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Toxoplasma gondiiis a major food-borne parasite and undercooked meat of infected pigs represents an important source of infection for humans. Since infections in pigs are mostly subclinical, adequate diagnostic tests for use at the farm level are pursued. Oral fluid (OF) was shown to be a promising matrix for direct and indirect detection of infections with various pathogens in pigs. The objective of this study was to assess whetherT. gondiiinfections in pigs could be diagnosed using an indirect ELISA kit adapted for OF samples (OF-ELISA). Routine serology and OF-immunoblot (IB) were used as standards for the comparison. For this, serial OF samples from sows (n=8) and fatteners (n=3) experimentally inoculated withT. gondiioocysts, individual field samples from potentially exposed sows (n=9) and pooled OF samples from potentially exposed group-housed fatteners (n=195 pig groups, including 2,248 animals) were analysed for antibodies againstT. gondiiby ELISA. For individual animals, OF-ELISA exhibited a relative diagnostic specificity of 97.3% and a relative diagnostic sensitivity of 78.8%. In experimentally infected animals, positive OF-ELISA results were observed from 1.5weeks post inoculation (pi) until the end of the experimental setup (8 to 30weeks pi); however, values below the estimated cut-off were occasionally observed in some animals despite constant seropositivity. In potentially exposed individual animals, OF- and serum-ELISA results showed 100% agreement. In group-housed fatteners, antibodies againstT. gondiicould be reliably detected by OF-ELISA in groups in which at least 25% of the animals were seropositive. This OF-ELISA, based on a commercially available serum-ELISA, may represent an interesting non-invasive screening tool for detecting pig groups with a high exposure toT. gondiiat the farm level. The OF-ELISA may need further adjustments to consistently detect individual infected pigs, probably due to variations in OF antibody concentration over time.

Major climatic changes in conjunction with animal movement may be associated with the spread of parasites and their vectors into new populations, with potentially important consequences for population persistence. Parasites can evolve to adapt to unsuitable ecological conditions and take up refuge within new host species, with consequences for the population growth of the new host species. One parasite species that has likely been increasing its geographic range, and potentially infecting new hosts, is the recently described air sac nematode Serratospiculoides amaculata, in great tits (Parus major) in Slovakia. In this study, we screened wild birds for potential air sac nematode infection in a woodland area of southern Germany. We identified four additional host species: Eurasian nuthatch, great spotted woodpecker, greenfinch and robin. As infection by this group of nematodes can be highly pathogenic, we recommend further investigation into its potential risk to these populations., International Journal for Parasitology. Parasites and Wildlife, 21, ISSN:2213-2244

The diagnosis of eimeriosis in calves mainly relies on the presence of diarrhoea and the excretion of Eimeria oocysts in the faeces. Restraining the animals to collect rectal samples for diagnostic purposes is stressful and time-consuming. The aim of this study was to evaluate a method for the quantification of oocysts in environmental barn straw samples. To investigate the recovery rate of the method, straw and Eimeria negative faeces were spiked with Eimeria oocysts in plastic bags and mixed with water and 0.05% Tween 20 (v/v); the liquids were filtered twice through sieves (mesh size 300 and 52 μm), centrifuged and the number of oocysts in the sediment determined using a McMaster counting chamber. A recovery rate of 52.4% (95% confidence interval: 48.2–56.5%) was obtained. In the following, field straw (n = 156) and individual faecal samples (n = 195, also analysed by McMaster counting chambers) were collected on four different farms. Eimeria oocysts were present on all farms in faecal (84/195, 43.1%) and straw samples (119/156, 76.3%). In 37 (23.7%) straw samples, sporulated oocysts were observed, with a sporulation rate ranging from 0 to 40%. Despite high variability between farms and examination days, mean numbers of oocysts in the straw positively correlated with mean numbers of oocysts excreted in the faeces (ρSpearman = 0.60). The examination of environmental straw samples may represent an easy-to-perform, non-invasive, inexpensive preliminary diagnostic approach for surveillance of eimeriosis at group level, having the potential to assess the infection pressure.

Background The lungworm Aelurostrongylus abstrusus infects wild and domestic feline species worldwide and is considered a primary respiratory parasite of cats. Definitive diagnosis is based on identification of first stage larvae (L1s) released in faeces approximately 5 to 6 weeks after infection. More recently, serology has been shown to be a diagnostic alternative for A. abstrusus infection in cats. The present study was aimed at evaluating the diagnostic performance of serological antibody detection compared to faecal examination for A. abstrusus infection in a population of cats with known infection status from endemic areas in Italy and to identify factors (larval scores, age, co-infections with other helminths) that may influence test sensitivity and specificity. Methods All cats resulting positive in the Baermann technique (n=78) were tested with the A. abstrusus ELISA An additional 90 serum samples from cats living in three geographical areas with infection prevalence greater than 10%, but that resulted negative in Baermann, were also tested. Results Among 78 cats copromicroscopically positive for L1s of A. abstrusus (Group 1), 29 (37.2%) were seropositive in ELISA. Of the 90 cats from Group 2 (cats living in three geographical areas in Italy with A. abstrusus prevalence greater than 10%, but negative in Baermann examination), 11 (12.2%) were positive in ELISA. The overall seroprevalence was 23.8%. There was no statistical difference either between average optical density (OD) values of cats excreting >100 L1s vs. cats excreting P value = 0.3247) or comparing the OD values with age of infected cats. Few Baermann-negative cats positive for Toxocara cati or hookworms were seropositive, supporting lack of cross reactivity to these nematodes. Conclusions Results from the present study suggest that relying solely on faecal examination may underestimate prevalence of A. abstrusus infection in cats and that field surveys based on antibody detection is useful for establishing true prevalence of infected and/or exposed animals.

The faecal egg count reduction test (FECRT) remains the method of choice for establishing the efficacy of anthelmintic compounds in the field, including the diagnosis of anthelmintic resistance. We present a guideline for improving the standardization and performance of the FECRT that has four sections. In the first section, we address the major issues relevant to experimental design, choice of faecal egg count (FEC) method, statistical analysis, and interpretation of the FECRT results. In the second section, we make a series of general recommendations that are applicable across all animals addressed in this guideline. In the third section, we provide separate guidance details for cattle, small ruminants (sheep and goats), horses and pigs to address the issues that are specific to the different animal types. Finally, we provide overviews of the specific details required to conduct an FECRT for each of the different host species. To address the issues of statistical power vs. practicality, we also provide two separate options for each animal species; (i) a version designed to detect small changes in efficacy that is intended for use in scientific studies, and (ii) a less resource-intensive version intended for routine use by veterinarians and livestock owners to detect larger changes in efficacy. Compared to the previous FECRT recommendations, four important differences are noted. First, it is now generally recommended to perform the FECRT based on pre- and post-treatment FEC of the same animals (paired study design), rather than on post-treatment FEC of both treated and untreated (control) animals (unpaired study design). Second, instead of requiring a minimum mean FEC (expressed in eggs per gram (EPG)) of the group to be tested, the new requirement is for a minimum total number of eggs to be counted under the microscope (cumulative number of eggs counted before the application of a conversion factor). Third, we provide flexibility in the required size of the treatment group by presenting three separate options that depend on the (expected) number of eggs counted. Finally, these guidelines address all major livestock species, and the thresholds for defining reduced efficacy are adapted and aligned to host species, anthelmintic drug and parasite species. In conclusion, these new guidelines provide improved methodology and standardization of the FECRT for all major livestock species.

Background Cytauxzoon spp. infection is believed to be a newly emerging tick-borne disease in felids in Europe, with three species of the haemoparasite having recently been differentiated in wild felids. In Switzerland, rare infections have been documented in domestic cats in the west and northwest of the country, the first of which was in 2014. The aims of the present study were: (i) to characterize a Cytauxzoon spp. hotspot in domestic cats in central Switzerland; (ii) to elucidate the geographic distribution of Cytauxzoon spp. in domestic cats in Switzerland; (iii) to assess suspected high-risk populations, such as stray and anaemic cats; and (iv) to investigate the newly emerging nature of the infection. Cytauxzoon spp. were further differentiated using mitochondrial gene sequencing. Methods The overall study included samples from 13 cats from two households in central Switzerland (study A), 881 cats from all regions of Switzerland (study B), 91 stray cats from a hotspot region in the northwest of Switzerland and 501 anaemic cats from across Switzerland (study C), and 65 Swiss domestic cats sampled in 2003 and 34 European wildcats from eastern France sampled in the period 1995–1996 (study D). The samples were analysed for Cytauxzoon spp. using real-time TaqMan quantitative PCR, and positive samples were subjected to 18S rRNA, cytochrome b (CytB) and cytochrome c oxidase subunit I (COI) gene sequencing. Results In study A, six of 13 cats from two neighbouring households in central Switzerland tested postive for Cytauxzoon spp.; two of the six infected cats died from bacterial infections. In studies B and C, only one of the 881 cats (0.1%; 95% confidence interval [CI]: 0–0.3%) in the countrywide survey and one of the 501 anaemic cats (0.2%; 95% CI: 0–0.6%) tested postive for Cytauxzoon spp. while eight of the 91 stray cats in the northwest of Switzerland tested positive (8.8%; 95% CI: 3.0–14.6%). In study D, Cytauxzoon spp. was detected in one of the 65 domestic cat samples from 2003 (1.5%; 95% CI: 0–4.5%) and in ten of the 34 European wildcat samples from 1995 to 1996 (29%; 95% CI: 14.2–44.7%). The isolates showed ≥ 98.6% sequence identities among the 18S rRNA, CytB and COI genes, respectively, and fell in the subclade Cytauxzoon europaeus based on CytB and COI gene phylogenetic analyses. Conclusions The study challenges the newly emerging nature of Cytauxzoon spp. in central Europe and confirms that isolates from domestic cats in Switzerland and European wild felids belong to the same species. Graphical Abstract

The skin microbiota plays an essential role in the protection against pathogens. It is our skin microbiota that makes us smell different from each other, rendering us more or less attractive to mosquitoes. Mosquitoes exploit skin bacterial odours to locate their hosts and are vectors of pathogens that can cause severe diseases such as malaria and dengue fever. A novel solution for long-lasting protection against insect vectors of disease could be attained by manipulating the bacterial commensals on human skin. The current options for protection against biting insects usually require topical application of repellents that evaporate within hours. We discuss possible routes for the use of commensal bacteria to create a microbial-based repellent.

Anthelmintic resistance (AR) has thus far only rarely been reported for intestinal helminths of dogs and cats, in contrast to parasites of livestock and horses. We highlight possible reasons for this striking and important discrepancy, including ecological, biological and genetic factors and/or intervention regimens of key intestinal helminths concerning both host groups. In view of the current knowledge related to the genetics, mechanisms and principles of AR development, we point at issues which in our view contribute to a comparatively lower risk of AR development in intestinal helminths of dogs and cats. Finally, we specify research needs and provide recommendations by which, based on the available information about AR in ruminant and equine helminths, the development of AR in dog and cat helminths may best be documented, prevented or at least postponed., Graphical abstract Image 1, Highlights • Identifying causes of differences in anthelmintic resistance between livestock and dogs & cats. • Discussion of roles of AR mechanisms, genetics and husbandry in delayed AR evolution in dogs & cats. • Research needs for improved tools to monitor AR development in dogs & cats.

Genetically modified maize DP4114 × MON 89034 × MON 87411 × DAS-40278-9 was developed by crossing to combine four single events: DP4114, MON 89034, MON 87411 and DAS-40278-9. The GMO Panel previously assessed the four single maize events and two of the subcombinations and did not identify safety concerns. No new data on the single maize events or the assessed subcombinations were identified that could lead to modification of the original conclusions on their safety. The molecular characterisation, comparative analysis (agronomic, phenotypic and compositional characteristics) and the outcome of the toxicological, allergenicity and nutritional assessment indicate that the combination of the single maize events and of the newly expressed proteins in the four-event stack maize does not give rise to food and feed safety and nutritional concerns. Therefore, no post-market monitoring of food/feed is considered necessary. In the case of accidental release of viable four-event stack maize grains into the environment, this would not raise environmental safety concerns. The GMO Panel assessed the likelihood of interactions among the single events in eight of the maize subcombinations not previously assessed and concludes that these are expected to be as safe as the single events, the previously assessed subcombinations and the four-event stack maize. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of maize DP4114 × MON 89034 × MON 87411 × DAS-40278-9. Post-market monitoring of food/feed is not considered necessary. The GMO Panel concludes that the four-event stack maize and its subcombinations are as safe as its non-GM comparator and the tested non-GM maize varieties with respect to potential effects on human and animal health and the environment.

In 2012, EFSA issued an opinion on plants developed through cisgenesis and intragenesis. With the development of New Genomic Techniques (NGTs) in the last decade, cisgenic and intragenic plants can now be obtained with the insertion of a desired sequence in a precise location of the genome. EFSA has been requested by European Commission to provide an updated scientific opinion on the safety and the risk assessment of plants developed through cisgenesis and intragenesis, in order to (i) identify potential risks, comparing them with those posed by plants obtained by conventional breeding and Established Genomic Techniques (EGTs) and (ii) to determine the applicability of current guidelines for the risk assessment of cisgenic and intragenic plants. The conclusions of the previous EFSA opinion were reviewed, taking into consideration the new guidelines and the recent literature. The GMO panel concludes that no new risks are identified in cisgenic and intragenic plants obtained with NGTs, as compared with those already considered for plants obtained with conventional breeding and EGTs. There are no new data since the publication of the 2012 EFSA opinion that would challenge the conclusions raised in that document. The conclusions of the EFSA 2012 Scientific Opinion remain valid. The EFSA GMO Panel reiterates from these conclusions that with respect to the source of DNA and the safety of the gene product, the hazards arising from the use of a related plant-derived gene by cisgenesis are similar to those from conventional plant breeding, whereas additional hazards may arise for intragenic plants. Furthermore, the EFSA GMO Panel considers that cisgenesis and intragenesis make use of the same transformation techniques as transgenesis, and therefore, with respect to the alterations to the host genome, cisgenic, intragenic and transgenic plants obtained by random insertion do not cause different hazards. Compared to that, the use of NGTs reduces the risks associated with potential unintended modifications of the host genome. Thus, fewer requirements may be needed for the assessment of cisgenic and intragenic plants obtained through NGTs, due to site-directed integration of the added genetic material. Moreover, the GMO panel concludes that the current guidelines are partially applicable and sufficient. On a case-by-case basis, a lesser amount of data might be needed for the risk assessment of cisgenic or intragenic plants obtained through NGTs.

Malaria transmission-blocking vaccines induce antibodies that target Plasmodium in the mosquito vector. We recently reported that Pfs230 vaccine achieves activity superior to Pfs25 in humans. Here, we describe clonal expansion in the variable region of immunoglobulin heavy chains (VH) of antigen-specific single B cells collected from humans immunised with Pfs230D1-EPA or Pfs25-EPA conjugate vaccines formulated in Alhydrogel®. Based on studies of CD27+ memory B cells following Pfs230 vaccination, clonal expansion and somatic hypermutation was seen in four of five subjects. Pfs25 did not induce sufficient CD27+ cells for sorting; based instead on CD19+ Pfs25-reactive B cells, clonal expansion was only seen in two of five subjects. Clonal expansions and mutations in Pfs230-specific single B cells combined with the enhanced activity of Pfs230 antibodies by complement, might justify the outstanding activity of Pfs230D1 as a TBV candidate.

The larval stage of the helminthic cestode Echinococcus multilocularis can inflict tumor-like hepatic lesions that cause the parasitic disease alveolar echinococcosis in humans, with high mortality in untreated patients. Opportunistic properties of the disease have been established based on the increased incidence in immunocompromised patients and mouse models, indicating that an appropriate adaptive immune response is required for the control of the disease. However, cellular interactions and the kinetics of the local hepatic immune responses during the different stages of infection with E. multilocularis remain unknown. In a mouse model of oral infection that mimics the normal infection route in human patients, the networks of the hepatic immune response were assessed using single-cell RNA sequencing (scRNA-seq) of isolated hepatic CD3+ T cells at different infection stages. We observed an early and sustained significant increase in natural killer T (NKT) cells and regulatory T cells (Tregs). Early tumor necrosis factor (TNF)- and integrin-dependent interactions between these two cell types promote the formation of hepatic lesions. At late time points, downregulation of programmed cell death protein 1 (PD-1) and ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1)-dependent signaling suppress the resolution of parasite-induced pathology. The obtained data provide fresh insight into the adaptive immune responses and local regulatory pathways at different infection stages of E. multilocularis in mice.

With the current expansion of vector-based research and an increasing number of facilities rearing arthropod vectors and infecting them with pathogens, common measures for containment of arthropods as well as manipulation of pathogens are becoming essential for the design and running of such research facilities to ensure safe work and reproducibility, without compromising experimental feasibility. These guidelines and comments were written by experts of the Infravec2 consortium, a Horizon 2020-funded consortium integrating the most sophisticated European infrastructures for research on arthropod vectors of human and animal diseases. They reflect current good practice across European laboratories with experience of safely handling different mosquito species and the pathogens they transmit. As such, they provide experience-based advice to assess and manage the risks to work safely with mosquitoes and the pathogens they transmit. This document can also form the basis for research with other arthropods, for example, midges, ticks or sandflies, with some modification to reflect specific requirements. info:eu-repo/semantics/publishedVersion

The endangered Bengal tiger (Panthera tigris tigris) is a keystone species playing an essential role in ecology as well as in the social and spiritual lives of the Himalayan people. The latest estimate of the Bengal tiger population in Bhutan accounts for 103 individuals. Infectious organisms, including zoonotic parasites causing high burden in human health, have received little attention as a cause of mortality in tigers. Taeniosis/cysticercosis, caused by the cestode Taenia solium, is considered one of the major neglected tropical diseases in Southeast Asia. We present here a case of neurocysticercosis in a Bengal tiger showing advanced neurological disease outside Thimphu, the capital city of Bhutan. After palliative care, the animal died, and necropsy revealed multiple small cysts in the brain. Here we show the presence of two genetic variants of T. solium in the parasite material collected based on PCR and sequencing of the complete cox1 and cytB genes. The sequences form a discrete branch within the Asia plus Madagascar cluster of the parasite. On other hand, tests for feline morbillivirus, feline calicivirus, canine distemper virus, Nipah, rabies, Japanese encephalitis, feline leukaemia and feline immunodeficiency virus were negative. In contrast, PCR for feline herpesvirus was positive and a latex agglutination test revealed an elevated antibody titer against Toxoplasma gondii (titer 1:256). The molecular examination of taeniid eggs isolated from the tiger faeces produced sequences for which the highest homology in GenBank is between 92% and 94% with T. regis and T. hydatigena. This fatal case of T. solium neurocysticercosis, a disease previously unrecorded in tigers or other non-domestic felids, demonstrates an anthropogenically driven transmission of a deadly pathogen which could become a serious threat to the tiger population., Graphical abstract Image 1, Highlights • This is the first report of a fatal infection with Taenia solium in a Bengal tiger. • There is a need for research into infectious disease threats to tigers in Bhutan. • The importance of a ‘One Health’ approach is demonstrated and recommended. • Non-domestic felids.

Metastrongylus sp. is a lungworm with worldwide distribution in wild boars and other suids. In Switzerland, two wild boar populations are geographically divided by the Alps. We investigated 84 wild boars, 52 from north and 32 from south of the Alps, different in sex and age (juveniles, subadults, adults), shot between September and December 2018. The lungs were macroscopically examined and dissected for the detection of Metastrongylus specimens. Additionally, faecal samples were obtained from 55 animals and analysed by sedimentation/flotation and the mini-FLOTAC® method. Overall, 12′774 Metastrongylus sp. specimens were isolated: prevalence was 77.4% and mean burden 196.5 (range: 1–2589), with no significant differences between north (80.8%, 218.0) and south (71.9%, 157.4) nor between sexes and age groups. Macroscopically, dense nodular lesions associated with Metastrongylus sp. were present in 19 out of 65 (33.9%) positive lungs. Five Metastrongylus sp. were detected: M. pudendotectus (67.9%), M. salmi (63.1%), M. confusus (56%), M. apri (44%) and M. asymmetricus (17.9%), with a significant difference (p = 0.012) between north (32.7%) and south (62.5%) for M. apri. The lungworm population was female biased. The number of Metastrongylus sp. eggs in faecal samples did not correlate with worm burdens. Furthermore, the following endoparasites were detected: Isospora suis/Eimeria sp. (74.5%), Strongyloides suis (27.3%%), Trichuris suis (20.0%), Hyostrongylus rubidus/Oesophagostomum sp. (18.2%), Globocephalus sp. (9.1%), Capillaria sp. (7.3%), Ascaris suum (3.6%), Giardia sp. (3.6%) and Balantidium coli (1.8%), with significant differences for S. suis (north 36.1% > south 10.5%) and Globocephalus sp. (only south, 26.3%). Although geographically separated, both Swiss wild boar populations share similar parasite spectra, while also showing some regional idiosyncrasies partially explained by ecological and climatic factors. Despite their clinical relevance being unknown, accurate knowledge concerning the distribution of endoparasites in the wildlife reservoir is relevant to better understand risk factors for the domestic pig population., Graphical abstract Image 1, Highlights • 12′774 Metastrongylus sp. were isolated from wild boars north and south of the Alps. • Metastrongylus prevalence was 77.4%, mean burden 196.5, five species were detected. • No significant differences between wild boar sexes and age groups. • Overall, similar parasite spectra with regional idiosyncrasies were identified. • Known parasite occurrence is relevant to evaluate risk factors for domestic pigs.

Background Cronobacter sakazakii is a foodborne pathogen that causes septicemia, meningitis, and necrotizing enterocolitis in neonates and infants. The current research details the full genome sequences of two extremely persistent C. sakazakii strains (H322 and GK1025B) isolated from powdered infant formula (PIF) manufacturing settings. In addition, the genetic attributes associated with five plasmids, pH322_1, pH322_2, pGK1025B_1, pGK1025B_2, and pGK1025B_3 are described. Materials and Methods Using PacBio single-molecule real-time (SMRT®) sequencing technology, whole genome sequence (WGS) assemblies of C. sakazakii H322 [Sequence type (ST)83, clonal complex [CC] 83) and GK1025B (ST64, CC64) were generated. Plasmids, also sequenced, were aligned with phylogenetically related episomes to determine, and identify conserved and missing genomic regions. Results A truncated ~ 13 Kbp type 6 secretion system (T6SS) gene cluster harbored on virulence plasmids pH322_2 and pGK1025B_2, and a second large deletion (~ 6 Kbp) on pH322_2, which included genes for a tyrosine-type recombinase/integrase, a hypothetical protein, and a phospholipase D was identified. Within the T6SS of pH322_2 and pGK1025B_2, an arsenic resistance operon was identified which is in common with that of plasmids pSP291_1 and pESA3. In addition, PHASTER analysis identified an intact 96.9 Kbp Salmonella SSU5 prophage gene cluster in pH322_1 and pGK1025B_1 and showed that these two plasmids were phylogenetically related to C. sakazakii plasmids: pCS1, pCsa767a, pCsaC757b, pCsaC105731a. Plasmid pGK1025B_3 was identified as a novel conjugative Cronobacter plasmid. Furthermore, WGS analysis identified a ~ 16.4 Kbp type 4 secretion system gene cluster harbored on pGK1025B_3, which contained a phospholipase D gene, a key virulence factor in several host–pathogen diseases. Conclusion These data provide high resolution information on C. sakazakii genomes and emphasizes the need for furthering surveillance studies to link genotype to phenotype of strains from previous investigations. These results provide baseline data necessary for future in-depth investigations of C. sakazakii that colonize PIF manufacturing facility settings and genomic analyses of these two C. sakazakii strains and five associated plasmids will contribute to a better understanding of this pathogen's survival and persistence within various “built environments” like PIF manufacturing facilities.

International audience; As part of the human microbiota, the fungus Candida albicans colonizes the oral cavity and other mucosal surfaces of the human body. Commensalism is tightly controlled by complex interactions of the fungus and the host to preclude fungal elimination but also fungal overgrowth and invasion, which can result in disease. As such, defects in antifungal T cell immunity render individuals susceptible to oral thrush due to interrupted immunosurveillance of the oral mucosa. The factors that promote commensalism and ensure persistence of C. albicans in a fully immunocompetent host remain less clear. Using an experimental model of C. albicans oral colonization in mice we explored fungal determinants of commensalism in the oral cavity. Transcript profiling of the oral isolate 101 in the murine tongue tissue revealed a characteristic metabolic profile tailored to the nutrient poor conditions in the stratum corneum of the epithelium where the fungus resides. Metabolic adaptation of isolate 101 was also reflected in enhanced nutrient acquisition when grown on oral mucosa substrates. Persistent colonization of the oral mucosa by C. albicans also correlated inversely with the capacity of the fungus to induce epithelial cell damage and to elicit an inflammatory response. Here we show that these immune evasive properties of isolate 101 are explained by a strong attenuation of a number of virulence genes, including those linked to filamentation. De-repression of the hyphal program by deletion or conditional repression of NRG1 abolished the commensal behaviour of isolate 101, thereby establishing a central role of this factor in the commensal lifestyle of C. albicans in the oral niche of the host.Author summary : The oral microbiota represents an important part of the human microbiota and includes several hundreds to several thousands of bacterial and fungal species. One of the most prominent fungus colonizing the oral cavity is the yeast Candida albicans. While the presence of C. albicans usually remains unnoticed, the fungus can under certain circumstances cause lesions on the lining of the mouth referred to as oral thrush or contribute to other common oral diseases such as caries. Maintaining C. albicans commensalism in the oral mucosa is therefore of utmost importance for oral health and overall wellbeing. While overt fungal growth and disease is limited by immunosurveillance mechanisms during homeostasis, C. albicans strives to survive and evades elimination from the host. Here, we show that while commensalism in the oral cavity is characterized by a restricted fungal virulence and hyphal program, enforcing filamentation in a commensal isolate is sufficient for driving pathogenicity and fungus-induced inflammation in the oral mucosa thwarting persistent colonization. Our results further support a critical role for specialized nutrient acquisition allowing the fungus to thrive in the nutrient poor environment of the squamous epithelium. Together, this work revealed key determinants of C. albicans commensalism in the oral niche.

Infections with intravascular digenean trematodes of the Spirorchiidae family (spirorchiidoses) are of great conservation concern both in marine and freshwater turtles due to their pathogenic potential. Between 2014 and 2021, Spirorchis sp. infections associated with granulomatous inflammation and sudden death were detected in European pond turtles (Emys orbicularis) from three conservation breeding facilities in Switzerland. Blood fluke eggs associated with lesions were found in the intestine, spleen, testis, skeletal musculature, heart, kidneys, stomach, pancreas, liver, lung, and meninges from nine pond turtles submitted for necropsy and in the intestinal content from five of these animals. Two novel polymerase chain reactions (PCRs) targeting the 28S ribosomal RNA gene and the ITS2 region and subsequent sequencing revealed 100% nucleotide identity with a Spirorchis sp. previously isolated from an Escambia map turtle (Graptemys ernsti) in the USA. Our findings suggest a spill-over event secondary to direct or indirect contact with invasive North American turtle species in Switzerland. We describe the clinical, haematological, ultrasonographical, endoscopical, parasitological, pathological, and molecular findings associated with spirorchiid blood fluke infections of the Spirorchis genus in E. orbicularis, as well as the biosecurity measures that were developed to prevent the spread of this parasite among breeding and highly endangered free-ranging E. orbicularis populations in Switzerland., Graphical abstract Image 1, Highlights • Spirorchiidosis was recorded in three Swiss Emys orbicularis breeding facilities. • Spirorchis sp. infection was detected by parasitological and molecular methods. • Spill-over events from invasive American turtle species are suspected. • Biosecurity measures are essential to avoid Spirorchis sp. spread into wild populations.

Red deer (Cervus elaphus) carcasses showing grey-greenish discolouration have been increasingly observed in the canton of Grisons, Switzerland. We investigated whether Sarcocystis infections were associated with this pathology, and whether wild and domestic canids were involved in their transmission. Meat from affected red deer (n = 26), faeces and intestines from red foxes (Vulpesvulpes) (n = 126), and faeces from hunting dogs (n = 12) from the region, were analysed. Eosinophilic myositis and/or fasciitis were diagnosed in 69% of the deer, and sarcocysts were observed in 89% of the animals. Molecular typing targeting a ~700bp variable region of the 18S rRNA gene revealed Sarcocystis hjorti in 73%, S. venatoria/S. iberica in 54%, S. linearis/S. taeniata in 12%, S. pilosa in 8% and S. ovalis in 4% of the deer samples. No inflammatory changes were observed in red deer carcasses with normal appearance (n = 8); however, sarcocysts were observed in one sample, and S. hjorti, S. venatoria/S. iberica or S. silva DNA was detected in five samples. Sarcocystis oocysts/sporocysts were observed in 11/106 faecal and 6/20 intestinal fox samples, and in 2/12 canine samples. Sarcocystis tenella (n = 8), S. hjorti (n = 2), S. gracilis (n = 2), and S. miescheriana (n = 1) were identified in foxes, and S. gracilis (n = 2), S. capreolicanis (n = 1) and S. linearis/S. taeniata (n = 1) in dogs. This study provides first molecular evidence of S. pilosa and S. silva infection in red deer and S. linearis/S. taeniata in dogs and represents the first record of S. ovalis transmitted by corvids in Central Europe. Although Sarcocystis species infecting red deer are not regarded as zoonotic, the affected carcasses can be declared as unfit for human consumption due to the extensive pathological changes., Graphical abstract Image 1, Highlights • Red deer with eosinophilic myositis/fasciitis were infected with Sarcocystis. • Red foxes and hunting dogs in Grisons transmit Sarcocystis spp. to cervids, ruminants and swine. • Sarcocystis ovalis, transmitted by corvid birds, infect red deer in Central Europe. • Sarcocystis silva and S. pilosa were first detected in red deer by molecular methods. • Red foxes are natural definitive hosts of Sarcocystis hjorti.

In a previous scientific opinion on application EFSA-GMO-BE-2016-138, the EFSA Panel on Genetically Modified Organisms (GMO Panel) could not conclude on the comparative analysis and on the food/feed safety assessment of genetically modified (GM) oilseed rape (OSR) MS11 because of the lack of an appropriate compositional data set. Following a request from the European Commission, the GMO Panel assessed additional information related to OSR MS11 to complement the original scientific opinion. The GMO Panel concluded that the information submitted (on the composition of the two-event stack MS11 × RF3) could not be used for the assessment of the composition of OSR MS11 and requested the applicant to perform a complementary set of field trials to generate additional data. The applicant did not perform the requested field trials and did not provide any new experimental data on the composition of OSR MS11. Hence, the GMO Panel is still not in the position to conclude on either the compositional analysis or the toxicological, allergenicity or nutritional assessment of OSR MS11. Therefore, the previous conclusions of the GMO Panel still hold.

Hepatozoon spp. is the causative agent of a vector-borne parasitic disease in many animal species. In felids, Hepatozoon felis, Hepatozoon canis and Hepatozoon silvestris have been molecularly isolated. Hepatozoonosis usually causes asymptomatic infections in domestic cats, but clinical cases have recently been reported in Europe. We describe the first Italian case of hepatozoonosis in a cat with an unusual presentation. An 11-year-old neutered European shorthair cat was urgently hospitalized for intestinal intussusception. Hematology, biochemistry, FIV-FeLV tests, blood smears and molecular investigation targeting the 18S rRNA gene of Hepatozoon spp. were performed on blood samples; in addition, histological and molecular investigations were performed to analyze surgical samples to identify Hepatozoon infection. Hepatozoon gamonts were detected in granulocytes in the blood smear, and Hepatozoon spp. DNA was confirmed by PCR on blood. The intussusception was caused by a sessile endoluminal nodule that was surgically removed. Histologically, many elements referring to parasitic tissue forms were identified in the intestinal cells, and then the specimens were molecularly confirmed to harbor H. silvestris. This is the first description of symptomatic hepatozoonosis in a domestic cat in Italy. Hepatozoon silvestris has been described in wild felids, which are usually resilient to the infection, whereas the domestic cat seems to be more susceptible. Indeed, H. silvestris in cats usually presents tropism for skeletal muscle and myocardium with subsequent clinical manifestations. This is the first description of a domestic cat with H. silvestris localized in the intestinal epithelium and associated with intussusception. Graphical abstract

Selective vulnerability is an enigmatic feature of neurodegenerative diseases (NDs), whereby a widely expressed protein causes lesions in specific cell types and brain regions. Using the RiboTag method in mice, translational responses of five neural subtypes to acquired prion disease (PrD) were measured. Pre-onset and disease onset timepoints were chosen based on longitudinal electroencephalography (EEG) that revealed a gradual increase in theta power between 10- and 18-weeks after prion injection, resembling a clinical feature of human PrD. At disease onset, marked by significantly increased theta power and histopathological lesions, mice had pronounced translatome changes in all five cell types despite appearing normal. Remarkably, at a pre-onset stage, prior to EEG and neuropathological changes, we found that 1) translatomes of astrocytes indicated reduced synthesis of ribosomal and mitochondrial components, 2) glutamatergic neurons showed increased expression of cytoskeletal genes, and 3) GABAergic neurons revealed reduced expression of circadian rhythm genes. These data demonstrate that early translatome responses to neurodegeneration emerge prior to conventional markers of disease and are cell type-specific. Therapeutic strategies may need to target multiple pathways in specific populations of cells, early in disease. Funding: German Center for Neurodegenerative Diseases; Knut and Alice Wallenberg foundation; Deutsche Forschungsgemeinschaft [DI 1718/3-1]; Helmholtz-Alberta Initiative-Neurodegenerative Disease Research [HAI-NDR SO-083]

The stable fly Stomoxys calcitrans (Diptera: Muscidae) is considered as the main mechanical vector of the lumpy skin disease virus (LSDV). In addition, the mosquito species Aedes aegypti (Diptera: Culicidae) was shown to transmit the virus from donor to receptor animals. Retention of the virus for several days was shown for two additional tropical mosquito species and the biting midge Culicoides nubeculosus (Diptera: Ceratopogonidae). In the present study, viral retention for 10- or 7-days post feeding on virus-spiked blood through a membrane was shown for field-collected Aedes japonicus and laboratory-reared Culex pipiens, two widely distributed mosquito species in temperate regions. Viral DNA could be detected from honey-coated Flinders Technology Associates (FTA) cards and shedded faeces for 1 or 4 days after an infectious blood meal was given to Ae. aegypti. Virus increase over time and virus dissemination was observed in laboratory-reared C. nubeculosus, but the virus could be isolated from field-collected biting midges only from the day of exposure to the blood meal. Thus, mosquitoes might serve as mechanical vectors of LSDV in case of interrupted feeding. A putative biological virus transmission by Culicoides biting midges, as suspected from field observations, deserves further investigations.

EFSA was asked by the european Commission to develop criteria as advice for consideration for the risk assessment of plants produced by targeted mutagenesis, cisgenesis and intragenesis. EFSA proposes in this statement six main criteria to assist the risk assessment of these plants. The first four criteria are related to the molecular characterisation of the genetic modification introduced in the recipient plant. The four criteria evaluate whether any exogenous DNA sequence(s) is/are present (Criterion 1), whether such sequence derives from the breeders' gene pool (Criterion 2), the type of integration (Criterion 3) and whether any endogenous plant gene is interrupted (Criterion 4). Depending on the evaluation of the above criteria, the product can be a genome edited plant where no exogenous DNA sequence is present, or a cisgenic or intragenic plant where the cisgenic and intragenic sequence are introduced by targeted insertion and no plant endogenous genes are interrupted. In these cases, two more criteria are assessed to evaluate the history of safe use (Criterion 5) and the structure and function of the new allele (Criterion 6). If cisgenic and intragenic sequence are introduced by random integration without interruption of an endogenous gene, or when no risk is identified when an endogenous gene is interrupted, the criteria 5 and 6 will also be assessed. Evaluating the history of safe use is an important part of the proportionate risk assessment of cisgenic, intragenic and genome-edited plants since the newly introduced allele may already be present in nature. However, when the history of safe use cannot be sufficiently demonstrated, the function and structure of the introduced allele should be carefully assessed. Recommendations are also included on the aspects that need further elaboration for full applicability of the criteria proposed herein are also included.

Synthetic biology (SynBio) is an interdisciplinary field at the interface of molecular engineering and biology aiming to develop new biological systems and impart new functions to living cells, tissues and organisms. EFSA has been asked by the European Commission to evaluate SynBio developments in agri-food with the aim of identifying the adequacy and sufficiency of existing guidelines for risk assessment and determine if updated guidance is needed. In this context, the GMO Panel has previously adopted an Opinion evaluating the SynBio developments in agri-food/feed and the adequacy and sufficiency of existing guidelines for the molecular characterisation and environmental risk assessment of genetically modified plants (GMPs) obtained through SynBio and reaching the market in the next decade. Complementing the above, in this Opinion, the GMO Panel evaluated the adequacy and sufficiency of existing guidelines for the food and feed risk assessment of GMPs obtained through SynBio. Using selected hypothetical case studies, the GMO Panel did not identify novel potential hazards and risks that could be posed by food and feed from GMPs obtained through current and near future SynBio approaches; considers that the existing guidelines are adequate and sufficient in some Synbio applications; in other cases, existing guidelines may be just adequate and hence need updating; areas needing updating include those related to the safety assessment of new proteins and the comparative analysis. The GMO Panel recommends that future guidance documents provide indications on how to integrate the knowledge available from the SynBio design and modelling in the food and feed risk assessment and encourages due consideration to be given to food and feed safety aspects throughout the SynBio design process as a way to facilitate the risk assessment of SynBio GMPs and reduce the amount of data required.

Background Infection with the cardiopulmonary nematode Angiostrongylus vasorum may cause severe disease in dogs, therefore prophylactic treatments are necessary to prevent infection in dogs at risk. A clinical field study was conducted to demonstrate the efficacy and safety of an oral combination of sarolaner, moxidectin and pyrantel (Simparica Trio®) for the prevention of A. vasorum infection in dogs (prevention study). A survey study was conducted concurrently to determine the infection pressure in the same areas. Methods Prevention and survey studies were both conducted at the same veterinary clinics in endemic hot spots for A. vasorum in Denmark and Italy. The prevention study was a randomized, placebo controlled, double masked study where 622 client-owned dogs were treated and tested at 30 days intervals for 10 months. In the survey study 1628 dogs that were at risk of infection and/or were suspected to be infected were tested by fecal and/or serological methods, and the percent of dogs positive for A. vasorum was calculated. Results In the prevention study, there were no adverse events related to treatment with Simparica Trio®. Two placebo-treated animals became infected with A. vasorum during the 10-month study period, while none of the dogs in the combination product-treated group became infected. In the survey study, 12.2% of the study dogs were found positive to A. vasorum, indicating high exposure to the parasite during the period of the prevention study. Conclusions Monthly oral treatment with the combination of sarolaner, moxidectin and pyrantel (Simparica Trio®) was 100% effective in the prevention of natural infection with A. vasorum in dogs in highly endemic areas. In endemic areas, A. vasorum occurrence in dogs at risk is considerable.

This guidance document provides harmonised and flexible methodologies to apply scientific criteria and prioritisation methods for grouping chemicals into assessment groups for human risk assessment of combined exposure to multiple chemicals. In the context of EFSA’s risk assessments, the problem formulation step defines the chemicals to be assessed in the terms of reference usually through regulatory criteria often set by risk managers based on legislative requirements. Scientific criteria such as hazard‐driven criteria can be used to group these chemicals into assessment groups. In this guidance document, a framework is proposed to apply hazard‐driven criteria for grouping of chemicals into assessment groups using mechanistic information on toxicity as the gold standard where available (i.e. common mode of action or adverse outcome pathway) through a structured weight of evidence approach. However, when such mechanistic data are not available, grouping may be performed using a common adverse outcome. Toxicokinetic data can also be useful for grouping, particularly when metabolism information is available for a class of compounds and common toxicologically relevant metabolites are shared. In addition, prioritisation methods provide means to identify low‐priority chemicals and reduce the number of chemicals in an assessment group. Prioritisation methods include combined risk‐based approaches, risk‐based approaches for single chemicals and exposure‐driven approaches. Case studies have been provided to illustrate the practical application of hazard‐driven criteria and the use of prioritisation methods for grouping of chemicals in assessment groups. Recommendations for future work are discussed., This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2021.EN-7029/full

Background Bluetongue is a serious disease of ruminants caused by the bluetongue virus (BTV). BTV is transmitted by biting midges (Culicoides spp.). Serological evidence from livestock and the presence of at least one competent vector species of Culicoides suggests that transmission of BTV is possible and may have occurred in Kazakhstan. Methods We estimated the risk of transmission using a mathematical model of the reproduction number R0 for bluetongue. This model depends on livestock density and climatic factors which affect vector density. Data on climate and livestock numbers from the 2466 local communities were used. This, together with previously published model parameters, was used to estimate R0 for each month of the year. We plotted the results on isopleth maps of Kazakhstan using interpolation to smooth the irregular data. We also mapped the estimated proportion of the population requiring vaccination to prevent outbreaks of bluetongue. Results The results suggest that transmission of bluetongue in Kazakhstan is not possible in the winter from October to March. Assuming there are vector-competent species of Culicoides endemic in Kazakhstan, then low levels of risk first appear in the south of Kazakhstan in April before spreading north and intensifying, reaching maximum levels in northern Kazakhstan in July. The risk declined in September and had disappeared by October. Conclusion These results should aid in surveillance efforts for the detection and control of bluetongue in Kazakhstan by indicating where and when outbreaks of bluetongue are most likely to occur. The results also indicate where vaccination efforts should be focussed to prevent outbreaks of disease. Graphical abstract

Canine angiostrongylosis is a potentially lethal parasitic disease that can manifest itself with a broad spectrum of clinical signs, including respiratory distress, neurological and bleeding disorders, or non-specific signs. The occurrence of Angiostrongylus vasorum is widely reported in Europe, but very little is known about its presence in Austria. In this first large-scale survey, 1279 sera were collected from Austrian dogs and tested by an ELISA for the detection of circulating antigen of A. vasorum (sensitivity: 95.7%, specificity 94.0%) and by a separate ELISA detecting specific antibodies (sensitivity 81.0%, specificity 98.8%). Furthermore, 1040 faecal samples were tested for the presence of lungworm first stage larvae (L1). One dog (0.1%, 95% confidence intervals [CI]: 0.0–0.4%) was positive in both ELISAs, while 1.2% (n = 15, CI: 0.7–1.9%) of the tested dogs were antigen-positive and 1.5% (n = 19, CI: 0.9–2.3%) were positive for specific antibodies. Overall, 13 dogs (1.3%; CI: 0.7–2.1%) were positive for A. vasorum L1 while 31 dogs were positive for Crenosoma vulpis L1 (3.0%; CI: 2.0–4.2%). One dog shed L1 from both A. vasorum and C. vulpis (0.1%, CI: 0.0–0.5%). Dogs positive for A. vasorum originated from northeast, southeast and south Austria (antigen and/or antibody detection), but also from north, west and southwest Austria (antibody detection) and from northeast and west Austria (L1 detection). One of 88 blood samples (1.1%, CI: 0.0–6.2%) submitted from the eastern part of Austria was positive by a rapid assay for A. vasorum antigen detection (Angio Detect™). Crenosoma vulpis positive samples originated from northwest, north, northeast, south and west Austria. These results confirm the very sporadic occurrence of A. vasorum in the investigated areas of the country. However, due to the substantial infectious pressure from the surrounding countries and the free circulation of dogs and foxes acting as wildlife reservoirs and due to clinical relevance for infected dogs, it is crucial to maintain disease awareness also in areas where the parasite has not yet been detected.

Maize NK603 × T25 × DAS-40278-9 (three-event stack maize) was produced by conventional crossing to combine three single events: NK603, T25 and DAS-40278-9. The GMO Panel previously assessed the three single maize events and two of the subcombinations and did not identify safety concerns. No new data on the single maize events or the two subcombinations were identified that could lead to modification of the original conclusions on their safety. The molecular characterisation, comparative analysis (agronomic, phenotypic and compositional characteristics) and the outcome of the toxicological, allergenicity and nutritional assessment indicate that the combination of the single maize events and of the newly expressed proteins in the three-event stack maize does not give rise to food and feed safety and nutritional concerns. The GMO Panel concludes that the three-event stack maize, as described in this application, is as safe as the non-GM comparator and the selected non-GM reference varieties. In the case of accidental release of viable grains of the three-event stack maize into the environment, this would not raise environmental safety concerns. The GMO Panel assessed the likelihood of interactions among the single events in one of the maize subcombinations not previously assessed and concludes that these are expected to be as safe as the single events, the previously assessed subcombinations and the three-event stack maize. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of the three-event stack maize. Post-market monitoring of food/feed is not considered necessary. The GMO Panel concludes that the three-event stack maize and its subcombinations are as safe as the non-GM comparator and the selected non-GM reference varieties with respect to potential effects on human and animal health and the environment., European Commission: EFSA-Q-2019-00808.

Mutations are changes in the genetic material that may be transmitted to subsequent generations. Mutations appear spontaneously in nature and are one of the underlying driving forces of evolution. In plants, in vivo and in vitro random mutagenesis relies on the application of physical and chemical mutagens to increase the frequency of mutations thus accelerating the selection of varieties with important agronomic traits. The European Commission has requested EFSA to provide a more detailed description of in vivo and in vitro random mutagenesis techniques and the types of mutations and mechanisms involved, to be able to conclude on whether in vivo and in vitro random mutagenesis techniques are to be considered different techniques. To address the European Commission request, a literature search was conducted to collect information on the random mutagenesis techniques used in plants both in vivo and in vitro, on the type of mutations generated by such techniques and on the molecular mechanisms underlying formation of those mutations. The GMO Panel concludes that most physical and chemical mutagenesis techniques have been applied both in vivo and in vitro; the mutation process and the repair mechanisms act at cellular level and thus there is no difference between application of the mutagen in vivo or in vitro; and the type of mutations induced by a specific mutagen are expected to be the same, regardless of whether such mutagen is applied in vivo or in vitro. Indeed, the same mutation and the derived trait in a given plant species can be potentially obtained using both in vivo and in vitro random mutagenesis and the resulting mutants would be indistinguishable. Therefore, the GMO Panel concludes that the distinction between plants obtained by in vitro or in vivo approaches is not justified., European Commission: EFSA-Q-2020-00445