Inter-strain Comparisons Research Articles

Comparative analysis of multiple consensus genomes of the same strain of Marek's disease virus reveals intrastrain variation.

Current strategies to understand the molecular basis of Marek's disease virus (MDV) virulence primarily consist of cataloging divergent nucleotides between strains with different phenotypes. However, most comparative genomic studies of MDV rely on previously published consensus genomes despite the confirmed existence of MDV strains as mixed viral populations. To assess the reliability of interstrain genomic comparisons relying on published consensus genomes of MDV, we obtained two additional consensus genomes of vaccine strain CVI988 (Rispens) and two additional consensus genomes of the very virulent strain Md5 by sequencing viral stocks and cultured field isolates. In conjunction with the published genomes of CVI988 and Md5, this allowed us to perform three-way comparisons between multiple consensus genomes of the same strain. We found that consensus genomes of CVI988 can vary in as many as 236 positions involving 13 open reading frames (ORFs). By contrast, we found that Md5 genomes varied only in 11 positions involving a single ORF. Notably, we were able to identify 3 single-nucleotide polymorphisms (SNPs) in the unique long region and 16 SNPs in the unique short (US) region of CVI988GenBank.BAC that were not present in either CVI988Pirbright.lab or CVI988USDA.PA.field. Recombination analyses of field strains previously described as natural recombinants of CVI988 yielded no evidence of crossover events in the US region when either CVI988Pirbright.lab or CVI988USDA.PA.field were used to represent CVI988 instead of CVI988GenBank.BAC. We were also able to confirm that both CVI988 and Md5 populations were mixed, exhibiting a total of 29 and 27 high-confidence minor variant positions, respectively. However, we did not find any evidence of minor variants in the positions corresponding to the 19 SNPs in the unique regions of CVI988GenBank.BAC. Taken together, our findings suggest that continued reliance on the same published consensus genome of CVI988 may have led to an overestimation of genomic divergence between CVI988 and virulent strains and that multiple consensus genomes per strain may be necessary to ensure the accuracy of interstrain genomic comparisons.

C57Bl/6N mice have an attenuated lung inflammatory response to dsRNA compared to C57Bl/6J and BALB/c mice

BackgroundLower respiratory infections caused by ssRNA viruses are a major health burden globally. Translational mouse models are a valuable tool for medical research, including research on respiratory viral infections. In in vivo mouse models, synthetic dsRNA can be used as a surrogate for ssRNA virus replication. However, studies investigating how genetic background of mice impacts the murine lung inflammatory response to dsRNA is lacking. Hence, we have compared lung immunological responses of BALB/c, C57Bl/6N and C57Bl/6J mice to synthetic dsRNA.MethodsdsRNA was administered intranasally to BALB/c, C57Bl/6N and C57Bl/6J mice once/day for three consecutive days. Lactate dehydrogenase (LDH) activity, inflammatory cells, and total protein concentration were analyzed in bronchoalveolar lavage fluid (BALF). Pattern recognition receptors levels (TLR3, MDA5 and RIG-I) were measured in lung homogenates using RT-qPCR and western blot. Gene expression of IFN-β, TNF-α, IL-1β and CXCL1 was assessed in lung homogenates by RT-qPCR. ELISA was used to analyze protein concentrations of CXCL1 and IL-1β in BALF and lung homogenates.ResultsBALB/c and C57Bl/6J mice showed infiltration of neutrophils to the lung, and an increase in total protein concentration and LDH activity in response to dsRNA administration. Only modest increases in these parameters were observed for C57Bl/6N mice. Similarly, dsRNA administration evoked an upregulation of MDA5 and RIG-I gene and protein expression in BALB/c and C57Bl/6J, but not C57Bl/6N, mice. Further, dsRNA provoked an increase in gene expression of TNF-α in BALB/c and C57Bl/6J mice, IL-1β only in C57Bl/6N mice and CXCL1 exclusively in BALB/c mice. BALF levels of CXCL1 and IL-1β were increased in BALB/c and C57Bl/6J mice in response to dsRNA, whereas the response of C57Bl/6N was blunt. Overall, inter-strain comparisons of the lung reactivity to dsRNA revealed that BALB/c, followed by C57Bl/6J, had the most pronounced respiratory inflammatory responses, while the responses of C57Bl/6N mice were attenuated.ConclusionsWe report clear differences of the lung innate inflammatory response to dsRNA between BALB/c, C57Bl/6J and C57Bl/6N mice. Of particular note, the highlighted differences in the inflammatory response of C57Bl/6J and C57Bl/6N substrains underscore the value of strain selection in mouse models of respiratory viral infections.

Rapid loss of virulence during submergence of Z. tritici asexual spores

Zymoseptoria tritici, the causal agent of Septoria tritici blotch, is a notable pathogen of temperate-grown wheat. To better understand the mechanisms underpinning pathogenicity, leaf infection assays are commonly used to compare either the virulence of Z. tritici wildtype or mutant strains, or the susceptibility of wheat cultivars. These assays, which control for many biotic, abiotic and experimental variables, involve the application of known spore numbers to leaves. To achieve this, spore numbers are quantified during a period of aqueous suspension. Published methods rarely state the period in which spores are held in suspension, suggesting that this variable may be uncontrolled. Using simple, agar-based plating experiments, this work firstly demonstrates that blastospore culturability (the ability to form a colony when plated on appropriate agar) decreases rapidly over time during maintenance in aqueous suspension. It is subsequently shown that this reduction in culturability correlates to a reduction in the virulence of the blastospore population. This is shown in three wild type Z. tritici strains. From this, it is concluded that suspension time is a variable of major importance in experimental design and one which, if not controlled, may lead to erroneous conclusions from inter-strain comparisons. The conidia of the unrelated fungus Magnaporthe oryzae also rapidly lose culturability when stored in aqueous suspension, whereas the microspores of Fusarium oxysporum f. sp. cubense do not, suggesting that this phenomenon occurs in some but not all other fungi. Finally, a droplet method of inoculations is proposed to decrease the variability in the numbers of spores applied, within and between experiments.

Multi-omics analysis of thermal stress response in a zooxanthellate cnidarian reveals the importance of associating with thermotolerant symbionts.

Corals and their endosymbiotic dinoflagellates of the genus Symbiodinium have a fragile relationship that breaks down under heat stress, an event known as bleaching. However, many coral species have adapted to high temperature environments such as the Red Sea (RS). To investigate mechanisms underlying temperature adaptation in zooxanthellate cnidarians we compared transcriptome- and proteome-wide heat stress response (24 h at 32°C) of three strains of the model organism Aiptasia pallida from regions with differing temperature profiles; North Carolina (CC7), Hawaii (H2) and the RS. Correlations between transcript and protein levels were generally low but inter-strain comparisons highlighted a common core cnidarian response to heat stress, including protein folding and oxidative stress pathways. RS anemones showed the strongest increase in antioxidant gene expression and exhibited significantly lower reactive oxygen species (ROS) levels in hospite. However, comparisons of antioxidant gene and protein expression between strains did not show strong differences, indicating similar antioxidant capacity across the strains. Subsequent analysis of ROS production in isolated symbionts confirmed that the observed differences of ROS levels in hospite were symbiont-driven. Our findings indicate that RS anemones do not show increased antioxidant capacity but may have adapted to higher temperatures through association with more thermally tolerant symbionts.

Identification of new genomospecies in the Mycobacterium terrae complex.

Members of the Mycobacterium terrae complex are slow-growing, non-chromogenic acid-fast bacilli found in the natural environment and occasionally in clinical material. These genetically closely-related members are difficult to differentiate by conventional phenotypic and molecular tests. In this paper we describe the use of whole genome data for the identification of four strains genetically similar to Mycobacterium sp. JDM601, a newly identified member of the M. terrae complex. Phylogenetic information from the alignment of genome-wide orthologous genes and single nucleotide polymorphisms show consistent clustering of the four strains together with M. sp. JDM601 into a distinct clade separate from other rapid and slow growing mycobacterial species. More detailed inter-strain comparisons using average nucleotide identity, tetra-nucleotide frequencies and analysis of synteny indicate that our strains are closely related to but not of the same species as M. sp. JDM601. Besides the 16S rRNA signature described previously for the M. terrae complex, five more hypothetical proteins were found that are potentially useful for the rapid identification of mycobacterial species belonging to the M. terrae complex. This paper illustrates the versatile utilization of whole genome data for the delineation of new bacterial species and introduces four new genomospecies to add to current members in the M. terrae complex.

Central corneal thickness does not correlate with TonoLab-measured IOP in several mouse strains with single transgenic mutations of matricellular proteins

Accurate and reliable measurement of intraocular pressure (IOP) is crucial in the study of glaucoma using the mouse model. The purpose of this study was to determine the relationship between TonoLab-measured IOP and central corneal thickness (CCT) in mouse strains with single gene mutations of matricellular proteins. Wild-type (WT) and transgenic mouse strains with single gene mutations (KO) of thrombospondin-1 (TSP-1), thrombospondin-2 (TSP-2), osteopontin (OPN), hevin, and secreted protein acidic rich in cysteine (SPARC) were imaged at six weeks using optical coherence tomography (Stratus, Zeiss) to determine CCT. IOP was measured between 11am and 3pm using TonoLab, one week later. For all measurements, mice were anesthetized using intraperitoneal injection ketamine:xylazine. CCT and IOP were measured in 583 mice (TSP-1 n = 71 and 41, TSP-2 n = 60 and 32, OPN n = 81 and 50, hevin n = 59 and 76, SPARC n = 54 and 59, WT and KO, respectively). Mean CCT was 5–6% lower in three KO strains—TSP-1, OPN, and SPARC—compared to their corresponding WT (p = 1.55 × 10−7, 1.63 × 10−11, and 1.91 × 10−7, respectively). The mean IOP was 8.3%, 6.6%, and 15.1% lower in three KO strains—TSP-1, TSP-2, and SPARC—compared to corresponding WT (p = 2.11 × 10−5, 2.93 × 10−3, and 3.76 × 10−9, respectively. Linear regression of IOP versus CCT yielded no statistically significant within-strain correlations for TSP-1 (p = 0.12 and 0.073), TSP-2 (p = 0.473 and 0.92), OPN (p = 0.212 and 0.916), Hevin (p = 0.746 and 0.257), and SPARC (p = 0.080 and 0.056), reported as p-values considering a null hypothesis of zero slope (WT and KO, respectively). Neither C57-derived strains (TSP-1 and OPN) nor 129-derived strains (TSP-2, hevin, SPARC) demonstrated a correlation between mean IOP and mean CCT across different strains (p = 0.75 and p = 0.53, respectively). Taken together, these results indicate that CCT is not required to interpret TonoLab IOP readings in the mice when CCT varies 10% about the mean. This does not exclude the possibility of an IOP-CCT correlation for CCT values outside this range or for inter-strain comparisons where the mean CCT differs more than 10%.

Comparative Geno-Plasticity Analysis of Mycoplasma bovis HB0801 (Chinese Isolate)

Mycoplasma bovis pneumonia in cattle has been epidemic in China since 2008. To investigate M. bovis pathogenesis, we completed genome sequencing of strain HB0801 isolated from a lesioned bovine lung from Hubei, China. The genomic plasticity was determined by comparing HB0801 with M. bovis strain ATCC® 25523™/PG45 from cow mastitis milk, Chinese strain Hubei-1 from lesioned lung tissue, and 16 other Mycoplasmas species. Compared to PG45, the genome size of HB0801 was reduced by 11.7 kb. Furthermore, a large chromosome inversion (580 kb) was confirmed in all Chinese isolates including HB0801, HB1007, a strain from cow mastitis milk, and Hubei-1. In addition, the variable surface lipoproteins (vsp) gene cluster existed in HB0801, but contained less than half of the genes, and had poor identity to that in PG45, but they had conserved structures. Further inter-strain comparisons revealed other mechanisms of gene acquisition and loss in HB0801 that primarily involved insertion sequence (IS) elements, integrative conjugative element, restriction and modification systems, and some lipoproteins and transmembrane proteins. Subsequently, PG45 and HB0801 virulence in cattle was compared. Results indicated that both strains were pathogenic to cattle. The scores of gross pathological assessment for the control group, and the PG45- and HB0801-infected groups were 3, 13 and 9, respectively. Meanwhile the scores of lung lesion for these three groups were 36, 70, and 69, respectively. In addition, immunohistochemistry detection demonstrated that both strains were similarly distributed in lungs and lymph nodes. Although PG45 showed slightly higher virulence in calves than HB0801, there was no statistical difference between the strains (P>0.05). Compared to Hubei-1, a total of 122 SNP loci were disclosed in HB0801. In conclusion, although genomic plasticity was thought to be an evolutionary advantage, it did not apparently affect virulence of M. bovis strains in cattle.

Inter-strain heterogeneity in rat hepatic transcriptomic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The biochemical and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been the subject of intense study for decades. It is now clear that essentially all TCDD-induced toxicities are mediated by DNA–protein interactions involving the Aryl Hydrocarbon Receptor (AHR). Nevertheless, it remains unknown which AHR target genes cause TCDD toxicities. Several groups, including our own, have developed rodent model systems to probe these questions. mRNA expression profiling of these model systems has revealed significant inter-species heterogeneity in rodent hepatic responses to TCDD. It has remained unclear if this variability also exists within a species, amongst rodent strains. To resolve this question, we profiled the hepatic transcriptomic response to TCDD of diverse rat strains (L-E, H/W, F344 and Wistar rats) and two lines derived from L-E×H/W crosses, at consistent age, sex, and dosing (100μg/kg TCDD for 19h). Using this uniquely consistent dataset, we show that the majority of TCDD-induced alterations in mRNA abundance are strain/line-specific: only 11 genes were affected by TCDD across all strains, including well-known dioxin-responsive genes such as Cyp1a1 and Nqo1. Our analysis identified two novel universally dioxin-responsive genes as well as 4 genes induced by TCDD in dioxin-sensitive rats only. These 6 genes are strong candidates to explain TCDD-related toxicities, so we validated them using 152 animals in time-course (0 to 384h) and dose–response (0 to 3000μg/kg) experiments. This study reveals that different rat strains exhibit dramatic transcriptional heterogeneity in their hepatic responses to TCDD and that inter-strain comparisons can help identify candidate toxicity-related genes.

A Comparative Survey of the Frequency and Distribution of Polymorphism in the Genome of Xenopus tropicalis

Naturally occurring DNA sequence variation within a species underlies evolutionary adaptation and can give rise to phenotypic changes that provide novel insight into biological questions. This variation exists in laboratory populations just as in wild populations and, in addition to being a source of useful alleles for genetic studies, can impact efforts to identify induced mutations in sequence-based genetic screens. The Western clawed frog Xenopus tropicalis (X. tropicalis) has been adopted as a model system for studying the genetic control of embryonic development and a variety of other areas of research. Its diploid genome has been extensively sequenced and efforts are underway to isolate mutants by phenotype- and genotype-based approaches. Here, we describe a study of genetic polymorphism in laboratory strains of X. tropicalis. Polymorphism was detected in the coding and non-coding regions of developmental genes distributed widely across the genome. Laboratory strains exhibit unexpectedly high frequencies of genetic polymorphism, with alleles carrying a variety of synonymous and non-synonymous codon substitutions and nucleotide insertions/deletions. Inter-strain comparisons of polymorphism uncover a high proportion of shared alleles between Nigerian and Ivory Coast strains, in spite of their distinct geographical origins. These observations will likely influence the design of future sequence-based mutation screens, particularly those using DNA mismatch-based detection methods which can be disrupted by the presence of naturally occurring sequence variants. The existence of a significant reservoir of alleles also suggests that existing laboratory stocks may be a useful source of novel alleles for mapping and functional studies.

The interaction between Listeria monocytogenes and the host gastrointestinal tract

Listeria monocytogenes is a ubiquitous bacterium that causes significant foodborne disease with high mortality rates in immunocompromised adults. In pregnant women foodborne infection can give rise to infection of the fetus resulting in miscarriage. In addition, the bacterium has recently been demonstrated to cause localized gastrointestinal symptoms, predominantly in immunocompetent individuals. The murine model of systemic L. monocytogenes infection has provided numerous insights into the mechanisms of pathogenesis of this organism. However, recent application of transcriptomic and proteomic approaches as well as the development of new model systems has allowed a focus upon factors that influence adaptation to gastrointestinal environments and adhesion to and invasion of the gastrointestinal mucosa. In addition, the availability of a large number of complete L. monocytogenes genome sequences has permitted inter-strain comparisons and the identification of factors that may influence the emergence of 'epidemic' phenotypes. Here we review some of the exciting recent developments in the analysis of the interaction between L. monocytogenes and the host gastrointestinal tract.

Deciphering the genetic basis for polyketide variation among mycobacteria producing mycolactones.

BackgroundMycolactones are immunosuppressive and cytotoxic polyketides, comprising five naturally occurring structural variants (named A/B, C, D, E and F), produced by different species of very closely related mycobacteria including the human pathogen, Mycobacterium ulcerans. In M. ulcerans strain Agy99, mycolactone A/B is produced by three highly homologous type I polyketide megasynthases (PKS), whose genes (mlsA1: 51 kb, mlsA2: 7.2 kb and mlsB: 42 kb) are found on a 174 kb plasmid, known as pMUM001.ResultsWe report here comparative genomic analysis of pMUM001, the complete DNA sequence of a 190 kb megaplasmid (pMUM002) from Mycobacterium liflandii 128FXT and partial sequence of two additional pMUM replicons, combined with liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. These data reveal how PKS module and domain differences affecting MlsB correlate with the production of mycolactones E and F. For mycolactone E these differences from MlsB in M. ulcerans Agy99 include replacement of the AT domain of the loading module (acetate to propionate) and the absence of an entire extension module. For mycolactone F there is also a reduction of one extension module but also a swap of ketoreductase domains that explains the characteristic stereochemistry of the two terminal side-chain hydroxyls, an arrangement unique to mycolactone FConclusionThe mycolactone PKS locus on pMUM002 revealed the same large, three-gene structure and extraordinary pattern of near-identical PKS domain sequence repetition as observed in pMUM001 with greater than 98.5% nucleotide identity among domains of the same function. Intra- and inter-strain comparisons suggest that the extreme sequence homogeneity seen among the mls PKS genes is caused by frequent recombination-mediated domain replacement. This work has shed light on the evolution of mycolactone biosynthesis among an unusual group of mycobacteria and highlights the potential of the mls locus to become a toolbox for combinatorial PKS biochemistry.

Morphological correlates of emotional and cognitive behaviour: insights from studies on inbred and outbred rodent strains and their crosses

Every study in rodents is also a behavioural genetic study even if only a single strain is used. Outbred strains are genetically heterogeneous populations with a high intrastrain variation, whereas inbred strains are based on the multiplication of a unique individual. The aim of the present review is to summarize findings on brain regions involved in three major components of rodent behaviour, locomotion, anxiety-related behaviour and cognition, by paying particular attention to the genetic context, genetic models used and interstrain comparisons. Recent trends correlating gene expression in inbred strains with behavioural data in databases, morpho-behavioural-haplotype analyses and problems arising from large-scale multivariate analyses are discussed. Morpho-behavioural correlations in multiple strains are presented, including correlations with projection neurons, interneurons and fibre systems in the striatum, midbrain, amygdala, medial septum and hippocampus, by relating them to relevant transmitter systems. In addition, brain areas differentially activated in different strains are described (hippocampus, prefrontal cortex, nucleus accumbens, locus ceruleus). Direct interstrain comparisons indicate that strain differences in behavioural variables and neuronal markers are much more common than usually thought. The choice of the appropriate genetic model can therefore contribute to an interpretation of positive results in a wider context, and help to avoid misleading interpretations of negative results.

QUALITY CONTROL METHOD TO MEASURE PREDATOR EVASION IN WILD AND MASS-REARED MEDITERRANEAN FRUIT FLIES (DIPTERA: TEPHRITIDAE)

Sterile male insects, mass-reared and released as part of sterile insect technique (SIT) programs, must survive long enough in the field to mature sexually and compete effectively with wild males for wild females. An often reported problem in Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) SIT programs is that numbers of released sterile males decrease rapidly in the field for various reasons, including losses to different types of predators. This is a serious issue in view that most operational programs release sterile flies at an age when they are still immature. Previous field and field-cage tests have confirmed that flies of laboratory strains are less able to evade predators than wild flies. Such tests involve, however, considerable manipulation and observation of predators and are therefore not suitable for routine measurements of predator evasion. Here we describe a simple quality control method with aspirators to measure agility in medflies and show that this parameter is related to the capacity of flies to evade predators. Although further standardization of the test is necessary to allow more accurate inter-strain comparisons, results confirm the relevance of measuring predator evasion in mass-reared medfly strains. Besides being a measure of this sterile male quality parameter, the described method could be used for the systematic selection of strains with a higher capacity for predator evasion.

Genotype-Dependent Activity of Tryptophan Hydroxylase-2 Determines the Response to Citalopram in a Mouse Model of Depression

Polymorphism of tryptophan hydroxylase, the rate-limiting enzyme in the synthesis of brain serotonin (5-HT), is associated with less synthesis of brain 5-HT in DBA/2J and BALB/c than in C57BL/6J and 129/Sv mice. We selected the forced swimming test, a mouse model used to assess the antidepressant potential of drugs, and neurochemical techniques to study strain differences in the response to citalopram, a selective 5-HT reuptake inhibitor. Citalopram reduced immobility time in C57BL/6J and 129/Sv mice but had no such effect in DBA/2J and BALB/c mice. The drug reduced accumulation of 5-hydroxytryptophan (5-HTP), an indicator of 5-HT synthesis, in C57BL/6J and 129/Sv mice but much less in DBA/2J and BALB/c mice. Pretreatment with tryptophan raised 5-HTP accumulation and reinstated the antidepressant-like effect of citalopram in DBA/2J and BALB/c mice, whereas pharmacological inhibition of 5-HT synthesis prevented the effect of citalopram in C57BL/6J and 129/Sv mice. Because there were no strain differences in catecholamine synthesis, locomotor activity, and brain levels of citalopram at the end of the behavioral test, the results suggest that the failure of citalopram to reduce immobility time in DBA/2J and BALB/c mice is attributable to genotype-dependent impairment of 5-HT synthesis. Interstrain comparisons could probably be a useful strategy for understanding the mechanisms underlying the response to selective serotonin reuptake inhibitors.

Common Evolutionary Origin for the Unstable Virulence Plasmid pMUM Found in Geographically Diverse Strains ofMycobacterium ulcerans

The 174-kb virulence plasmid pMUM001 in Mycobacterium ulcerans epidemic strain Agy99 harbors three very large and homologous genes that encode giant polyketide synthases (PKS) responsible for the synthesis of the lipid toxin mycolactone. Deeper investigation of M. ulcerans Agy99 resulted in identification of two types of spontaneous deletion variants of pMUM001 within a population of cells that also contained the intact plasmid. These variants arose from recombination between two 8-kb sections of the same plasmid sequence, resulting in the loss of a 65-kb region bearing two of the three mycolactone PKS genes. Investigation of nine diverse M. ulcerans strains by using PCR and Southern hybridization for eight pMUM001 gene sequences confirmed the presence of pMUM001-like elements (collectively called pMUM) in all M. ulcerans strains. Physical mapping of these plasmids revealed that like M. ulcerans Agy99, three strains had undergone major deletions in their mycolactone PKS loci. Online liquid chromatography-sequential mass spectrometry analysis of lipid extracts confirmed that strains with PKS deletions were unable to produce mycolactone or any related cometabolites. Interstrain comparisons of the plasmid gene sequences revealed greater than 98% nucleotide identity, and the phylogeny inferred from these sequences closely mimicked the phylogeny from a previous multilocus sequence typing study in which chromosomally encoded loci were used, a result that is consistent with the hypothesis that M. ulcerans diverged from the closely related organism Mycobacterium marinum by acquiring pMUM. Our results suggest that pMUM is a defining characteristic of M. ulcerans but that in the absence of purifying selection, deletion of plasmid sequences and a corresponding loss of mycolactone production readily arise.

Population genetic structure and cladistic analysis of Trypanosoma brucei isolates

Using a novel multilocus DNA marker analysis method, we studied the population genetic structure of Trypansoma brucei stocks and derived clones isolated from animal and rhodesiense sleeping sickness patients during a national sleeping sickness control program in Mukono district, Uganda. We then performed a cladistic analysis to trace relationships and evolution, using stocks and clones recovered from geographically and temporally matched hosts, including inter-strain comparisons with T. b. gambiense stocks and clones. Our results show that while there was close genetic relatedness among parasite populations from the same geographical region, micro-heterogeneities exist between different stocks. Data are presented that indicate that not every human sleeping sickness focus may be associated with a particular human-infective trypanosome strain responsible for long-term stability of the reference focus. We provide evidence of genetic sub-structuring among type 1 T. b. gambiense stocks, which has potentially important implications for molecular epidemiology of T. brucei.

Intra- and interstrain differences in models of “behavioral despair”

In the present studies, base line and drug-induced performance of two mouse strains (C57Bl/6 and NIH-Swiss) was evaluated in the forced swim test (FST) and tail suspension test (TST). Intra- and interstrain comparisons indicate that the biological substrates mediating performance in these behavioral procedures are not identical. For example, in NIH-Swiss mice, a sevenfold difference in base line immobility was observed between the FST and TST. By contrast, the base line immobility in C57Bl/6 mice was similar in both procedures. Further, in C57Bl/6 mice, imipramine produced a “U-shaped” dose–response curve in the FST, whilst no evidence of a biphasic response was present in the TST at doses up to 45 mg/kg. In the FST, the AMPA receptor potentiator LY451646 produced a similar dose–response relationship in C57Bl/6 and NIH-Swiss mice, but the minimum effect dose (MED) was fivefold higher in NIH-Swiss mice. This potency difference appears due to both pharmacokinetic and pharmacodynamic factors. These intra- and interstrain differences in performance indicate that despite a face value similarity, the neurochemical pathways involved in mediating performance in these two widely used tests are not identical.

Curie-Point Pyrolysis Mass Spectrometry as a Tool in Clinical Microbiology

Pyrolysis mass spectrometry is a well established analytical tool that has received a considerable boost from the development of low cost, dedicated instruments and sophisticated statistical analyses on personal computers. Further analytical developments, especially in the area of neural networks, are pushing the technology to the forefront of methods for the discrimination and identification of microorganisms and their products. The speed and reproducibility of pyrolysis mass spectrometry and its applicability to a wide range of microorganisms make it an attractive method for epidemiological studies. For inter-strain comparisons, the method is at least as discriminatory as conventional typing systems and usually gives discrimination similar to that of nucleic acid fingerprinting techniques. There has been some success in using neural networks to make identifications across pyrolysis mass spectrometric batches. Further development of methods used to handle data from multiple PyMS analyses can be expected to extend the value of pyrolysis mass spectrometry in clinical microbiology.

Electrospray Ionization-Mass Spectrometry as a Tool for Characterization of Glutathione S-Transferase Isozymes

Reversed-phase high-performance liquid chromatography coupled to electrospray ionization-mass spectrometry (ESI-MS) was used for the first time to determine the molecular masses of nine rat liver cytosolic glutathione S-transferase (GST) subunits. The precision of the measurements was ±3-4 mass units which, in practice, allowed discrimination between monomers differing by more than 8 Da. Mass accuracy was improved by replicates in the measurements. Comparison of experimental values to cDNA or protein-deduced data available reveals slight differences. N-terminal sequence analyses and interstrain mass comparisons tend to show that primary structures of liver cytosolic GSTs are well conserved from Sprague-Dawley to Wistar rats. Moreover, ESI-MS analysis enabled identification of two minor additional subunits present in both strains, one of which belongs to the μ-class. In addition to rapid and accurate mass determination of GST monomers, and direct determinations achieved on heterodimeric forms, this technique provides precise information on minor structural differences or modifications of these proteins. As such, it constitutes a useful tool for rapid characterization of purified GSTs in comparative studies.

Application of pyrolysis mass spectrometry to the investigation of outbreaks of food poisoning and non-gastrointestinal infection associated with Bacillus species and Clostridium perfringens

Eighteen isolates of Bacillus species and 15 of Clostridium perfringens, all of which had been associated with outbreaks of either food poisoning or non-gastrointestinal infection (NGI), were examined for relatedness by pyrolysis mass spectrometry (PyMS). The PyMS-analysis correctly clustered all the groups of epidemiologically related isolates of both genera, and distinguished all the single, epidemiologically unrelated isolates of the same species. PyMS is a simple, rapid and inexpensive technique which can provide useful and accurate inter-strain comparisons within both the Bacillus and Clostridium genera in complete accord with conventional serological typing results.