Norwegian Isolates Research Articles

Emergence of a colistin-resistant KPC-2-producing Klebsiella pneumoniae ST258 clone in Hungary

Nine Klebsiella pneumoniae isolates showing non-susceptibility to carbapenems were collected from three centres in the north-eastern region of Hungary. The minimum inhibitory concentrations (MICs) of antibiotics were determined by Etest. The putative production of a carbapenemase was tested by the modified Hodge test. The presence of bla (KPC) genes was verified by polymerase chain reaction (PCR) and sequencing. Furthermore, molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All isolates showed extensively drug-resistant (XDR) phenotype, and of these, eight isolates were highly resistant to colistin. The isolates carried bla (KPC-2), bla (SHV-12), bla (TEM-1) and bla (SHV-11). PFGE analysis of the nine KPC-2-producing Hungarian ST258 K. pneumoniae isolates, two KPC-2-producing Norwegian ST258 isolates and 33 CTX-M-15-producing ST11 isolates revealed the existence of one genetic cluster at an 88% similarity level. The overall results of the PFGE clustering, MLST and the presence of SHV-11 in both ST11 and ST258 suggest that this is the first hyperepidemic clonal complex of multidrug-resistant K. pneumoniae, probably CC258/CC340, possibly undergoing worldwide spread.

Dynamics of serum antibodies to and load of porcine circovirus type 2 (PCV2) in pigs in three finishing herds, affected or not by postweaning multisystemic wasting syndrome

BackgroundDespite that PMWS commonly affects pigs aged eight to sixteen weeks; most studies of PMWS have been conducted during the period before transfer to finishing herds. This study focused on PCV2 load and antibody dynamics in finishing herds with different PMWS status.MethodsSequentially collected blood samples from 40 pigs in each of two Swedish (A and B) and one Norwegian (C) finishing herds were analysed for serum PCV2-load and -antibodies and saliva cortisol. The two Swedish herds differed in PMWS status, despite receiving animals from the same sow pool (multi-site production). However, the PMWS-deemed herd (A) had previously also received pigs from the spot market. ResultsThe initial serum PCV2 load was similar in the two Swedish herds. In herd A, it peaked after two weeks in the finishing herd and a high number of the pigs had serum PCV2 levels above 107 per ml. The antibody titres increased continually with exception for the pigs that developed PMWS, that had initially low and then declining antibody levels. Pigs in the healthy herd B also expressed high titres of antibodies to PCV2 on arrival but remained at that level throughout the study whereas the viral load steadily decreased. No PCV2 antibodies and only low amounts of PCV2 DNA were detected in serum collected during the first five weeks in the PMWS-free herd C. Thereafter a peak in serum PCV2 load accompanied by an antibody response was recorded. PCV2 from the two Swedish herds grouped into genotype PCV2b whereas the Norwegian isolate grouped into PCV2a. Cortisol levels were lower in herd C than in herds A and B.ConclusionsThe most obvious difference between the Swedish finishing herds and the Norwegian herd was the time of infection with PCV2 in relation to the time of allocation, as well as the genotype of PCV2. Clinical PMWS was preceded by low levels of serum antibodies and a high load of PCV2 but did not develop in all such animals. It is notable that herd A became affected by PMWS after errors in management routine, emphasising the importance of proper hygiene and general disease-preventing measures.

Outbreak of viral haemorrhagic septicaemia (VHS) in seawater-farmed rainbow trout in Norway caused by VHS virus Genotype III

We describe the finding of a novel viral haemorrhagic septicaemia virus (VHSV) Genotype III strain that caused disease of both a neurological and septicaemic nature in seawater-farmed rainbow trout Oncorhynchus mykiss in Storfjorden, Norway. In November 2007, an outbreak of VHS associated with slightly elevated mortality was confirmed at a seawater site rearing rainbow trout (90 to 440 g). Within 3 to 4 mo, the disease was recognised in 3 neighbouring sea sites with ongrowing rainbow trout. The clinical, gross pathological and histopathological findings were in accordance with VHS, and the diagnosis was confirmed by the detection of VHSV in brain and internal tissues by immunohistochemistry, cell culture and reverse transcriptase PCR (RT-PCR). Sequence analysis of the G-gene revealed that the isolated virus clustered with VHSV Genotype III and that the Norwegian isolate represents a unique strain of VHSV. The pathogenicity of the virus strain to rainbow trout and Atlantic salmon Salmo salar was examined using infection experiments. In immersion trials, the Norwegian isolate produced a cumulative mortality of 70% in rainbow trout, while nearly 100% mortality was obtained after intraperitoneal injection of the virus. For Atlantic salmon, no mortality was observed in immersion trials, whereas 52% mortality was observed after intraperitoneal injection. The Norwegian isolate thus represents the first VHSV of Genotype III pathogenic to rainbow trout.

Emergence of clonally related Klebsiella pneumoniae isolates of sequence type 258 producing plasmid-mediated KPC carbapenemase in Norway and Sweden

The class A carbapenemase KPC has disseminated rapidly worldwide, challenging the treatment of Gram-negative infections. This report describes the first KPC-producing Klebsiella pneumoniae isolates identified in Norway (n=6) and the second isolate from Sweden. Antimicrobial susceptibility profiles were determined using Etest. PCR and sequencing were used to determine the bla(KPC) variant, the surrounding genetic structure and the presence of AmpC and extended-spectrum beta-lactamase genes. PFGE and multilocus sequence typing (MLST) were used for epidemiological comparisons. Localization of bla(KPC) was investigated by S1 nuclease digestion, followed by PFGE and Southern blot hybridization. All isolates expressed a multidrug-resistant phenotype with some variability in the carbapenem susceptibility profile. The Norwegian isolates carried bla(KPC-2), while the Swedish isolate carried bla(KPC-3). All isolates carried TEM-1, but were negative for bla(CTX-M) and bla(AmpC) genes. SHV-11 and SHV-12 were detected in the Norwegian isolates, while the Swedish isolate carried only SHV-11. Isolates from four patients were associated with import from Greece (n=3) and Israel. The other isolates were probably associated with local transmissions. PFGE and MLST showed that the isolates were clonally related, with three isolates displaying ST258, a single locus variant of ST11 previously associated with the clonal spread of CTX-M-15-producing K. pneumoniae in Hungary. In all isolates, bla(KPC) was located on plasmids as part of isoform a of Tn4401. The emergence of KPC-producing isolates of K. pneumoniae in Norway and Sweden is associated with multiple import events and probable local transmission of a successful multiresistant ST258 clone, closely related to the CTX-M-15-producing ST11 clone previously described in Hungary.

Epidemiology of enterovirus types causing neurological disease in Austria 1999–2007: Detection of clusters of echovirus 30 and enterovirus 71 and analysis of prevalent genotypes

Between 1999 and 2007 1,388 stool specimens from patients with acute flaccid paralysis or aseptic meningitis were submitted to the Austrian reference laboratory for poliomyelitis. Samples (201) yielded non-poliovirus enterovirus in culture. One hundred eighty-one viruses were available for typing and 78 isolates which remained serologically untyped were further analyzed by CODEHOP-PCR and sequencing of the VP1 gene and the 5'-untranslated region (5'-UTR). Typing revealed an Echovirus 30 outbreak in northwestern Austria in 2000, which was in accordance with the situation in Europe, and no dramatic seasonal changes of Coxsackie viruses were observed. In 2002/2003 a small outbreak of enterovirus 71 (EV71), affected 12 patients in the province of Styria. This virus was identified as genotype C1 and appeared to be genetically distinct from the isolates observed in 2001/2002 in Vienna. In 2004 two unrelated cases occurred in Lower Austria, which were identified as genotype C4, which has been described associated with high mortality most recently in China. In contrast to the situation in Asia the detected EV71 cases were not associated with hand-foot-mouth disease, but with serous meningitis only. This was surprising as a recent publication suggested a reduced neurovirulence of C1 genotype in children in Norway, presumably due to alterations in 5'-UTR and polymerase gene. However, comparing the 5'-UTR of the Austrian isolates and established virulent reference strains to the Norwegian isolate and an attenuated EV71 laboratory strain we did not find an indication that the genotype C1 possesses a RNA structure in its 5'-UTR leading to reduced neurovirulence.

Poxvirus infection in Hungarian great tits ( Parus major ): Case report

From a total of 1819 great tits (Parus major) ringed in 2007 in Pilis Mountains, Hungary, 15 birds presented nodular proliferative lesions on different areas of the head and eyelids, suggesting a poxvirus infection. Three birds were submitted for analysis. The presence of avipoxvirus infection was confirmed by histopathology, electron microscopy (EM) and a polymerase chain reaction (PCR) based technique. Nucleotide sequence analysis of a 428 base pairs (bp) fragment of the viral 4b core protein gene revealed 100% identity between two of the Hungarian isolates (PM9 HUN, PM33 HUN) and two great tit poxvirus strains isolated in Norway in 1973 (GTV A256, GTV A311). The third Hungarian isolate (PM34 HUN) was more closely related to a different Norwegian isolate (GTVA310) than to the Hungarian isolates. The nucleotide sequence analysis of a shorter fragment of the viral 4b core protein (227 bp) gene revealed 100% identity between the Hungarian isolates, the same Norwegian isolates and a great tit poxvirus strain detected in Austria in 2007.

The AmpC phenotype in Norwegian clinical isolates of Escherichia coli is associated with an acquired ISEcp1-like ampC element or hyperproduction of the endogenous AmpC

The aim of the study was to examine resistance mechanisms associated with an AmpC phenotype in Norwegian clinical isolates of Escherichia coli. Clinical E. coli isolates (n = 106) with reduced susceptibility to third-generation cephalosporins without clavulanic acid synergy were collected from 12 Norwegian laboratories from 2003 to 2005. Twenty-two isolates with an AmpC phenotype were selected for further characterization by PFGE, isoelectric focusing, different PCR-based techniques, DNA sequencing, AmpC qRT-PCR, transfer studies and plasmid analyses. The 22 isolates were not clonally related by the PFGE analysis. All isolates expressed a beta-lactamase with a pI of 9.0-9.2. Ten isolates contained a bla(CMY) gene, which was linked to an ISEcp1-like element in all cases. Twelve isolates had mutations or insertions in the promoter or the attenuator regions, leading to increased expression of the chromosomal ampC gene. One of these isolates had an ISEc10 element inserted upstream of the chromosomal ampC gene. This is the first molecular study of Norwegian clinical E. coli isolates with an AmpC phenotype. Resistance was mediated either by expression of bla(CMY) from acquired ISEcp1-like-bla(CMY) elements, or by mutations or insertions in the chromosomal ampC gene control region leading to hyperproduction of the endogenous AmpC enzyme. There was no correlation between the level of ampC mRNA and the MICs of cephalosporins.

Symptomless infectious cDNA clone of a Norwegian isolate of Poinsettia mosaic virus

An infectious cDNA clone of a Norwegian isolate of Poinsettia mosaic virus (PnMV) was generated. It consisted of 6,098 nucleotides and encoded a polyprotein of 219.5 kDa. Sequence comparisons indicated that this isolate shared 98.6% (nucleotide) and 97.1% (amino acid) identity with the previously sequenced isolate from Germany. RNA transcripts derived from this cDNA were infectious in Nicotiana benthamiana. However, plants did not present typical PnMV symptoms. Furthermore, RNA transcripts from this cDNA clone were not infectious in poinsettia. Serial propagation of this cDNA clone in N. benthamiana plants restored symptom induction in this host but did not re-establish infectivity in poinsettia.

Bacteriocins (meningocins) in Norwegian isolates of Neisseria meningitidis: possible role in the course of a meningococcal epidemic

In the Neisseria meningitidis strain MC58 (serogroup B; ET-5 complex) genome three putative islands of horizontally transferred DNA (IHTs) have been identified. IHT-A2 codes for eight hypothetical proteins and two disrupted open reading frames with similarity to a secretion protein (NMB0097) and an ABC transporter (NMB0098). The strains MC58 and 44/76 (shown here) are meningocin resistant/weakly sensitive. None of these strains are meningocin producers. However, NMB0097 and NMB0098 homologues with open reading frames are found in meningocin producers (N. meningitidis P241 (serogroup A; systemic isolate) and BT878 (serogroup B; carrier isolate), and also in strain FAM18 (serogroup C; ET-37 complex). Knocking out either of the two genes in the strain BT878 yielded mutants that did not secrete meningocin. A similarly disrupted tolC mutant in strain BT878 still released meningocin. Among systemic meningococcal isolates prior to and at the onset (mid-1973 to the end of 1974) of the epidemic peaking in 1975 in North Norway, 12 of 30 (40%) isolates of serogroup A were meningocin producers. However, the rate for serogroup B was 1 of 45 (2.2%). Serogroup B meningocin-resistant/weakly sensitive non-producers dominated in the region from mid-1975 and spread to the rest of the country from then on. No producers were found in selected pharyngeal isolates from healthy carriers collected in Svalbard in the early spring of 1975. Our results suggest that meningocinogeny has played a part in the change from serogroup A to serogroup B among isolates in North Norway during the first half of 1975.

Evolutionary mechanisms involved in the virulence of infectious salmon anaemia virus (ISAV), a piscine orthomyxovirus

Infectious salmon anaemia virus (ISAV) is an orthomyxovirus causing a multisystemic, emerging disease in Atlantic salmon. Here we present, for the first time, detailed sequence analyses of the full-genome sequence of a presumed avirulent isolate displaying a full-length hemagglutinin-esterase (HE) gene (HPR0), and compare this with full-genome sequences of 11 Norwegian ISAV isolates from clinically diseased fish. These analyses revealed the presence of a virulence marker right upstream of the putative cleavage site R 267 in the fusion (F) protein, suggesting a Q 266 → L 266 substitution to be a prerequisite for virulence. To gain virulence in isolates lacking this substitution, a sequence insertion near the cleavage site seems to be required. This strongly suggests the involvement of a protease recognition pattern at the cleavage site of the fusion protein as a determinant of virulence, as seen in highly pathogenic influenza A virus H5 or H7 and the paramyxovirus Newcastle disease virus.

Multiple-locus variable-number tandem-repeats analysis of Listeria monocytogenes using multicolour capillary electrophoresis and comparison with pulsed-field gel electrophoresis typing

The multiple-locus variable-number tandem-repeats analysis (MLVA) method for genotyping has proven to be a fast and reliable typing tool in several bacterial species. MLVA is in our laboratory the routine typing method for Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli O157. The gram-positive bacteria Listeria monocytogenes, while not isolated as frequent as S. Typhimurium and E. coli, causes severe illness with an overall mortality rate of 30%. Thus, it is important that any outbreak of this pathogen is detected early and a fast trace to the source can be performed. In view of this, we have used the information provided by two fully sequenced L. monocytogenes strains to develop a MLVA assay coupled with high-resolution capillary electrophoresis and compared it to pulsed-field gel electrophoresis (PFGE) in two sets of isolates, one Norwegian (79 isolates) and one Swedish (61 isolates) set. The MLVA assay could resolve all of the L. monocytogenes serotypes tested, and was slightly more discriminatory than PFGE for the Norwegian isolates (28 MLVA profiles and 24 PFGE profiles) and opposite for the Swedish isolates (42 MLVA profiles and 43 PFGE profiles).

A comparison of phylogenetic group, virulence factors and antibiotic resistance in Russian and Norwegian isolates of Escherichia coli from urinary tract infection

Isolates of Escherichia coli from 31 Norwegian and 31 Russian females with significant bacteruria who presented with clinical signs of urinary tract infection (UTI) were tested for antimicrobial sensitivity, the presence of virulence genes, phylogroup distribution and clonal affinity. Twenty isolates, representing the full clonal diversity of a collection of 138 intestinal isolates of E. coli from healthy Norwegian females, served as a reference group. Russian UTI isolates belonged more often to phylogroup A and possessed fewer virulence genes than did Norwegian isolates. UTI isolates of E. coli were genetically heterogeneous and had a high degree of antimicrobial sensitivity.

Sequence analysis of the fusion protein gene from infectious salmon anemia virus isolates: evidence of recombination and reassortment

Studies of infectious salmon anemia virus (ISAV; genus Isavirus, family Orthomyxoviridae) haemagglutinin-esterase (HE) gene sequences have shown that this gene provides a tool for genotyping and, hence, a tool to follow the dissemination of ISAV. The problem with using only the HE gene is that ISAV has a segmented genome and one segment may not tell the whole story about the origin and history of ISAV from outbreaks. To achieve a better genotyping system, the present study has focused on segment 5, the fusion (F) protein gene, which contains sequence variation at about the same level as the HE gene. The substitution rates of the HE and F gene sequences, based on 54 Norwegian ISAV isolates, are 6.1(+/-0.3)x10(-6) and 8.6(+/-5.0)x10(-5) nt per site per year, respectively. The results of phylogenetic analysis of the two gene segments have been compared and, with the exception of a few cases of reassortment, they tell the same story about the ISAV isolates. A combination of the two segments is recommended as a tool for future genotyping of ISAV. Inserts (INs) of 8-11 aa may occur close to the cleavage site of the precursor F(0) protein in some ISAV isolates. The nucleotide sequence of two of these INs shows 100% sequence identity to parts of the 5' end of the F protein gene, whilst the third IN is identical to a part of the nucleoprotein gene. This shows that recombination is one of the evolutionary mechanisms shaping the genome of ISAV. The possible importance of the INs with respect to virulence remains uncertain.

Genetic diversity of small-ruminant lentiviruses: characterization of Norwegian isolates of Caprine arthritis encephalitis virus

Small-ruminant lentiviruses (SRLVs), including Caprine arthritis encephalitis virus (CAEV) in goats and maedi-visna virus (MVV) in sheep, are lentiviruses that, despite overall similarities, show considerable genetic variation in regions of the SRLV genome. To gain further knowledge about the genetic diversity and phylogenetic relationships among field isolates of SRLVs occurring in geographically distinct areas, the full-length genomic sequence of a CAEV isolate (CAEV-1GA) and partial env sequences obtained from Norwegian CAEV-infected goats were determined. The genome of CAEV-1GA consisted of 8,919 bp. Alignment studies indicated significant diversity from published SRLV sequences. Deletions and hypervariability in the 5' part of the env gene have implications for the size of the proposed CAEV-1GA Rev protein and the encoded surface glycoprotein (SU). The variable regions in the C-terminal part of SU obtained from Norwegian CAEV isolates demonstrate higher sequence divergence than has been described previously for SRLVs. Phylogenetic analysis based on SU sequences gives further support for a unique group designation. The results described here reveal a distant genetic relationship between Norwegian CAEV and other SRLVs and demonstrate that there is more geographical heterogeneity among SRLVs than reported previously.

Antimicrobial resistance in Campylobacter jejuni from humans and broilers in Norway

In this study comprising isolates from 2001 to 2003, resistance was considerably more widespread among Campylobacter jejuni from humans infected abroad than infected within Norway. The discrepancy was particularly notable for fluoroquinolone resistance (67.4% vs. 6.5%). This is probably a reflection of a low resistance prevalence in Norwegian broiler isolates (1.2% fluoroquinolone resistant).

EnterotoxigenicStaphylococcus aureusin bulk milk in Norway

To investigate the presence of enterotoxigenic Staphylococcus aureus in bulk milk and in a selection of raw milk products. Samples of bovine (n = 220) and caprine (n = 213) bulk milk, and raw milk products (n = 82) were analysed for S. aureus. Isolates were tested for staphylococcal enterotoxin (SE) production (SEA-SED) by reversed passive latex agglutination and for SE genes (sea-see, seg-sej) by multiplex PCR. Staphylococcus aureus was detected in 165 (75%) bovine and 205 (96.2%) caprine bulk milk samples and in 31 (37.8%) raw milk product samples. Enterotoxin production was observed in 22.1% and 57.3% of S. aureus isolates from bovine and caprine bulk milk, respectively, while SE genes were detected in 52.5% of the bovine and 55.8% of the caprine bulk milk isolates. SEC and sec were most commonly detected. A greater diversity of SE genes were observed in bovine vs caprine isolates. Staphylococcus aureus seems highly prevalent in Norwegian bulk milk and isolates frequently produce SEs and contain SE genes. Enterotoxigenic S. aureus were also found in raw milk products. Staphylococcus aureus in Norwegian bovine and caprine bulk milk may constitute a risk with respect to staphylococcal food poisoning from raw milk products.

IS900 Restriction Fragment Length Polymorphism (RFLP) Analysis of Mycobacterium avium subsp. paratuberculosis Isolates from Goats and Cattle in Norway

In Norway, paratuberculosis has been frequently diagnosed in goats, while cattle have been almost free of the infection. This difference in prevalence between goats and cattle has led to speculations about the existence of a Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) isolate that is non-pathogenic for cattle. There is little information available on genotypic variation of M. a. paratuberculosis isolated from animals in Norway. In the present study, genotypic information on 51 isolates from goats and four isolates from cattle in Norway was obtained by use of IS900 restriction fragment length polymorphism (RFLP) analysis. All isolates from cattle and 84% of the isolates from goats had the same RFLP pattern (B-C1). Five RFLP patterns not previously detected were found. No genotypic variation that could explain a difference in host origin was found between the isolates from cattle and the majority of the Norwegian goat isolates. This lack of difference indicates that the most common M. a. paratuberculosis isolates in Norway may infect both cattle and goats.

Morphological, chemical and molecular differentiation of Fusarium equiseti isolated from Norwegian cereals

The morphological variation, secondary metabolite profiles and restriction fragment length polymorphisms (RFLPs) of PCR amplified intergenic spacer (IGS) ribosomal DNA (rDNA) were studied in 27 isolates of Fusarium equiseti, 25 isolated from Norwegian cereals and 2 from soil obtained from the IBT culture collection (BioCentrum, Technical University of Denmark). All 27 isolates were tested for production of fusarochromanone (FUSCHR), zearalenone (ZEA) and the trichothecenes: 15-monoacetoxy-scirpentriol (MAS), diacetoxy-scirpenol (DAS), T-2 and HT-2 toxins, T2-triol, neosolaniol (NEO), deoxynivalenol (DON), nivalenol (NIV) and 4-acetylnivalenol (Fus-X). The trichothecenes were analysed by GC-MS in a selected ion monitoring mode, while FUSCHR was determined by ion pair HPLC with fluorometric detection and production of ZEA by TLC. For amplification of IGS rDNA primers CNL12 and CNS1 were applied. IGS rDNA was digested with the four restriction enzymes: AvaII, CfoI, EcoRI and Sau3A. In addition, we sequenced the IGS rDNA region of three of the Norwegian isolates. There were two morphological types among the Norwegian strains of F. equiseti, type I with short apical cells (dominating) and type II with long apical cells, with four haplotypes identified based on the RFLP data. Variation in secondary metabolite profiles within and between the morphological groups was observed and the levels of produced toxins were: FUSCHR 3000–42,500 and 25–30 ng/g, NIV 20–2500 and 120–700 ng/g, FUS-X 20–15,000 and 0 ng/g, DAS 30–7500 and 0–600 ng/g, and MAS 10–600 and 0–500 ng/g, for strains with short and long apical cells, respectively. NEO was detected in 16/27 strains tested (all morphotype I). All but four strains of type I (these four lacked a restriction site for EcoRI) had identical RFLP profiles. The isolates of type II had two haplotypes. The IGS sequence similarity data indicated differences between these morphotypes corresponding to two separate lineages apparently at the species level.

Isolates of Piscirickettsia salmonis from Scotland and Ireland show evidence of clonal diversity.

Salmonid rickettsial septicemia, caused by Piscirickettsia salmonis, causes major mortalities in Chilean salmonid aquaculture and is an increasing problem in Atlantic salmon in Ireland and Scotland. Analysis of 16S-to-23S internal transcribed sequences and 16S ribosomal DNA (rDNA) shows that Irish isolates of P. salmonis form two new groups of the organism while Scottish isolates cluster together with Norwegian and Canadian isolates from Atlantic salmon.

Use of ELISA to identify Protoceratium reticulatum as a source of yessotoxin in Norway

Picked cells of Protoceratium reticulatum collected from five locations in Norway were shown by ELISA analysis to contain yessotoxins (YTXs). The production of yessotoxin (YTX) was verified by culturing followed by LC-MS analysis of one of the Norwegian isolates. This is the first report of the biogenic origin of YTXs in Norway. The sensitivity of the ELISA method made it possible to quantitate YTXs in algal cultures, net-hauls, and in single cells of P. reticulatum. The cells picked from cultures and net-hauls contained 18–79 pg YTXs per cell. Dilution series and analyses of cells from non-YTX-producing algal species demonstrated the presence of only minimal matrix effects on the ELISA, probably attributable to the presence of salts. The sensitivity of this method makes it possible to search for other possible producers of YTXs, and might also make it possible to follow the YTXs through the food chain. This method allows, for the first time, measurement of the variability in toxin content within a population of dinoflagellate cells—rather than just the average amount of toxin per cell.