed luciferase production induced by poly I:C. RT-PCR analysis of IPNV infected cells showed IFN gene transcription from 6h post-infection with increasing expression up to 24h. Housekeeping genes beta-actin and GAPDH were also expressed along with upregulation of IRF1 and slight upregulation of STAT1. When RTG-P1 cells were stimulated with IFN, Mx transcripts, measured by qRT-PCR, peaked at 3-6h and thereafter fell to low levels, but in the presence of IPNV, Mx transcription at this time was significantly suppressed but continued to rise gradually. Luciferase production was lower in infected cells at 12h post-infection but not significantly after 24h. These results indicate that, in non-stimulated RTG-P1 cells, while IPNV induces IFN transcription, activation of Mx expression is suppressed. Furthermore, when stimulated by IFN, the rate of Mx transcription is significantly suppressed by the virus. This would probably give time for the virus to replicate rapidly in the early phases of infection. Contrary to the fibroblastic cell line, IPNV stimulated IFN production by salmon macrophages in vitro at least as strongly as poly I:C, with no suppression of the IFN response to poly I:C, and the virus persisted for up to 9 days without causing CPE.

Down-regulation of the cytoglobin gene, located on 17q25, in tylosis with oesophageal cancer (TOC): evidence for trans-allele repression.

Tylosis (focal non-epidermolytic palmoplantar keratoderma) is an autosomal dominant skin disorder that is associated with the early onset of squamous cell oesophageal cancer (SCOC) in three families. Our previous linkage and haplotype analyses have mapped the tylosis with oesophageal cancer (TOC) locus to a 42.5 kb region on chromosome 17q25 that has also been implicated in the aetiology of sporadically occurring SCOC from a number of different geographical populations. Oesophageal cancer is one of the 10 leading causes of cancer mortality worldwide. No inherited disease-causing mutations have been identified in the genes located in the 42.5 kb minimal region. We now show that cytoglobin gene expression in oesophageal biopsies from tylotic patients is dramatically reduced by approximately 70% compared with normal oesophagus. Furthermore, both alleles are equally repressed. Given the autosomal dominant nature of the disease, these results exclude haploinsufficiency as a mechanism of the disease and instead suggest a novel trans-allele interaction. We also show that the promoter is hypermethylated in sporadic oesophageal cancer samples: this may constitute the 'second hit' of a gene previously implicated in this disease by allelic imbalance studies.

Effect of systemic corticosteroid therapy on risk for intra-abdominal or pelvic abscess in non-operated Crohn's disease.

BACKGROUND & AIMS: Systemic corticosteroid therapy increases risk of postoperative sepsis in Crohn's disease. This study investigates its effect on risk for sepsis in non-operated patients. METHODS: A retrospective case-control study was performed in 432 patients with Crohn's disease (the 94% of our database for whom adequate documentation could be retrieved). Two analyses were performed. The first tested the hypothesis that patients with perforating Crohn's disease (n = 86) were more likely to develop intra-abdominal or pelvic abscess (n = 29) if they had received systemic corticosteroids during the previous 3 months. The second analysis, confined to interventions since 1998, tested the hypothesis that corticosteroid therapy was more common during the 3 months before presentation with intra-abdominal or pelvic abscess (n = 12) than during the 3 months after presentation with a relapse of nonperforating disease (n = 24 consecutive patients). In both analyses adjustment was made for any other significant variable. RESULTS: Systemic corticosteroid therapy was associated with an adjusted odds ratio (OR) for intra-abdominal or pelvic abscess of 9.03 (95% confidence interval [CI], 2.40-33.98) in patients with perforating Crohn's disease. Patients receiving prednisolone > or = 20 mg per day had an OR of 2.81 (95% CI, 0.99-7.99) for abscess compared with those receiving a lower dose. In patients with relapsed active disease, corticosteroid therapy was associated with an unadjusted OR of 9.31 (95% CI, 1.03-83.91) for intra-abdominal or pelvic abscess. Neither smoking nor azathioprine usage was associated with increased risk for abscess. CONCLUSIONS: Systemic corticosteroid therapy for Crohn's disease is associated with increased risk for intra-abdominal or pelvic abscess.

AIP56, a novel plasmid-encoded virulence factor of Photobacterium damselae subsp. piscicida with apoptogenic activity against sea bass macrophages and neutrophils.

A strategy used by extracellular pathogens to evade phagocytosis is the utilization of exotoxins that kill host phagocytes. We have recently shown that one major pathogenicity strategy of Photobacterium damselae subsp. piscicida (Phdp), the agent of the widespread fish pasteurellosis, is the induction of extensive apoptosis of sea bass macrophages and neutrophils that results in lysis of these phagocytes by post-apoptotic secondary necrosis. Here we show that this unique process is mediated by a novel plasmid-encoded apoptosis inducing protein of 56 kDa (AIP56), an exotoxin abundantly secreted by all virulent, but not avirulent, Phdp strains tested. AIP56 is related to an unknown protein of the enterohemorrhagic Escherichia coli O157:H7 and NleC, a Citrobacter rodentium type III secreted effector of unknown function. Passive immunization of sea bass with a rabbit anti-AIP56 serum conferred protection against Phdp challenge, indicating that AIP56 represents a key virulence factor of that pathogen and is a candidate for the design of an anti-pasteurellosis vaccine.

Novel microsatellite markers and single nucleotide polymorphisms refine the tylosis with oesophageal cancer (TOC) minimal region on 17q25 to 42.5 kb: sequencing does not identify the causative gene.

Tylosis (focal non-epidermolytic palmoplantar keratoderma) is associated with the early onset of squamous cell oesophageal cancer in three families. Linkage and haplotype analyses have previously mapped the tylosis with oesophageal cancer ( TOC) locus to a 500-kb region on chromosome 17q25 that has also been implicated in sporadically occurring squamous cell oesophageal cancer. In the current study, 17 additional putative microsatellite markers were identified within this 500-kb region by using sequence data and seven of these were shown to be polymorphic in the UK and US families. In addition, our complete sequence analysis of the non-repetitive parts of the TOC minimal region identified 53 novel and six known single nucleotide polymorphisms (SNPs) in one or both of these families. Further fine mapping of the TOC disease locus by haplotype analysis of the seven polymorphic markers and 21 of the 59 SNPs allowed the reduction of the minimal region to 42.5 kb. One known and two putative genes are located within this region but none of these genes shows tylosis-specific mutations within their protein-coding regions. Alternative mechanisms of disease gene action must therefore be considered.

Effect of intraperitoneally administered IL-1beta-derived peptides on resistance to viral haemorrhagic septicaemia in rainbow trout Oncorhynchus mykiss.

The present work provides the first information concerning the immunostimulatory activity of trout interleukin (IL)-1beta-derived peptides in vivo. Previous studies have demonstrated the ability of 2 such peptides, referred to as P1 and P3, to up-regulate a range of important immune parameters in vitro. P1 corresponds to fragment 146-157 (YVTPVPIETEAR) of the trout sequence and is analogous to a biologically active mammalian IL-1beta-derived peptide, whilst P3 was synthesised to complex with the IL-1 receptor and corresponds to fragment 197-206 (YRRNTGVDIS) of the trout sequence. Optimal migration of peritoneal leucocytes, peptide induced phagocytosis and intracellular respiratory burst activity occurred following intraperitoneal injection of 3.0 micromol of P3. Furthermore, resistance to viral haemorrhagic septicaemia virus (VHSV) was soon augmented (2 d) post-injection of P3.

Autosomal recessive colorectal adenomatous polyposis due to inherited mutations of MYH.

Familial adenomatous polyposis (FAP) and attenuated FAP are autosomal dominant disorders characterised by multiple colorectal adenomas and cancers. Both are caused by inherited mutations in the APC gene, and management includes genetic testing, colonoscopic surveillance, and prophylactic surgery for the relatives of index cases. Among 614 families recorded in six regional registers of polyposis in the UK, we identified 111 with neither dominant transmission nor evidence of APC mutation. Molecular genetic analysis showed that 25 had biallelic mutations of the MYH gene. Since our data show that MYH polyposis can be transmitted as an autosomal recessive trait, a change in genetic counselling, testing, and surveillance is needed.

Physical and transcript map of the minimally deleted region III on 17p implicated in the early development of Barrett's oesophageal adenocarcinoma.

Allelic imbalance (AI) studies on chromosome 17 (C17) in Barrett's oesophageal adenocarcinoma (BOA) tumours strongly suggest that a minimally deleted region on C17p harbours a BOA-associated gene with tumour suppressor function. This deleted region, designated minimal region III (MRIII), lies between the two microsatellite markers D17S1852 and D17S954. Computational sequence analysis techniques, BLAST and NIX, were used to assemble a physical map of MRIII, consisting of three overlapping bacterial artificial chromosome (BAC) clones, 297N7, 963H4 and 795F17, from the RPCI-11 library. The 270 kb genomic sequence of MRIII was analysed using the computational gene prediction methods NIX and TAP to identify putative BOA genes. A transcript map of MRIII has been generated and contains 25 candidate BOA genes, four of which are the named genes MYH3, SCO1, x006 and MAGOH-LIKE. The other candidates consist of seven genes predicted by TAP with associated ESTs identified by NIX, two genes predicted by TAP alone and 12 genes/ESTs (or pairs of ESTs) identified by NIX alone. No disease-specific mutations were identified in x006 or MAGOH-LIKE, although expression analysis of these genes suggests that they may show alternative splicing or be altered epigenetically or in regulatory regions in oesophageal cancer.

Characterization of a 500 kb region on 17q25 and the exclusion of candidate genes as the familial Tylosis Oesophageal Cancer (TOC) locus.

The locus for a syndrome of focal palmoplantar keratoderma (Tylosis) associated with squamous cell oesophageal cancer (TOC) has been mapped to chromosome 17q25, a region frequently deleted in sporadic squamous cell oesophageal tumours. Further haplotype analysis described here, based on revised maps of marker order, has reduced the TOC minimal region to a genetic interval of 2 cM limited by the microsatellite markers D17S785 and D17S751. Partial sequence data and complete physical maps estimate the actual size of this region to be only 0.5 Mb. This analysis allowed the exclusion of proposed candidate tumour suppressor genes including MLL septin-like fusion (MSF), survivin, and deleted in multiple human cancer (DMC1). Computer analysis of sequence data from the minimal region identified 13 candidate genes and the presence of 50-70 other 'gene fragments' as ESTs and/or predicted exons and genes. Ten of the characterized genes were assayed for mutations but no disease-specific alterations were identified in the coding and promoter sequences. This region of chromosome 17q25 is, therefore, relatively gene-rich, containing 13 known and possibly as many as 50 predicted genes. Further mutation analysis of these predicted genes, and others possibly residing in the region, is required in order to identify the elusive TOC locus.

Antibody increases phagocytosis and killing of Lactococcus garvieae by rainbow trout (Oncorhynchus mykiss, L.) macrophages.

The present study reports that specific antibody increased the bactericidal activity of rainbow trout head-kidney macrophages against virulent capsulated Lactococcus garvieae in the absence of complement. The observed increased bactericidal activity appeared to result from increased phagocytosis of capsulated L. garvieae in the presence of specific antibody and may in part explain the protective effect of antibody previously reported against this disease.

Invasion of fish epithelial cells by Photobacterium damselae subsp. piscicida: evidence for receptor specificity, and effect of capsule and serum.

Photobacterium damselae subsp. piscicida is a fish pathogen which causes serious disease in commercial warmwater fish species. Because information on the initial stages of the infection is scarce, an investigation of the invasion ability of this pathogen was undertaken utilizing a fish epithelial cell line (epithelioma papillosum carpio, EPC), a virulent capsulated strain of P. damselae (MT1415), an avirulent non-capsulated strain of P. damselae (EPOY-8803-ii) and Escherichia coli HB101 as a non-invasive control. P. damselae was found to be able to adhere to and invade fish epithelial cells and remain inside them for 6-9 h. There were no significant differences in invasiveness between the capsulated and non-capsulated strains. A kinetics study demonstrated that P. damselae invasiveness was more efficient at low m.o.i., reaching saturation at higher m.o.i., suggesting internalization may be receptor-mediated. Invasion efficiency (IE) was significantly higher than in the control E. coli HB101. Engulfment of bacteria was possibly by an endocytic process and was unaffected by killing the bacteria with UV light. However, heat-killed bacteria had significantly reduced invasion capability. Ultrastructural studies showed that inside the epithelial cells, the bacteria remained within large vacuoles for a few hours and no evidence of intracellular replication was found, by either fluorescence or electron microscopic studies. Normal sea bass serum slightly reduced the invasion capability of the MT1415 strain, but heat-inactivated normal serum had no effect. On the other hand, heat-inactivated fish antiserum raised against the same strain reduced the percentage of invaded epithelial cells by 50%. As for other pathogens, an intracellular phase of P. damselae may be a mechanism to delay or avoid phagocytosis and host immune responses, favouring the spread of infection.

Utilization of transferrin and salmon serum as sources of iron by typical and atypical strains of Aeromonas salmonicida.

The ability of typical and atypical strains of Aeromonas salmonicida to utilize non-haem sources of protein-bound iron was evaluated. (i) In a plate bioassay, the suppression of growth imposed on typical and atypical A. salmonicida by addition of the high-affinity iron chelator ethylenediamine-di(o- hydroxyphenylacetic acid) (EDDA) to the growth medium was reversed by the addition of 30% or 90% iron-saturated bovine or human transferrin (Tf) or lactoferrin (Lf) to the growth medium. (ii) The mechanism of obtaining iron from Tf was investigated by the addition of bovine Tf contained within a dialysis bag. The reversal of iron-restricted growth suppression differed between the strains in that the atypical strains were unable to utilize Tf contained within a dialysis bag while the typical strains were able to do so. This suggested a siderophore-mediated uptake of iron from Tf by the typical strains, which are known to produce siderophores while atypical strains do not. (iii) A solid-phase binding assay using horseradish-peroxidase-conjugated or biotinylated Tf or Lf failed to detect Tf/Lf-binding activity using whole typical or atypical cells. (iv) When atypical extracellular products (ECP) plus bovine Tf or salmon serum were enclosed in a dialysis bag, diffusible products were released which could reverse the EDDA-imposed growth suppression of an atypical strain. This reversal was negated by inhibition of the ECP metalloprotease with EDTA. (v) Purified 70 kDa serine protease of a typical strain was able to digest bovine Tf to low molecular mass fragments as observed in SDS-PAGE. These results indicate that typical and atypical strains of A. salmonicida differ in their mechanism of utilization of non-haem protein- bound sources of iron. Typical strains utilize Tf via a siderophore-mediated mechanism and are also able to digest Tf with the extracellular serine protease. Atypical strains utilize Tf by a siderophore-independent mechanism probably involving the proteolytic degradation of Tf by the extracellular metalloprotease.