Low-virulence Strain Research Articles

New insights about the complexity of Pseudomonas syringae pv. actinidiae across the world

The bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa), is an emblematic example of a catastrophic disease of fruit crops. In 2008, after decades of limited damages, a new, extremely virulent form emerged and devastated thousands of hectares of Actinidia spp. orchards all over the world with impressive economic losses. To elucidate the diffusion of different Psa types on world scale and their presumptive movements, it is necessary to recognize genetic diversities within its populations. We analyzed an exhaustive collection of 142 Psa strains, that included a conspicuous number isolated in the Asiatic area of origin of Actinidia spp. MLVA has been chosen as the investigative technique for its efficacy and reproducibility, as well as its simplicity and low cost. A panel of 13 VNTR loci was identified and used to study the Psa complex. Results indicate that this MLVA assay can reveal a much wider diversity among Psa populations throughout the world than previously assessed. The congruence of results with those of other molecular approaches was demonstrated and the VNTRs loci were divided in two different panels having an increasing discriminatory power. Ten main clonal complexes were depicted that correlate repeatedly with geographic origin of strains, but also with their virulence and isolation time. The recently originated hypervirulent type of Psa, in which, beside the known strains from Europe, New Zealand and Chile, only few strains from Shaanxi Province in China are also included, confirmed to be genetically very homogeneous. China proved to be the area where the broadest genetic variability resides but, interestingly, different Psa types are retrievable in both Japan and Korea. The low virulent strains group together and are deeply different from any other Psa, so that they can no longer be considered as a real Psa variant. The effectiveness of MLVA in resolving differences between Psa strains and related implications on pathogen spread are discussed.

Increased frequency of travel in the presence of cross-immunity may act to decrease the chance of a global pandemic.

The high frequency of modern travel has led to concerns about a devastating pandemic since a lethal pathogen strain could spread worldwide quickly. Many historical pandemics have arisen following pathogen evolution to a more virulent form. However, some pathogen strains invoke immune responses that provide partial cross-immunity against infection with related strains. Here, we consider a mathematical model of successive outbreaks of two strains-a low virulence (LV) strain outbreak followed by a high virulence (HV) strain outbreak. Under these circumstances, we investigate the impacts of varying travel rates and cross-immunity on the probability that a major epidemic of the HV strain occurs, and the size of that outbreak. Frequent travel between subpopulations can lead to widespread immunity to the HV strain, driven by exposure to the LV strain. As a result, major epidemics of the HV strain are less likely, and can potentially be smaller, with more connected subpopulations. Cross-immunity may be a factor contributing to the absence of a global pandemic as severe as the 1918 influenza pandemic in the century since. This article is part of the theme issue 'Modelling infectious disease outbreaks in humans, animals and plants: approaches and important themes'. This issue is linked with the subsequent theme issue 'Modelling infectious disease outbreaks in humans, animals and plants: epidemic forecasting and control'.

Experimental infection with high- and low-virulence strains of border disease virus (BDV) in Pyrenean chamois (Rupicapra p. pyrenaica) sheds light on the epidemiological diversity of the disease.

Since 2001, Pyrenean chamois (Rupicapra pyrenaica pyrenaica) populations have been affected by border disease virus (BDV) causing mortalities of more than 80% in some areas. Field studies carried out in France, Andorra, and Spain have shown different epidemiological scenarios in chamois populations. This study was designed to confirm the presence of BDV strains of a high and low virulence in free-ranging chamois populations from Pyrenees and to understand the implications of these findings to the diverse epidemiological scenarios. An experimental infection of Pyrenean chamois with a high-virulence (Cadí-6) and low-virulence (Freser-5) BDV strains was performed. Pregnant and non-pregnant animals with and without antibodies against BDV were included in each group. Cadí-6 BDV strain was confirmed to be of high virulence for seronegative adults and their foetuses. The antibody negative chamois infected with Freser-5 BDV strain did not show symptoms, presented less viral distribution and RNA load in tissues than Cadí-6 group, and cleared the virus from the serum. However, foetuses died before the end of the experiment and RNA virus was detected in sera and tissues although with lower RNA load than the Cadí-6 group. Chamois from both groups presented lesions in brain but the ones infected with the low-virulence Freser-5 BDV strain were mild and most likely transient. In both groups, seropositive pregnant females and all but one of their foetuses did not present viraemia or viral RNA in tissues. The existence of a low-virulence strain has been confirmed experimentally and related to chamois population infection dynamics in the area where it was isolated. Such strain may persist in the chamois population through PI animals and may induce cross-protection in chamois against high-virulence strains. This study demonstrates that viral strain diversity is a significant factor in the heterogeneity of epidemiological scenarios in Pyrenean chamois populations.

Effect of Satureja khuzistanica essential oil (SKEO) extract on expression of lasA and lasB genes in Pseudomonas aeruginosa

Expressions of lasA and lasB genes of Pseudomonas aeruginosa are associated with bacterium pathogenicity. The present study was aimed to assess the effect of Satureja khuzistanica essential oil (SKEO) extract on expression of lasA and lasB genes in P. aeruginosa. Pseudomonas aeruginosa isolates were cultured in Mueller Hinton broth containing sub-inhibitory concentrations of SKEO and total RNA extracted using Trizol method. cDNA was synthesized using random Hexamer primer and finally the expression of lasA and lasB genes carried out by real-time PCR. The MICs of SKEO extract for PA9, PA10, PA11, PA13, PA41 and PA42 isolates were 8, 8, 8, 9, 7 and 12 μg/ml, respectively. Statistical analysis for 6 isolates revealed that the reduction in expression of lasA and lasB genes under SKEO treatment was significant (P<0.05). The insignificantly increasing of lasB gene expression may lead to low virulent strains, for probably reason that the strain's exotoxin A are destroyed in the high amount of protease. In conclusion, using of SKEO in burned patients infected with P. aeruginosa may be effective; however, it is better to assess the spectrum activity of SKEO, pharmacokinetics, potency and its toxicity in human cells.

IMMUNOGENIC PROPERTIES OF CELLULAR AND EXTRACELLULAR PROTEIN-CONTAINING ANTIGENS OF STAPHYLOCOCCUS AUREUS

Aim. Comparative study of immunobiological properties of cell wall surface antigens and extracellular protein-containing antigens of Staphylococcus aureus. Materials and methods. Preparations: surface antigens of the cell wall (peptidoglycan, teichoic acids, protein antigens) of the strains of S. aureus containing in the staphylococcal vaccine «Staphylovac» (SV) and extracellular protein-containing antigens of S. aureus (EPCA). The parameters of innate immunity were evaluated by the effect of preparations on the immunophenotype of mononuclear leukocytes (ML) of the spleen of mice, expression of Toll-like receptors (using flow cytometry), phagocytic activity of macrophages of peritoneal exudate of mice after the introduction of SV and EPCA; the protective activity of preparations was studied in experiments of active protection of BALB/c mice. Results. To isolate EPCA, we used the virulent strain of S. aureus №6, and for obtaining surface antigens of the cell wall, 4 strains were selected, the most immunogenic of which was the low virulent strain of S. aureus №1991. Both preparations increased the number of TLR2 and MHC II expressing cells; CV administration caused an increase in the number of cells with CD25 marker, reflecting the early activation of immunocompetent cells, and EPCA immunization — led to a shorter expression of this marker. On the other hand, an increased number of CD19 positive cells were detected for a longer period of time during EPCA immunization. The longest activation of phagocytosis under the action of SV was established. High protective activity of bo-th types of the studied preparations was noted. Conclusion. The antigens of the cell wall and extracellular protein-containing experimental drugs possessed comparable immunological properties, however, the surface antigens to a greater extent activate innate immunity, and extracellular — adaptive immunity.

Virulence evaluation of classical swine fever virus subgenotype 2.1 and 2.2 isolates circulating in China

Classical swine fever (CSF) remains an important pig disease in China, where it usually presents with mild or atypical clinical manifestations, with large scale outbreaks rarely seen. This has led to speculation about the possible circulation of viral strains of low virulence. To investigate this possibility, five field isolates within the predominant genotype 2 (2.1b, 2.1c, 2.1 h and 2.2) were evaluated and compared by experimental infection of naturally farrowed but colostrum-deprived piglets. All infected piglets displayed clinical signs, including persistent high fever, depression, anorexia, dyspnea, conjunctivitis, constipation, and hesitant gait. Typical pathological lesions, including pulmonary edema, hemorrhagic or cellulosic exudation, and swelling and hemorrhage of lymph nodes, were observed. Viremia and Erns protein expression in the blood of all infected animals were detectable from 3 to 5 days post infection (DPI), their presence correlating with the onset of fever, clinical signs and leukopenia. E2 antibody did not develop in any of the field CSFV-infected piglets during the disease course, while Erns antibody was detectable in 4–56% of infected animals at various time points. Mortalities ranged from 20 to 80% within 21 DPI, progressing to 100% by 43 DPI. Based on clinical scores and fatalities within 21 DPI, 2 of the 5 field isolates were classified as of moderate virulence and 3 of high virulence; i.e., no field isolates of low virulence were identified. The study has provided data supporting the use of these isolates as challenge viruses to evaluate the efficacy of current CSF vaccines.

Bordetella pertussis Infection in Infants and Young Children in Shanghai, China, 2016-2017: Clinical Features, Genotype Variations of Antigenic Genes and Macrolides Resistance.

The global resurgence of pertussis in countries with high vaccination coverage has been a concern of public health. Nasopharyngeal swabs were collected for Bordetella pertussis culture from children with suspected pertussis. Clinical and vaccination information were reviewed through electronic medical chart and immunization record. Antibiotics susceptibility was evaluated using E-test for erythromycin, azithromycin, clarithromycin and sulfamethoxazole/trimethoprim. The MLST genotypes and 7 antigenic genes (ptxP, ptxA, ptxC, Prn, fim3, fim2 and tcfA) of Bordetella pertussis were identified by polymerase chain reaction amplification and sequencing. During January 2016 to September 2017, a total of 141 children 1-48 months of age were culture-confirmed with pertussis, of whom 98 (69.5%) were younger than 6 months, 25 (17.7%) had completed at least 3 doses of DTaP and 75 (53.2%) had a clear exposure to household members with persistent cough. Fully vaccinated cases manifested milder disease than unvaccinated and not-fully vaccinated cases. All strains were MLST2. High-virulent strains characteristic of ptxP3/prn2/ptxC2 constituted 41.1% (58/141) and were all susceptible to macrolides while low-virulent strains characteristic of ptxP1/prn1/ptxC1 constituted 58.9% (83/141) and 97.6% (81/83), respectively, were highly resistant to macrolides. Pertussis is resurging among infants and young children in Shanghai, and household transmission is the main exposure pathway. The high-virulent strains harboring ptxP3/prn2/ptxC2 and the macrolide-resistant Bordetella pertussis strains are quite prevalent. These issues impose a public health concern in Shanghai. Our findings are important to modify the DTaP vaccination strategy and the management guideline of pertussis in China.

Meningococcal strain evolution, dusty dry air and respiratory tract infections: An explosive relationship.

Meningococcal strain evolution, dusty dry air and respiratory tract infections: An explosive relationship.

High-Load Reovirus Infections Do Not Imply Physiological Impairment in Salmon.

The recent ubiquitous detection of PRV among salmonids has sparked international concern about the cardiorespiratory performance of infected wild and farmed salmon. Piscine orthoreovirus (PRV) has been shown to create substantial viremia in salmon by targeting erythrocytes for principle replication. In some instances, infections develop into heart and skeletal muscle inflammation (HSMI) or other pathological conditions affecting the respiratory system. Critical to assessing the seriousness of PRV infections are controlled infection studies that measure physiological impairment to critical life support systems. Respiratory performance is such a system and here multiple indices were measured to test the hypothesis that a low-virulence strain of PRV from Pacific Canada compromises the cardiorespiratory capabilities of Atlantic salmon. Contrary to this hypothesis, the oxygen affinity and carrying capacity of erythrocytes were unaffected by PRV despite the presence of severe viremia, minor heart pathology and transient cellular activation of antiviral response pathways. Similarly, PRV-infected fish had neither sustained nor appreciable differences in respiratory capabilities compared with control fish. The lack of functional harm to salmon infected with PRV in this instance highlights that, in an era of unprecedented virus discovery, detection of viral infection does not necessarily imply bodily harm and that viral load is not always a suitable predictor of disease within a host organism.

The modulation of autophagy by African swine fever virus

African swine fever virus (ASFV) causes a lethal haemorrhagic disease of domestic pigs with mortality rates of up to 100 %. An outbreak in Russia in 2007 has since spread into Europe. There is currently no vaccine available, however infection with attenuated strains of ASFV can protect against infection with closely related virulent strains. Autophagy is a conserved, essential cell process that regulates multiple pathways that are critical for mounting an effective immune response. Experiments have shown that inhibiting the ability of viruses to regulate autophagy can lead to enhanced immune responses. We have shown that ASFV does not require autophagy for replication and that autophagosome formation is inhibited during infection. In addition, through analysis of key proteins in the upstream autophagy pathway, we describe a novel mechanism of ASFV inhibition of autophagy. This research will expand our understanding of the interaction between ASFV and the autophagy pathway with the potential that a low virulent ASFV strain with an altered ability to modulate autophagy will provide enhanced immunity against virulent isolates.

Early Detection of Toxoplasma gondii Infection in Mongolian Gerbil by Quantitative Real-Time Pcr

Toxoplasmosis, caused by Toxoplasma gondii, is associated with several clinical syndromes, including encephalitis, chorioretinitis, and congenital infection. Toxoplasma gondii is a ubiquitous apicomplexan parasite found in both humans and animals. Mongolian gerbils, which are more susceptible to both high- and low-virulence Toxoplasma strains compared with mice, are considered useful models for assessing diagnosis and treatment methods for toxoplasmosis, as well as infection by and host defense to this organism. Here we established a quantitative real-time polymerase chain reaction (qPCR) method targeting the B1 gene for early and specific detection of T. gondii infection in Mongolian gerbil. The detection limit of the developed qPCR was approximately 1 T. gondii tachyzoite. This method was also applied to detect T. gondii genomic DNA in experimentally infected Mongolian gerbils, with positive results in blood (66.7%), liver (73.3%), lung (80.0%), spleen (80.0%), and peritoneal fluid (66.7%) samples as early as 1 day postinfection. Specificity tests confirmed no cross-reactivity with DNA templates of Neospora caninum, Cryptosporidium parvum, Eimeria tenella, Trypanosoma evansi, Schistosoma japonicum, Angiostrongylus cantonensis, and Strongyloides stercoralis. This study first reports the use of Mongolian gerbils as an animal model for early diagnosis of toxoplasmosis by qPCR.

Developing a risk management framework to improve public health outcomes by enumerating Salmonella in ground turkey.

Salmonella spp. continue to be a leading cause of foodborne morbidity worldwide. To assess the risk of foodborne disease, current national regulatory schemes focus on prevalence estimates of Salmonella and other pathogens. The role of pathogen quantification as a risk management measure and its impact on public health is not well understood. To address this information gap, a quantitative risk assessment model was developed to evaluate the impact of pathogen enumeration strategies on public health after consumption of contaminated ground turkey in the USA. Public health impact was evaluated by using several dose-response models for high- and low-virulent strains to account for potential under- or overestimation of human health impacts. The model predicted 2705-21 099 illnesses that would result in 93-727 reported cases of salmonellosis. Sensitivity analysis predicted cooking an unthawed product at home as the riskiest consumption scenario and microbial concentration the most influential input on the incidence of human illnesses. Model results indicated that removing ground turkey lots exceeding contamination levels of 1 MPN/g and 1 MPN in 25 g would decrease the median number of illnesses by 86-94% and 99%, respectively. For a single production lot, contamination levels higher than 1 MPN/g would be needed to result in a reported case to public health officials. At contamination levels of 10 MPN/g, there would be a 13% chance of detecting an outbreak, and at 100 MPN/g, the likelihood of detecting an outbreak increases to 41%. Based on these model prediction results, risk management strategies should incorporate pathogen enumeration. This would have a direct impact on illness incidence linking public health outcomes with measurable food safety objectives.

An O-Antigen Glycoconjugate Vaccine Produced Using Protein Glycan Coupling Technology Is Protective in an Inhalational Rat Model of Tularemia.

There is a requirement for an efficacious vaccine to protect people against infection from Francisella tularensis, the etiological agent of tularemia. The lipopolysaccharide (LPS) of F. tularensis is suboptimally protective against a parenteral lethal challenge in mice. To develop a more efficacious subunit vaccine, we have used a novel biosynthetic technique of protein glycan coupling technology (PGCT) that exploits bacterial N-linked glycosylation to recombinantly conjugate F. tularensis O-antigen glycans to the immunogenic carrier protein Pseudomonas aeruginosa exoprotein A (ExoA). Previously, we demonstrated that an ExoA glycoconjugate with two glycosylation sequons was capable of providing significant protection to mice against a challenge with a low-virulence strain of F. tularensis. Here, we have generated a more heavily glycosylated conjugate vaccine and evaluated its efficacy in a Fischer 344 rat model of tularemia. We demonstrate that this glycoconjugate vaccine protected rats against disease and the lethality of an inhalational challenge with F. tularensis Schu S4. Our data highlights the potential of this biosynthetic approach for the creation of next-generation tularemia subunit vaccines.

OK-432 Administration Inhibits Murine Allergic Rhinitis at the Induction Phase, through the Macrophage Activation with TLR2 Signaling Pathway.

OK-432, a preparation of a low-virulence strain (Su) of Streptococcus pyogenes (Group A) killed by a penicillin and lyophilized, is a stiff inducer of Th1 cytokines, and exerts anti-cancer effects in tumor-bearing mice. OK-432 has been reported to consist of many bacterial components, such as peptidoglycan, M-protein, etc. However, it is yet to be ascertained which bacterial component induces T helper 1 (Th1) responses. For the last decade, Toll-like receptor (TLR) family proteins are well elucidated to play a role in recognizing bacterial components and inducing interleukin (IL)-12 from macrophages. Above all, peptidoglycan seems to be the agonist of TLR2 rather than the obverse. In our present study, the role of TLR2 for the recognition of OK-432 by macrophages and the effects of OK-432 are examined on murine allergic rhinitis model. Interestingly, results show IL-12 production by macrophages derived from TLR2 knock-out (ko) mice was significantly decreased, in comparison with that of macrophages derived from wild-type mice. Moreover, in TLR2 ko mice, no regulatory effect of OK-432 was observed on an allergic rhinitis model. These data indicate that TLR2 signaling is involved in regulating OK-432-induced anti-T helper 2 (Th2) immunity, and may offer a new prophylactic and therapeutic approach using OK-432 to downregulate allergic disorders, such as allergic rhinitis.

Host and microbial factors in kidney transplant recipients with Escherichia coli acute pyelonephritis or asymptomatic bacteriuria: a prospective study using whole-genome sequencing.

Urinary tract infection is the most common infection among kidney transplant recipients (KTRs). Many transplant physicians fear that host compromise will allow low-virulence strains to cause pyelonephritis in KTRs, so they often treat asymptomatic bacteriuria with antibiotics. Identification of the host/microbe factors that determine the clinical presentation (i.e. pyelonephritis versus asymptomatic bacteriuria) once an Escherichia coli strain enters a KTRs bladder could inform management decisions. We prospectively collected all E. coli isolates causing either pyelonephritis or asymptomatic bacteriuria in KTRs at our institution (December 2012-June 2015). Whole-genome sequencing was used to assess bacterial characteristics (carriage of 48 virulence genes and phylogenetic and clonal background). Host parameters were also collected. We analysed 72 bacteriuria episodes in 54 KTRs (53 pyelonephritis, 19 asymptomatic bacteriuria). The pyelonephritis and asymptomatic bacteriuria isolates exhibited a similar total virulence gene count per isolate [median 18 (range 5-33) and 18 (5-30), respectively; P = 0.57] and for individual virulence genes differed significantly only for the prevalence of the pap operon (pyelonephritis 39%,versus asymptomatic bacteriuria 0%; P = 0.002). No other significant between-group differences were apparent for 86 other bacterial and host variables. Our findings suggest that bacterial adherence plays a role in the pathogenesis of pyelonephritis in KTRs despite significantly altered host urinary tract anatomy and weakened immunity. Whether KTRs might benefit from targeted therapies (e.g. vaccination or inhibitors of fimbrial adhesion) has yet to be studied.

Protection efficacy of Argentinian isolates of Mycobacterium avium subsp. paratuberculosis with different genotypes and virulence in a murine model

Paratuberculosis is a chronic disease caused by Mycobacterium avium subsp. paratuberculosis (Map). The disease causes economic losses and, therefore, it is imperative to follow proper control strategies, which should include an effective vaccine. Several strategies have assessed the virulence and immune response of Map strains that could be used as a vaccine. This study evaluates the degree of virulence, immune response, and protection of Argentinian strains of Map with different genotype in a murine model. Four local isolates (Cattle type) with different genotypes (analyzed by MIRU-VNTR and SSRs) were selected and evaluated in a virulence assay in BALB/c mice. This assay allowed us to differentiate virulent and low-virulence Map strains. The less virulent strains (1543/481 and A162) failed to induce a significant production of the proinflammatory cytokine IFNg, whereas the virulent strain 6611 established infection along with a proinflammatory immune response. On the other hand, the virulent strain 1347/498 was efficient in establishing a persistent infection, but failed to promote an important Th1 response compared with 6611 at the evaluated time. We selected the low-virulence strain 1543/498 as a live vaccine and the virulent strain 6611 as a live and inactivated vaccine in a protection assay in mice. Strain 1543/481 failed to protect the animals from challenge, whereas strain 6611, in its live and inactivated form, significantly reduced the CFUs count in the infected mice, although they had different immunological response profiles. The inactivated virulent strain 6611 is a potential vaccine candidate against paratuberculosis to be tested in cattle.

Comparison of Highly and Weakly Virulent Dickeya solani Strains, With a View on the Pangenome and Panregulon of This Species.

Bacteria belonging to the genera Dickeya and Pectobacterium are responsible for significant economic losses in a wide variety of crops and ornamentals. During last years, increasing losses in potato production have been attributed to the appearance of Dickeya solani. The D. solani strains investigated so far share genetic homogeneity, although different virulence levels were observed among strains of various origins. The purpose of this study was to investigate the genetic traits possibly related to the diverse virulence levels by means of comparative genomics. First, we developed a new genome assembly pipeline which allowed us to complete the D. solani genomes. Four de novo sequenced and ten publicly available genomes were used to identify the structure of the D. solani pangenome, in which 74.8 and 25.2% of genes were grouped into the core and dispensable genome, respectively. For D. solani panregulon analysis, we performed a binding site prediction for four transcription factors, namely CRP, KdgR, PecS and Fur, to detect the regulons of these virulence regulators. Most of the D. solani potential virulence factors were predicted to belong to the accessory regulons of CRP, KdgR, and PecS. Thus, some differences in gene expression could exist between D. solani strains. The comparison between a highly and a low virulent strain, IFB0099 and IFB0223, respectively, disclosed only small differences between their genomes but significant differences in the production of virulence factors like pectinases, cellulases and proteases, and in their mobility. The D. solani strains also diverge in the number and size of prophages present in their genomes. Another relevant difference is the disruption of the adhesin gene fhaB2 in the highly virulent strain. Strain IFB0223, which has a complete adhesin gene, is less mobile and less aggressive than IFB0099. This suggests that in this case, mobility rather than adherence is needed in order to trigger disease symptoms. This study highlights the utility of comparative genomics in predicting D. solani traits involved in the aggressiveness of this emerging plant pathogen.

Clinical and infection dynamics of foot-and-mouth disease in beef feedlot cattle: An expert survey

Parameterizing mathematical models of foot-and-mouth disease virus (FMDv) transmission is challenging due to knowledge gaps on the variable dynamics in susceptible populations. Expert opinion surveys are an approach to gather data on topics where no data have been reported. The objective of this study was to collect—via an expert-opinion survey—key parameter values of the potential FMD natural history and transmissibility in beef feedlot cattle in the U.S. Experts with experience working with FMD in endemic and non-endemic settings were targeted. Parameters surveyed were: duration of infection and disease stages, proportions of animals with specific clinical manifestations, duration and extent of the reduction in feed consumption, and probabilities of severe clinical disease and FMDv transmission. We surveyed the parameter values for infections by strains of different virulence, different infection doses, and routes of transmission. Twenty-seven experts from around the world agreed to participate and 16 (59%) completed the survey. The expert responses to individual questions were resampled via Monte Carlo simulations; to the resulting distributions, candidate theoretical distributions were fitted using the maximum likelihood method and the sought parameter values estimated based on the best-fit distributions. Of the infection stages, the estimates of the expected FMD latent period in beef feedlot ranged from 1.7 to 5.3 days and the infectious period from 5.6 to 10.9 days. Of the disease stages, the estimated incubation period ranged from 2.9 to 6.1 days, subclinical period from 1.2 to 2.8 days, and clinical period from 4.2 to 7.5 days. Probability of developing clinical disease after infection varied from 82% (IQ range 90–70%) with high-virulent to 63% (IQ range 89–60%) with low-virulent strains. Reduction in feed consumption was estimated to last 5 (SD ± 2) days in cattle infected by a low-virulent FMDv strain and 7 (SD ± 2) days for high virulent strains. The study results can be used in combination with experimental and outbreak investigation data to parameterize FMDv-transmission models to evaluate intervention responses during hypothetical FMD epidemics in beef feedlot populations in the U.S.

The effect of entomopathogenic fungi Metarhizium robertsii of different virulence on the generation of reactive oxygen species in Galleria mellonella larvae

The toxicity of reactive oxygen species (ROS) plays a significant role in the immune response of insects. Little is known about the effect of the virulence of entomopathogenic fungi on the generation of ROS in a host. The aim of the study was to reveal whether the different levels of virulence cause the different ROS production in insects. The half-lethal dosages of two Metarhizium robertsii strains, of high and low virulence, which have shown a similar survival to Galleria mellonella larvae after treatment, were used in the study. The rates of ROS generation were determined in the cuticle, hemocytes and cell-free hemolymph of G. mellonella larvae 1, 3, 5 days after fungi treatment. We have shown that the level of ROS production in the cuticle and hemolymph of G. mellonella larvae depends on the virulence of the M. robertsii strains. The influence of both fungal strains on the rate of ROS formation in hemocytes was the same throughout the observation period. The host’s defense mechanism was activated in the cuticle under the treatment of both low and high virulent fungi strains. In the hemolymph, the activation of the immune response occurred only after treatment with low virulent strain.

Up-regulated RxLR effector genes of Plasmopara viticola in synchronized host-free stages and infected leaves of hosts with different susceptibility

Fast recognition of host signals and early activation of infection mechanisms in Plasmopara viticola are decisive for successful infestation of Vitis vinifera. To better understand interactive processes at the first front line of combat between the pathogen and its host, a specific pre-infective stage was generated in a host-free system. Zoospore encystment was triggered within minutes after treatment with CaCl2. Subsequently, high rates of germ tube formation occurred in a synchronized manner. This method was employed to compare development-related gene expression in strains of different virulence. Soon after germination, spores showed strong up-regulation of two effector genes, PvRxLR18 and PvRxLR28, particularly in the high virulence strain. On infected grapevine leaf-discs of cultivars with different susceptibility, a similar up-regulation was found at 6 hours post inoculation (hpi). This effect was much more evident in the high virulence than in the low virulence strain and was significantly higher on leaves of the tolerant cultivar Regent than on Müller-Thurgau. In addition, PvRxLR67 was up-regulated 24 hpi in the high virulence strain indicating that different effectors are active in later infection stages. Differences in the expression pattern of RxLR effector genes between the two strains corroborated with infection symptoms visible by sporulation.