Albanian j. agric. sci. ISSN: 2218-2020, (2012), (Special Edition) Copyright © Agricultural University of Tirana

ISOLATION CHLAMYDOPHILA PSITTACI IN TIRANA ORNAMENTAL BIRDS GËZIME SHEHU1*, LULJETA QAFMOLLA2, RUZHDI KEÇI3, XHELIL KOLECI3, ZAÇE MALAJ3, YMER ELEZI4 1

Protection of Animal Health, Department of Agriculture, Tirana, Albania

2.

Virologe at the Institute of Food Safety and Veterinary, Tirana, Albania;

3.

Lecturers in the Faculty of Veterinary Medicine, Tirana, Albania;

4.

Researcher in the field of ornamental birds, Tirana, Albania.

*Corresponding author (e-mail): gezimes@yahoo. com  

Abstract This study is the first of this kind conducted in Albania, which was based on the serological control of 573 blood samples taken from pigeons, parrots and canaries in Tirana. Sampling was carried out in all four seasons of 2011 year. Their control was carried out by Institute of Veterinary Food Safety (FSV) Tirana. In order to check the s samples it was used an indirect immunofluorescence test (IFT), combined with chlamydia’s isolation in chicken’s egg embryo cells, as a comparative method. According to the data from the Public Health Institute (PHI) in Tirana, it has been observed an increase in the vulnerability of humans, mainly in to those in young age, who have been in contact with decorative birds. Relative specificity of fluorescent antibody’s test in the serum was approximately 95. 3% and the relative sensitivity was about 60. 3%. The results of this study, which as mentioned above were conducted for the first time in Albania, showed that immunofluorescence test performed using diagnostic kits of Medical service-2000, combined with primary isolation of chlamydia in embryos of chicken eggs, was very specific, in which there were identified 51 cases with Ch. Psittaci and very valuable for the identification of beneficial options to the veterinary service. Keywords words: immunofluorescence overhead, relative sensitivity, fluorescent antibody, immunodepresion, seropozitivity

1. Introduction Chlamydiosis in pigeons, parrots and canaries has become a distressing disease, increasing not only because of its economic impact in the industry of decorative and domestic birds, but also for its zoonotic nature [8]. Even in Albania from year to year, this category of birds has been increasing, serving as a potential risk in spreading the chlamydial infection not only in batches of birds, but also to humans. [19]. Evidence shows that during the period 2001-2011 there have been reported about 635 cases of individuals infected with chlamydia [5] who mostly were pople in their young age or individuals involved in decorative bird breeding. Expressed clinical signs were depending on chlamydia’s pathogenicity, type, race, physiological condition, age of the bird, route and time frame of exposure, stressful factors, immunodepresive and individual situation of the birds [17, 18] and the presence of other infections in an interaction. Psittacosis soft “explosion’s “ can often go unnoticed because there are no clear symptoms, the International Conference 31 October 2012, Tirana

most obvious are those of airways and diarrhea [21]. In adult wild birds, Chlamydia is often seen without clinical signs and they can serve as asymptomatic carriers. The infection can develop in the acute form, sub-acute, or it can be chronic. In the acute form the disease can cause severe damages, which can be fatal, especially for some species, while at younger birds, it appears to be highly sensitive. The most typical sings are seen on young birds, which often appear to be weak, showing anorexia, they faint, lie in a special position, from their eyes and nose there is noticed a purulent flow, they usually are contracted and stay with disheveled feathers [13]. Diagnostic methods used for detection of chlamydic infection in the birds are numerous. For specificity, sensitivity, speed, relativity and its simplicity, the identification of chlamydial antibodies, esepcially in sub-clinical cases, is made by using the indirect immunofluorescence test which is considered to be the most effecive test. On the other side the primary isolation of chlymidia of chicken embryos [2, 4] can also be used as comperative method [16]. In different species such as, pigeons, parrots and canaries, it has

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also been seen that their immunological responses towards their organism can be different [11]. The study showed that organs taken from dead birds which were infected with Ch. psittaci were important in identifying the infection by using diagnostic kits. The aim of the study was to develop and evaluate a rapid antemortem diagnostic technique for detection of C. psittaci in the serum of birds. Also as a quick and accurate method it can allow a veterinary doctor to immediately begin treating the sickening birds thus, preventing the infection to spread from domestic birds to humans, [13] which is the basic objective of the veterinary medicine. 2. Material and methods 2.1 Identify chlamydia with method indirect immunofluorescence test(IFT) To conduct the study there were selected 5 areas, with comprehensive coverage for monitioring all decorative birds that live in Tirana, such as; parrots and canaries in cages and sales outlets; from pigeons the racing ones were also selected, which served alerts in cases of people affected by this infection, mostly amatour youngsters, who had been in contact with these birds. Perennial scope had as an objective the study of the dynamics of the disease, as well as climatic factors correlating with other environmental issues[6]. The process of taking the blood samples from decorative birds was carried out in batches, at 0. 5 ml / head, which were then marked and have been monitored throughout the year; 573 samples which prevailed according to their species were: 118 heads of parrots, 348 pigeons and 107 canaries, of which 30% were selected from young birds, varying from 2-4 months old. As a laboratory method it was selected the indirect immunofluorescence test, which because of its high specificity assists in all sub-clinical cases, when a seropositive birds lack a complete clinical framework. It is fast and simple to implement in the field and laboratory procedures, [2] and also combined with primary isolation method of chlamydia in the chicken’s embryo it can obtain rapid and accurated data [4]. The IFT control procedure was based on the principle of reaction of 573 blood samples, using IgG Antibody Kit, imported by Medical Servicce-2000. The separation of serum was made using the usual method, by following rigorously all steps to preserve the kit, dilution, International Conference 31 October 2012, Tirana

incubation, testing and control of the material prepared. At the end the reading of the small droplet shaped spots was made using a 400x magnification for each tile, and then they were compared with the visual intensity of basic troops, shown in the positive control well and negative. The tiles were stored in a dark room in a temp 2-8 degree celsius for a period of 24 hours. In the positive responses appeared a fluorescent glow, sharp, regular elementary troops and the were stained, which was rated at 1+, 2+. 2.2 Identification of chlamydia directly with histopathological control Birds 'bodies, lung, trachea, liver, spleen and air sacs in parrots, pigeons, canaries of different ages, young adult, clinically healthy, [21] but including those seropositive after being separated from the herd, sacrificed, histological incisions were made and stained with May-Grunwald-Giemsa method. Control became usig an ordinary microscope with magnification 1000 x. 2.3 Isolation of chlamydia in chicken embryos The isolation of chlamydia cells in chicken embryos was carried out according to standard procedures, injected into the viteline sac, up 0. 5 ml. emulsion prepared with positive material from positive birds and suspected, from the lungs, liver, spleen, trachea and air sacs injured birds and sacrificed, in chicken embryos aged 6-7 days. Then they were placed for incubation at 39 degree celsius[15]. After that it was carefully observed the replication of chlamydia, and evaluation of results was done from the fetus state, which in case it results positive, usually dies within 5-12 days after inoculation. After the histopathological control of infected embryos with the typical chlamydie infection, the material was collected and homogenised with a 20% suspension sacus vitelinus. The identification of the agent was carried out by preparation of an ifected sakus vitelinus antigen[21]. 3. Results In the capital city the chlamydia infection circulating level in the decorative birds is considered relatively low: in parrots: 12. 7%, pigeons: 8. 4%, and canaries: 7. 47%. Chlamydia focuses on birds' bodies, however more sensitive and higher concentrations of it is found in the lungs. The IFT

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Isolation Chlamydophiila psittaci in Tirana T ornameental birds

method, com m mbined with thhe isolation of o chlamydiees in e embryos of chicken c eggs,, as well as histopatholog h gical c control of thhe heads daamaged, hass proven too be e effective forr the disease control, on o the practtical im mplementatiion of field and laborattory proceduures. C Chlamydia c chicken embbryo grew annd multiplied in thhe first pass,, where the largest conceentration of them is in the coree of an egg. Death D of em mbryos, underr the a action of chllamydia is inn high perceentage, the most m p pathogenic w were chlamyddia separatedd from the paarrot a less was those of dovve and canariies. and Identificcation of Ch. C psittaci is done by c clinically heealthy birds,, and thosee coupled other o p pathologies, where the intensity of o touching the b bodies, in thee first case has h been mucch lower thaan in c cases when one pathoology was combined with w a another pathhology, i. e aspergilosiss, parasites etc.. M Morbidity caases of birdds only by chlamydia are o observed in total 10, (7 parrots and 3 pigeons), but a always accom mpanied witth other causes, which have h 45

A Amazona

40

On ndule

Calopssitoe

In total

28.5

35

mydia, wheere after histopatholog h gy enaabled Chlam "Baasic corps" are a localized in the brain ns of damageed bird ds. Clinic diisease has bbeen evidentt and obviouus maiinly in youung birds, which make possible a veteerinary service, whereas adults show a sub-clinicaal form m, serving as asymthom matic carriees, helping to t keeep permantly recycling off infection. The datta collected ffrom the surv vey show thaat chlaamydia are ordinarily rresident in the t bodies of o bird ds, which are activateed when lowered theeir susttainability ass a result of stress and otther resourcees thatt accompanny infectioon, finding gs that arre com mpatible witth those off many foreeign authorrs. Reccognition of favorable facctors and thee developmennt cyccle chlamydiees can serve as options for f preventivve meaasures by thhe veterinarry service to o control thhe diseease in pooultry in ggeneral and d particularlly deccorative, andd as a zooonosis, preevention annd protection of huuman health.

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6.6

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8.8

15

10

20

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12.5

16.6

25

16.6

23

30

0 S Spring

S Summer

A Autumn

Winter

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15.9

Figure 1-Informationn about the poositivity of chlamydiosis in parrots by thee breeds(in%))

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6.15

6.3

8

In total 10.05

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12

11.3

14

2.5 3.3

5

6

8 04 8.04

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4 2 0 Spring

Su ummer

Autumn

Winter

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F Figure 2: Innformation about a the poositivity of chlamidiosiis in pigeonns by breedss (in%) International Conference C 3 October 2012, Tirana 31

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8

7.5 7.5

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Shehu et al

6 4

0 0 0

2 0 Spring

Summer

Autumn

Winter

Annual

p of chhlamydiosis in n canaries by breeds (in%) Figure 3 - Information about the positivity

4 Discussioon 4. From the presentatioon of data, inn Figure no. 1, 2, 33, 4 in overrall it has beeen identifieed a total off 51 h heads of serropositive biirds, of whiich 15 heads of p parrots, 28 heeads of pigeoons, 8 heads of canaries, and b using the primary isoolation methood of chlamyydia by inn chickens embryos, there weree prepared 15 s samples, of which w 5 weree parrots, 7 were w pigeons and 3 canaries. Selecting the methood of indiirect im mmunofluorrescence in determininng the posiitive s samples resuulted to be veery useful esspecially in subc clinical casees, when coomplete clinical signs were w m missing, mosstly in adult birds, b and coombined withh the m method of issolation chlaamydia in chhicken embrryos; b because the death of em mbryos occuurred when they w were at the age of 12-18 days, makking compleetely c compatible w IFT scoore. [4]. From with m the histologic c control of tyypical chlam mydia infecttion in chiccken e embryos, in general g for all a species itt was observed a v vascular conngestion of membranes m s sacus -viteliinus, [1, 4, ] An im mportant eleement was observed o andd the d dynamics of infection, whhich accordinng to the speecies s stands as follows : parroot’s, average annual leveel of innfection hass been 12. 7%, in sprring 14. 2% %, in s summer 23% %, in fall 8.. 8%, and inn winter 6. 6% [Figure 1]. Pigeon’s, average annnual level of innfection waas about 8. 04%, 8. 5% % in springg, in s summer 12. 3%, 3 in fall 6.. 3%, while in i winter, 3. 3%, w where is alm most is exttint complettely [Figure 2]. C Canarie’s, avverage annuaal level of infection i waas 7. 4 47%, in sprinng 10. 3%, inn summer 111. 5%, in autuumn 6 89%, whille in the winter it was 0, which meeans 6. thhat is completely extinctt [Figure 3]. s as parrrots, Althouggh in differeent species such p pigeons, canaaries the freqquency was different, it was s strictly relateed to seasonaal conditionss of the weatther. International Conference C 3 October 2012, Tirana 31

t In spring the infection haas shown a tendency to incrrease, in sum mmer where the weatherr has been so s hot it has shoown it maxximum valuee whereas in i umn, with the t weatherr cooling, th he value haas autu tend ded to decrrease, while in the win nter when thhe weaather has been b cold thhe infection n is reducedd, hidd den or wiped out, facts which coinccide with thaat of the t foreign authors a [17] etc. To be considered as a sup pport for inccreasing thee frequency of infectioon during the monnths with waarm and hot weather therre werre also manyy other factoors, individu ually or in thhe corrrelations bettween them have contrributed to thhe situ uation. Thosee can be from m the: increaase of contacct betw ween birds with warm m weather, activation of o arth hropods andd hematophhag insects which hellp spreead the movement m vvectors of infection in i deccorative birdss, which havve also been observed o on a casee by case basis on thhe infected birds. Whille mon nitoring the incidence oof pigeons (n not decorativve onees) and seroppositive racees, which make m up abouut 30% % of the saamples, as a source of infection we w sho ould also evaaluate the coontact with water weedds, whiich have beeen polluted with eksemeents of porteer bird ds. On the otther hand unnfamiliar areaas affected by b infeection, and the t transit dduring the raaces [20], buut also o the use of o uncontrolled food products p witth unssafe origin, low hygiene standards of breedingg, streesses and strrains circulaation with hiigh virulence, etc.. should alsso be taken more into consideratioon [11] During moonitoring, it w was noted thaat infection to t ung people is organizedd in acute form causinng you dam mage to form mer companny because of their higgh sensitivity. Yooung birds have show wn signs of o nte from eyees weaakness typicaal, anorexia, leak purulen and d nose, inacctivity, stayy in position to collecct, dish heveled feaathers[13]. In cases with milld dev velopments birds b lacked clear sympto oms, the most

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obvious would be those of the respiratory tract and diarrhea [8, 9] making them serve as asymptomatic carriers, recycling permanently the infection. Localizing chlamidieve bird bodies, was conducted microscopic research in lung, trachea, spleen, liver, kidney, and air sacs 60 heads of birds, parrots, pigeons, canaries, including those races, clinically sound, suspected, inclusion and those that resulted seropositive IFT method, which after being separated from the herd, was sacrificed. During histopathological control was found that chlamydia were present in clinically healthy birds, as well as for other patients, where the percentage of affected birds was higher, and sensitive organs. Cases with combined pathologies are observed frequently during examination, but they had the advantage of younger ages, in relation to adult birds. Thus, in 10 chapters birds parrot affected by aspergilosis, chlamydia were present in all heads, 15 heads dove birds affected by mykoplazmosis, heteroksidosis, capilariosis and histomonosis, chlamydia were present in 12 chapters, and in canaries it was not identified such cases. It is noted that the intensity of colonies chlamydic in organs of birds was many times higher than in healthy ones, where in a microscopic field in the affected organs were seen on 50-60 chlamidic colony. It was noticed that their condition and the bird’s race played a role in the intensity of the disease. The isolation of chlymidia in chicken egg embryos aged 6-7 days, were made from 15 samples, of which 5 parrots, 7 pigeons and 3 canaries, which resulted seropositive of the IFT method. To avoid horizontal transmission of infection through eggs, before infecting, 6 embryos aged 6-7 days, two for each species where checked with IFT method, which resulted in negative form of chlamydia. Control of the embryos was carried out for 14-15 days in a row and the death rate of embryos infected with suspension by parrots was 100%. Infected embryos suspension in doves was 4, or 57. 7%, whereas embryos infected with suspension in canary bodies was 1, or 33%. Mortality dynamics was during the period 3-14 days after infection, and in parrots 7080% of mortality rate occurred between 3-8 days after infection, in pigeons it was 10-12 days after infection, while the canaries 13 days after the infection. Histopathological control of dead embryos dominated by the presence of hemorrhage in the body, in the head region of the feet, thickening of the lining of an egg and the slowdown in their growth International Conference 31 October 2012, Tirana

and development. Surviving embryos, especially those with suspension by pigeons and canaries, microscopic research have been identified the "Basic corpus". By monitoring the people affected with C. psittaci, in the past three years, there have been a total of 132 cases, and after controlling almost 45 of them, it was found that the disease has been correlated directly from their contact with seropositive birds, parrots, canaries and doves, and its frequency was variable, with age, the level of exposure and patogjenociteti were the determining factors for the occurrence and form infection clinic [19, 21

Figure

4-Direct

immunofluorescence

staining of Ch. Psittaci, the green chlamydia inclusions inside the cells (bar 20 μm).

Figure 5:-Modified Giménez stainingof Ch. psittaci, the red inclusions inside the cells (bar 20 μm References

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antibodies with the mcro-immunofluorescence test Journal of clinical. Microbiology 1991, 29, 707-711 3. Andersen AA: Two new serovar of Chlamidia psittaci from North Amerirican birds. Journal of Veterinary Diagnostic Investigation 1997, 9, 159-164 4. Andersen AA: Avian chlamidiosis In OIE Manual of Diagnostic tests and Vaccines for terrestrial Animals. Sixth Edition OIE, Paris, France 2008, 431-442 5. Instituti i Shëndetit Publik –Frekuenca e zoonozave në Shqipëri, 2011, 15- Tiranë, 6. Batta MK, Dhingra PN, Mangat APS: Chlamydiosis in birds from serological survey. Ind. J. Anim. Sci. 1993, 63:526-527. 7. Bourke SJ, Carrington D, Frew CE, McSharry G: A comparison of the seroepidemiology of chlamydial infection in pigeon fanciers and farmers in the U. K. J. Infect. 1992, 25 Suppl. 1:91-98. 8. Barnes RC: Laboratory diagnosis of human Chlamydial infections. Clin. Microbiol. Rev. 1989-2:119-135. 9. Bejleri J, Berxholi K : Klamidiet në shpendëBuletini i Shkencave Zooteknike dhe Veterinare, 1987, 2, 63-70 10. El-Halawani ME, Waibel PE, Appel JR, Good AL: Effects of temperature stress on catecholamines and corticosterone of male turkeys. American. J. Physiol. 1973, 224:384388. 11. Geens T, Dewitte A, Boon N, Vanrompay D: Development of a Chlamydophila psittaci species specific and genotype specific real-time PCR. Veterinary Research 2005, 36, 787-797 12. Grimes J: Chlamydia psittaci latex agglutination antigen for rapid detection of antibody activity in avian sera: comparison with direct complement fixation and isolation results. Avian Diseases 1986, 30:60-66.

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13. Greub G: Minutes of the Subcommittee on the Taxonomy of the Chlamidiae. International Journal of Systamatic and Evolutionare Microbiology, 2010a, 60, 2691-2693; 2694 14. Harrison GJ: A practitioner’s view of the problem of avian chlamidiosis. J. Amer. Vet. Med. Assoc. 1989, 195:1525-1528. 15. Hobson D, Johnson F, Byng R: The growth of the ewe abortion Chlamydia1 agent in McCoy cell cultures. J Comp Pathol 1977, 87:155-l59. 16. Kennedy G, Taylor F, Werdin R et al. : Laboratory diagnosis of chlamydial diseases. Proc Annu Meet Am Assoc Vet Lab Diagnostic 1985, 28:421-435. 17. Lublin A, Mechani S, Malkinson M, Bendheim U, Weisman Y: A 3-year survey in different avian species of frequency of detection Chlamydia psittaci antigens. Proceed. 1993 Europ. Conf. Avian Med. Surg. Utrecht. The Netherlands. 1993. 478-492. 18. Lublin A, Mechani S, Malkinson M, Weisman, Y and Bendheim U: A 4-year survey of the distribution of Chlamydia psittaci in 19 orders of birds in Israel with emphasis on seasonal variability. Proceed. 3rd Conf. Europ. Comm. Assoc. Avian Vet. Jerusalem. Israel. ECAMS. 1995. 1-2 19. Pospisil L, Veznik Z, Hirt M, Svecova D, Diblikova I, Pejcoch M: Detection of Chlamydia in the intestines and lungs in pigeons and humans. Epidemiol., Microbiol., Immunologic 1996, 45:123-126. 20. Salinas J, Caro MR and Cuello F: Antibody prevalence and isolation of Chlamydia psittaci from pigeons (Columba livia). Avian Diseases 1993, 37:523-527. 21. Woods L, Woods D: Evaluation and development of a new antemortem diagnostic test of Chlamydia psittaci infection in psittacine birds. Proc Assoc Avian Vet. 1986 75-79.

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