Assessment of African swine fever vaccine candidate ASFV-G-∆MGF in a reversion to virulence study.

African swine fever (ASF) has gained panzootic dimensions and commercial vaccines are still unavailable. Recently, a series of live attenuated vaccines has raised hope for an efficacious and safe vaccine, among them "ASFV-G-∆MGF". We tested the latter in an in vivo reversion to virulence study in accordance with International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products guidelines. Upon forced animal passaging, a virus variant emerged that was associated with transient fever and an increased replication and shedding. However, all animals were healthy upon completion of the study and reversion to significant virulence was not observed. The genomic changes did not affect the recombination site but involved deletions and reorganizations in the terminal regions of the genome. Thus, our study underscores that in-depth safety characterization is needed for live ASF vaccines. For this particular candidate, additional studies should target long-term effects and transmission characteristics before thorough benefit-risk analysis can be carried out.

Pathology of African Swine Fever in Reproductive Organs of Mature Breeding Boars.

African swine fever (ASF) is a severe, globally important disease in domestic and wild pigs. The testing of alternative transmission routes has proven that the ASF virus (ASFV) can be efficiently transmitted to sows via semen from infected boars through artificial insemination. Boars intramuscularly inoculated with the ASFV strain "Estonia 2014" showed grossly and microscopically visible changes in the testis, epididymis, prostate, and vesicular gland. The gross lesions included hemorrhages on the scrotum, testicular membranes, and parenchyma; edema; hydroceles; and proliferations of the tunica vaginalis. Histopathologically, vasculitis and perivasculitis was detected in the testis and epididymis. Subacutely infected animals further revealed a degeneration of the testicular and epididymal tubules, pointing to the destruction of the blood-testis and blood-epididymis barriers upon disease progression. This was confirmed by evidence of semen round cells and sperm abnormalities at later time points after the infection. The histopathology was associated with the presence of viral DNA and the infectious virus, and in a limited amount with viral antigens. In most scenarios, the impact of these changes on the reproductive performance and long-term persistence of the virus is probably negligible due to the culling of the animals. However, under backyard conditions and in wild boar populations, infected males will remain in the population and the long-term fate should be further evaluated.

Composting of Wild Boar Carcasses in Lithuania Leads to Inactivation of African Swine Fever Virus in Wintertime.

African swine fever (ASF) continues to spread and persist in the Eurasian wild boar population. The infection pressure resulting from infected carcasses in the environment can be a major contributor to disease persistence and spread. For this reason, it is crucial to find a safe and efficient method of carcass disposal under different circumstances. In the presented study, we investigated open-air composting of carcasses under winter conditions in northeastern Europe, i.e., Lithuania. We can demonstrate that the ASF virus (ASFV) is inactivated in both entire wild boar carcasses and pieces thereof in a time- and temperature-dependent manner. Composting piles reached up to 59.0 °C, and ASFV was shown to be inactivated. However, the ASFV genome was still present until the end of the 112-day sampling period. While further studies are needed to explore potential risk factors (and their mitigation), such as destruction of composting piles by scavengers or harsh weather conditions, composting seems to present a valid method to inactivate the ASFV in wild boar carcasses where rendering or other disposal methods are not feasible. In summary, composting provides a new tool in our toolbox of ASF control in wild boar and can be considered for carcass disposal.

Artificial Insemination as an Alternative Transmission Route for African Swine Fever Virus.

The rapid spread of the African swine fever virus (ASFV), causing severe disease with often high fatality rates in Eurasian suids, prevails as a threat for pig populations and dependent industries worldwide. Although advancing scientific progress continually enhances our understanding of ASFV pathogenesis, alternative transmission routes for ASFV have yet to be assessed. Here, we demonstrate that ASFV can efficiently be transferred from infected boars to naïve recipient gilts through artificial insemination (AI). In modern pig production, semen from boar studs often supplies many sow herds. Thus, the infection of a boar stud presents the risk of rapidly and widely distributing ASFV within or between countries. Daily blood and semen collection from four boars after intramuscular inoculation with ASFV strain 'Estonia 2014' resulted in the detection of ASFV genomes in the semen as early as 2 dpi, in blood at 1 dpi while semen quality remained largely unaffected. Ultimately, after insemination with extended semen, 7 of 14 gilts were ASFV positive by 7 days post insemination, and all gilts were ASFV positive by 35 days post insemination. Twelve out of 13 pregnant gilts aborted or resorbed at the onset of fever. A proportion of fetuses originating from the remaining gilt showed both abnormalities and replication of ASFV in fetal tissues. Thus, we present evidence for the efficient transmission of ASFV to gilts via AI and also to implanted embryos. These results underline the critical role that boar semen could play in ASFV transmission.

Taking a Promising Vaccine Candidate Further: Efficacy of ASFV-G-ΔMGF after Intramuscular Vaccination of Domestic Pigs and Oral Vaccination of Wild Boar.

African swine fever (ASF) is a pandemic threat to the global pig industry and wild suids. A safe and efficacious vaccine could monumentally assist in disease eradication. In the past years, promising live attenuated vaccine candidates emerged in proof-of-concept experiments, among which was "ASFV-G-∆MGF". In our study, we tested the vaccine candidate in three animal experiments intramuscularly in domestic pigs and orally in wild boar. Further, a macrophage-grown vaccine virus and a virus grown on permanent cells could be employed. Irrespective of the production system of the vaccine virus, a two-dose intramuscular immunization could induce close-to-sterile immunity with full clinical protection against challenge infection. After oral immunization, 50% of the vaccinees seroconverted and all responders were completely protected against subsequent challenge. All nonresponders developed ASF upon challenge with two acute lethal infections and two mild and transient courses. The latter results show a lower efficiency after oral administration that would have to be taken into consideration when designing vaccination-based control measures. Overall, our findings confirm that "ASFV-G-∆MGF" is a most promising vaccine candidate that could find its way into well-organized and controlled immunization campaigns. Further research is needed to characterize safety aspects and define possible improvements of oral efficiency.

Impact of ASFV Detergent Inactivation on Biomarkers in Serum and Saliva Samples.

African swine fever (ASF) is a notifiable viral disease of domestic and wild suids. Despite intensive research efforts, the pathogenesis of the disease is still far from being understood. Analysis of biomarkers in different body fluids may supplement traditional pathogenesis studies. As reliable protocols are often established in laboratories with lower biosafety, the reliable inactivation of samples is crucial. The objective of this study was to find a procedure that inactivates the virus while preserving the biomarkers for downstream analyses. To this means, three different inactivation protocols were employed, namely Tergitol-type NP-40 (NP-40), polyoxyethylene-p-t-octylphenol (Triton X-100) and one with 95 °C heating. It could be demonstrated that all samples treated with 0.5% (v/v) concentration of both detergents showed an absence of virus infectivity. The same was true for heated samples. However, heated serum was not suitable for analyses. Next, the impact of treatment on biomarker readouts was assessed. While all protocols had an impact on the detection of biomarkers, correlation was retained. In particular, NP-40 may be the desired detergent for more accurate measurements while achieving efficient virus inactivation. Based on these studies, samples can be reliably inactivated for most biomarker analyses, and thus broader interdisciplinary cooperation is possible.

Efficacy of Liming Forest Soil in the Context of African Swine Fever Virus.

Since September 2020, Germany has experienced the first ever outbreak of African swine fever (ASF). The first known cases occurred exclusively in wild boar in forest areas in Brandenburg and Saxony; in July 2021, infected domestic pigs were also confirmed for the first time. As wild boar are considered the main reservoir for the virus in the European region, an effective interruption of this infection chain is essential. In particular, the removal and safe disposal of infected carcasses and the direct disinfection of contaminated, unpaved ground are priorities in this regard. For the disinfection, highly potent as well as environmentally compatible disinfectants must be used, which are neither influenced in their effectiveness by the soil condition nor by increased organic contamination. Thus, in this study, slaked lime, milk of lime and quicklime (1% to 10% solutions) were selected for efficacy testing against the test virus recommended by the German Veterinary Society (DVG), Modified Vaccinia Ankara virus (MVAV), and ASF virus (ASFV) in conjunction with six different forest soils from Saxony in two different soil layers (top soil and mineral soil) each. In summary, 10% of any tested lime type is able to inactivate both MVAV and ASFV under conditions of high organic load and independent of the water content of the soil. At least a 4 log reduction of the virus titer in all tested forest soil types and layers and by all applied lime types was observed. In conclusion, the high efficacy and suitability of all tested lime products against both viruses and in the presence of high organic load in forest soil can be confirmed and will help to control ASF spread.

Performance Characteristics of Real-Time PCRs for African Swine Fever Virus Genome Detection-Comparison of Twelve Kits to an OIE-Recommended Method.

African swine fever (ASF) is a major threat to pig production, and real-time PCR (qPCR) protocols are an integral part of ASF laboratory diagnosis. With the pandemic spread of ASF, commercial kits have risen on the market. In Germany, the kits have to go through an approval process and thus, general validation can be assumed. However, they have never been compared to each other. In this study, 12 commercial PCR kits were compared to an OIE-recommended method. Samples representing different matrices, genome loads, and genotypes were included in a panel that was tested under diagnostic conditions. The comparison included user-friendliness, internal controls, and the time required. All qPCRs were able to detect ASFV genome in different matrices across all genotypes and disease courses. With one exception, there were no significant differences when comparing the overall mean. The overall specificity was 100% (95% CI 87.66-100), and the sensitivity was between 95% and 100% (95% CI 91.11-100). As can be expected, variability concerned samples with low genome load. To conclude, all tests were fit for purpose. The test system can therefore be chosen based on compatibility and prioritization of the internal control system.

The Cellular Innate Immune Response of the Invasive Pest Insect Drosophila suzukii against Pseudomonas entomophila Involves the Release of Extracellular Traps.

Drosophila suzukii is a neobiotic invasive pest that causes extensive damage to fruit crops worldwide. The biological control of this species has been unsuccessful thus far, in part because of its robust cellular innate immune system, including the activity of professional phagocytes known as hemocytes and plasmatocytes. The in vitro cultivation of primary hemocytes isolated from D. suzukii third-instar larvae is a valuable tool for the investigation of hemocyte-derived effector mechanisms against pathogens such as wasp parasitoid larvae, bacteria, fungi and viruses. Here, we describe the morphological characteristics of D. suzukii hemocytes and evaluate early innate immune responses, including extracellular traps released against the entomopathogen Pseudomonas entomophila and lipopolysaccharides. We show for the first time that D. suzukii plasmatocytes cast extracellular traps to combat P. entomophila, along with other cell-mediated reactions, such as phagocytosis and the formation of filopodia.

Epidemiological approach to nematode polyparasitism occurring in a sympatric wild ruminant multi-host scenario.

The epidemiology behind multi-host/multi-parasite systems is particularly interesting to investigate for a better understanding of the complex dynamics naturally occurring in wildlife populations. We aimed to approach the naturally occurring polyparasitism of gastrointestinal nematodes in a sympatric wild ruminant scenario present in south-east Spain. To this end, the gastrointestinal tract of 252 wild ruminants of four different species (red deer, Cervus elaphus; mouflon, Ovis aries musimon; Iberian ibex, Capra pyrenaica and fallow deer, Dama dama) were studied in Cazorla, Segura y Las Villas Natural Park (Andalusia, Spain). Of the analysed animals, 81.52% were positive for parasite infection and a total of 29 nematode species were identified. Out of these, 25 species were detected in at least two host species and 11 parasitized all ruminant species surveyed. The multi-host interaction between these nematodes and the four host species is discussed under the perspective of host family-based differences.

Organization of the Structural Protein Region of La Jolla Virus Isolated from the Invasive Pest Insect Drosophila suzukii.

Drosophila suzukii (Ds) is an invasive pest insect that infests ripening fruit, causing severe economic losses. Control measures based on chemical pesticides are inefficient and undesirable, so biological alternatives have been considered, including native Ds viruses. We previously isolated a strain of La Jolla virus (LJV-Ds-OS20) from Ds in Germany as a candidate biopesticide. Here we characterized the new strain in detail, focusing on the processing of its capsid proteins. We tested LJV growth during Ds development to optimize virus production, and established a laboratory production system using adult flies. This system was suitable for the preparation of virions for detailed analysis. The LJV-Ds-OS20 isolate was cloned by limiting dilution and the complete nucleotide sequence was determined as a basis for protein analysis. The terminal segments of the virus genome were completed by RACE-PCR. LJV virions were also purified by CsCl gradient centrifugation and analyzed by SDS-PAGE and electron microscopy. The capsid proteins of purified LJV virions were resolved by two-dimensional SDS-PAGE for N-terminal sequencing and peptide mass fingerprinting. The N-terminal sequences of VP1 and VP2, together with MS data representing several capsid proteins, allowed us to develop a model for the organization of the LJV structural protein region. This may facilitate the development of new viral strains as biopesticides.

The Role of Male Reproductive Organs in the Transmission of African Swine Fever-Implications for Transmission.

African swine fever (ASF) has evolved from an exotic animal disease to a threat to global pig production. An important avenue for the wide-spread transmission of animal diseases is their dissemination through boar semen used for artificial insemination. In this context, we investigated the role of male reproductive organs in the transmission of ASF. Mature domestic boars and adolescent wild boars, inoculated with different ASF virus strains, were investigated by means of virological and pathological methods. Additionally, electron microscopy was employed to investigate in vitro inoculated sperm. The viral genome, antigens and the infectious virus could be found in all gonadal tissues and accessory sex glands. The viral antigen and viral mRNAs were mainly found in mononuclear cells of the respective tissues. However, some other cell types, including Leydig, endothelial and stromal cells, were also found positive. Using RNAScope, p72 mRNA could be found in scattered halo cells of the epididymal duct epithelium, which could point to the disruption of the barrier. No direct infection of spermatozoa was observed by immunohistochemistry, or electron microscopy. Taken together, our results strengthen the assumption that ASFV can be transmitted via boar semen. Future studies are needed to explore the excretion dynamics and transmission efficiency.

Identification of entomopathogenic bacteria associated with the invasive pest Drosophila suzukii in infested areas of Germany.

The invasive insect pest Drosophila suzukii causes extensive damage to soft-skinned fruit crops as they ripen. Current control methods involve the application of chemical pesticides, but this approach is ineffective and environmentally hazardous. To investigate the potential of bacterial pathogens carried by D. suzukii as biocontrol agents, we characterized bacteria associated with D. suzukii larvae in two parts of Hesse, Germany, by collecting infested fruits and culturing individual bacteria from moribund specimens for taxonomic classification by 16S rDNA sequencing. Among the bacteria we detected, some had a detrimental effect on the host whereas others were neutral or beneficial. When the detrimental and beneficial bacteria were presented simultaneously, we observed complex tripartite interactions that modulated the insect's innate immune response. Our study provides insight into the complex relationships within the microbiome and pathobiome of D. suzukii and may lead to the isolation of bacteria that can be used as biological control agents.

The infection of Harmonia axyridis by a parasitic nematode is mediated by entomopathogenic bacteria and triggers sex-specific host immune responses.

The harlequin ladybird Harmonia axyridis is native to Asia but has been introduced into many countries as a biological control agent. It is now considered an invasive pest, threatening the biodiversity of native ladybirds globally, in part because of its superior immune system. H. axyridis is infected and killed by the parasitic nematode Parasitylenchus bifurcatus, which could therefore be developed as a biological strategy to counter the spread of this insect pest. However, effective control requires an understanding of the tripartite relationship between H. axyridis, P. bifurcatus and their potential bacterial mutualists. Here we describe the isolation of two species of nematode-associated bacteria (Serratia marcescens and Providencia rettgeri) which were highly virulent against H. axyridis in survival experiments. In addition, contact between the nematodes and beetles led to the sex-specific modulation of multiple host immunity-related genes after 24 and 48 h, with many genes encoding antimicrobial peptides rapidly and stably repressed in females whereas the same genes were initially induced in males before suppression at the later time point. These data provide evidence that the female immune system responds much more strongly to the nematodes and provokes, in turn, a more robust invasion strategy involving the bacterial mutualists.

Identification and characterization of natural viruses associated with the invasive insect pest Drosophila suzukii.

The invasive insect pest Drosophila suzukii infests ripening fruits and causes extensive damage to crops in the northern hemisphere. Novel, environmentally friendly strategies to control the spread of this species are urgently needed, and one promising approach is the deployment of entomopathogenic viruses. Here we report the identification and characterization of two natural viruses associated with D. suzukii: Drosophila A virus (DAV) and La Jolla virus (LJV). Our work provides new tools for the development of biological control agents that protect crops against D. suzukii without a harmful impact on biodiversity.

Leukocytes coincubated with human sperm trigger classic neutrophil extracellular traps formation, reducing sperm motility.

OBJECTIVE: To determine whether the human spermatozoon is a sufficient stimulus to trigger the release of neutrophil extracellular traps (NETs) in a time- and dose-dependent manner. DESIGN: Experimental study. SETTING: University-based laboratory. PATIENT(S): Semen samples from four men and blood samples from six healthy female donors. INTERVENTION(S): Polymorphonuclear neutrophils (PMN) isolated from peripheral blood were incubated with fresh human spermatozoa for 60, 90, 120, and 180 minutes and at different PMN/sperm concentrations (1:1 [25 × 104], 1:3 [25 × 104:75 × 104], 1:6 [25 × 104:15 × 105], 1:18 [25 × 104:45 × 105]). MAIN OUTCOME MEASURE(S): During coincubation of PMN/sperm, the release of NETs was measured by PicoGreen. Immunofluorescence for histone H3, neutrophil elastase (NE), and myeloperoxidase (MPO) was performed. Different NETs inhibitors were tested: diphenylene iodonium, Suc-Ala- Ala-Pro-Val chloromethyl ketone (CMK), and 4-aminobenzoic acid hydrazide (ABAH) inhibitors of NADPH oxidase, NE, and MPO. Progressive mobility was assessed at increasing doses of neutrophils (1:18 [25 × 104:45 × 105], 6:18 [15 × 105:45 × 105], 9:18 [252 × 104:45 × 105]). RESULT(S): The quantity of NETs increased at the ratio of 1:6 after 2 hours and continued to increase subsequently. A ratio of 1:18 showed significant increases in NETs production at all times. Assessment of the inhibitors showed that CMK and ABAH inhibit NETs formation. Scanning and transmission electron microphotographs and immunofluorescence confirmed NETs formation induced by the spermatozoa. After 1 hour, progressive motility diminished in the two groups with the highest proportion of neutrophils and after 2 hours in all groups exposed to neutrophils. CONCLUSION(S): We show that the stimulus of the human spermatozoon triggers the release of NETs; this response is dose dependent and increases with exposure time. The motility of affected spermatozoa diminishes, suggesting that this interaction on a larger scale would decrease the probability of successful fertilization.

First description of an in vitro culture system for Eimeria ovinoidalis macromeront formation in primary host endothelial cells.

The apicomplexan parasite Eimeria ovinoidalis is distributed worldwide and causes clinical ovine coccidiosis. As one of the most pathogenic species in sheep, the principal clinical sign is profuse diarrhoea in young animals, which leads to important economic losses in the ovine industry. We here aimed to establish an in vitro culture system for the development of E. ovinoidalis macromeronts, as no suitable systems are currently available for any ovine Eimeria species. Faecal samples containing more than 90% of E. ovinoidalis oocysts were collected from naturally infected lambs and ewes in Murcia Region (Spain). E. ovinoidalis oocysts were collected, left to sporulate in potassium dichromate and stored at 4°C until further studies were conducted. Moreover, a suitable excystation protocol was effectively established, resulting in the release of viable sporozoites, which were allowed to infect primary bovine umbilical vein endothelial cells (BUVEC) and permanent bovine colonic epithelial cells (BCEC). In vitro first merogony was successfully accomplished exclusively in BUVEC leading to macromeront formation (up to 100µm) and the release of fully developed and viable merozoites I stages. Given that we were able to establish a suitable in vitro system for the first merogony of such pathogenic Eimeria species in sheep, advances might be further made not only on studies regarding the control of ovine coccidiosis, such as drug screenings, but also on the better understanding of molecular parasite-host cell interactions as already demonstrated for other ruminant Eimeria species.