The role of Mycoplasma canis in the pathogenesis of infertility and urogenital diseases in dogs

Namen doktorske naloge je bil razjasniti pomembnost okužbe z M. canis pri psih v patogenezi obolenj sečil in spolovil in z njimi povezanih plodnostnih motenj. V klinično študijo smo vključili 52 psov, od tega 7 psov z znaki obolenj sečil in spolovil, 14 psov s kliničnimi znaki plodnostnih motenj in 31 psov brez kliničnih znakov okužbe. Povzročitelja smo dokazovali s pomočjo PCR (angl. polymerase chain reaction) metode v vaginalnih in prepucialnih brisih psov. Analizo sestave celotne bakterijske raznovrstnosti v vzorcih brisov smo nadgradili z metagenomskim pristopom. Prisotnost specifičnih protiteles smo ugotavljali z metodo DIBA (angl. dot immunobinding assay) in rezultate potrdili z metodo po Westernu. Pri pacientih smo poleg kliničnih znakov bolezni ugotavljali odstopanja v hematoloških in izbranih biokemijskih parametrih v krvi, odstopanja v analizi urina ter specifičnih parametrih analize plodnostnih motenj in skupine medsebojno primerjali. Z metodo PCR smo M. canis dokazali pri 3/7 (43 %) pacientov z obolenji sečil in spolovil, pri 6/14 (43 %) pacientih s plodnostnimi motnjami in pri značilno nižjem deležu 6/31 (19 %) zdravih psov iz kontrolne skupine. Z metagenomsko analizo bakterijske sestave med skupinami nismo ugotovili značilnih razlik. Prisotnost specifičnih protiteles na vsaj 1 od 5 izbranih sevov smo z metodo DIBA ugotovili pri 54/55 (98 %) vseh vzorcev. Pri vseh pacientih, razen pri 2 iz kontrolne skupine, smo zabeležili reakcijo na sev M. canis Larissa. Specifičnost reakcije smo potrdili z metodo po Westernu. Rezultati doktorske naloge so nam v pomoč pri izboru ustreznih diagnostičnih metod, planu zdravljenja in spremljanju odgovora nanj pri pacientih s ponavljajočimi se vnetnimi obolenji sečil in spolovil ali plodnostnimi motnjami. Serološki presejalni testi so smiselni, vsaj pri pacientih s plodnostnimi motnjami. Za dokaz okužbe pa je najprimernejša uporaba specifčne metode PCR za dokazovanje M. canis.The aim of this dissertation was to investigate the role of M. canis infection in dogs with urogenital diseases and with fertility problems. The clinical study included 52 dogs7 dogs with urogenital disease symptoms, 14 dogs with fertility problems, and 31 healthy dogs. The pathogen was detected by PCR (polymerase chain reaction) method on vaginal and preputial swabs. Swab samples were analysed to determine the full bacterial spectrum, and these analyses were extended by metagenomic analysis. The presence of antibodies was tested by the Dot-immunobinding assay (DIBA) method, while the results were confirmed by the Western Blot method. In addition to clinical signs, we looked for abnormalities in haematological and biochemical parameters in blood, urinalysis, and specific parameters of fertility disorders, and compared the groups with each other. M. canis was detected by PCR method in 3/7 (43 %) patients with urogenital disorders, in 6/14 (43 %) patients with fertility disorders, and in a statistically significantly lower proportion of healthy dogs in the control group (6/31, 19 %). Metagenomic analysis of bacterial composition showed no significant differences between the groups. The DIBA method showed the presence of specific antibodies against at least one of the five selected strains in 54/55 samples (98 %). With all patients, except two from the control group, we detected a reaction to the M. canis Larissa strain. The results of the DIBA test were confirmed by the Western Blot method. The results of the thesis will help us to select appropriate diagnostic methods and treatment plan and to monitor the response to treatment in canine patients with chronic urinary tract diseases or fertility disorders. The results show that serological screening tests are useful at least for patients with fertility disorders. The specific PCR method is the most suitable for the detection of M. canis infections

MOLECULAR DETECTION AND SEROPREVALENCE OF MYCOPLASMAS IN CLINICALY HEALTHY WORKING DOGS

In this study seroprevalence and prevalence of mycoplasmas in clinically healthy dogs were studied. Thirty-four working dogs of various breeds, gender and age were included in this research. Among them, 27 were working dogs from Slovene armed forces and 7 were working sheepdogs. We used dot-immunobinding assay (DIBA) as a serological test for the detection of specific antibodies to Mycoplasma cynos, Mycoplasma canis and Mycoplasma molare and consensus PCR for detection of genes for 16S rRNA or 16S/23S IGS region of mycoplasmas. Specific antibodies against at least one of the canine mycoplasmas were detected in 94.1% dogs. Of them 23.5% samples showed positive reaction only to M. cynos, 20.6% were positive only to M. canis and none of the samples were positive only to M. molare. Altogether 47.0% of samples were positive to M. cynos and M. canis whereas only one dog (2.9%) had specific antibodies to all three mycoplasmas tested. The presence of mycoplasmas detected by PCR was 57.14% in younger dogs (≤ 1 year) and 18.52% to 35.29% in older dogs, depending on year of the sampling. Genital swabs were positive in more cases (60%) in comparison with oral swabs (46.67%). M. canis was detected in 40% of positive cases, in the same percent of samples mixed not determined mycoplasma infections were confirmed. Mycoplasma species such as:  M. cynos, M. edwardii, M. maculosum, M. spumans were determined each in single cases and in one case mixed ureaplasma infection was confirmed.   MOLEKULARNA DETEKCIJA IN SEROPREVALENCA MIKOPLAZEM PRI KLINIČNO ZDRAVIH DELOVNIH PSIH Namen raziskave je bil določiti seroprevalenco in prevalenco mikoplazem pri klinično zdravih delovnih psih. V raziskavo je bilo vključenih 34 delovnih psov različnih pasem in starosti, od tega 27 psov iz Slovenske vojske in 7 ovčarskih psov. Za dokazovanje specifičnih protiteles proti bakterijam Mycoplasma cynos, Mycoplasma canis in Mycoplasma molare smo uporabili metodo točkastega imunskega odtisa (ang. Dot Immuno Binding Assay- DIBA) in konvencionalni PCR, ki temelji na pomnoževanju odseka gena za ribosomalno RNK 16s ali intergenskega odseka genoma med genoma ribosomalnih RNK 16s in 23s. Specifična protitelesa proti vsaj eni od izbranih vrst mikoplazem so bila ugotovljena pri 94.1 % psov. Med njimi je 23.5 % vzorcev reagiralo pozitivno samo na M. canis, 20.6 % samo na M. canis in noben od vzorcev ni reagiral pozitivno samo na M. molare. Skupno je 47.0 % vzorcev reagiralo pozitivno na M. canis in M. canis hkrati, en pes (2.9 %) je imel specifična protitelesa proti vsem trem testiranim mikoplazmam. Z metodo PCR smo mikoplazme dokazali v vzorcih 57.1 % psov mlajših od enega leta, in pri 18.5 % do 35.3 % starejših od enega leta, odvisno od leta vzorčenja. Genitalni brisi so bili pozitivni v 60 % primerov v primerjavi z oralnimi kjer je bil delež 46.7 %. M. canis je bila ugotovljena v 40 % pozitivnih primerov, v enakem deležu so bile ugotovljene tudi mešane nedeterminirane mikoplazemske okužbe. Mikoplazme, kot so M. cynos, M. edwardii, M. maculosumin, M. spumans so bile ugotovljene posamično. V enem primeru je bila ugotovljena mešana okužba z ureaplazmami. Ključne besede: delovni psi; pasje mikoplazme; Mycoplasma canis; Mycoplasma cynos; DIBA; PC