Virulent Group Research Articles

Molecular characterization of circulating infectious bursal disease viruses in chickens from different Egyptian governorates during 2023

Infectious bursal disease virus (IBDV) induces severe immunosuppression in chickens, leading to significant economic losses in the global poultry industry. This study investigated 52 chicken flocks, including commercial broilers, layers, and baladi, from various Egyptian governorates in 2023. These flocks exhibited symptoms of depression, along with kidney and bursa lesions, indicative of IBDV infection. Pooled Bursal homogenates were tested using RT-PCR with VP2-specific primers, revealing that 20 flocks tested positive for IBDV. Six representative samples were selected from 20 positive flocks for isolation in embryonated chicken eggs. The embryonic lesions observed included haemorrhage, skull swelling, and liver necrosis with a pale-yellow appearance, in addition to congestion and thickening in the chorioallantoic membrane (CAM). Partial amplification of the VP2 gene from the harvested embryo suspensions of the six IBDV isolates was performed for sequencing. Phylogenetic analysis of the sequences revealed that five IBDV isolates (VV4, VV5, VV6, VV10, and VV16) belonged to the very virulent strain group A3 cluster, whereas one isolate (VV2) clustered with Chinese Variant strains in the A2d group. Sequence analysis of the hypervariable region (HVR) of VP2 compared to that of Egypt-USC-IBD-1-2019 and vvIBDV/Beh21/Egypt/18 highly virulent IBDV strains revealed several amino acid mutations. The VP2 HVR of all isolates maintained the serine-rich heptapeptide sequence SWSASGS, which is adjacent to the major hydrophilic peak B and serves as a virulence marker. Histopathological examination revealed that bursae from chickens infected with vvIBDV exhibited marked interlobular oedema and lymphoid depletion. In contrast, bursae from chickens infected with Variant IBDV showed massive lymphoid depletion, with hyperplasia of the bursal capsule. These findings highlight the circulation of both virulent and Variant IBDV strains in Egyptian chicken flocks, complicating disease control. Consequently, there is a need to update vaccination programs and vaccine strains for IBDV in Egypt.

Phylogenetic Diversity, Antibiotic Resistance, and Virulence of Escherichia coli Strains from Urinary Tract Infections in Algeria.

Urinary tract infections (UTIs) caused by Escherichia coli represent a significant public health concern due to the high virulence and antimicrobial resistance exhibited by these pathogens. This study aimed to analyze the phylogenetic diversity and antibiotic resistance profiles of Uropathogenic E. coli (UPEC) strains isolated from UTI patients in Algeria, focusing on virulence factors such as extended β-lactamase (ESBL) production, biofilm formation, and hemolytic activity. Phylogenetic grouping of 86 clinical imipenem resistant E. coli isolates showed the prevalence of group B2 (48.9%), followed by groups E (22.1%), unknown (12.8%), A (8.1%), and B1 (4.7%), and Clade I, D, Clade I, or Clade II (1.2%). The highest resistance rates were observed towards amoxicillin (86.04%), ticarcillin (82.55%), piperacillin (73.25%), nitrofurantoin (84.88%), and trimethoprim-sulfamethoxazole (51.16%). Notably, 69.8% of UPEC strains were multidrug-resistant (MDR) and 23.2% were extensively drug-resistant (XDR). Additionally, 48.9%, 42%, and 71% of strains demonstrated ESBL production, hemolytic activity, and weak biofilm production, respectively. Continuous monitoring and characterization of UPEC strains are essential to track the spread of the most resistant and virulent phylogenetic groups over time, facilitating rapid therapeutic decisions to treat infections and prevent the emergence of new resistant organisms, helping choose the most effective antibiotics and reducing treatment failure.

Treated wastewater: A hotspot for multidrug- and colistin-resistant Klebsiella pneumoniae

Wastewater treatment plants are hotspots for the release of antimicrobial resistant pathogenic bacteria into aquatic ecosystems, significantly contributing to the cycle of antimicrobial resistance. Special attention should be paid to antimicrobial resistant ESKAPE bacteria, which have been identified as high-priority targets for control measures. Among them, Klebsiella pneumoniae is particularly noteworthy. In this study, we collected wastewater samples from the inlet, sedimentation tank, and effluent water of a wastewater treatment plant in June, July, October, and November of 2018. We detected and characterized 42 K. pneumoniae strains using whole genome sequencing (15 from the inlet, 8 from the sedimentation tank, and 19 from the effluent). Additionally, the strains were tested for their antimicrobial resistance phenotype. Using whole genome sequencing no distinct patterns were observed in terms of their genetic profiles. All strains were resistant to tetracycline, meanwhile 60%, 47%, and 37.5% of strains isolated from the inlet, sedimentation tank, and effluent, respectively, were multidrug resistant. Some of the multidrug resistant isolates were also resistant to colistin, and nearly all tested positive for the eptB and arnT genes, which are associated with polymyxin resistance. Various antimicrobial resistance genes were linked to mobile genetic elements, and they did not correlate with detected virulence groups or defense systems. Overall, our results, although not quantitative, highlight that multidrug resistant K. pneumoniae strains, including those resistant to colistin and genetically unrelated, being discharged into aquatic ecosystems from wastewater treatment plants. This suggests the necessity of monitoring aimed at genetically characterizing these pathogenic bacteria.

Rhizoctonia species, anastomosis groups, and pathogenicity isolated from common bean in Lake Van Basin, Turkiye

Common bean (Phaseolus vulgaris L.) is an important vegetable crop grown in Lake Van Basin. Local genotypes are widely grown in the region. Rhizoctonia root rot induced by Rhizoctonia solani Kühn is an important soilborne plant disease that leads to global economic losses as well as in Turkey. The present study was conducted to determine anastomosis groups and pathogenicity of Rhizoctonia spp. obtained from bean plants in Lake Van Basin in 2013 and 2014. A total of 236 Rhizoctonia isolates in 5 anastomosis groups were obtained from bean plant roots. It was observed that AG- 4 (112) was the most isolated group in beans, followed by AG- 2 (41), AG- 3 (28), AG- 5 (33), and binucleate AG- K (22) isolates. Pathogenicity test conducted in thirty isolates in 5 anastomosis groups was analyzed for A64 (Bitlis/ Adilcevaz), TR68557 genotypes, and Gina (cv.) under growth chamber conditions. The study findings demonstrated that all tested isolates could infect the bean plant with different degrees of severity; however, the most virulent group was AG- 4. It was determined that the most virulent isolate was Isolate No. 19 in the A64 genotype, Isolate No. 2 in TR68557, and Isolate No. 18 in Gina cv. in in vivo tests. The identification and pathogenicity determination of Rhizoctonia isolates are the first steps towards an efficient control strategy for bean diseases caused by Rhizoctonia species. In order to obtain quality and productive products in the Van Lake Basin, where intensive bean production is carried out, precautions should be taken by considering the damage caused by Rhizoctonia spp. on plants.

Characterizing genetic diversity of Sclerotium rolfsii isolates by biomapping of mycelial compatibility groupings and multilocus sequence analysis.

Genetic diversity in Sclerotium rolfsii is useful for understanding its population structure, identifying different mycelial compatibility groups (MCGs), and developing targeted strategies for disease management in affected crops. In our study, a comprehensive genetic analysis was conducted on 50 isolates of S. rolfsii, collected from various geographic regions and host plants. Two specific genes, TEF1α and RPB2, were utilized to assess the genetic diversity and relationships among these isolates. Notably, out of 1225 pairings examined, only 154 exhibited a compatible reaction, while the majority displayed antagonistic reactions, resulting in the formation of a barrier zone. The isolates were grouped into 10 distinct MCGs. These MCGs were further characterized using genetic sequencing. TEF1α sequences distinguished the isolates into 17 distinct clusters, and RPB2 sequences classified them into 20 clusters. Some MCGs shared identical gene sequences within each gene, while others exhibited unique sequences. Intriguingly, when both TEF1α and RPB2 sequences were combined, all 10 MCGs were effectively differentiated, even those that appeared identical with single-gene analysis. This combined approach provided a comprehensive understanding of the genetic diversity and relationships among the S. rolfsii isolates, allowing for precise discrimination between different MCGs. The results shed light on the population structure and genetic variability within this plant pathogenic fungus, providing valuable insights for disease management and control strategies. This study highlights the significance of comprehending the varied virulence characteristics within S. rolfsii isolates, categorizing them into specific virulence groups based on disease severity index (DSI) values. The association with MCGs provides additional insights into the genetic underpinnings of virulence in this pathogen. Furthermore, the identification of geographical patterns in virulence implies the influence of region-specific factors, with potential implications for disease control and crop protection strategies.Please confirm if the author names are presented accurately and in the correct sequence (given name, middle name/initial, family name). Author 1 Given name: [G. M. Sandeep] Last name [Kumar]. Author 2 Given name: [Praveen Kumar] Last name [Singh]. Also, kindly confirm the details in the metadata are correct.I confirm that the given names are accurate and presented in the correct sequence.

Comparative Study of Staphylococcus epidermidis Virulence Profiles in an animal Model of Endophthalmitis

Introduction: Gram-positive staphylococci are responsible for over 90% of cases of postoperative infectious endophthalmitis, with Staphylococcus epidermidis accounting for approximately 70% of isolated microorganisms. Objective: To evaluate the relationship between S. epidermidis virulence and severity of endophthalmitis in an animal model. Methodology: New Zealand albino rabbits were divided into two groups and administered 100 colony-forming units of S. epidermidis strains. In the virulent group, four rabbits received a multi-antibiotic resistant, mecA, ica, and atlE gene carrying S. epidermidis strain, from a patient´s conjuntival microbiota and three a biofilm-forming S. epidermidis ATCC 35984. In the non-virulent group, five rabbits were inoculated with a strain sensitive to all tested antibiotics and lacking mecA, ica, and atlE genes, also from a patient and three rabbits received the non-producer biofilm S. epidermidis ATCC 29122. Clinical and ultrasound examinations were conducted every three hours until endophthalmitis symptoms appeared, followed by daily clinical assessments. Histological evaluations were performed 15 days post-inoculation. Results: The less virulent group displayed milder inflammation and reduced intraocular damage in comparison to the more virulent group based on clinical and ultrasound observations. Nevertheless, histopathological analysis revealed similar inflammation in all groups, 15 days post-inoculation. Discussion: Less virulent S. epidermidis strains induced less severe inflammation as observed through clinical and ultrasound assessments. However, long-term histopathological assessments showed effects comparable to those seen with the more virulent strain.

Defense Regulatory Network Associated with circRNA in Rice in Response to Brown Planthopper Infestation

The brown planthopper (BPH), Nilaparvata lugens (Stål), a rice-specific pest, has risen to the top of the list of significant pathogens and insects in recent years. Host plant-mediated resistance is an efficient strategy for BPH control. Nonetheless, BPH resistance in rice cultivars has succumbed to the emergence of distinct virulent BPH populations. Circular RNAs (circRNAs) play a pivotal role in regulating plant–environment interactions; however, the mechanisms underlying their insect-resistant functions remain largely unexplored. In this study, we conducted an extensive genome-wide analysis using high-throughput sequencing to explore the response of rice circRNAs to BPH infestations. We identified a total of 186 circRNAs in IR56 rice across two distinct virulence groups: IR-IR56-BPH (referring to IR rice infested by IR56-BPH) and IR-TN1-BPH, along with a control group (IR-CK) without BPH infestation. Among them, 39 circRNAs were upregulated, and 43 circRNAs were downregulated in the comparison between IR-IR56-BPH and IR-CK. Furthermore, in comparison with IR-CK, 42 circRNAs exhibited upregulation in IR-TN1-BPH, while 42 circRNAs showed downregulation. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the targets of differentially expressed circRNAs were considerably enriched in a multitude of biological processes closely linked to the response to BPH infestations. Furthermore, we assessed a total of 20 randomly selected circRNAs along with their corresponding expression levels. Moreover, we validated the regulatory impact of circRNAs on miRNAs and mRNAs. These findings have led us to construct a conceptual model that circRNA is associated with the defense regulatory network in rice, which is likely facilitated by the mediation of their parental genes and competing endogenous RNA (ceRNA) networks. This model contributes to the understanding of several extensively studied processes in rice-BPH interactions.

Detection of Resistance, Susceptibility, Tolerance, and Virulence in Plant-Nematode Interactions: Part I-Sedentary Endoparasitic Nematodes.

The use of nonhost, tolerant, or resistant plants, to manage plant parasitic nematodes (PPNs), is an appealing, economic, and environmentally friendly agronomic practice, which is effective when precise information on the identification of PPN species and their virulence to target host crops is available. This chapter describes suggested protocols to evaluate the reaction of the most important crops and fruit trees to infestation by the most damaging PPN with sedentary endoparasitic habits, with the aim of assessing resistance and tolerance traits, sources of resistance in progenies from breeding programs, the reaction to nematodes of newly released cultivars, and the virulence of the most noxious PPNs. These protocols consist of classical screening techniques not involving biochemical and molecular analyses. PPN species and genera considered in this chapter include (i) the most importantspecies of root-knot nematodes Meloidogyne spp., including alsoM. chitwoodi, M. enterolobii, and M. graminicola, and (ii) the cyst-forming nematodes of the genera Globodera and Heterodera, such as the potato cyst nematodes (PCNs) Globodera rostochiensis and G. pallida, and also Heterodera avenae group, H. ciceri, H. glycines, and H. schachtii. Schemes are given to identify virulence groups for most of these nematodes.

Isolation and pathogenicity of a highly virulent group III porcine Getah virus in China.

Getah virus (GETV) is a multi-host virus found in pigs, horses, and blue foxes. Clinically, GETV can cause fever, diarrhea, and reproductive disorders in pigs, representing significant threats to pig breeding. At present, few studies have examined the pathogenicity of GETV in pigs of different ages. In the present study, a new strain, named GETV-QJ, was isolated from clinically ill pigs, and whole genome sequencing analysis was performed. Besides the pathogenicity of piglets and pregnant sows of this strain was further studied. the results illustrated that the strain belonged to group III. The strain had 93.6%-96.3% homology with other subtypes, and its homology with the same subtype strain ranged 96.5%-99%. Further studies on the pathogenicity of the virus indicated that this strain caused severe diarrhea, fever, and intestinal and lung damage in 7-day-old piglets, resulting in their death. The piglet survival rate was 0%. In pregnant sows, this strain did not cause fever, death, or abortion, but it induced viremia, which affected the farrowing performance of sows and led to reduced piglet survival. In this study, we isolated a highly virulent group III and comprehensively established a pathogenic model of GETV in piglets and sows, providing a reference and guidance for the prevention and control of this infection.

Characterization of field isolates of Fusarium spp. from eggplant in India for species complexity and virulence

Eggplant wilt, despite emerging as a severe disease in India, the etiology must be better studied for its species' complexity and variability. The identity of fungal isolates associated with eggplants of India was established morphologically followed by sequencing and phylogenetic analysis. Three species, Fusarium falciforme, Fusarium incarnatum and Fusarium proliferatum, were observed for the first time in India. The isolates were tested for pathogenicity. Though all of them were pathogenic, the isolates displayed varying degrees of virulence. In further studies, the genetic relatedness of the isolates for virulence was assessed with candidate avirulent (SIX effectors), virulent (Fow1 and Fow2) and SSR markers. The SIX effector genes could not delineate the virulent isolates and were expressed in some non-F. oxysporum isolates for the first time. Likewise, the virulent genes, Fow1 for expression across the isolates and Fow2 for random expression across the isolates, were unsuitable markers for identifying the virulent groups. Hence, the F. oxysporum and F. solani isolates were genotyped with SSR markers. Though the clustering did not correlate with their virulence levels, the dendrogram grouping revealed variability among the F. oxysporum and F. solani isolates. This study concludes that although multiple species of Fusarium are associated with eggplant wilt in India, only F. oxysporum and F. solani are widespread in the surveyed areas. Though the three markers could not delineate the race specificity of the isolates, only the SSR makers could identify the genetic variability and hence, would help screen eggplant germplasm for fusarium wilt resistance.

Clinical Characteristics and Molecular Epidemiology of ST23 Klebsiella pneumoniae in China.

In clinical settings, CG23 Klebsiella pneumoniae (Kp) is the most virulent clonal group of Kp. Continuous fusions of hypervirulent (Hv) and highly resistant strains have been reported; however, few studies have analysed the molecular epidemiology and clinical characteristics of CG23 strains, especially MDR-sequence type ST23 strains. In this study, we investigated the molecular characteristics of ST23 Kp and analysed the clinical characteristics of ST23 Kp infections in a large teaching hospital of the third class in China. ST23 Kp isolates were screened using whole-genome sequencing data from a large single centre. We compared the clinical characteristics of ST23 strains isolated from community-acquired infections (CAI) and hospital acquired infection (HAI). In addition, the infection characteristics of MDR and poor-prognosis isolates were investigated. We analysed genetic characteristics of ST23 Kp and further investigated the evolutionary relationship based on single-nucleotide polymorphism phylogenetic trees. We detected 184 ST23 strains between 2013 and July of 2018. There were no significant differences between the isolation rates of pulmonary, bloodstream, urinary tract, and cutaneous soft tissue infections in the community and hospitals, except for abscess infections. MDR strains primarily cause pulmonary infections and abscesses; infections with a poor prognosis are typically bloodstream and pulmonary infections. Fourteen MDR strains producing extended-spectrum or class C beta-lactamases, resulting in resistance to third-generation cephalosporins. In 3.8% of ST23 Kp strains, the clb locus was absent. The phylogenetic tree revealed that the isolates were primarily divided into three clades, and based on clinical data, it is inferred that three clonal transmission events have occurred, mainly in ICU causing lung infection. This study demonstrates that virulence and drug-resistance fusion events of ST23 strains occur gradually, and that the hypervirulent clones facilitate the widespread dissemination of CAI and HAI, particularly pulmonary. Monitoring genomics and developing antivirulence strategies are essential.

Morphological, pathogenic and genetic diversity in Diplodia seriata associated with black rot canker of apple in India

Apple cankers are extremely destructive diseases threatening the global apple industry through direct and indirect losses. The population structure of the pathogens is of paramount significance for the development of efficient management strategies. Therefore, phenotypic, pathogenic, and genetic diversity of Diplodia seriata causing black rot canker of apple was investigated in this study. All the isolates were included for investigating the in vitro mycelial growth, conidial dimensions, and pathogenic variability on two-year-old potted apple seedlings. The ISSR approach was used to investigate the molecular diversity of D. seriata. Mycelial growth rates were found to vary significantly amongst the isolates; however, there were no major variations seen between the different geographical groupings of isolates. Pathogenicity tests revealed variations in the size of cankers among the isolates indicating the presence of virulence variability. The isolates were segregated into three virulence groups based on canker length. The Bayesian analyses of ISSR data divided the isolates into two genetic clusters. The genetic clustering of the isolates revealed no relationship with geographical origin of the isolates. Furthermore, no direct relationship of genetic clustering was observed with morphological or pathogenic variability. The ISSR primers revealed very high level of variability in D. seriata; however, no distinct populations of the pathogen existed which is an indication of high level of gene flow between the diverse geographical populations. According to our knowledge, this is the first thorough investigation on the diversity of D. seriata associated with apple black rot canker in India.

Anastomosis grouping and phylogenetic analysis of Rhizoctonia isolates on wheat in Türkiye

This study aims to determine the species and evaluate the genetic diversity of the pathogenic and nonpathogenic Rhizoctonia spp. and anastomosis groups (AG) from wheat plants and rhizosphere soils in Turkey. Rhizoctonia species were isolated from plants and rhizosphere soils in wheat fields in 5 provinces in the Central Anatolian Region of Türkiye. As a result of the isolations, a total of 88 multinucleate (MN) and binucleate (BN) Rhizoctonia isolates were obtained. Identifications of the isolates were determined by rDNA-ITS sequence analyses. The identified isolates belonged to MN Waitea circinata var. zeae, W. circinata var. oryzae, W. circinata var. circinata, MN Rhizoctonia solani AG 2-1, AG 2-2, AG 3, AG 4-HGII, AG 4-HGIII, AG 5, AG 8, AG 11 and BN AG A, AG DI, AG E, AG G, AG H, AG I, AG I-like and AG K. The most isolated group was W. circinata var. circinata. In the pathogenicity studies, the most virulent group was determined as R. solani AG 4. Among the binucleate isolates, groups other than R. cerealis AG DI were not found to be pathogenic. Neighbor-joining phylogenetic trees of isolates were constructed from rDNA-ITS sequences. As a result of this study, the regional distribution of MN and BN Rhizoctonia AG isolates in important wheat production areas in the Central Anatolia Region, Türkiye was determined. In addition, this study is the first comprehensive study in which the genetic diversity of Rhizoctonia AGs isolates obtained from wheat and rhizosphere soils in the region was evaluated with a molecular approach.

Sequencing and analysis of the Meq oncogene of MDV causing marek’s disease in Bac Ninh province from 2019 to 2022

Marek’s disease is a contagious avian viral disease that is caused by a Marek’s disease virus serotype 1 (MDV-1) or gallid herpesvirus 2 (GaHV-2), a member of the genus Alphaherpesvirus, family Herpesviridae. Up to now, the disease has caused significant losses to the chicken farming industry in many provinces and cities across the country. In this study, four MDV-1 (GaHV-2) strains causing the disease in Bac Ninh province during the 2019–2022 period were identified by multiplex Polymerase Chain Reaction (PCR). Subsequently, the segments of 1020 bp encoding 339 amino acids of Meq gene (Marek’s disease virus EcoRI fragment Q) from four samples were collected and sequenced. The rate of identity and homology between the four Vietnamese strains and other global strains ranged from 89.4% to 100% and 87.6% to 100%, respectively. These rates were higher between the four Vietnamese strains and MDV-1 Chinese strains, ranging from 99.2% to 100% and 98.2% to 100%, respectively. The molecular characterization of the Meq gene revealed that all four of the GaHV-2 strains infecting chickens in the Bac Ninh province belonged to the highly virulent group, with a low proline ratio in Meq protein (ranging from 20.59–21.18%), consisting of three PPPP motifs and three interrupted motifs that contain mutations at the second position of the proline rich region (PRR): PPPP>P (Q/A/R)PP. Overall, this study provides valuable information on molecular characteristics of MDV-1 strains in Bac Ninh province during the 2019–2022 period.

Children with autism show differences in the gut DNA virome compared to non-autistic children: a case control study

BackgroundSeveral previous studies have identified a potential role that the gut microbiome can play in autism spectrum disorder (ASD) in children, but little is known about how variations in the virome may be involved in ASD. We aimed to understand the changes in the gut DNA virome of children with ASD.MethodsA case–control study was presented, in which 13 two-children families were observed while considering the age, mode of birth, history of antibiotic use, and vaccination history to minimize the influence of confounding factors. DNA viral metagenomic sequencing was successfully performed on stool samples from 11 children with ASD and 12 healthy non-ASD children. The basic composition and gene function of the participants' fecal DNA virome were detected and analyzed. Finally, the abundance and diversity of the DNA virome of children with ASD and their healthy siblings were compared.ResultsThe gut DNA virome in children aged 3–11 years was found to be dominated by the Siphoviridae family of Caudovirales. The proteins encoded by the DNA genes mainly carry out the functions of genetic information transmission and metabolism. Compared the gut DNA virome of ASD and healthy non-ASD children, their abundance of Caudovirales and Petitvirales both showed a significant negative correlation (r = -0.902, P < 0.01), there was no statistically significant difference in the relative abundance of viruses at the order and family levels, and a difference in the relative abundance at the genus level for Skunavirus (Ζ = -2.157, P = 0.031). Viral α diversity was reduced in children with ASD, but α diversity and β diversity did not differ statistically between groups.ConclusionsThis study indicates that elevated Skunavirus abundance and decreased α diversity in the gut DNA virulence group of children with ASD, but no statistically significant difference in the change in alpha and beta diversity. This provides preliminary cumulative information on virological aspects of the relationship between the microbiome and ASD, and should benefit future multi-omics and large sample studies on the gut microbes in children with ASD.

Vibrio vulnificus, an Underestimated Zoonotic Pathogen.

V. vulnificus, continues being an underestimated yet lethal zoonotic pathogen. In this chapter, we provide a comprehensive review of numerous aspects of the biology, epidemiology, and virulence mechanisms of this poorly understood pathogen. We will emphasize the widespread role of horizontal gene transfer in V. vulnificus specifically virulence plasmids and draw parallels from aquaculture farms to human health. By placing current findings in the context of climate change, we will also contend that fish farms act as evolutionary drivers that accelerate species evolution and the emergence of new virulent groups. Overall, we suggest that on-farm control measures should be adopted both to protect animals from Vibriosis, and also as a public health measure to prevent the emergence of new zoonotic groups.

Culture-Independent Detection and Identification of Leptospira Serovars.

Pathogenic Leptospira, the causative agents of leptospirosis, comprise >200 serotypes (called serovars). Most have a restricted reservoir-host range, and some, e.g., serovar Copenhageni, are cosmopolitan and of public health importance owing to their propensity to produce severe, fatal disease in humans. Available serotyping approaches-such as multilocus sequence typing, core genome sequence typing, pulsed-field gel electrophoresis, and the cross-agglutination absorption test-are tedious and expensive, and require isolation of the organisms in culture media-a protracted and incredibly inefficient process-precluding their use in prospective studies or outbreak investigations. The unavailability of culture-independent assays capable of distinguishing Leptospira serotypes remains a crucial gap in the field. Here, we have developed a simple yet specific real-time qPCR assay-targeting a Leptospira-unique gene encoding a putative polysaccharide flippase-that provides intraspecies, serotype-defining (i.e., epidemiologically useful) information, and improves upon the sensitivity of preferred lipL32-based qPCR-based diagnostic tests. The assay, dubbed RAgI ("rage one"), is rapid and affordable, and reliably and specifically detects group I pathogenic Leptospira in culture, serum, and urine, with no detectable off-target amplification-even of the genetically related but low virulence group II pathogenic (formerly "intermediate") or nonpathogenic Leptospira. It retained 100% diagnostic specificity when tested against difficult sample types, including field-collected dog urine samples and environmental samples containing varied and complex microbial species-consortia. This assay holds considerable promise in the clinical setting, and for routine epidemiological and environmental surveillance studies. IMPORTANCE Leptospirosis is caused by a diverse group of pathogenic spirochetes comprising over 200 different serotypes. Some are widely reported and of public health importance owing to their propensity to produce severe, fatal disease in humans. Apart from their tedium and expense, current serotyping approaches require isolation of the organisms in culture media-a protracted and incredibly inefficient process-rendering them useless clinically and limiting their utilization in prospective studies or outbreak investigations. The unavailability of culture-independent assays capable of distinguishing Leptospira serotypes remains a crucial gap in the field. The 11108 qPCR-assay overcomes this barrier to progress via direct taxonomic and serotype classification of Leptospira from urine and serum samples, and hence, is the first qPCR-based prognostic test for human leptospirosis.

Virulence Diversity of Puccinia striiformis f. sp. Tritici in Common Wheat in Russian Regions in 2019–2021

Yellow (stripe) rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a major disease of common wheat worldwide. Disease epidemics in Russia have been frequent and destructive, mostly in the North Caucasus. However, over the last 5 years, the significance of Pst has markedly increased in other Russian regions. Therefore, the Pst virulence diversity was investigated in Triticum aestivum in six geographically distant regions of the European (North Caucasus, North-West, Low Volga, Central Black Earth region, and Volga-Vyatka) and Asian (West Siberia) parts of Russia, with strongly different climates, environmental conditions, and growing wheat genotypes. Seventy-nine virulence pathotypes among 117 isolates were identified using the 12 Avocet Yr gene lines (Yr1, Yr5, Yr6, Yr7, Yr8, Yr9, Yr10, Yr15, Yr17, Yr24, Yr27, and YrSp) and eight supplemental wheat differentials (Heines VII, Vilmorin 23, Hybrid 46, Strubes Dickkopf, Carstens V, Suwon 92/Omar, Nord Desprez, and Heines Peko). Only four pathotypes occurred in two or more regions. High variability was detected within Pst populations from Dagestan, Central, North-West, and West Siberia that postulated to form an intrapopulation subdivision of each of them into several subgroups. Most regional virulence groups of pathotypes were closely related, except for several small subgroups of pathotypes from West Siberia, Dagestan, North-West, and Central European regions. All Pst isolates were avirulent in lines with Yr5, Yr10, Yr15, and Yr24 genes. Virulence to Yr17 was detected for several isolates of two pathotypes, one each from the North-West and Low Volga regions. Variation in virulence frequency was observed in other differential lines.

Evaluation of the Protective Immune Response Induced by an rfbG-Deficient Salmonella enterica Serovar Enteritidis Strain as a Live Attenuated DIVA (Differentiation of Infected and Vaccinated Animals) Vaccine in Chickens.

Salmonella enterica serovar Enteritidis (S. Enteritidis), one of the zoonotic pathogens, not only results in significant financial losses for the global poultry industry but also has the potential to spread to humans through poultry and poultry products. Vaccination is an effective method to prevent Salmonella infections. In this study, we constructed a live attenuated DIVA (differentiation of infected and vaccinated animals) vaccine candidate, Z11ΔrfbG, and evaluated its protective effectiveness and DIVA potential in chickens. Compared to that of the virulent wild-type strain, the 50% lethal dose (LD50) of the rfbG mutant strain increased 56-fold, confirming its attenuation. High serum levels of S. Enteritidis-specific IgG titers indicated that a significant humoral immune response was induced in the vaccinated group. After challenge, the nonvaccinated group showed serious clinical symptoms (diarrhea, depression, decreased appetite, ruffled feathers, and weight loss), pathological changes (white nodules in the liver and fatty lesions in liver cells), and death. In contrast, there were no clinical symptoms, pathological changes, or death in the 5 × 106- and 5 × 107-CFU-vaccinated groups. Z11ΔrfbG vaccination significantly reduced S. Enteritidis colonization in the spleen, liver, and cecum. In addition, the Z11ΔrfbG-vaccinated group exhibited a negative response to the serological test, whereas the virulent wild-type Z11 infection group was strongly positive for the serological test, showing a DIVA capability of Z11ΔrfbG vaccination. Overall, our findings demonstrate the viability of the rfbG mutant as a live attenuated chicken vaccine that can discriminate between animals that have been immunized and those that have been infected. IMPORTANCE S. Enteritidis is a highly adapted pathogen that causes significant economic losses in the poultry industry around the world. Vaccination is an effective method of controlling S. Enteritidis infections. Here, we demonstrated that S. Enteritidis Z11ΔrfbG has the potential to be a safe, immunogenic, and DIVA vaccine candidate for the control of Salmonella infections in chickens. Z11ΔrfbG not only provided effective protection in chickens but also distinguished between infected and vaccinated chickens by serological tests.

Expression of SIX1b and SIX1c effector genes and banana resistance genes during Foc TR4 infection on banana cultivars

Abstract. Ulilalbab AR, Widinugraheni S, Masanto, Subandiyah S, Wibowo A. 2022. Expression of SIX1b and SIX1c effector genes and banana resistance genes during Foc TR4 infection on banana cultivars. Biodiversitas 23: 5314-5322. The relative expression SIX1 genes isolates of Foc TR4 and banana resistance genes have not been intensively studied. This study aimed to determine the expression of SIX1 genes of Foc TR4 and banana resistance genes. Twelve Foc TR4 isolates were collected from several regions in Indonesia. DSI analysis was performed to categorize their virulence levels. The representative Foc TR4 isolates from different virulence levels were artificially inoculated on the Musa acuminata Colla Cavendish subgroup and cv. Barangan and incubated for 48, 72, and 96 HPI. The expressed genes were quantitatively analyzed using the qPCR technique using primers to amplify SIX1b, SIX1c, chitinase, and PR-protein 1 genes. The results categorized the virulence of Foc TR4 isolates into moderate, virulent, and high virulent. The isolates of KJG and Batu-4 were selected for gene expression study representing moderate and high virulent groups, respectively. The results of in planta assay found that the expression of SIX1b, SIX1c, chitinase, and PR-protein1 was upregulated on the inoculated plants during the incubation period. However, the expression of these genes was increasingly upregulated in both bananas at early-stage inoculation. We assumed that plant defense genes of bananas might actively encounter the common virulence mechanisms of Foc TR4 at the initial stage of inoculation.