Prospects For the Use of Loop Isothermal Amplification in the Diagnosis of Particularly Dangerous Infectious Diseases Caused by the Viruses of the Pathogenicity Group I

Dangerous viral infectious diseases pose a serious threat to human life and health, as their uncontrolled spread leads to the development of major outbreaks and epidemics. Rapid and accurate detection of the pathogen is an essential component of the fight against infectious diseases. This review is devoted to loop-mediated isothermal amplification (LAMP), which is one of the simplest and most reliable methods of molecular-genetic research that meets modern requirements. The simplicity of the analysis and registration of the obtained results, which is necessary under conditions with minimal laboratory capacities, makes it possible to consider this type of diagnostic technology as the most promising, which allows us to identify genetic markers (DNA or RNA) of pathogens of dangerous infectious diseases in the shortest possible time. Objective of the review is to summarize and systematize the data available to date on the use of LAMP for detecting RNA of dangerous infectious diseases caused by the Ebola,Marburg and Lassa viruses. The paper discusses the basic principles of the loop isothermal amplification reaction, the components that make up the reaction mixture and are used for the analysis, as well as methods for detecting the results obtained. When studying the information available in the literature sources about the advantages and disadvantages of LAMP, it is shown that in many cases, isothermal amplification is not inferior in sensitivity and specificity to the main molecular-genetic diagnostic methods currently used. Modifications that can be used for accelerated diagnostics of RNA-containing viruses are also considered.

Pathotype Grouping of Cercospora sojina Isolates on Soybean and Sensitivity to QoI Fungicides

Frogeye leaf spot (FLS), caused by Cercospora sojina, is a common disease of soybean in the southern and northern United States and causes significant yield loss. The use of the current race scheme for classification for C. sojina does not take into account the range of disease severity reactions within each differential. The objective of this research was to better understand the diversity among C. sojina isolates through the development and use of pathogenicity groups. In this study, 83 isolates acquired from 2006 to 2009 were screened using 12 soybean (Glycine max) differentials. Disease severity on the 12 differentials ranged from 0 to 9, where 0 is immune and 9 is very susceptible. The average severity for each isolate across differentials ranged from 1 to 7. The 83 isolates were grouped into five pathogenicity groups (PG): PG1, PG2, PG3, PG4, and PG5, reflecting the severity grouping. Using the 12 differentials, PG1 isolates were differentiated by the lack of infection on Davis, Peking, Kent, Palmetto, Hood, CNS, Tracy, and Richland. PG2 had a range of infections on a scale of 1 to 2 on all differentials except on Davis; PG3 isolates had severity ranging from 3 to 4 except on Davis. PG4 isolates caused no infection on Davis, a maximum disease severity of 5 on Peking, while the rest of differentials had severities from 5 to 6. PG5 isolates caused no infection on Davis, severity of 7 on CNS, and severity of 8 on Kent, Hood, and Palmetto. The remaining seven differentials had severities of 9. Across the geographical locations, the predominant pathotypes were PG3 and PG4 and represented 84% of the tested isolates. Azoxystrobin fungicide sensitivity tests showed that 88% of the isolates were sensitive and dominated the population, while only 6% had a high level of fungicide resistance, suggesting that FLS resistance to the QoI fungicide group was not yet completely developed and had not spread to other areas at the time when these isolates were acquired. The overall virulence profile of the isolates indicated that there was variation in disease severity, suggesting that selection of resistance for each PG may produce lines with more precisely defined interactions to specific pathotypes of C. sojina. This may improve the screening and selection of useful resistance genes that could be pyramided for resistance to each pathogenicity group.

Identification of Sources of Resistance to Blackleg (Leptosphaeria maculans) in a Brassica napus Germplasm Collection

The increased prevalence of strains of pathogenicity group 4 of Leptosphaeria maculans, causal agent of blackleg, represents a serious threat to the canola (Brassica napus L.) industry in North Dakota, a state that contributes >85% of U.S. canola production. The objective of this study was to identify sources of resistance to PG-4 in a collection of 559 B. napus plant introduction materials (PIs). Replicated trials were conducted twice in the greenhouse using a mixture of five PG-4 isolates to evaluate the reaction at seedling stage; then, an elite group of PIs was evaluated in replicated field trials between 2014 and 2016 at Langdon, ND, using a combination of lab-produced inoculum and blackleg-infested canola residues. Combined analysis of field data indicated three PIs had on average 73 to 80% less (P < 0.05) internal stem tissue discoloration than the commercial hybrids used as the controls. These PIs could be used in canola breeding programs as good sources of resistance against PG-4; in addition, efforts should be made to characterize the resistance they carry.

Effect of urethral meatus cleansing on midstream urine contamination rate in boys

Background Clean-catch midstream urine (MSU) remains thestandard procedure for urine collection even if it's role to reducebacterial contamination rate is unclear.Objective To compare bacterial contamination rate betweenclean-catch (cleaning urethral meatus with medicated soap) andnon clean-catch MSU among boys.Methods An experimental study with parallel groups and blockrandomization was conducted. Toilet-trained boys aged 3 to 18years, without symptoms or signs of urinary tract infection wererecruited from the Pediatric Outpatient Clinic at Sardjito Hospitaland from a local elementary school. Subjects with history of renaldisease, those who were on under antibiotic treatment in thepreceding week, or with meatal abnormality or non-cooperativewere excluded. Urine specimen was collected by a trained nurse,and was cultured within one hour by personnel blinded to theassignment. Significant bacteriuria was defined as growth of asingle pathogenic organism (degree of pathogenicity group I-III)with colony count 2: 105 colony forming unit/mi. Contaminationwas defined as any growth not fulfilling criteria for significantbacteriuria or growth of multiple organisms.Results A total of 80 boys were enrolled. The contamination ratein the clean-catch group was 13% (5 out of 40) compared with10% (4 out of 40) in the non clean-catch group (P=l.O). Theadjusted risk ratio for contamination in the clean-catch MSUgroup, adjusted to age and circumcision status, was 1.37 (95%CI 0.42; 4.51).Conclusion Clean-catch method does not reduce bacterial con-tamination rate of midstream urine cultures in boys

Identification of Leptosphaeria maculans Pathogenicity Group 4 Causing Blackleg on Winter Canola in Oklahoma

Winter canola (Brassica napus L.) is a relatively new crop to Oklahoma and other southern U.S. states where it is considered a desirable rotation crop with wheat. In 2009, approximately 15,000 ha of winter canola were harvested in Oklahoma (3); that area is expected to almost double in 2010. Blackleg, a potentially devastating canola disease, was detected in Oklahoma in 2009. Blackleg is caused by Leptosphaeria maculans (Desmaz.) Ces. & de Not (anamorph = Phoma lingam (Tode:Fr.) Desmaz.). In early 2010, leaf samples showing typical symptoms of blackleg were collected from four canola fields near the town of Enid in Garfield County, OK. Small portions of infected tissues were surface disinfested in an aqueous solution of NaOCl (0.5% a.i.) for 1 min, rinsed twice in sterile distilled water, and plated on V8 medium. Seven colonies were isolated and when grown in pure culture, all produced 2 × 4.5 μm guttulate, unicellular, hyaline spores in pycnidia that ranged from 200 to 480 μm in diameter. These morphological characteristics correspond with those of P. lingam (1). To verify the pathogenic nature of the isolates and establish the pathogenicity group (PG) to which they belong, a standard inoculation protocol was followed on a set of three differential cultivars, Quinta, Glacier, and Westar (2). Briefly, for each isolate, tiny puncture wounds were made with sterile needles on the cotyledons of six 10-day-old plants of each differential and a 10-μl aliquot of a pycnidiospore suspension (1 × 107 spores ml–1) was deposited on the wounds. Also, a set of differentials were inoculated with distilled water (mock inoculation). Inoculated plants were incubated overnight in a misting chamber at 21°C in the dark and returned the next day to the greenhouse. Disease severity was recorded 10 days after inoculation using a 0 to 9 scale in which 0 to 2 = resistant, 3 to 6 = intermediate, and 7 to 9 = susceptible. This process was repeated three times. Two of the seven isolates evaluated were highly virulent on all three differentials, an indication they belong to pathogenicity group 4 (2). The other five isolates produced small lesions on Westar (resistant reaction) but failed to develop symptoms on the other two differentials. This phenotypic reaction has been associated with strains of PG-1. Mock-inoculated plants did not develop lesions. To our knowledge, this is the first time blackleg isolates from Oklahoma have been identified to the PG level. While this information will assist breeders in the development of both spring and winter canola lines with resistance to blackleg, additional studies are necessary to determine the relative prevalence and diversity of the various PG in Oklahoma. References: (1) G. H. Boerema. Trans. Br. Mycol. Soc. 67:289, 1976. (2) A. Mengistu et al. Plant Dis. 75:1279, 1991. (3) USDA. National Agricultural Statistics Service. Retrieved from http://www.nass.usda.gov/Statistics_by_State/Ag_Overview/ AgOverview_OK.pdf , September 20, 2010.

Sanitary Protection of the Territory from Importation and Spread of Particularly Dangerous Infections. Communication 6. Argentine and Bolivian Hemorrhagic Fevers

Presented are the results of examination of Argentine and Bolivian hemorrhagic fevers in accordance with previously proposed categories, signs and criteria of particularly dangerous viral infections (PDVI), actual for sanitary protection of the territory. Argentine and Bolivian hemorrhagic fevers are contagious PDVI of pathogenicity group I, capable of epidemic spread. Anti-epidemic measures are necessary in case of Argentine and Bolivian hemorrhagic fevers importation onto non-endemic territory to prevent the epidemiologic complications.

Current State of Collection Activity Relative to the Use of Infectious Agents of I-II Pathogenicity Groups

Subject of present survey is the regulatory background specifying collection activity relative to the use of infectious agents of I-II pathogenicity group. Revised are the preferred activities which are to be realized by the leading collections of pathogenic microorganisms. Suggested are promising approaches for the improvement of the work of collection centers that are to be solved at the modern organizational, legal, technical and scientific-methodological level.

Development of a SCAR Marker to Track Canola Resistance Against Blackleg Caused by Leptosphaeria maculans Pathogenicity Group 3

Blackleg of rapeseed and canola (Brassica napus) is caused by various pathogenicity groups (PG) of Leptosphaeria maculans. The disease occurring in the Canadian prairies for the last two decades was caused by PG2 and was controlled by host resistance. PG3 and PG4 isolates have been found recently in Canada, but there is no resistance available against these pathogenicity groups in commercial Canadian varieties. This study sought to identify canola cultivars that could be used as sources of resistance to PG3 and to develop molecular markers for marker-assisted selection. Resistance to PG3 specifically was found in B. napus ‘Dunkeld’ and ‘Quinta’, while B. juncea ‘Cutlass’ and ‘Domo’ proved to be resistant to PG2, PG3, and PG4. A set of F2 progeny of ‘Westar’ (susceptible) × ‘Dunkeld’ was used to identify genetic markers linked to PG3 resistance. These markers were physically located on a BAC clone from B. rapa subsp. pekinensis containing a homolog to a serine threonine 20 (ste20)-like kinase in Arabidopsis thaliana. Thus, we have developed a sequence characterized amplified region (SCAR) marker available for marker-assisted selection in breeding canola for resistance against blackleg caused by L. maculans PG3. This work has received a provisional patent (serial # 60/977,933 – Oct. 5, 2007).