Protein post-translational modifications (PTMs) are an important battleground in the evolutionary arms races that are waged between the host innate immune system and viruses. One such PTM, ADP-ribosylation, has recently emerged as an important mediator of host antiviral immunity. Important for the host–virus conflict over this PTM is the addition of ADP-ribose by PARP proteins and removal of ADP-ribose by macrodomain-containing proteins. Interestingly, several host proteins, known as macroPARPs, contain macrodomains as well as a PARP domain, and these proteins are both important for the host antiviral immune response and evolving under very strong positive (diversifying) evolutionary selection. In addition, several viruses, including alphaviruses and coronaviruses, encode one or more macrodomains. Despite the presence of the conserved macrodomain fold, the enzymatic activity of many of these proteins has not been characterized. Here, we perform evolutionary and functional analyses to characterize the activity of macroPARP and viral macrodomains. We trace the evolutionary history of macroPARPs in metazoans and show that PARP9 and PARP14 contain a single active macrodomain, whereas PARP15 contains none. Interestingly, we also reveal several independent losses of macrodomain enzymatic activity within mammalian PARP14, including in the bat, ungulate, and carnivore lineages. Similar to macroPARPs, coronaviruses contain up to three macrodomains, with only the first displaying catalytic activity. Intriguingly, we also reveal the recurrent loss of macrodomain activity within the alphavirus group of viruses, including enzymatic loss in insect-specific alphaviruses as well as independent enzymatic losses in two human-infecting viruses. Together, our evolutionary and functional data reveal an unexpected turnover in macrodomain activity in both host antiviral proteins and viral proteins. Full article

Hepatitis E virus (HEV), a common cause of viral hepatitis in developing countries, is mainly transmitted via the fecal–oral route, but also may be a prevalent hospital-transmitted agent among patients on regular hemodialysis due to parenteral transmission. Previous epidemiological studies among hemodialysis patients in Greece, using different diagnostic techniques, gave conflicting results. Τhe present study aimed to measure the exposure rate of hemodialysis patients of north-eastern Greece to HEV by estimating the overall seroprevalence, and to identify potential risk factors. Serum samples from all patients attending the hemodialysis centers of north-eastern Greece (n = 6) were tested for the presence of anti-HEV IgG antibodies using a modern and sensitive ELISA (Enzyme-linked Immunosorbent Assay) technique (Wantai). In total, 42 out of 405 hemodialysis patients were positive for anti-HEV IgG (10.4%), while all samples were negative for HEV RNA when tested using nested RT-PCR. HEV seropositivity among hemodialysis patients was significantly associated with area of residence and contact with specific animals (pork, deer). No association was found with religion, gender distribution and hemodialysis duration. This study showed an increased seroprevalence of HEV among hemodialysis patients in Greece. Agricultural or livestock occupation and place of residence seem to be independent factors that increase the risk of HEV infection. In conclusion, HEV infection calls for the regular screening of hemodialysis patients regardless of the hemodialysis duration or clinical symptoms. Full article

Introduction: Periprosthetic joint infections (PJI) represent a devastating consequence following total joint arthroplasty (TJA). In this study, the authors describe a modified surgical technique developed to enhance the classical irrigation and debridement procedure (DAIR) to improve the possibilities of retaining an acutely infected TJA. Materials and Methods: This technique, debridement antibiotic pearls and retention of the implant (DAPRI), aims to remove the intra-articular biofilm allowing a higher and prolonged local antibiotic concentration by using calcium sulphate antibiotic-added beads in a setting of acute (<4 weeks from symptoms onset) PJI with pathogen identification. The combination of three different surgical techniques (tumor-like synovectomy, argon beam/acetic acid application and chlorhexidine gluconate brushing) aims to remove the bacterial biofilm from the implant without explanting the original hardware. Results: In total, 62 patients met the acute infection criteria (<4 weeks of symptoms); there were 57 males and five females. The patients’ average age at the time of treatment was 71 years (62–77) and the average BMI was 37 kg/m². The micro-organism, always identified through synovial fluid analysis (culture, multiplex PCR or Next Generation Sequencing), was an aerobic Gram + in 76% (S. Coag-Neg 41%; S. aureus 16%), Gram—in 10% (E. coli 4%) and anaerobic Gram + in 4%. The DAPRI treatment was performed at an average of 3 days from symptoms onset (1–7 days). All patients underwent a 12-week course of post-operative antibiotic therapy (6 weeks I.V. and 6 weeks oral). All patients were available at the 2-year minimum FU (24–84 months). A total of 48 (77.5%) patients were infection-free at the final FU, while 14 patients underwent 2-stage revision for PJI recurrence. In total, four patients (6.4%) had a prolonged drainage from the wound after placement of the calcium sulphate beads. Conclusions: This study suggests that the DAPRI technique could represent a valid alternative to the classic DAIR procedure. The current authors do not recommend this procedure outside of the main inclusive criteria (acute scenario micro-organism identification). Full article

Candida albicans is the leading cause of vulvovaginal yeast infections; however, other species are becoming relevant in this niche. The spatial distribution of these fungi in the female genital tract remains poorly understood. In this study, swab samples were collected from 33 patients, first from the anterior vulva and then from the upper third and right lateral wall of the vagina: 16 were with symptoms of vulvovaginal candidiasis and 17 were without characteristic symptoms; furthermore, the genus and species of each isolate were identified. In vitro susceptibility testing for fluconazole and clotrimazole was performed for all isolates. Candida albicans was the most common species (63.6%), followed by Rhodotorula spp. (51.5%), and then Candida parapsilosis (15.2%). Rhodotorula spp. and C. parapsilosis were more commonly associated with colonization, and C. albicans with infection. Rhodotorula spp. isolates presented a low susceptibility to fluconazole, with the MIC ranging from 32 to >64 µg/mL. Differences in susceptibility to fluconazole and clotrimazole between the pairs of vaginal and vulvar isolates were found for Candida albicans, Rhodotorula spp., and Nakaseomyces glabratus. The results suggest that different niches may impact the susceptibility profiles of the isolates, as well as their different clinical behaviors. Full article

The rat lungworm, Angiostrongylus cantonensis, is an emerging parasite that can cause eosinophilic meningitis in humans. Over the past 60 years, the worm has greatly expanded its original Asian distribution to most tropical and subtropical areas of the world, mostly by traveling on ships with its definitive hosts, rats. Angiostrongylus cantonensis was recently found for the first time in Continental Europe, specifically in 3 (2 Rattus norvegicus and 1 Rattus rattus) out of 27 rats trapped in the sewer system of the city of Valencia, Spain. Updating the investigation, the parasite has subsequently been found in 8 (5 R. norvegicus and 3 R. rattus) out of 94 rats analyzed. The highest prevalence of infection (20%) was obtained in rats trapped in the orchards that surround the city, where both snails and slugs (intermediate hosts) abound, and where vegetables consumed in Valencia, other parts of Spain, and abroad, are produced. The presence of the parasite in rats does not necessarily mean that the disease it causes becomes a relevant public health concern since it strongly depends on the food habits of the population at risk. If proper precautions are taken, the risk of acquiring neuroangiostrongylosis should be minimal. Full article

The ongoing global emergence of arthropod-borne (arbo) viruses has accelerated research into the interactions of these viruses with the immune systems of their vectors. Only limited information exists on how bunyaviruses, such as Rift Valley fever virus (RVFV), are sensed by mosquito immunity or escape detection. RVFV is a zoonotic phlebovirus (Bunyavirales; Phenuiviridae) of veterinary and human public health and economic importance. We have shown that the infection of mosquitoes with RVFV triggers the activation of RNA interference pathways, which moderately restrict viral replication. Here, we aimed to better understand the interactions between RVFV and other vector immune signaling pathways that might influence RVFV replication and transmission. For this, we used the immunocompetent Aedes aegypti Aag2 cell line as a model. We found that bacteria-induced immune responses restricted RVFV replication. However, virus infection alone did not alter the gene expression levels of immune effectors. Instead, it resulted in the marked enhancement of immune responses to subsequent bacterial stimulation. The gene expression levels of several mosquito immune pattern recognition receptors were altered by RVFV infection, which may contribute to this immune priming. Our findings imply that there is a complex interplay between RVFV and mosquito immunity that could be targeted in disease prevention strategies. Full article

Theileria orientalis Ikeda has caused an epidemic of bovine anemia and abortion across several U.S. states. This apicomplexan hemoparasite is transmitted by Haemaphysalis longicornis ticks; however, it is unknown if other North American ticks are competent vectors. Since the disease movement is largely determined by the host tick range(s), the prediction of the T. orientalis spread among U.S. cattle populations requires determination of additional competent tick vectors. Although Rhipicephalus microplus has mostly been eradicated from the U.S., outbreaks in populations occur frequently, and the U.S. remains at risk for reintroduction. Since R. microplus is a vector of Theileria equi and T. orientalis DNA has been detected in R. microplus, the goal of this study was to determine whether R. microplus is a competent vector of T. orientalis. Larval R. microplus were applied to a splenectomized, T. orientalis Ikeda-infected calf for parasite acquisition, removed as molted adults, and applied to two T. orientalis naïve, splenectomized calves for transmission. After 60 days, the naïve calves remained negative for T. orientalis by PCR and cytology. Additionally, T. orientalis was not detected in the salivary glands or larval progeny of acquisition-fed adults. These data suggest that R. microplus is not a competent vector of the U.S. T. orientalis Ikeda isolate. Full article

Since tick-borne diseases (TBDs) incidence, both in human and animal populations, is increasing worldwide, there is the need to assess the presence, distribution and prevalence of tick-borne pathogens. Reliable estimates on tick-borne pathogens (TBPs) prevalence represent the public health foundation to create risk maps and take effective prevention and control actions against TBDs. Tick surveillance consists of collecting and testing (usually in pools) thousands of specimens. Construction and analysis of tick pools represent a challenge due to the complexity of tick-borne pathogens and tick-borne diseases ecology. The aim of this study is to provide a practical guideline on appropriate pooling strategies and statistical analysis of infection prevalence through: (i) reporting the different pooling strategies and statistical methodologies commonly used to calculate pathogen prevalence in tick populations and (ii) practical comparison between statistical methods utilising a real dataset of infection prevalence in ticks collected in Northern Italy. Reporting detailed information on tick pool composition and size is as important as the correct TBPs prevalence estimation. Among the prevalence indexes, we suggest using maximum-likelihood estimates of pooled prevalence instead of minimum infection rate or pool positivity rate given the merits of the method and availability of software. Full article

The coronavirus disease 2019 (COVID-19) pandemic has significantly impacted global public health safety and the economy. Multiple antiviral drugs have been developed, and some have received regulatory approval and/or authorization. The use of nutraceuticals can be beneficial for preventing and treating COVID-19 complications. AHCC is a standardized, cultured extract of an edible mushroom Lentinula edodes of the Basidiomycete family of fungi that is enriched in acylated α-1,4-glucans. Here, we evaluated the effects of the oral administration of AHCC on the host response to SARS-CoV-2 infection in two murine models, K18-hACE2 transgenic mice and immunocompetent BALB/c mice. Oral administration of AHCC every other day for one week before and one day post SARS-CoV-2 infection in both strains of mice decreased the viral load and attenuated inflammation in the lungs. AHCC treatment also significantly reduced SARS-CoV-2-induced lethality in the K18-hACE2 mice. AHCC administration enhanced the expansion of γδ T cells in the spleen and lungs before and after viral infection and promoted T helper 1-prone mucosal and systemic T cell responses in both models. In AHCC-fed BALB/c mice, SARS-CoV-2 specific IgG responses were also enhanced. In summary, AHCC supplementation enhances host resistance against mild and severe COVID-19 infection primarily via the promotion of innate and adaptive T cell immune responses in mice. Full article

The impact of malaria-associated acute kidney injury (MAKI), one of the strongest predictors of death in children with severe malaria (SM), has been largely underestimated and research in this area has been neglected. Consequently, a standard experimental mouse model to research this pathology is still lacking. The purpose of this study was to develop an in vivo model that resembles the pathology in MAKI patients. In this study, unilateral nephrectomies were performed on wild-type mice prior to infection with Plasmodium berghei NK65. The removal of one kidney has shown to be an effective approach to replicating the most common findings in humans with MAKI. Infection of nephrectomized mice, compared to their non-nephrectomized counterparts, resulted in the development of kidney injury, evident by histopathological analysis and elevated levels of acute kidney injury (AKI) biomarkers, including urinary neutrophil gelatinase-associated lipocalin, serum Cystatin C, and blood urea nitrogen. Establishment of this in vivo model of MAKI is critical to the scientific community, as it can be used to elucidate the molecular pathways implicated in MAKI, delineate the development of the disease, identify biomarkers for early diagnosis and prognosis, and test potential adjunctive therapies. Full article

Proteus mirabilis (PM) is a Gram-negative, rod-shaped bacterium that causes catheter-associated urinary tract infections (CAUTIs). The specific roles of bacterial surface components (BSCs) in PM pathogenicity and CAUTIs remain unknown. To address this knowledge gap, we utilized relevant in vitro adhesion/invasion models and a well-established murine model of CAUTI to assess the ability of wildtype (WT) and seven mutant strains (MSs) of PM with deficiencies in various genes encoding BSCs to undergo the infectious process (including adhesion to catheters) in both model systems. Overall, MSs adhesion to catheters and the different cell types tested was significantly reduced compared to WT, while no invasion of cells was evident at 24 h. In vivo, WT showed a greater number of planktonic (urine) bacteria, bacteria adherent to catheters, and bacteria adherent to/invading bladder tissue when compared to the MSs. Bacterial counts in urine for PMI3191 and waaE mutants were lower than that for WT and other MSs. The complementation of mutated BSC genes resulting in the biggest defects restored the invasion phenotype both in vitro and in vivo. BSCs play a critical role at various steps in the pathogenicity of PM including adhesion to indwelling medical devices and adhesion/invasion of urinary tissue in vivo. Full article

Autochthonous hepatitis E (HEV) cases have been increasingly recognized and reported in Europe, caused predominantly by the zoonotic HEV genotype 3. The clinical picture is highly variable, from asymptomatic to acute severe or prolonged hepatitis in immunocompromised patients. The main route of transmission to humans in Europe is the ingestion of undercooked pork meat. Transfusion-transmitted HEV infections have also been reported. The aim of the study was to determine the HEV epidemiology and risk in the Finnish blood donor population. A total of 23,137 samples from Finnish blood donors were screened for HEV RNA from individual samples and 1012 samples for HEV antibodies. Additionally, laboratory-confirmed hepatitis E cases in 2016–2022 were extracted from national surveillance data. The HEV RNA prevalence data was used to estimate the risk of transfusion transmission of HEV in the Finnish blood transfusion setting. Four HEV RNA-positive were found, resulting in 1:5784 (0.02%) RNA prevalence. All HEV RNA-positive samples were IgM-negative, and genotyped samples represented genotype HEV 3c. HEV IgG seroprevalence was 7.4%. From the HEV RNA rate found in this study and data on blood component usage in Finland in 2020, the risk estimate for a severe transfusion-transmitted HEV infection is 1:1,377,000 components or one in every 6–7 years. In conclusion, the results indicate that the risk of transfusion-transmitted HEV (HEV TTI) in Finland is low. However, continuous follow-up of the HEV epidemiology in relation to the transfusion risk landscape in Finland is necessary, as well as promoting awareness in the medical community of the small risk for HEV TTI, especially for immunocompromised patients. Full article

Background: In 2007, Australia introduced a national human papillomavirus (HPV) vaccination program. In 2017, the onset of cervical screening changed from 18 to 25 years of age, utilising human papillomavirus (HPV) nucleic acid testing. The objective of the study is to describe the HPV genotypes and HPV16 variants in biopsies from women ≤ 25 years of age with cervical carcinoma (CC) (cases), compared with those aged >25 years (controls), in a pre-vaccination cohort. Methods: HPV genotyping of archival paraffin blocks (n = 96) was performed using the INNO-LiPA HPV Genotyping assay. HPV16-positive samples were analysed for variants by type-specific PCR spanning L1, E2 and E6 regions. Results: HPV16 was the commonest genotype in cases (54.5%, 12/22) and controls (66.7%, 46/69) (p = 0.30), followed by HPV18 (36.3%, 8/22 vs. 17.3% 12/69, respectively) (p = 0.08). Furthermore, 90% (20/22) of cases and 84.1% (58/69) of controls were positive for HPV16 or 18 (p = 0.42); 100% (22/22) of cases and 95.7% (66/69) of controls had at least one genotype targeted by the nonavalent vaccine (p = 0.3). The majority of HPV16 variants (87.3%, 48/55) were of European lineage. The proportion of unique nucleotide substitutions was significantly higher in cases (83.3%, 10/12) compared with controls (34.1%, 15/44), (p < 0.003, χ², OR 9.7, 95%CI 1.7–97.7). Conclusions: Virological factors may account for the differences in CCs observed in younger compared with older women. All CCs in young women in this study had preventable 9vHPV types, which is important messaging for health provider adherence to new cervical screening guidelines. Full article

Rose rosette disease (RRD) caused by the rose rosette emaravirus (RRV) and transmitted by the eriophyid mite Phyllocoptes fructiphilus (Pf), both native to North America, has caused significant damage to roses over the last several decades. As cultural and chemical control of this disease is difficult and expensive, a field trial was established to systematically screen rose germplasm for potential sources of resistance. One hundred and eight rose accessions representing the diversity of rose germplasm were planted in Tennessee and Delaware, managed to encourage disease development, and evaluated for symptom development and viral presence for three years. All major commercial rose cultivars were susceptible to this viral disease to varying levels. The rose accessions with no or few symptoms were species accessions from the sections Cinnamomeae, Carolinae, Bracteatae, and Systylae or hybrids with these. Among these, some were asymptomatic; they displayed no symptoms but were infected by the virus. Their potential depends on their ability to serve as a source of viruses. The next step is to understand the mechanism of resistance and genetic control of the various sources of resistance identified. Full article

To forge a path towards livestock disease emergency preparedness in Denmark, 15 different strategies to mitigate foot-and-mouth disease (FMD) were examined by modelling epidemics initiated in cattle, pig or small ruminant herds across various production systems located in four different Danish regions (Scenario 1), or in one specific livestock production system within each of the three species geographically distributed throughout Denmark (Scenario 2). When additional mitigation strategies were implemented on top of basic control strategies in the European foot-and-mouth disease spread model (EuFMDiS), no significant benefits were predicted in terms of the number of infected farms, the epidemic control duration, and the total economic cost. Further, the model results indicated that the choice of index herd, the resources for outbreak control, and the detection time of FMD significantly influenced the course of an epidemic. The present study results emphasise the importance of basic mitigation strategies, including an effective back-and-forward traceability system, adequate resources for outbreak response, and a high level of awareness among farmers and veterinarians concerning the detection and reporting of FMD at an early stage of an outbreak for FMD control in Denmark. Full article

In Europe, raccoons are invasive neozoons with their largest population in Germany. Globally, this mesocarnivore acts as a wildlife reservoir for many (non-)zoonotic (re-)emerging pathogens, but very little epidemiological data is available for southwest Germany. This exploratory study aimed to screen free-ranging raccoons in Baden-Wuerttemberg (BW, Germany) for the occurrence of selected pathogens with One Health relevance. Organ tissue and blood samples collected from 102 animals, obtained by hunters in 2019 and 2020, were subsequently analysed for two bacterial and four viral pathogens using a qPCR approach. Single samples were positive for the carnivore protoparvovirus-1 (7.8%, n = 8), canine distemper virus (6.9%, n = 7), pathogenic Leptospira spp. (3.9%, n = 4) and Anaplasma phagocytophilum (15.7%, n = 16). West Nile virus and influenza A virus were not detected. Due to their invasive behaviour and synanthropic habit, raccoons may increase the risk of infections for wildlife, domestic animals, zoo animals and humans by acting as a link between them. Therefore, further studies should be initiated to evaluate these risks. Full article

Monkeypox virus (MPXV), a member of the Orthopoxvirus (OPXV) genus, is a zoonotic virus, endemic to central and western Africa that can cause smallpox-like symptoms in humans with fatal outcomes in up to 15% of patients. The incidence of MPXV infections in the Democratic Republic of the Congo, where the majority of cases have occurred historically, has been estimated to have increased as much as 20-fold since the end of smallpox vaccination in 1980. Considering the risk global travel carries for future disease outbreaks, accurate epidemiological surveillance of MPXV is warranted as demonstrated by the recent Mpox outbreak, where the majority of cases were occurring in non-endemic areas. Serological differentiation between childhood vaccination and recent infection with MPXV or other OPXVs is difficult due to the high level of conservation within OPXV proteins. Here, a peptide-based serological assay was developed to specifically detect exposure to MPXV. A comparative analysis of immunogenic proteins across human OPXVs identified a large subset of proteins that could potentially be specifically recognized in response to a MPXV infection. Peptides were chosen based upon MPXV sequence specificity and predicted immunogenicity. Peptides individually and combined were screened in an ELISA against serum from well-characterized Mpox outbreaks, vaccinee sera, and smallpox sera collected prior to eradication. One peptide combination was successful with ~86% sensitivity and ~90% specificity. The performance of the assay was assessed against the OPXV IgG ELISA in the context of a serosurvey by retrospectively screening a set of serum specimens from the region in Ghana believed to have harbored the MPXV-infected rodents involved in the 2003 United States outbreak. Full article

Historically, the seals and harbour porpoises of the Baltic Sea and North Sea have been subjected to hunting, chemical pollutants and repeated mass mortalities, leading to significant population fluctuations. Despite the conservation implications and the zoonotic potential associated with viral disease outbreaks in wildlife, limited information is available on the circulation of viral pathogens in Baltic Sea seals and harbour porpoises. Here, we investigated the presence of the influenza A virus (IAV), the phocine distemper virus (PDV) and the cetacean morbillivirus (CeMV) in tracheal swabs and lung tissue samples from 99 harbour seals, 126 grey seals, 73 ringed seals and 78 harbour porpoises collected in the Baltic Sea and North Sea between 2002–2019. Despite screening 376 marine mammals collected over nearly two decades, we only detected one case of PDV and two cases of IAV linked to the documented viral outbreaks in seals in 2002 and 2014, respectively. Although we find no evidence of PDV and IAV during intermediate years, reports of isolated cases of PDV in North Sea harbour seals and IAV (H5N8) in Baltic and North Sea grey seals suggest introductions of those pathogens within the sampling period. Thus, to aid future monitoring efforts we highlight the need for a standardized and continuous sample collection of swabs, tissue and blood samples across Baltic Sea countries. Full article

The worldwide public health and socioeconomic consequences caused by the COVID-19 pandemic highlight the importance of increasing preparedness for viral disease outbreaks by providing rapid disease prevention and treatment strategies. The NSP3 macrodomain of coronaviruses including SARS-CoV-2 is among the viral protein repertoire that was identified as a potential target for the development of antiviral agents, due to its critical role in viral replication and consequent pathogenicity in the host. By combining virtual and biophysical screening efforts, we discovered several experimental small molecules and FDA-approved drugs as inhibitors of the NSP3 macrodomain. Analogue characterisation of the hit matter and crystallographic studies confirming binding modes, including that of the antibiotic compound aztreonam, to the active site of the macrodomain provide valuable structure–activity relationship information that support current approaches and open up new avenues for NSP3 macrodomain inhibitor development. Full article

Recent field surveys conducted in five common alder ecosystems in Portugal have shown the occurrence of severe canopy dieback, bleeding canker and root rot symptoms indicative of Phytophthora infections. Isolations from symptomatic tissues, rhizosphere and water samples yielded a total of 13 Phytophthora species belonging to 6 phylogenetic clades, including P. lacustris (13 isolates), P. multivora (10), P. amnicola (9), P. chlamydospora (6), P. polonica (6), P. bilorbang (4), P. plurivora (4), P. cinnamomi (3), P. asparagi (2), P. cactorum (2), P. pseudocryptogea (2), P. gonapodyides (1) and P. rosacearum (1). Results of the pathogenicity test confirmed the complex aetiology of common alder decline and the additional risk posed by Phytophthora multivora to the riparian habitats in Portugal. At the same time, the diversity of Phytophthora assemblages detected among the investigated sites suggests that different species could contribute to causing the same symptoms on this host. Two species, P. amnicola and P. rosacearum, are reported here for the first time in natural ecosystems in Europe. Full article

We aim to provide an evidence-based evaluation of whole genome sequence (WGS) methods, employed at the Salmonella reference laboratory in England, in terms of its impact on public health and whether these methods remain a fit for purpose test under UKAS ISO 15189. The evaluation of the genomic methods were mapped against the value of detecting microbiological clusters to support the investigation of food-borne outbreaks of Salmonella in England between 2012–2020. The analysis of WGS with both SNP- and allelic-based methods provided an unprecedented level of strain discrimination and detection of additional clusters when comparing to all of the previous typing methods. The robustness of the routine genomic sequencing at the reference laboratory ensured confidence in the microbiological identifications, even in large outbreaks with complex international food distribution networks. There was evidence that the phylogeny derived from the WGS data can be used to inform the provenance of strains and support discrimination between domestic and non-domestic transmission events. Further insight on the evolutionary context of the emerging pathogenic strains was enabled with a deep dive of the phylogenetic data, including the detection of nested clusters. The public availability of the WGS data linked to the clinical, epidemiological and environmental context of the sequenced strains has improved the trace-back investigations during outbreaks. The global expansion in the use of WGS-based typing in reference laboratories has shown that the WGS methods are a fit for purpose test in public health as it has ensured the rapid implementation of interventions to protect public health, informed risk assessment and has facilitated the management of national and international food-borne outbreaks of Salmonella. Full article

Tick-borne encephalitis (TBE) is Eurasia’s most important tick-borne viral disease. Rodents play an important role as natural hosts. Longitudinal studies on the dynamics of the seroprevalence rates in wild rodents in natural foci over the year are rare, and the dynamics of the transmission cycle still need to be understood. To better understand the infection dynamics, rodents were captured in a capture-mark-release-recapture-study in two natural foci in Bavaria, Germany, monthly from March 2019 to October 2022. Overall, 651 blood and thoracic lavage samples from 478 different wild rodents (Clethrionomys glareolus and Apodemus flavicollis) were analyzed for antibodies against tick-borne encephalitis virus (TBEV) by indirect immunofluorescence assay (IIFA) and confirmed using a serum neutralization test (SNT). Furthermore, a generalized linear mixed model (GLMM) analysis was performed to investigate ecological and individual factors for the probability of infection in rodents. Clethrionomys glareolus (19.4%) had a higher seroprevalence than A. flavicollis (10.5%). Within Cl. glareolus, more males (40.4%) than females (15.6%) were affected, and more adults (25.4%) than juveniles (9.8%). The probability of infection of rodents rather depends on factors such as species, sex, and age than on the study site of a natural focus, year, and season. The high incidence rates of rodents, particularly male adult bank voles, highlight their critical role in the transmission cycle of TBEV in a natural focus and demonstrate that serologically positive rodents can be reliably detected in a natural focus regardless of season or year. In addition, these data contribute to a better understanding of the TBEV cycle and thus could improve preventive strategies for human infections. Full article

Wild carnivore species infected with highly pathogenic avian influenza (HPAI) virus subtype H5N1 during the 2021–2022 outbreak in the Netherlands included red fox (Vulpes vulpes), polecat (Mustela putorius), otter (Lutra lutra), and badger (Meles meles). Most of the animals were submitted for testing because they showed neurological signs. In this study, the HPAI H5N1 virus was detected by PCR and/or immunohistochemistry in 11 animals and was primarily present in brain tissue, often associated with a (meningo) encephalitis in the cerebrum. In contrast, the virus was rarely detected in the respiratory tract and intestinal tract and associated lesions were minimal. Full genome sequencing followed by phylogenetic analysis demonstrated that these carnivore viruses were related to viruses detected in wild birds in the Netherlands. The carnivore viruses themselves were not closely related, and the infected carnivores did not cluster geographically, suggesting that they were infected separately. The mutation PB2-E627K was identified in most carnivore virus genomes, providing evidence for mammalian adaptation. This study showed that brain samples should be included in wild life surveillance programs for the reliable detection of the HPAI H5N1 virus in mammals. Surveillance of the wild carnivore population and notification to the Veterinary Authority are important from a one-heath perspective, and instrumental to pandemic preparedness. Full article

Ginger (Zingiber officinale Roscoe) is an important horticultural crop, valued for its culinary and medicinal properties. Fusarium yellows of ginger, caused by Fusarium oxysporum f. sp. zingiberi (Foz), is a devastating disease that has significantly reduced the quality and crop yield of ginger worldwide. The compatible interaction between ginger and Foz leading to susceptibility is dissected here. The pathogenicity of two Foz isolates on ginger was confirmed by their ability to colonise ginger and in turn induce both internal and external plant symptoms typical of Fusarium yellows. To shed light on Foz susceptibility at the molecular level, a set of defense-responsive genes was analysed for expression in the roots of ginger cultivars challenged with Foz. These include nucleotide-binding site (NBS) type of resistant (R) genes with a functional role in pathogen recognition, transcription factors associated with systemic acquired resistance, and enzymes involved in terpenoid biosynthesis and cell wall modifications. Among three R genes, the transcripts of ZoNBS1 and ZoNBS3 were rapidly induced by Foz at the onset of infection, and the expression magnitude was cultivar-dependent. These expression characteristics extend to the other genes. This study is the first step in understanding the mechanisms of compatible host–pathogen interactions in ginger. Full article

Tenacibaculum is a genus of Gram-negative filamentous bacteria with a cosmopolitan distribution. The research describing Tenacibaculum genomes stems primarily from Norway and Chile due to their impacts on salmon aquaculture. Canadian salmon aquaculture also experiences mortality events related to the presence of Tenacibaculum spp., yet no Canadian Tenacibaculum genomes are publicly available. Ribosomal DNA sequencing of 16S and four species-specific 16S quantitative-PCR assays were used to select isolates cultured from Atlantic salmon with mouthrot in British Columbia (BC), Canada. Ten isolates representing four known and two unknown species of Tenacibaculum were selected for shotgun whole genome sequencing using the Oxford Nanopore’s MinION platform. The genome assemblies achieved closed circular chromosomes for seven isolates and long contigs for the remaining three isolates. Average nucleotide identity analysis identified T. ovolyticum, T. maritimum, T. dicentrarchi, two genomovars of T. finnmarkense, and two proposed novel species T. pacificus sp. nov. type strain 18-2881-A^T and T. retecalamus sp. nov. type strain 18-3228-7B^T. Annotation in most of the isolates predicted putative virulence and antimicrobial resistance genes, most-notably toxins (i.e., hemolysins), type-IX secretion systems, and oxytetracycline resistance. Comparative analysis with the T. maritimum type-strain predicted additional toxins and numerous C-terminal secretion proteins, including an M12B family metalloprotease in the T. maritimum isolates from BC. The genomic prediction of virulence-associated genes provides important targets for studies of mouthrot disease, and the annotation of the antimicrobial resistance genes provides targets for surveillance and diagnosis in veterinary medicine. Full article

Toxoplasma gondii is a zoonotic parasite of importance to both human and animal health. The parasite has various transmission routes, and the meat of infected animals appears to be a major source of human infections in Europe. We aimed to estimate T. gondii prevalence in a selection of animal host species. A systematic literature review resulting in 226 eligible publications was carried out, and serological data were analyzed using an age-dependent Bayesian hierarchical model to obtain estimates for the regional T. gondii seroprevalence in livestock, wildlife, and felids. Prevalence estimates varied between species, regions, indoor/outdoor rearing, and types of detection methods applied. The lowest estimated seroprevalence was observed for indoor-kept lagomorphs at 4.8% (95% CI: 1.8–7.5%) and the highest for outdoor-kept sheep at 63.3% (95% CI: 53.0–79.3%). Overall, T. gondii seroprevalence estimates were highest within Eastern Europe, whilst being lowest in Northern Europe. Prevalence data based on direct detection methods were scarce and were not modelled but rather directly summarized by species. The outcomes of the meta-analysis can be used to extrapolate data to areas with a lack of data and provide valuable inputs for future source attribution approaches aiming to estimate the relative contribution of different sources of T. gondii human infection. Full article

West Nile virus (WNV) transmission rate is shaped by the interaction between virus reservoirs and vectors, which may be maximized in farm environments. Based on this hypothesis, we screened for WNV in wild birds in three scenarios with decreasing gradient of interaction with horses: (i) the farm (A₁); (ii) the neighborhood (A₂); and (iii) a wild area (A₃). We captured wild birds and analyzed their sera for WNV antibodies by blocking ELISA and micro-virus neutralization test. Flavivirus infections were tested with generic and specific PCR protocols. We parameterized linear mixed models with predictors (bird abundance and diversity, vector abundance, vector host abundance, and weather quantities) to identify Flavivirus spp. and WNV exposure risk factors. We detected a low rate of Flavivirus infections by PCR (0.8%) and 6.9% of the birds were seropositive by ELISA. Exposure to Flavivirus spp. was higher in A₁ (9%) than in A₂ and A₃ (5.6% and 5.8%, respectively). Bird diversity was the most relevant predictor of exposure risk and passerines dominated the on-farm bird community. Our results suggest that measures deterring the use of the farm by passerines should be implemented because the environmental favorability of continental Mediterranean environments for WNV is increasing and more outbreaks are expected. Full article

Different E. coli pathotypes are common zoonotic agents. Some of these pathotypes cause recurrent and widespread calf diarrhea and contribute to significant economic losses in the livestock sector worldwide in addition to putting humans at risk. Here, we investigated the occurrence of E. coli pathotypes in diarrheic calves in Ethiopia kept under various calf management practices. One hundred fecal samples were collected from diarrheic calves in 98 different farms. E. coli was isolated in the samples from 99 of the diarrheic calves, and virulence genes were detected in 80% of the samples. The occurrence of E. coli pathotypes in the samples was 32% ETEC, 23% STEC, 18% STEC/ETEC, 3% EPEC, 2% EAEC, and 1% EHEC. No diarrheic calves were positive for the EIEC and DAEC pathotypes. The occurrence of pathotypes was positively associated with female calves (EPEC, p = 0.006), aged less than 2 weeks (STEC, p = 0.059), and calves fed colostrum via the hand method (STEC, p = 0.008 and EAEC, p = 0.003). This study revealed that several E. coli pathotypes occurred among calves affected with diarrhea. Moreover, the presence of a mixed STEC/ETEC pathotypes infection was present in the studied low-income setting. These findings indicate a considerable risk for the zoonotic transmission from calves to humans and the options to provide the better management for younger calves in order to reduce the economic loss. Full article

Prophages are abundant elements integrated into bacterial genomes and contribute to inter-strain genetic variability and, in some cases, modulate the environmental behavior of bacteria, such as pathogen virulence. Here, we described prophage occurrence and diversity in publicly available Erwinia genome assemblies, a genus containing plant pathogens. Prophage-like sequences were identified and taxonomically classified. Sequence diversity was analyzed through intergenomic similarities. Furthermore, we searched for anti-phage defense systems in Erwinia spp., such as DISARM, BREX, and CRISPR-Cas systems, and identified the putative targets of CRISPR spacers. We identified 939 prophage-like sequences in 221 Erwinia spp. genome assemblies. Only 243 prophage-like sequences were classified, all belonging to the Caudoviricetes class. The set of putative Erwinia prophages was mostly unique since only three sequences showed more than 70% intergenomic similarities to known Erwinia phages. Overall, the number and type of CRISPR-Cas systems were conserved within Erwinia species, with many spacers directed to the putative prophages identified. This study increased the knowledge of the diversity and distribution of Erwinia prophages, contributing to the characterization of genetic and ecological factors influencing Erwinia spp. environmental fitness. Full article

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. Full article

Wind-supported transport of particle matter (PM) contaminated with excreta from highly pathogenic avian influenza virus (HPAIv)-infected wild birds may be a HPAIv-introduction pathway, which may explain infections in indoor-housed poultry. The primary objective of our study was therefore to measure the nature and quantity of PM entering poultry houses via air-inlets. The air-inlets of two recently HPAIv-infected poultry farms (a broiler farm and a layer farm) were equipped with mosquito-net collection bags. PM was harvested every 5 days for 25 days. Video-camera monitoring registered wild bird visits. PM was tested for avian influenza viruses (AIV), Campylobacter and Salmonella with PCR. Insects, predominantly mosquitoes, were tested for AIV, West Nile, Usutu and Schmallenberg virus. A considerable number of mosquitoes and small PM amounts entered the air-inlets, mostly cobweb and plant material, but no wild bird feathers. Substantial variation in PM entering between air-inlets existed. In stormy periods, significantly larger PM amounts may enter wind-directed air-inlets. PM samples were AIV and Salmonella negative and insect samples were negative for all viruses and bacteria, but several broiler and layer farm PM samples tested Campylobacter positive. Regular wild (water) bird visits were observed near to the poultry houses. Air-borne PM and insects—potentially contaminated with HPAIv or other pathogens—can enter poultry air-inlets. Implementation of measures limiting this potential introduction route are recommended. Full article

Staphylococcus aureus is an important pathogen associated with hospital, community, and livestock-acquired infections, with the ability to develop resistance to antibiotics. Nasal carriage by hospital inpatients is a risk for opportunistic infections. Antibiotic susceptibility patterns, virulence genes and genetic population structure of S. aureus nasal isolates, from inpatients at Busia County Referral Hospital (BCRH) were analyzed. A total of 263 inpatients were randomly sampled, from May to July 2015. The majority of inpatients (85.9%) were treated empirically with antimicrobials, including ceftriaxone (65.8%) and metronidazole (49.8%). Thirty S. aureus isolates were cultured from 29 inpatients with a prevalence of 11% (10.3% methicillin-susceptible S. aureus (MSSA), 0.8% methicillin resistant S. aureus (MRSA)). Phenotypic and genotypic resistance was highest to penicillin-G (96.8%), trimethoprim (73.3%), and tetracycline (13.3%) with 20% of isolates classified as multidrug resistant. Virulence genes, Panton-Valentine leukocidin (pvl), toxic shock syndrome toxin-1 (tsst-1), and sasX gene were detected in 16.7%, 23.3% and 3.3% of isolates. Phylogenetic analysis showed 4 predominant clonal complexes CC152, CC8, CC80, and CC508. This study has identified that inpatients of BCRH were carriers of S. aureus harbouring virulence genes and resistance to a range of antibiotics. This may indicate a public health risk to other patients and the community. Full article

On the African continent, a large number of arthropod-borne viruses (arboviruses) with zoonotic potential have been described, and yet little is known of most of these pathogens, including their actual distribution or genetic diversity. In this study, we evaluated as a proof-of-concept the effectiveness of the nonspecific sequencing technique sequence-independent single primer amplification (SISPA) on third-generation sequencing techniques (MinION sequencing, Oxford Nanopore Technologies, Oxford, UK) by comparing the sequencing results from six different samples of arboviruses known to be circulating in Africa (Crimean–Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), Dugbe virus (DUGV), Nairobi sheep disease virus (NSDV), Middleburg virus (MIDV) and Wesselsbron virus (WSLV)). All sequenced samples were derived either from previous field studies or animal infection trials. Using this approach, we were able to generate complete genomes for all six viruses without the need for virus-specific whole-genome PCRs. Higher Cq values in diagnostic RT-qPCRs and the origin of the samples (from cell culture or animal origin) along with their quality were found to be factors affecting the success of the sequencing run. The results of this study may stimulate the use of metagenomic sequencing approaches, contributing to a better understanding of the genetic diversity of neglected arboviruses. Full article

The public health threat from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to intensify with emerging variants of concern (VOC) aiming to render COVID-19 vaccines/infection-induced antibodies redundant. The SARS-CoV-2 spike protein is responsible for receptor binding and infection of host cells making it a legitimate antibody target. Antibodies mostly target epitopes in the receptor binding domain (RBD). Mutations occurring within epitopes influence antibody specificity and function by altering their 3D architecture. However, the mechanisms by which non-epitope mutations in the RBD influence antibody specificity and function remain a mystery. We used Protein Data Bank (PDB) deposited 3D structures for the original, Beta, Delta, BA.1, and BA.2 RBD proteins in complex with either neutralizing antibodies or Angiotensin-Converting Enzyme 2 (ACE2) to elucidate the structural and mechanistic basis for neutralizing antibody evasion driven by non-epitope amino acid substitutions in the RBD. Since the mechanism behind the extensively reported functional discrepancies between the same antibody when used individually and when used in an antibody cocktail is lacking, we explored the structural basis for this inconsistency. Finally, since SARS-CoV-2 antibodies are viral mutagens, we deciphered determinants for antibody-pressured amino acid substitutions. On the one hand, we show that non-epitope mutations in the RBD domain of SARS-CoV-2 VOC influence the formation of hydrogen bonds in the paratope-epitope interface by repositioning RBD amino-acid sidechains (AASCs). This increases the distance between complementary donor/acceptor atoms on paratope and epitope AASCs leading to weaker or the complete prevention of the formation of hydrogen bonds in the paratope-epitope interface. On the other hand, we show that SARS-CoV-2 VOC employ the same strategy to simultaneously search for complementary donor/acceptor atoms on ACE2 AASCs to form new interactions, potentially favoring increased viral transmission. Additionally, we illustrate that converting the spike protein to an RBD, a deletion mutation, also repositions epitope AASCs and that AASC interactions in the paratope-epitope interface vary when an antibody is used individually versus when utilized as a cocktail with other antibodies. Finally, we show that the process of substituting immunogenic RBD amino acids begins with the repositioning of their AASCs induced by immune/antibody pressure. We show that donor/acceptor atoms from any amino acid can determine cross-reactivity instead, provided they possess and present spatially pairing donor/acceptor atoms. By studying structural alignments for PDB deposited antibody-RBD 3D structures and relating them to published binding and neutralization profiles of the same antibodies, we demonstrate that minor structural alterations such as epitope AASC repositioning have a major impact on antibody effectiveness and, hence, should receive adequate attention given that protein structure dictates protein function. Full article

Lactic acid bacteria (LAB) are an essential part of the microbiota of the digestive tract of honeybees (Apis mellifera L.). Antagonistic activity of 103 LAB strains (isolates from different environments) against 21 honeybee pathogens/opportunistic pathogens (with agar slab method) was screened. The growth of Paenibacillus genus was inhibited to the most extent. The highest antagonistic activity was demonstrated by Lacticaseibacillus casei 12AN, while the lowest by Apilactobacillus kunkeei DSM 12361, a species naturally inhabiting the honeybee gut. LAB isolated from the honeybee environment demonstrated stronger antagonism against pathogens than collection strains. The antagonistic activity of cell-free supernatants (CFSs) from 24 LAB strains against 7 honeybee pathogens was additionally assessed at physiological pH with the microtitration method. The same was determined for selected CFSs at neutralized pH. CFSs with physiological pH showed significantly stronger antibacterial activity than CFSs with neutralized pH. The results confirmed that the mechanism of antimicrobial activity of LAB is acidification of the environment. The obtained results may, in the future, contribute to a better understanding of the antagonistic properties of LAB and the construction of a probiotic preparation to increase the viability of honeybee colonies. Full article

Infectious pancreatic necrosis (IPN), caused by IPNV, affects several species of farmed fish, particularly Atlantic salmon, and is responsible for significant economic losses in salmon aquaculture globally. Despite the introduction of genetically resistant farmed Atlantic salmon and vaccination strategies in the Chilean salmon industry since 2019, the number of IPN outbreaks has been increasing in farmed Atlantic salmon in the freshwater phase. This study examined gross and histopathological lesions of IPNV-affected fish, as well as the IPNV nucleotide sequence encoding the VP2 protein in clinical cases. The mortality reached 0.4% per day, and the cumulative mortality was from 0.4 to 3.5%. IPNV was isolated in the CHSE-214 cell line and was confirmed by RT-PCR, and VP2 sequence analysis. The analyzed viruses belong to IPNV genotype 5 and have 11 mutations in their VP2 protein. This is the first report of IPN outbreaks in farmed Atlantic salmon genetically resistant to IPNV in Chile. Similar outbreaks were previously reported in Scotland and Norway during 2018 and 2019, respectively. This study highlights the importance of maintaining a comprehensive surveillance program in conjunction with the use of farmed Atlantic salmon genetically resistant to IPNV. Full article

Salmonella is one of the most important foodborne pathogens. Fifty-three strains of Salmonella deposited in the Culture Collection of Industrial Microorganisms—Microbiological Resources Center (IAFB) were identified using molecular and proteomic analyses. Moreover, the genetic similarity of the tested strains was determined using the PFGE method. Main virulence genes were identified, and phenotypical antibiotic susceptibility profiles and prevalence of resistance genes were analyzed. Subsequently, the occurrence of the main mechanisms of β-lactam resistance was determined. Virulence genes, invA, fimA, and stn were identified in all tested strains. Phenotypic tests, including 28 antibiotics, showed that 50.9% of the strains were MDR. The tet genes associated with tetracyclines resistance were the most frequently identified genes. Concerning the genes associated with ESBL-producing Salmonella, no resistance to the TEM and CTX-M type was identified, and only two strains (KKP 1597 and KKP 1610) showed resistance to SHV. No strains exhibited AmpC-type resistance but for six Salmonella strains, the efflux-related resistance of PSE-1 was presented. The high number of resistant strains in combination with multiple ARGs in Salmonella indicates the possible overuse of antibiotics. Our results showed that it is necessary to monitor antimicrobial resistance profiles in all food chain links constantly and to implement a policy of proper antibiotic stewardship to contain or at least significantly limit the further acquisition of antibiotic resistance among Salmonella strains. Full article

Despite an increased incidence of Clostridioides difficile infections, data on the reservoirs and dissemination routes of this bacterium are limited. This study examined the prevalence and characteristics of C. difficile isolates in spinach fields. C. difficile was detected in 2/60 (3.3%) of spinach and 6/60 (10%) of soil samples using culture-based techniques. Whole genome sequencing (WGS) analysis identified the spinach isolates as belonging to the hypervirulent clade 5, sequence type (ST) 11, ribotypes (RT) 078 and 126 and carried the genes encoding toxins A, B and CDT. The soil isolates belonged to clade 1 with different toxigenic ST/RT (ST19/RT614, ST12/RT003, ST46/RT087, ST16/RT050, ST49/RT014/0) strains and one non-toxigenic ST79/RT511 strain. Antimicrobial resistance to erythromycin (one spinach isolate), rifampicin (two soil isolates), clindamycin (one soil isolate), both moxifloxacin and rifampicin (one soil isolate), and multi-drug resistance to erythromycin, vancomycin and rifampicin (two soil isolates) were observed using the E test, although a broader range of resistance genes were detected using WGS. Although the sample size was limited, our results demonstrate the presence of C. difficile in horticulture and provide further evidence that there are multiple sources and dissemination routes for these bacteria. Full article

The aim of this research was to describe the incidence and treatments of mastitis and other common bovine diseases using one year of retrospective observational data (n = 50,329 cow-lactations) obtained from herd management software of 37 large dairy farms in Wisconsin. Incidence rate (IR) was defined as the number of first cases of each disease divided by the number of lactations per farm. Clinical mastitis (CM) remains the most diagnosed disease of dairy cows. Across all herds, the mean IR (cases per 100 cow-lactations) was 24.4 for clinical mastitis, 14.5 for foot disorders (FD), 11.2 for metritis (ME), 8.6 for ketosis (KE), 7.4 for retained fetal membranes (RFM), 4.5 for diarrhea (DI), 3.1 for displaced abomasum (DA), 2.9 for pneumonia (PN) and 1.9 for milk fever (MF). More than 30% of cows that had first cases of CM, DA, RFM, DI, and FD did not receive antibiotics. Of those treated, more than 50% of cows diagnosed with PN, ME and CM received ceftiofur as a treatment. The IR of mastitis and most other diseases was greater in older cows (parity ≥ 3) during the first 100 days of lactation and these cows were more likely to receive antibiotic treatments (as compared to younger cows diagnosed in later lactation). Cows of first and second parities in early lactation were more likely to remain in the herd after diagnosis of disease, as compared to older cows and cows in later stages of lactation. Most older cows diagnosed with CM in later lactation were culled before completion of the lactation. These results provide baseline data for disease incidence in dairy cows on modern U.S. dairy farms and reinforce the role of mastitis as an important cause of dairy cow morbidity. Full article

In the course of 2022, an epidemic of monkeypox (MPX) arose worldwide. In order to assess the level of knowledge of the Italian adults regarding the new emerging disease, its prevention and their level of worrying and the acceptance of a possible vaccination, a web questionnaire was spread nationwide. A total of 1352 individuals (mean age 54.5 ± 13.4, 52.4% males) completed the questionnaire. Only 26. 7% of the sample were aware of the burden of the epidemic at the moment of the investigation; 47.1% were not able to identify the transmission route, nor the main symptoms (48.9%); and 54.2% were hesitant toward a possible MPXV vaccination, mainly because of a lack of confident in vaccines (38.5%). A low level of worrying about the disease was registered (mean score 2.3 ± 1.2 on a 5-point scale). In the regression analysis performed considering MPX knowledge as outcome, a lower level of knowledge was associated with higher age (OR 1.378, CI95% 0.998–1.904), working or studying in a nonhealthcare setting (OR 0.046, CI95% 0.033–0.066), being single (OR 0.624, CI95% 0.455–0.856) and having mass media as the main source of information (OR 0.332, CI95% 0.158–0.696). These findings indicate as of the time of this investigation, the communication about the MPXV epidemic was not effective in determining a good level of knowledge about the disease and its transmission among Italian adults. This highlights the need to improve risk communication strategies. Full article

Feline infectious peritonitis (FIP) is a complex and historically fatal disease, though recent advances in antiviral therapy have uncovered potential treatments. A newer therapeutic option, unlicensed molnupiravir, is being used as a first-line therapy for suspect FIP and as a rescue therapy to treat cats who have persistent or relapsed clinical signs of FIP after GS-441524 and/or GC376 therapy. Using owner-reported data, treatment protocols for 30 cats were documented. The 26 cats treated with unlicensed molnupiravir as a rescue therapy were treated with an average starting dosage of 12.8 mg/kg and an average ending dosage of 14.7 mg/kg twice daily for a median of 12 weeks (IQR = 10–15). In total, 24 of 26 cats were still living disease-free at the time of writing. One cat was euthanized after completing treatment due to a prolonged seizure, and the other cat underwent retreatment for relapsed clinical signs. Few adverse effects were reported, with the most notable—folded ears (1), broken whiskers (1), and severe leukopenia (1)—seen at dosages above 23 mg/kg twice daily. This study provides a proof of principle for the use of molnupiravir in cats and supports the need for future studies to further evaluate molnupiravir as a potentially safe and effective therapy for FIP. Full article

Salmonella is estimated to cause over a million infections and ~400 deaths annually in the U.S. Salmonella enterica serotype Javiana strains (n = 409) that predominantly originated from the State of Arkansas over a six-year period (2003 to 2008) were studied. This period coincided with a rapid rise in the incidence of S. Javiana infections in the U.S. Children under the age of 10 displayed the highest prevalence of S. Javiana infections, regardless of sex or year of detection. Antimicrobial susceptibility to 15 different antimicrobials was assessed and 92% (n = 375) were resistant to at least one of the antimicrobials. Approximately 89% of the isolates were resistant to sulfisoxazole alone and 3% (n = 11) were resistant to different antimicrobials, including gentamicin, ciprofloxacin or ceftiofur. The pulsed-field gel electrophoresis (PFGE) analyses assessed the genotypic diversity and distribution of S. Javiana strains using XbaI restriction. Nine major clusters were identified and isolates from each group were digested with the restriction enzyme AvrII. Isolates with identical profiles of XbaI and AvrII were found to be disseminated in human populations. These distinct “types” of S. Javiana were persistent in human populations for multiple years. A subset of isolates (n = 19) with unique resistance phenotypes underwent plasmid and incompatibility (Inc) type analyses and the isolates resistant to more than one antimicrobial harbored multiple plasmids (<3 to 165 kb). Furthermore, these strains possessed 14 virulence genes, including pagC, cdtB, and iroN. The whole genome sequences (WGS) of 18 isolates that mostly originated from Arkansas from 2003 to 2011 were compared with isolates collected from different areas in the U.S. in 1999, indicating the perseverance of S. Javiana in disseminating antimicrobial resistance and virulence genes. Full article

Toxoplasma gondii is one of the most common protozoan parasites and is widely present in all warm-blooded animals. Although clinical disease is uncommon, some species, including ring-tailed lemurs (Lemur catta), have been found to develop acute and lethal toxoplasmosis. The aim of this study was to describe the pathologic, immunohistochemical, serological, and molecular findings of an outbreak of fatal toxoplasmosis in three captive ring-tailed lemurs in Central Italy in 2009. The animals died acutely within few days. The necropsy was immediately performed; necrotic lesions in the spleen, liver, and kidney, as well as interstitial pneumonia, were found histologically. All animals had high titers of anti–T. gondii-specific antibodies (1:1280 IgM and 1:640 IgG) according to a modified agglutination test (MAT) and immunohistochemistry showed scattered tachyzoites in the target organs. Diagnosis was confirmed by PCR and clonal type II was identified. In addition, the seven co-habiting lemurs were seronegative. This paper reports the first outbreak of acute disseminated toxoplasmosis in captive ring-tailed lemurs in Italy. These findings confirm the high susceptibility of this endangered species to toxoplasma infection, which may be considered a further threat to captive population viability. Full article

Severe mastitis can lead to considerable disturbances in the cows’ general condition and even to septicemia and death. The aim of this cross-sectional study was to identify factors associated with the severity of the clinical expression of mastitis. Streptococcus (Str.) uberis (29.9%) was the most frequently isolated pathogen, followed by coliform bacteria (22.3%). The majority of all mastitis cases (n = 854) in this study were either mild or moderate, but 21.1% were severe. It can be deduced that the combination of coliform pathogens and increasing pathogen shedding of these showed associations with severe mastitis. Furthermore, animal-related factors associated with severe disease progression were stages of lactation, and previous diseases in the period prior to the mastitis episode. Cows in early lactation had more severe mastitis. Ketosis and uterine diseases in temporal relation to the mastitis were associated with more severe mastitis in the diseased cows. Hypocalcemia was significantly associated with milder mastitis. As another factor, treatment with corticosteroids within two weeks before mastitis was associated with higher severity of mastitis. Knowledge of these risk factors may provide the basis for randomized controlled trials of the exact influence of these on the severity of mastitis. Full article

Ascaris suum is present in traditionally managed indoor pig herds and in industrialized farms, especially in older fatteners and sows. The increasing resistance to common antihelminthic drugs redirected research towards alternative and traditional therapies, which also include medicinal plants. This study comparatively evaluated the in vitro antiparasitic effects of Allium sativum L., Artemisia absinthium L., Cucurbita pepo L., Coriandrum sativum L., Satureja hortensis L. and Calendula officinalis L. against A. suum egg hatching and larval development. A. suum eggs were sampled from randomized fecal specimens collected from traditionally raised swine. The egg suspension (ES, 12 × 10³/mL) was divided into two controls (C) (1C—1 mL ES + 1 mL distilled water, 2C—five plates of 1 mL ES + 1 mL ethanol of 70%, 35%, 17.5%, 8.75%, and 4.375%, respectively) and six experimental groups, and placed in 3 mL cell plates. The experimental groups (EG, 1–6) included ES + each alcoholic plant extract (10%, 5%, 2.5%, 1.25%, 0.625%). Both C and EG were performed in quintuplicate. All variants were incubated at 27 °C for a total of 21 days. A. suum eggs were examined after 2, 14 (L1), and 21 (L2/L3) days of incubation. The efficacy of all tested plant extracts increased with concentration. Anti-embryogenic effects on A. suum eggs were expressed by all plants. A superior influence was observed in A. sativum L., A. absinthium L., C. pepo L. and S. hortensis L. extracts, at all concentrations tested. A. sativum L. and A. absinthium L. extracts showed the strongest antihelminthic activity, while C. sativum L. and C. officinalis L. were the weakest ascaricids. Future in-depth phytochemical studies are required to identify the compounds responsible for the anthelminthic properties of these plant species. Full article

The global pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to efforts in developing effective vaccine approaches. Currently, approved coronavirus disease 2019 (COVID-19) vaccines are administered through an intramuscular (I.M.) route. Here, we show that the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD), when displayed on immunogenic liposomes, can be intranasally (I.N.) administered, resulting in the production of antigen-specific IgA and antigen-specific cellular responses in the lungs. Following I.N. immunization, antigen-presenting cells of the lungs took up liposomes displaying the RBD. K18 human ACE2-transgenic mice that were immunized I.M or I.N with sub-microgram doses of RBD liposomes and that were then challenged with SARS-CoV-2 had a reduced viral load in the early course of infection, with I.M. achieving complete viral clearance. Nevertheless, both vaccine administration routes led to full protection against lethal viral infection, demonstrating the potential for the further exploration and optimization of I.N immunization with liposome-displayed antigen vaccines. Full article

Candida-macrophage interactions are important immune defense responses associated with disseminated and deep-seated candidiasis in humans. Cells of Candida spp. express functional amyloids on their surfaces during the pathogenesis of disseminated candidiasis. These amyloids become decorated with serum amyloid P-component (SAP) that binds to Candida cells and macrophages and downregulates the cellular and cytokine response to the fungi. In this report, further characterization of the interactions of SAP and fungal functional amyloid are demonstrated. Blocking the binding of SAP to macrophage FcγR1 receptors increases phagocytosis of yeast cells; seeding a pro-amyloid-forming peptide on the yeast cell surface also increases phagocytosis of yeasts by macrophages; and, lastly, miridesap, a small palindromic molecule, prevents binding of SAP to yeasts and removes SAP that is bound to C. albicans thus, potentially increasing phagocytosis of yeasts by macrophages. Some, or all, of these interventions may be useful in boosting the host immune response to disseminated candidiasis. Full article

Anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines may trigger immune-mediated adverse events, including myocarditis. Evidence of vaccine safety in patients with rheumatic disorders and underlying autoimmune myocarditis is scarce. To address this issue, we studied 13 patients with systemic lupus erythematosus (SLE) and allied conditions with a history of myocarditis and receiving mRNA-based vaccines. Data about general and cardiac laboratory tests, treatment, and disease status were collected during routine consultations before and after the primary vaccination course and after each vaccine dose administration, while myocarditis symptoms were closely monitored. A significant increase in troponin levels from baseline was found after 13 (6–20) days from the first (p = 0.046) and 17 (4–29) days after the second dose (p = 0.013). Troponin levels progressively decreased within 3 (1–6) months in the absence of typical symptoms or signs of myocarditis. A significant increase in the constitutional domain of the British Isles Lupus Assessment Group (BILAG) index (p = 0.046) was observed in SLE patients. However, no patient needed any treatment change. mRNA-based anti-SARS-CoV-2 vaccines can apparently be safely administered to patients with SLE and lupus-like disorders with previous myocarditis despite potential subclinical and transient rises in cardiac damage markers. Full article

Monkeypox is re-emerging and spreading over the world, posing a serious threat to human life, especially in non-endemic countries, including Saudi Arabia. Due to the paucity of research on knowledge about monkeypox in Saudi Arabia, this study aimed to evaluate the general population’s knowledge of monkeypox in a sample of the country. A web-based cross-sectional survey was conducted from 25 May 2022 to 15 July 2022. Participants’ knowledge about monkeypox on a 23-item scale and socio-demographic characteristics were gathered in the survey. Pearson’s Chi-square test was used to compare knowledge level (categorized into high and low) and explanatory variables. Out of 480, only 48% of the respondents had high knowledge (mean score > 14). Participants’ age, marital status, residential region, living in the urban area, education level, employment status, being a healthcare worker, income, and smoking status were significantly associated with the level of knowledge about monkeypox (p < 0.01). Overall, social media (75.0%) was the most frequently reported source from where participants obtained monkeypox-related information followed by TV and radio (45.6%), family or friend (15.6%), and healthcare provider (13.8%). We found that overall knowledge of monkeypox infection was slightly poor among the Saudi population. These findings highlight the urgent need for public education on monkeypox to promote awareness and engage the public ahead of the outbreak. Full article

Due to its economic impact on livestock and its zoonotic effect, Q fever is a public and animal health problem. Information on this infection in Italy is presently supported by reports of reproductive problems in livestock farms and is, therefore, insufficient to properly understand the impact of the disease. This study aimed to describe for the first time the seroprevalence of Q fever in dairy cows and water buffalos in the Campania region (Southern Italy). A total of 424 dairy cattle and 214 water buffalo were tested using a commercial indirect ELISA kit. An overall seroprevalence of 11.7% confirmed the wide distribution of C. burnetii in this region. Several factors were positively associated with higher seroprevalence, such as species (higher in cattle than in water buffalo), age, and coexistence with other ruminant species. The final model of logistic regression included only age (older) and species (cattle), which were positively associated with the presence of Q fever antibodies. Our findings support the widespread presence of Coxiella burnettii in Campania and show a seroprevalence similar to that observed in previous studies in other Italian regions and European countries. Since human cases are typically linked to contact with infected ruminants, there is a need to improve surveillance for this infection. Full article