Objectives: The recent detection and expansion of West Nile virus (WNV) and Usutu virus (USUV) in the Netherlands, Germany, and Austria point to the likelihood of the viruses spreading to Northern Europe. Migratory birds and ornithophilic mosquitoes may spread these viruses to new areas. We sampled birds during the spring and autumn bird migration of 2021 in Southern Sweden to investigate the risk of the introduction of mosquito-borne zoonotic avian viruses like WNV and USUV.

Methods: We collected blood samples from 1775 birds comprising 59 species and determined the seroprevalence of WNV using a competitive enzyme-linked immunosorbent assay (ELISA). WNV and USUV belong to the Japanese encephalitis serocomplex, and antibodies against both viruses are detected in the WNV ELISA. Focus-forming assays or fluorescence-based neutralization assays were performed to verify ELISA results and to differentiate between antibodies against WNV and USUV.

Results: We found nine (0.51%) samples to be WNV-antibody-positive. Cross-neutralization experiments with WNV and USUV confirmed that seven (0.41%) had WNV-neutralizing antibodies and two (0.11%) had USUV-neutralizing antibodies. Interestingly, the two samples had neutralizing antibodies of both viruses. All samples but one with anti-flavivirus antibodies came from long-distance migrants wintering in sub-Saharan Africa. Antibodies were detected in samples taken during spring and autumn and only in adult birds.

Conclusion: The findings show that migratory birds in Sweden have been exposed to WNV and USUV. © 2025 Elsevier B.V., All rights reserved.

Ducks have recently received a lot of attention from the research community due to their importance as natural reservoirs of avian influenza virus (AIV). Still, there is a lack of tools to efficiently determine the immune status of ducks. The purpose of this work was to develop an automated differential blood count for the mallard duck (Anas platyrhynchos), to assess reference values of white blood cell (WBC) counts in this species, and to apply the protocol in an AIV field study. We established a flow cytometry-based duck WBC differential based on a no-lyse no-wash single-step one-tube technique, applying a combination of newly generated monoclonal antibodies with available duck-specific as well as cross-reacting chicken markers. The blood cell count enables quantification of mallard thrombocytes, granulocytes, monocytes, B cells, CD4(+) T cells (T helper) and CD8(+) cytotoxic T cells. The technique is reproducible, accurate, and much faster than traditional evaluations of blood smears. Stabilization of blood samples enables analysis up to 1 week after sampling, thus allowing for evaluation of blood samples collected in the field. We used the new technique to investigate a possible influence of sex, age, and AIV infection status on WBC counts in wild mallards. We show that age has an effect on the WBC counts in mallards, as does sex in juvenile mallards. Interestingly, males naturally infected with low pathogenic AIV showed a reduction of lymphocytes (lymphocytopenia) and thrombocytes (thrombocytopenia), which are both common in influenza A infection in humans.IMPORTANCE Outbreaks of avian influenza in poultry and humans are a global public health concern. Aquatic birds are the primary natural reservoir of avian influenza viruses (AIVs), and strikingly, AIVs mainly cause asymptomatic or mild infection in these species. Hence, immunological studies in aquatic birds are important for investigating variation in disease outcome of different hosts to AIV and may aid in early recognition and a better understanding of zoonotic events. Unfortunately, immunological studies in these species were so far hampered by the lack of diagnostic tools. Here, we present a technique that enables high-throughput white blood cell (WBC) analysis in the mallard and report changes in WBC counts in wild mallards naturally infected with AIV. Our protocol permits large-scale immune status monitoring in a widespread wild and domesticated duck species and provides a tool to further investigate the immune response in an important reservoir host of zoonotic viruses. Outbreaks of avian influenza in poultry and humans are a global public health concern. Aquatic birds are the primary natural reservoir of avian influenza viruses (AIVs), and strikingly, AIVs mainly cause asymptomatic or mild infection in these species.

Background: The tick-borne bacterium Neoehrlichia mikurensis can cause persistent asymptomatic bloodstream infections, but transfusion-mediated transmission has not been reported. This study aimed to investigate the prevalence of N. mikurensis in blood donors, and recipients of blood components from N. mikurensis-positive donors were traced. Methods: In 2019 and 2021, 1007 blood donors were recruited. Participants completed a questionnaire and additional blood samples were collected during blood donation. Detection of N. mikurensis was performed by PCR followed by sequencing. Positive donors were interviewed and retested. Look-back was performed on positive donations and on all subsequent donations. Results: N. mikurensis was detected in 7/1006 (0.7%) donors. A total of 380/1005 (38%) donors reported at least one noticed tick bite during the current season. The questionnaire could not detect any differences between negative and positive N. mikurensis-donors. Two of the positive donors were still positive on days 318 and 131 after the index donation, respectively. One donor with persistent N. mikurensis in blood experienced slight fatigue. All other had no symptoms attributable to neoehrlichiosis. Look-back included ten donations and 20 blood components. Eight components were discarded, and 12 recipients of N. mikurensis-positive donations were identified. PCR was negative in seven recipients. Five recipients had died, but their medical records gave no evidence for neoehrlichiosis. Conclusions: Although N. mikurensis was found in 0.7% of blood donors, transfusion-mediated infection was not detected, despite several recipients being at high risk for severe neoehrlichiosis. The results warrant further studies as well as raised clinical awareness. © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

Domestic mallards (Anas platyrhynchos domesticus) are traditionally used as a model to investigate infection dynamics and immune responses to low pathogenic avian influenza viruses (LPAIVs) in free-living mallards. However, it is unclear whether the immune response of domestic birds reflects the response of their free-living counterparts naturally exposed to these viruses. We investigated the extent to which the innate humoral immune response was similar among (i) wild-type domestic mallards in primary and secondary infection with LPAIV H4N6 in a laboratory setting (laboratory mallards), (ii) wild-type domestic mallards naturally exposed to LPAIVs in a semi-natural setting (sentinel mallards), and (iii) free-living mallards naturally exposed to LPAIVs. We quantified innate humoral immune function by measuring non-specific natural antibodies (agglutination), complement activity (lysis), and the acute phase protein haptoglobin. We demonstrate that complement activity in the first 3 days after LPAIV exposure was higher in primary-exposed laboratory mallards than in sentinel and free-living mallards. LPAIV H4N6 likely activated the complement system and the acute phase response in primary-exposed laboratory mallards, as lysis was higher and haptoglobin lower at day 3 and 7 post-exposure compared to baseline immune function measured prior to exposure. There were no differences observed in natural antibody and haptoglobin concentrations among laboratory, sentinel, and free-living mallards in the first 3 days after LPAIV exposure. Our study demonstrates that, based on the three innate humoral immune parameters measured, domestic mallards seem an appropriate model to investigate innate immunology of their free-living counterparts, albeit the innate immune response of secondary-LPAIV exposed mallards is a better proxy for the innate immune response in pre-exposed free-living mallards than that of immunologically naive mallards.

The Gram-negative bacterium Campylobacter jejuni is able to enter, survive and multiply within the free living amoeba Acanthamoeba polyphaga, but the molecular mechanisms behind these events are still unclear. We have studied the uptake and intracellular trafficking of viable and heat killed bacterial cells of the C. jejuni strain 81-176 in A. polyphaga. We found that viable bacteria associated with a substantially higher proportion of Acanthamoeba trophozoites than heat killed bacteria. Furthermore, the kinetics of internalization, the total number of internalized bacteria as well as the intracellular localization of internalized C. jejuni were dramatically influenced by bacterial viability. Viable bacteria were internalized at a high rate already after 1 h of co-incubation and were observed in small vacuoles tightly surrounding the bacteria. In contrast, internalization of heat killed C. jejuni was low at early time points and did not peak until 96 h. These cells were gathered in large spacious vacuoles that were part of the degradative pathway as determined by the uptake of fluorescently labeled dextran. The amount of heat killed bacteria internalized by A. polyphaga did never reach the maximal amount of internalized viable bacteria. These results suggest that the uptake and intracellular survival of C. jejuni in A. polyphaga is bacterially induced.

Low concentrations of Campylobacter jejuni cells in environmental samples make them difficult to study with conventional culture methods. Here, we show that enrichment by amoeba cocultures works well with low-concentration samples and that this method can be combined with molecular techniques without loss of genetic specificity.

Several species of free-living amoebae can cause disease in humans. However, in addition to the direct pathogenicity of e.g. Acanthamoebae and Naegleria species, they are recognized as environmental hosts, indirectly involved in the epidemiology of many pathogenic bacteria. Although several studies have demonstrated intracellular survival of many different bacteria in these species, the extent of such interactions as well as the implications for the epidemiology of the bacterial species involved, are largely unknown and probably underestimated. In this study, we evaluated eight different unicellular eukaryotic organisms, for their potential to serve as environmental hosts for Campylobacter species. These organisms include four amoebozoas (Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba rhysodes and Hartmanella vermiformis), one alveolate (Tetrahymena pyriformis), one stramenopile (Dinobryon sertularia), one eugoenozoa (Euglena gracilis) and one heterolobosea (Naegleria americana). Campylobacter spp. including Campylobacter jejuni, Campylobacter coli and Campylobacter lari are the most common cause of gastroenteritis in the western world. Survival and replication of these three species as well as Campylobacter hyointestinalis were assessed in co-cultures with the eukaryotic organisms. Campylobacter spp. generally survived longer in co-cultures, compared to when incubated in the corresponding growth media. The eukaryotic species that best promoted bacterial survival was the golden algae D. sertularia. Three species of amoebozoas, of the genus Acanthamoeba promoted both prolonged survival and replication of Campylobacter spp. The high abundance in lakes, ponds and water distribution networks of these organisms indicate that they might have a role in the epidemiology of campylobacteriosis, possibly contributing to survival and dissemination of these intestinal pathogens to humans and other animals. The results suggest that not only C. jejuni, but a variety of Campylobacter spp. can interact with different eukaryotic unicellular organisms.

Campylobacter jejuni is a recognized and common gastrointestinal pathogen in most parts of the world. Human infections are often food borne, and the bacterium is frequent among poultry and other food animals. However, much less is known about the epidemiology of C. jejuni in the environment and what mechanisms the bacterium depends on to tolerate low pH. The sensitive nature of C. jejuni stands in contrast to the fact that it is difficult to eradicate from poultry production, and even more contradictory is the fact that the bacterium is able to survive the acidic passage through the human stomach. Here we expand the knowledge on C. jejuni acid tolerance by looking at protozoa as a potential epidemiological pathway of infection. Our results showed that when C. jejuni cells were coincubated with Acanthamoeba polyphaga in acidified phosphate-buffered saline (PBS) or tap water, the bacteria could tolerate pHs far below those in their normal range, even surviving at pH 4 for 20 h and at pH 2 for 5 h. Interestingly, moderately acidic conditions (pH 4 and 5) were shown to trigger C. jejuni motility as well as to increase adhesion/internalization of bacteria into A. polyphaga. Taken together, the results suggest that protozoa may act as protective hosts against harsh conditions and might be a potential risk factor for C. jejuni infections. These findings may be important for our understanding of C. jejuni passage through the gastrointestinal tract and for hygiene practices used in poultry settings.

We present a novel and simple technique for storing live Acanthamoeba for long periods of time. The amoebae are maintained at refrigerator temperatures in a peptone-yeast extract-glucose (PYG) medium normally used for cultivation. Using this method, we obtained survival rates of at least 4 years for Acanthamoeba polyphaga and 3 years for Acanthamoeba castellanii and Acanthamoeba rhysodes. Advantages of this storage method are: (1) it is quick and simple, (2) inexpensive, (3) does not require encystment before storage, (4) resuscitation of cysts can be achieved within a week of culture in PYG medium at 27A degrees C, and does not require co-culture with bacteria or any special equipment.