Plasmodiophora brassicae in Mexico, from anecdote to fact

For years, the presence of clubroot disease and its causal agent, Plasmodiophora brassicae, in Mexico has been given by granted. However, after a long search in the scientific literature in English and Spanish, as well as grey literature including thesis and government reports, we were not able to find any information regarding the actual detection of the pathogen, hosts affected, areas with the disease, or any real information about clubroot ('hernia de la col', in Mexico). To confirm if P. brassicae was indeed in Mexico, we started a true detective adventure. First, we identified agricultural communities in south-east Mexico known to grow cruciferous crops. Second, we asked to the growers if they have ever seen clubroot symptoms, showing them during the inquires pictures of the characteristic galls that might have been present in their crops. Third, we collected soil from two of the communities with positive response and grew an array of cruciferous in the soil as baits to 'fish' the clubroot pathogen. We detected the presence of galls in the roots of 32 plants and observed the presence of resting spores. Through a P. brassicae specific PCR assay, we were able to confirm the presence of the clubroot pathogen in the samples and in Mexico for the very first time. This study is the first report and identification of P. brassicae in Mexico, opening the doors to understand the genetic diversity of this elusive and devastating plant pathogen.

Species-specific PCR assay for the detection of Babesia odocoilei

We developed a PCR assay for the detection of Babesia odocoilei based on the 18S rRNA gene. Multiple specimens of B. odocoilei were examined, and the assay consistently produced a small specific PCR product of 306 bp. The PCR assay was also challenged with DNA from 13 other Babesia species and 2 Theileria species, originating from 10 different host species; however, nonspecific DNA amplification and multiple banding patterns were observed, and the amplicon banding patterns varied between different isolates of the same species. Sensitivity was determined to be 6.4 pg of DNA, and an estimated 0.0001% parasitism. This assay can be utilized for species-specific differential detection of B. odocoilei.

Are members of the Anopheles fluviatilis complex conspecific?

Anopheles fluviatilis sensu lato, a primary malaria vector in India, was identified to be comprised of four cryptic species, provisionally designated as species S, T, U and V. However, Kumar et al. (Mol Ecol Resour, 2013;13:354-61) considered all of the then known three members of this species complex (S, T and U) conspecific. The specific status of species S and T was refuted based on the lack of sufficient barcode gap in mitochondrial-CO1 and the perceived presence of heterozygotes in populations as detected through one of the two species-specific PCR assays employed for the cryptic species identification. The existence of species U was refuted claiming that earlier investigations have already refuted their existence. This conclusion is concerning because of the differential public health implications of members of the Fluviatilis Complex. Here we discuss problems associated with the CO1-based barcode approach for delimitation of cryptic species, the perceived heterozygosity between species S and T based on a species-specific PCR assay, and interpretation of published reports. We demonstrated that fixed differences do exist in the ITS2-rDNA sequence of species S and T with no evidence of heterozygotes in sympatric populations and, that the observed heterozygosity by Kumar et al. in the ITS2-based species diagnostic PCR is due to the high mispriming tendency of the T-specific primer with species S. We infer that mitochondrial DNA-based barcoding-gap, an arbitrary threshold recommended for species delimitation, alone, is inadequate to delimit the members of An. fluviatilis complex.

A New Specific Sequence to Distinguish B.canis From Other Brucella by PCR

Background: Brucellosis is a zoonotic disease worldwide. The increasing number of pet dogs has raised new risk of people getting canine brucella with absent or mild symptom. Besides, the canine brucellosis can be caused by other brucella species, so their infection could be omitted by the PCR method. The present PCR methods can only detect canine brucella, by which cases infected with other brucella would appear negative. It’s an urge to develop a specific PCR assay for detecting canine Brucellosis, Whether the pathogen was B.canis or any other Brucella.Resaults: a differential sequence of B.canis were found by genome comparison analysis and were analyzed by BLAST. Then a PCR method was established using specific primers in the sequence and tested for clinical application. It could detect canine brucellosis caused by B.canis or other Brucella species with 310-bp and 413-bp product, respectively. The developed PCR method had specificity for non-brucella and a sensitivity of 100 copies of Brucella DNA. The detection accuracy verified with spiked samples was 95.5% (21/22) for B.canis and 100% (22/22) for other brucella. Conclusions: The study found a specific sequence of B.canis and developed a PCR detection method to detect canine brucellosis caused by B.canis or other Brucella species. The method established in this study will more comprehensively detect the pathogen of canine brucellosis and provide important methods and means for preventing and controlling this disease.

PharmFrag: An Easy and Fast Multiplex Pharmacogenetics Assay to Simultaneously Analyze 9 Genetic Polymorphisms Involved in Response Variability of Anticancer Drugs

Regarding several cytotoxic agents, it was evidenced that genetic polymorphisms in genes encoding enzymes involved in their metabolism are associated with higher risk of toxicity. Genotyping these genes before treatment is a valuable strategy to prevent side effects and to predict individual response to drug therapy. This pharmacogenetic approach is recommended for chemotherapies such as thiopurines (azathioprine, 6-mercaptopurine, thioguanine), irinotecan, and fluoropyrimidines (capecitabine and 5-fluorouracil). In this study, we aimed at developing and validating a fast, cost-effective, and easily implementable multiplex genotyping method suitable for analyzing a panel of nine variants involved in the pharmacogenetics of widely prescribed anticancer drugs. We designed a multiplex-specific PCR assay where fragments were labeled by two different fluorescent dye markers (HEX/FAM) identifiable by fragment analysis. These two labels were used to discriminate bi-allelic variants, while the size of the fragment allowed the identification of a particular polymorphism location. Variants of interest were TPMT (rs1800462, rs1142345, rs1800460), NUDT15 (rs116855232), DPYD (rs55886062, rs3918290, rs67376798, rs75017182), and UGT1A1 (rs8175347). The assay was repeatable, and genotypes could be determined when DNA sample amounts ranged from 25 to 100 ng. Primers and dye remained stable in a ready-to-use mixture solution after five freeze–thaw cycles. Accuracy was evidenced by the consistency of 187 genotyping results obtained with our multiplex assay and a reference method. The developed method is fast and cost-effective in simultaneously identifying nine variants involved in the pharmacological response of anticancer drugs. This assay can be easily implemented in laboratories for widespread access to pharmacogenetics in clinical practice.

Improvement of New PCR Assay Targeting Mutations for Fast Recognition of Azole Resistant Mould Fungus, Aspergillus fumigatus

Allergenic mould fungus, Aspergillus fumigatus well known for its invasive aspergillosis disease is intrinsically resistant to most of the fluconazole group of medicines. It was studied that the mutations encoding 14a-sterol demethylase has the capacity to make triazole-resistant to this clinical fungal isolates. The present study is a multiplex allele-specific PCR assay targeting mutations for rapid detection of azole-resistant A. fumigatus for suitable patient management. During the present work clinical and wild isolates of A. fumigatus were employed to evaluate MAS-PCR, which was confirmed by direct DNA sequencing of cyp5 IA98 region from selected isolates of A. fumigatus. MAS-PCR assay was successfully developed to simplify the rapid detection of L98H mutation in cyp51A gene that confers the resistance to triazoles in A. fumigatus strains. The assay would help in primary acknowledgement of triazole-resistant A. fumigatus strains for proper management of patients with invasive aspergillosis.

Recombination between African and Asian lineages of Zika virus in vitro and its consequences for viral phenotype

Recombination is a process of extensive genetic exchange that is known to contribute to virus evolution and has been frequently observed in positive-sense RNA viruses. Zika virus (ZIKV) is an emerging arbovirus of the family Flaviviridae with two distinct lineages – African and Asian. While some phylogenetic evidence suggests that recombination in the envelope-encoding region of the ZIKV genome has occurred during evolution, there has been no experimental evidence for ZIKV recombination to date. We conducted co-infections of mammalian and insect cells, using the prototype African ZIKV strain (MR766) and an Asian isolate from the 2015-16 ZIKV outbreak in Brazil (BeH819015), and used a recombinant-specific PCR assay to detect recombinant sequences from total cell RNA extracts. In brief, a 564bp fragment spanning the boundary between the structural and the non-structural genes of the viral genome was amplified using a primer pair consisting of an Asian-specific and an African-specific primer. A total of 24 individual sequences were screened. All were in-frame recombinants and they formed 10 unique junctions. Several of the detected recombinant sequences were chosen for construction of full-length infectious clones to test the viability and phenotype of the recombinant viruses. This study represents the first isolation of recombinant ZIKV sequences from co-infected cultured cells and demonstrates the capacity of ZIKV to recombine in an experimental system. Further investigation is required to better understand the evolutionary potential of this mechanism and its putative role in the emergence of ZIKV.

Characterization of nontypeable Actinobacillus pleuropneumoniae isolates

Two Actinobacillus pleuropneumoniae isolates from clinical cases of porcine pleuropneumonia in Japan were positive in the capsular serovar 15–specific PCR assay, but nontypeable (NT) in the agar gel precipitation (AGP) test. Nucleotide sequence analysis of gene clusters involved in the biosynthesis of capsular polysaccharide (CPS) and lipopolysaccharide O-polysaccharide (O-PS) revealed that both clusters contained transposable element IS Apl1 of A. pleuropneumoniae belonging to the IS30 family. Immunoblot analysis revealed that these 2 isolates could not produce O-PS. We conclude that the IS Apl1 of A. pleuropneumoniae can interfere in the biosynthesis of both CPS and O-PS.

Genetically Distinct Acidovorax citrulli Strains Display Cucurbit Fruit Preference Under Field Conditions

Strains of Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) of cucurbits, can be assigned to two groups, I and II. The natural association of group I and II strains with different cucurbit species suggests host preference; however, there are no direct data to support this hypothesis under field conditions. Hence, the objective of this study was to assess differences in the prevalence of group I and II A. citrulli strains on cucurbit species in the field. From 2017 to 2019, we used group I and II strains to initiate BFB outbreaks in field plots planted with four cucurbit species. At different times, we collected symptomatic tissues and assayed them for group I and II strains using a group-specific PCR assay. Binary distribution data analysis revealed that the odds of melon, pumpkin, and squash foliage infection by group I strains were 21.7, 11.5, and 22.1 times greater, respectively, than the odds of watermelon foliage infection by the group I strain (P < 0.0001). More strikingly, the odds of melon fruit infection by the group I strain were 97.5 times greater than watermelon fruit infection by the same strain (P < 0.0001). Unexpectedly, some of the group II isolates recovered from the 2017 and 2019 studies were different from the group II strains used as inocula. Overall, data from these experiments confirm that A. citrulli strains exhibit a preference for watermelon and melon, which is more pronounced in fruit tissues.