Molecular genetic variation within and among isolates of QPX (Thraustochytridae), a parasite of the hard clam Mercenaria mercenaria

The thraustochytrid known as QPX (Quahog Parasite Unknown) has sporadically caused disease in the hard clam Mercenaria mercenaria along the east coast of North America since the 1960s. We hypothesized that genetically distinct QPX strains might be responsible for outbreaks of QPX disease in different areas and tested this hypothesis by comparing several QPX isolates recovered from the recent outbreak in Raritan Bay, New York with QPX strains isolated from 2 outbreaks in Massachusetts, USA. There was no variation in small subunit rDNA (SSU rDNA), 5.8S rDNA, or 4 mitochondrial gene sequences. In contrast, both of the ribosomal ribonucleic acid (rRNA) operon intergenic spacers, internal transcribed spacers 1 and 2 (ITS1 and ITS2), revealed substantial sequence variation. However, strain-specific sequences were not detected because the ITS sequence variation within QPX isolates was comparable to the variation between isolates. ITS1 sequences recovered from an infected clam by amplification with a QPX ITS2-specific primer were identical to those recovered from the QPX isolates.     

A PCR-based diagnostic assay for the detection of Roseovarius crassostreae in Crassostrea virginica affected by juvenile oyster disease (JOD)

We have developed a PCR-assay for the diagnosis of juvenile oyster disease (JOD) based on the detection of Roseovarius crassostreae directly from affected oysters. Species-specific primers are used to amplify the 16S-23S rDNA internal transcribed spacer (ITS) of R. crassostreae, and confirmation of product identity is accomplished by restriction enzyme analysis. No false positives were obtained with either closely related bacterial species or from other DNAs present in oyster samples. The assay has the potential to detect as few as 10 cells of R. crassostreae per oyster when samples are taken from the inner valve surfaces of the animal. Inclusion of material from soft body surfaces is not necessary, and may reduce sensitivity approximately 10-fold. In a JOD-affected population, a positive PCR result was obtained from all oysters from which these bacteria were subsequently cultured. The assay also detected the presence of R. crassostreae in 2 oysters from which no R. crassostreae isolates were recovered. No R. crassostreae was detected by either PCR or bacteriology in oysters from a population that was not exhibiting JOD-signs. This assay is expected to advance regional disease management efforts and provide valuable insights into the disease process and epizootiology of JOD.     

Genetic evaluation of a proposed introduction: the case of the greater prairie chicken and the extinct heath hen

Population introduction is an important tool for ecosystem restoration. However, before introductions should be conducted, it is important to evaluate the genetic, phenotypic and ecological suitability of possible replacement populations. Careful genetic analysis is particularly important if it is suspected that the extirpated population was unique or genetically divergent. On the island of Martha's Vineyard, Massachusetts, the introduction of greater prairie chickens (Tympanuchus cupido pinnatus) to replace the extinct heath hen (T. cupido cupido) is being considered as part of an ecosystem restoration project. Martha's Vineyard was home to the last remaining heath hen population until its extinction in 1932. We conducted this study to aid in determining the suitability of greater prairie chickens as a possible replacement for the heath hen. We examined mitochondrial control region sequences from extant populations of all prairie grouse species (Tympanuchus) and from museum skin heath hen specimens. Our data suggest that the Martha's Vineyard heath hen population represents a divergent mitochondrial lineage. This result is attributable either to a long period of geographical isolation from other prairie grouse populations or to a population bottleneck resulting from human disturbance. The mtDNA diagnosability of the heath hen contrasts with the network of mtDNA haplotypes of other prairie grouse (T. cupido attwateri, T. pallidicinctus and T. phasianellus), which do not form distinguishable mtDNA groupings. Our findings suggest that the Martha's Vineyard heath hen was more genetically isolated than are current populations of prairie grouse and place the emphasis for future research on examining prairie grouse adaptations to different habitat types to assess ecological exchangeability between heath hens and greater prairie chickens.     

Duck plague epizootics in the United States, 1967-1995

In 1967, the first confirmed diagnosis of duck plague (DP) in the USA was made from pekin ducks (Anas platyrhynchos domesticus) on commercial duck farms on Long Island, New York. Within 10 mo, DP was confirmed as the cause of death in migratory waterfowl on a Long Island bay. This paper reviews 120 DP epizootics reported from 1967 to 1995 that involved waterfowl species native to North America or were reported in areas with free-flying waterfowl at risk. Duck plague epizootics occurred in 21 states with the greatest number reported in Maryland (29), New York (18), California (16), and Pennsylvania (13). The greatest frequency of epizootics (86%) was detected during the months of March to June. At least 40 waterfowl species were affected with the highest frequency of epizootics reported in captive or captive-reared ducks including muscovy ducks (Cairina moschata) (68%), mallard ducks (A. platyrhynchos) (18%) and black ducks (A. rubripes) (14%). The greatest number of waterfowl died in three epizootics that involved primarily migratory birds in 1967 and 1994 in New York (USA) and 1973 in South Dakota (USA). The greatest number of DP epizootics reported since 1967 appear to have involved flocks of non-migratory rather than migratory waterfowl; therefore, in our opinion it remains unknown if DP is enzootic in either non-migratory or migratory waterfowl.     

Genotypes of Candida dubliniensis in clinical isolates

Amplification of specific sequences of the ITS1 and ITS2 regions and the intervening 5.8S rRNA gene has lead to the identification of four separate genotypes in Candida dubliniensis. Using primers specific for each genotype, we have studied the prevalence of these genotypes among 68 clinical isolates, mostly from Spanish patients infected by HIV. The majority of the isolates tested belonged to genotype 1 (97%), while only one isolate each from genotypes 2 (1.5%) and 3 (1.5%) were detected in the oral cavity of two patients with HIV infection.     

Genotypic characteristics in populations of Sclerotinia sclerotiorum from New York State,USA

White mould, caused by the fungus Sclerotinia sclerotiorum, is one of the most destructive diseases of beans globally. In New York State, USA, white mould causes substantial losses in soybean, snap, dry and succulent baby lima beans, which are grown successively in intensive crop rotations. Management strategies for white mould in these crops are reliant upon the prophylactic use of fungicides. No complementary information on the genetic structure of the populations of S. sclerotiorum in New York State, USA is available. Twenty isolates of S. sclerotiorum were collected from symptomatic bean plants within each of 10 fields across New York State, USA in 2014. Eight microsatellite (SSR) markers were used to characterise the genotypic diversity of the hyphal‐tipped isolates. Twenty‐four multilocus genotypes (MLGs) were detected within the population but one MLG was most prevalent. Although STRUCTURE analysis identified two subpopulations, these subpopulations were not associated with geographic location, suggesting no spatial structure to the population. In addition, the pathogen populations were predominantly clonal, with some evidence of infrequent outcrossing. These findings may assist in understanding the durability of management strategies for white mould and support the selection of representative isolates for host resistance screening for pathogen populations in the sampling area.     

Characterization of Whitney's Clethrionomy gapperi virus isolates from Massachusetts.

Six strains of virus were recovered from the blood and/or liver of five Clethrionomys gapperi ochraceus trapped in southeastern Massachusetts during 1969. Biological, antigenic and physiochemical properties of these isolates are reported. USA M-2268a was selected as the reference strain. This strain was identical by complement-fixation and neutralization tests to Whitneys C. gapperie virus (USA 64-7855) from New York State and was related to, but distinct from, an unpublished agent (Johnson's Microtus montanus enterovirus USA M-1146) isolated in June, 1962 from voles trapped in Klamath County, Oregon. USA M-2268a was resistant to lipid solvents and acid pH and was stable at temperatures of 4 C, 22 C, and 37 C. Virus was detected over a 10-day observation period in four species of mosquitoes inoculated with USA M-2268a, although there was no evidence of infection or replication, and transmission attempts by bite failed. Neutralizing antibody was detected in C.g. gapperi and C. g. ochraceus in various habitats throughout the state.     

Genetic and phenotypic intraspecific variation in the microsporidian Encephalitozoon hellem.

Encephalitozoon hellem is a microsporidian species that causes disseminated infections in HIV-positive patients. Identical genotypes of E. hellem, as assessed by the sequence of the rDNA internal transcribed spacer, have been identified in isolates from humans and from a psittacine bird. However, by analysing the rDNA ITS of four E. hellem isolates from Switzerland (three) and Tanzania (one), two new genotypes were identified. Differences among the E. hellem isolates were also detected by Western blot analysis, but there was no absolute match between ITS genotype and antigen profile. Hence, strain variation exists in E. hellem and the ITS sequence seems a valuable marker in obtaining further insight into the epidemiology of this pathogen.     

Characterization of Genotypes of Enterocytozoon bieneusi in Immunosuppressed and Immunocompetent Patient Groups

ABSTRACT. A retrospective phylogenetic analysis was performed on isolates of Enterocytozoon bieneusi to characterize the genotypes in different patient cohorts. Fifty-seven isolates, collected from patients living in Malawi and the Netherlands, were classified by age and immune status of the hosts. Sequence analysis of the internal transcribed spacer (ITS) region identified 16 genotypes; nine have not previously been described. Genotypes K and D were most prevalent among patient groups, whereas genotype C was restricted to transplantation patients receiving immunosupressives and genotype B showed a predisposition toward patients living with HIV/AIDS. Different genotypes showed more dispersion among isolates from Malawi compared with those from the Netherlands. A constructed map estimating the genealogy of the ITS region reveals a dynamic evolutionary process between the genotypes.     

ITS1 intra-individual variability of Ascaris isolates from Brazil

The zoonotic potential of Ascaris infecting pigs has stimulated studies of molecular epidemiology with internal transcribed spacer 1 (ITS1) as the target. The aim of this study was to determine the value of Ascaris ITS1 as a molecular marker through assessing the intra-individual genetic diversity of Ascaris isolates from two geographical areas of Brazil. DNA was extracted from single isolated eggs, ITS1 PCR was performed, and the PCR products were cloned and sequenced. Clone analysis showed high ITS1 intra-individual variability revealed by 2–4 ITS1 genotypes/haplotypes per sample (egg). Two genotypes, G1 and G6, and 13 new haplotypes were detected and characterized. The most prevalent in humans, G1 and/or the Brazilian G6, were detected in all samples. Except for genotype G1, no relationship was observed between Brazilian ITS1 genotypes/haplotypes and those previously described in China, Bangladesh, Japan, United Kingdom, Australia, and Denmark, with respect to geographic origin or host affiliation. However, an association between the two geographically separated Brazilian ITS1 isolates was observed. The ITS1 intra-individual variability revealed in this study indicated that the use of this genetic region to discriminate human and pig Ascaris genotypes should be reconsidered.     

Genetic and historical evidence disagree on likely sources of the Atlantic amethyst gem clam Gemma gemma (Totten, 1834) in California

Aim Historical information about source populations of invasive species is often limited; therefore, genetic analyses are used. We compared inference about source populations from historical and genetic data for the oyster‐associated clam, Gemma gemma that invaded California from the USA Atlantic coast. Location Mid‐Atlantic (North Carolina, Maryland), Northeastern (New Jersey, New York, Massachusetts) and the California coasts (Elkhorn Slough, San Francisco Bay, Bolinas Lagoon, Tomales Bay, Bodega Harbor). Methods The documented history of transplantation of Eastern oysters to California was reviewed. Cytochrome c oxidase subunit I (COI) sequences from recent and archived clams were examined in a haplotype network. We used AMOVA to detect geographic genetic structure and a permutation test for significant reductions in diversity. Results Chesapeake Bay oysters were transplanted to New York prior to shipment to San Francisco Bay and from there to peripheral bays. Gemma in the Northeastern and Mid‐Atlantic regions were genetically differentiated. In California, populations in Bodega Harbor and Tomales Bay were genetically similar to those in the Mid‐Atlantic area while clams in San Francisco Bay, Elkhorn Slough and Bolinas Lagoon resembled populations in the Northeastern region. In California, genetic variation was not highest in San Francisco Bay despite greater magnitude of oyster plantings. Haplotypes varied over time in native and introduced populations. Main Conclusions Historical records and inferences from genetics agree that both Northeastern and Mid‐Atlantic regions were sources for Gemma in California. Only complex genetic hypotheses reconcile the strong segregation of haplotypes in California to the historical evidence of mixing in their proximate source (New York). These hypotheses include sorting of mixtures of haplotypes or selection in non‐native areas. Haplotype turnover in San Francisco and Massachusetts samples over time suggests that the sorting hypothesis is plausible. We suggest, however, that Gemma was introduced independently and recently to Tomales Bay and Bodega Harbor.     

Molecular epidemiology of anthrax cases associated with recreational use of animal hides and yarn in the United States

To determine potential links between the clinical isolate to animal products and their geographic origin, we genotyped (MLVA-8, MVLA-15, and canSNP analysis) 80 environmental and 12 clinical isolates and 2 clinical specimens from five cases of anthrax (California in 1976 [n = 1], New York in 2006 [n = 1], Connecticut in 2007 [n = 2], and New Hampshire in 2009[n = 1]) resulting from recreational handling of animal products. For the California case, four clinical isolates were identified as MLVA-8 genotype (GT) 76 and in the canSNP A.Br.Vollum lineage, which is consistent with the Pakistani origin of the yarn. Twenty eight of the California isolates were in the A.Br.Vollum canSNP lineage and one isolate was in the A.Br. 003/004 canSNP sub-group. All 52 isolates and both clinical specimens related to the New York and Connecticut cases were MLVA-8 GT 1. The animal products associated with the NY and CT cases were believed to originate from West Africa, but no isolates from this region are available to be genotyped for comparison. All isolates associated with the New Hampshire case were identical and had a new genotype (GT 149). Isolates from the NY, CT and NH cases diverge from the established canSNP phylogeny near the base of the A.Br.011/009. This report illustrates the power of the current genotyping methods and the dramatically different epidemiological conditions that can lead to infections (i.e., contamination by a single genotype versus widespread contamination of numerous genotypes). These cases illustrate the need to acquire and genotype global isolates so that accurate assignments can be made about isolate origins.     

Population structures of two genotypes of Vibrio vulnificus in oysters (Crassostrea virginica) and seawater

Vibrio vulnificus biotype 1 strains can be classified into two genotypes based on the PCR analysis of variations in the virulence-correlated gene (vcg). Genotype has been correlated with human infection for 90% of isolates from human cases having the vcgC sequence type and 87% of environmental strains having the vcgE variant. In this study we examined the dynamics of V. vulnificus populations and the distribution of the two genotypes recovered from oysters and surrounding estuarine wasters. Analysis of 880 isolates recovered from oysters showed a disparity in the ratio of the two genotypes, with those of the vcgE (E) genotype accounting for 84.4% of the population. In contrast, 292 isolates recovered from the waters surrounding the oyster sites revealed an almost equal distribution of the two genotypes. The levels of vcgC (C genotype) strains from both sources increased as a percentage of the population as water temperatures increased, while no culturable V. vulnificus cells were recovered from December through February. Our results suggest that there is a selective advantage for strains of the E genotype within oysters while survival of the C genotype strains may be favored by increased water column temperatures. These data suggest that the low incidence of infections may be due to the comparatively rare consumption of an oyster that contains a greater number of V. vulnificus vcgC genotype strains than of vcgE genotype strains. Levels of the two genotypes as well as seasonal dynamics within both oyster tissue and the surrounding waters may aid in identifying risk factors associated with human infection.     

Epizootiology and pathology of juvenile oyster disease in the Eastern oyster, Crassostrea virginica.

Juvenile Oyster Disease (JOD) causes mortalities of small cultured oysters, Crassostrea virginica. The present study was an intensive epizootiological and pathological investigation of JOD in eight sequentially deployed cohorts at sites on Long Island, New York. JOD symptoms and mortalities began in all groups at about the same time. Lesions on the mantle were detected histologically about 1 week before the principal symptom, a conchiolin deposit on the inner shell, appeared. Mortality began about 1 week later and reached 60-90% in oysters <25 mm. Mantle lesions were highly correlated with subsequent conchiolin-deposit prevalence and with total mortality. Larger juveniles (25-40 mm) were affected by the disease and produced conchiolin deposits, but mortalities did not exceed 30%. Mortalities were consistently related to size, but not necessarily to age or length of "exposure" in the field. There was no indication that JOD was linked to a particular broodstock or hatchery. Wild spat deployed at experimental sites showed JOD symptoms before the hatchery-produced groups did and cohorts maintained inside a hatchery experienced essentially no JOD. Histological examination of cohorts experiencing high mortalities failed to reveal an obvious etiological agent, but showed a disease pattern similar to that described for other bivalve diseases with a bacterial etiology. Similarities and differences between this and other studies of JOD suggest that one or more bacterial species is responsible for JOD, but that a trigger, probably temperature, is also involved and may vary from site to site.     

Evidence for Zoonotic Potential of Enterocytozoon bieneusi in Its First Molecular Characterization in Captive Mammals at Bangladesh National Zoo

To determine the occurrence and genotypes of Enterocytozoon bieneusi in captive mammals at Bangladesh National Zoo and to assess their zoonotic significance, 200 fecal samples from 32 mammalian species were examined using a nested PCR and sequencing of internal transcribed spacer (ITS) gene. Enterocytozoon bieneusi was detected in 16.5% (33/200) of the samples. Seven different ITS genotypes were identified, including two known genotypes (D and J) and five new ones (BAN4 to BAN8). Genotype D was the most common genotype being observed in 19 isolates. In phylogenetic analysis, four genotypes (D, BAN4, BAN5, and BAN6), detected in 30 isolates (90.9%), belonged to Group 1 having zoonotic potential. The sequence of genotype J found in a Malayan pangolin was clustered in so‐called ruminant‐specific Group 2. The other two genotypes BAN7 and BAN8 were clustered in primate‐specific Group 5. To our knowledge, this is the first report of molecular characterization of E. bieneusi in Bangladesh, particularly in captive‐bred wildlife in this country. The potentially zoonotic genotypes of E. bieneusi are maintained in zoo mammals that may transmit among these animals and to the humans through environmental contamination or contact.     

Metapopulation structure for perpetuation of Francisella tularensis tularensis

Background  

Outbreaks of Type A tularemia due to Francisella tularensis tularensis are typically sporadic and unstable, greatly hindering identification of the determinants of perpetuation and human risk. Martha's Vineyard, Massachusetts has experienced an outbreak of Type A tularemia which has persisted for 9 years. This unique situation has allowed us to conduct long-term eco-epidemiologic studies there. Our hypothesis is that the agent of Type A tularemia is perpetuated as a metapopulation, with many small isolated natural foci of transmission. During times of increased transmission, the foci would merge and a larger scale epizootic would occur, with greater likelihood that humans become exposed.     

The occurrence and rapid discrimination of Fomes fomentarius genotypes by ITS-RFLP analysis

Sequence comparison of available Fomes fomentarius (L.) J. Kickx f. internal transcribed spacer (ITS) of ribosomal DNA sequences demonstrated genetic non-homogeneity of the species. Multiple sequence alignment indicated the presence of two genotypes with overall similarity of about 97% and a strong statistics support. Rapid and reliable method for discrimination of F. fomentarius genotypes based on restriction digestion of polymerase chain reaction (PCR)-amplified ITS sequences was developed. BseNI and SchI restriction endonucleases were found to clearly discriminate between two F. fomentarius genotypes. The method was used to study the variability in F. fomentarius isolates collected from natural forest reserves in Vihorlat Mountains (East Slovakia). In most localities both genotypes occur concurrently. The isolates belonging to the genotype A were found to be prevalent on beech (Fagus sylvatica), while genotype B tends to be found mainly on other hosts. The grouping of selected isolates was confirmed by sequence analysis. Our results indicate that F. fomentarius includes at least two sympatric cryptic species.     

Genotyping of Escherichia coli from environmental and animal samples

The triplex PCR of Clermont et al. [Clermont, O., Bonacorsi, S., Bingen, E., 2000. Rapid and simple determination of the Escherichia coli phylogenetic groups. Appl. Environ. Microbiol. 66, 4555-4558.] was used to genotype E. coli isolates from the Mid-Atlantic region of the USA, obtained from freshwater, animal internal organs, and feces. Of 445 isolates subjected to genotyping, 118 isolates (26%) were genotype A, 111 (25%) genotype D, 140 (31%) genotype B1, and 76 (17%) genotype B2. All four genotypes were present in three sets of freshwater stream samples. When isolates from chicken cecal ingesta, cecal mucosa, and tracheal mucosa were screened, there was selective distribution of genotypes in these organs. Genotype D was rarely encountered in feces, milk, and intestinal tissues of dairy cows, while all four genotypes were represented in goose feces. Isolates from the feces of zoo animals reared in the US demonstrated a predominance of genotype B1. Thirty-six of the A isolates in our overall collection were subgenotype A(0), in which none of the three amplicons are observed; confirmation that these isolates were E. coli was done using an ancillary lacZ PCR assay. We conclude that the genotyping triplex PCR assay, used in combination with traditional culture methods, can be useful in categorizing E. coli from environmental and veterinary sources in the Mid-Atlantic region of the USA.