Best Practices for Population Genetic Analyses.

Population genetic analysis is a powerful tool to understand how pathogens emerge and adapt. However, determining the genetic structure of populations requires complex knowledge on a range of subtle skills that are often not explicitly stated in book chapters or review articles on population genetics. What is a good sampling strategy? How many isolates should I sample? How do I include positive and negative controls in my molecular assays? What marker system should I use? This review will attempt to address many of these practical questions that are often not readily answered from reading books or reviews on the topic, but emerge from discussions with colleagues and from practical experience. A further complication for microbial or pathogen populations is the frequent observation of clonality or partial clonality. Clonality invariably makes analyses of population data difficult because many assumptions underlying the theory from which analysis methods were derived are often violated. This review provides practical guidance on how to navigate through the complex web of data analyses of pathogens that may violate typical population genetics assumptions. We also provide resources and examples for analysis in the R programming environment.

Genomic Analyses of Dominant U.S. Clonal Lineages of Phytophthora infestans Reveals a Shared Common Ancestry for Clonal Lineages US11 and US18 and a Lack of Recently Shared Ancestry Among All Other U.S. Lineages.

Populations of the potato and tomato late-blight pathogen Phytophthora infestans are well known for emerging as novel clonal lineages. These successions of dominant clones have historically been named US1 through US24, in order of appearance, since their first characterization using molecular markers. Hypothetically, these lineages can emerge through divergence from other U.S. lineages, recombination among lineages, or as novel, independent lineages originating outside the United States. We tested for the presence of phylogenetic relationships among U.S. lineages using a population of 31 whole-genome sequences, including dominant U.S. clonal lineages as well as available samples from global populations. We analyzed ancestry of the whole mitochondrial genome and samples of nuclear loci, including supercontigs 1.1 and 1.5 as well as several previously characterized coding regions. We found support for a shared ancestry among lineages US11 and US18 from the mitochondrial genome as well as from one nuclear haplotype on each supercontig analyzed. The other nuclear haplotype from each sample assorted independently, indicating an independent ancestry. We found no support for emergence of any other of the U.S. lineages from a common ancestor shared with the other U.S. lineages. Each of the U.S. clonal lineages fit a model where populations of new clonal lineages emerge via migration from a source population that is sexual in nature and potentially located in central Mexico or elsewhere. This work provides novel insights into patterns of emergence of clonal lineages in plant pathogen genomes.

Loop-mediated isothermal amplification for detection of the tomato and potato late blight pathogen, Phytophthora infestans.

AIMS: To design and validate a colorimetric loop-mediated isothermal amplification assay for rapid detection of Phytophthora infestans DNA. METHODS AND RESULTS: Two sets of loop-mediated isothermal amplification (LAMP) primers were designed and evaluated for their sensitivity and specificity for P. infestans. ITSII primers targeted a portion of the internal transcribed spacer region of ribosomal DNA. These primers had a limit of detection of 2 pg P. infestans DNA and cross-reacted with the closely related species Phytophthora nicotianae. Rgn86_2 primers, designed to improve assay specificity, targeted a portion of a conserved hypothetical protein. These primers had a limit of detection of 200 pg P. infestans DNA and did not cross-react with P. nicotianae. The specificity of the Rgn86_2 assay was tested further using the closely related species P. andina, P. ipomoeae, P. mirabilis and P. phaseoli. Cross-reactions occurred with P. andina and P. mirabilis, but neither species occurs on tomato or potato. Both primer sets were able to detect P. infestans DNA extracted from tomato late blight leaf lesions. CONCLUSIONS: Two colorimetric LAMP assays detected P. infestans DNA from pure cultures as well as infected leaf tissue. The ITSII primers had higher sensitivity, and the Rgn86_2 primers had higher specificity. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a LAMP assay for the detection of P. infestans, the causal organism of potato and tomato late blight. These assays have potential for immediate utility in plant disease research and diagnostic laboratories.

Diversity of Foliar Phytophthora Species on Rhododendron in Oregon Nurseries.

The genus Phytophthora contains some of the most notorious plant pathogens affecting nursery crops. Given the recent emergence of the sudden oak death pathogen Phytophthora ramorum, particularly in association with Rhododendron spp., characterization of Phytophthora communities associated with this host in nursery environments is prudent. Many taxa may present symptoms similar to P. ramorum but we do not necessarily know their identity, frequency, and importance. Here, we present a survey of Phytophthora taxa observed from seven nurseries in the U.S. state of Oregon. Incidence and diversity of Phytophthora communities differed significantly among nurseries and among seasons within nursery. The taxa P. syringae and P. plurivora were widespread and detected at most of the nurseries sampled. Nine other taxa were also detected but were found either in a single nursery or were shared among only a few nurseries. Characterization of the Phytophthora communities present in nurseries is an important step toward understanding the ecology of these organisms as well as an aid to nursery managers in determining what risks may be present when symptomatic plants are observed. This study builds on an increasing literature, which characterizes Phytophthora community structure in nurseries.

Multiplexed microsatellite recovery using massively parallel sequencing.

Conservation and management of natural populations requires accurate and inexpensive genotyping methods. Traditional microsatellite, or simple sequence repeat (SSR), marker analysis remains a popular genotyping method because of the comparatively low cost of marker development, ease of analysis and high power of genotype discrimination. With the availability of massively parallel sequencing (MPS), it is now possible to sequence microsatellite-enriched genomic libraries in multiplex pools. To test this approach, we prepared seven microsatellite-enriched, barcoded genomic libraries from diverse taxa (two conifer trees, five birds) and sequenced these on one lane of the Illumina Genome Analyzer using paired-end 80-bp reads. In this experiment, we screened 6.1 million sequences and identified 356,958 unique microreads that contained di- or trinucleotide microsatellites. Examination of four species shows that our conversion rate from raw sequences to polymorphic markers compares favourably to Sanger- and 454-based methods. The advantage of multiplexed MPS is that the staggering capacity of modern microread sequencing is spread across many libraries; this reduces sample preparation and sequencing costs to less than $400 (USD) per species. This price is sufficiently low that microsatellite libraries could be prepared and sequenced for all 1373 organisms listed as 'threatened' and 'endangered' in the United States for under $0.5 M (USD).

[Epidemiological findings of Toxoplasma gondii infections of humans in the area of Cottbus]. / Epidemiologische Befunde zur Toxoplasma gondii-Infektion des Menschen im Raum Cottbus.

Using the indirect fluorescence antibody test (IFAT) results concerning the infection of people by Toxoplasma in dependence on age, sex, settlement area and consumption of raw meat in the region of (State Brandenburg) Cottbus are represented as well as on frequency of primoinfections during pregnancy. Infections by Toxoplasma have been found out in 35.6 of 100 cases (n = 3665). With 39.2% women are infected more often than men with 27.1%. Probands of the urbane settlement area show with 37.9% (n = 3402) an higher degree of the infections than probands living in the country with 30.9% (n = 2656). Consumption of raw meat increases the risk of an infection significant. The frequency of primoinfections - found by prospective study with 22559 pregnant women - is placed by 6%, that of connatale infections by 2.6%.

[Head lice infestation in the Cottbus district]. / Untersuchungen zum Kopflausbefall im Bezirk Cottbus.

670 children of infant-schools and 109 children of schools were examined for head-lice. Also the existence of nits was valued. The identified infestation of 9.9% is to stimulate examinations in other districts of GDR and measures to form the search and the control of these ectoparasites with more effects.

[Toxoplasma gondii infections and oocyst shedding in domestic cats and the significance of this for the epidemiology and epizootiology of toxoplasmosis]. / Toxoplasma gondii-Infektionen und Oozystenausscheidung bei Hauskatzen und ihre Bedeutung für die Epidemiologie und Epizootiologie der Toxoplasmose.

Because of the dissemination of oocysts the cat and other felines have a central position within Toxoplasma gondii epidemiology and epizootiology. From 1984 to 1986, 267 cats between 2 months and 15 years old were serologically examined (SFT) for Toxoplasma antibodies. The rate of infection was 59.9 per cent. The infection rate of stray cats (72.5%) was significantly higher than that of domestic cats (40%). The high infection rate of herbivores results from behaviour of stray cats. In faeces of 264 animals Toxoplasma oocysts could not be proved by flotation technique. But in 39 samples of faeces Toxocara mystax eggs were found, in one sample Toxascaris leonina eggs. Coproscopical examinations did not reflect the real degree of parasitization of cats. The rate of disseminators of oocysts is drainable by Toxoplasma gondii infection found serologically. About 40% of the animals at the age of up to one year were found to excrete Toxoplasma oocysts.

[The effect of anthelmintics on the viability of Taenia saginata oncospheres]. / Uber den Einfluss von Anthelminthika auf die Lebensfähigkeit von Taenia saginata-Onkosphären.

The viability of taeniid eggs has been investigated by means of methylene blue staining. In patients with niclosamide treatment the degree of dead taeniid eggs was 90-100% (19 of 25 cases). In patients without therapy the taeniid eggs were vital in 22 of 25 cases. Therefore recognition and treatment of all taeniid hosts is a way to reduce the taeniosis-cysticercosis extent.

[Cryptosporidium detection in humans]. / Kryptosporidiennachweis beim Menschen.

Cryptosporidium is known as an important cause of gastroenteritis in several animal species, especially in newborn calves and lambs. In our investigations we tested faeces samples of 78 diarrhoeic patients by the method of Ziehl-Neelsen modified after Henriksen. We could find Cryptosporidium oocysts in a boy of one and a half year. Cryptosporidia also should be considered as a cause of diarrhoeas in man.

[Larva migrans visceralis--occurrence of ascarid eggs in sandboxes in the East German district city of Cottbus]. / Larva migrans visceralis--Vorkommen von Ascarideneiern in Spielsandkästen der DDR-Bezirksstadt Cottbus.

Between 1982 and 1985 18 sand-pits were examined for Toxocara canis eggs. Eggs were found in 4 sand-pits. This implies a risk of larva migrans visceralis infection for children playing in these sand-pits. Some measures for diminishing the contamination are recommended.

[Larva migrans visceralis--occurrence of Toxocara canis in dogs in the East German district of Cottbus]. / Larva migrans visceralis--Vorkommen von Toxocara canis bei Hunden im DDR-Bezirk Cottbus.

Larvae of the nematode genus Toxocara are the main causative agents of the L.m.v. syndrom. 5,002 dogs from the Cottbus district aged between 2 and 36 months were examined. 994 animals (19.9%) were found to be infected with Toxocara canis. Dogs from a rural area have a higher infection rate (25.6%) than dogs from an urban area (15.2%). The relatively high infection rate of dogs, high egg-production and the tenacity of the development stages are factors dangerous to human health.

[Value of microprecipitation in agar (MPA) in the diagnosis of human toxoplasmosis]. / Untersuchungen zum Wert der Mikropräzipitation im Agar (MPA) bei der Diagnostik der menschlichen Toxoplasmose.

Results of researches on the value of the MPA for the diagnosis of toxoplasmosis are explained. They are based upon epicritical reports of toxoplasmosis cases and comparison researches by means of IFAR. The author supposes the hypothesis that the MPA is able to point out the "nonsterile immunity" of Toxoplasma infection.