A Comparison of the Performance of Exfoliative Cytopathology, Polymerase Chain Reaction (PCR), Culture and ELISA in the Detection of Brucella canis

Abstract

This study was aimed to determine the performance of cytopathology for the diagnosis of B. canis on the basis of a comparison with culture, PCR and ELISA results. For this purpose, swabs for cytological, molecular and bacteriological examinations blood serum samples for serological tests were taken from 147 dogs which are suspected to have genital infection.. While the cytological samples were stained with May-Grünwald Giemsa solution, the swab samples were tested by bacteriologically and PCR and the serum samples were assayed by ELISA. According to the results, while ELISA yielded a seropositivity rate of 11.5%, PCR demonstrated a positivity rate of 1.8%. No B. canis could be isolated from the samples were taken for bacteriological examinations. At cytological examination, it was observed that the samples contained coccobacilli presented with abundant neutrophil leukocytes and macrophages. Some of these immune cells and epithelial cells were observed to contain round/oval shaped bacteria in their cytoplasm. The results of this study suggest that cytopathology can be used for auxiliary test for the diagnosis of brucellosis.

Keywords

• Brucella canis
• Cytopathology
• PCR
• ELISA

Brucella canis’in Tanısında Eksfolyatif Sitopatoloji Verilerinin Polimeraz Zincir Reaksiyonu (PZR), Kültür ve ELISA Yöntemleri ile Karşılaştırması

Abstract

Bu çalışmada, B. canis’in tanısında sitopatolojik yöntemin kullanılabilirliğinin PCR, kültür ve ELISA yöntemleri ile belirlenmesi amaçlandı. Bu amaçla toplam 147 genital sistem enfeksiyonu şüphesi olan köpekten alınan sitolojik, swap ve kan serumu örnekleri incelendi. Sitolojik örneklere May Grünwald Giemsa boyaması yapılırken, mikrobiyolojik incelemeler amacıyla elde edilen swaplar bakteriyolojik kültür ve PCR ile test edildiler. Serum örnekleri ELISA ile test edildi. Yapılan çalışmalar sonucu ELISA yöntemiyle yapılan incelemelerde %11.5 oranında seropozitiflik, PCR yönteminde ise %1.8 oranında pozitiflik tespit edildi. Bakteriyolojik muayene için alınan örneklerde B. canis izole edilemedi. Sitolojik incelemelerde kokobasil ile beraber çok sayıda nötrofil lökosit ve makrofajla karşılaşıldı. Yer yer bu hücrelerin ve epitel hücrelerin sitoplazmasında yuvarlak-oval şekilli etkenlerle karşılaşıldı. Yapılan bu çalışmanın sonuçları sitopatolojinin brusellanın tanısında yardımcı bir test olarak kullanılabilirliğini ortaya koymuştur.

Keywords

• Brusella canis
• Sitopatoloji
• PCR
• ELISA.

References

1. Abdissa K, Tadesse M, Bezabih M, Bekele A, Apers L, Rigouts L, Abebe G, 2014: Bacteriological methods as add on tests to fine-needle aspiration cytology in diagnosis of tuberculous lymphadenitis: can they reduce the diagnostic dilemma?. BMC Infect Dis, 31, 14,720.
2. Alton, GG & Institut national agronomique Paris-Grignon, 1988: Techniques for the brucellosis laboratory. Institut Nacional de la Recherche Agronomique, Paris.
3. Barouin-Melo SM, Poester FP, Riberio MB, Alcantara AC, Aguiar PHP, Nascimento IL, Schaer RE, Nascimento RM, Freire SM, 2007: Diagnosis of canine brucellosis by ELISA using an antigen obtained from wild Brucella canis. Res Vet Sci, 83, 340-346.
4. Boon GD, Rebar AH, Denicola DB, 1982: A cytologic comparison of Romanowsky stains and Papanicolaou-type stains. I. Introduction, methodology and cytology of normal tissues. Vet Clin Path, 11, 22-30.
5. Carmichael LE, 1990: Brucella canis. In: Animal brucellosis, Ed; Nielsen K, Duncan JR. Boca Raton: CRC Press; 335-350.
6. Corbel MJ, Bracewell CD, Thomas EL, Gill KPW, 1979: Techniques in the identification of Brucella species. In: Identification Methods for Microbiologists, 2nd ed., Ed; Skinner FA & Lovelock DW, pp:86-89, Academic Press, London, UK and New York, USA.
7. Corbel MJ, Hendry DMFD, 1983: Methods for the identification of Brucella. Booklet 2085. Ministry of Agriculture, Fisheries and Food, Lion House, Alnwick, Northumberland, UK.
8. Diker KS, Aydin N, Erdeger J, Ozyurt M, 1987: A serologic survey of dogs for Brucella canis and Brucella abortus and evaluation of mercapto ethanol microagglutination test. Ankara Univ Vet Fak Derg, 34, 268-277.

9. Farrell ID, 1974: The development of new selective medium for the Isolation of Brucella abortus from Contaminated Sources. Res Vet Sci, 16, 280-286.
10. Flores-Castro R, Carmichael LE, 1978: Canine brucellosis. Current status of methods for diagnosis. Cornell Vet, 68, 76-88.
11. Ghisleni G, Roccabianca P, Ceruti R, Stefanello D, Bertazzolo W, Bonfanti U, Caniatti M, 2006: Correlation between fine-needle aspiration cytology and histopathology in the evaluation of cutaneous and subcutaneous masses from dogs and cats. Vet Clin Pathol, 35, 1, 24-30.
12. Hollett RB, 2006: Canine brucellosis: Outbreaks and compliancei. Theriogenology, 66, 575-587.
13. Istanbulluoglu E, Diker S, 1983: An serological analysis of Brucella canis. Ankara Univ Vet Fak Derg, 30, 14-18.
14. Johnson CA, Walker RD, 1992: Clinical signs and diagnosis of Brucella canis infection. The Comp Cont Educ, 14, 763-772.
15. Keid LB, Soares RM, Vasconcellos SA, Chiebao DP, Salgado VR, Megid J, Richtzenhain LJ, 2007: A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches. Theriogenology, 68, 1260-1270.
16. Kustritz MVR, 2005: Pregnancy diagnosis and abnormalities of pregnancy in the dog. Theriogenology, 64, 755-765.
17. Madoz LV, Giuliodori MJ, Migliorisi AL, Jaureguiberry M, Sota RL, (2014): Endometrial cytology, biopsy, and bacteriology for the diagnosis of subclinical endometritis in grazing dairy cows. J Dairy Sci, 97, 1-7.
18. Moore JA, Gupta BN, 1970: Epizootiology, diagnosis, and control of Brucella canis. J Am Vet Med Assoc, 156, 1737-1740.
19. Nielsen K, Smith P, Yu WL, Rojas X, Perez B, Conde S, Samartino L, Robles C, 2007: Detection of ovine antibody to Brucella ovis by indirect enzyme ımmunoassay. J Immunoass Immunoch, 28, 243-250.
20. OIE, 2009: Manual of Standards for Diagnostic tests and Vaccines. 3rd Edition, Caprine and ovinebrucellosis chapter (2.7.2), Bovine Brucellosis chapter (2.4.3). Office International of Epizooties, Paris, France.
21. Oliveira MZD, Vale V, Keid L, Freire SM, Meyer R, Portela RW, Barrouin-Melo SM, 2011: Validation of an ELISA method for the serological diagnosis of canine brucellosis due to Brucella canis. Res Vet Sci, 90, 425-431.
22. Oncel T, Akan M, Sareyyupoglu B, Tel OY, Ciftci A, 2005: Seroprevalance of Brucella canis infection of dogs in two provinces in Turkey. Turk J Vet Anim Sci, 29, 779-783.
23. Power W, 1985: Cerebrospinal fluid lyphocytosis in acute bacterial meningitis. Am J Med, 79, 2, 216-220.
24. Sayan M, Erdenlig S, Stack J, Kilic S, Guducuoglu H, Aksoy HY, Baklan EA, Etiler N, 2011: A serological Diagnostic Survey for Brucella canis infection in Turkish Patients with brucellosis-Like symptoms. Jpn J Infect Dis, 64, 516-519.
25. Watchel EG, 1964: Exfoliative Cytology in Gynaecological Practice: Butterworth & Co.
26. Wellman ML, 1990: The cytologic diagnosis of neoplasia. Vet Clin North Am Small Anim Prac, 20, 919-937.