Experimental Vaccine Research Articles

The Effect of Saccharomyces cerevisiae Fermentation Product Supplementation on Pro-Inflammatory Cytokines in Holstein Friesian Cattle Experimentally Inoculated with Digital Dermatitis

Digital dermatitis (DD) poses a major animal welfare concern for the dairy industry, with even broader economic implications for the agricultural industry worldwide. The postbiotic, a Saccharomyces cerevisiae fermentation product (SCFP), has had a positive influence on the innate immune system of cattle, which makes it a potential candidate as a feed supplement as part of a prevention strategy for DD. This study investigated the effect of a commercial SCFP feed supplement compared to a control feed supplement on the production of pro-inflammatory cytokines (IL-1β and IL-6) by peripheral blood mononuclear cells (PBMCs) in Holstein Friesian steers experimentally infected with DD. The results showed that SCFP supplementation was associated with an overall reduced IL-1β production (p = 0.005), particularly prior to experimental inoculation with a DD lesion homogenate. However, the results of the analysis suggest that the innate immune system in the SCFP group became prepared to respond more rapidly to DD infection post-inoculation. During active (M2), chronic (M4), and focal flare-ups (M4.1) of DD, SCFP supplementation resulted in a more rapid secretion of IL-1β (M2: p = 0.038; M4/M4/1: p = 0.034). A more rapid response to DD infection for IL-6 was only found for chronic (M4) and focal flare-ups (M4.1) of DD (p = 0.006). These findings emphasize the difference in cytokine response between various stages of DD in the SCFP group compared to the control, highlighting implications for DD prevention and treatment.

Multiomics dissection of Brassica napus L. lateral roots and endophytes interactions under phosphorus starvation

Many plants associate with endophytic microbes that improve root phosphorus (P) uptake. Understanding the interactions between roots and endophytes can enable efforts to improve P utilization. Here, we characterize the interactions between lateral roots of endophytes in a core collection of 50 rapeseed (Brassica napus L.) genotypes with differing sensitivities to low P conditions. With the correlation analysis result between bacterial abundance and plant physiological indices of rapeseeds, and inoculation experiments on plates and soil, we identify one Flavobacterium strain (C2) that significantly alleviates the P deficiency phenotype of rapeseeds. The underlying mechanisms are explored by performing the weighted gene coexpression network analysis (WGCNA), and conducting genome-wide association studies (GWAS) using Flavobacterium abundance as a quantitative trait. Under P-limited conditions, C2 regulates fatty acid and lipid metabolic pathways. For example, C2 improves metabolism of linoleic acid, which mediates root suberin biosynthesis, and enhances P uptake efficiency. In addition, C2 suppresses root jasmonic acid biosynthesis, which depends on α-linolenic acid metabolism, improving C2 colonization and activating P uptake. This study demonstrates that adjusting the endophyte composition can modulate P uptake in B. napus plants, providing a basis for developing agricultural microbial agents.

Cattle, sheep, and goat humoral immune responses against SARS-CoV-2

Following the emergence of SARS-CoV-2 in late 2019, several species of domestic and wild animals have been found to be susceptible to SARS-CoV-2 infection through experimental inoculation and animal surveillance activities. Detection of SARS-CoV-2 specific antibodies in animals is an important surveillance tool since viral shedding in animals can only be detected for a short period of time. In this study, convenience serum samples were collected from 691 cattle, 698 sheep, and 707 goats from several regions in the United States, between 2019 to 2022. The samples were evaluated for the presence of SARS-CoV-2 specific antibodies using two commercial enzyme-linked immunosorbent assays (ELISA); one based on the inhibition of the SARS-CoV-2 receptor-binding domain (sVNT) and the other based on the nucleocapsid protein (N-ELISA) of SARS-CoV-2. Positive samples from the sVNT were additionally evaluated using a conventional virus neutralization test (VNT) employing the Wuhan-like SARS-CoV-2 USA/WA1/2020 isolate. Our results indicate that ∼1% (6/691) of cattle, ∼2% (13/698) of sheep, and ∼2.5% (18/707) of goat serum samples were positive when using the sVNT, whereas ∼4% of cattle (25/691) and sheep (27/698), and 2.5% (18/707) of goat serum samples tested positive with the N-ELISA. None of the sVNT positive cattle, sheep, or goat serum samples had detectable neutralizing antibody activity (<1:8) against the SARS-CoV-2 USA/WA1/2020 isolate by the VNT. Our results indicate low seropositivity in cattle, sheep, and goats in the U.S., indicating the importance to continue monitoring for SARS-CoV-2 prevalence in animal species that are in close contact with humans.

Isolation and Identification of Pear Ring Rot Fungus and Resistance Evaluation of Different Pear Varieties

Botryosphaeria dothidea is a significant plant pathogen responsible for causing ulcers, wilt, and fruit decay across a wide range of host plants. One notable fungal disease attributed to B. dothidea is pear tree ring rot, which currently ranks among the most severe diseases affecting pear trees in China. This pathogen primarily targets branches and fruits, occasionally impacting leaves as well, leading to tree weakening, fruit rot, and leaf drop. The annual repercussions of this disease severely affect both the yield and quality of pear fruits, thereby impeding the healthy development of the pear industry. Recent studies have indicated that other species within the B. dothidea complex can also induce pear ring rot; however, specific physiological strains of B. dothidea remain unreported. Consequently, this study collected tissues from pear trees infected with ring rot from orchards located in Liaoning, Hebei, Shandong, and other regions throughout China. Through morphological characterization combined with pathogenicity assessments and DNA sequence comparisons involving partial internal transcribed spacer (ITS), translation elongation factor (TEF), and β-tubulin (TUB) genes, 21 strains belonging to the Botryosphaeria spp. were identified. These 21 strains served as research subjects for inoculating dormant annual branches from 30 germplasm resources of pear trees in vitro. The results demonstrated that all tested strains could induce lesions on the branches which were characterized by dark brown spots. Furthermore, inoculation experiments involving these 21 strains were conducted to evaluate the resistance levels of various pear varieties against ring rot disease. The resistance was assessed by inoculating different isolates onto distinct pear varieties; this approach established the criteria for evaluating resistance while minimizing identification errors stemming from the variable responses exhibited by certain varieties towards individual strains. Ultimately, this study aims to provide a theoretical foundation for effective prevention and treatment strategies against pear ring rot.

Specific anti-toxic immunotherapy: use in medical practice and perspectives

The results of use of approaches and means of antitoxic immunotherapy in medical practice are summarized: passive transfer of various variants of antibodies specific to toxic compounds, vaccination – specific active immunization with vaccines carrying determinants of the immunochemical specificity of target toxic compounds. The practical effectiveness of the known approaches of passive transfer and vaccination is associated with the ability of specific antibodies, by binding target bioactive compounds with pronounced toxicity, to change the availability of corresponding structures in the body-targets and, in the presence of a sufficient number of specific antibodies with high binding ability to the target compounds, neutralize their toxicity. In medical practice, polyspecific heterologous antisera or the gamma globulin fraction of antisera (extremely rare monoclonal antibodies of narrow specificity) are widely used for passive transfer as antidotes in the treatment of victims in order to prevent deaths and extensive necrosis of soft tissues at the site of the bite of poisonous snakes and insects. Active immunization – vaccination with appropriate antigenic drugs should create in immunized individuals a state of humoral immunity with the corresponding characteristics of antibody formation specific to the target compound. When a target toxic compound enters an immunized organism, it is also possible to neutralize its toxicity. The most successful experience in using the principles of active immunization as a technology for specific antitoxic therapy is associated with the practice of using toxoids for toxinemic infections. Specific practical techniques used to achieve the effectiveness of possible approaches to specific antitoxic immunotherapy in the form of passive transfer of specific antibodies or their fragments are considered: to combat lethal infections in the pathogenesis of which the toxic effects of bacterial exotoxins are significant; when treating victims of snake and insect bites, exposure to poisons of marine organisms, algae and plant toxins; in the treatment of severe intoxication with certain low molecular weight toxic substances: digoxin, colchicine, tricyclic antidepressants. The most successful experiences of using the principles of active immunization as a technology for specific antitoxic therapy, based on the use of toxoids with specificity for diphtheria, tetanus, botulism, cholera, typhoid fever, dysentery, gas gangrene and other toxinemic infections, are also considered. The fairly high immunogenicity of toxoids with the possibility of activating both constitutional and adaptive immunity has become the basis for their use as macromolecular carriers of hapten analogues of narcotic substances – a promising direction in drug addiction, in the implementation of which a number of experimental molecular and combined vaccines of opiates, methamphetamine, cocaine, and nicotine. This variant of practical efforts in medicine can be regarded as a new direction of specific antitoxic immunotherapy – an option to combat drug addiction by vaccinating drug addicts.

Specificity determinants of pathogens in forest

Abstract Host‐specific pathogens have long been suggested to act as a major driver of species diversity in tropical forests. However, determining the degree of host specificity of potential pathogens coupled to key cellular characteristics of infection is difficult and time‐consuming. These challenges have delayed progress in relating the functional specificity of pathogenic fungi to ecological consequences. We tested the pathogenicity of 27 (of 215) fungi that were isolated from surface sterilized roots of seedlings from four common tree species in a diverse subtropical forest. Inoculation experiments showed that six fungi exhibited strong pathogenicity on the host seedlings. Five of these only infected their specific hosts (i.e. host‐specific pathogen species). Green fluorescent protein labelling revealed that in three host‐specific pathogens fungal hyphae were able to grow into the vascular tissues only in their specific host plant, in contrast to other fungal‐host combinations that exhibit non‐pathogenic interactions. This fluorescent labelling technique allows direct tracking of the progress of fungal infection in different host tissues. Synthesis. By coupling the green fluorescent protein technique with standard host inoculation experiments, we determined the developmental differences between infections by pathogenic and non‐pathogenic fungi in a relatively simple and straightforward way. Our work provides a useful tool for rapidly screening and categorizing host–fungal interactions, reflecting the functional basis of host specificity of pathogenic fungi. More broadly, this technique can contribute to understanding the roles of pathogens in species coexistence and biodiversity maintenance in forest communities.

Defense strategies against sweet potato chlorotic stunt and pakakuy virus coinfection unraveled

AbstractThis study into the response of two Ipomoea batatas (L.) Lam cultivars, Melinda and Tio Joe, to coinfection with sweet potato chlorotic stunt virus (SPCSV) and sweet potato pakakuy virus (SPPV), employed a comprehensive approach encompassing symptomatology, real‐time quantitative polymerase chain reaction, metagenomics, and transcriptomics. SPCSV is a quarantine virus with synergistic effects, which decimate yields. SPPV is the most prevalent DNA virus in sweet potato germplasm, with a tendency to persist in meristems, posing a significant risk for germplasm transfer between territories. Graft inoculation experiments revealed that while Tio Joe remained asymptomatic for 12 weeks and suppressed virus replication, Melinda displayed symptoms early on and exhibited high virus titers. Metagenomic analyses corroborated these observations and confirmed that SPCSV and SPPV were responsible. Transcriptomic analysis unveiled disparities in gene expression between Melinda and Tio Joe. Differential gene expression was heightened and altered in Melinda as the viruses disrupted its gene expression. Its defense strategies, such as inducing abscisic acid signaling, were insufficient to overcome disruptive viral effects like oxidative stress, rendering it susceptible. In contrast, Tio Joe had relatively stable differential gene expression, indicating resistance to SPPV–SPCSV coinfection. Overexpressed genes such as sirtuin, rapid alkalization factor, and nuclear pore anchor triggered quantitative resistance, supported with enriched organelles such as mitochondrion and pathways such as proteasome and cutin, suberine, and wax biosynthesis. Tio Joe maintained its genome integrity and inhibited viral replication by tightly controlling gene expression and preventing reactive oxygen species accumulation.

VmSpm1: a secretory protein from Valsa mali that targets apple's abscisic acid receptor MdPYL4 to suppress jasmonic acid signaling and enhance infection.

Pathogenic fungi such as Valsa mali secrete effector proteins to manipulate host defenses and facilitate infection. Subtilases are identified as potential virulence factors, yet their specific roles in fruit tree pathogens, such as those affecting apple trees, are poorly understood. Our research shows VmSpm1 as a virulence factor in V. mali. Knocking it out decreased virulence, whereas its heterologous expression in apple led to reduced disease resistance. Using Y2H, BiFC, SLC, and Co-IP techniques, we demonstrated an interaction between VmSpm1 and MdPYL4. MdPYL4 levels increased during V. mali infection. The stable transgenic apple lines inoculation experiment showed that MdPYL4 correlates with enhanced resistance to Apple Valsa canker when overexpressed in apples. Furthermore, through in vitro and in vivo assays, we showed the degradative role of VmSpm1 on MdPYL4. MdPYL4 promotes the synthesis of jasmonic acid (JA) in apples in an abscisic acid-dependent manner. The degradation of MdPYL4 leads to a reduction in JA content in apples during V. mali infection, thereby impairing JA signal transduction and decreasing disease resistance in apple plants. In summary, this study reveals how V. mali utilizes VmSpm1 to subvert JA signaling, shedding light on fungal manipulation of plant hormones to disrupt immunity.

Novel Porcine Getah Virus from Diarrheal Piglets in Jiangxi Province, China: Prevalence, Genome Sequence, and Pathogenicity.

Getah virus (GETV) is a mosquito-borne virus belonging to the genus Alphavirus in the family Togaviridae. Its infection poses an increasing threat to animals and public health in China. In this study, an epidemiological survey of GETV on 46 pig farms in Jiangxi Province, China, was performed; GETV isolation and characterization were carried out, including a complete sequence determination and phylogenetic analysis; and pathogenicity of the GETV was experimentally investigated by inoculating newborn piglets with the isolated GETV strain. Epidemiological studies conducted on the organs of infected pigs, aborted piglets, and the blood of aborted sows sampled from pig farms in Jiangxi Province, China, demonstrated that 44 out of the 46 pig farms were positive for GETV, which is a positivity rate of 95.65% (44/46). Of the 411 samples tested, 47.93% (197/411) were found positive for GETV. A GETV strain called GETV-JX-CHN-22 was obtained, which showed stable proliferation in Vero cells. One-step growth curve results showed that the GETV-JX-CHN-22-P7 (passage 7) isolate reached a peak titer of 108.3 TCID50/mL at 24 hpi. An analysis of the whole-genome sequencing results showed that GETV-JX-CHN-22 (prototype) and GETV-JX-CHN-22-P7 shared nucleotide sequence similarities of 95.3% to 99.6% with 73 reference strains of GETV in GenBank. Genetic evolution analysis revealed that GETV-JX-CHN-22 and GETV-JX-CHN-22-P7 belonged to the GIII group, the same group members of most strains were reported in China. Animal inoculation experiments indicated that piglets exhibited typical symptoms and pathological changes of GETV infection after 24 h inoculation, which reproduced the pathogenicity of GETV field strain infections in piglets. To our knowledge, this study is the first report on the detection and isolation of porcine GETV associated with diarrhea from pig farms in Jiangxi Province, China. It is of great importance to study the infection spectrum, transmission mechanism, and public health significance of GETV. The results provide foundations for the genomic and biological (pathogenic) characteristics of the circulating GETV in Jiangxi Province, China.

Plant growth promoting bacteria promote rice growth cultivated in two different sandy soils subjected distinct climates conditions.

Sandy soils contain around 70% sand in their composition, making them highly fragile and susceptible to land degradation. Practices such as no-tillage cultivation, the use of bioinoculants, and the application of organic amendments can restore the organic matter in these soils, ensuring sustainable production. In this context, this work aimed to study the microbiological aspects of two sandy soil areas (Brazilian Northeast and South) under contrasting climatic conditions (tropical and temperate). With this purpose, prokaryotic communities were evaluated, and the plant growth-promoting potential of isolated bacteria was assessed by rice inoculation in sandy soil. Despite the high sand content in both soils, soil from the NE was related to the highest phosphorous, calcium, potassium, copper, sodium, zinc, magnesium, and manganese contents, organic matter percentage, and pH. The Shannon diversity index indicated that prokaryotic communities in NE were more diverse than in SU, and PCA revealed that microbial composition exhibited distinct patterns. The rice inoculation experiments were executed to verify if the bacterial isolates displayed a similar growth promotion potential when inoculated in sandy soil areas subjected to different climatic conditions. When all PGP characteristics evaluated were pooled in a PCA, a similar pattern was observed for SU and NE. Burkholderia sp. SU94 was related to highest PGP characteristics evaluated. Paraburkholderia sp. NE32 showed similar results to those of the non-inoculated control. This similar effect of rice growth in the Northeast and South of Brazil suggests that isolate SU94 adapts to different environmental conditions.

The zoonotic LCK-3110 strain of Rocahepevirus ratti leads to mild infection in chickens after experimental inoculation

Rocahepevirus ratti [rat hepatitis E virus (HEV)] was originally isolated from rats and found to be non-infectious to nonhuman primates, suggesting humans were not a susceptible host. However, in 2018, rat HEV infections were identified in human patients. High seroprevalence for rat HEV in rats in many countries necessitates studying this emerging zoonotic outbreak. Lack of a human derived rat HEV infectious clone, cell culture systems, and animal models have hindered this effort. In response to the increase in human infection cases by rat HEV, we utilized an infectious clone of the zoonotic rat HEV LCK-3110 strain originally reported from human cases. Capped RNA transcripts of the rat HEV LCK-3110 strain were synthesized, and replication was assessed in both cell culture via transfection and chickens via intrahepatic inoculation. Naive chickens were cohoused together with inoculated chickens. Our results demonstrated that although chickens were susceptible, virus replication was inefficient with only a few of the chickens inoculated with rat HEV having low levels of viremia and fecal virus shedding. However, LCK-3110 HEV was able to transmit between chickens as several naive cohoused chickens became infected as evidenced by viremia, fecal shedding, and the presence of viral protein upon histopathology of the liver. Rat HEV is an emerging zoonotic virus with an ability to spillover across species. Chickens have potential to serve as intermediary hosts, possibly playing a role in rat HEV spread and exposure to humans.

At Least 55 Undeclared Chemical Elements Found in COVID-19 Vaccines from AstraZeneca, CanSino, Moderna, Pfizer, Sinopharm and Sputnik V, with Precise ICP-MS

The experimental vaccines supposedly invented to combat COVID-19 were coercively forced upon the global population beginning late in 2020. They have precipitated innumerable and varied disease conditions ranging from mild to lethal. This increase in health disorders and sudden deaths began to manifest concomitantly with the number of people inoculated and doses administered per person. By the end of 2023, 24 undeclared chemical elements had been detected by Scanning Electron Microscopy Coupled with Energy-Dispersive X-Ray Spectroscopy (SEM-EDX), in the COVID-19 vaccines of the different brands, by various research groups from different countries around the world. In this paper, we report laboratory results from high precision Inductively Coupled Plasma Mass Spectrometry (ICP-MS) that confirm and expand previous results by SEM-EDX. To this end, the contents of vials from different lots of the brands AstraZeneca/Oxford, CanSino Biologics, Pfizer/BioNTech, Sinopharm, Moderna and Sputnik V were analyzed. Among the undeclared chemical elements were detected 11 of the 15 cytotoxic lanthanides used in electronic devices and optogenetics. In addition, among the undeclared elements were all 11 of the heavy metals: chromium was found in 100% of the samples; arsenic 82%; nickel 59%; cobalt and copper 47%; tin 35%; cadmium, lead and manganese in 18%; and mercury in 6%. A total of 55 undeclared chemical elements were found and quantified with ICP-MS. Combining these findings with results from SEM-EDX, altogether 62 undeclared chemical elements have been found in the various products. In all brands, we found boron, calcium, titanium, aluminum, arsenic, nickel, chromium, copper, gallium, strontium, niobium, molybdenum, barium and hafnium. With ICP-MS, we found that the content of the samples is heterogeneous, the elemental composition varies in different aliquots extracted from the same vial.

Multifaceted roles of NADPH oxidases in the growth and pathogenicity of Beauveria bassiana

ABSTRACT Reactive oxygen species (ROS), synthesized by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, are vital molecules in biological cells, influencing various physiological processes such as fungal growth, development, and virulence. Beauveria bassiana, an entomopathogenic fungus, is a promising biopesticide for agricultural, forestry, and urban pest control. This study focuses on the characterization of NADPH oxidases (Noxs) in B. bassiana. Gene expression profiles of Noxs in B. bassiana (BbNoxs) were analysed using RT-qPCR. Knockout strains of single BbNoxA, BbNoxB, BbNoxR, and double BbNoxA and BbNoxB were constructed via homologous recombination, and their phenotypic characteristics were examined. Fungal virulence was evaluated using Galleria mellonella larvae, and infection structures formation and penetration ability were assessed on cicada wings. ROS production and actin assembly during fungal growth and infection were detected using staining and marker methods. Expression analysis revealed significant upregulation of BbNoxs during fungal growth and infection. Compared to the wild-type strain, single knockouts (ΔBbNoxA/B/R) and double knockout (ΔBbNoxAB) of BbNoxs exhibited reduced conidial yields, accelerated conidial germination rates. Deletion of BbNoxB or BbNoxR decreased fungal virulence compared to the WT strain in topical inoculation experiments. Additionally, loss of BbNoxB or BbNoxR impaired infection structures formation, penetration ability, ROS production, and actin aggregation during fungal infection. BbNoxs are crucial for fungal growth, development, and virulence in B. bassiana, playing essential roles in infection structures formation, penetration, ROS production, and actin assembly. Understanding their functions provides insights into B. bassiana’s pathogenic mechanisms.

Study on the feasibility of using livestock blood as a fetal bovine serum substitute for cultured meat.

Fetal bovine serum (FBS) accounts for the largest portion of the cost of cultured meat production or cell culture experiments and is highly controversial in terms of animal welfare because it is taken from the fetus of a pregnant cow during slaughtering. Nevertheless, FBS is the most important supplement in the cell culture manufacturing process. This study aimed to develop an FBS substitute from slaughterhouse waste blood to reduce the cost of FBS in cultured meat production through various experiments. Our study successfully demonstrated that adult livestock blood obtained from slaughterhouses can effectively replace FBS. Our substitute, when cultured with bovine myosatellite cells, demonstrated cell growth that was either equivalent to or superior to that of commercial FBS. In the process of muscle generation through differentiation, the substitutes from bovine and chicken formed 70%-75% more bovine muscle compared to the control group using FBS. Furthermore, using the FBS substitute can reduce cell culture costs by approximately 61% compared to using commercial FBS. Therefore, the groundbreaking FBS substitute will not only contribute to the development of technology to mass-produce cultured meat using livestock byproducts but will also lower the production cost of media for experimental cell culture or vaccine production.

A chimeric strain of porcine reproductive and respiratory syndrome virus 2 derived from HP-PRRSV and NADC30-like PRRSV confers cross-protection against both strains

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant swine viral infectious diseases worldwide. Vaccination is a key strategy for the control and prevention of PRRS. At present, the NADC30-like PRRSV strain has become the predominant epidemic strain in China, superseding the HP-PRRSV strain. The existing commercial vaccines offer substantial protection against HP-PRRSV, but their efficacy against NADC30-like PRRSV is limited. The development of a novel vaccine that can provide valuable cross-protection against both NADC30-like PRRSV and HP-PRRSV is highly important. In this study, an infectious clone of a commercial MLV vaccine strain, GD (HP-PRRSV), was first generated (named rGD). A recombinant chimeric PRRSV strain, rGD-SX-5U2, was subsequently constructed by using rGD as a backbone and embedding several dominant immune genes, including the NSP2, ORF5, ORF6, and ORF7 genes, from an NADC30-like PRRSV isolate. In vitro experiments demonstrated that chimeric PRRSV rGD-SX-5U2 exhibited high tropism for MARC-145 cells, which is of paramount importance in the production of PRRSV vaccines. Moreover, subsequent in vivo inoculation and challenge experiments demonstrated that rGD-SX-5U2 confers cross-protection against both HP-PRRSV and NADC30-like PRRSV, including an improvement in ADG levels and a reduction in viremia and lung tissue lesions. In conclusion, our research demonstrated that the chimeric PRRSV strain rGD-SX-5U2 is a novel approach that can provide broad-spectrum protection against both HP-PRRSV and NADC30-like PRRSV. This may be a significant improvement over previous MLV vaccinations.

Effect of Initial Inoculum on the Temporal and Spatial Dynamics of Wheat Blast Under Field Conditions in Bolivia.

Epidemiological studies to better understand wheat blast (WB) spatial and temporal patterns were conducted in three field environments in Bolivia between 2019 and 2020. The temporal dynamics of wheat leaf blast (WLB) and spike blast (WSB) were best described by the logistic model compared with the Gompertz and exponential models. The nonlinear logistic infection rates were higher under defined inoculation in experiments two and three than under undefined inoculation in experiment one, and they were also higher for WSB than for WLB. The onset of WLB began with a spatial clustering pattern according to autocorrelation analysis and Moran's index values, with higher severity and earlier onset for defined than for undefined inoculation until the last sampling time. The WSB onset did not start with a spatial clustering pattern; instead, it was detected later until the last sampling date across experiments, with higher severity and earlier onset for defined than for undefined inoculation. Maximum severity (Kmax) was 1.0 for WSB and less than 1.0 for WLB. Aggregation of WLB and WSB was higher for defined than for undefined inoculation. The directionality of hotspot development was similar for both WLB and WSB, mainly occurring concentrically for defined inoculation. Our results show no evidence of synchronized development but suggest a temporal and spatial progression of disease symptoms on wheat leaves and spikes. Thus, we recommend that monitoring and management of WB should be considered during early growth stages of wheat planted in areas of high risk.

Leucoanthocyanidin Reductase 3 (PaLAR3) Locus in Norway Spruce (Picea abies) and Its Link to Resistance Against Heterobasidion parviporum

ABSTRACTHeterobasidion parviporum is a fungal pathogen that is drastically damaging Norway spruce (Picea abies) in Europe. The infections will result in root and stem rot, causing significant economic losses for forest owners. Previous studies have shown that the PaLAR3 gene, which encodes the leucoanthocyanidin reductase enzyme, can increase resistance to H. parviporum in Norway spruce. The presence of the B allele at the PaLAR3 locus has been associated with higher (+)‐catechin concentrations and increased enzyme production in inoculation experiments, resulting in inhibited pathogen growth. The control of H. parviporum involves a multifaceted approach, including silvicultural and sustainable forestry practices, genetic resistance and chemical/biological control. In this study, we determined the PaLAR3 genotypes in a representative sample of Norway spruce breeding materials from southern Finland and examined their effect on necrosis caused by H. parviporum in spruce stems. The results showed that the homozygous PaLAR3BB genotype was present in only 9% of the trees. However, the necrotic area interacted with homozygous PaLAR3BB under low‐water treatment. These findings support the idea that the PaLAR3 locus may be a valuable marker for identifying P. abies resistance to different strains of Heterobasidion parviporum.

Rpv10.2: A Haplotype Variant of Locus Rpv10 Enables New Combinations for Pyramiding Downy Mildew Resistance Traits in Grapevine.

In viticulture, pathogens like the oomycete Plasmopara viticola, the causal agent of downy mildew, can cause severe yield loss and require extensive application of plant protection chemicals. Breeders are generating pathogen-resistant varieties exploiting American and Asian wild Vitis germplasm as sources of resistance. Several loci mediating resistance to P. viticola have been identified in the past but may be overcome by specifically adapted strains of the pathogen. Aiming to find and characterize novel loci, a cross population with Vitis amurensis ancestry was investigated searching for resistance-correlated quantitative trait loci (QTL). As a prerequisite, a genetic map was generated by analyzing the 244 F1 individuals derived from a cross of the downy mildew susceptible Vitis vinifera cultivar 'Tigvoasa' and the resistant V. amurensis pBC1 breeding line We 90-06-12. This genetic map is based on the information from 627 molecular markers including 56 simple sequence repeats and 571 rhAmpSeq markers. A phenotypic characterization of the progeny showed a clear segregation of the resistance traits in the F1 population after an experimental inoculation of leaf discs with downy mildew. Combining genetic and phenotypic data, an analysis for QTL revealed a major locus on linkage Group 9 that correlates strongly with the resistance to downy mildew. The locus was mapped to a region of about 80 kb on the PN40024 (12x.V2) grapevine reference genome. This genomic region co-localizes with the formerly identified locus Rpv10 from the grapevine cultivar 'Solaris'. As we found different allele sizes of the locus-linked SSR markers than those characterizing the known Rpv10 locus and differences in the sequence of a candidate gene, it was regarded as a haplotype variant and named Rpv10.2.

Replication of the Venezuelan Equine Encephalitis Vaccine from a Synthetic PCR Fragment.

There is no approved human vaccine for Venezuelan equine encephalitis (VEE), a life-threatening disease caused by the VEE virus (VEEV). In previous studies, plasmid DNA encoding the full-length RNA genome of the VEE V4020 vaccine was used for the preparation of experimental live virus VEE vaccines in the plasmid-transfected cell culture. Here, we used the high-fidelity polymerase chain reaction (PCR) to prepare synthetic, transcriptionally active PCR (TAP) fragments encoding the V4020 genome. TAP fragment initiated the replication of the V4020 live virus vaccine in TAP fragment-transfected cells. A transfection of less than 1 ug of TAP fragment resulted in the replication of the V4020 vaccine virus in CHO cells. We conclude that not only plasmid DNA but also synthetic PCR-generated DNA fragments can be used for the manufacturing of live vaccines for VEEV and, potentially, other viruses.

Multi-omics analysis reveals regime shifts in the gastrointestinal ecosystem in chickens following anticoccidial vaccination and Eimeria tenella challenge.

Coccidiosis, caused by Eimeria parasites, significantly impacts poultry farm economics and animal welfare. Beyond its direct impact on health, Eimeria infection disrupts enteric microbial populations leading to dysbiosis and increases vulnerability to secondary diseases such as necrotic enteritis, caused by Clostridium perfringens. The impact of Eimeria infection or anticoccidial vaccination on host gastrointestinal phenotypes and enteric microbiota remains understudied. In this study, the metabolomic profiles and microbiota composition of chicken caecal tissue and contents were evaluated concurrently during a controlled experimental vaccination and challenge trial. Cobb500 broilers were vaccinated with a Saccharomyces cerevisiae-vectored anticoccidial vaccine and challenged with 15,000 Eimeria tenella oocysts. Assessment of caecal pathology and quantification of parasite load revealed correlations with alterations to caecal microbiota and caecal metabolome linked to infection and vaccination status. Infection heightened microbiota richness with increases in potentially pathogenic species, while vaccination elevated beneficial Bifidobacterium. Using a multi-omics factor analysis, data on caecal microbiota and metabolome were integrated and distinct profiles for healthy, infected, and recovering chickens were identified. Healthy and recovering chickens exhibited higher vitamin B metabolism linked to short-chain fatty acid-producing bacteria, whereas essential amino acid and cell membrane lipid metabolisms were prominent in infected and vaccinated chickens. Notably, vaccinated chickens showed distinct metabolites related to the enrichment of sphingolipids, important components of nerve cells and cell membranes. Our integrated multi-omics model revealed latent biomarkers indicative of vaccination and infection status, offering potential tools for diagnosing infection, monitoring vaccination efficacy, and guiding the development of novel treatments or controls.IMPORTANCEAdvances in anticoccidial vaccines have garnered significant attention in poultry health management. However, the intricacies of vaccine-induced alterations in the chicken gut microbiome and its subsequent impact on host metabolism remain inadequately explored. This study delves into the metabolic and microbiotic shifts in chickens post-vaccination, employing a multi-omics integration analysis. Our findings highlight a notable synergy between the microbiome composition and host-microbe interacted metabolic pathways in vaccinated chickens, differentiating them from infected or non-vaccinated cohorts. These insights pave the way for more targeted and efficient approaches in poultry disease control, enhancing both the efficacy of vaccines and the overall health of poultry populations.