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High-throughput zebrafish histology.

The morphological effects of mutation and disease are often critical to our understanding of normal and abnormal function. The power and popularity of zebrafish as a forward and reverse genetic vertebrate model system, combined with its small size, have made it an ideal model in which to study the genetics of histologically scorable phenotypes. The presence of multiple tissue types in this organism's small larvae also makes it a potentially important model for toxicological analysis. Studying histological phenotypes is greatly enhanced by high-throughput methods of histology. Here, we describe details of high-throughput histology of the zebrafish using larval arrays, along with recent advances in mold design and discussion of work in progress that will lead to easier ways for people in the field to more rapidly score phenotypes in arrays. These detailed descriptions, together with the troubleshooting guide, should enable any laboratory with ties to a histology facility to perform high-throughput histology of zebrafish.

Animals↗

Correlation between male and female reproduction in the subdioecious herb Astilbe biternata (Saxifragaceae).

Genotypic trade-offs between male and female reproduction are commonly assumed in theoretical studies of the evolution of gender specialization. Although these trade-offs are supported by higher seed production of females than hermaphrodites in natural populations of gynodioecious species, comparisons between male and female reproductive allocation among hermaphrodite individuals under controlled conditions are rare. We assessed phenotypic and genotypic correlations between stamen and fruit production in fruiting males of the near-dioecious herb Astilbe biternata. In the field, we found a significant negative phenotypic correlation between stamen production and fruit production within individuals that produced both stamens and fruit as well as higher fruit set in females than fruiting males. The negative correlation between fruit and stamen production that was observed in the field was also apparent across clonally propagated genotypes. These results suggest that negative genetic correlations between male and female reproduction may limit the independent evolution of fruit and stamen production in A. biternata.

Journal Article↗

Drug sensitivity screening in vitro of populations of Trypanosoma congolense originating from cattle and tsetse flies at Nguruman, Kenya.

An in vitro assay that utilises in vitro-derived metacyclic trypanosomes was used to determine the drug sensitivity of 7 populations of Trypanosoma congolense collected from cattle and tsetse flies at Nguruman; a trypanosomiasis-endemic area in southwest Kenya. The metacyclic trypanosomes used in the assay were obtained from cultures initiated directly from either the blood of cattle with low levels of parasitaemia or from guts of infected tsetse flies. Sensitivities to isometamidium chloride, diminazene aceturate and homidium salts were assessed at various drug concentrations (0.5 ng-50 micrograms/ml). The results were compared with those obtained with two characterised laboratory populations. In spite of the fact that isometamidium chloride had not been widely used at Nguruman, two of the stocks (KE 3302 and KE 3303) expressed high levels of resistance to this drug (resistant to 100 ng/ml and 1000 ng/ml, respectively). In contrast, all of the populations examined were as sensitive, or more so, to diminazene aceturate than the sensitive laboratory clone IL 1180; two stocks (KE 3305 and KE 3306) were more sensitive (resistant to 0.5 microgram/ml, sensitive to 1 microgram/ml) and three stocks expressed the same level of sensitivity as IL 1180 (resistant to 1 microgram/ml, sensitive to 5 micrograms/ml). Since the results of the in vitro assay correlated well with field observations it was concluded that the assay would be a useful tool in epidemiological studies to determine the resistance phenotypes of trypanosome populations in the field, thereby enabling development of appropriate control measures for particular areas.

Animals↗

Surface-induced changes in protein adsorption and implications for cellular phenotypic responses to surface interaction.

Understanding external factors that determine cellular phenotypic responses is of key interest in the field of biomaterials. Currently, material surface characteristics, protein adsorption and cellular phenotypic responses are all considered to be interrelated and ultimately determine the biocompatibility of materials. The exact nature of the relationship between these distinct, yet related, phenomena still remains to be elucidated. Through the use of a series of thermoresponsive N-isopropylacrylamide-based co-polymer films, we aimed to shed light on the relationship between surface hydrophobicity, protein adsorption and subsequent cellular response. Despite changes in co-polymer hydrophobicity mediated by altered ratios of constituent monomers, differential cellular response was only apparent in the presence of serum. Co-polymer films displayed alterations with respect to the amount of protein adsorbed on the surface, with individual serum proteins (albumin and fibronectin) displaying contrasting adsorption characteristics. Changes in protein adsorption corresponded to changes in cell adhesion, cytoskeletal organisation and cell morphology, as well as to changes in cell movement and intracellular signalling events. Examination of focal adhesion kinase (FAK), and extracellular signal-regulated kinase (ERK 1/2), important mediators of adhesion and growth factor-related signalling events, revealed a comparative reduction in phosphorylation of these signalling proteins in cells grown on co-polymers in comparison to those cultured on tissue culture polystyrene (TCP; used as a control surface). We also associated surface-mediated phenotypic alterations of cells grown on TCP and co-polymer films with particular changes in gene expression. These results indicate that cellular response to interaction with our series of co-polymer films is determined by the surface-adsorbed protein layer, which in turn is determined by the changing surface chemistry as the ratio of the co-monomers is altered.

Acrylamides↗

Phenotypic and genotypic differentiation of Campylobacter spp. isolated from Austrian broiler farms: a comparison.

An identification scheme based on restriction fragment length polymorphism of polymerase chain reaction products (PCR-RFLP) was developed to differentiate isolates of the genera Campylobacter, Arcobacter and Helicobacter. Based on the 16S rRNA gene of these genera, PCR amplified a 1216-bp fragment. The amplicons were digested with the restriction enzymes RsaI and EcoRV. Additional differentiation was obtained using a PCR-assay based on the hippuricase gene. Genotyping was performed on several reference strains from the National Collection of Typing Culture (NCTC), London, and on 130 field isolates. In parallel, a phenotypic differentiation was performed, in order to compare the results. In 119 cases (91.5%) the results obtained from the genotypic characterization were concordant with those from phenotypic testing. Co-infections with Campylobacter jejuni and Campylobacter coli in two samples and seven hippurate-negative C. jejuni-strains were identified by the genotypic method. Furthermore, PCR-RFLP assays identified an atypical isolate as Campylobacter fetus/hyointestinalis.

Animals↗

High frequency transfer and horizontal spread of apramycin resistance in calf faecal Escherichia coli.

OBJECTIVES: The aminoglycoside apramycin has been used extensively in animal husbandry in the UK since 1978. This study aimed to determine both whether calves that had never been treated with aminoglycoside antibiotics harboured apramycin-resistant (apr(R)) commensal Escherichia coli, and the mode of spread of the resistance gene. METHODS: Apr(R) E. coli from weekly calf faecal samples were typed by pulsed-field gel electrophoresis, antibiotic resistance phenotype, plasmid restriction profiles and plasmid transfer frequencies. RESULTS: During 4 months of weekly sampling, six of 11 calves were found to harbour apr(R) E. coli. All apr(R) E. coli (45) were cross-resistant to gentamicin and tobramycin, which are both used in human medicine. Resistance was conferred by the aac(3)IV gene, present on three different conjugative plasmids. Two of these plasmids also mediated tetracycline and streptomycin resistance. One plasmid demonstrated very high transfer frequencies and was found in three different genotypes. CONCLUSIONS: We report the presence of apr(R) commensal E. coli in cattle that have never been treated with aminoglycosides. The presence of one conjugative plasmid in three different genotypes is evidence of horizontal spread of this plasmid. This is the first report of a very high transfer frequency of apr(R) plasmid, demonstrating horizontal spread in the commensal flora of food animals.

Acetyltransferases↗

Characterization of strains of Mycoplasma mycoides subsp. mycoides small colony type isolated from recent outbreaks of contagious bovine pleuropneumonia in Botswana and Tanzania: evidence for a new biotype.

Four strains of Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) isolated from recent outbreaks of contagious bovine pleuropneumonia (CBPP) in Africa have been investigated. One Botswanan strain, M375, displayed numerous and significant phenotypic differences from both contemporary field isolates and older field and vaccine strains (African, Australian, and European strains dating back to 1936). Differences include altered morphology, reduced capsular polysaccharide production, high sensitivity to MmmSC rabbit hyperimmune antisera in vitro, and unique polymorphisms following immunoblotting. While insertion sequence analysis using IS1634 clearly indicates a close evolutionary relationship to west African strains, hybridization with IS1296 shows the absence of a band present in all other strains of MmmSC examined. The data suggest that a deletion has occurred in strain M375, which may explain its altered phenotype, including poor growth in vitro and a relative inability to cause septicemia in mice. These characteristics are also exhibited by Mycoplasma capricolum subsp. capripneumoniae (causal agent of contagious caprine pleuropneumonia [CCPP]), against which M375 antiserum exhibited some activity in vitro (unique among the various MmmSC antisera tested). These findings may have evolutionary implications, since CCPP is believed to be lung specific and without a septicemic phase (unlike CBPP). Since M375 was isolated from a clinical case of CBPP, this novel biotype may be fairly widespread but not normally isolated due to difficulty of culture and/or a potentially altered disease syndrome. Bovine convalescent antisera (obtained from contemporary naturally infected cattle in Botswana) were active against strain M375 in an in vitro growth inhibition test but not against any other strains of MmmSC tested. There exists the possibility therefore, that strain M375 may possess a set of protective antigens different from those of other strains of MmmSC (including vaccine strains). These findings have implications for the control of the current CBPP epidemic in Africa.

Animals↗

Macrolide resistance mechanisms and expression of phenotypes among Streptococcus pneumoniae circulating in Italy.

In Italy, macrolide-resistant pneumococci have been isolated at a rate increasing from 6% in 1993 to 31.7% in 1998. A collection of 161 erythromycin-resistant Streptococcus pneumoniae recovered between 1993 and 1997 has now been phenotypically and genotypically characterized. Approximately 90% of these microorganisms possessed a constitutive MLS(B) mechanism of resistance. PCR detected ermB and mefE genes in strains showing MLS(B) and M phenotypes, respectively. Using pulsed-field gel electrophoresis of chromosomal DNA, one dominant restriction profile and its variations were detected in 51 S. pneumoniae isolates collected from different locations, indicating the circulation of a clone characterized by the possession of a great ability to spread.

Anti-Bacterial Agents↗

Something's rotten: a nosocomial outbreak of malodorous Pseudomonas aeruginosa.

From July 1994 through November 1996, a phenotypically unique strain of Pseudomonas aeruginosa producing a pungent, "rotten-potato" odor and a positive lysine decarboxylase reaction was isolated from 39 patients at UCLA Medical Center (Los Angeles). Most cases (95%) were in intensive care units and had clinical infections (72%). Most isolates (74%) were recovered from cultures of respiratory secretions. To determine risk factors for acquisition of the organism, 23 cases were compared with 23 randomly selected controls matched by service and isolate date. Multivariate analysis revealed that isolation of malodorous P. aeruginosa was associated with mechanical ventilation of > 24 hours' duration (odds ratio [OR] = 9.4; P = .001) and transfer from an outside hospital (OR = 5.7; P = .04). DNA from outbreak strains hybridized to P. aeruginosa-specific toxin A and phospholipase C gene probes and all outbreak isolates tested were found to be identical by use of pulsed-field gel electrophoresis. An unusual phenotypic characteristic of the strain led to the recognition of a nosocomial outbreak of P. aeruginosa infection associated with mechanical ventilation.

Carboxy-Lyases↗

[Genotyping of EsBLs-producing Escherichia coli by pulsed-field gel electrophoresis in nosocomial infection].

OBJECTIVE: Using a genotying method which is accurate and rapid to study the molecular epidemiology of EsBLs-producing Escherichia coli in nosocomial infection. METHODS: After an analysis of clinical information to ascertain nosocomial infection, EsBLs were detected by phenotype confirmation test and pulsed-field gel electrophoresis (PFGE). RESULTS: Three EsBLs-producing strains in ICU were verified as epidemiological-related because their dice coefficient index was higher than 95%. The others were verified as non-epidemiological-related since their different lanes were more than 6. CONCLUSION: PFGE is a perfect molecular typing method to study epidemiolgy and analyze homology.

China↗

Enhanced responsiveness to parathyroid hormone and induction of functional differentiation of cultured rabbit costal chondrocytes by a pulsed electromagnetic field.

Pulsed electromagnetic fields promote healing of delayed united and ununited fractures by triggering a series of events in fibrocartilage. We examined the effects of a pulsed electromagnetic field (recurrent bursts, 15.4 Hz, of shorter pulses of an average of 2 gauss) on rabbit costal chondrocytes in culture. A pulsed electromagnetic field slightly reduced the intracellular cyclic adenosine 3',5'-monophosphate (cAMP) level in the culture. However, it significantly enhanced cAMP accumulation in response to parathyroid hormone (PTH) to 140% of that induced by PTH in its absence, while it did not affect cAMP accumulation in response to prostaglandin E1 or prostaglandin I2. The effect on cAMP accumulation in response to PTH became evident after exposure of the cultures to the pulsed electromagnetic field for 48 h, and was dependent upon the field strength. cAMP accumulation in response to PTH is followed by induction of ornithine decarboxylase, a good marker of differentiated chondrocytes, after PTH treatment for 4 h. Consistent with the enhanced cAMP accumulation, ornithine decarboxylase activity induced by PTH was also increased by the pulsed electromagnetic field to 170% of that in cells not exposed to a pulsed electromagnetic field. Furthermore, stimulation of glycosaminoglycan synthesis, a differentiated phenotype, in response to PTH was significantly enhanced by a pulsed electromagnetic field. Thus, a pulsed electromagnetic field enhanced a series of events in rabbit costal chondrocytes in response to PTH. These findings show that exposure of chondrocytes to a pulsed electromagnetic field resulted in functional differentiation of the cells.

Alprostadil↗

Adaptation to Plant Defence in an Agricultural Insect Pest: Integrating Genome Scans and Gene Expression in the Soybean Aphid Reveals Multi-Genic Pathways.

In agroecosystems, intense selection pressures cause species to adapt and spread, often leading to the evolution and persistence of pests. Understanding how pests rapidly adapt can help develop sustainable strategies for their management and improve agroecosystem health. Pest adaptation involves stable variations in DNA sequence, as well as dynamic shifts in gene expression, often mediated by non-coding regulatory elements. We examined adaptation to plant defences in the soybean aphid, Aphis glycines, in which virulent aphids have overcome plant defences and avirulent aphids have not. Previous data with laboratory colonies suggested that virulent aphids have higher overall gene expression, including transposable elements, some of which influence gene regulation. However, we lack information on how genetic variation in natural populations impacts adaptation and potentially gene regulation. We integrated population genome scans of field-collected, soybean aphid populations with gene expression profiles of virulent and avirulent laboratory colonies to uncover connections between genetic differentiation and gene regulation for virulence. Genome scan methods found 2144 single nucleotide polymorphisms (SNPs) with significant genetic differentiation (i.e., outliers) in field-collected populations. These SNPs were near 1004 genes, representing 5.16% of the effective number of genes. Based on previous RNA-Seq data with laboratory colonies, we found 3160 genes and 147 long non-coding RNAs (lncRNAs) with differential expression among virulent and avirulent biotypes. By integrating both data sets, we identified 16 genes and 5 long non-coding RNAs with differential expression and that were associated with an outlier SNP (within 10 kbp). We validated SNPs with additional field collected aphids and found an aphid clone with stronger virulence than our laboratory virulent colony, surviving on 2 different aphid-resistant soybean varieties. This new virulent clone had fixed allele differences at 9 SNPs compared to our avirulent and other virulent colony. Field collected soybean aphids matching the phenotype of this new virulent clone had significant genetic differentiation with 3 outlier SNPs near genes related to zinc transport and lachesin compared to field collected avirulent aphids. Our entire data reinforced the importance of a potential multi-genetic response to overcome plant defence and generates new insights into complex genetic and regulatory mechanisms involved in insect-plant interactions.

Animals↗

Molecular epidemiological study of Haemophilus influenzae serotype b strains obtained from children with meningitis in Japan.

We report an epidemiological study of 30 Haemophilus influenzae serotype b (Hib) strains derived from the cerebrospinal fluid of children with meningitis. The Hib strains were biotyped, tested for beta-lactamase production, and genotyped by long PCR-ribotyping, random amplified polymorphic DNA (RAPD) analysis, and genomic DNA restriction fragment length polymorphism (RFLP) analysis by pulsed-field gel electrophoresis (PFGE). The phenotypic study characterized 22 of the strains (73%) as biotype I. A genotypic study using long PCR-ribotyping with HaeIII restriction digestion showed no polymorphisms among these 30 Hib strains, but RAPD analysis with two sets of primers demonstrated two distinctive subtypes: one typical of the strains of biotype group II and the second characteristic of the strains of biotype groups I and IV. Each RAPD group was subtyped into several genotypic groups by PFGE-RFLP with SmaI digestion. The genotyping of clinically isolated Hib strains may help to elucidate transmission routes in community infections, endemicity, and the reasons for vaccine failure.

Child, Preschool↗

Macular dystrophy with protan genotype and phenotype studied with cone type specific ERGs.

PURPOSE: To determine the L- and M-cone driven ERG responses in a male patient with macular dystrophy and a protan phenotype. METHODS: We measured large field ERG thresholds to stimuli which modulated exclusively the L- or the M-cones or the two in various combinations (both in-phase and in counterphase). In none of the stimuli, the S-cones were modulated. Additionally, standard and multifocal ERGs were measured. Analysis of the L- and M-cone pigment genes was performed by means of PCR, RFLP analysis and DNA sequencing techniques. RESULTS: Macular dystrophy was revealed by the markedly abnormal multifocal ERGs in presence of near normal standard ERGs. The large field ERG responses were exclusively driven by the M-cones with enlarged thresholds when compared with otherwise normal protanopes. In addition, the M-cone driven ERG response phases were abnormal. Pigment gene analysis confirmed a protan genotype with the presence of a single 5'red/3'green hybrid pigment gene. CONCLUSIONS: Our novel stimulus technique allows a reliable analysis of the separate cone pathways even in cases with macular dysfunction. The increased thresholds and the abnormal phase behavior of the M-cone driven ERGs reflect altered mechanisms of the retinal physiology in this patient. The data strongly suggest that the macular dystrophy and the protanopia have independent origins.

Adult↗

Naturally occurring variability in some phenotypic markers and correlates of haemonchotolerance in West African Dwarf goats in a subhumid zone of Nigeria.

West African Dwarf (WAD) goats of the Nigerian subhumid zone generally show strong resistance and resilience to Haemonchus contortus in laboratory experiments, although a relatively small proportion are susceptible to infection. Little is known about these extremes of response phenotype in nature. Therefore, a survey was carried out of gastrointestinal nematode infections in WAD goats, with emphasis on abomasal worms, at three goat markets in Southern Nigeria during the rainy season. Faecal samples (n=1070) were collected weekly from goats between April and September, and 352 abomasa and small intestines from local abattoirs were examined. Total strongyle (prevalence=65.0%) and H. contortus (prevalence=64.3%) faecal egg counts (FEC) varied between the three markets, being highest throughout at Opi. FEC increased from April to peak in August. Based on raw FEC, 76.1% of goats had FEC of <100, and 4.7%>500. Adjustment of these figures for monthly and between-market differences, gave figures of 78.8 and 3.4%, respectively. H. contortus worm burdens (WB) showed a similar pattern with 67.9% of goats harbouring <200 worms and 8.2% >1000, and after adjustment 69.6 and 6.0%, respectively. Fecundity, based on eggs in the uterus, did not vary between markets or monthly, but fell with increasing WB. Trichostrongylus colubriformis was less frequent (prevalence=42.4%) but goats from Opi also carried higher WB, and worms were similarly highly aggregated in hosts. When the between-market and monthly differences for both species were controlled, a highly significant positive correlation between the species emerged. Therefore, although a small subset of goats, highly susceptible to H. contortus, exists in this breed, the majority show resistance under field conditions and the resistant phenotype is also resistant to T. colubriformis. Both species are highly aggregated in the susceptible subset of the population. While, we cannot yet exclude alternative explanations, our data are compatible with a strong genetic basis for this phenomenon.

Abomasum↗

Phenotypic and genetic differentiation between native and introduced plant populations.

Plant invasions often involve rapid evolutionary change. Founder effects, hybridization, and adaptation to novel environments cause genetic differentiation between native and introduced populations and may contribute to the success of invaders. An influential idea in this context has been the Evolution of Increased Competitive Ability (EICA) hypothesis. It proposes that after enemy release plants rapidly evolve to be less defended but more competitive, thereby increasing plant vigour in introduced populations. To detect evolutionary change in invaders, comparative studies of native versus introduced populations are needed. Here, we review the current empirical evidence from: (1) comparisons of phenotypic variation in natural populations; (2) comparisons of molecular variation with neutral genetic markers; (3) comparisons of quantitative genetic variation in a common environment; and (4) comparisons of phenotypic plasticity across different environments. Field data suggest that increased vigour and reduced herbivory are common in introduced plant populations. In molecular studies, the genetic diversity of introduced populations was not consistently different from that of native populations. Multiple introductions of invasive plants appear to be the rule rather than the exception. In tests of the EICA hypothesis in a common environment, several found increased growth or decreased resistance in introduced populations. However, few provided a full test of the EICA hypothesis by addressing growth and defence in the same species. Overall, there is reasonable empirical evidence to suggest that genetic differentiation through rapid evolutionary change is important in plant invasions. We discuss conceptual and methodological issues associated with cross-continental comparisons and make recommendations for future research. When testing for EICA, greater emphasis should be put on competitive ability and plant tolerance. Moreover, it is important to address evolutionary change in characteristics other than defence and growth that could play a role in plant invasions.

Adaptation, Physiological↗

Characterisation of Listeria ivanovii isolates from the UK using pulsed-field gel electrophoresis.

Forty-three Listeria ivanovii isolates were collected in the UK between 1991 and 1997 from: 35 animal infections; two human infections; five foods; and one environmental source. A further two type strains of L. ivanovii (subsp. ivanovii and subsp. londoniensis) were obtained from a culture collection. These bacteria were characterised by conventional phenotypic methods and by pulsed-field gel electrophoresis (PFGE) using ApaI and SmaI. Forty-two of the isolates from the UK were identified as L. ivanovii subsp. ivanovii and the remaining culture as L. ivanovii subsp. londoniensis. Six and four PFGE profiles were obtained using ApaI and SmaI digestion respectively; six composite profiles were obtained combining the results for both enzymes. The PFGE profile of the UK L. ivanovii subsp. londoniensis (isolated from processed shrimps) was similar to the type strain of this subspecies and differed from all of the L. ivanovii subsp. ivanovii tested. The majority of isolates (38 out of 45) belonged to one profile showing that the UK population of this bacterium is much less genetically diverse than similar studies have shown for Listeria monocytogenes.

Animals↗

Host adaptation of pigeon isolates of Salmonella enterica subsp. enterica serovar Typhimurium variant Copenhagen phage type 99 is associated with enhanced macrophage cytotoxicity.

Phage type 99 of Salmonella enterica subsp. enterica serovar Typhimurium variant Copenhagen strains isolated from pigeons were examined for the presence of genotypic and phenotypic characteristics. The pulsed-field gel electrophoresis patterns obtained with XbaI and BlnI from 38 pigeon strains were compared with those obtained from 89 porcine, poultry, and human strains of variant Copenhagen. Identical patterns with XbaI and four closely related patterns with BlnI were obtained with the pigeon strains, whereas 16 XbaI patterns were found with the other strains. The XbaI patterns of the pigeon strains showed a low genetic similarity to the patterns of the porcine, poultry, and human strains and invariably showed a low-molecular-weight band that was absent in the majority of the other strains. The virulence genes shdA, spvR, pefA, sopE, and spvB were uniformly present in six pigeon isolates representing the genetic diversity found with BlnI. These six pigeon-derived strains were highly cytotoxic for pigeon macrophages compared to three porcine strains. After experimental infection of pigeons with a pigeon strain, clinical symptoms, fecal shedding, and colonization of internal organs were more pronounced than those after infection with a porcine strain. These data suggest that the phage type 99 strains used in this study are highly adapted to pigeons and should be classified as a host-restricted lineage of the serovar Typhimurium.

Adaptation, Physiological↗