Detection of avian influenza viruses from shorebirds: evaluation of surveillance and testing approaches.

Although influenza A viruses have been isolated from numerous shorebird species (Family: Scolopacidae) worldwide, our understanding of natural history of these viruses in this diverse group is incomplete. Gaining this information can be complicated by sampling difficulties related to live capture, the need for large sample sizes related to a potentially low prevalence of infection, and the need to maintain flexibility in diagnostic approaches related to varied capabilities and resources. To provide information relevant to improving sampling and testing of shorebirds for influenza A viruses, we retrospectively evaluated a combined data set from Delaware Bay, USA, collected from 2000 to 2009. Our results indicate that prevalence trends and subtype diversity can be effectively determined by either direct sampling of birds or indirect sampling of feces; however, the extent of detected subtype diversity is a function of the number of viruses recovered during that year. Even in cases where a large number of viruses are identified, an underestimate of true subtype diversity is likely. Influenza A virus isolation from Ruddy Turnstones can be enhanced by testing both cloacal and tracheal samples, and matrix real-time PCR can be used as an effective screening tool. Serologic testing to target species of interest also has application to shorebird surveillance. Overall, all of the sampling and diagnostic approaches have utility as applied to shorebird surveillance, but all are associated with inherent biases that need to be considered when comparing results from independent studies.

Is the occurrence of avian influenza virus in Charadriiformes species and location dependent?

Birds in the order Charadriiformes were sampled at multiple sites in the eastern half of the continental USA, as well as at Argentina, Chile, and Bermuda, during 1999-2005, and tested for avian influenza virus (AIV). Of more than 9,400 birds sampled, AIV virus was isolated from 290 birds. Although Ruddy Turnstones (Arenaria interpres) comprised just 25% of birds sampled, they accounted for 87% of isolates. Only eight AIV isolations were made from birds at four locations outside of the Delaware Bay, USA, region; six of these were from gulls (Laridae). At Delaware Bay, AIV isolations were predominated by hemagglutinin (HA) subtype H10, but subtype diversity varied each year. These results suggest that AIV infection among shorebirds (Scolopacidae) may be localized, species specific, and highly variable in relation to AIV subtype diversity.

White-tailed deer (Odocoileus virginianus) develop spirochetemia following experimental infection with Borrelia lonestari.

Borrelia lonestari is considered a putative agent of southern tick-associated rash illness (STARI) and is known to occur naturally only in lone star ticks (Amblyomma americanum) and white-tailed deer (Odocoileus virginianus). We used a low passage isolate of B. lonestari (LS-1) to inoculate white-tailed deer, C3H mice, Holstein cattle, and beagles. Animals were monitored via examination of Giemsa and acridine orange stained blood smears, polymerase chain reaction (PCR), indirect fluorescent antibody (IFA) test, and/or culture isolation. Spirochetes were visualized in blood smears of both deer on days post-inoculation (DPI) 6, 8, 12 and one deer on DPI 15. Whole blood collected from deer tested PCR positive starting on DPI 4 and remained positive as long as DPI 28. Both deer developed antibody titers of >64, with a maximum IFA titer of 1024. The organism was reisolated from the blood of both deer on DPI 6 and one deer on DPI 12. All isolation attempts from mice, calves, or dogs were negative, although one of seven mice was transiently PCR positive. Mice and dogs developed an IFA titer > or =64, while calves lacked a detectable antibody response. These preliminary experimental infection trials show that white-tailed deer are susceptible to infection with B. lonestari and develop a spirochetemia following needle-inoculation, while C3H mice, calves, and dogs do not. Results suggest that deer may serve as a vertebrate reservoir host. Tick transmission studies are needed to confirm that this organism can be maintained in a natural cycle involving deer and A. americanum.

Mycoplasma gallisepticum in house finches (Carpodacus mexicanus) and other wild birds associated with poultry production facilities.

Since 1994, an epidemic of conjunctivitis caused by Mycoplasma gallisepticum (MG) has spread throughout the eastern population of house finches (Carpodacus mexicanus). The adaptation of MG to a free-flying avian species presents potential problems for the control of mycoplasmosis in commercial poultry. To evaluate risks associated with this emerging problem, a field survey was conducted to assess prevalence of MG infection in house finches and other passerine birds associated with poultry farms. Between November 1997 and March 1999, 1058 birds were captured by mist net or trap at 17 farms and at 10 feeder stations in northeast Georgia. Birds were bled and screened by serum plate agglutination (SPA) for antibodies to MG. Birds with negative or weak positive SPA results were released at capture sites, and those with strong positive SPA reactions were kept for further evaluation. Necropsies were performed on selected house finches and individuals of 11 other passerine species, and samples were collected for MG testing by culture, polymerase chain reaction (PCR), hemagglutination inhibition, and histopathology. Testing revealed 19.1% of 671 birds caught at farms and 11.6% of 387 birds caught at feeder sites were SPA positive for MG. Three house finches captured on farms were positive for MG by culture and PCR, whereas three from feeder sites were positive only by PCR. No MG isolates were made from tufted titmice (Baeolophus bicolor), but 40% were positive by PCR. Individuals from 10 additional species were SPA positive only. Results suggest that MG persists at low levels in house finches in northeast Georgia and that tufted titmice may be nonclinical carriers of MG or a related mycoplasma. Positive SPA reactions in other species may be caused by nonspecific reactions or contact exposure. Current biosecurity recommendations should be sufficient to minimize risks of transmission between wild and domestic birds.

Potential for transmission of the finch strain of Mycoplasma gallisepticum between house finches and chickens.

Although Mycoplasma gallisepticum (MG) is established in house finch (Carpodacus mexicanus) populations in at least 33 states, the potential risk of MG introduction to domestic poultry by infected finches currently is unknown. The objectives of this study were to determine if chickens could be infected with the finch strain of MG via direct, across-wire, and proximity (across-room) contact with naturally infected house finches and to determine if house finches could be infected through direct contact with experimentally infected chickens. Chickens were infected with the finch strain of MG through direct contact with naturally infected house finches, a determined by seroconversion (80%), polymerase chain reaction (PCR) (20%), and culture of MG (30%). Clinical disease was not observed in infected chickens. Isolates from chickens were identified as the original finch strain by arbitrary primed PCR. Transmission required an extended period of direct contact (10 wk) with infected finches, and no evidence of MG infection was detected in chickens exposed to infected finches across wire or across the room. Evidence of contact transmission of MG from infected chickens to house finches was limited to positive serum plate agglutination results, and infection could not be confirmed by PCR or culture. Results suggest that minimal biosecurity measures that restrict direct contact between chickens and house finches should significantly reduce the potential for MG transmission between these species.

Natural Mycoplasma gallisepticum infection in a captive flock of house finches.

Naturally-occurring mycoplasmal conjunctivitis is described among 104 wild-caught, and initially seronegative, house finches (Carpodacus mexicanus) maintained in captivity for 12 wk during November 1995 through January 1996. Finches housed in three pens were monitored for clinical signs, and > or = 10 birds were euthanatized for necropsy and mycoplasma testing every 2 wk. Within 2 to 4 wk following initial detection of lesions, > 50% of the birds in each of three pens developed a debilitating disease characterized by mild to severe ocular swelling, conjunctivitis, and ocular and nasal discharge. Microscopic lesions in affected finches consisted of mild to severe lymphoplasmacytic inflammation with epithelial and lymphoid hyperplasia in conjunctivae, nasal turbinates, and trachea. Mycoplasma gallisepticum infection was confirmed by culture or polymerase chain reaction (PCR) in all birds with conjunctival lesions and in 43% of birds without lesions. An arbitrary primer PCR was used to confirm M. gallisepticum isolates as identical to a field strain previously associated with house finch conjunctivitis. Most birds (89%) with conjunctivitis developed a concurrent antibody response detectable by serum plate agglutination (SPA) within 2 wk of lesion development. Hemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) tests were less sensitive than the SPA test. The clinical severity of this disease and high proportion of affected birds suggests that M. gallisepticum may have a negative impact on free-flying house finch populations.

Mycoplasmas in wild turkeys living in association with domestic fowl.

One hundred and nineteen Merriam's wild turkeys (Meleagris gallopavo merriami) and 31 domestic chickens coexisting on a ranch in west-central Colorado (USA) were surveyed for mycoplasmosis by serologic and cultural methods. Although no clinical signs were apparent in any wild turkeys tested, 51 (43%) had positive rapid plate agglutination (RPA) reactions for M. gallisepticum (MG) and/or M. synoviae (MS); 37% of 56 adults and 48% of 63 subadults were classified as positive reactors to MG and/or MS. No turkeys tested in 1992 (n = 61) and 17 (29%) of 58 turkeys tested in 1993 were RPA-positive for M. meleagridis (MM). Hemagglutination inhibition (HI) test results were negative for MG, MS and MM as were most enzyme-linked immunosorbent assay (ELISA) test reactions (MG = 99%, MS = 93%, MM = 87%). Immunoblotting showed mild to moderate reactivity to MG proteins in 49% of 41 samples tested. Most chickens were strongly positive for MS by RPA (81%), HI (58%) and ELISA (87%); 48% also were positive for MG by RPA but all were MG-negative by HI and ELISA. No pathogenic mycoplasmas were isolated from either group of birds. Mycoplasma gallopavonis was commonly identified from the wild turkeys, and M. gallinaceum was isolated from both the chickens and wild turkeys. In a transmission study conducted in 1994, disease-free domestic turkeys failed to seroconvert when co-housed with wild turkeys from this population that were RPA-positive for MG. Collectively, the results of this study were inconclusive regarding the status of pathogenic mycoplasmas within this wild turkey population.

Hemorrhagic disease in white-tailed deer in Texas: a case for enzootic stability.

Although antibodies to viruses in both the epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) sero-groups have been reported from white-tailed deer (Odocoileus virginianus) in Texas (USA), there are few reports of hemorrhagic disease (HD) in these populations. To understand the extent and diversity of exposure to the North American EHDV and BTV serotypes in these deer populations, we serologically tested 685 white-tailed deer collected from November 1991 through March 1992 throughout their range in Texas. Overall, 574 (84%) of deer had antibodies to EHDV or BTV. Prevalence estimates varied according to ecological region, from 57% in the Gulf Prairies to 100% in the northwest Edwards Plateau. Based on serum neutralization tests, the deer had evidence of previous exposures to multiple EHDV and BTV serotypes, with evidence of exposure to two to five serotypes detected in each ecological region. The apparent lack of HD in relation to this high antibody prevalence cannot be explained, but may be related to enzootic stability in which a near perfect host-virus relationship exists.

Field investigation of Mycoplasma gallisepticum infections in house finches (Carpodacus mexicanus) from Maryland and Georgia.

A field study investigating the occurrence of Mycoplasma gallisepticum (MG) in house finches (Carpodacus mexicanus) was conducted in Maryland and Georgia. Eighty-eight finches were captured and examined grossly and microscopically for MG-related conjunctivitis. Serum samples were obtained for serum plate agglutination (SPA) and hemagglutination inhibition (HI) testing. Swabs from conjunctiva, sinus, and choanal cleft were inoculated into two mycoplasma broth media for culture and polymerase chain reaction (PCR) testing. From Maryland, 12 of 57 birds examined had gross conjunctival lesions. MG was isolated from 9 of the 12 affected birds and from three birds without gross lesions. Fourteen of 22 finches tested by PCR were positive for MG. Sixteen of 38 birds were positive for MG by SPA, and 9 of these had HI titers of 1:40 or 1:80. From Georgia, 3 of 31 finches examined had gross lesions; two of these were both culture and PCR positive for MG. Twelve birds were positive by SPA, and two of these had HI titers of 1:80. Histologic findings in birds with gross conjunctivitis from both locations were characterized by extensive epithelial and lymphoid hyperplasia as well as lymphoplasmacytic inflammation in conjunctival tissues; keratitis was rarely present. The source of MG infection in house finches is unknown, and further research is warranted to determine the prevalence and impact of this newly described disease.

Avian tick paralysis caused by Ixodes brunneus in the southeastern United States.

Between 1988 and 1994, 16 definitive and 26 presumptive cases of tick paralysis were diagnosed in 10 species of birds from five southeastern states in the USA. All birds had engorged adult female Ixodes brunneus ticks on the head region and were partially paralyzed or dead. Cases occurred in the winter and early spring months, and most birds were passerines found in private yards or near feeders. All stages of I. brunneus feed exclusively on birds, and this species previously has been associated with avian tick paralysis. Little is known concerning the life cycle of this ixodid tick and its impact on wild bird populations.

Toxoplasmosis in wild turkeys: a case report and serologic survey.

Toxoplasmosis was diagnosed in a free-ranging wild turkey (Meleagris gallopavo) from West Virginia (USA) in June 1993. Gross findings included emaciation, splenomegaly, multifocal necrotizing hepatitis and splenitis, and crusting dermatitis on the head and neck. Histologically, multifocal necrosis with mononuclear inflammation was present in kidney, liver, spleen, heart, lungs, and pancreas. Toxoplasma gondii was confirmed in sections of liver by avidin-biotin immunohistochemical analysis. Subsequently, a retrospective serosurvey of wild turkeys for T. gondii antibodies was conducted using turkey sera collected between 1984 and 1989. An antibody prevalence of 10% was detected in 130 birds from 21 locations in the southeastern United States. While wild turkeys in the Southeast have T. gondii antibodies, this is only the second natural case of fatal toxoplasmosis reported; it appears that wild turkeys infrequently develop clinical disease when infected with T. gondii.

Experimental infection of Borrelia burgdorferi in white-tailed deer.

Four white-tailed deer (Odocoileus virginianus) were experimentally inoculated with Borrelia burgdorferi to determine serologic response by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Deer had antibodies by ELISA by 2 to 3 wk post-inoculation (PI) and remained positive for 10 wk. Deer demonstrated immunoblotting reactivity between 10 and 14 days PI and consistently showed antibody response to nine B. burgdorferi antigens. Attempts were made to recover the spirochete from blood and tissues; B. burgdorferi was isolated from an ear punch biopsy from one of the inoculated deer.

Isolation and transmission of the Lyme disease spirochete from the southeastern United States.

The isolation of the Lyme disease spirochete (Borrelia burgdorferi) from the southeastern United States is reported. Three isolates, two from cotton mice (Peromyscus gossypinus) and one from the black-legged tick (Ixodes scapularis), were recovered from Sapelo Island, Georgia, in July and September 1991. The spirochetes were characterized by indirect fluorescent antibody assay using a battery of five monoclonal antibodies, by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) of whole cell lysates, and by the polymerase chain reaction (PCR) assay using primers for three DNA target sequences found in B. burgdorferi reference strain B-31. Transmission experiments indicate that the three Georgia isolates can infect experimentally inoculated hamsters and mice. Tick transmission of one of the isolates has been attempted so far; I. scapularis transmitted isolate SI-1 from hamsters to mice, but the lone-star tick, Amblyomma americanum, did not.

Mycoplasma gallopavonis in eastern wild turkeys.

Serum samples and tracheal cultures were collected from eastern wild turkeys (Meleagris gallopavo sylvestris) trapped for relocation in South Carolina (USA) during 1985 to 1990. Sera were tested for Mycoplasma gallisepticum and M. synoviae by the rapid plate agglutination and hemagglutination inhibition tests and were found to be negative. Tracheal cultures were negative for all pathogenic Mycoplasma spp., including M. gallisepticum, M. synoviae, M. meleagridis, and M. iowae. However, M. gallopavonis was isolated from every group of wild turkeys tested in 1986 to 1990. These data suggest that M. gallopavonis, which is generally considered nonpathogenic, may be a common microorganism in eastern wild turkeys.

Mycoplasma synoviae in a released pen-raised wild turkey.

Mycoplasma synoviae (MS) was isolated from the sinus of an adult female "wild-type" turkey found feeding with backyard chickens at a private residence in Randolph County, N.C. Clinical signs included sinusitis, dyspnea, emaciation, diarrhea, and nasal discharge. The bird was seropositive for MS and M. gallisepticum (MG) on the rapid plate agglutination test and had titers of 1:160 for MS and 1:20 for MG on the hemagglutination-inhibition test. Isolations of MS and M. gallopavonis were confirmed by the fluorescent antibody test. This case represents the first and only report of MS in a free-ranging "wild-type" turkey in the eastern United States. Behavioral and other evidence suggests that the bird was a released pen-raised turkey.

An investigation of the persistence of Mycoplasma gallisepticum in an Eastern population of wild turkeys.

Mycoplasma gallisepticum infection had been confirmed by culture and serology among wild turkeys (Meleagris gallopavo) in close association with domestic fowl on Cumberland Island, Georgia (USA) in 1980. In 1988, wild turkeys were surveyed by serologic and cultural methods for evidence of M. gallisepticum. Chickens (Gallus gallus) and guinea fowl (Numida meleagris) from the site where the disease was originally detected also were tested by serologic and cultural methods for M. gallisepticum infections. There was no conclusive evidence that M. gallisepticum was present in wild turkeys or guinea fowl. In contrast, most chickens were strongly seropositive for M. gallisepticum, suggesting that they had been infected, although the organism was not recovered by cultural or bioassay methods. Other species of Mycoplasma isolated were M. gallopavonis from wild turkeys, M. gallinaceum and M. pullorum from chickens, and M. gallinaceum from guinea fowl. It appears that M. gallisepticum has not persisted or spread in the wild turkey population on Cumberland Island, despite continued contact by some wild turkeys with suspected carrier chickens.