HEALTH STATUS OF FREE-LIVING PIGEONS. (Columba livia domestica) IN THE CITY OF LJUBLJANA

HEALTH STATUS OF FREE-LIVING PIGEONS (Columba livia domestica) IN THE CITY OF LJUBLJANA Alenka Dovč 1, Olga Zorman-Rojs, Aleksandra Vergles Rataj, Vojka Bole-Hribovšek, U. Krapež, M. Dobeic University of Ljubljana, Veterinary faculty, Gerbičeva 60, 1000 Ljubljana, Slovenia ABSTRACT In the year 2000 an epidemiological research on the health status of free-living pigeons in the city of Ljubljana, Slovenia was undertaken. With regard to the most common bacterial, viral, and parasitic diseases, a total of 139 pigeons were captured. Serum samples, oropharyngeal and cloacal swabs as well as samples of faeces and feather were taken from captured birds. Antibodies to paramyxovirus type 1 were found in 84.2% of the sera examined and 23.7% of birds were serologically positive to Chlamydophila psittaci. Antibodies to avian influenza virus were not detected. Salmonella spp. was isolated from 5.7% of the cloacal swabs. Trichomonas gallinae was clinically suspected and then microscopically confirmed using oropharyngeal swabs in 7.9% of examined birds. Eimeria spp. was identified in 71.9%, Capillaria sp. in 26.6% and Ascaridia columbae in 4.3% of faecal samples examined. Among ectoparasites Columbicola columbae and Campanulotes bidentatus compar were found. KEY WORDS Pigeons, health, bird diseases, epidemiology, serology, parasites, Ljubljana, Slovenia 1 Institute for Health care of Poultry, Veterinary faculty, Cesta v Mestni log 47, 1000 Ljubljana, Slovenia Alenka.Dovc@vf.uni-lj.si 00386 1 4779 339

INTRODUCTION The population of free-living pigeons has increased in large cities worldwide. In Ljubljana, a regulation programme to control the pigeon population was started three years ago. The estimated number of feral pigeons in Ljubljana is about 5000. It is important to determine the potential diseases that may be carried by this avian species, because they could be a source of infection for other animals and even to the human population. Although many birds do not show any clinical sings of disease, they may be a potential carriers of many bacterial, viral and parasitic diseases. The role of pigeons in the transmission of diseases to humans and to domestic animals, especially in connection with intensive poultry production, has been well documented. The presence of Salmonella spp., Campylobacter jejuni and Chlamydophila psittaci in birds living close to humans, as well as a potential interspecies transmission to animals (Salinas et al., 1993; Casanovas et al., 1995; Toro et al., 1999) was stressed. Newcastle disease (ND) and avian influenza (AI) are the most important viral diseases that could be transmitted to poultry farms by free-living pigeons. Although pigeon paramyxovirus type 1 (PMV-1) normally does not cause as severe course of the disease as an infection with velogenic PMV strains, pigeon PMV-1 strains have clearly been shown to be pathogenic for chickens (Alexander and Parson, 1986; Werner et al., 1999). A wide range of ectoparasites and endoparasites in feral pigeons has been reported. Among endoparasites coccidia, Trichomonas gallinae, Ascaridia columbae, Capillaria sp. and Raillietina columbae, were found very often by examining the faeces (Toro et al, 1999; Mushi et al., 2000). Haemoproteus columbae was confirmed in blood (Mushi et al., 2000) as well as serum antibodies against Toxoplasma gondii were detected (Mushi et al., 2001). Ectoparasites cause lesions of feather and skin and can impact on the general condition status of infested birds. They also play an important role in the transmission of bacteria and viruses to other birds (Toro et al., 1999; Mushi et al., 2000). The zoonotic potential of soft ticks (Argas sp.) isolated from pigeons which could be carriers of Coxiella burnetii has been reported (Stein (1999). Veraldi et al. (1998) described skin manifestation of the disease caused by Argas reflexus in human patients. In this paper we describe the results of monitoring of some diseases in free-living pigeons Columba livia domestica in Ljubljana.

MATERIALS AND METHODS Birds In the year 2000, altogether 139 free-living pigeons, of different sex and age, were sampled from ten different locations in Ljubljana. The birds were handled in the same manner by using keep net. All captured pigeons were included in examinations. Each bird was weighed and clinically examined. Two birds were euthanased for ethical reasons; one bird had an open fracture of the right tibia and fibula and the other one had a shot wound. Both animals were necropsied and samples were taken for further bacteriological examinations. Sample collection Blood samples were obtained by the wing-vein puncture and blood was collected using Microtainer tubes (Becton Dickinson, Heidelberg, Germany) with serum separator. For serological examinations serum samples were stored at 20 o C in eppendorf tubes until testing. Cloacal swabs were used for the bacteriological examinations. Wings, a neck and a cloaca region were examined for the presence of ectoparasites. Visible ectoparasites were removed for the determination. For the faeces collection each pigeon was isolated in pigeon box for six hours. Individual faecal samples were examined for the presence of endoparasites. Oropharyngeal swabs were taken only from eleven birds, out of 139 examined, because swabs were taken only from pigeons, which were clinically suspected for trichomonosis. Swabs were submerged in saline (0,9% NaCl). Serological examinations The collected sera were tested for the antibodies (IgG) against Chlamydophila psittaci (CP), using indirect immunofluorescence test (CP spot BioMerieux, France). Antigen spots on substrate slides were covered with diluted sera (1:40, 80, 160, 320, 640 and 1280) for 30 minutes at 37 o C. Afterwards slides were twice washed in PBS, each time for 5 minutes. FITC conjugated rabbit anti-chicken IgG (whole molecule, Sigma Chemical Company, USA) was added for 30 minutes at 37 o C. Slides were washed again, like before, and mounted in mounting medium.

The serums were tested for the presence of antibodies against avian PMV-1 by the haemagglutination inhibition (HI) test. For HI test two-fold serial dilutions in 96-well microtitre plates were prepared and tested with 4 units of ND virus strain La Sota and 1% of washed chicken red blood cells (CEC, 1992). For the detection of anti-ai antibodies an agar gel immunodiffusion test was used. Test was done by standard procedure (O.I.E. Manual, 2000) using avian influenza antigen and positive control sera supplied by Veterinary Laboratories Agency Weighbridge, New Haw, Addle stone, United Kingdom. Bacteriology Cloacal swabs were used for bacteriological examinations for Salmonella spp. The swabs were incubated in tetrathionate broth at 37 C for 20 to 24 hours. After incubation a loopful of broth was transferred to XLD and Rambach agar and incubated for another 20 to 24 hours at 37 C. Suspicious colonies were transferred to Drigalski agar for pure culture. The blue S colonies were determinated to species level with API 20 E (BioMerieux). Serovars were identified by slide agglutination for O and H antigens. Parasitology If an ectoparasite was detected on a bird, it was isolated on the slide then covered with a drop of lactic acid and observed for its morphological characteristics by bright-field microscopy. The faecal samples were examined for the presence of endoparasites using the flotation method and the sedimentation method (Thienpont et al., 1979). The oropharyngeal swabs were used for the isolation of Trichomonas gallinae. Trichomonas gallinae was then detected microscopically in freshly prepared wet mount. If there were not any trichomonas in observed sample a drop of iodine solution was added and sample was checked again for the presence of trichomonas. After addition of iodine solution trichomonas flagella were clearly seen.

RESULTS Serology Antibodies against CP were found in 23.7% of examined sera (Table 1) as well as antibodies (HI titre > 4 log 2 ) against PMV-1, which were detected in 84.2% of blood sera examined (Table 2). All samples were negative for antibodies against AI. Bacteriological findings Salmonella enterica serovar Sinthia was isolated from seven (5.0%) cloacal swabs. Salmonella enterica serovar Typhimurium was isolated from lung and liver of one bird (0.7%), which was euthanased (Table 3). Together Salmonellae were isolated from 5,7 % of examined birds. Detection of ecto and endoparasites Ectoparasites were found on 72,7% of examined birds. Spread wings and neck region were exactly examined. From those region two species of ectoparasites Columbicola columbae and Campanulotes bidentatus compar were confirmed from feather samples. The incidence of endoparasites in faecal samples is shown in Table 4. Trichomonas gallinae were detected in eleven pigeons (7,9%) from which swabs were taken. We noticed that these pigeons had oropharyngeal diphtheroid plaque characteristic for trichomonosis. We also found that all infested birds were younger than half year. The age of pigeons was determined by their appearance. They had grey soft nostril, supple upper beak and their feathers on wings and tail apexes were uninjured. Other birds (92,1%) didn t show any clinically visible changes in beak cavity and pharyngeal region, that s why samples were not taken from these pigeons.

DISCUSSION Free-living pigeons are usually in a rather close contact with humans and animals. Although many birds do not show any clinical signs of diseases, they may be a permanent source of infection. Several authors have reported the possibility of transmission of some bacterial diseases from free-living pigeons to humans (Glunder, 1989; Mushi et al., 2001). In our study among bacterial zoonosis salmonellosis and avian chlamydiosis were investigated. Salmonella enterica serovar Sinthia was isolated in 5.0% of the cloacal swabs sampled. Salmonella enterica serovar Typhimurium was isolated only in the case when internal organs of euthanased bird were examined. Similar results were reported o also by other authors. Salmonella spp. were isolated by Casanovas et al. (1995) in 1.5% of examined birds and by Adesiyun et al. (1998) in 0.8% of examined birds. Toro et al. (1999) detected Salmonella sp. of groups B and D in 3.0% of examined birds. In our investigation the presence of specific antibodies against CP was confirmed in 23.7% of the sera tested. The absence of clinical signs specific for chlamydophilosis and relatively low titres of IgG antibodies indicate the chronic form of the disease. The present findings indicate much more favourable epidemiological situation in comparison with our previous investigations of chlamydophilosis in pigeons. A seroprevalence rate of 66.7% was obtained in the period between years 1991 and 1997. During the years 1991, 1994 and 1997 we detected even higher percentage of seropositive birds then average of 66.7% for the six years period. The titers of specific antibodies were also higher during these years (Dovč, 1998). Salinas et al. (1993) found 34.4% seropositivity by using the microimmunofluorescence test for detection of antibodies against CP. Vlahović et al. (1998) reported 40.9% of serologically positive pigeons detected by a complement fixation test in the period between 1992 and 1997. Similar results (47,7%) were also reported by Pavlak et al. (2000). A high percentage (84.2%) of antibodies against avian PMV-1 and their relatively high titres revealed the presence of virus in the pigeon population. The results are not surprising. In the same period pigeon PMV-1 was isolated in four cases of racing and fancy pigeons (Krapež et al., 2001; Zorman-Rojs et al., 2002). All the tests for anti-ai antibodies were negative. Currently no cases of AI had been diagnosed in Slovenia.

Protozoa Eimeria spp. (71.9%) and nematodes Capillaria sp. (26.6%) were often detected in contrary to low detection of Ascaridia columbae (4.3%). Mushi et al. (2000) found the presence of coccidia oocysts in 40.0% and Ascaridia columbae in 30.0% of the faecal samples examined. In 2001, the same author described positive antibody response against Toxoplasma gondii in 100.0% of examined serums and the presence of Haemoproteus columbae in the 80,0% of blood smears. Trichomonas gallinae caused clinical signs in 7.9% of the population tested. The severe clinical signs were observed only in young pigeons. Lethargy, dyspnoea and discharge resulted from an inflammation and caseous lesions, which had developed in the beak cavity and pharyngeal region. Bright-field microscopy proved to be a very helpful method, but only when fresh taken samples had been used. The present findings are very similar to those reported by Toro et al. (1999). In our research only Columbicola columbae and Campanulotes bidentatus compar were found among the monitored ectoparasites. Columbicola columbae was found more frequently than Campanulotes bidentatus compar. A higher prevalence of ectoparasites Columbicola columbae (30%) and pigeon fly - Pseudolynchia canariensis infestation (50%) has been described (Mushi et al., 2000). Infestation with Columbicola columbae in 41.0% and chewing lice in 68.0% of examined birds were described by Toro (1999). Haag and Gurdan (1990) detected infestation with chewing lice in 85.3% of feral pigeons. This is the first epizootiological report focusing on the health of free-living pigeons in Slovenia. The prevalence of pathogens, found in our study, shows that free-living pigeons could be important in the transmission of these diseases among different animal species as well as from a zoonotic point of view. ACKNOWLEDGEMENT The city municipality of Ljubljana, the Republic of Slovenia, supported the work. The number of the permission for biological experiment is: 323-02-11/00, 27. 3. 2000.

REFERENCES Adesiyun, A.A., Seepersadsingh, N., Inder, L. and Caesar K. (1998): Some bacterial enteropathogens in wildlife and racing pigeons from Trinidad. J. Wildl. Dis. 34, 73-80. Alexander, D.J. and Parson, G. (1986): Pathogenicity for chickens of avian paramyxovirus type 1 isolates obtained from pigeons in Great Britain during 1983-85. Avian. Pathol. 15, 487-493. Casanovas, L., de Simon, M., Ferrer, M.D., Arques, J. and Monzon, G. (1995): Intestinal carriage of campylobacters, salmonellas, yersinias, and listerias in pigeons in the city of Barcelona. J. Appl. Bacteriol. 78(1), 11-13. CEC (1992): Council directive 92/66/EEC of 14 July 1992 introducing Community measures for the control of Newcastle. Off. J. Eur. Comm. L260, 1-20. Dovč, A. (1998): Chlamydiosis - Chlamydia psittaci infection in domestic and wild birds in Slovenia. Vet. Nov., 10, 419-422. Dovč, A. (1995): [Uvajanje imunofluorescenčnih metod v diagnostiko klamidioze (Chlamydia psittaci) ter epizootiologija pri papigah in golobih.] Slov. Vet. Res. 32, 257-276. Fukata, T., Uemura T., Baba E., Horiuchi S. and Arakawa A. (1986): Isolation of clostridia, salmonellae and coccidia from wild pigeons in Japan. Br. Vet. J. 142, 291-293. Glunder, G. (1989): Infections of pigeons as a risk to the health of humans and animals. Dtsch. Tierärztl. Wochenschr. 96, 112-116. Haag, D. and Gurdan G. (1990): [Űber den hygienischen Zustand der Strassentauben in Basel]. Swiss. Vet. J. 7, 19-21. Krapež, U., Dovč A., Zorman-Rojs O., Barlič-Maganja D. and Mrzel I. (2001): [Zaražanje paramiksovirusima u golubova]. Zbornik Savjetovanje Peradarski dani, Poreč. Hrvatski veterinarski institut, Centar za peradarstvo, Zagreb, pp. 103-104. Mushi, EZ, Binta M.G., Chabo R.G., Ndebele R., Panzirah R. (2000): Parasites of domestic pigeons (Columba livia domestica) in Sebele, Gaborone, Botswana. J. S. Afr. Vet. Assoc. 71(4), 249-250. Mushi, E.Z., Binta M.G., Chabo R.G., Ndebele R. and Panzirah R. (2001): Seroprevalence of Toxoplasma gondii and Chlamydia psittaci in domestic pigeons (Columba livia domestica) at Sebele, Gaborone, Botswana. Onderst. J. Vet. Res. 68(2), 159-161. O.I.E. manual of standards for diagnostic tests and vaccines (2000): Lists A and B diseases of mammals, birds and bees. 4 th ed. Office International des Epizooties, Paris, pp. 212-220.

Pavlak, M., Vlahović K., Gregurić J., Župančić Z., Jerčić J. and Bozikov J. (2000): An epidemiological study of Chlamydia spp. in feral pigeons (Columba livia var. domestica). Z. Jagdwiss. 46, 84-95. Salinas, J., Caro M.R. and Cuello F. (1993): Antibody prevalence and isolation of Chlamydia psittaci from pigeons (Columba livia). Avian. Dis. 37, 523-727. Stein, A. and Raoult D. (1999): Pigeon pneumonia in Provence: a bird-borne Q fever outbreak. Clin. Infect. Dis. 29(3), 617-620. Thienpont, D., Rochette F., Vanparijs O.F.J. (1979): Diagnosing helminthiasis through coprological examination. Janssen Research Foundation, Beerse, pp. 31-34. Toro, H., Saucedo C., Borie C., Gough R.E. and Alcaino H. (1999): Health status of free-living pigeons in the city of Santiago. Avian. Pathol. 28(6), 619-623. Veraldi, S., Barbareschi M., Zerboni R., Scarabelli G. (1998). Skin manifestations caused by pigeon tiks (Argas reflexus). Cutis. 61(1), 38-40. Vlahović, K., Župančić Z., Gregurić J. and Pavlak M. (1998): [Zur Zuverlässigkeit diagnostischer Verfahren beim Nachweis von Infektionen mit Chlamydia spp. bei Vögeln] Reliability of diagnostic methods in proving infections of Chlamydia spp. in birds. Z. Jagdwiss. 44, 133-139. Zorman-Rojs, O., Krapež, U., Grom, J., Barlič Magajna, D. (2002): Molecular detection pathotyping of paramyxovirus type 1 isolates (New castle disease virus). Slov. Vet. Res. 39, 39-45. Werner, O., Römer-OberdörferA., Köllner B., Manvell R.J. and Alexander D.J. (1999): Characterization of avian paramyxovirus type 1 strains isolated in Germany during 1992 to 1996. Avian. Pathol. 28, 79-88.

Table 1 Indirect immunofluorescence for antibodies against Chlamydophila psittaci (CP). Antibodies titre against CP N* % Negative 106 76.3 Positive 1:40 24 17.3 1:80 5 3.6 1:160 2 1.4 1:320 2 1.4 Total positive 33 23.7 Total 139 100.0 * N = number of pigeons

Table 2 Haemagglutination inhibition test (HI) for avian paramyxoviruses type-1 antibodies. HI antibodies titre to PMV-1 N* % 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 Negative 1 1 1 6 13 22 15.8 Positive 77 32 7 0 1 117 84.2 Total 139 100.0 * N = number of pigeons

Table 3 Salmonella spp. isolated from pigeon droppings. Bacteria N* (Total = 139) Incidence %** Salmonella enterica serovar Sinhtia 7 5.0 Salmonella enterica serovar Typhimurium 1 0.7 Total 8 5.7 * N = number of pigeons ** Percentage of pigeons from which salmonella serovar was isolated.

Table 4 Incidence of parasite species detected in pigeon droppings. Parasite species N* (Total = 139) Incidence %** Eimeria spp. 100 71.9 Capillaria sp. 37 26.6 Ascaridia columbae 6 4.3 * N = number of pigeons ** Percentage of pigeons in which each parasite species was detected.