A single Haemoproteus plataleae haplotype is widespread in white ibis (Eudocimus albus) from urban and rural sites in southern Florida.

The American white ibis (Eudocimus albus), a common bird species in Florida, has become increasingly urban, with many populations relying heavily on urban and suburban habitats, which may alter parasite transmission. Parasites of ibis, especially haemosporidians, are understudied. Avian haemosporidia can have a wide range of impacts on birds, including decreased reproductive success or increased mortality. Because southern Florida is subtropical and has a high diversity of potential vectors for haemosporidia, we hypothesized that there will be a high prevalence and genetic diversity of haemosporidia in white ibis. A total of 636 ibis from South Florida were sampled from 2010 to 2022, and blood samples were tested for haemosporidia by examination of Giemsa-stained thin blood smears and/or nested PCRs targeting the cytochrome b gene. A total of 400 (62.9%, 95% CI 59-66.7%) ibis were positive for parasites that were morphologically identified as Haemoproteus plataleae. Sequences of 302 positives revealed a single haplotype of Haemoproteus (EUDRUB01), which was previously reported from white ibis in South Florida and captive scarlet ibis (E. ruber) in Brazil. No Plasmodium or Leucocytozoon infections were detected. Parasitemias of the 400 positive birds were very low (average 0.084%, range 0.001%-2.16% [although only 2 birds had parasitemias >1%]). Prevalence and parasitemias were similar for males and females (68% vs. 61.6% and 0.081% vs. 0.071%, respectively). Prevalence in juveniles was lower compared with adults (52% vs. 67.4%) but parasitemias were higher in juveniles (0.117% vs. 0.065%). This data shows that H. plataleae is common in ibis in South Florida. Although parasitemias were generally low, additional research is needed to determine if this parasite has subclinical effects on ibis, if additional haplotypes or parasite species infect ibis in other regions of their range, or if H. plataleae is pathogenic for other sympatric avian species.

Diversity and prevalence of hemoparasites of wading birds in southern Florida, USA.

Relatively few studies on hemoparasites have been conducted on wading birds in the families Ardeidae and Threskiornithidae (order Pelecaniformes), especially in the United States. In this study, we obtained baseline data on the prevalence and genetic diversity of haemosporidian parasites in wading birds opportunistically sampled from southern Florida, USA. We detected blood parasites in White Ibis (Eudocimus albus), Glossy Ibis (Plegadis falcinellus), Green Heron (Butorides virescens), and Roseate Spoonbill (Platalea ajaja) with several novel host-parasite relationships. Infected birds had low parasitemias (average 0.77%, range 0-4%) suggesting that infections were chronic. Despite the low sample sizes for several of our sampled species, these data highlight the diversity of parasites in this understudied group of birds and suggest that additional studies are needed to investigate the potential impacts of these parasites on their health, especially since southern Florida is becoming increasingly urbanized which can alter parasite transmission or host susceptibility.

A Quantitative PCR Protocol for Detection of Oxyspirura petrowi in Northern Bobwhites (Colinus virginianus).

Oxyspirura petrowi is a parasitic nematode that infects wild birds. This parasite has a broad host range, but has recently been reported in high prevalences from native Galliformes species in the United States. In order to better understand the impact O. petrowi has on wild bird populations, we developed a quantitative PCR protocol to detect infections in wild northern bobwhites (Colinus virginianus). We used paired fecal and cloacal swab samples from wild caught and experimentally infected northern bobwhites and matching fecal float data from experimentally infected birds to validate our assay. Overall we detected more positive birds from fecal samples than the paired cloacal swabs and there was strong agreement between the qPCR results from fecal samples and from fecal flotation (84%; κ = 0.69 [0.53-0.84 95% CI]). We also detected O. petrowi DNA in ten replicates of samples spiked with one O. petrowi egg. This qPCR assay is an effective assay to detect O. petrowi infections in wild birds. Our results suggest that fecal samples are the most appropriate sample for detecting infections; although, cloacal swabs can be useful for determining if O. petrowi is circulating in a population.

Urbanized White Ibises (Eudocimus albus) as Carriers of Salmonella enterica of Significance to Public Health and Wildlife.

Worldwide, Salmonella spp. is a significant cause of disease for both humans and wildlife, with wild birds adapted to urban environments having different opportunities for pathogen exposure, infection, and transmission compared to their natural conspecifics. Food provisioning by people may influence these factors, especially when high-density mixed species flocks aggregate. White Ibises (Eudocimus albus), an iconic Everglades species in decline in Florida, are becoming increasingly common in urbanized areas of south Florida where most are hand-fed. We examined the prevalence of Salmonella shedding by ibises to determine the role of landscape characteristics where ibis forage and their behavior, on shedding rates. We also compared Salmonella isolated from ibises to human isolates to better understand non-foodborne human salmonellosis. From 2010-2013, 13% (n = 261) adult/subadult ibises and 35% (n = 72) nestlings sampled were shedding Salmonella. The prevalence of Salmonella shedding by ibises significantly decreased as the percent of Palustrine emergent wetlands and herbaceous grasslands increased, and increased as the proportion of open-developed land types (e.g. parks, lawns, golf courses) increased, suggesting that natural ecosystem land cover types supported birds with a lower prevalence of infection. A high diversity of Salmonella serotypes (n = 24) and strain types (43 PFGE types) were shed by ibises, of which 33% of the serotypes ranked in the top 20 of high significance for people in the years of the study. Importantly, 44% of the Salmonella Pulsed-Field Gel Electrophoresis patterns for ibis isolates (n = 43) matched profiles in the CDC PulseNet USA database. Of these, 20% came from Florida in the same three years we sampled ibis. Importantly, there was a negative relationship between the amount of Palustrine emergent wetland and the number of Salmonella isolates from ibises that matched human cases in the PulseNet database (p = 0.056). Together, our results indicate that ibises are good indicators of salmonellae strains circulating in their environment and they have both the potential and opportunity to transmit salmonellae to people. Finally, they may act as salmonellae carriers to natural environments where other more highly-susceptible groups (nestlings) may be detrimentally affected.

A survey of neonicotinoid use and potential exposure to northern bobwhite (Colinus virginianus) and scaled quail (Callipepla squamata) in the Rolling Plains of Texas and Oklahoma.

Northern bobwhite (quail) (Colinus virginianus) and scaled quail (Callipepla squamata) populations have declined dramatically in the Rolling Plains ecoregion of Texas and Oklahoma (USA). There is rising concern about potential toxicity of neonicotinoids to birds. To investigate this concern, the authors examined crops of 81 northern bobwhite and 17 scaled quail to determine the presence or absence of seeds treated with 3 neonicotinoids (clothianidin, imidacloprid, and thiamethoxam). No treated seeds were found in the 98 crops examined. Liver samples from all 98 quail were collected and analyzed for neonicotinoid residues. Analysis revealed very low concentrations of neonicotinoids within the quail liver samples. The results suggest there is little to no risk of direct toxicity to quail from neonicotinoids. Environ Toxicol Chem 2016;35:1511-1515. © 2015 SETAC.

Subtype-specific influenza A virus antibodies in Canada geese (Branta canadensis).

Historically, surveillance for influenza A viruses (IAVs) in wild birds has relied on viral detection assays. This was largely due to poor performance of serological assays in wild birds; however, recently developed commercial serological assays have improved the ability to detect IAV antibodies in wild birds. Serological surveillance for IAV antibodies in Canada geese (Branta canadensis) has shown that, despite a low prevalence of virus isolations, Canada geese are frequently exposed to IAVs and that exposure increases with latitude, which follows virus isolation prevalence patterns observed in dabbling ducks. The objectives of this study were to further evaluate IAV antibodies in Canada geese using a subtype-specific serological assay to determine if Canada geese are exposed to subtypes that commonly circulate in dabbling ducks. We collected serum samples from Canada geese in Minnesota, New Jersey, Pennsylvania, and Wisconsin and tested for antibodies to IAVs using a blocking ELISA. Positive samples were further tested by hemagglutination inhibition for 10 hemagglutinin IAV subtypes (H1-H10). Overall, we detected antibodies to NP in 24% (714/2919) of geese. Antibodies to H3, H4, H5, and H6 subtypes predominated, with H5 being detected most frequently. A decrease in H5 HI antibody prevalence and titers was observed from 2009 to 2012. We also detected similar exposure pattern in Canada geese from New Jersey, Minnesota, Washington and Wisconsin. Based on the published literature, H3, H4, and H6 viruses are the most commonly reported IAVs from dabbling ducks. These results indicate that Canada geese also are frequently exposed to viruses of the same HA subtypes; however, the high prevalence of antibodies to H5 viruses was not expected as H5 IAVs are generally not well represented in reported isolates from ducks.

Influenza A virus H5-specific antibodies in mute swans (Cygnus olor) in the USA.

The use of serologic assays for influenza A virus (IAV) surveillance in wild birds has increased because of the availability of commercial enzyme-linked immunosorbent assays (ELISAs). Recently, an H5-specific blocking ELISA (bELISA) was shown to reliably detect H5-specific antibodies to low- and high-pathogenic H5 viruses in experimentally infected waterfowl. Mute Swans (Cygnus olor) were frequently associated with highly pathogenic H5N1 outbreaks in Europe and may have a similar role if highly pathogenic H5N1 is introduced into North America. We measured the prevalence of antibodies to the nucleoprotein and H5 protein in Mute Swans using three serologic assays. We collected 340 serum samples from Mute Swans in Michigan, New Jersey, New York, and Rhode Island, US. We detected antibodies to the IAV nucleoprotein in 66.2% (225/340) of the samples. We detected H5-specific antibodies in 62.9% (214/340) and 18.8% (64/340) using a modified H5 bELISA protocol and hemagglutination inhibition (HI) assay, respectively. The modified H5 bELISA protocol detected significantly more positive samples than did the manufacturer's protocol. We also tested 46 samples using virus neutralization. Neutralization results had high agreement with the modified H5 bELISA protocol and detected a higher prevalence than did the HI assay. These results indicate that North American Mute Swans have high nucleoprotein and H5 antibody prevalences.

Wood ducks (Aix sponsa) as potential reservoirs for avian influenza and avian paramyxoviruses.

Influenza A viruses (IAVs) and avian paramyxoviruses (APMVs) are important pathogens of poultry worldwide, and both commonly occur in wild waterfowl, especially ducks in the family Anatidae. Although wood ducks (Aix sponsa) are members of the Anatidae, their behaviour differs from most other species in this family, which could affect the transmission of IAVs and APMVs. We collected cloacal and oropharyngeal swab and blood samples from more than 700 wood ducks across nine states in the eastern United States of America. No IAVs were isolated, and based on blocking enzyme-linked immunoassay ELISA results, antibodies to IAVs were only detected in 0.2% of samples. In contrast, 23 (3%) APMVs were isolated (22 Newcastle disease virus and 1 APMV-6), and antibodies to multiple serotypes of APMVs were detected in more than 60% of the samples. After-hatch-year birds were more likely to be antibody positive for APMV-4 and APMV-6 compared to hatch-year birds. Female birds were more likely to be antibody positive for APMV-4 than were male birds. Our results indicate that wood ducks are probably not an important host for IAV but are frequently infected with APMVs.

First report of Angiostrongylus vasorum and Hepatozoon from a red fox (Vulpes vulpes) from West Virginia, USA.

Angiostrongylus vasorum was identified in the lungs of a red fox (Vulpes vulpes) from West Virginia, United States (US), indicating a new geographical location for this metastrongylid nematode. The fox was euthanized and submitted for necropsy after displaying erratic behavior. We did not detect rabies virus or canine distemper virus from the fox. We observed bronchopneumonia associated with A. vasorum infection disseminated in both lungs. In addition, protozoal meronts were observed in the liver, spleen, and mesenteric lymph node, and were identified as Hepatozoon canis. Lymphoid depletion was also observed in the spleen and mesenteric lymph node. In addition to A. vasorum and H. canis infections, Eucoleus aerophilus eggs and adult worms were observed in the lungs of the fox. Severe lesions associated with A. vasorum infection were observed in the lungs and these were determined to be the likely cause of morbidity; however, synergistic effects among the multiple infections detected in this fox cannot be ruled out. This is the first report of an autochthonous A. vasorum infection in the US and from outside of Newfoundland Canada, the only place in North America where the parasite is known to be endemic. Additionally, this is the first report of a H. canis infection in a red fox from the US.

Evaluation of a commercial enzyme-linked immunosorbent assay for detection of antibodies against the H5 subtype of Influenza A virus in waterfowl.

The ID Screen Influenza H5 Antibody Competition enzyme-linked immunosorbent assay was tested for the detection of antibodies to the H5 subtype of influenza A (IA) virus in waterfowl. Assays were conducted with sera obtained from Mallards (Anas platyrhynchos) and Pekin Ducks (Anas platyrhynchos domestica), experimentally infected with eight low pathogenic (LP) and nine highly pathogenic (HP) H5N1 IA viral strains. Three incubation periods (1, 4 and 18 hours) and two dilutions (1:2 and 1:5) were tested. All serum samples from LP H5-infected birds tested positive; however, improved detection rates were observed for viruses belonging to the HP H5N1 clade 2.2.1 as compared with those belonging to clade 2.1.3.

Evaluation of a restriction fragment length enzyme assay for differentiation of Haemoproteus and Plasmodium across a standard region of the mitochondrial genome.

Avian hemosporidian parasites are a genetically diverse group of parasites with a near cosmopolitan distribution. Over the past 2 decades, several PCR protocols have been designed to detect these parasites. The majority of these protocols amplify part of or the entire mitochondrial cytochrome b gene. However, many of these protocols co-amplify 2 genera (Haemoproteus and Plasmodium), making it impossible to determine which genus is amplified without post-PCR analysis. A uniform database (MalAvi), containing sequences amplified with the primers HAEMF and HAEMR2, has been developed to increase comparability across studies. We analyzed sequences from the MalAvi database and new sequences and found that digestion with EcoRV could be used to distinguish Haemoproteus from the majority of Plasmodium sequences. In addition, we tested 220 wild birds from Costa Rica and the United States for avian hemosporidians and assessed the ability of EcoRV to distinguish these 2 genera. Thirty-six positive samples were sequenced to confirm the restriction profiles, and we also analyzed 63 new hemosporidian sequences from ongoing studies in the United States for the restriction site. Among these new samples, all of the 85 Haemoproteus (subgenus Parahaemoproteus) and 14 Plasmodium were distinguishable. Overall, 887 of 898 (98.8%) sequences from our studies and the MalAvi database were assigned to the correct genus. Of these samples, all Haemoproteus samples were correctly identified and all but 11 Plasmodium samples were correctly identified by the EcoRV assay. Overall, this restriction enzyme protocol is able to quickly and efficiently classify these 2 genera of avian malarial parasites and would be useful for researchers interested in identifying parasites to genus-level, studies focused on sequence analysis of only a single genus, or for detecting co-infections that would need cloning prior to sequence analysis.

Spatial, temporal, molecular, and intraspecific differences of haemoparasite infection and relevant selected physiological parameters of wild birds in Georgia, USA.

The prevalence of five avian haemoparasite groups was examined for effects on health and associations with extrinsic factors. Overall, 786 samples were examined from six sites in two Georgia (USA) watersheds, during breeding and non-breeding periods in 2010 and 2011. Among the four most commonly infected species, Haemoproteus prevalence was significantly higher in Northern Cardinals (Cardinalis cardinalis) compared to Indigo Buntings (Passerina cyanea) and Tufted Titmice (Baeolophus bicolor) while prevalence in White-throated Sparrows (Zonotrichia albicollis) was significantly higher than in Indigo Buntings. Higher prevalence of Plasmodium was noted in Tufted Titmice and Northern Cardinals. While Leucocytozoon prevalence was highest in White-throated Sparrows, Trypanosoma prevalence was highest in Tufted Titmice. Interesting differences in infection probabilities were noted between foraging guilds with Haemoproteus associated with low-middle level strata and birds in the middle-upper strata were more likely to be infected with Plasmodium and Trypanosoma. In contrast, ground-foraging birds were more likely to be infected with Leucocytozoon. Breeding season was correlated with higher polychromasia counts and higher prevalence of Haemoproteus, Plasmodium and Trypanosoma. In addition, prevalence of infection with certain haemoparasite genera and packed cell volume (PCV) were different among host species. Body mass index was inversely correlated with prevalence of microfilaria infection but positively related to Haemoproteus infection. However, we found no relationship between PCV or polychromasia levels with haemoparasite infection. Molecular characterization of 61 samples revealed 19 unique Haemoproteus (n = 7) and Plasmodium (n = 12) haplotypes with numerous new host records. No differences were noted in haplotype diversity among birds with different migratory behaviors or foraging heights, thus additional studies are needed that incorporate molecular analysis, host biology, and vector biology into comprehensive models on parasite ecology. Detailed morphological examination of these parasites is also necessary to determine if closely related haplotypes represent single species or morphologically distinct, but closely related, haplotypes.

Searching Before It Is Too Late: A Survey of Blood Parasites in Ctenosaura melanosterna, a Critically Endangered Reptile of Honduras.

For species at risk of extinction, any parasites they have would be expected to face a similar fate. In such cases, time is running out for efforts to identify and study their parasitic fauna before they are gone. We surveyed the hemoparasite fauna of 50 black-chested, spiny-tailed iguanas (Ctenosaura melanosterna), a critically-endangered species, on an island off the coast of Honduras. Blood samples from captured animals were tested for hemoparasites by thin blood smear and molecular analyses. Based on microscopy, two parasites were identified, a Plasmodium sp. in 14% of iguanas and a Hepatozoon sp. in 32%. For both parasites, parasitemia levels were <0.1%. Prevalence and parasitemias of Hepatozoon declined with increasing host size, a pattern differing from most prior studies of saurian reptiles. From a subset of iguanas with microscopy-confirmed Plasmodium infections, sequence analysis of 454 bp of the cytochrome b gene indicated that the Plasmodium species was distinct from known Plasmodium and was most closely related to P. chiricahuae (96.5% similarity) followed by P. mexicanum (95.8% similarity). Efforts to amplify the Hepatozoon parasite using PCR were not successful. Additional surveys and studies of this host-parasite system would be valuable, both to science and to the management of this endangered animal.

Antibodies to avian influenza viruses in Canada geese (Branta canadensis): a potential surveillance tool?

Traditionally, the epidemiology of avian influenza viruses (AIVs) in wild birds has been defined by detection of virus or viral RNA through virus isolation or reverse-transcription polymerase chain reaction. Our goals were to estimate AIV antibody prevalence in Canada geese (Branta canadensis) and measure effects of age and location on these estimates. We collected 3,205 samples from nine states during June and July 2008 and 2009: Georgia, Massachusetts, Minnesota, Mississippi, New Jersey, North Carolina, Pennsylvania, Washington, and West Virginia. Serum samples were tested for AIV antibodies with the use of a commercial blocking enzyme-linked immunosorbent assay. Overall, 483 (15%) Canada geese had detectable antibodies to AIV. Significantly higher prevalences were detected in geese collected from northeastern and upper midwestern states compared with southeastern states. This trend is consistent with results from virus isolation studies reporting AIV prevalence in North American dabbling ducks. Within Pennsylvania, significantly higher antibody prevalences were detected in goose flocks sampled in urban locations compared to flocks sampled in rural areas. Antibody prevalence was significantly higher in after-hatch-year geese compared to hatch-year geese. No significant differences in prevalence were detected from 10 locations sampled during both years. Results indicate that Canada geese are frequently exposed to AIVs and, with resident populations, may potentially be useful as sentinels to confirm regional AIV transmission within wild bird populations.

Detection of stx and stx genes in Pennsylvanian white-tailed deer.

Shiga toxin-producing E. coli carrying the stx(1) and/or stx(2) genes can cause multi-symptomatic illness in humans. A variety of terrestrial and aquatic environmental reservoirs of stx have been described. Culture based detection of microbes in deer species have found a low percentage of samples that have tested positive for Stx-producing microbes, suggesting that while deer may contain these microbes, their overall abundance in deer is low. In this study, quantitative PCR (qPCR) was utilized to test for the presence of stx genes in white-tailed deer fecal matter in western Pennsylvania. In this culture independent screening, nearly half of the samples tested positive for the stx(2) gene, with a bias towards samples that were concentrated with stx(2). This study, while limited in scope, suggests that deer may be a greater reservoir for stx than was previously thought.

Prevalence of antibodies to type a influenza virus in wild avian species using two serologic assays.

Serologic testing to detect antibodies to avian influenza (AI) virus has been an underused tool for the study of these viruses in wild bird populations, which traditionally has relied on virus isolation and reverse transcriptase-polymerase chain reaction (RT-PCR). In a preliminary study, a recently developed commercial blocking enzyme-linked immunosorbent assay (bELISA) had sensitivity and specificity estimates of 82% and 100%, respectively, for detection of antibodies to AI virus in multiple wild bird species after experimental infection. To further evaluate the efficacy of this commercial bELISA and the agar gel immunodiffusion (AGID) test for AI virus antibody detection in wild birds, we tested 2,249 serum samples collected from 62 wild bird species, representing 10 taxonomic orders. Overall, the bELISA detected 25.4% positive samples, whereas the AGID test detected 14.8%. At the species level, the bELISA detected as many or more positive serum samples than the AGID in all 62 avian species. The majority of positive samples, detected by both assays, were from species that use aquatic habitats, with the highest prevalence from species in the orders Anseriformes and Charadriiformes. Conversely, antibodies to AI virus were rarely detected in the terrestrial species. The serologic data yielded by both assays are consistent with the known epidemiology of AI virus in wild birds and published reports of host range based on virus isolation and RT-PCR. The results of this research are also consistent with the aforementioned study, which evaluated the performance of the bELISA and AGID test on experimental samples. Collectively, the data from these two studies indicate that the bELISA is a more sensitive serologic assay than the AGID test for detecting prior exposure to AI virus in wild birds. Based on these results, the bELISA is a reliable species-independent assay with potentially valuable applications for wild bird AI surveillance.

Evaluation of a commercial blocking enzyme-linked immunosorbent assay to detect avian influenza virus antibodies in multiple experimentally infected avian species.

Wild birds of the orders Anseriformes and Charadriiformes are the natural reservoirs for avian influenza (AI) viruses. Traditionally, AI virus surveillance in wild birds has relied on virus identification strategies, including virus isolation and detection. To evaluate the accuracy of a commercial blocking enzyme-linked immunosorbent assay (bELISA) and the agar gel immunodiffusion (AGID) test for detection of antibodies in wild birds, which is indicative of AI virus infection, we tested 281 serum samples from various wild avian species that were experimentally infected with AI viruses. Included in these samples were 178 samples from birds with confirmed AI virus infections (122 infected with low-pathogenic AI [LPAI] viruses and 56 infected with highly pathogenic AI [HPAI] viruses) and 103 samples from birds that were uninfected, negative controls. The sensitivities of the bELISA and the AGID test were 0.820 (95% confidence interval [95% CI], 0.756 to 0.874) and 0.674 (95% CI, 0.600 to 0.742), respectively. Both tests had an estimated specificity of 1.00 (95% CI, 0.965 to 1.00). The bELISA was significantly more sensitive than the AGID test for both LPAI virus- and HPAI virus-infected birds. Both assays, however, had a higher sensitivity for birds infected with HPAI virus than for birds infected with LPAI virus. These results demonstrate the potential utility of the bELISA for detection of antibodies to both LPAI and HPAI viruses in multiple avian species, representing five avian orders and 17 genera. Additional studies are warranted to further evaluate the utility of the bELISA for use with naturally infected birds.