Alenka Dovč

Chlamydial infections in feral pigeons in Europe: Review of data and focus on public health implications.

Feral pigeons (Columba livia domestica), which thrive in most European towns and cities, are commonly infected with the zoonotic bacterium Chlamydophila psittaci, the agent of psittacosis (also known as ornithosis) in humans. A number of surveys carried out over the last thirty years across Europe have detected high seropositivity values and high percentages of infection in feral pigeon populations. Overall, when considering data from 11 European countries, seropositivity values to C. psittaci in the sampled populations ranged from 19.4% to 95.6%. In most surveys, the complement fixation test was used, and antibodies were detected in 19.4-66.3% of the samples, with a median of 46.1%. Indirect immunofluorescence and ELISA tests were employed less frequently, but led to the detection of higher percentages of seropositivity (23.7-67.7% and 35.9-95.6%, respectively). Attempts to grow C. psittaci in cell culture or embryonated chicken eggs were successful in 2-42.3% and 0-57.1% of samples, respectively, antigen detection methods were positive in 2.3-40% of samples, while conventional PCR and real-time PCR using different genomic targets detected the organism in 3.4-50% of samples. Twenty-five C. psittaci isolates from pigeons were typed as ompA genotype B (n=14), E (n=10) and E/B (n=1). The huge increase of feral pigeon populations in Europe is a major cause of concern for the detrimental effect of pigeon droppings on environmental hygiene, in addition to the extensive damage due to the fouling of buildings and monuments. The most important pathogenic organism transmissible from feral pigeons to humans is C. psittaci, with 101 cases of disease reported in the literature. Exposure to C. psittaci-contaminated dust, direct contact with pigeons through handling and, to a lesser extent, through pigeon feeding have been identified as hazardous exposures in more than half of the human cases, while loose or transient contacts with feral pigeons have been mentioned in about 40% of the cases. Education initiatives as to the communication of a health risk resulting from contact with pigeons and pigeon excreta should primarily be targeted at individuals who may be exposed to C. psittaci-contaminated dust, such as demolition/construction workers. Recommendations to this category of workers include wearing protective clothes with hoods, boots, gloves and air filter face masks when removing pigeon faeces from roofs, garrets and buildings, especially if working indoors. Monitoring for C. psittaci infections in these workers over time should also be considered. Children should be warned not to handle sick or dead pigeons, and immunocompromised individuals should be advised to carefully limit their contact to feral pigeons. Culling of pigeons by shooting or poisoning is both unethical and ineffective as the place of the killed birds in the population is quickly filled by new juveniles or immigrating birds from neighbouring areas. Pigeon-deterring systems, such as nets and plastic or metal spikes applied to buildings and monuments will prevent their fouling, and the administration of contraceptive drugs may allow size regulation of the pigeon populations. Nevertheless, the measure that will ultimately lead to permanent reduction and will establish healthy sustainable populations is the restriction of indiscriminate feeding by pigeon lovers. The erection of dovecotes and artificial breeding facilities should be considered for providing shelter and a balanced diet to the birds, as well as a chance of interaction for pigeon lovers in a hygienically controlled environment.

Parasites in pet reptiles

Exotic reptiles originating from the wild can be carriers of many different pathogens and some of them can infect humans. Reptiles imported into Slovenia from 2000 to 2005, specimens of native species taken from the wild and captive bred species were investigated. A total of 949 reptiles (55 snakes, 331 lizards and 563 turtles), belonging to 68 different species, were examined for the presence of endoparasites and ectoparasites. Twelve different groups (Nematoda (5), Trematoda (1), Acanthocephala (1), Pentastomida (1) and Protozoa (4)) of endoparasites were determined in 26 (47.3%) of 55 examined snakes. In snakes two different species of ectoparasites were also found. Among the tested lizards eighteen different groups (Nematoda (8), Cestoda (1), Trematoda (1), Acanthocephala (1), Pentastomida (1) and Protozoa (6)) of endoparasites in 252 (76.1%) of 331 examined animals were found. One Trombiculid ectoparasite was determined. In 563 of examined turtles eight different groups (Nematoda (4), Cestoda (1), Trematoda (1) and Protozoa (2)) of endoparasites were determined in 498 (88.5%) animals. In examined turtles three different species of ectoparasites were seen. The established prevalence of various parasites in reptiles used as pet animals indicates the need for examination on specific pathogens prior to introduction to owners.

Campylobacter, salmonella and chlamydia in free-living birds of Croatia

Campylobacteriosis, salmonellosis and avian chlamydiosis are zoonotic diseases in which birds have been suggested to play an important role as reservoirs. We have investigated the prevalence of Campylobacter and Salmonella spp. and Chlamydophila sp. in 107 free-living birds belonging to 25 species from 13 families from Croatia in order to examine the natural infections caused by these agents. Campylobacter jejuni-like organisms were isolated from 2 of 107 free-living bird species examined (1.9%). Salmonella was isolated from 8 fresh fecal specimens from free-living bird species (7.4%). These isolates were identified as S. typhimurium in 4 (3.7%), and S. enteriditis in 4 (3.7%) free-living birds. These samples originated from feral pigeons (Columba livia domesticus; n=14; 28.6%), rook (Corvus frugilegus; n=13; 15.4%), buzzard (Buteo buteo; n=12; 16.7%), black-headed gull (Larus ridibundus; n=8; 12.5%) and tawny owl (Strix aluco; n=8; 12.5%). The presence of Chlamydophila sp. was not detected in the free-living birds examined during this study. Epidemiological aspects and possible significance of the examined birds as a source of infections for domestic animals and humans are discussed.

Molecular characterization of atypical Chlamydia and evidence of their dissemination in different European and Asian chicken flocks by specific real-time PCR.

Chlamydia psittaci is a zoonotic pathogen associated primarily with avian chlamydiosis. New chlamydial agents with suspected zoonotic potential were recently detected from domestic poultry in Germany and France indicating that the spectrum of Chlamydiaceae encountered in birds is not confined to a single chlamydial species. For further characterization, a specific real-time PCR targeting the conserved 16S rRNA gene was developed and validated for a specific detection of these atypical Chlamydiaceae. In order to address the epidemiological importance of the new chlamydial agents and their distribution, Chlamydiaceae-positive chicken samples collected from flocks from five different countries were examined. The results confirmed that C.psittaci is not the predominant chlamydial species among chickens examined and suggested that the new chlamydial agents could putatively be widespread in poultry flocks (France, Greece, Croatia, Slovenia and China at least) justifying their systematic investigation when poultry samples are submitted to laboratories for avian chlamydiosis diagnosis. Besides, 16S rRNA-based dendrogram, including sequences from both isolates of the new chlamydial agents or positive samples as well as representative sequences from species belonging to the order Chlamydiales, showed the new chlamydial agents to form a distinct line of descent separated from those of other chlamydial species, but clearly grouped within the family Chlamydiaceae. Finally, the phylogenetic tree inferred from the multi-locus sequence typing based on four housekeeping fragments (gatA, gidA, enoA and hflX) and the ompA-based dendrogram showed an almost identical topology of the new chlamydial agents with that recovered by 16S rRNA-based dendrogram. Interestingly, partial ompA gene sequences displayed considerable diversity among isolates.

Long-term study of chlamydophilosis in Slovenia

Immune reactivity for Chlamydophila (C.) psittaci in Slovenia was monitored in parrots, canaries, finches and nine species of recently captured free-living birds (house sparrows, Eurasian goldfinches, tree sparrows, chaffinches, European greenfinches, European serines, Eurasian siskins, Eurasian linnets and Eurasian bullfinches) in the period from 1991 to 2001. In subsequent years, specific IgG antibodies were found using immunofluorescence in parrots (0.7– 53.6%), canaries (0.0–3.5%), finches (0.0–5.7%) and in captured free-living birds (33.3% of Eurasian goldfinches in 1994). An experimental infection with C. psittaci was performed in order to study clinical signs and pathological changes in canaries and finches. The C. psittaci strain used for experimental infection was isolated from a cockatiel (Nymphicus hollandicus). Chlamydial DNA was extracted from clinical material followed by RFLP-PCR analysis. Infection of canaries and finches was confirmed in organ smears by direct immunofluorescence and a modified Gimenez staining method. In addition, serological tests of indirect immunofluorescence and complement fixation were applied. However, in spite of positive immunological reaction there were no clinical signs three weeks after infection. The present study includes results of a serological survey of persons belonging to the most important risk groups (breeders, pet shopkeepers and veterinarians). The results of microimmunofluorescence to identify the presence of specific antibodies and correlation between appearance of infection in birds and important risk groups are presented. Out of 143 persons belonging to the high-risk group we found 10 (7%) persons who were immunologically positive. Testing of two successive samples was used to demonstrate an increase in IgG and IgA titres in human sera. However, IgM, which is indicative of acute infection, could not be detected.

Characterisation of Mycoplasma gallisepticum strains involved in respiratory disease in pheasants and peafowl

Two cases of Mycoplasma gallisepticum infection in different avian species in backyard gamebird operations in Slovenia were investigated. In the first case, M gallisepticum was associated with severe respiratory disease with almost 20 per cent mortality in pheasants, whereas the infection was less pathogenic for chickens and turkeys reared at the same site. The M gallisepticum isolates from pheasants had a unique pMGA gene sequence containing a repeat of 12 nucleotides, and they contained only small amounts of the cytadhesins MGC1 and MGC3 and no PvpA protein. However, they expressed some typical M gallisepticum proteins and several proteins which were immunogenic for pheasants, chickens and turkeys. A strain of M gallisepticum isolated from the sinus of a pheasant was highly pathogenic for chicken embryos. In the second case, the M gallisepticum strain that was associated with respiratory disease and mortality in peafowl also affected chickens. M gallisepticum strain ULB 992 was isolated from the infraorbital sinus of a dead peafowl. The ULB 992 strain synthesised a small amount of MGC3, a truncated form of MGC1 and lacked PvpA. However, it expressed several proteins which were immunogenic for the birds infected with M gallisepticum at both gamebird operations.

Age, sexual and seasonal differences of haematological values and antibody status to Chlamydophila sp. in feral and racing pigeons (Columba livia forma domestica) from an urban environment (Zagreb, Croatia)

The aim of this study was to determine the basic haematological parameters in feral and racing pigeons and to compare these parameters according to age, sex and season in healthy feral pigeons as well as between Chlamydophila-serologically positive and negative feral pigeons. Red blood cells (RBC), packed cell volume (PCV), haemoglobin (Hb), mean cell volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), white blood count (WBC), thrombocyte count and differential WBC, were determined in 366 pigeons (Columba livia forma domestica) captured in the City of Zagreb between 1999 and 2002. Of these, 232 feral (179 adult and 53 juvenile, 104 male and 75 female) and 57 racing pigeons (25 male and 32 female) were clinically healthy and bacteriologically and serologically negative, but 77 birds had antibody titres against Chlamydophila sp. Significantly lower values of RBC, PCV, Hb, MCH, WBC and thrombocyte (P<0.05) were observed in young compared to adult pigeons, while the differences in MCV and MCH were not significant between age classes. In differential WBC of young pigeons, a significantly higher percentage of heterophils, eosinophils, basophils and monocytes and a significantly smaller percentage of lymphocytes (P<0.01) was found than in adult pigeons. Significant sex-related differences were seen only in MCV values and in the percentage of lymphocytes (higher in females) and neutrophils (higher in males). PCV, Hb, MCV and MCH increased, while WBC decreased during wintertime (P<0.05). In differential WBC, percentage of heterophils was low in summer and autumn. At the same time, a higher percentage of basophils was found. Low numbers of monocytes were found in summer and low values of eosinophils in winter. In racing pigeons, values of eosinophils and basophils were significantly lower than in feral pigeons. Pigeons which had antibodies against Chlamydophila sp. possessed a higher percentage of monocytes and less lymphocytes than sero-negative animals, while WBC was significant lower than in sero-negative feral pigeons.

Surveillance of Influenza A Viruses in Wild Birds in Slovenia from 2006 to 2010

SUMMARY. Within the framework of the surveillance program for the early detection of H5 and H7 subtypes of avian influenza (AI) viruses, samples from 2547 wild birds of different species that were collected between 2006 and 2010 were examined by PCR-based methods. AI viruses of various subtypes were detected in 4.4% of birds from four different orders: Anseriformes, Ciconiiformes, Charadriiformes, and Pelecaniformes. Highly pathogenic avian influenza (HPAI) H5N1 viruses were detected only in 2006. HPAI H5N1 virus was confirmed in 1.9% of birds from four different species. Comparison of nucleotide sequences of the H5N1 hemagglutinin gene indicated that two different HPAI H5N1 viruses from the European–Middle Eastern–African clade 1 had been introduced into Slovenia, despite the relatively short duration of the HPAI outbreak. Low pathogenic avian influenza (LPAI) viruses were detected in 2.5% of birds during a 5-yr period. The subtypes H1, H2, H3, H4, H5, H7N7, H8, H10, H11, and H13N6 were determined in 18 out of 64 cases. The highest prevalence (81%) of LPAI viruses, including the H5 subtype, were found in birds sampled as a part of the “active” surveillance system.

Prevalence of Pigeon Circovirus Infections in Feral Pigeons in Ljubljana, Slovenia

SUMMARY. Pigeon circovirus (PiCV) was detected by real-time PCR in cloacal swabs, pharyngeal swabs, and serum samples taken from 74 feral pigeons (Columba livia var. domestica) that were caught at various locations in the city of Ljubljana, Slovenia. PiCV infections were detected in the majority of the tested birds. The highest (74.3%) detection rate was observed in the cloacal swabs and the lowest (31.1%) in serum samples. PiCV DNA was more readily detected in the cloacal swabs, pharyngeal swabs, and serum samples of birds younger than 1 yr. Molecular analysis of partial open reading frame V1 sequences showed that PiCV strains detected in feral pigeons share high nucleotide and amino acid sequence identities with PiCV strains detected in ornamental, racing, meat, and feral pigeons. RESUMEN. Reporte de Caso—Prevalencia de las infecciones asociadas con circovirus de las paloma en las palomas en libertad en Ljubljana, Eslovenia. El circovirus de las palomas (PiCV) fue detectado por PCR en tiempo real en hisopos cloacales, hisopos faríngeos y en muestras de suero tomadas de 74 palomas en libertad (Columba livia var. domestica) que fueron capturadas en distintos lugares de la ciudad de Ljubljana, Eslovenia. Se detectó la infección por el circovirus de las palomas en la mayoría de las aves analizadas. El porcentaje de detección más alto (74.3%) se observó en los hisopos cloacales y la más baja (31.1%) en muestras de suero. El ADN del circovirus de las palomas fue detectado más fácilmente en los hisopos cloacales, hisopos faríngeos y en muestras de suero de las aves menores de un año. El análisis molecular de las secuencias parciales del marco de lectura continua V1 mostró que las cepas de circovirus detectadas en las palomas en libertad comparten altas identidades en las secuencias de nucleótidos y de aminoácidos con las cepas de circovirus detectadas en palomas ornamentales, de competencia, productoras de carne y salvajes.

Evidence of avian influenza virus and paramyxovirus subtype 2 in wild-living passerine birds in Slovenia

A total of 670 cloacal swabs were taken from 37 species of wild-living passerine birds in years 2004, 2005, and 2006. The cloacal swabs were pooled into pools of three swabs for analysis. Isolation of avian influenza virus (AIV) and avian paramyxoviruses (APMV) was done on chicken embryos. One-step reverse-transcription polymerase chain reaction (RT-PCR) was used to detect AIV RNA. AIV nucleic acid was detected by RT-PCR in a sample of one common starling (Sturnus vulgaris). All attempts of AIV isolation from wild-living passerine birds’ samples were negative. APMV 2 was isolated in one robin (Erithacus rubecula) sample. In light of the presented results and literature data, passerines appear to play a minor role as potential disseminators of AIV and APMV; however, it seems that some wild-living passerine species could act as occasional carriers.