Pathogen Surveillance in Swallows (family Hirundinidae): Investigation into Role as Avian Influenza Vector in Eastern Canada Agricultural Landscapes.

First detected in Atlantic Canada in December 2021, highly pathogenic avian influenza virus (HPAIV) subtype H5N1 clade 2.3.4.4b, A/Goose/Guangdong/1/96 lineage, has caused massive mortality in wild birds and domestic poultry in North America. Swallows (Hirundinidae), abundant in North American agricultural ecosystems, have been proposed as possible (bridge) species for HPAIV transmission between wild and domestic birds. We aimed to seek evidence of the potential role of swallows in bridging AIV infection between wild bird reservoirs and poultry flocks in eastern Canada. During a wide-scale outbreak of HPAIV in wild birds and poultry farms across eastern Canada, 200 samples were collected from swallow breeding sites in the Canadian provinces of New Brunswick, Nova Scotia, Ontario, and Quebec, June-August 2022. Samples came from Barn Swallow (Hirundo rustica; n=142), Tree Swallow (Tachycineta bicolor; n=56), and Cliff Swallow (Petrochelidon pyrrhonota; n=2) nests. All samples tested negative for AIV, suggesting that HPAIV and low pathogenic AIV (LPAIV) strains were probably not circulating widely in swallows during the 2022 breeding season in eastern Canada; thus swallows may present a low risk of transmitting AIV. Within a management context, these findings suggest that removing nests of Barn Swallows, a species at risk in Canada, from the exterior of biosecure domestic poultry facilities may not significantly reduce risks of HPAI transmission to poultry.

High costs of infection: Alphavirus infection reduces digestive function and bone and feather growth in nestling house sparrows (Passer domesticus).

Increasingly, ecoimmunology studies aim to use relevant pathogen exposure to examine the impacts of infection on physiological processes in wild animals. Alphaviruses are arthropod-borne, single-stranded RNA (ssRNA) viruses ("arboviruses") responsible for millions of cases of human illnesses each year. Buggy Creek virus (BCRV) is a unique alphavirus that is transmitted by a cimicid insect, the swallow bug, and is amplified in two avian species: the house sparrow (Passer domesticus) and the cliff swallow (Petrochelidon pyrrhonota). BCRV, like many alphaviruses, exhibits age-dependent susceptibility where the young are most susceptible to developing disease and exhibit a high mortality rate. However, alphavirus disease etiology in nestling birds is unknown. In this study, we infected nestling house sparrows with Buggy Creek virus and measured virological, pathological, growth, and digestive parameters following infection. Buggy Creek virus caused severe encephalitis in all infected nestlings, and the peak viral concentration in brain tissue was over 34 times greater than any other tissue. Growth, tissue development, and digestive function were all significantly impaired during BCRV infection. However, based on histopathological analysis performed, this impairment does not appear to be the result of direct tissue damage by the virus, but likely caused by encephalitis and neuronal invasion and impairment of the central nervous system. This is the first study to examine the course of alphavirus diseases in nestling birds and these results will improve our understanding of age-dependent infections of alphaviruses in vertebrate hosts.

Avian Viral Pathogens in Swallows, Zimbabwe : Infectious Diseases in Hirundinidae: A Risk to Swallow?

We sampled 417 swallows in a wetland ecosystem of Zimbabwe in February 2010 and October 2011. RT-PCR tests revealed circulation of avian paramyxovirus type I, avian influenza and West Nile disease viruses in these populations. We discuss the relevance of these findings in relation to what is known on the epidemiology of these viruses in these hosts and in relation to the host ecology. We conclude with recommendations to focus more research on Passeriformes in disease ecology and in particular on the hirundinidae family.

Immune responses of a native and an invasive bird to Buggy Creek Virus (Togaviridae: Alphavirus) and its arthropod vector, the swallow bug (Oeciacus vicarius).

Invasive species often display different patterns of parasite burden and virulence compared to their native counterparts. These differences may be the result of variability in host-parasite co-evolutionary relationships, the occurrence of novel host-parasite encounters, or possibly innate differences in physiological responses to infection between invasive and native hosts. Here we examine the adaptive, humoral immune responses of a resistant, native bird and a susceptible, invasive bird to an arbovirus (Buggy Creek virus; Togaviridae: Alphavirus) and its ectoparasitic arthropod vector (the swallow bug; Oeciacus vicarius). Swallow bugs parasitize the native, colonially nesting cliff swallow (Petrochelidon pyrrhonota) and the introduced house sparrow (Passer domesticus) that occupies nests in cliff swallow colonies. We measured levels of BCRV-specific and swallow bug-specific IgY levels before nesting (prior to swallow bug exposure) and after nesting (after swallow bug exposure) in house sparrows and cliff swallows in western Nebraska. Levels of BCRV-specific IgY increased significantly following nesting in the house sparrow but not in the cliff swallow. Additionally, house sparrows displayed consistently higher levels of swallow bug-specific antibodies both before and after nesting compared to cliff swallows. The higher levels of BCRV and swallow bug specific antibodies detected in house sparrows may be reflective of significant differences in both antiviral and anti-ectoparasite immune responses that exist between these two avian species. To our knowledge, this is the first study to compare the macro- and microparasite-specific immune responses of an invasive and a native avian host exposed to the same parasites.

An enzootic vector-borne virus is amplified at epizootic levels by an invasive avian host.

Determining the effect of an invasive species on enzootic pathogen dynamics is critical for understanding both human epidemics and wildlife epizootics. Theoretical models suggest that when a naive species enters an established host-parasite system, the new host may either reduce ('dilute') or increase ('spillback') pathogen transmission to native hosts. There are few empirical data to evaluate these possibilities, especially for animal pathogens. Buggy Creek virus (BCRV) is an arthropod-borne alphavirus that is enzootically transmitted by the swallow bug (Oeciacus vicarius) to colonially nesting cliff swallows (Petrochelidon pyrrhonota). In western Nebraska, introduced house sparrows (Passer domesticus) invaded cliff swallow colonies approximately 40 years ago and were exposed to BCRV. We evaluated how the addition of house sparrows to this host-parasite system affected the prevalence and amplification of a bird-associated BCRV lineage. The infection prevalence in house sparrows was eight times that of cliff swallows. Nestling house sparrows in mixed-species colonies were significantly less likely to be infected than sparrows in single-species colonies. Infected house sparrows circulated BCRV at higher viraemia titres than cliff swallows. BCRV detected in bug vectors at a site was positively associated with virus prevalence in house sparrows but not with virus prevalence in cliff swallows. The addition of a highly susceptible invasive host species has led to perennial BCRV epizootics at cliff swallow colony sites. The native cliff swallow host confers a dilution advantage to invasive sparrow hosts in mixed colonies, while at the same sites house sparrows may increase the likelihood that swallows become infected.

Persistence of Buggy Creek virus (Togaviridae, Alphavirus) for two years in unfed swallow bugs (Hemiptera: Cimicidae: Oeciacus vicarius).

Alphaviruses (Togaviridae) have rarely been found to persist for long in the adult insects that serve as their vectors. The ectoparasitic swallow bug (Hemiptera: Cimicidae: Oeciacus vicarius Horvath), the vector for Buggy Creek virus (BCRV; Togaviridae, Alphavirus), lives year-round in the mud nests of its host, the cliff swallow (Petrochelidon pyrrhonota Vieillot). We measured the prevalence of BCRV in swallow bugs at sites with cliff swallows present and at the same sites after cliff swallows had been absent for 2 yr. We collected bugs directly from cliff swallow nests in the field and screened bug pools with BCRV-specific real-time-polymerase chain reaction (RT-PCR) and plaque assay. At two colony sites last occupied by birds 2 yr earlier, we found 12.5 and 55.6% of bug pools positive for BCRV RNA by RT-PCR. Infection rates (per 1,000 bugs) for these sites were 1.32 and 7.39. RNA prevalence in the unfed bugs was not significantly different from that in fed bugs 2 yr earlier at the same sites. The RNA-positive samples from unfed bugs failed to yield cytopathic BCRV by Vero-cell plaque assay. However, viral RNA concentrations did not differ between unfed bugs and bugs at active sites, and over 84% of positive bug pools were cytopathic to Vero cells 4-5 wk later, after cliff swallows moved into one of the colony sites. These data demonstrate the persistence of potentially infectious BCRV in unfed swallow bugs for at least 2 yr in nature.

Natural infection of vertebrate hosts by different lineages of Buggy Creek virus (family Togaviridae, genus Alphavirus).

Buggy Creek virus (BCRV; family Togaviridae, genus Alphavirus) is an arbovirus transmitted by the ectoparasitic swallow bug (Hemiptera: Cimicidae: Oeciacus vicarius) to cliff swallows (Petrochelidon pyrrhonota) and house sparrows (Passer domesticus). BCRV occurs in two lineages (A and B) that are sympatric in bird nesting colonies in the central Great Plains, USA. Previous work on lineages isolated exclusively from swallow bugs suggested that lineage A relies on amplification by avian hosts, in contrast to lineage B, which is maintained mostly among bugs. We report the first data on the BCRV lineages isolated from vertebrate hosts under natural conditions. Lineage A was overrepresented among isolates from nestling house sparrows, relative to the proportions of the two lineages found in unfed bug vectors at the same site at the start of the summer transmission season. Haplotype diversity of each lineage was higher in bugs than in sparrows, indicating reduced genetic diversity of virus amplified in the vertebrate host. BCRV appears to have diverged into two lineages based on different modes of transmission.

Cliff swallows, swallow bugs, and West Nile virus: an unlikely transmission mechanism.

The cliff swallow (Petrochelidon pyrrhonota) could play an important role in the transmission of West Nile virus (WNV) because of its breeding ecology, reservoir competence status, and potentially high natural exposure rates. Cliff swallows nest within colonies and their nests are occupied year-round by swallow bugs (Oeciacus vicarius), hematophagus ectoparasites that feed primarily on cliff swallows. These parasites are likely exposed to WNV while feeding on infectious blood of nesting cliff swallow adults and nestlings and thus, if competent vectors, could contribute to seasonal elevations in WNV transmission. In addition, swallow bugs remain within nests year-round and therefore could provide a potential overwintering mechanism for WNV if persistently infected. To test the hypotheses that swallow bugs are competent vectors and become persistently infected with WNV, we experimentally inoculated cliff swallow nestlings, allowed swallow bugs to feed on these birds during the acute phase of infection, and then exposed naive cliff swallow nestlings to the same swallow bugs. In addition, a subset of swallow bugs that fed on infectious swallow nestlings was maintained through a simulated overwintering period. Although swallow bugs ingested infectious blood (up to 10(6.8) plaque-forming units of WNV/mL serum) and subsequently blood-fed on naive swallows, no WNV transmission was detected, and all bugs tested WNV negative after the simulated overwintering period. Although many ecologic scenarios exist beyond the present study, our results suggest that swallow bugs may be unlikely to serve as competent biological vectors for WNV during active transmission periods or to reinitiate seasonal transmission.

Phylogeographical structure and evolutionary history of two Buggy Creek virus lineages in the western Great Plains of North America.

Buggy Creek virus (BCRV) is an unusual arbovirus within the western equine encephalitis complex of alphaviruses. Associated with cimicid swallow bugs (Oeciacus vicarius) as its vector and the cliff swallow (Petrochelidon pyrrhonota) and house sparrow (Passer domesticus) as its amplifying hosts, this virus is found primarily in the western Great Plains of North America at spatially discrete swallow nesting colonies. For 342 isolates collected in Oklahoma, Nebraska, Colorado and North Dakota, from 1974 to 2007, we sequenced a 2076 bp region of the 26S subgenomic RNA structural glycoprotein coding region, and analysed phylogenetic relationships, rates of evolution, demographical histories and temporal genetic structure of the two BCRV lineages found in the Great Plains. The two lineages showed distinct phylogeographical structure: one lineage was found in the southern Great Plains and the other in the northern Great Plains, and both occurred in Nebraska and Colorado. Within each lineage, there was additional latitudinal division into three distinct sublineages. One lineage is showing a long-term population decline. In comparing sequences taken from the same sites 8-30 years apart, in one case one lineage had been replaced by the other, and in the other cases there was little evidence of the same haplotypes persisting over time. The evolutionary rate of BCRV is in the order of 1.6-3.6x10(-4) substitutions per site per year, similar to that estimated for other temperate-latitude alphaviruses. The phylogeography and evolution of BCRV could be better understood once we determine the nature of the ecological differences between the lineages.

Host and vector movement affects genetic diversity and spatial structure of Buggy Creek virus (Togaviridae).

Determining the degree of genetic variability and spatial structure of arthropod-borne viruses (arboviruses) may help in identifying where strains that potentially cause epidemics or epizootics occur. Genetic diversity in arboviruses is assumed to reflect relative mobility of their vertebrate hosts (and invertebrate vectors), with highly mobile hosts such as birds leading to genetic similarity of viruses over large areas. There are no empirical studies that have directly related host or vector movement to virus genetic diversity and spatial structure. Using the entire E2 glycoprotein-coding region of 377 Buggy Creek virus isolates taken from cimicid swallow bugs (Oeciacus vicarius), the principal invertebrate vector for this virus, we show that genetic diversity between sampling sites could be predicted by the extent of movement by transient cliff swallows (Petrochelidon pyrrhonota) between nesting colonies where the virus and vectors occur. Pairwise F(ST) values between colony sites declined significantly with increasing likelihood of a swallow moving between those sites per 2-day interval during the summer nesting season. Sites with more bird movement between them had virus more similar genetically than did pairs of sites with limited or no bird movement. For one virus lineage, Buggy Creek virus showed greater haplotype and nucleotide diversity at sites that had high probabilities of birds moving into or through them during the summer; these sites likely accumulated haplotypes by virtue of frequent virus introductions by birds. Cliff swallows probably move Buggy Creek virus by transporting infected bugs on their feet. The results provide the first empirical demonstration that genetic structure of an arbovirus is strongly associated with host/vector movement, and suggest caution in assuming that bird-dispersed arboviruses always have low genetic differentiation across different sites.

Bird movement predicts Buggy Creek virus infection in insect vectors.

Predicting the spatial foci of zoonotic diseases is a major challenge for epidemiologists and disease ecologists. Migratory birds are often thought to be responsible for introducing some aviozoonotic pathogens such as West Nile and avian influenza viruses to a local area, but most information on how bird movement correlates with virus prevalence is anecdotal or indirect. We report that the prevalence of Buggy Creek virus (BCRV) infection in cimicid swallow bugs (Oeciacus vicarius), the principal invertebrate vector for this virus, was directly associated with the likelihood of movement by cliff swallows (Petrochelidon pyrrhonota), an amplifying host for the virus, between nesting colonies. The prevalence of BCRV in bugs was also directly correlated with the number of swallows immigrating into a site. Birds that move into a site are often transient individuals that may have more often encountered virus elsewhere. These results indicate that the magnitude and direction of daily bird movement in a local area can accurately predict transmission foci for this virus and provide rare quantitative evidence that birds can play a critical role in the dispersal of certain vector-borne viruses.

Phylogenetic analysis of Buggy Creek virus: evidence for multiple clades in the Western Great Plains, United States of America.

We present the first detailed phylogenetic analysis of Buggy Creek virus (BCRV), a poorly known alphavirus with transmission cycles involving a cimicid swallow bug (Oeciacus vicarius) vector and cliff swallows (Petrochelidon pyrrhonota) and house sparrows (Passer domesticus) as the principal avian hosts. Nucleotide sequences of a 2,075-bp viral envelope glycoprotein-coding region, covering the entire PE2 gene, were determined for 33 BCRV isolates taken from swallow bugs at cliff swallow colonies in Nebraska and Colorado in the summer of 2001 and were compared with the corresponding region of BCRV isolates collected from Oklahoma in the 1980s. We also analyzed isolates of the closely related Fort Morgan virus (FMV) collected from Colorado in the 1970s. Phylogenetic analysis indicated that BCRV falls into the western equine encephalomyelitis complex of alphaviruses, in agreement with antigenic results and a previous alphavirus phylogeny based on the E1 coding region. We found four distinct BCRV/FMV clades, one each unique to Nebraska, Colorado, and Oklahoma and one containing isolates from both Nebraska and Colorado. BCRV isolates within the two clades from Nebraska showed 5.7 to 6.2% nucleotide divergence and 0.7 to 1.9% amino acid divergence, and within these clades, we found multiple subclades. Nebraska subclades tended to be confined to one or a few cliff swallow colonies that were close to each other in space, although in some cases, near-identical isolates were detected at sites up to 123 km apart. Viral gene flow occurs when cliff swallows move (bugs) between colony sites, and the genetic structure of BCRV may reflect the limited dispersal abilities of its insect vector.