Bird Hosts Research Articles

Evolution of the CRISPR-Cas9 defence system in Mycoplasma gallisepticum following colonization of a novel bird host.

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are bacterial defences that target bacteriophages and mobile genetic elements. How these defences evolve in novel host environments remains largely unknown. We studied the evolution of the CRISPR-Cas system in Mycoplasma gallisepticum (also named Mycoplasmoides gallisepticum), a bacterial pathogen of poultry that jumped into a passerine host ~30 years ago. Over the decade following the host shift, all isolates displaying a functional CRISPR-Cas system were found not only to harbour completely new sets of spacers, but the DNA protospacer adjacent motif recognized by the main effector M. gallisepticum Cas9 (MgCas9) was also different. These changes in CRISPR-Cas diversity and specificity are consistent with a change in the community of phages and mobile elements infecting M. gallisepticum as it colonized the novel host. In the years following the host shift, we also detected a gradual rise in isolates displaying non-functional MgCas9. After 12 years, all circulating isolates harboured inactive forms only. This loss of CRISPR-Cas function comes at a time when the passerine host is known to have evolved widespread resistance, which in turn drove the evolution of increasing M. gallisepticum virulence through antagonistic coevolution. Such striking concordance in the rise of inactivated forms of CRISPR-Cas and the evolution of host resistance suggests that the inactivation of the CRISPR-Cas system was necessary for enabling adaptive bacterial responses to host-driven selection. We highlight the need to consider both host and pathogen selection pressures on bacteria for understanding the evolution of CRISPR-Cas systems and the key factors driving the emergence of a pathogenic bacterium in a novel host.

Распределение очагов инвазии литоральных моллюсков партенитами и личинками трематод в губах и заливах Мурманского побережья в мае–июне 2021 года

Species composition and geographical variation in the distribution of digenean parthenite and larvae in intertidal snails in the littoral zones along the Murmansk coast were examined. Four species of littoral gastropods (Littorina saxatilis, L. obtusata, L. littorea and Nucella lapillus) were investigated. We found 10 species of digeneans, of which 9 have marine birds as a final host. The exception was Podocotyle atomon which uses fish as a definitive host. Larvae of trematode Microphallus pygmaeus, M. piriformes and Podocotyle atomon are found everywhere on the Murmansk coast. The richest helminth fauna were recorded in the Kola Bay and in Ivanovskaya Bay, the poorest helminth fauna were recorded in Dvorovaya Bay. No pronounced trends in distribution of infection foci were noted. Trematode prevalence appears to be predominantly determined by local site condition favoring or hindering final host (birds and fishes) abundance.

A new species of Rhinonyssidae (Mesostigmata) nasal mite (Ptilonyssus simplex n. sp.) from the Bohemian waxwing in Denmark.

A new species of nasal mite, Ptilonyssus simplex n. sp. is described and illustrated based on material from Bohemian waxwing, Bombycilla garrulus (Linnaeus, 1758) (Bombycillidae). The bird host was collected in a residental area in Hirtshals, Denmark, in October 2022. A set of diagnostic characters is presented in order to differentiate the new species from the overall similar species Ptilonyssus bombycillae Pence, 1973 which has been recorded from Bombycilla spp. in Europe and Northern America.

First Record of Two Nasal Mites Genus Rhinonyssus (Mesostigmata, Rhinonyssidae) Parasitizing Birds from Estonia

Rhinonyssids are obligate hematophagous mites that parasitize the nasal cavity of domestic and wilds birds worldwide. For the first time, two species of nasal mites of the genus Rhinonyssus from Estonia are described. One species of nasal mite, Rhinonyssus pluvialis Fain et Johnston, 1966, is described and illustrated based on material from Pluvialis apricaria Linnaeus, 1758 (Charadriiformes, Charadriidae). Another species of nasal mite, Rhinonyssus tringae Fain, 1963, is described and illustrated based on material from Tringa glareola Linnaeus, 1758 (Charadriiformes, Scolopacidae). The bird hosts were collected in Estonia, Puhato järv.

Seasonal variation in the stomach microbiota of two sympatrically breeding Pygoscelis penguin species at Signy Island, South Orkney Islands.

The gut microbiomes of Antarctic penguins are important for the fitness of the host birds and their chicks. The compositions of microbial communities in Antarctic penguin guts are strongly associated with the birds' diet, physiological adaptation and phylogeny. Whilst seasonal changes in food resources, distribution and population parameters of Antarctic penguins have been well addressed, little research is available on the stability or variability of penguin stomach microbiomes over time. Here, we focused on two Pygoscelis penguin species breeding sympatrically in the maritime Antarctic and analysed their stomach contents to assess whether penguin gut microbiota differed over three austral summer breeding seasons. We used a high-throughput DNA sequencing approach to study bacterial diversity in stomach regurgitates of Adélie (Pygoscelis adeliae) and chinstrap (Pygoscelis antarctica) penguins that have a similar foraging regime on Signy Island (South Orkney Islands). Our data revealed significant differences in bacterial alpha and beta diversity between the study seasons. We also identified bacterial genera that were significantly associated with specific breeding seasons, diet compositions, chick-rearing stages and sampling events. This study provides a baseline for establishing future monitoring of penguin gut microbiomes in a rapidly changing environment.

Implementation of a Cuckoo Search intelligent simulation system for node placement problem in Wireless Mesh Networks: Performance evaluation for tuning hyperparameters [formula omitted] and [formula omitted]

In recent years, Internet of Things (IoT) is the hottest topics in the research and implementation of wireless networks. Most of the research of IoT-based applications is related with the data collecting and processing using sensors and actuators. As the network environment for connecting IoT devices are considered WLAN or Bluetooth. The Wireless Mesh Networks (WMNs) are the most suitable networks for the Internet of Things (IoT) due to their numerous benefits. However, they have several issues related to wireless communications. One solution is the optimization of position and location of mesh routers. But finding the best location of mesh routers is an NP-hard problem. To deal with this issue, we propose and implement an intelligent simulation system based on the Cuckoo Search (CS) algorithm called WMN-CS. In the implemented system, the hyperparameters should be tuned to find better solutions. This paper evaluates the WMN-CS performance for various combination of hyperparameters by tuning host bird discovery rate (pa) and scale parameter (γ). The simulation results show that a better performance is achieved when the scale parameter (γ) is between 0.07 and 0.09 and the host bird recognition rate (pa) is between 0.900 and 0.925.

Ungrazed seminatural habitats around farms benefit bird conservation without enhancing foodborne pathogen risks

ContextConservation in working landscapes is critical for halting biodiversity declines and ensuring farming system sustainability. However, concerns that wildlife may carry foodborne pathogens has created pressure on farmers to remove habitat and reduce biodiversity, undermining farmland conservation. Nonetheless, simplified farming landscapes may host bird communities that carry higher foodborne disease risks.ObjectivesWe analyzed the effects of local farming practices and surrounding landscapes on bird communities and food-safety risks across 30 California lettuce farms. Specifically, we sought to determine how farmland diversification affects bird diversity, fecal contamination, and foodborne pathogen incidences, thereby identifying potential tradeoffs between managing farms for bird conservation versus food safety.MethodsWe surveyed birds at 227 point-count locations, quantified fecal contamination along 120 transects, and assayed 601 bird feces for pathogenic E. coli, Campylobacter spp., and Salmonella spp. We then used hierarchical models to quantify effects of farm management and landscape context on bird communities and food-safety risks.ResultsSurrounding ungrazed seminatural areas were associated with higher bird diversity, more species of conservation concern, and fewer flocks that may increase risks from foodborne pathogens. In contrast, on-farm diversification practices and surrounding grazing lands offered weaker bird conservation benefits. Surrounding grazed lands were associated with more potentially pathogenic bird feces in crop fields.ConclusionsOur results suggest that habitat conservation around produce farms could support bird conservation without increasing foodborne pathogens, especially on farms further from grazing lands. Thus, interventions that diversify farming systems offer potential to simultaneously conserve biodiversity and provide safe food for human consumption.

Spatiotemporally Explicit Epidemic Model for West Nile Virus Outbreak in Germany: An Inversely Calibrated Approach

Since the first autochthonous transmission of West Nile Virus was detected in Germany (WNV) in 2018, it has become endemic in several parts of the country and is continuing to spread due to the attainment of a suitable environment for vector occurrence and pathogen transmission. Increasing temperature associated with a changing climate has been identified as a potential driver of mosquito-borne disease in temperate regions. This scenario justifies the need for the development of a spatially and temporarily explicit model that describes the dynamics of WNV transmission in Germany. In this study, we developed a process-based mechanistic epidemic model driven by environmental and epidemiological data. Functional traits of mosquitoes and birds of interest were used to parameterize our compartmental model appropriately. Air temperature, precipitation, and relative humidity were the key climatic forcings used to replicate the fundamental niche responsible for supporting mosquito population and infection transmission risks in the study area. An inverse calibration method was used to optimize our parameter selection. Our model was able to generate spatially and temporally explicit basic reproductive number (R0) maps showing dynamics of the WNV occurrences across Germany, which was strongly associated with the deviation from daily means of climatic forcings, signaling the impact of a changing climate in vector-borne disease dynamics. Epidemiological data for human infections sourced from Robert Koch Institute and animal cases collected from the Animal Diseases Information System (TSIS) of the Friedrich-Loeffler-Institute were used to validate model-simulated transmission rates. From our results, it was evident that West Nile Virus is likely to spread towards the western parts of Germany with the rapid attainment of environmental suitability for vector mosquitoes and amplifying host birds, especially short-distance migratory birds. Locations with high risk of WNV outbreak (Baden-Württemberg, Bavaria, Berlin, Brandenburg, Hamburg, North Rhine-Westphalia, Rhineland-Palatinate, Saarland, Saxony-Anhalt and Saxony) were shown on R0 maps. This study presents a path for developing an early warning system for vector-borne diseases driven by climate change.

Chewing lice (Psocodea, Phthiraptera: Amblycera and Ischnocera) in wild birds in Zungarococha, Iquitos (Peru)

The objective of this work was to identify the species of chewing lice collected from thirty wild birds from the town of Zungarococha, Iquitos (Peru). We identified 11 specimens from a total of 36 collected. The species Columbicola passerinae (Wilson, 1941) presented the largest number of hosts, and Motmotnirmus marginellus (Nitzsch, 1874) and Docophorus rufus (Kellogg, 1899) presented the highest prevalence, intensity, and average abundance. This study reports 11 species of lice for the first time, and new host associations, expanding the list of host birds in Peru.

Phylogenetic relationships and systematics of tapeworms of the family Davaineidae (Cestoda, Cyclophyllidea), with emphasis on species in rodents.

The present study aims at clarifying the poorly known phylogenetic relationships and systematics of cestodes of the family Davaineidae Braun, 1900 (Cyclophyllidea), primarily the genus Raillietina Fuhrmann, 1920 and of the subfamily Inermicapsiferinae (Anoplocephalidae) from mammals (mostly rodents, 31 new isolates) and birds (eight new isolates). Phylogenetic analyses are based on sequences of the large subunit ribosomal RNA gene (28S) and mitochondrial NADH dehydrogenase subunit 1 gene (nad1). The main phylogenetic pattern emerging from the present analysis is the presence of three independent lineages within the main clade of the subfamily Davaineinae, one of which is almost entirely confined to species from rodents and the other two show a mixture of species from birds and mammals. It is suggested that the major diversification of the main clade took place in birds, possibly in galliforms. The subsequent diversification included repeated host shifts from birds to mammals and to other birds, and from rodents to other mammals, showing that colonisation of new host lineages has been the main driver in the diversification of davaineine cestodes. It is also shown that all isolates of Inermicapsifer Janicki, 1910, mainly from rodents, form a monophyletic group positioned among Raillietina spp. in the "rodent lineage", indicating that the genus Inermicapsifer is a member of the family Davaineidae. This means that the subfamily Inermicapsiferinae and the family Inermicapsiferidae should be treated as synonyms of the Davaineidae, specifically the subfamily Davaineinae. Three additional genera generally included in the Inermicapsiferinae, i.e. Metacapsifer Spasskii, 1951, Pericapsifer Spasskii, 1951 and Thysanotaenia Beddard, 1911, are also assigned here to the Davaineidae (subfamily Davaineinae). Raillietina spp. were present in all three main lineages and appeared as multiple independent sublineages from bird and mammalian hosts, verifying the non-monophyly of the genus Raillietina and suggesting a presence of multiple new species and genera.

Unregulated introduced fish (Perca fluviatilis Linnaeus, 1758) is host to zoonotic parasites in a small Mediterranean island

Introduced species have a major impact on freshwater ecosystems, particularly on islands. Numerous fish species have been introduced in Corsica (Mediterranean island, southern France) as part of planned programs or clandestinely. The introduction of non-native freshwater fish species can have a range of impacts on the recipient ecosystem, including through the co-introduction of its pathogens. A sample of introduced perch Perca fluviatilis Linnaeus, 1758 from the artificial reservoir of Padula was examined following a report of parasites by an angler. The analyses revealed the occurrence of Eustrongylides sp. (Nematoda) and Clinostomum complanatum (Digenea), two zoonotic parasites in P. fluviatilis. Both parasites are reported for the first time in France. Eustrongylides sp. and C. complanatum may have been introduced with their fish intermediate hosts or through their final bird hosts. The occurrence of the two parasites raises concerns from both a veterinary and human health perspective as they can use a wide range of amphibians as intermediate hosts and can be acquired in humans through the consumption of raw or undercooked fish.

Infectivity of Wild-Bird Origin Influenza A Viruses in Minnesota Wetlands across Seasons.

The environmental tenacity of influenza A viruses (IAVs) in the environment likely plays a role in their transmission; IAVs are able to remain infectious in aquatic habitats and may have the capacity to seed outbreaks when susceptible wild bird hosts utilize these same environments months or even seasons later. Here, we aimed to assess the persistence of low-pathogenicity IAVs from naturally infected ducks in Northwestern Minnesota through a field experiment. Viral infectivity was measured using replicate samples maintained in distilled water in a laboratory setting as well as in filtered water from four natural water bodies maintained in steel perforated drums (hereafter, mesocosms) within the field from autumn 2020 to spring 2021. There was limited evidence for the extended persistence of IAVs held in mesocosms; from 65 initial IAV-positive samples, only six IAVs persisted to at least 202 days in the mesocosms compared to 17 viruses persisting at least this long when held under temperature-controlled laboratory settings in distilled water. When accounting for the initial titer of samples, viruses detected at a higher concentration at the initiation of the experiment persisted longer than those with a lower starting titer. A parallel experimental laboratory model was used to further explore the effects of water type on viral persistence, and the results supported the finding of reduced tenacity of IAVs held in mesocosms compared to distilled water. The results of this investigation provide evidence that many factors, including temperature and physicochemical properties, impact the duration of viral infectivity in natural settings, further extending our understanding of the potential and limitations of environmental-based methodologies to recover infectious IAVs.

Large-scale spatial drivers of avian schistosomes in Northern Michigan inland lakes.

Avian schistosomes are snail-borne trematode parasites (Trichobilharzia spp.) that can cause a nasty skin rash in humans when their cercariae mistake us for their normal bird hosts. We sought to investigate drivers of the spatial distribution of Trichobilharzia cercaria abundance throughout Northern Michigan lakes. For 38 sites on 16 lakes, we assessed several dozen potential environmental predictors that we hypothesized might have direct or indirect effects on overall cercaria abundance, based on known relationships between abiotic and biotic factors in wetland ecosystems. We included variables quantifying local densities of intermediate hosts, temperature, periphyton growth rates, human land use and hydrology. We also measured daily abundance of schistosome cercariae in the water over a 5-week period, supported by community scientists who collected and preserved filtered water samples for qPCR. The strongest predictor of cercaria abundance was Lymnaea host snail density. Lymnaea density was higher in deeper lakes and at sites with more deciduous tree cover, consistent with their association with cool temperature habitats. Contrary to past studies of human schistosomes, we also found a significant negative relationship between cercaria abundance and submerged aquatic vegetation, possibly due to vegetation blocking cercaria movement from offshore snail beds. If future work shows that these effects are indeed causal, then these results suggest possible new approaches to managing swimmer's itch risk in northern MI lakes, such as modifying tree cover and shallow-water vegetation at local sites.

Prevalence, Diversity, and Virulence of Campylobacter Carried by Migratory Birds at Four Major Habitats in China.

Campylobacter species, especially C. jejuni and C. coli, are the main zoonotic bacteria causing human gastroenteritis. A variety of Campylobacter species has been reported in wild birds, posing a potential avian-human transmission pathway. Currently, there has been little surveillance data on Campylobacter carriage in migratory birds in China. In the current work, fresh fecal droppings from individual migratory birds were collected at four bird wintering/stopover sites in China from May 2020 to March 2021. Nucleic acid was extracted and tested for Campylobacter with PCR-based methods. Overall, 73.8% (329/446) of the samples were positive for Campylobacter, demonstrating location and bird host specificity. Further speciation revealed the presence of C. jejuni, C. coli, C. lari, C. volucris, and an uncharacterized species, which all harbored a variety of virulence factors. Phylogenetic analysis performed on concatenated 16S rRNA-atpA-groEL genes elucidated their genetic relationship, demonstrating both inter- and intra-species diversity. The wide distribution and high diversity of Campylobacter spp. detected in migratory birds in China indicated potential transmission across territories. The existence of virulence factors in all of these species highlighted their public health importance and the necessity of monitoring and controlling Campylobacter and other pathogens carried by migratory birds.

EFFECT OF ECOLOGICAL CONDITIONS ON DESI AND FAYOUMI CHICKEN TRAITS AT DISTRICT KASHMORE, SINDH-PAKISTAN

The present study was undertaken to evaluate the different body organs measurement and multiple phenotypic traits of desi and fayoumi selected chicken breeds under free range and intensive management conditions during, 2021. Host birds were collected from local peoples and hatcheries of district Kashmore and brought through intensive care management system in Animal Husbandry, Department of Zoology, Shah Abdul Latif University Khairpur Mirs. Both chicken male and female populations were reared separately in the same ecology and F1 generation chickens were obtained. The feeding and rearing procedures were followed by NRC and ISA recommended guidelines. The average values of phenotypic traits like neck length of desi and fayoumi males measured in cm (20.4±1.9), (16.5±1.4) body height (43.4±3.2), (33.7±3.2) shank length (12.5±0.82) (8.3±0.72) beak length (3.6±0.21) (2.7±0.16) and wings span (23.7±1.6) (18.3±1.5) in intensive conditions. While as, in the free range conditions the neck length of desi and fayoumi males was recorded in cm (18.7±1.6) (16.4±1.3) body height (38.7±3.2) (34.6±3.1), shank length (10.3±0.97) (9.4±0.94), length of beak (3.3±0.24) (2.7±0.18), and wing span (20.4±1.5) (18.5±1.4), in both chicken breeds. The different phenotype traits of desi and fayoumi chicken breeds viz., the color of eyes, comb, plumage, feather, barbell, mumps, and beak colors percentage were found with variation in intensive and free range conditions. Concludingly, desi chickens were found with larger body parts with dark color variants. The present study suggests, more work should be carried on other characteristics to enhance poultry quality and quantity in future endeavors.

Mitochondrial and apicoplast genome copy abundances of haemosporidian parasites are explained by host species and parasitic lineage

Endosymbiotic organelles, such as mitochondria and plastids, contain own remnant genomes (nucleoids), whose variable abundance in cells may be adaptive to the physiological necessities and functions of the cells. Unicellular apicomplexan parasites contain one mitochondrium and one apicoplast with variable genome copy numbers. We measured the abundance of mitochondrial, apicoplast and nuclear genome copies in a set of avian blood samples infected with haemosporidian blood parasites, belonging to the three main genera Plasmodium, Haemoproteus and Leucocytozoon. We designed general primers suitable for qPCR, amplifying fragments of the mitogenome, plastome and nuclear genomes of avian malaria and related haemosporidian parasites. We measured the amplification of these fragments in 153 samples of 23 avian host species and infected with 33 parasitic lineages. We estimate on average several hundred mitochondrial genome copies and several tens of apicoplast copies per haploid gametocyte cell with substantial variation among samples. Host species appeared to differ in their mitogenome abundance while parasitic lineages differed in plastome abundance per cell (per nuclear copy signal). We did not find consistent differences between parasite genera or higher avian taxa. Parasite lineages and host bird species did not differ consistently in infection intensity, estimated from parasite to host nuclear signals, which may indicate that samples were taken at different stages of infection. However, this and similar results remain to be cross-validated with in-situ imaging techniques. The novel molecular tools introduced here offer avenues for the characterization of nucleoid abundance of haemosporidian parasites over environmental conditions and parasitic developmental stages. Such measures will improve our understanding of parasite physiology, ecology, the coadaptation and coevolution with hosts and suggest possible augmentations to standard methods in the research field.

Stem chewing lice on Cretaceous feathers preserved in amber

Phthirapteran lice (true lice or parasitic lice) are a major group of ectoparasitic insects living on their bird or mammal hosts during their entire life cycle.1 Due to their highly specialized lifestyles, they are extremely poorly represented in fossil records.2 Molecular clock estimations have speculated extensively about the origin time of parasitic lice,3,4 yet none have been confirmed unequivocally. Herein, we report a new family of stem chewing lice, based on two adult insects associated with several semiplume feathers preserved within a piece of Kachin amber from the mid-Cretaceous. They display some defining characteristics of the Amblycera, an early-diverging lineage of the crown lice group. These features include a wingless body, chewing mouthparts, narrow and small thorax, and short tarsus with elongated euplantulae. Our phylogenetic analysis places the new taxa in the Amblycera, and the discovery thus pushes back the lice fossil records by at least 55 million years. Furthermore, the new specimens show primitive characters such as compressed and club-shaped terminal segments of antennae, maxillary and labial palps, and unmodified femora of hind legs, providing key information for the evolutionary relationship between free-living booklice and parasitic lice. This suggests that some ectoparasitic characters defining the crown lice group might have evolved among amblyceran and non-amblyceran lice in parallel. These newly described fossil specimens imply at least a Cretaceous age of Phthiraptera.

Prevalence of Avian Influenza Virus in Atypical Wild Birds Host Groups during an Outbreak of Highly Pathogenic Strain EA/AM H5N1

The global outbreak of highly pathogenic avian influenza (HPAI) H5N1 Eurasian lineage goose/Guangdong clade 2.3.4.4b virus that was detected in North America in 2021 is the largest in history and has significantly impacted wild bird populations and domestic poultry across the continent. Synanthropic birds may play an important role in transmitting the virus laterally to other wild bird species and domestic poultry. Understanding the dynamics of HPAI in atypical, or nonreservoir, wild bird hosts may help inform management decisions and potential risk factors to both wild and domestic bird populations. Following the confirmation of infections of HPAI H5N1 in domestic poultry at two commercial premises in Indiana, United States, we sampled and tested 266 Columbiformes and Passeriformes birds and found no detections of the virus at either location. We further queried laboratories within the National Animal Health Laboratory Network for avian influenza (AI) virus diagnostic test results for wild birds submitted from morbidity/mortality events, for a total of 9,368 birds tested across eight orders and 1,543 avian influenza virus detections between February 2022 and March 2023. Query results were assessed for viral prevalence by taxonomic group and suggested that the virus most often was observed in predatory and scavenging birds. The highest prevalence was observed in raptors (0.2514), with prevalence rates in exclusively scavenging Cathartidae reaching up to 0.5333. There is evidence that the consumption of infected tissues is a key pathway for transmission of AI viruses in predatory and scavenging birds. Although detections were found in nonpredatory synanthropic birds, including orders Columbiformes and Passeriformes, the risk of transmission from and between these groups appears comparatively low. Understanding the dynamics of AI viruses in synanthropic bird orders during the global HPAI H5N1 outbreak in wild bird populations can provide pertinent information on viral transmission, disease ecology, and risk to humans and agriculture.

Oriental Reed Warblers do not abandon Common Cuckoo chicks during prolonged nestling periods

The Oriental Reed Warbler (Acrocephalus orientalis) is one of the most commonly used hosts for the parasitic Common Cuckoo (Cuculus canorus). However, as hosts that feed unrelated parasitic nestlings may suffer extra reproductive costs, they may be less willing to care for nestlings that have prolonged nestling periods. To test this hypothesis, the duration of feeding by Oriental Reed Warblers under natural conditions for their own nestlings was compared with the duration of feeding under natural conditions for Common Cuckoo nestlings and for artificially prolonged cuckoo nestlings. The results showed that Oriental Reed Warblers did not starve, drive away, or desert any of the nestlings in the experiment, and neither parent was left alone. Our experimental study indicates that both Oriental Reed Warbler parents were willing to care for nestlings with a prolonged nestling period (up to 30 days, twice the average duration time that the Oriental Reed Warblers fed their own chicks in natural conditions). However, further experiments and observations are required in other host bird species to examine whether both parents or one of the parents may exhibit the behavior of abandoning nestlings with a prolonged nestling period.

Photodynamic Inactivation of Bovine Coronavirus with the Photosensitizer Toluidine Blue O.

Coronaviruses (CoVs) belong to the group of enveloped positive-sense single-strand RNA viruses and are causative agents of respiratory, gastro-intestinal, and central nervous systems diseases in many host species, i.e., birds, mammals, and humans. Beta-CoVs revealed a great potential to cross the barrier between species by causing three epidemics/pandemics among humans in the 21st century. Considering the urgent need for powerful antiviral agents for decontamination, prevention, and treatment of BCoV infections, we turned our attention to the possibility of photodynamic inactivation with photosensitizers in combination with light irradiation. In the present study, we evaluated, for the first time, the antiviral activity of toluidine blue O (TBO) against Beta-coronavirus 1 (BCoV) in comparison to methylene blue (MB). First, we determined the in vitro cytotoxicity of MB and TBO on the Madin-Darby bovine kidney (MDBK) cell line with ISO10993-5/Annex C. Thereafter, BCoV was propagated in MDBK cells, and the virus titer was measured with digital droplet PCR, TCID50 assay and plaque assay. The antiviral activity of non-toxic concentrations of TBO was estimated using the direct inactivation approach. All effects were calculated in MAPLE 15® mathematical software by developing programs for non-linear modeling and response surface analysis. The median inhibitory concentration (IC50) of TBO after 72 h of incubation in MDBK cells was 0.85 µM. The antiviral activity of TBO after the direct inactivation of BCoV (MOI = 1) was significantly stronger than that of MB. The median effective concentration (EC50) of TBO was 0.005 µM. The cytopathic effect decreased in a concentration-dependent manner, from 0.0025 to 0.01 µM, and disappeared fully at concentrations between 0.02 and 0.3 µM of TBO. The number of virus particles also decreased, depending on the concentration applied, as proven by ddPCR analysis. In conclusion, TBO exhibits significant potential for direct inactivation of BCoV in vitro, with a very high selectivity index, and should be subjected to further investigation, aiming at its application in veterinary and/or human medical practice.