Pathogens

African swine fever virus (ASFv) is one of the most challenging pathogens to affect both domestic and wild pigs. The disease has now spread to Europe and Asia, causing great damage to the pig industry. Although no commercial vaccine with which to control the disease is, as yet, available, some potential vaccine candidates have shown good results in terms of protection. However, little is known about the host immune mechanisms underlying that protection, especially in wild boar, which is the main reservoir of the disease in Europe. Here, we study the role played by two cytokines (IL-10 and IFN-γ) in wild boar orally inoculated with the attenuated vaccine candidate Lv17/WB/Rie1 and challenged with a virulent ASFv genotype II isolate. A group of naïve wild boar challenged with the latter isolate was also established as a control group. Our results showed that both cytokines play a key role in protecting the host against the challenge virus. While high levels of IL-10 in serum may trigger an immune system malfunctioning in challenged animals, the provision of stable levels of this cytokine over time may help to control the disease. This, together with high and timely induction of IFN-γ by the vaccine candidate, could help protect animals from fatal outcomes. Further studies should be conducted in order to support these preliminary results and confirm the role of these two cytokines as potential markers of the evolution of ASFV infection. Full article

Domestic rabbits are commercially farmed for their meat whilst gastrointestinal diseases can hinder their production. Passalurus ambiguus and Eimeria spp. are two common rabbit intestinal parasites that can cause diarrhoea, among other symptoms, and in severe cases, death. C. guttulatus is a commensal yeast of the rabbits’ stomach that is considered apathogenic but can worsen symptoms in rabbits suffering from coccidiosis. In the present case report, we describe an outbreak of deaths in three different age groups (A: lactating does, B: 58 days old and C: 80 days old) in an industrial rabbit farm in Greece. Symptoms included depression, diarrhoea, inappetence, weight loss, dehydration and ruffled furs. Using a faecal flotation technique, sick rabbits were found to be moderately to heavily infected with P. ambiguus, Eimeria spp. and C. guttulatus. Treatment with fenbendazole and oregano oil combined with hygiene control measures successfully controlled the infections and resolved clinical symptoms. A faecal flotation method or other reliable diagnostic technique should be used regularly in industrial rabbit farms to screen for gastrointestinal parasitic infections. Early diagnosis and control will help to maintain production levels and, therefore, limit financial losses for the farmer while ensuring animal welfare. Full article

Salpingitis is manifested as hemorrhagic follicular inflammation exudations and peritonitis, leading to reduced egg production and high culling of breeder flocks. From 2018 to 2021, increasing salpingitis during egg peak is threatening the poultry industry post-artificial insemination, both in breeder layers and breeder ducks across China. In our study, Escherichia coli (E. coli), Enterococcus faecalis (E. faecalis) and Chlamydia psittaci (C. psittaci) were isolated and identified from the diseased oviducts using biochemical tests and PCR. To identify and isolate pathogenicity, we inoculated the isolates into laying hens via an intravaginal route. Later, laying hens developed typical salpingitis after receiving the combination of the aforementioned three isolates (1 × 10⁵ IFU/mL of C. psittaci and 1 × 10⁶ CFU/mL of E. faecalis and E. coli, respectively), while less oviduct inflammation was observed in the layers inoculated with the above isolate alone. Furthermore, 56 breeder ducks were divided into seven groups, eight ducks per group. The birds received the combination of three isolates, synergic infection of E. coli and E. faecalis, and C. psittaci alone via vaginal tract, while the remaining ducks were inoculated with physiological saline as the control group. Egg production was monitored daily and lesions of oviducts and follicles were determined post-infection on day 6. Interestingly, typical salpingitis, degenerated follicles and yolk peritonitis were obviously found in the synergic infection of three isolates and the birds inoculated with C. psittaci alone developed hemorrhagic follicles and white exudates in oviducts, while birds with E. faecalis or E. coli alone did not develop typical salpingitis. Finally, higher E. coli loads were determined in the oviducts as compared to E. faecalis and C. psittaci infection. Taken together, the combination of E. coli and E. faecalis, and C. psittaci could induce typical salpingitis and yolk peritonitis both in laying hens and breeder ducks. Secondary infection of E. coli and E. faecalis via artificial insemination is urgently needed for investigation against salpingitis. Full article

More than 1 year has passed since social activities have been restricted due to the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). More recently, novel SARS-CoV-2 variants have been spreading around the world, and there is growing concern that they may have higher transmissibility and that the protective efficacy of vaccines may be weaker against them. Immediate measures are needed to reduce human exposure to the virus. In this study, the antiviral efficacy of deep-ultraviolet light-emitting diode (DUV-LED) irradiation (280 ± 5 nm, 3.75 mW/cm²) against three SARS-CoV-2 variants was evaluated. For the B.1.1.7, B.1.351, and P.1 variant strains, irradiation of the virus stocks for 1 s resulted in infectious titer reduction rates of 96.3%, 94.6%, and 91.9%, respectively, and with irradiation for 5 s, the rates increased to 99.9%, 99.9%, and 99.8%, respectively. We also tested the effect of pulsed DUV-LED irradiation (7.5 mW/cm², duty rate: 50%, frequency: 1 kHz) under the same output conditions as for continuous irradiation and found that the antiviral efficacy of pulsed and continuous irradiation was the same. These findings suggest that by further developing and optimizing the DUV-LED device to increase its output, it may be possible to instantly inactivate SARS-CoV-2 with DUV-LED irradiation. Full article

Chagas disease is caused by the parasite Trypanosoma cruzi, which is transmitted by insect-vectors in the taxonomic subfamily Triatominae and affects approximately 8,000,000 people world-wide. Current mitigation strategies for Chagas focus on insecticides, infrastructure improvements, and management of symptoms, which are largely unsustainable in underserved communities where the disease is widespread. Transmission patterns of vector-borne diseases are known to adaptively respond to habitat change; as such, the objective of our study was to evaluate how the physical characteristics of Triatoma dimidiata would vary in relation to land use in El Salvador. We hypothesized that the color and morphology of T. dimidiata would change with municipal levels of urban and natural green space, natural green space, and agricultural space, as well as municipal diversity, richness, and evenness of land use types. Our results characterize how T. dimidiata color and morphology vary directly with anthropogenic changes to natural and agricultural environments, which are reflective of a highly adaptable population primed to respond to environmental change. Mitigation studies of Chagas disease should exploit the relationships between anthropogenic land use and T. dimidiata morphology to evaluate how the transmission pattern of T. cruzi and Chagas disease symptomology are impacted. Full article

A total of 15 RT-PCR confirmed COVID-19 patients were admitted to our hospital during the in-itial outbreak in Taiwan. The average time of virus clearance was delayed in seven patients, 24.14 ± 4.33 days compared to 10.25 ± 0.56 days post-symptom onset (PSO) in the other eight pa-tients. There was strong antibody response in patients with viral persistence at the pharynx, with peak values of serum antibody 677.2 ± 217.8 vs. 76.70 ± 32.11 in patients with delayed versus rapid virus clearance. The patients with delayed viral clearance had excessive antibodies of compromised quality in an early stage with the delay in peak virus neutralization efficacy, 34.14 ± 7.15 versus 12.50 ± 2.35 days PSO in patients with rapid virus clearance. Weak antibody re-sponse of patients with rapid viral clearance was also effective, with substantial and comparable neutralization efficacy, 35.70 ± 8.78 versus 41.37 ± 11.49 of patients with delayed virus clearance. Human Cytokine 48-Plex Screening of the serial sera samples revealed elevated concentrations of proinflammatory cytokines and chemokines in a deceased patient with delayed virus clear-ance and severe disease. The levels were comparatively less in the other two patients who suf-fered from severe disease but eventually survived. Full article

Recently, recombinant monoclonal antibodies (mAbs) of three Ig isotypes (IgG, IgA, and IgM) sharing the same anti-spike protein Fab region were developed; we evaluated their neutralizing abilities using a pseudo-typed lentivirus coated with the SARS-CoV-2 spike protein and ACE2-transfected Crandell–Rees feline kidney cells as the host cell line. Although each of the anti-SARS-CoV-2 mAbs was able to neutralize the spike-coated lentiviruses, IgM and IgA neutralized the viral particles at 225-fold and 125-fold lower concentrations, respectively, than that of IgG. Our finding that the neutralization ability of Igs with the same Fab domain was dramatically higher for IgM and IgA than IgG mAbs suggests a strategy for developing effective and affordable antibody therapies for COVID-19. The efficient neutralization conferred by IgM and IgA mAbs can be explained by their capacity to bind multiple virions. While several IgG mAbs have been approved as therapeutics by the FDA, there are currently no IgM or IgA mAbs available. We suggest that mAbs with multiple antigen-binding sites such as IgM and IgA could be developed as the new generation of therapy. Full article

The possibilities for diagnosing prion diseases have shifted significantly over the last 10 years. The RT-QuIC assay option has been added for neuropsychiatric symptoms, supporting biomarkers and final post-mortem confirmation. Samples of brain homogenates used for final diagnosis, archived for many years, provide the possibility for retrospective studies. We used a second-generation RT-QuIC assay to detect seeding activity in different types of sporadic and genetic prion diseases in archival brain homogenates and post-mortem CSF samples that were 2 to 15 years old. Together, we tested 92 archival brain homogenates: 39 with definite prion disease, 28 with definite other neurological disease, and 25 with no signs of neurological disorders. The sensitivity and specificity of the assay were 97.4% and 100%, respectively. Differences were observed in gCJD E200K, compared to the sporadic CJD group. In 52 post-mortem CSF samples—24 with definite prion disease and 28 controls—we detected the inhibition of seeding reaction due to high protein content. Diluting the samples eliminated such inhibition and led to 95.8% sensitivity and 100% specificity of the assay. In conclusion, we proved the reliability of archived brain homogenates and post-mortem CSF samples for retrospective analysis by RT-QuIC after long-term storage, without changed reactivity. Full article

Fasciola hepatica is a widespread pathogen that is known for its harmful effects on the health and productivity of ruminant animals. To identify the proteins present in all periods of infection with F. hepatica but not in those with Fasciola gigantica by shotgun liquid chromatography–tandem mass spectrometry (LC–MS/MS), we collected the ESPs and sera of F. hepatica and F. gigantica. In this study, the sheep were artificially infected with F. hepatica and the sera were collected at five different periods: 3 days post-infection (dpi), 7 dpi, 21 dpi, 63 dpi, and 112 dpi. The interacting proteins were pulled down from the sheep sera of all five periods and the sera with F. gigantica by co-immunoprecipitation (Co-IP) assay, before being identified by LC–MS/MS analysis. Thirty, twenty-two, twenty-three, twenty-seven, and twenty-two proteins were pulled down by the infected sera at 3 dpi, 7 dpi, 21 dpi, 63 dpi, and 112 dpi, respectively. Among them, 12 proteins existed in all periods, while six proteins could be detected in all periods in F. hepatica but not in F. gigantica. Protein relative pathway analysis revealed that these proteins mainly refer to the metabolism, regulation of genetic activity, and signal transduction of F. hepatica. In conclusion, this study provides meaningful data for the diagnosis of fasciolosis and to understand the interactions between F. hepatica and the host. Full article

West Nile virus (WNV) is the most widespread flavivirus in the world with a wide vertebrate host range. Its geographic expansion and activity continue to increase with important human and equine outbreaks and local bird mortality. In a previous experiment, we demonstrated the susceptibility of 7-week-old red-legged partridges (Alectoris rufa) to Mediterranean WNV isolates Morocco/2003 and Spain/2007, which varied in virulence for this gallinaceous species. Here we study the pathogenesis of the infection with these two strains to explain the different course of infection and mortality. Day six post-inoculation was critical in the course of infection, with the highest viral load in tissues, the most widespread virus antigen, and more severe lesions. The most affected organs were the heart, liver, and spleen. Comparing infections with Morocco/2003 and Spain/2007, differences were observed in the viral load, virus antigen distribution, and lesion nature and severity. A more acute and marked inflammatory reaction (characterized by participation of microglia and CD3+ T cells) as well as neuronal necrosis in the brain were observed in partridges infected with Morocco/2003 as compared to those infected with Spain/2007. This suggests a higher neurovirulence of Morocco/2003, probably related to one or more specific molecular determinants of virulence different from Spain/2007. Full article

Here we report on an EHV-1 outbreak investigation caused by a novel genotype H₇₅₂ (histidine in amino acid position 752 of the ORF 30 gene). The outbreak involved 31 performance horses. Horses were monitored over a period of 35 days for clinical signs, therapeutic outcome and qPCR results of EHV-1 in blood and nasal secretions. The morbidity of the EHV-1 outbreak was 84% with 26 clinically infected horses displaying fever and less frequently anorexia and distal limb edema. Four horses showed mild transient neurological deficits. Clinically diseased horses experienced high viral load of EHV-1 in blood and/or nasal secretions via qPCR, while subclinically infected horses had detectable EHV-1 mainly in nasal secretions. The majority of infected horses showed a rise in antibody titers to EHV-1 during the outbreak. All 31 horses were treated with valacyclovir, while clinically infected horses further received flunixin meglumine and sodium heparin. This investigation highlights various relevant aspects of an EHV-1 outbreak caused by a new H₇₅₂ genotype: (i) importance of early detection of EHV-1 infection; (ii) diagnostic challenge to assess H₇₅₂ genotype; (iii) apparent benefit of valacyclovir use in the early stage of the outbreak; and (iv) weekly testing of blood and nasal secretions by qPCR in order to monitor individual infection status and lift quarantine. Full article

Endophytic, saprobic, and pathogenic fungi have evolved elaborate strategies to obtain nutrients from plants. Among the diverse plant-fungi interactions, the most crucial event is the attachment and penetration of the plant surface. Appressoria, specialized infection structures, have been evolved to facilitate this purpose. In this review, we describe the diversity of these appressoria and classify them into two main groups: single-celled appressoria (proto-appressoria, hyaline appressoria, melanized (dark) appressoria) and compound appressoria. The ultrastructure of appressoria, their initiation, their formation, and their function in fungi are discussed. We reviewed the molecular mechanisms regulating the formation and function of appressoria, their strategies to evade host defenses, and the related genomics and transcriptomics. The current review provides a foundation for comprehensive studies regarding their evolution and diversity in different fungal groups. Full article

Rickettsioses are arthropod-borne zoonotic diseases, several of which occur in Australia. This study aimed to assess the exposure levels and risk factors for Rickettsia spp. among Australian wildlife rehabilitators (AWRs) using serology, PCR and a questionnaire. Antibody titres against Spotted Fever Group (SFG), Typhus Group (TG) and Scrub Typhus Group (STG) antigens were determined using an immunofluorescence assay. PCR targeting the gltA gene was performed on DNA extracts from whole blood and serum. Logistic regression was used to identify risk factors associated with seropositivity. Of the 27 (22.1%; 27/122) seropositive participants all were seropositive for SFG, with 5/27 (4.1%) also positive for TG. Of the 27 positive sera, 14.8% (4/27) were further classified as exposure to R. australis, 3.7% (1/27) to R. honei, 3.7% (1/27) to R. felis and 77.8% (21/27) were classified as ‘indeterminate’—most of which (85.7%; 18/21) were indeterminate R. australis/R. honei exposures. Rickettsia DNA was not detected in whole blood or serum. Rehabilitators were more likely to be seropositive if more than one household member rehabilitated wildlife, were older than 50 years or had occupational animal contact. These findings suggest that AWRs are at increased risk of contracting Rickettsia-related illnesses, however the source of the increased seropositivity remains unclear. Full article

The potato cyst nematode (PCN) Globodera pallida has acquired significant importance throughout Europe due to its nefarious effects on potato production. Rapid and reliable diagnosis of PCN is critical during the surveillance programs and for the implementation of control measures. Molecular DNA-based methods are available, but they require expensive laboratory facilities, equipment and trained technicians. Moreover, there is an additional need of time for sample shipment and testing. In this work, we have developed a new and simple assay which reliably discriminates G. pallida from other cyst nematodes in less than 40 min. This assay may be applied either on cysts or juveniles with the ability to detect a single juvenile of G. pallida in a sample of at least 40 juveniles of the non-target species G. rostochiensis. This test should be a tool to improve the performance of the laboratory and has the potential to be performed on-site. Full article

Malaria is the most lethal parasitic disease in the world. Mortality and severity in symptoms are higher in men than women, suggesting that oestrogens, which are in higher concentration in females than in males, may regulate the immune response against malaria. Tamoxifen, a selective oestrogen receptor modulator used in breast cancer treatment due to its antagonistic effect on oestrogen receptors α and β, is also studied because of its potential therapeutic use for several parasitic diseases. However, most studies, including one in malaria, have not addressed the immunomodulatory role of tamoxifen. In this work, we evaluated the effect of tamoxifen on the immune response of CBA/Ca mice against Plasmodium berghei ANKA. This study showed for the first time that tamoxifen increased parasite load, aggravated symptoms by decreasing body temperature and body weight, and worsened anaemia. Additionally, tamoxifen significantly increased the splenic index and the percentages of CD4⁺ and NK⁺ cells on day eight post-infection. By contrast, tamoxifen decreased both CD8⁺ and B220⁺ populations in the spleen and decreased the serum levels of IL-2, IL-6, and IL-17. Our findings support the notion that tamoxifen is a potent immunomodulator in malaria-infected mice and suggest caution when administering it to malaria-infected women with breast cancer. Full article

The objective of this paper is to provide an overview of the current status of rabies in Europe, with special emphasis on Croatia and Southeast and East Europe. Due to the systematic implementation of a rabies eradication program by oral vaccination of wild animals, by the end of the 20th century, most West and Central European countries were rabies-free. The EU goal was to eradicate rabies in wildlife and domestic animals by 2020. No matter how achievable the goal seemed to be, the disease is still present in the eastern part of the EU, as was notified in 2020 by two member states—Poland and Romania. Croatia has been rabies-free for the last seven years but given that it borders a non-EU country in which a case of rabies was confirmed in 2020, it will continue to contribute to the maintenance of the rabies-free region. A rabies-free EU can only be achieved by continuous oral vaccination, coordination and a regional approach. The prevention of reintroductions from bordering countries in which rabies has not been eradicated yet, and the support for the eradication efforts made by these countries, are goals still pending. Full article

Coxiella burnetii is the causative agent of Q fever. It can infect animals, humans, and birds, as well as ticks, and it has a worldwide geographical distribution. To better understand the epidemiology of C. burnetii in Somalia, ticks infesting camels were collected from five different regions, including Bari, Nugaal, Mudug, Sool, and Sanaag, between January and March 2018. Collected ticks were tested for C. burnetii and Coxiella-like endosymbiont DNA by using IS1111, icd, and Com1-target PCR assays. Moreover, sequencing of the 16S-rRNA was conducted. Molecular characterization and typing were done by adaA-gene analysis and plasmid-type identification. Further typing was carried out by 14-marker Multi-Locus Variable-Number Tandem Repeats (MLVA/VNTR) analysis. The investigated ticks (n = 237) were identified as Hyalomma spp. (n = 227, 95.8%), Amblyomma spp. (n = 8, 3.4%), and Ripicephalus spp. (n = 2, 0.8%), and 59.1% (140/237) of them were positive for Coxiella spp. While Sanger sequencing and plasmid-type identification revealed a C. burnetii that harbours the QpRS-plasmid, MLVA/VNTR genotyping showed a new genotype which was initially named D21. In conclusion, this is the first report of C. burnetii in ticks in Somalia. The findings denote the possibility that C. burnetii is endemic in Somalia. Further epidemiological studies investigating samples from humans, animals, and ticks within the context of “One Health” are warranted. Full article

Research on alien and invasive species focuses on the direct effects of invasion on native ecosystems, and the possible positive effects of their presence are most often overlooked. Our aim was to check the suitability of selected alien species (the snail Physa acuta, the bivalve Dreissena polymorpha, and the gammarid Dikerogammarus villosus) as diluents for infectious bird schistosome cercariae—the etiological factor of swimmer’s itch. It has been hypothesized that alien species with different feeding habits (scrapers, filterers and predators) that cohabit the aquatic environment with intermediate hosts of the schistosomatid trematodes are capable of feeding on their free-swimming stages—cercariae. In the laboratory conditions used, all experimental animals diluted the cercariae of bird schistosome. The most effective diluents were P. acuta and D. villosus. However, a wide discrepancy in the dilution of the cercariae between replicates was found for gammarids. The obtained results confirm the hypothesis that increased biodiversity, even when alien species are involved, creates the dilution effect of the free-living stages of parasites. Determining the best diluent for bird schistosome cercariae could greatly assist in the development of current bathing areas protection measures against swimmer’s itch. Full article

Human milk oligosaccharides (HMOs), the third largest solid fraction in human milk, can modulate inflammation through Toll-like receptor signaling, but little is known about their immunomodulatory potential in the oral cavity. In this study, we determined whether the HMOs 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL) regulate human-beta defensin (hBD)-2 and -3, cathelicidin (hCAP18/LL-37), and cytokine responses in human gingival cells using a three-dimensional oral mucosal culture model. The model was incubated with 0.1% or 1% 2′-FL and 3-FL, alone and in combination, for 5 or 24 h, and hBD-2, hBD-3, and hCAP18/LL-37 were analyzed by immunohistochemistry. The expression profiles of interleukin (IL)-1, IL-1RA, IL-8, and monocyte chemoattractant protein (MCP)-1 were determined by LUMINEX immunoassay. The combination of 1% 2′-FL and 1% 3-FL, and 1% 3-FL alone, for 24 h upregulated hBD-2 protein expression significantly (p < 0.001 and p = 0.016, respectively). No changes in the other antimicrobial peptides or proinflammatory cytokines were observed. Thus, 3-FL, alone and in combination with 2′-FL, stimulates oral mucosal secretion of hBD-2, without effecting a proinflammatory response when studied in an oral mucosal culture model. Full article