Seroprevalence of Paramyxoviruses in Synanthropic and Semi–Free-Range Birds Author(s): Fernando Esperón, Belén Vázquez, Azucena Sánchez, Jovita Fernández-Piñero, María Yuste, Elena Neves, Verónica Nogal, and María Jesús Muñoz Source: Avian Diseases, 58(2):306-308. 2014. Published By: American Association of Avian Pathologists DOI: http://dx.doi.org/10.1637/10689-101113-ResNote.1 URL: http://www.bioone.org/doi/full/10.1637/10689-101113-ResNote.1
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
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AVIAN DISEASES 58:306–308, 2014
Research Note— Seroprevalence of Paramyxoviruses in Synanthropic and Semi–Free-Range Birds Fernando Espero´n,AC Bele´n Va´zquez,A Azucena Sa´nchez,B Jovita Ferna´ndez-Pin˜ero,A Marı´a Yuste,A Elena Neves,A Vero´nica Nogal,A and Marı´a Jesu´s Mun˜ozA B
A Centro de Investigacio´n en Sanidad Animal (CISA-INIA), 28130 Valdeolmos, Spain Department of Avian Pathology, Laboratorio Central de Veterinaria, 28110 Algete, Spain
Received 11 October 2013; Accepted 27 January 2014; Published ahead of print 27 January 2014 SUMMARY. Avian paramyxoviruses (APMVs) are classified into nine different serotypes (APMV 1–9). Virulent strains of APMV-1 are already well characterized as the etiologic agent of Newcastle disease (ND), an important disease in poultry that is potentially capable of infecting all orders of avian species. However, very little is known about the other eight serotypes, the majority of which can cause disease in domestic birds. The role of synanthropic and semi–free-range birds as reservoirs of avian paramyxoviruses is not well understood and the main objective of this work was to evaluate the seroprevalence of APMV 1–9 in these kind of birds. A total of 296 sera, oropharyngeal swabs, and cloacal enemas were collected from semi–free-range birds belonging to four different species: feral pigeons (Columba livia var. domestica), hybrid ducks (Anas sp.), domestic geese (Anser anser domesticus), and white storks (Ciconia ciconia). Antibodies against NDV were found in 56.3% of domestic geese, 42.9% of feral pigeons, and 30.4% of hybrid ducks. Antibodies for other APMVs (-3, -4, -6, -7, -8, -9) were also found. Seven positive individuals were positive to real-time RT-PCR detection, all of them feral pigeons captured in 2006 and 2007. The results obtained reinforce the idea that semi–free-range birds may be good sentinels for the detection of NDV and other avian paramyxoviruses. RESUMEN. Nota de Investigacio´n—Seroprevalencia de paramixovirus en aves sinantro´picas y en aves mantenidas en semilibertad. Los paramixovirus aviares (APMVs) se clasifican en nueve serotipos diferentes (APMV 1–9). Las cepas virulentas del paramixovirus aviar serotipo 1 (APMV-1) ya esta´n bien caracterizadas como el agente etiolo´gico de la enfermedad de Newcastle (ND), una enfermedad importante en la avicultura que es potencialmente capaz de infectar a todos los o´rdenes de las especies aviares. Sin embargo, se sabe muy poco acerca de los otros ocho serotipos, la mayorı´a de los cuales pueden causar enfermedades en aves dome´sticas. El papel como reservorios de paramixovirus aviares de las aves sinantro´picas y en condiciones de semilibertad no se ha comprendido adecuadamente y el objetivo principal de este trabajo fue evaluar la seroprevalencia de los paramyxovirus aviares serotipos 1 al 9 en este tipo de aves. Se recolectaron un total de 296 sueros, hisopos orofarı´ngeos y enemas cloacales de aves en semilibertad pertenecientes a cuatro especies diferentes: palomas dome´sticas (Columba livia var. domestica), patos hı´bridos (Anas sp.), gansos dome´sticos (Anser anser domesticus) y cigu¨en˜as blancas (Ciconia ciconia). Se encontraron anticuerpos contra el virus de Newcastle en el 56.3% de los gansos dome´sticos, en el 42.9% de las palomas y en el 30.4% de los patos hı´bridos. Tambie´n se encontraron anticuerpos para otros paramyxovirus aviares (-3, -4, -6, -7, -8, -9). Siete individuos positivos tambie´n fueron positivos para deteccio´n por transcripcio´n reversa y PCR en tiempo real, todas estas aves fueron palomas capturadas entre los an˜os 2006 y 2007. Los resultados obtenidos refuerzan la idea de que las aves en semilibertad pueden ser buenos centinelas para la deteccio´n del virus de la enfermedad de Newcastle y de otros paramixovirus. Key words: NDV, avian paramyxovirus, hybrid ducks, domestic geese, white storks, feral pigeons Abbreviations: APMV 5 avian paramyxovirus; HI 5 hemagglutination inhibition; HIA 5 hemagglutination inhibition assay; ND 5 Newcastle disease; NDV 5 Newcastle disease virus; NI 5 neuraminidase inhibition; PPMV 5 pigeon paramyxovirus; ROC 5 receiver operating characteristic
Avian paramyxoviruses (APMVs) (genus Avulavirus) can be classified into nine serotypes (APMV 1–9) according to the results of hemagglutination inhibition (HI) and neuraminidase inhibition (NI) assays. The best-known avian paramyxovirus is APMV-1, the virulent strains of which are the etiologic agent of Newcastle disease (ND), an important disease in poultry that has the potential to infect all orders of avian species, both wild and domestic. Despite the use of vaccination as a control method, ND outbreaks still occur in many countries (9). Although APMV-1 is well characterized, very little is known about the other serotypes (11). Pigeon paramyxovirus (PPMV) is a variant of APMV-1 and is considered enzootic in racing pigeons in C Corresponding author. Centro de Investigacio´n en Sanidad Animal (CISA-INIA), Ctra. Algete a El Casar s/n, 28130, Valdeolmos, Madrid, Spain. E-mail: [email protected]
the majority of European Union countries. APMV-2 and APMV-3 have been shown to infect and cause disease in poultry and have a serious economic impact (4,6). APMV-4 has been isolated from chickens, ducks, and geese (21). APMV-5 was initially isolated in budgerigars (Melopsittacus undulatus) (16) but is often not easily available from reference laboratories. APMV-6 and APMV-7 are associated with clinical disease in turkeys (2) while APMV-8 and APMV-9 are apparently restricted to infections in ducks and geese (5,7). Between 2000 and 2009 in Europe, highly significant numbers of virulent Newcastle disease virus (NDV) strains have been isolated from chickens and wild birds (3). Wild birds mainly act as asymptomatic carriers and reservoirs that are potentially capable of spreading the disease to other susceptible bird populations (10). However, very few studies have reported the seroprevalence of the other paramyxoviruses in wild birds, especially in synanthropic birds. These species, which live in close contact with humans, could act as
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Paramyxoviruses in synanthropic and semi–free-ranging birds
Table 1.
Seroprevalence (%) for the different avian paramyxoviruses from the studied species.A
Species
ELISA
1B
PPMV
2B
3B
4B
6B
7B
8B
9B
FP HD DG WS Overall
53.2 31.7 76.7 29.4 49.7
42.9 30.4 56.3 0.0 38.8
22.2 n.d. n.d. n.d. n.d.
0.0 0.0 0.0 0.0 0.0
0.0 0.0 3.1 0.0 0.3
0.0 1.4 6.3 0.0 1.0
0.0 5.8 6.3 0.0 1.9
12.2 2.9 0.0 0.0 8.1
0.0 7.2 0.0 10.5 2.3
0.0 0.0 0.0 10.5 0.6
A
FP 5 feral pigeon; HD 5 hybrid duck; n.d. 5 not done; DG 5 domestic goose; WS 5 white stork. Numbers 1–9 refer to the number of the avian paramyxovirus (APMV).
B
‘‘bridge species’’ or as sentinels for disease emergence. In this work 296 sera, oropharyngeal swabs, and cloacal enemas from four bird species in regular contact with humans were analyzed to investigate the presence of APMV in their populations. MATERIALS AND METHODS During 2006–2011, 296 sera, oropharyngeal swabs, and cloacal enemas—obtained as previously described (22)—were collected from adult birds belonging to four different species: feral pigeons (Columba livia; 186 samples), hybrid ducks (Anas sp., probably a product of mallards and domestic ducks; 63 samples), domestic geese (Anser anser domesticus; 30 samples), and white storks (Ciconia ciconia; 17 samples). In all, 205 samples were collected from birds living in urban areas (Madrid, Spain, 40.41669–3.700346; 128 feral pigeons, 60 hybrid ducks, and 17 white storks), whilst 91 samples were taken from birds present in rural environments (58 feral pigeons, 30 domestic geese, and 3 hybrid ducks). The domestic geese and the three hybrid ducks live in an area with a high density of poultry farms (Almoguera, Spain, 40.29783–2.98217). The samples were analyzed using a hemagglutination inhibition assay (HIA) for avian paramyxoviruses (APMV-1 to APMV-9) (18). AMPV-5 was not tested for, given that it lacks the capacity to agglutinate red blood cells (16). Pigeon samples were also analyzed using HIA for PPMV-1. Control samples were kindly provided by the Istituto Zooprofilattico Sperimentale delle Venezie (Padova, Italy). The HIA was carried out using a total volume of 25 ml of 4 HA units of the following APMV antigens: APMV-1/ULSTER 2C, PPMV-1/Pigeon/It/4400/00, APMV-2/CK/ YUCAIPA/56, APMV-3/Ty/1087/82, APMV-4/P/Duck/Hong KongD3/ 75, APMV-6/Duck/Hong Kong/199/77, APMV-7/Dove/UK/4/75, APMV-8/Goose/1053/76, and APMV-9/Pintail/Italy/493/04. All samples with titers equal to or greater than 1/16 were considered positive (18). In order to avoid overinterpretation of the results we decided, when a sample is positive simultaneously against different AMPV serotypes, to consider only the infection by AMPV with the highest titer (co-infection is only considered if the highest titer is shared by two or more viruses). In addition, a commercial indirect competition ELISA test (SVANOVIRH NDV-Ab, SVANOVA, Uppsala, Sweden) was used following the manufacturer’s instructions to evaluate wild bird serum samples. Sensitivity, specificity, and the area under the receiver operating characteristic (ROC) curve were calculated. In order to determine the presence of viral RNA, a real-time RT-PCR test (23) was applied in oropharyngeal swabs and cloacal enemas. RNA was extracted by a pressure filtration method (QuickGeneH DNA tissue kit S, FujiFilm Lifescience, Tokyo, Japan), adding an RNA carrier in the step of lysate preparation. Positive samples were inoculated in embryonated chicken eggs. These two isolates were further characterized following a molecular pathotyping procedure which is based on the amino acid sequencing of the fusion protein cleavage site (17).
RESULTS AND DISCUSSION
Prevalence results are summarized in Table 1. Antibodies were found for all the APMV serotypes except APMV-2. However, a
previous study about seroprevalences of APMVs in Spain revealed the presence of antibodies against APMV-2 in domestic and wild birds (14). This discrepancy could be derived from differences about the interpretation of the results. In our work, due to the fact of crossreactions among APMV, we only considered the highest titer as positive whereas co-infection is only considered when the same titer is observed simultaneously in two or more APMVs. In fact, we have several samples positive against APMV-2 but with higher titers against APMV-1 and, thus, we only considered those positive against APMV-1 (data not shown). The most seroprevalent APMV was APMV-1, with an overall score of 38.8%, followed by PPMV-1 (only analyzed in pigeons) with a score of 22.2%. The species with the greatest prevalence against APMV-1 was the domestic goose (56.3%) followed by feral pigeons (42.9%) and hybrid ducks (30.4%). No white stork had antibodies against APMV-1. The results for the first three species are relatively high. In the case of the pigeons, the prevalence is similar to that obtained from pigeons in Taiwan (43.3%) (20), while for geese and ducks our results were similar to those described in ducks from New Zealand (46.4% of samples with titers equal to or greater than 1/16) (19) and much higher than those published previously from Spain (0% and 3.4% for geese and ducks, respectively) (14,15). All seropositive species in this study are considered as potential reservoirs of APMV-1. Neither pigeons nor ducks are highly susceptible to APMV-1 but can still be infected and spread the virus to other moresusceptible species (8). On the other hand, geese are considered to be susceptible to infection but their clinical response to the disease varies (8). The geese analyzed in this study were from an area with a high density of poultry farms and so there is a potential risk of APMV-1 introduction in poultry. Seven feral pigeons were positive to RNA detection, all from urban environments and captured in 2007. All of these individuals showed seropositivity against AMPV1. Only two samples grew in embryonated chicken eggs and, therefore, molecular characterization of the NDV was attempted in these samples. The retrieved sequence of the amino acid positions no. 112–117 was RRQKR*F in both isolates, being identified as virulent NDV. These results are in consonance with those previously described in Europe and specifically in Spain (3). No viral RNA was detected in the rest of species. Most ND outbreaks in pigeons are due to a pigeon-specific virus known as PPMV-1 that is closely related to NDV (8). In all, 22.2% of the pigeons were seropositive against PPMV-1; however, none showed clinical signs of paramyxovirus infection. Clinical signs in pigeons vary mainly with age because young birds are more susceptible (12). In the present study, only adult pigeons were sampled. In adults, mortality is minimal and morbidity is approximately 10%. APMV-7 was present in feral pigeons (12.2%) and hybrid ducks (2.9%). Although we still lack epidemiologic information about this serotype, it seems that it is naturally present in pigeons, turkeys (Meleagridis spp.), and chickens (Gallus gallus) (1). APMV-7 was also found in mallards from New Zealand, in which 2.8% of the 315
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F. Espero´n et al.
sera were positive for the antibody to APMV-7 (titers equal to or greater than 1/16) (19). Domestic geese also had antibodies against APMV-3 (3.1%), APMV-4, and APMV-6 (both 6.3%); hybrid ducks were positive to APMV-4 (1.4%) and APMV-6 (5.8%). APMV-3 can affect a number of different bird species including turkeys, poultry chicks, Psittaciformes and Passeriformes. In terms of its pathogenicity, APMV-3 is probably second only in importance to NDV (13). Antibodies against APMV-8 and APMV-9 were detected in white storks (10.5%) and against APMV-8 in hybrid ducks (7.2%). APMV-8 and APMV-9 have only been isolated very rarely (1). On the other hand, antibodies against APMV-8 have been detected previously in a number of duck species (overall 11.6% for Anas spp.) from Spain (15) and in mallards from New Zealand (4.4% of titers equal to or greater than 1/16) (19). This is the first report of APMV8 and APMV-9 antibodies in white storks. Prevalence results for APMV-1 antibodies obtained using ELISA were greater than those obtained using HIA in all species. Although the specificity of the test was relatively low (76.2%), its sensibility was high (90.4%) and the area under the ROC curve was moderately high (0.833). These results indicate that the ELISA test can be used for screening APMV-1 antibodies but that further confirmation of positives still needs to be made for HIA tests. However, in the case of the white storks, 29.4% of the samples tested positive with the ELISA in contrast to the 0.0% of positive samples using the APMV-1 HIA. This thus renders the ELISA an unsuitable test for this species, and it is important to highlight that this assay should be tested for paramyxovirus species other than those studied in this work. In conclusion, the results obtained in this study reveal that avian paramyxoviruses—especially APMV-1—are circulating in the tested bird populations, including virulent strains of NDV in the case of feral pigeons, a finding that emphasizes the interest of synanthropic and semi–free-range birds as sentinels for NDV and other avian paramyxoviruses. REFERENCES 1. Alexander, D. J. Paramyxoviruses infection. In: Virus infection of birds. J. B. McFerran, and M. S. McNully, eds. Elsevier Science, Amsterdam. pp. 321–340. 1993. 2. Alexander, D. J. Newcastle disease and other avian paramyxoviruses. Rev. Sci. Tech. 19:443–462. 2000. 3. Alexander, D. J. Newcastle disease in the European Union 2000 to 2009. Avian Pathol. 40:547–558. 2011. 4. Alexander, D. J., and D. A. Senne. Newcastle disease, other avian paramyxoviruses, and pneumovirus infections. In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Blackwell Publishing, Ames, IA. pp. 75–116. 2008. 5. Alexander, D. J., M. Pattison, and I. Macpherson. Avian paramyxoviruses of PMV-3 serotype in British turkeys. Avian Pathol. 12:469–482. 1983. 6. Bankowski, R. A., J. Almquist, and J. Dombrucki. Effect of paramyxovirus yucaipa on fertility, hatchability, and poult yield of turkeys. Avian Dis. 25:517–520. 1981. 7. Capua, I., R. De Nardi, M. S. Beato, C. Terregino, M. Scremin, and V. Guberti. Isolation of an avian paramyxovirus type 9 from migratory waterfowl in Italy. Vet. Rec. 155:156. 2004. 8. Cattoli, G., L. Susta, C. Terregino, and C. Brown. Newcastle disease: a review of field recognition and current methods of laboratory detection. J. Vet. Diagn. Invest. 23:637–656. 2011.
9. Dortmans, J. C., B. P. Peeters, and G. Koch. Newcastle disease virus outbreaks: Vaccine mismatch or inadequate application? Vet. Microbiol. 160:17–22. 2012. 10. Dundon, W. G., A. Heidari, A. Fusaro, I. Monne, M. S. Beato, G. Cattoli, G. Koch, E. Starick, I. H. Brown, E. W. Aldous, F. X. Briand, G. Le Gall-Recule´, V. Jestin, P. H. Jørgensen, M. Berg, S. Zohari, G. Metreveli, M. Munir, K. Sta˚hl, E. Albina, A. Hammoumi, P. Gil, R. S. de Almeida, K. S´mietanka, K. Doman´ska-Blicharz, Z. Minta, S. Van Borm, T. van den Berg, A. M. Martin, I. Barbieri, and I. Capua. Genetic data from avian influenza and avian paramyxoviruses generated by the European network of excellence (EPIZONE) between 2006 and 2011—review and recommendations for surveillance. Vet. Microbiol. 27:209–221. 2012. 11. Kim, S. H., S. Xiao, H. Shive, P. L. Collins, and S. K. Samal. Replication, neurotropism, and pathogenicity of avian paramyxovirus serotypes 1–9 in chickens and ducks. PLoS One 7:e34927. 2012. 12. King, D. J. Avian paramyxovirus type 1 from pigeons: isolate characterization and pathogenicity after chicken or embryo passage of selected isolates. Avian Dis. 40:707–714. 1996. 13. Kumar, S., B. Nayak, P. L. Collins, and S. K. Samal. Complete genome sequence of avian paramyxovirus type 3 reveals an unusually long trailer region. Virus Res. 137:189–197. 2008. 14. Maldonado, A., A. Arenas, M. C. Tarradas, J. Carranza, I. Luque, A. Miranda, and A. Perea. Prevalence of antibodies to avian paramyxoviruses 1, 2 and 3 in wild and domestic birds in southern Spain. Avian Pathol. 23:145–152. 1994. 15. Maldonado, A., A. Arenas, M. C. Tarradas, I. Luque, R. Astorga, A. Perea, and A. Miranda. Serological survey for avian paramyxoviruses from wildfowl in aquatic habitats in Andalusia. J. Wildl. Dis. 31:66–69. 1995. 16. Nerome, K., M. Nakayama, M. Ishida, and H. Fukumi. Isolation of a new avian paramyxovirus from budgerigar (Melopsittacus undulatus). J. Gen. Virol. 38:293–301. 1978. 17. Oberdorfer, A., and O. Werner. Newcastle disease virus: detection and characterization by PCR of recent German isolates differing in pathogenicity. Avian Pathol. 27:237–243. 1998. 18. [O.I.E.] World Organisation for Animal Health. Manual of diagnostic tests and vaccines for terrestrial animals. 2012 [modified May 2012]. Available from: http://www.oie.int/fileadmin/Home/eng/Health_ standards/tahm/2.03.14_NEWCASTLE_DIS.pdf 19. Stanislawek, W. L., C. R. Wilks, J. Meers, G. W. Horner, D. J. Alexander, R. J. Manvell, J. A. Kattenbelt, and A. R. Gould. Avian paramyxoviruses and influenza viruses isolated from mallard ducks (Anas platyrhynchos) in New Zealand. Arch. Virol. 147:1287–1302. 2002. 20. Tsai, H. J., and C. Y. Lee. Serological survey of racing pigeons for selected pathogens in Taiwan. Acta Vet. Hung. 54:179–189. 2006. 21. Tumova, B., J. H. Robinson, and B. C. Easterday. A hitherto unreported paramyxovirus of turkeys. Res. Vet. Sci. 27:135–140. 1979. 22. Va´zquez, B., F. Espero´n, E. Neves, J. Lo´pez, C. Ballesteros, and M. J. Mun˜oz. Screening for several potential pathogens in feral pigeons (Columba livia) in Madrid. Acta Vet. Scand. 52:45. 2010. 23. Wise, M. G., D. L. Suarez, B. S. Seal, J. C. Pedersen, D. A. Senne, D. J. King, D. R. Kapczynski, and E. Spackman. Development of a real-time reverse-transcription PCR for detection of Newcastle disease virus RNA in clinical samples. J. Clin. Microbiol. 42:329–338. 2004.
ACKNOWLEDGMENTS This work was co-financed by INIA-MAGRAMA AEG-11-053, FAU 2008-00001-C02-01, and CAM S2009/AGR-1489. We would like to thank the staff of the Animal Facility and the Department of Reproduction (INIA) for their assistance in the collection and preservation of blood samples from chicken donors, and Mike Lockwood for the English revision of the manuscript.
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
AVIAN DISEASES 58:306–308, 2014
Research Note— Seroprevalence of Paramyxoviruses in Synanthropic and Semi–Free-Range Birds Fernando Espero´n,AC Bele´n Va´zquez,A Azucena Sa´nchez,B Jovita Ferna´ndez-Pin˜ero,A Marı´a Yuste,A Elena Neves,A Vero´nica Nogal,A and Marı´a Jesu´s Mun˜ozA B
A Centro de Investigacio´n en Sanidad Animal (CISA-INIA), 28130 Valdeolmos, Spain Department of Avian Pathology, Laboratorio Central de Veterinaria, 28110 Algete, Spain
Received 11 October 2013; Accepted 27 January 2014; Published ahead of print 27 January 2014 SUMMARY. Avian paramyxoviruses (APMVs) are classified into nine different serotypes (APMV 1–9). Virulent strains of APMV-1 are already well characterized as the etiologic agent of Newcastle disease (ND), an important disease in poultry that is potentially capable of infecting all orders of avian species. However, very little is known about the other eight serotypes, the majority of which can cause disease in domestic birds. The role of synanthropic and semi–free-range birds as reservoirs of avian paramyxoviruses is not well understood and the main objective of this work was to evaluate the seroprevalence of APMV 1–9 in these kind of birds. A total of 296 sera, oropharyngeal swabs, and cloacal enemas were collected from semi–free-range birds belonging to four different species: feral pigeons (Columba livia var. domestica), hybrid ducks (Anas sp.), domestic geese (Anser anser domesticus), and white storks (Ciconia ciconia). Antibodies against NDV were found in 56.3% of domestic geese, 42.9% of feral pigeons, and 30.4% of hybrid ducks. Antibodies for other APMVs (-3, -4, -6, -7, -8, -9) were also found. Seven positive individuals were positive to real-time RT-PCR detection, all of them feral pigeons captured in 2006 and 2007. The results obtained reinforce the idea that semi–free-range birds may be good sentinels for the detection of NDV and other avian paramyxoviruses. RESUMEN. Nota de Investigacio´n—Seroprevalencia de paramixovirus en aves sinantro´picas y en aves mantenidas en semilibertad. Los paramixovirus aviares (APMVs) se clasifican en nueve serotipos diferentes (APMV 1–9). Las cepas virulentas del paramixovirus aviar serotipo 1 (APMV-1) ya esta´n bien caracterizadas como el agente etiolo´gico de la enfermedad de Newcastle (ND), una enfermedad importante en la avicultura que es potencialmente capaz de infectar a todos los o´rdenes de las especies aviares. Sin embargo, se sabe muy poco acerca de los otros ocho serotipos, la mayorı´a de los cuales pueden causar enfermedades en aves dome´sticas. El papel como reservorios de paramixovirus aviares de las aves sinantro´picas y en condiciones de semilibertad no se ha comprendido adecuadamente y el objetivo principal de este trabajo fue evaluar la seroprevalencia de los paramyxovirus aviares serotipos 1 al 9 en este tipo de aves. Se recolectaron un total de 296 sueros, hisopos orofarı´ngeos y enemas cloacales de aves en semilibertad pertenecientes a cuatro especies diferentes: palomas dome´sticas (Columba livia var. domestica), patos hı´bridos (Anas sp.), gansos dome´sticos (Anser anser domesticus) y cigu¨en˜as blancas (Ciconia ciconia). Se encontraron anticuerpos contra el virus de Newcastle en el 56.3% de los gansos dome´sticos, en el 42.9% de las palomas y en el 30.4% de los patos hı´bridos. Tambie´n se encontraron anticuerpos para otros paramyxovirus aviares (-3, -4, -6, -7, -8, -9). Siete individuos positivos tambie´n fueron positivos para deteccio´n por transcripcio´n reversa y PCR en tiempo real, todas estas aves fueron palomas capturadas entre los an˜os 2006 y 2007. Los resultados obtenidos refuerzan la idea de que las aves en semilibertad pueden ser buenos centinelas para la deteccio´n del virus de la enfermedad de Newcastle y de otros paramixovirus. Key words: NDV, avian paramyxovirus, hybrid ducks, domestic geese, white storks, feral pigeons Abbreviations: APMV 5 avian paramyxovirus; HI 5 hemagglutination inhibition; HIA 5 hemagglutination inhibition assay; ND 5 Newcastle disease; NDV 5 Newcastle disease virus; NI 5 neuraminidase inhibition; PPMV 5 pigeon paramyxovirus; ROC 5 receiver operating characteristic
Avian paramyxoviruses (APMVs) (genus Avulavirus) can be classified into nine serotypes (APMV 1–9) according to the results of hemagglutination inhibition (HI) and neuraminidase inhibition (NI) assays. The best-known avian paramyxovirus is APMV-1, the virulent strains of which are the etiologic agent of Newcastle disease (ND), an important disease in poultry that has the potential to infect all orders of avian species, both wild and domestic. Despite the use of vaccination as a control method, ND outbreaks still occur in many countries (9). Although APMV-1 is well characterized, very little is known about the other serotypes (11). Pigeon paramyxovirus (PPMV) is a variant of APMV-1 and is considered enzootic in racing pigeons in C Corresponding author. Centro de Investigacio´n en Sanidad Animal (CISA-INIA), Ctra. Algete a El Casar s/n, 28130, Valdeolmos, Madrid, Spain. E-mail: [email protected]
the majority of European Union countries. APMV-2 and APMV-3 have been shown to infect and cause disease in poultry and have a serious economic impact (4,6). APMV-4 has been isolated from chickens, ducks, and geese (21). APMV-5 was initially isolated in budgerigars (Melopsittacus undulatus) (16) but is often not easily available from reference laboratories. APMV-6 and APMV-7 are associated with clinical disease in turkeys (2) while APMV-8 and APMV-9 are apparently restricted to infections in ducks and geese (5,7). Between 2000 and 2009 in Europe, highly significant numbers of virulent Newcastle disease virus (NDV) strains have been isolated from chickens and wild birds (3). Wild birds mainly act as asymptomatic carriers and reservoirs that are potentially capable of spreading the disease to other susceptible bird populations (10). However, very few studies have reported the seroprevalence of the other paramyxoviruses in wild birds, especially in synanthropic birds. These species, which live in close contact with humans, could act as
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Table 1.
Seroprevalence (%) for the different avian paramyxoviruses from the studied species.A
Species
ELISA
1B
PPMV
2B
3B
4B
6B
7B
8B
9B
FP HD DG WS Overall
53.2 31.7 76.7 29.4 49.7
42.9 30.4 56.3 0.0 38.8
22.2 n.d. n.d. n.d. n.d.
0.0 0.0 0.0 0.0 0.0
0.0 0.0 3.1 0.0 0.3
0.0 1.4 6.3 0.0 1.0
0.0 5.8 6.3 0.0 1.9
12.2 2.9 0.0 0.0 8.1
0.0 7.2 0.0 10.5 2.3
0.0 0.0 0.0 10.5 0.6
A
FP 5 feral pigeon; HD 5 hybrid duck; n.d. 5 not done; DG 5 domestic goose; WS 5 white stork. Numbers 1–9 refer to the number of the avian paramyxovirus (APMV).
B
‘‘bridge species’’ or as sentinels for disease emergence. In this work 296 sera, oropharyngeal swabs, and cloacal enemas from four bird species in regular contact with humans were analyzed to investigate the presence of APMV in their populations. MATERIALS AND METHODS During 2006–2011, 296 sera, oropharyngeal swabs, and cloacal enemas—obtained as previously described (22)—were collected from adult birds belonging to four different species: feral pigeons (Columba livia; 186 samples), hybrid ducks (Anas sp., probably a product of mallards and domestic ducks; 63 samples), domestic geese (Anser anser domesticus; 30 samples), and white storks (Ciconia ciconia; 17 samples). In all, 205 samples were collected from birds living in urban areas (Madrid, Spain, 40.41669–3.700346; 128 feral pigeons, 60 hybrid ducks, and 17 white storks), whilst 91 samples were taken from birds present in rural environments (58 feral pigeons, 30 domestic geese, and 3 hybrid ducks). The domestic geese and the three hybrid ducks live in an area with a high density of poultry farms (Almoguera, Spain, 40.29783–2.98217). The samples were analyzed using a hemagglutination inhibition assay (HIA) for avian paramyxoviruses (APMV-1 to APMV-9) (18). AMPV-5 was not tested for, given that it lacks the capacity to agglutinate red blood cells (16). Pigeon samples were also analyzed using HIA for PPMV-1. Control samples were kindly provided by the Istituto Zooprofilattico Sperimentale delle Venezie (Padova, Italy). The HIA was carried out using a total volume of 25 ml of 4 HA units of the following APMV antigens: APMV-1/ULSTER 2C, PPMV-1/Pigeon/It/4400/00, APMV-2/CK/ YUCAIPA/56, APMV-3/Ty/1087/82, APMV-4/P/Duck/Hong KongD3/ 75, APMV-6/Duck/Hong Kong/199/77, APMV-7/Dove/UK/4/75, APMV-8/Goose/1053/76, and APMV-9/Pintail/Italy/493/04. All samples with titers equal to or greater than 1/16 were considered positive (18). In order to avoid overinterpretation of the results we decided, when a sample is positive simultaneously against different AMPV serotypes, to consider only the infection by AMPV with the highest titer (co-infection is only considered if the highest titer is shared by two or more viruses). In addition, a commercial indirect competition ELISA test (SVANOVIRH NDV-Ab, SVANOVA, Uppsala, Sweden) was used following the manufacturer’s instructions to evaluate wild bird serum samples. Sensitivity, specificity, and the area under the receiver operating characteristic (ROC) curve were calculated. In order to determine the presence of viral RNA, a real-time RT-PCR test (23) was applied in oropharyngeal swabs and cloacal enemas. RNA was extracted by a pressure filtration method (QuickGeneH DNA tissue kit S, FujiFilm Lifescience, Tokyo, Japan), adding an RNA carrier in the step of lysate preparation. Positive samples were inoculated in embryonated chicken eggs. These two isolates were further characterized following a molecular pathotyping procedure which is based on the amino acid sequencing of the fusion protein cleavage site (17).
RESULTS AND DISCUSSION
Prevalence results are summarized in Table 1. Antibodies were found for all the APMV serotypes except APMV-2. However, a
previous study about seroprevalences of APMVs in Spain revealed the presence of antibodies against APMV-2 in domestic and wild birds (14). This discrepancy could be derived from differences about the interpretation of the results. In our work, due to the fact of crossreactions among APMV, we only considered the highest titer as positive whereas co-infection is only considered when the same titer is observed simultaneously in two or more APMVs. In fact, we have several samples positive against APMV-2 but with higher titers against APMV-1 and, thus, we only considered those positive against APMV-1 (data not shown). The most seroprevalent APMV was APMV-1, with an overall score of 38.8%, followed by PPMV-1 (only analyzed in pigeons) with a score of 22.2%. The species with the greatest prevalence against APMV-1 was the domestic goose (56.3%) followed by feral pigeons (42.9%) and hybrid ducks (30.4%). No white stork had antibodies against APMV-1. The results for the first three species are relatively high. In the case of the pigeons, the prevalence is similar to that obtained from pigeons in Taiwan (43.3%) (20), while for geese and ducks our results were similar to those described in ducks from New Zealand (46.4% of samples with titers equal to or greater than 1/16) (19) and much higher than those published previously from Spain (0% and 3.4% for geese and ducks, respectively) (14,15). All seropositive species in this study are considered as potential reservoirs of APMV-1. Neither pigeons nor ducks are highly susceptible to APMV-1 but can still be infected and spread the virus to other moresusceptible species (8). On the other hand, geese are considered to be susceptible to infection but their clinical response to the disease varies (8). The geese analyzed in this study were from an area with a high density of poultry farms and so there is a potential risk of APMV-1 introduction in poultry. Seven feral pigeons were positive to RNA detection, all from urban environments and captured in 2007. All of these individuals showed seropositivity against AMPV1. Only two samples grew in embryonated chicken eggs and, therefore, molecular characterization of the NDV was attempted in these samples. The retrieved sequence of the amino acid positions no. 112–117 was RRQKR*F in both isolates, being identified as virulent NDV. These results are in consonance with those previously described in Europe and specifically in Spain (3). No viral RNA was detected in the rest of species. Most ND outbreaks in pigeons are due to a pigeon-specific virus known as PPMV-1 that is closely related to NDV (8). In all, 22.2% of the pigeons were seropositive against PPMV-1; however, none showed clinical signs of paramyxovirus infection. Clinical signs in pigeons vary mainly with age because young birds are more susceptible (12). In the present study, only adult pigeons were sampled. In adults, mortality is minimal and morbidity is approximately 10%. APMV-7 was present in feral pigeons (12.2%) and hybrid ducks (2.9%). Although we still lack epidemiologic information about this serotype, it seems that it is naturally present in pigeons, turkeys (Meleagridis spp.), and chickens (Gallus gallus) (1). APMV-7 was also found in mallards from New Zealand, in which 2.8% of the 315
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sera were positive for the antibody to APMV-7 (titers equal to or greater than 1/16) (19). Domestic geese also had antibodies against APMV-3 (3.1%), APMV-4, and APMV-6 (both 6.3%); hybrid ducks were positive to APMV-4 (1.4%) and APMV-6 (5.8%). APMV-3 can affect a number of different bird species including turkeys, poultry chicks, Psittaciformes and Passeriformes. In terms of its pathogenicity, APMV-3 is probably second only in importance to NDV (13). Antibodies against APMV-8 and APMV-9 were detected in white storks (10.5%) and against APMV-8 in hybrid ducks (7.2%). APMV-8 and APMV-9 have only been isolated very rarely (1). On the other hand, antibodies against APMV-8 have been detected previously in a number of duck species (overall 11.6% for Anas spp.) from Spain (15) and in mallards from New Zealand (4.4% of titers equal to or greater than 1/16) (19). This is the first report of APMV8 and APMV-9 antibodies in white storks. Prevalence results for APMV-1 antibodies obtained using ELISA were greater than those obtained using HIA in all species. Although the specificity of the test was relatively low (76.2%), its sensibility was high (90.4%) and the area under the ROC curve was moderately high (0.833). These results indicate that the ELISA test can be used for screening APMV-1 antibodies but that further confirmation of positives still needs to be made for HIA tests. However, in the case of the white storks, 29.4% of the samples tested positive with the ELISA in contrast to the 0.0% of positive samples using the APMV-1 HIA. This thus renders the ELISA an unsuitable test for this species, and it is important to highlight that this assay should be tested for paramyxovirus species other than those studied in this work. In conclusion, the results obtained in this study reveal that avian paramyxoviruses—especially APMV-1—are circulating in the tested bird populations, including virulent strains of NDV in the case of feral pigeons, a finding that emphasizes the interest of synanthropic and semi–free-range birds as sentinels for NDV and other avian paramyxoviruses. REFERENCES 1. Alexander, D. J. Paramyxoviruses infection. In: Virus infection of birds. J. B. McFerran, and M. S. McNully, eds. Elsevier Science, Amsterdam. pp. 321–340. 1993. 2. Alexander, D. J. Newcastle disease and other avian paramyxoviruses. Rev. Sci. Tech. 19:443–462. 2000. 3. Alexander, D. J. Newcastle disease in the European Union 2000 to 2009. Avian Pathol. 40:547–558. 2011. 4. Alexander, D. J., and D. A. Senne. Newcastle disease, other avian paramyxoviruses, and pneumovirus infections. In: Diseases of poultry, 12th ed. Y. M. Saif, A. M. Fadly, J. R. Glisson, L. R. McDougald, L. K. Nolan, and D. E. Swayne, eds. Blackwell Publishing, Ames, IA. pp. 75–116. 2008. 5. Alexander, D. J., M. Pattison, and I. Macpherson. Avian paramyxoviruses of PMV-3 serotype in British turkeys. Avian Pathol. 12:469–482. 1983. 6. Bankowski, R. A., J. Almquist, and J. Dombrucki. Effect of paramyxovirus yucaipa on fertility, hatchability, and poult yield of turkeys. Avian Dis. 25:517–520. 1981. 7. Capua, I., R. De Nardi, M. S. Beato, C. Terregino, M. Scremin, and V. Guberti. Isolation of an avian paramyxovirus type 9 from migratory waterfowl in Italy. Vet. Rec. 155:156. 2004. 8. Cattoli, G., L. Susta, C. Terregino, and C. Brown. Newcastle disease: a review of field recognition and current methods of laboratory detection. J. Vet. Diagn. Invest. 23:637–656. 2011.
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ACKNOWLEDGMENTS This work was co-financed by INIA-MAGRAMA AEG-11-053, FAU 2008-00001-C02-01, and CAM S2009/AGR-1489. We would like to thank the staff of the Animal Facility and the Department of Reproduction (INIA) for their assistance in the collection and preservation of blood samples from chicken donors, and Mike Lockwood for the English revision of the manuscript.
