Editor’s Choice Articles

Mass mortality events due to disease outbreaks have recently affected almost every healthy population of fan mussel, Pinna nobilis in Mediterranean Sea. The devastating mortality of the species has turned the interest of the research towards the causes of these events. After the haplosporidan infestation and the infection by Mycobacterium sp., new emerging pathogens have arisen based on the latest research. In the present study, a metagenomic approach of 16S rRNA next generation sequencing (NGS) was applied in order to assess the bacterial diversity within the digestive gland of diseased individuals as well as to carry out geographical correlations among the biodiversity of microbiome in the endangered species Pinna nobilis. The specimens originated from the mortalities occurred in 2019 in the region of Greece. Together with other bacterial genera, the results confirmed the presence of Vibrio spp., assuming synergistic effects in the mortality events of the species. Alongside with the presence of Vibrio spp., numerous bacterial genera were detected as well, including Aliivibrio spp., Photobacterium spp., Pseudoalteromonas spp., Psychrilyobacter spp. and Mycoplasma spp. Bacteria of the genus Mycoplasma were in high abundance particularly in the sample originated from Limnos island representing the first time recorded in Pinna nobilis. In conclusion, apart from exclusively the Haplosporidan and the Mycobacterium parasites, the presence of potentially pathogenic bacterial taxa detected, such as Vibrio spp., Photobactrium spp. and Alivibrio spp. lead us to assume that mortality events in the endangered Fan mussel, Pinna nobilis, may be attributed to synergistic effects of more pathogens. Full article

Streptococcus uberis is a common cause of intramammary infection and mastitis in dairy cattle. Unlike other mammary pathogens, S. uberis evades detection by mammary epithelial cells, and the host–pathogen interactions during early colonisation are poorly understood. Intramammary challenge of dairy cows with S. uberis (strain 0140J) or isogenic mutants lacking the surface-anchored serine protease, SUB1154, demonstrated that virulence was dependent on the presence and correct location of this protein. Unlike the wild-type strain, the mutant lacking SUB1154 failed to elicit IL-1β from ex vivo CD14+ cells obtained from milk (bovine mammary macrophages, BMM), but this response was reinstated by complementation with recombinant SUB1154; the protein in isolation elicited no response. Production of IL-1β was ablated in the presence of various inhibitors, indicating dependency on internalisation and activation of NLRP3 and caspase-1, consistent with inflammasome activation. Similar transcriptomic changes were detected in ex vivo BMM in response to the wild-type or the SUB1154 deletion mutant, consistent with S. uberis priming BMM, enabling the SUB1154 protein to activate inflammasome maturation in a transcriptionally independent manner. These data can be reconciled in a novel model of pathogenesis in which, paradoxically, early colonisation is dependent on the innate response to the initial infection. Full article

Understanding African swine fever virus (ASFV) transmission is essential for strategies to minimize virus spread during an outbreak. ASFV can survive for extended time periods in animal products, carcasses, and the environment. While the ASFV genome was found in environments around infected farms, data on the virus survival in soil are scarce. We investigated different soil matrices spiked with ASFV-positive blood from infected wild boar to see if ASFV can remain infectious in the soil beneath infected carcasses. As expected, ASFV genome detection was possible over the entire sampling period. Soil pH, structure, and ambient temperature played a role in the stability of infectious ASFV. Infectious ASFV was demonstrated in specimens originating from sterile sand for at least three weeks, from beach sand for up to two weeks, from yard soil for one week, and from swamp soil for three days. The virus was not recovered from two acidic forest soils. All risk mitigation experiments with citric acid or calcium hydroxide resulted in complete inactivation. In conclusion, the stability of infectious ASFV is very low in acidic forest soils but rather high in sandy soils. However, given the high variability, treatment of carcass collection points with disinfectants should be considered. Full article

Mycobacterial infections are a major concern in veterinary medicine because of the difficulty achieving an etiological diagnosis, the challenges and concerns of treatment, and the potential zoonotic risk. Mycobacterium kansasii, a slow-growing non-tuberculous mycobacteria, causes disease in both humans and animals. While infections have been well described in humans, where it may be misdiagnosed as tuberculosis, there are fewer reports in animals. Only four cases have been reported in the domestic cat. This case report describes systemic M. kansasii infection in two sibling indoor-only cats that presented two and half years apart with cutaneous disease that was found to be associated with osteolytic and pulmonary pathology. Infection with M. kansasii was confirmed in both cats by polymerase chain reaction on fine-needle aspirate of a lumbosacral soft tissue mass in one cat and on a tissue punch biopsy of a skin lesion in the other; interferon-gamma release assay inferred M. avium-complex and M. tuberculosis-complex infection in the two cats, respectively. Both patients made a full recovery following antimicrobial therapy with rifampicin, azithromycin, and pradofloxacin (plus N-acetyl cysteine in cat 2). This report highlights successful treatment of systemic M. kansasii mycobacteriosis in the cat and the challenge of accurately diagnosing this infection. Full article

Tick-borne encephalitis virus (TBEV) depends mainly on a fragile mode of transmission, the co-feeding between infected nymphs and larvae on rodents, and thus persists under a limited set of biotic and abiotic conditions. If these conditions change, natural TBEV foci might be unstable over time. We conducted a longitudinal study over seven years in a mountain forest in Alsace, Eastern France, located at the western border of known TBEV distribution. The objectives were (i) to monitor the persistence of TBEV circulation between small mammals and ticks and (ii) to discuss the presence of TBEV circulation in relation to the synchronous activity of larvae and nymphs, to the densities of questing nymphs and small mammals, and to potential changes in meteorological conditions and deer densities. Small mammals were trapped five times per year from 2012 to 2018 to collect blood samples and record the presence of feeding ticks, and were then released. Questing nymphs were collected twice a year. Overall, 1344 different small mammals (Myodes glareolus and Apodemus flavicollis) were captured and 2031 serum samples were tested for the presence of antibodies against TBEV using an in-house ELISA. Seropositive rodents (2.1%) were only found from 2012 to 2015, suggesting that the virus disappeared afterwards. In parallel, we observed unusual variations in inter-annual nymph abundance and intra-annual larval activity that could be related to exceptional meteorological conditions. Changes in the densities of questing nymphs and deer associated with the natural stochastic variations in the frequency of contacts between rodents and infected ticks may have contributed to the endemic fadeout of TBEV on the study site. Further studies are needed to assess whether such events occur relatively frequently in the area, which could explain the low human incidence of TBE in Alsace and even in other areas of France. Full article

Preventive chemotherapy (PC) is a WHO-recommended strategy to control and eliminate schistosomiasis and soil-transmitted helminths (STHs). We assessed the prevalence, intensity, and correlates of schistosomiasis and STH infection after five rounds of PC in southern Ethiopia. A total of 3162 school children from four schools in Wondo Gennet and Hawella Tula districts were screened for Schistosoma mansoni and STHs infection. The overall prevalence of S. mansoni infection was 25.8% (range between schools 11.6% to 54.1%), with light (19.1%), moderate (5.3%), and heavy (1.4%) infection intensities. A total of 61.6% S. mansoni-infected children were STH co-infected. The overall prevalence of STHs infection was 54.7% (range between schools 30.6–71.0%), with moderate-to-heavy intensity infections being 16.3%. Ascaris lumbricoides was the most prevalent 45% (95% CI, 43.5–47) followed by Trichuris trichiura 25.3% (95% CI, 23.8–26.9) and hookworm 6.1% (95% CI, 5.3–7). A total of 33.7% of STHs-infected children had A. lumbricoides and T. trichiura co-infections. S. mansoni infection was significantly associated with school and STHs co-infection (p < 0.001). STH infection was correlated with school and younger age (p < 0.001). Despite repeated PC, S. mansoni and STH infection remain significant health problems, and the WHO target to control schistosomiasis and eliminate STH by 2020 may not be achieved. Intensified control and prevention measures, including drug efficacy surveillance, is recommended. Full article

Synthetic peptide vaccines were designed to target the neuropeptides innervating Ixodes ricinus salivary glands and hindgut and they were tested for their capacity to afford protective immunity against nymphs or larvae and Anaplasma phagocytophilum-infected nymph infestation, in mice and sheep, respectively. In both models, the assembly of SIFamide (SIFa) or myoinhibitory peptide (MIP) neuropeptides into multiple antigenic peptide constructs (MAPs) elicited a robust IgG antibody response following immunization. Nevertheless, no observable detrimental impact on nymphs was evidenced in mice, and, unfortunately, the number of engorged nymphs on sheep was insufficient for firm conclusions to be drawn, including for bacterial transmission. Regarding larvae, while vaccination of the sheep did not globally diminish tick feeding success or development, analyses of animals at the individual level revealed a negative correlation between anti-SIFa and MIP antibody levels and larva-to-nymph molting success for both antigens. Our results provide a proof of principle and precedent for the use of MAPs for the induction of immunity against tick peptide molecules. Although the present study did not provide the expected level of protection, it inaugurates a new strategy for protection against ticks based on the immunological targeting of key components of their nervous system. Full article

The cattle industry is suffering economic losses caused by bovine leukemia virus (BLV) and enzootic bovine leukosis (EBL), the clinical condition associated with BLV infection. This pathogen spreads easily without detection by farmers and veterinarians due to the lack of obvious clinical signs. Cattle movement strongly contributes to the inter-farm transmission of BLV. This study quantified the farm-level risk of BLV introduction using a cattle movement analysis. A generalized linear mixed model predicting the proportion of BLV-infected cattle was constructed based on weighted in-degree centrality. Our results suggest a positive association between weighted in-degree centrality and the estimated number of introduced BLV-infected cattle. Remarkably, the introduction of approximately six cattle allowed at least one BLV-infected animal to be added to the farm in the worst-case scenario. These data suggest a high risk of BLV infection on farms with a high number of cattle being introduced. Our findings indicate the need to strengthen BLV control strategies, especially along the chain of cattle movement. Full article

African swine fever (ASF) causes huge economic losses and is one of most dangerous diseases of pigs. The disease is known for almost 100 years, an effective vaccine or treatment is still unavailable, only proper biosecurity measures, including disinfection, are being applied, in order to prevent disease outbreaks. Eight active substances, i.e., formaldehyde, sodium hypochlorite, caustic soda, glutaraldehyde, phenol, benzalkonium chloride, potassium peroxymonosulfate and acetic acid, were tested, in order to confirm their effectiveness against African swine fever virus (ASFV). This specific selection was done based on the World Organisation for Animal Health (OIE)’s recommendation and previous disinfectant studies on surfaces. The result of our study shows that most of them inactivate the virus, in recommended concentrations. In order to reduce the cytotoxicity of the four substances, Microspin S-400 HR columns were applied, therefore making it possible to demonstrate four logarithms virus titer reduction. Sodium hypochlorite, glutaraldehyde, caustic soda and potassium peroxymonosulfate showed the best ASFV inactivation rates, achieving titer reductions over 5 logs. Despite microfiltration, the virucidal activity of formaldehyde was not assessable, due to its high cytotoxicity. Our results showed that cleaning is particularly important, because removal of the soiling provides improved effectiveness of the tested chemical compounds. Full article

Ischemia reperfusion injury (IRI) during liver transplantation increases morbidity and contributes to allograft dysfunction. There are no therapeutic strategies to mitigate IRI. We examined a novel hypothesis: caspase 1 and caspase 11 serve as danger-associated molecular pattern (DAMPs) sensors in IRI. By performing microarray analysis and using caspase 1/caspase 11 double-knockout (Casp DKO) mice, we show that the canonical and non-canonical inflammasome regulators are upregulated in mouse liver IRI. Ischemic pre (IPC)- and post-conditioning (IPO) induce upregulation of the canonical and non-canonical inflammasome regulators. Trained immunity (TI) regulators are upregulated in IPC and IPO. Furthermore, caspase 1 is activated during liver IRI, and Casp DKO attenuates liver IRI. Casp DKO maintained normal liver histology via decreased DNA damage. Finally, the decreased TUNEL assay-detected DNA damage is the underlying histopathological and molecular mechanisms of attenuated liver pyroptosis and IRI. In summary, liver IRI induces the upregulation of canonical and non-canonical inflammasomes and TI enzyme pathways. Casp DKO attenuate liver IRI. Development of novel therapeutics targeting caspase 1/caspase 11 and TI may help mitigate injury secondary to IRI. Our findings have provided novel insights on the roles of caspase 1, caspase 11, and inflammasome in sensing IRI derived DAMPs and TI-promoted IRI-induced liver injury. Full article

Objectives: Assess the predictive accuracy of the WHO COVID-19 severity classification on COVID-19 hospitalized patients. The secondary aim was to compare its predictive power with a new prediction model, named COVID-19 EPI-SCORE, based on a Bayesian network analysis. Methods: We retrospectively analyzed a population of 295 COVID-19 RT-PCR positive patients hospitalized at Epicura Hospital Center, Belgium, admitted between March 1st and April 30th, 2020. Results: Our cohort’s median age was 73 (62–83) years, and the female proportion was 43%. All patients were classified following WHO severity classification at admission. In total, 125 (42.4%) were classified as Moderate, 69 (23.4%) as Severe, and 101 (34.2%) as Critical. Death proportions through these three classes were 11.2%, 33.3%, and 67.3%, respectively, and the proportions of critically ill patients (dead or needed Invasive Mechanical Ventilation) were 11.2%, 34.8%, and 83.2%, respectively. A Bayesian network analysis was used to create a model to analyze predictive accuracy of the WHO severity classification and to create the EPI-SCORE. The six variables that have been automatically selected by our machine learning algorithm were the WHO severity classification, acute kidney injury, age, Lactate Dehydrogenase Levels (LDH), lymphocytes and activated prothrombin time (aPTT). Receiver Operation Characteristic (ROC) curve indexes hereby obtained were 83.8% and 91% for the models based on WHO classification only and our EPI-SCORE, respectively. Conclusions: Our study shows that the WHO severity classification is reliable in predicting a severe outcome among COVID-19 patients. The addition to this classification of a few clinical and laboratory variables as per our COVID-19 EPI-SCORE has demonstrated to significantly increase its accuracy. Full article

Canine distemper virus (CDV) is a multi-host pathogen with variable clinical outcomes of infection across and within species. We used whole-genome sequencing (WGS) to search for viral markers correlated with clinical distemper in African lions. To identify candidate markers, we first documented single-nucleotide polymorphisms (SNPs) differentiating CDV strains associated with different clinical outcomes in lions in East Africa. We then conducted evolutionary analyses on WGS from all global CDV lineages to identify loci subject to selection. SNPs that both differentiated East African strains and were under selection were mapped to a phylogenetic tree representing global CDV diversity to assess if candidate markers correlated with documented outbreaks of clinical distemper in lions (n = 3). Of 54 SNPs differentiating East African strains, ten were under positive or episodic diversifying selection and 20 occurred in the clinical strain despite strong purifying selection at those loci. Candidate markers were in functional domains of the RNP complex (n = 19), the matrix protein (n = 4), on CDV glycoproteins (n = 5), and on the V protein (n = 1). We found mutations at two loci in common between sequences from three CDV outbreaks of clinical distemper in African lions; one in the signaling lymphocytic activation molecule receptor (SLAM)-binding region of the hemagglutinin protein and another in the catalytic center of phosphodiester bond formation on the large polymerase protein. These results suggest convergent evolution at these sites may have a functional role in clinical distemper outbreaks in African lions and uncover potential novel barriers to pathogenicity in this species. Full article

The West Bank can be considered a high-risk area for Legionnaires’ disease (LD) due to its hot climate, intermittent water supply and roof storage of drinking water. Legionella, mostly L. pneumophila, are responsible for LD, a severe, community-acquired and nosocomial pneumonia. To date, no extensive assessment of Legionella spp and L. pneumophila using cultivation in combination with molecular approaches in the West Bank has been published. Two years of environmental surveillance of Legionella in water and biofilms in the drinking water distribution systems (DWDS) of eight hospitals was carried out; 180 L. pneumophila strains were isolated, mostly from biofilms in DWDS. Most of the isolates were identified as serogroup (Sg) 1 (60%) and 6 (30%), while a minor fraction comprised Sg 8 and 10. Multilocus Variable number of tandem repeats Analysis using 13 loci (MLVA-8(12)) was applied as a high-resolution genotyping method and compared to the standard Sequence Based Typing (SBT). The isolates were genotyped in 27 MLVA-8(12) genotypes (Gt), comprising four MLVA clonal complexes (VACC 1; 2; 5; 11). The major fraction of isolates constituted Sequence Type (ST)1 and ST461. Most of the MLVA-genotypes were highly diverse and often unique. The MLVA-genotype composition showed substantial regional variability. In general, the applied MLVA-method made it possible to reproducibly genotype the isolates, and was consistent with SBT but showed a higher resolution. The advantage of the higher resolution was most evident for the subdivision of the large strain sets of ST1 and ST461; these STs were shown to be highly pneumonia-relevant in a former study. This shows that the resolution by MLVA is advantageous for back-tracking risk sites and for the avoidance of outbreaks of L. pneumophila. Overall, our results provide important insights into the detailed population structure of L. pneumophila, allowing for better risk assessment for DWDS. Full article

The ascomycete Trichoderma atroviride is well known for its mycoparasitic lifestyle. Similar to other organisms, light is an important cue for T. atroviride. However, besides triggering of conidiation, little is known on the physiological responses of T. atroviride to light. In this study, we analyzed how cultivation under different light wavelengths and regimes impacted the behavior of two T. atroviride wild-type strains: IMI206040 and P1. While colony extension of both strains was slightly affected by light, massive differences in their photoconidation responses became evident. T. atroviride P1 colonies conidiated under all conditions tested including growth in complete darkness, while IMI206040 required white, blue or green light to trigger asexual reproduction. Interestingly, deletion of the stress-activated MAP kinase-encoding gene tmk3 abolished the ability of strain P1 to conidiate in red and yellow light as well as in darkness. Furthermore, light-dependent differences in the mycoparasitic activity and in the biosynthesis of the secondary metabolite 6-pentyl-α-pyrone (6-PP) became evident. 6-PP production was highest upon dark incubation, while light, especially exposure to white light as light/dark cycles, had an inhibitory effect on its biosynthesis. We conclude that the response of T. atroviride to light is strain-dependent and impacts differentiation, mycoparasitism, and 6-PP production; hence, this should be considered in experiments testing the mycoparasitic activity of these fungi. Full article

Cytauxzoon felis is a hemoprotozoal tick-transmitted pathogen of felids. Felids that survive acute disease often remain infected and serve as reservoirs for subsequent tick transmission to other susceptible felines. States adjacent to Kansas have identified C. felis-domestic cat carriers while statewide awareness and concern of cytauxzoonosis have increased. The objective of this study was to determine the prevalence of C. felis-carriers in the eastern Kansas domestic cat population using a sensitive quantitative PCR assay targeting the C. felis Cox3 mitochondrial gene. An overall C. felis infection prevalence of 25.8% was determined for asymptomatic domestic cats in eastern Kansas. Significantly more C. felis-carrier cats were identified in spring and fall, suggesting a seasonal fluctuation of survivors. Additionally, a greater percentage of feral and owned cats were positive for C. felis compared to rescue/rescinded cats. This study demonstrates that C. felis-domestic cat carriers are common among cats that spend at least a portion of time outdoors in eastern Kansas, and that more cats likely survive cytauxzoonosis than expected. Understanding the role of domestic cat carriers of C. felis is essential in developing cytauxzoonosis mitigation strategies, including recommending year-round use of acaricide products for all cats that spend any time outdoors. Full article

Getah virus (GETV) is a mosquito-transmitted alphavirus primarily associated with disease in horses and pigs in Asia. GETV was also reported to have been isolated from mosquitoes in Australia in 1961; however, retrieval and sequencing of the original isolates (N544 and N554), illustrated that these viruses were virtually identical to the 1955 GETV_MM2021 isolate from Malaysia. K-mer mining of the >40,000 terabases of sequence data in the Sequence Read Archive followed by BLASTn confirmation identified multiple GETV sequences in biosamples from Asia (often as contaminants), but not in biosamples from Australia. In contrast, sequence reads aligning to the Australian Ross River virus (RRV) were readily identified in Australian biosamples. To explore the serological relationship between GETV and other alphaviruses, an adult wild-type mouse model of GETV was established. High levels of cross-reactivity and cross-protection were evident for convalescent sera from mice infected with GETV or RRV, highlighting the difficulties associated with the interpretation of early serosurveys reporting GETV antibodies in Australian cattle and pigs. The evidence that GETV circulates in Australia is thus not compelling. Full article

Bovine leukemia virus (BLV) is a deltaretrovirus infecting bovine B cells and causing enzootic bovine leucosis (EBL). The long terminal repeat (LTR) plays an indispensable role in viral gene expression. The BLV Tax protein acts as the main transactivator of LTR-driven transcription of BLV viral genes. The aim of this study was to analyze mutations in the BLV LTR region and tax gene to determine their association with transcriptional activity. LTRs were obtained from one hundred and six BLV isolates and analyzed for their genetic variability. Fifteen variants were selected and characterized based on mutations in LTR regulatory elements, and further used for in vitro transcription assays. Reporter vectors containing the luciferase gene under the control of each variant BLV promoter sequence, in addition to variant Tax expression vectors, were constructed. Both types of plasmids were used for cotransfection of HeLa cells and the level of luciferase activity was measured as a proxy of transcriptional activity. Marked differences in LTR promoter activity and Tax transactivation activity were observed amongst BLV variants. These results demonstrate that mutations in both the BLV LTR and tax gene can affect the promoter activity, which may have important consequences on proviral load, viral fitness, and transmissibility in BLV-infected cattle. Full article

Indwelling urinary catheters are common in health care settings and can lead to catheter-associated urinary tract infection (CAUTI). Long-term catheterization causes polymicrobial colonization of the catheter and urine, for which the clinical significance is poorly understood. Through prospective assessment of catheter urine colonization, we identified Enterococcus faecalis and Proteus mirabilis as the most prevalent and persistent co-colonizers. Clinical isolates of both species successfully co-colonized in a murine model of CAUTI, and they were observed to co-localize on catheter biofilms during infection. We further demonstrate that P. mirabilis preferentially adheres to E. faecalis during biofilm formation, and that contact-dependent interactions between E. faecalis and P. mirabilis facilitate establishment of a robust biofilm architecture that enhances antimicrobial resistance for both species. E. faecalis may therefore act as a pioneer species on urinary catheters, establishing an ideal surface for persistent colonization by more traditional pathogens such as P. mirabilis. Full article

Recently developed nanopore sequencing technologies offer a unique opportunity to rapidly close the genome and to identify complete sequences of mobile genetic elements (MGEs). In this study, 17 isolates of Listeria monocytogenes (Lm) epidemic clone II (ECII) from seven ready-to-eat meat or poultry processing facilities, not known to be associated with outbreaks, were shotgun sequenced, and among them, five isolates were further subjected to long-read sequencing. Additionally, 26 genomes of Lm ECII isolates associated with three listeriosis outbreaks in the U.S. and South Africa were obtained from the National Center for Biotechnology Information (NCBI) database and analyzed to evaluate if MGEs may be used as a high-resolution genetic marker for identifying and sourcing the origin of Lm. The analyses identified four comK prophages in 11 non-outbreak isolates from four facilities and three comK prophages in 20 isolates associated with two outbreaks that occurred in the U.S. In addition, three different plasmids were identified among 10 non-outbreak isolates and 14 outbreak isolates. Each comK prophage and plasmid was conserved among the isolates sharing it. Different prophages from different facilities or outbreaks had significant genetic variations, possibly due to horizontal gene transfer. Phylogenetic analysis showed that isolates from the same facility or the same outbreak always closely clustered. The time of most recent common ancestor of the Lm ECII isolates was estimated to be in March 1816 with the average nucleotide substitution rate of 3.1 × 10⁻⁷ substitutions per site per year. This study showed that complete MGE sequences provide a good signal to determine the genetic relatedness of Lm isolates, to identify persistence or repeated contamination that occurred within food processing environment, and to study the evolutionary history among closely related isolates. Full article

Pilchard orthomyxovirus (POMV) is an emerging pathogen of concern to the salmon industry in Australia. To explore the molecular events that underpin POMV infection, we challenged Atlantic salmon (Salmo salar) post-smolts in seawater via cohabitation. Tissue samples of the head kidney and liver were collected from moribund and surviving individuals and analyzed using transcriptome sequencing. Viral loads were higher in the head kidney compared to the liver, yet the liver presented more upregulated genes. Fish infected with POMV showed a strong innate immune response that included the upregulation of pathogen recognition receptors such as RIG-I and Toll-like receptors as well as the induction of interferon-stimulated genes (MX, ISG15). Moribund fish also presented a dramatic induction of pro-inflammatory cytokines, contributing to severe tissue damage and morbidity. An induction of major histocompatibility complex (MHC) class I genes (B2M) and markers of T cell-mediated immunity (CD8-alpha, CD8-beta, Perforin-1, Granzyme-A) was observed in both moribund fish and survivors. In addition, differential connectivity analysis showed that three key regulators (RELA/p65, PRDM1, and HLF) related to cell-mediated immunity had significant differences in connectivity in “clinically healthy” versus “clinically affected” or moribund fish. Collectively, our results show that T cell-mediated immunity plays a central role in the response of Atlantic salmon to the infection with POMV. Full article

Human Campylobacter jejuni infections are emerging, and constitute a significant health burden worldwide. The ubiquitously expressed pituitary adenylate cyclase-activating polypeptide (PACAP) is well-known for its cell-protective and immunomodulatory effects. In our actual intervention study, we used an acute campylobacteriosis model and assessed the potential disease-alleviating effects of exogenous PACAP. Therefore, secondary abiotic IL-10^−/− mice were perorally infected with C. jejuni and treated with synthetic PACAP38 intraperitoneally from day 2 until day 5 post-infection. Whereas PACAP did not interfere with the gastrointestinal colonization of the pathogen, mice from the PACAP group exhibited less severe clinical signs of C. jejuni-induced disease, as compared to mock controls, which were paralleled by alleviated apoptotic, but enhanced cell proliferative responses in colonic epithelia on day 6 post-infection. Furthermore, PACAP dampened the accumulation of macrophages and monocytes, but enhanced regulatory T cell responses in the colon, which were accompanied by less IFN-γ secretion in intestinal compartments in PACAP versus mock-treated mice. Remarkably, the inflammation-dampening properties of PACAP could also be observed in extra-intestinal organs, and strikingly, even the systemic circulation on day 6 post-infection. For the first time, we provide evidence that synthetic PACAP might be a promising candidate to combat acute campylobacteriosis and post-infectious sequelae. Full article

Siderophores produced in soil by plant growth-promoting rhizobacteria (PGPRs) play several roles, including nutrient mobilizers and can be useful as plants defense elicitors. We investigated the role of a synthetic mixed ligand bis-catechol-mono-hydroxamate siderophore (SID) that mimics the chemical structure of a natural siderophore, fimsbactin, produced by Acinetobacter spp. in the resistance against the phytopathogen Pseudomonas syringaepv tomato DC3000 (Pst DC3000), in Arabidopsis thaliana. We first tested the antibacterial activity of SID against Pst DC3000 in vitro. After confirming that SID had antibacterial activity against Pst DC3000, we tested whether the observed in vitro activity could translate into resistance of Arabidopsis to Pst DC3000, using bacterial loads as endpoints in a plant infection model. Furthermore, using quantitative polymerase chain reaction, we explored the molecular actors involved in the resistance of Arabidopsis induced by SID. Finally, to assure that SID would not interfere with PGPRs, we tested in vitro the influence of SID on the growth of a reference PGPR, Bacillus subtilis. We report here that SID is an antibacterial agent as well as an inducer of systemic priming of resistance in A. thaliana against Pst DC3000, and that SID can, at the same time, promote growth of a PGPR. Full article

The ongoing coronavirus disease 19 (COVID-19) pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a threat to human health. Despite this, many affected countries are now in the process of gradual lifting of COVID-19 restrictions that were initially implemented in response to the pandemic. The success of the so-called “exit strategy” requires continued surveillance of virus circulation in the community and evaluation of the prevalence of protective immunity among population. Serology tests are valuable tools for these purposes. Herein, SARS-CoV-2 full-length spike (S) recombinant protein was utilized to develop and optimize an indirect enzyme-linked immunoassay (ELISA) that enables a reliable detection of virus-specific IgG antibody in human sera. Importantly, the performance of this assay was evaluated utilizing micro-neutralization (MN) assay as a reference test. Our developed ELISA offers 100% sensitivity, 98.4% specificity, 98.8% agreement, and high overall accuracy. Moreover, the optical density (OD) values of positive samples significantly correlated with their MN titers. The assay specifically detects human IgG antibodies directed against SARS-CoV-2, but not those to Middle East respiratory syndrome coronavirus (MERS-CoV) or human coronavirus HKU1 (HCoV-HKU1). The availability of this in-house ELISA protocol would be valuable for various diagnostic and epidemiological applications. Full article

Infections with eggs of Echinococcus granulosus sensu lato (s.l.) can cause cystic echinococcosis in intermediate host animals and humans. Upon ingestion of viable eggs, oncospheres hatch from the eggs and subsequently develop into fluid-filled larval cysts, most frequently in the liver or the lungs. The slowly growing cysts progressively interfere with organ function. The risk of infection is determined by the host range of the parasite, its pathogenicity and other epidemiologically relevant parameters, which differ significantly among the five species within the E. granulosus s.l. complex. It is therefore essential to diagnose the correct species within E. granulosus s.l. to help understand specific disease epidemiology and to facilitate effective implementation of control measures. For this purpose, simple, fast and cost-effective typing techniques are needed. We developed quantitative real-time polymerase chain reactions (qPCRs) to target polymorphic regions in the mitochondrial genome of E. granulosus s.l. In a single-step typing approach, we distinguished E. granulosus s.l. members in four epidemiologically relevant subgroups. These were E. granulosus sensu stricto, E. equinus, E. ortleppi and the E. canadensis cluster. The technique also allowed identification and differentiation of these species from other Echinococcus or Taenia taxa for samples isolated from cysts or faeces. Full article

Both bacterial and fungal organisms display the ability to form biofilms; however, mixed bacterial/fungal biofilms are particularly difficult to control and eradicate. The opportunistic microbial pathogens Candida albicans and Staphylococcus aureus are among the most frequent causative agents of healthcare-acquired infections, and are often co-isolated forming mixed biofilms, especially from contaminated catheters. These mixed species biofilms display a high level of antibiotic resistance; thus, these infections are challenging to treat resulting in excess morbidity and mortality. In the absence of effective conventional antibiotic treatments, nanotechnology-based approaches represent a promising alternative for the treatment of highly recalcitrant polymicrobial biofilm infections. Our group has previously reported on the activity of pure positively charged silver nanoparticles synthesized by a novel microwave technique against single-species biofilms of C. albicans and S. aureus. Here, we have expanded our observations to demonstrate that that silver nanoparticles display dose-dependent activity against dual-species C. albicans/S. aureus biofilms. Moreover, the same nanoparticles were used to functionalize catheter materials, leading to the effective inhibition of the mixed fungal/bacterial biofilms. Overall, our results indicate the potent activity of silver nanoparticles against these cross-kingdom biofilms. More studies are warranted to examine the ability of functionalized catheters in the prevention of catheter-related bloodstream infections. Full article

Since the last decade, attention towards the occurrence of fungi in potable water has increased. Commensal and saprophytic microorganisms widely distributed in nature are also responsible for causing public health problems. Fungi can contaminate hospital environments, surviving and proliferating in moist and unsterile conditions. According to Italian regulations, the absence of fungi is not a mandatory parameter to define potable water, as a threshold value for the fungal occurrence has not been defined. This study evaluated the occurrence of fungi in potable water distribution systems in hospitals. The frequency of samples positive for the presence of fungi was 56.9%; among them, filamentous fungi and yeasts were isolated from 94.2% and 9.2% of the samples, respectively. The intensive care unit (87.1%) had the highest frequency of positive samples. Multivariable model (p < 0.0001), the variables of the period of the year (p < 0.0001) and type of department (p = 0.0002) were found to be statistically significant, suggesting a high distribution of filamentous fungi in the potable water of hospitals. Further studies are necessary to validate these results and identify the threshold values of fungi levels for different types of water used for various purposes to ensure the water is safe for consumption and protect public health. Full article

This study assessed the impact of a PRRSV (porcine reproductive and respiratory syndrome virus) recombinant strain (Horsens strain) on the reproductive performance of naïve pregnant sows in the last third of gestation. Fifteen sows were included: four negative reproductive controls (NTX), five infected with a PRRSV-1 field strain (Olot/91, T01), and six infected with the recombinant PRRSV-1 strain (Horsens strain, T02). Piglets were monitored until weaning. Reproductive performance was the primary variable. In sows, viremia and nasal shedding (T01 and T02 groups), and, in piglets, viral load in blood and in lungs, as well as macroscopic lung lesions (T01 and T02 groups), were the secondary variables. The reproductive performance results were numerically different between the two challenged groups. Moreover, viral loads in blood were 1.83 × 10⁶ ± 9.05 × 10⁶ copies/mL at farrowing, 1.05 × 10⁷ ± 2.21 × 10⁷ copies/mL at weaning from piglets born from T01 animals and 1.64 × 10³ ± 7.62 × 10³ copies/mL at farrowing, 1.95 × 10³ ± 1.17 × 10⁴ copies/mL at weaning from piglets born from T02 sows. Overall, 68.8% of T01 piglets and 38.1% of T02 piglets presented mild lung lesions. In conclusion, the results suggest that Horsens strain is less virulent than the field strain Olot/91 under these experimental conditions. Full article

COVID-19 disproportionately affects patients with medical comorbidities such as cardiovascular disease (CVD). Patients with CVD are widely prescribed 3-hydroxy-3-methyl-glutayl-CoA (HMG-CoA) reductase inhibitors (statins), a class of lipid-lowering medications known for their pleiotropic anti-inflammatory and immunomodulatory effects. However, the relationship between statin use and COVID-19 outcomes is not fully understood. In this preliminary study, we explored the association between statin use and severe COVID-19 outcomes in hospitalized patients, including intensive care unit (ICU) admission, the need for invasive mechanical ventilation (IMV), and in-hospital death. We performed a retrospective cohort study of 249 patients hospitalized with COVID-19 from 3 March 2020 to 10 April 2020 in Rhode Island, USA. Patient demographics, past medical history, current medications, and hospital course were recorded and analyzed. A multivariate logistic regression analysis was conducted to examine associations. After adjusting for age, sex, race, cardiovascular disease, chronic pulmonary disease, diabetes, and obesity, statin use was significantly associated with decreased risk for IMV (adjusted Odds Ratio (aOR) = 0.45, 95% Confidence Interval (CI): 0.20–0.99). Our results support the continued use of statins among COVID-19 patients and could have implications for future prospective studies on the management of COVID-19. Full article

Transient receptor potential (TRP) channel family proteins are sensors for pain, which sense a variety of thermal and noxious chemicals. Sensory neurons innervating the gut abundantly express TRPA1 and TRPV1 channels and are in close proximity of gut microbes. Emerging evidence indicates a bi-directional gut–brain cross-talk in several entero-neuronal pathologies; however, the direct evidence of TRP channels interacting with gut microbial populations is lacking. Herein, we examine whether and how the knockout (KO) of TRPA1 and TRPV1 channels individually or combined TRPA1/V1 double-knockout (dKO) impacts the gut microbiome in mice. We detect distinct microbiome clusters among the three KO mouse models versus wild-type (WT) mice. All three TRP-KO models have reduced microbial diversity, harbor higher abundance of Bacteroidetes, and a reduced proportion of Firmicutes. Specifically distinct arrays in the KO models are determined mainly by S24-7, Bacteroidaceae, Clostridiales, Prevotellaceae, Helicobacteriaceae, Rikenellaceae, and Ruminococcaceae. A1KO mice have lower Prevotella, Desulfovibrio, Bacteroides, Helicobacter and higher Rikenellaceae and Tenericutes; V1KO mice demonstrate higher Ruminococcaceae, Lachnospiraceae, Ruminococcus, Desulfovibrio and Mucispirillum; and A1V1dKO mice exhibit higher Bacteroidetes, Bacteroides and S24-7 and lower Firmicutes, Ruminococcaceae, Oscillospira, Lactobacillus and Sutterella abundance. Furthermore, the abundance of taxa involved in biosynthesis of lipids and primary and secondary bile acids is higher while that of fatty acid biosynthesis-associated taxa is lower in all KO groups. To our knowledge, this is the first study demonstrating distinct gut microbiome signatures in TRPA1, V1 and dKO models and should facilitate prospective studies exploring novel diagnostic/ therapeutic modalities regarding the pathophysiology of TRP channel proteins. Full article

Aim was to profile salivary total protease, Porphyromonas gingivalis gingipain, and neutrophil elastase activities in relation to the resolution of periodontal inflammation, salivary macrophage-derived chemokine (MDC), and macrophage inflammatory protein-1α concentrations. Nonsurgical periodontal treatment was performed in 24 periodontitis patients in a prospective interventional study design. Periodontal clinical parameters were recorded, and stimulated saliva samples were collected at baseline and 2, 6, and 12 weeks after treatment. Salivary total protease and gingipain activities were determined using fluorogenic substrates, elastase activity by chromogenic substrates, and cytokine concentrations by Luminex immunoassay. For statistical analyses, generalized linear mixed models for repeated measures were used. Salivary total protease activity elevated, while gingival inflammation and plaque accumulation decreased 2 and 6 weeks after periodontal therapy. Salivary MDC concentration was elevated 12 weeks after periodontal treatment. Patients with elevated protease activities at baseline in comparison to patients with low baseline total protease activities, had higher levels of gingival inflammation before and after periodontal treatment. In conclusion, elevations in salivary total protease activity seem to be part of periodontal healing at its early phases. Higher levels of salivary total protease activities before periodontal treatment may predict the severity and steadiness of unresolved gingival inflammation. Full article

Rocky Mountain spotted fever (RMSF) is a life-threatening tick-borne disease caused by Rickettsia rickettsii, which is widely distributed throughout the Americas. Over 4000 cases of RMSF are recorded annually in the United States, while only around 100 cases are reported in Brazil. Conversely, while case fatality rates in the United States oscillate around 5%, in Brazil they can surpass 70%, suggesting that differences in tick vectoring capacity, population sensitivity, and/or variability in virulence of the rickettsial strains may exist. In this study, we compared the susceptibility of C3H/HeN mice to two highly virulent strains of R. rickettsii, one from the United States (Sheila Smith) and the other from Brazil (Taiaçu). Animals inoculated with the Taiaçu strain succumbed to infection earlier and exhibited severe histological lesions in both liver and spleen sooner than mice infected with the Sheila Smith strain. These differences in survival and signs of the disease are not related to a greater proliferation of the Taiaçu strain, as there were no significant differences in the rickettsial load in mice tissues inoculated with either strain. The present study is the first step to experimentally assess differences in fatality rates of RMSF in two different regions of the American continent. Full article

Dengue outbreaks have regularly been recorded in Lao People’s Democratic Republic (PDR) since the first detection of the disease in 1979. In 2012, an integrated arbovirus surveillance network was set up in Lao PDR and an entomological surveillance has been implemented since 2016 in Vientiane Capital. Here, we report a study combining epidemiological, phylogenetic, and entomological analyzes during the largest DENV-4 epidemic ever recorded in Lao PDR (2015–2019). Strikingly, from 2015 to 2019, we reported the DENV-4 emergence and spread at the country level after two large epidemics predominated by DENV-3 and DENV-1, respectively, in 2012–2013 and 2015. Our data revealed a significant difference in the median age of the patient infected by DENV-4 compared to the other serotypes. Phylogenetic analysis demonstrated the circulation of DENV-4 Genotype I at the country level since at least 2013. The entomological surveillance showed a predominance of Aedesaegypti compared to Aedesalbopictus and high abundance of these vectors in dry and rainy seasons between 2016 and 2019, in Vientiane Capital. Overall, these results emphasized the importance of an integrated approach to evaluate factors, which could impact the circulation and the epidemiological profile of dengue viruses, especially in endemic countries like Lao PDR. Full article

The dynamics of Xylella fastidiosa infections in the context of the endophytic microbiome was studied in field-grown plants of the susceptible and resistant olive cultivars Kalamata and FS17. Whole metagenome shotgun sequencing (WMSS) coupled with 16S/ITS rRNA gene sequencing was carried out on the same trees at two different stages of the infections: In Spring 2017 when plants were almost symptomless and in Autumn 2018 when the trees of the susceptible cultivar clearly showed desiccations. The progression of the infections detected in both cultivars clearly unraveled that Xylella tends to occupy the whole ecological niche and suppresses the diversity of the endophytic microbiome. However, this trend was mitigated in the resistant cultivar FS17, harboring lower population sizes and therefore lower Xylella average abundance ratio over total bacteria, and a higher α-diversity. Host cultivar had a negligible effect on the community composition and no clear associations of a single taxon or microbial consortia with the resistance cultivar were found with both sequencing approaches, suggesting that the mechanisms of resistance likely reside on factors that are independent of the microbiome structure. Overall, Proteobacteria, Actinobacteria, Firmicutes, and Bacteriodetes dominated the bacterial microbiome while Ascomycota and Basidiomycota those of Fungi. Full article

Influenza A(H1N1)pdm09 viruses carrying a dual neuraminidase (NA) substitution were isolated from immunocompromised patients after administration of one or more NA inhibitors. These mutant viruses possessed an H275Y/I223R, H275Y/I223K, or H275Y/G147R substitution in their NA and showed enhanced cross-resistance to oseltamivir and peramivir and reduced susceptibility to zanamivir compared to single H275Y mutant viruses. Baloxavir could be a treatment option against the multidrug-resistant viruses because these dual H275Y mutant viruses showed susceptibility to this drug. The G147R substitution appears to stabilize the NA structure, with the fitness of the H275Y/G147R mutant virus being similar or somewhat better than that of the wild-type virus. Since the multidrug-resistant viruses may be able to transmit between humans, surveillance of these viruses must continue to improve clinical management and to protect public health. Full article

Japanese encephalitis virus (JEV) is the main cause of human viral encephalitis in Asia, with a mortality rate reaching 30%, mostly affecting children. The traditionally described cycle involving wild birds as reservoirs, pigs as amplifying hosts and Culex mosquitoes as vectors is questioned, with increasing evidence of a more complex multi-host system involved in areas where densities of pigs are low, such as in Cambodia. In 2018, we examined pigs, chickens, ducks and dogs from Kandal province, Cambodia, for antibody response against JEV by hemagglutination inhibition and virus neutralization assays. Forces of infection (FOI) for flaviviruses and JEV were estimated per species and per unit of body surface area (BSA). JEV seroprevalence reached 31% (95% CI: 23–41%) in pigs, 1% (95% CI: 0.1–3%) in chickens, 12% (95% CI: 7–19%) in ducks and 35% (95% CI: 28–42%) in dogs. Pigs were most likely to be infected (FOI: 0.09 per month), but the FOI was higher in ducks than in pigs for a given BSA (ratio of 0.13). Dogs had a lower FOI than ducks but a higher FOI than chickens (0.01 per month). For a given BSA, dogs were less likely to be infected than pigs (ratio of 1.9). In Cambodia, the virus may be circulating between multiple hosts. Dogs live in close contact with humans, and estimating their exposure to JEV infection could be a relevant indicator of the risk for humans to get infected, which is poorly known due to underdiagnosis. Understanding the JEV cycle and developing tools to quantify the exposure of humans is essential to adapt and support control measures for this vaccine-preventable disease. Full article

Poultry products are still an important cause of Salmonella infections worldwide, with an increasingly reported expansion of less-frequent serotypes or atypical strains that are frequently multidrug-resistant. Nevertheless, the ability of Salmonella to survive antimicrobials promoted in the context of antibiotic reducing/replacing and farming rethinking (e.g., organic acids and copper in feed/biocides) has been scarcely explored. We investigated Salmonella occurrence (conventional and molecular assays) among chicken meat at the processing stage (n = 53 batches/29 farms) and characterized their tolerance to diverse stress factors (antibiotics, copper, acid pH, and peracetic acid). Whole-genome sequencing was used to assess adaptive features and to perform comparative analysis. We found a low Salmonella occurrence (4%) and identified S. Enteritidis/ST11 plus atypical non-H₂S-producing S. 1,4,[5],12:i:-/ST3478. The ST3478 presented the ability to grow under diverse stresses (antibiotics, copper, and acid-pH). Comparative genomics among ST3478 isolates showed similar antibiotic/metal resistance gene repertoires and identical nonsense phsA thiosulfate reductase mutations (related to H₂S-negative phenotype), besides their close phylogenetic relationship by cgMLST and SNPs. This study alerts for the ongoing national and international spread of an emerging monophasic Salmonella Typhimurium clonal lineage with an enlarged ability to survive to antimicrobials/biocides commonly used in poultry production, being unnoticed by conventional Salmonella detection approaches due to an atypical non-H₂S-producing phenotype. Full article

Feline coronavirus (FCoV), the pathogen for feline infectious peritonitis, is a lethal infectious agent that can cause effusions in the pleural and abdominal cavities in domestic cats. To study the epidemiology of FCoV in Taiwan, 81 FIP-suspected sick cats with effusive specimens were recruited to test for FCoV infection using immunofluorescence staining and reverse transcription-polymerase chain reaction as detection methods, and viral RNAs were recovered from the specimens to conduct genotyping and phylogenetic analysis based on the spike (S) protein gene. The results revealed that a total of 47 (47/81, 58%) of the sick cats were positive for FCoV in the effusion samples, of which 39 were successfully sequenced and comprised of 21 type I strains, 9 type II strains, and 9 co-infections. The signalment analysis of these sick cats revealed that only the sex of cats showed a significant association (odds ratio = 2.74, 95% confidence interval = 1.06–7.07, p = 0.03) with the infection of FCoV, while age and breed showed no association. FCoV-positive cats demonstrated a significantly lower albumin to globulin ratio than negative individuals (p = 0.0004). The partial S gene-based phylogenetic analysis revealed that the type I strains demonstrated genetic diversity forming several clades, while the type II strains were more conserved. This study demonstrates the latest epidemiological status of FCoV infection in the northern part of Taiwan among sick cats and presents comparisons of Taiwan and other countries. Full article

The intestinal protozoan parasite, Giardia duodenalis, infects a large number of people in the world annually. Giardia infection has been considered a negative effect on intestinal epithelial cell growth, while the underlying mechanisms remain to be explored. Here we evaluated reactive oxygen species (ROS) production and apoptotic events in Giardia trophozoites-stimulated Caco-2 cells via fluorescence microscopy, transmission electron microscopy, flow cytometry, western blot, and cell counting kit-8 analyses. The results showed that Giardia trophozoite treatment could induce lactate dehydrogenase release and Caco-2 cell apoptosis. The ROS levels were increased post treatment. The observed typical characteristics of mitochondria damage include significant swelling and degeneration of matrix and cristae. After trophozoite treatment, the level of Bax protein expression was increased, while Bcl-2 protein decreased. Trophozoite stimulation also led to reduction of mitochondrial membrane potential and release of cytochrome c from the mitochondria to the cytoplasm, and this process was accompanied by activation of caspase-9 and caspase-3 and poly (ADP-ribose) polymerase 1 cleavage. Pretreatment with N-acetyl-L-cysteine, a ROS inhibitor, reversed G. duodenalis-induced Caco-2 cell apoptosis. Taken together, we indicated that G. duodenalis could induce Caco-2 cell apoptosis through a ROS- and mitochondria-mediated caspase-dependent pathway. This study furthers our understanding of the cellular mechanism of the interaction between Giardia trophozoites and host cells. Full article

Mosquitoes are the most important vectors for arthropod-borne viral diseases. Mixed viral infections of mosquitoes allow genetic recombination or reassortment of diverse viruses, turning mosquitoes into potential virologic mixing bowls. In this study, we field-collected mosquitoes of different species (Aedes aegypti and Culex pipiens complex), from different geographic locations and environments (central Europe and the Caribbean) for highly sensitive next-generation sequencing-based virome characterization. We found a rich virus community associated with a great diversity of host species. Among those, we detected a large diversity of novel virus sequences that we could predominately assign to circular Rep-encoding single-stranded (CRESS) DNA viruses, including the full-length genome of a yet undescribed Gemykrogvirus species. Moreover, we report for the first time the detection of a potentially zoonotic CRESS-DNA virus (Cyclovirus VN) in mosquito vectors. This study expands the knowledge on virus diversity in medically important mosquito vectors, especially for CRESS-DNA viruses that have previously been shown to easily recombine and jump the species barrier. Full article

Since it firstly emerged in China in 2013, clade 2.3.4.4 H5N6 highly pathogenic avian influenza viruses (HPAIVs) has rapidly replaced predominant H5N1 to become the dominant H5 subtype in China, especially in ducks. Not only endemic in China, it also crossed the geographical barrier and emerged in South Korea, Japan, and Europe. Here, we analyzed the genetic properties of the clade 2.3.4.4 H5N6 HPAIVs with full genome sequences available online together with our own isolates. Phylogenetic analysis showed that clade 2.3.4.4 H5N6 HPAIVs continuously reassorted with local H5, H6, and H7N9/H9N2. Species analysis reveals that aquatic poultry and migratory birds became the dominant hosts of H5N6. Adaption to aquatic poultry might help clade 2.3.4.4 H5N6 better adapt to migratory birds, thus enabling it to become endemic in China. Besides, migratory birds might help clade 2.3.4.4 H5N6 transmit all over the world. Clade 2.3.4.4 H5N6 HPAIVs also showed a preference for α2,6-SA receptors when compared to other avian origin influenza viruses. Experiments in vitro and in vivo revealed that clade 2.3.4.4 H5N6 HPAIVs exhibited high replication efficiency in both avian and mammal cells, and it also showed high pathogenicity in both mice and chickens, demonstrating high risk to public health. Considering all the factors together, adaption to aquatic poultry and migratory birds helps clade 2.3.4.4 H5N6 overcome the geographical isolation, and it has potential to be the next influenza pandemic in the world, making it worthy of our attention. Full article

Cryptococcosis, a mycosis presenting mostly as meningoencephalitis, affecting predominantly human immunodeficiency virus (HIV)-infected people, is mainly caused by Cryptococcus neoformans. The genetic variation of 48 C. neoformans isolates, recovered from 20 HIV-positive people in Lima, Peru, during the pre-highly active antiretroviral therapy (HAART) era, was studied retrospectively. The mating type of the isolates was determined by PCR, and the serotype by agglutination and CAP59-restriction fragment length polymorphism (RFLP). Genetic diversity was assessed by URA5-RFLP, PCR-fingerprinting, amplified fragment length polymorphism (AFLP), and multilocus sequence typing (MLST). All isolates were mating type alpha, with 39 molecular type VNI, seven VNII, corresponding to C. neoformans var. grubii serotype A, and two VNIII AD hybrids. Overall, the cryptococcal population from HIV-positive people in Lima shows a low degree of genetic diversity. In most patients with persistent cryptococcal infection, the same genotype was recovered during the follow-up. In four patients with relapse and one with therapy failure, different genotypes were found in isolates from the re-infection and from the isolate recovered at the end of the treatment. In one patient, two genotypes were found in the first cryptococcosis episode. This study contributes data from Peru to the ongoing worldwide population genetic analysis of Cryptococcus. Full article

Puumala orthohantavirus causes hemorrhagic fever with renal syndrome (HFRS) characterized by acute kidney injury (AKI), an abrupt decrease in renal function. Creatinine is routinely used to detect and quantify AKI; however, early AKI may not be reflected in increased creatinine levels. Therefore, kidney injury markers that can predict AKI are needed. The potential of the kidney injury markers urea, cystatin C, α1-microglobulin (A1M) and neutrophil gelatinase-associated lipocalin (NGAL) to detect early AKI during HFRS was studied by quantifying the levels of these markers in consecutively obtained plasma (P) and urine samples (U) for 44 HFRS patients. P-cystatin C and U-A1M levels were significantly increased during early HFRS compared to follow-up. In a receiver operating characteristic (ROC) curve analysis, P-cystatin C, U-A1M and P-urea predicted severe AKI with area under the curve 0.72, 0.73 and 0.71, respectively, whereas the traditional kidney injury biomarkers creatinine and U-albumin did not predict AKI. Nearly half of the HFRS patients (41%) fulfilled the criteria for shrunken pore syndrome, which was associated with the level of inflammation as measured by P-CRP. P-cystatin C and U-A1M are more sensitive and earlier markers compared to creatinine in predicting kidney injury during HFRS. Full article

A human-porcine reassortant strain, RVA/Human-wt/ZMB/UFS-NGS-MRC-DPRU4723/2014/G5P[6], was identified in a sample collected in 2014 from an unvaccinated 12 month old male hospitalised for gastroenteritis in Zambia. We sequenced and characterised the complete genome of this strain which presented the constellation: G5-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1. The genotype A8 is often observed in porcine strains. Phylogenetic analyses showed that VP6, VP7, NSP2, NSP4, and NSP5 genes were closely related to cognate gene sequences of porcine strains (e.g., RVA/Pig-wt/CHN/DZ-2/2013/G5P[X] for VP7) from the NCBI database, while VP1, VP3, VP4, and NSP3 were closely related to porcine-like human strains (e.g., RVA/Human-wt/CHN/E931/2008/G4P[6] for VP1, and VP3). On the other hand, the origin of the VP2 was not clear from our analyses, as it was not only close to both porcine (e.g., RVA/Pig-tc/CHN/SWU-1C/2018/G9P[13]) and porcine-like human strains (e.g., RVA/Human-wt/LKA/R1207/2009/G4P[6]) but also to three human strains (e.g., RVA/Human-wt/USA/1476/1974/G1P[8]). The VP7 gene was located in lineage II that comprised only porcine strains, which suggests the occurrence of independent porcine-to-human reassortment events. The study strain may have collectively been derived through interspecies transmission, or through reassortment event(s) involving strains of porcine and porcine-like human origin. The results of this study underline the importance of whole-genome characterisation of rotavirus strains and provide insights into interspecies transmissions from porcine to humans. Full article

Temperature response curves under diurnal oscillating temperatures differ from those under constant conditions for all stages of the Phytophthora infestans infection cycle on potatoes. We developed a mechanistic model (BLIGHTSIM) with an hourly time step to simulate late blight under fluctuating environmental conditions and predict late blight epidemics in potato fields. BLIGHTSIM is a modified susceptible (S), latent (L), infectious (I) and removed (R) compartmental model with hourly temperature and relative humidity as driving variables. The model was calibrated with growth chamber data covering one infection cycle and validated with field data from Ecuador. The model provided a good fit to all data sets evaluated. There was a significant interaction between average temperature and amplitude in their effects on the area under the disease progress curve (AUDPC) as predicted from growth chamber data on a single infection cycle. BLIGHTSIM can be incorporated in a potato growth model to study effects of diurnal temperature range on late blight impact under climate change scenarios. Full article

The plant-parasitic nematode Meloidogyne graminicola causes considerable damages to rice (Oryza sativa) culture. Resistance to M. graminicola in the related species Oryza glaberrima reduces root penetration by juveniles and stops further nematode development. M. graminicola genes expressed during O. sativa infection were previously characterized but no information is available about the molecular dialogue established with a resistant plant. We compared the M. graminicola transcriptomes of stage-two juveniles (J2s) before and during infection of susceptible or resistant rice. Among 36,121 M. graminicola genes surveyed, 367 were differentially expressed during infection of resistant or susceptible plants. Genes encoding cell wall-degrading enzymes, peptidases and neuropeptides were expressed for a longer time in resistant plants compared to susceptible plants. Conversely, genes related to nematode development were not activated in the resistant host. The majority of M. graminicola effector genes had similar expression patterns, whatever the host genotype. However, two venom allergen-like protein (VAP)-encoding genes were specifically induced in resistant plants and Mg-VAP1 silencing in J2s reduced their ability to colonize roots. This study highlighted that M. graminicola adapts its gene expression to the host susceptibility. Further investigation is required to assess the role of Mg-VAPs in the rice–nematode interaction. Full article

Mycoplasma bovis in cattle is difficult to diagnose. Recently, the ID screen^® mycoplasma bovis indirect ELISA (ID screen) was commercially released by IDVet. The objectives of this study were to: (1) gain and share experience of using the ID screen in adult dairy cows under field conditions; (2) determine the correlation between antibody levels in milk and serum and (3) compare the ID screen results with those of the Bio K 302 (BioX 302) ELISA from BioX Diagnostics. Paired serum and milk samples were collected from 270 cows from 12 Danish dairy herds with three categories of M. bovis disease history. The ID screen tested nearly all cows positive in all, but the three non-infected herds, while the BioX 302 tested very few cows positive. The ID screen is therefore a much more sensitive test than the BioX 302. However, cows in five exposed herds without signs of ongoing infection and two herds with no history of M. bovis infection also tested ID screen positive. Therefore, the performance and interpretation of the test must be investigated under field conditions in best practice test evaluation setups. A concordance correlation coefficient of 0.66 (95% CI: 0.59–0.72) between the ID screen serum and milk results indicates that milk samples can replace serum samples for the ID screen diagnosis of M. bovis in adult cows. Full article

Alveolar echinococcosis (AE) is caused by the intermediate stage of Echinococcus multilocularis. We aimed to correlate computed tomography (CT) data with histology to identify distinct characteristics for different lesion types. We classified 45 samples into five types with the Echinococcus multilocularis Ulm Classification for Computed Tomography (EMUC-CT). The various CT lesions exhibited significantly different histological parameters, which led us to propose a progression model. The initial lesion fit the CT type IV classification, which comprises a single necrotic area with the central located laminated layer, a larger distance between laminated layer and border zone, a small fibrotic peripheral zone, and few small particles of Echinococcus multilocularis (spems). Lesions could progress through CT types I, II, and III, characterized by shorter distances between laminated layer and border zone, more spems inside and surrounding the lesion, and a pronounced fibrotic rim (mostly in type III). Alternatively, lesions could converge to a highly calcified, regressive state (type V). Our results suggest that the CT types mark sequential stages of the infection, which progress over time. These distinct histological patterns advance the understanding of interactions between AE and human host; moreover, they might become prognostically and therapeutically relevant. Full article

The pathology of pigeon circovirus (PiCV) is still unknown, but it is regarded as an immunosuppressant. This study aimed to find a correlation between PiCV natural infection and immunosuppression. The study was conducted with 56 pigeons divided into the following groups: PiCV-positive but showing (group S) or not (group I) non-specific clinical symptoms and asymptomatic pigeons negative for PiCV (group H). The percentage and apoptosis of T CD3⁺ and B IgM⁺ splenocytes; the expression of CD4, CD8, and IFN-γ genes in splenic mononuclear cells; the number of PiCV viral loads in the bursa of Fabricius; and the level of anti-PiCV antibodies were analyzed. The results showed that the percentage of B IgM⁺ cells was almost two-fold lower in group S than in group H, and that ca. 20% of the lymphocytes were apoptotic. No increased apoptosis was detected in TCD3⁺ subpopulation. The PiCV viral loads were approximately one thousand and ten thousand times higher in group S than in groups I and H, respectively. Our results indicate a possible correlation between the number of PiCV viral loads and severity of PiCV infection and confirm that PiCV infection leads to the suppression of humoral immunity by inducing B lymphocyte apoptosis. Full article

Different helminths and protozoa are transmitted to humans by oral uptake of environmentally resistant parasite stages after hand-to-mouth contact or by contaminated food and water. The aim of this study was to develop and validate a method for the simultaneous detection of parasite stages from fresh produce (lettuce) by a one-way isolation test kit followed by genetic identification (PCR, sequencing). Three sentinel zoonotic agents (eggs of Toxocara canis, Echinococcus multilocularis and oocysts of Toxoplasma gondii) were used to investigate the practicability and sensitivity of the method. The detection limits (100% positive results) in the recovery experiments were four Toxocara eggs, two E. multilocularis eggs and 18 T. gondii oocysts (in 4/5 replicates). In a field study, helminth DNA was detected in 14 of 157 lettuce samples including Hydatigera taeniaeformis (Syn. Taenia taeniaeformis) (four samples), T. polyacantha (three), T. martis (one), E. multilocularis (two) and Toxocara cati (four). Toxoplasma gondii was detected in six of 100 samples. In vivo testing in mice resulted in metacestode growth in all animals injected with 40–60 E. multilocularis eggs, while infection rates were 20–40% with 2–20 eggs. The developed diagnostic strategy is highly sensitive for the isolation and genetic characterisation of a broad range of parasite stages from lettuce, whereas the sensitivity of the viability tests needs further improvement. Full article

Parasite monitoring is considered a necessary step for cetacean management and conservation. Between February 2013 and July 2015, 26 dolphins (15 Stenella coeruleoalba, 10 Tursiops truncatus, and one Grampus griseus) stranded along the Tuscan coastline of the protected marine area “Pelagos Sanctuary”, were examined. Organs, tissues, and faecal and blood samples taken from all animals were analysed by parasitological, immunological, and molecular techniques. Twenty-one out of 26 dolphins (80.77%) tested positive for at least one parasite species, and 13/15 (86.7%) S. coeruleoalba, 7/10 (70%) T. truncatus, and the single G. griseus were found positive. Identified parasites included the nematodes Skrjabinalius guevarai (7.69%, 2/26), Halocercus lagenorhynchi (3.85%, 1/26), Halocercus delphini (7.69%, 2/26), Stenurus ovatus (7.69%, 2/26), Crassicauda spp. (7.69%, 2/26); the trematodes Pholeter gastrophilus (26.92%, 7/26), Campula palliata (3.85%, 1/26); the cestodes Phyllobothrium delphini (42.31%, 11/26), Monorygma grimaldii (23.08%, 6/26), Tetrabothrium forsteri (7.69%, 2/26), Strobilocephalus triangularis (7.69%, 2/26), and the acanthocephalan Bolbosoma vasculosum (7.69%, 2/26). Moreover, 6/26 (23%) animals scored positive to Toxoplasma gondii at serology, but PCR confirmed the infection (T. gondii Type II genotype) in a single animal. In examined dolphins, obtained results showed a high prevalence of endoparasites, which included species considered as a cause of severe debilitation or death. Full article