Shamus P. Keeler

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.

Strain-related variation in the persistence of influenza A virus in three types of water: distilled water, filtered surface water, and intact surface water

BackgroundThe persistence of influenza A (IA) virus in aquatic habitats has been demonstrated to be a determinant for virus transmission dynamics in wild duck populations. In this study, we investigated virus strain-related variation in persistence in water for nine wild duck isolated IA viruses of three subtypes (H3N8, H4N6, and H8N4).ResultsWe experimentally estimated the loss of infectivity over time in three different types of water: distilled, filtered surface water, and intact surface water. All viruses persisted longest in distilled water followed by filtered surface water with markedly reduced durations of persistence observed in the intact surface water. Strain-related variations were observed in distilled and filtered surface water but limited variation was observed in the intact surface water.ConclusionsOur findings suggest that the role of surface water for long-term (between years) maintenance of AI viruses in the environment may be limited, and suggest that the physico-chemical characteristics of water, as well as microorganisms, may be of strong importance. Results also indicate that the extent of strain-related variation observed in distilled water may overestimate persistence abilities for IA viruses in the wild and supports the need to develop experiments that account for these effects to assess subtype, genotype, as well as spatial and temporal variation in the persistence of IA viruses in aquatic habitats.

PERSISTENCE OF LOW PATHOGENIC AVIAN INFLUENZA VIRUSES IN FILTERED SURFACE WATER FROM WATERFOWL HABITATS IN GEORGIA, USA

The natural reservoirs for avian influenza virus (AIV) are wild bird species of the orders Anseriformes and Charadriiformes. The primary route of transmission for wild birds is through fecally contaminated surface water on shared aquatic habitats. A distilled water model has shown that AIV remains infectious in water for weeks to months with pH, salinity, and temperature affecting stability. To evaluate the effect of pH, salinity, and temperature on AIV persistence in natural surface water, we measured the duration of infectivity for two common low pathogenic AIV subtypes in 15 filtered surface water samples collected from major waterfowl habitats in Georgia, USA. Trials were performed at three incubation temperatures 10, 17, and 28 C. Consistent with previous studies, pH and temperature had a significant effect on the stability of AIV in filtered surface water. Both viruses were less stable at warmer temperatures and in acidic water (pH<5.0). Due to the limited range of salinity of the field water samples, the role of salinity in AIV stability in surface water could not adequately be evaluated. Variations in persistence times between water samples with comparable pH and salinities indicated that other factors affect AIV stability in natural surface water. These results contribute to the current understanding of AIV persistence in aquatic habitats and may help in identifying areas with an increased likelihood of AIV persistence and potential transmission.

Abiotic factors affecting the persistence of avian influenza virus in surface waters of waterfowl habitats.

ABSTRACT Avian influenza (AI) virus can remain infectious in water for months, and virus-contaminated surface water is considered to be a source of infection within wild waterfowl populations. Previous work has characterized the effects of pH, salinity, and temperature on viral persistence in water, but most of that work was done with modified distilled water. The objective of this study was to identify the abiotic factors that influence the duration of AI virus persistence in natural surface water. Surface water samples were collected from 38 waterfowl habitats distributed across the United States. Samples were submitted to the U.S. Geological Survey National Water Quality Laboratory for chemical analysis and the University of Georgia for viral reduction time analysis. Samples were filtered with 0.22-μm filters, and the durations of persistence of three wild-bird-derived influenza A viruses within each water sample at 10, 17, and 28°C were determined. The effects of the surface water physicochemical factors on the duration of AI viral persistence in laboratory experiments were evaluated by multivariable linear regression with robust standard errors. The duration of AI virus persistence was determined to be longest in filtered surface water with a low temperature (<17°C), a neutral-to-basic pH (7.0 to 8.5), low salinity (<0.5 ppt), and a low ammonia concentration (<0.5 mg/liter). Our results also highlighted potential strain-related variation in the stability of AI virus in surface water. These results bring us closer to being able to predict the duration of AI virus persistence in surface water of waterfowl habitats.

Use of FTA® Sampling Cards for Molecular Detection of Avian Influenza Virus in Wild Birds

SUMMARY. Current avian influenza (AI) virus surveillance programs involving wild birds rely on sample collection methods that require refrigeration or low temperature freezing to maintain sample integrity for virus isolation and/or reverse-transcriptase (RT) PCR. Maintaining the cold chain is critical for the success of these diagnostic assays but is not always possible under field conditions. The aim of this study was to test the utility of Finders Technology Associates (FTA®) cards for reliable detection of AI virus from cloacal and oropharyngeal swabs of wild birds. The minimum detectable titer was determined, and the effect of room temperature storage was evaluated experimentally using multiple egg-propagated stock viruses (n  =  6). Using real time RT-PCR, we compared results from paired cloacal swab and samples collected on FTA cards from both experimentally infected mallards (Anas platyrhynchos) and hunter-harvested waterfowl sampled along the Texas Gulf Coast. Based on the laboratory trials, the average minimal detectable viral titer was determined to be 1 × 104.7 median embryo infectious dose (EID50)/ml (range: 1 × 104.3 to 1 × 105.4 EID50/ml), and viral RNA was consistently detectable on the FTA cards for a minimum of 20 days and up to 30 days for most subtypes at room temperature (23 C) storage. Real-time RT-PCR of samples collected using the FTA cards showed fair to good agreement in live birds when compared with both real-time RT-PCR and virus isolation of swabs. AI virus detection rates in samples from several wild bird species were higher when samples were collected using the FTA cards compared with cloacal swabs. These results suggest that FTA cards can be used as an alternative sample collection method when traditional surveillance methods are not possible, especially in avian populations that have historically received limited testing or situations in which field conditions limit the ability to properly store or ship swab samples.

Chapter 1. The biology of the caecal trematode Zygocotyle lunata.

This chapter examines the significant studies on the caecal paramphistomid Zygocotyle lunata from mainly 1941 to 2008. This digenean is one of two paramphistomid species in the family Zygocotylidae. Z. lunata has an almost global distribution being found in the wild in numerous waterfowl and various species of ruminants. It infects planorbid snails in the genera Helisoma and Biomphalaria. Because it may involve concurrent infections with Schistosoma mansoni in species of Biomphalaria snails, there is an interest in Z. lunata as a potential control agent against S. mansoni. Z. lunata may have some impact as a pathogen of birds in wildlife diseases, but its real assessment in this role is not fully understood. The cercariae of this paramphistomid when released from snails encyst on a substratum such as vegetation or the shells of aquatic invertebrates in the wild or in the laboratory on the glass or plastic of a container holding the snails. Most studies on the intra-molluscan parasitic stages are based on work from snails collected in the wild and experimental studies using laboratory-reared snails are sparse. Numerous experimental mammalian and avian hosts can be infected with the metacercarial cysts of this digenean, but quantitative experimental studies on the adult stages of this parasite using known numbers of cysts and well-defined strains of vertebrate hosts are sparse. Likewise, some studies on the immunology and pathology of this trematode have been done, but for the most part they are fragmentary and do not provide quantitative information on these topics. Published information on the molecular biology of this organism does not exist. The organism is in need of new research efforts at all levels of organization from the molecular to the community.

ISOLATION AND GENOTYPIC CHARACTERIZATION OF TRUEPERELLA (ARCANOBACTERIUM) PYOGENES RECOVERED FROM ACTIVE CRANIAL ABSCESS INFECTIONS OF MALE WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS)

Abstract:  Trueperella (Arcanobacterium) pyogenes is a causative agent of suppurative infections in domestic and wild animals. In some populations of captive or free-ranging white-tailed deer (Odocoileus virginianus), cranial abscess disease is an important source of mortality in adult males. Although the pathogenesis of this disease is poorly understood, T. pyogenes has been isolated from active infections with other opportunistic bacteria. In this study, bacteria associated with cranial abscess infections were identified, the prevalence of T. pyogenes associated with these infections was determined, and the presence of known virulence determinants in T. pyogenes isolates was ascertained. Using routine biochemical techniques seven bacterial species were identified from 65 samples taken from active cranial abscess infections of 65 male white-tailed deer. Trueperella pyogenes was recovered from 46 samples; in 32 samples it was the only bacterium species detected. Staphylococcus aureus was detected in 26 samples. From these samples, the presence of known and putative virulence genes of T. pyogenes—plo, nanH, nanP, cbpA, fimA, fimC, fimE, and fimG—was examined by conventional polymerase chain reaction. All T. pyogenes isolates were positive for the pyolysin genes plo, nanP, and fimA. Furthermore, nanH, fimA, fimC, and fimE were detected in over 70% of isolates. Of the isolates tested, 48% had genotypes containing all virulence genes except cbpA. The suggestive virulence potential of all isolates, coupled with the large number of pure cultures obtained, implies that T. pyogenes is a causative agent of cranial abscess disease.

A new Isospora species of passerines in the family Turdidae from Costa Rica.

abstract:  Seven thrush species (Turdidae) from Costa Rica were examined for intestinal parasites; 21 of the 84 (25%) birds sampled were positive for a new species of Isospora. Oocysts of Isospora zorzali n. sp. have thin, smooth, double, and colorless walls; they measure 19.7 ± 1.5 µm × 18.6 ± 1.4 µm (16–24 µm × 15–21 µm), with an average length–width ratio of 1.1 µm. Sporocysts are ovoid, measure 8.5 ± 1.1 µm × 14.5 ± 1.7 µm (7–11 µm × 11–18 µm) with an average length–width ratio of 1.7 µm. A nipple-like stieda body continuous with the sporocyst wall is present, but no substieda body was observed. A sporocyst residuum consisting of large equal sized granules was observed either clumped together or diffusely. The sporocysts fill the entire oocysts with little to no open space observed. This is the first report of Isospora species from any of the sampled host species and also the first report from any species of thrush in Costa Rica.

Epethelial Presence of Trueperella pyogenes Predicts Site-Level Presence of Cranial Abscess Disease in White-Tailed Deer (Odocoileus virginianus)

Cranial/intracranial abscess disease is an emerging source of significant mortality for male white-tailed deer (Odocoileus virginianus). Most cases of cranial/intracranial abscess disease are associated with infection by the opportunistic pathogen Trueperella pyogenes although the relationship between the prevalence of the bacteria and occurrence of disease is speculative. We examined 5,612 hunter-harvested deer from 29 sites across all physiographic provinces in Georgia for evidence of cranial abscess disease and sampled the forehead, lingual, and nasal surfaces from 692 deer. We used polymerase chain reaction (PCR) to determine presence of T. pyogenes from these samples. We found T. pyogenes prevalence at a site was a predictor for the occurrence of cranial abscess disease. Prevalence of T. pyogenes did not differ between samples from the nose or tongue although prevalence along the forehead was greater for males than females (p = 0.04), particularly at sites with high occurrence of this disease. Socio-sexual behaviors, bacterial prevalence, or physiological characteristics may predispose male deer to intracranial/cranial abscess disease. Determination of factors that affect T. pyogenes prevalence among sites may help explain the occurrence of this disease among populations.

A novel Isospora species (Apicomplexa: Eimeriidae) from warblers (Passeriformes: Parulidae) of Costa Rica.

Abstract:  Five of 16 (31%) rufous-capped warblers (Basileuterus rufifrons) and 2 of 5 (40%) ovenbirds (Seiurus aurocapilla) sampled from Costa Rica were positive for a novel species of Isospora. Oocysts have a thin, smooth, double-layered, colorless wall and measure 22.3 μm ± 1.6 μm × 24.3 μm ± 1.5 μm (19–25 μm × 21–28 μm) with an average length-width (L/W) ratio of 1.0 (1–1.3). Oocyst residuum and micropyle are absent, but 0–4 spherical to cigar-shaped polar granules (1–2.5 μm) are present. Sporocysts are ovoid and measure 11.8 μm ± 0.9 μm × 16 μm ± 1.7 μm (10–14 μm × 12–19 μm) with an average L/W ratio of 1.6 (1.0–1.9). A knob-like Stieda body continuous with the sporocyst wall and a trapezoidal compartmentalized substieda body are present. Each sporocyst contained 4 sporozoites and a diffuse sporocyst residuum consisting of many variable-sized granules, some as large as 2 μm. This is the second description of an Isospora species in New World warblers (Passeriformes: Parulidae) and the first report of Isospora from both the rufous-capped warbler and ovenbird.