Serološko istraživanje lajmske borelioze u pasa s područja grada Zagreba u Hrvatskoj.

Sera from 120 apparently healthy dogs, 74 purebred, 46 mixed-bred, 57 females and 63 males in the Zagreb area (Croatia) were examined by enzyme-linked immunoadsorbent assay (ELISA) for antibodies to Borrelia burgdorferi. During triple i/v immunization of dog at 1, 7 and 14 day, on each occasion with 5 ml 1.4×107 cells/ml of B. burgdorferi sensu stricto, strain B31 ATCC 35210, a positive control serum was obtained at day 28 p.i., while negative control serum was obtained prior to immunization. The antigen for ELISA was prepared from the same bacterial strain sonicated on ice. The sonicate was quantified by sodium dodecyl sulphate polyacrilamid gel electrophoresis (SDS PAGE). IgG antibodies to B. burgdorferi were estimated in 6 (5%) samples of dog sera in titre ranging from 1:100 to 1:2000 (Optical density-OD=0.650 + 36.49, n=6). Antibodies to B. burgdorferi were found in two females and four males, two Labrador retrievers and in one German shepherd, a Hungarian viszla, one cocker spaniel, and a German wirehaired pointer at 2.5 to 8 years of age. Of 114 seronegative samples three samples were found to be very close to the margin of optical density that determined seropositive result (OD=0.562). Estimated seroprevalence to B. burgdorferi in dog sera suggested that dogs in the Zagreb area are infected with B. burgdorferi and that the Zagreb area is part of a wider Central European Lyme boreliosis endemic area.Imunoenzimnim testom (ELISA) pretraženo je 120 uzoraka klinički zdravih pasa, 74 čistokrvna, 46 križanih, 57 ženki i 63 mužjaka područja grada Zagreba na prisutnost protutijela za B. burgdorferi. Trokratnom i/v imunizacijom psa 1., 7. i 14. dana s po 5 ml kulture B. burgdorferi sensu stricto soj B 31 ATCC 35210 u količini 1,4 × 107 bakterija/ml dobiven je 28. dan od početka imunizacije pozitivni kontrolni serum, a negativni je uzet prije imunizacije. Od istog soja načinjen je ultrazvučno lizirani antigen za imunoenzimni testž (ELISA) čiji je proteinski sastav određen elektroforezom u poliakrilamidnom gelu uz dodatak natrijevog dodecilsulfata (sodium dodecyl sulphate polyacrilamid gel electrophoresis - SDS PAGE). U 6 (5%) uzoraka seruma pasa ustanovljen je titar IgG protutijela od 1:500 do 1:2000 (ODp=0,650 + 36,49, n=6) u dvije kuje i četiri psa u dobi od 2,5 do 8 god., u dva labradora i po jednog njemačkog ovčara, mađarske vižle, koker španijela i njemačkog oštrodlakog ptičara. Od 114 seruma u kojih apsorpcijska vrijednost nije prelazila graničnu apsorpcijsku vrijednost za pozitivne (OD=0,562) u tri seruma apsorpcijska vrijednost je bila blizu granične apsorpcijske vrijednosti. Ustanovljena IgG protutijela za B. burgdorferi u serumima pasa ukazuju da psi područja grada Zagreba bivaju inficirani bakterijom B. burgdorferi i da je to područje dio šireg srednjeeuropskog endemskog žarišta lajmske borelioze

Uloga mišolikih sisavaca kao rezervoara leptospira u šumama hrasta lužnjaka Hrvatske.

In the year 2000, three hundred and eighty-one animals werw caught in 11 locations in the pedunculate oak forests extending across the wider region of the Sava River valley, an area known as an old natural focus of leptospires. Two hundred and twenty-seven animals were investigated for leptospiral carriership, 381 were tested for the existence of specific antibodies to leptospira. Through cultivation of kidney sections in the Korthof’s nutrient medium, 17 strains of leptospira were isolated from 3 species of myomorphous mammals, of which in the house mouse (Mus musculus) the serovar sejroe in 10 animals (71.4%), in the black-striped field mouse (Apodemus agrarius) the serovar pomona in 4 animals (5.4%) and in the yellownecked field mouse (Apodemus flavicollis) the serovars australis, pomona and one unindentified isolate in 3 animals (4.5%), representing a total of 7.5% of animals. Using the microscopic agglutination test in 47 animals (12.3%), antibodies to leptospira were found for 8 serovars, for the most part the serovar pomona, then (in decreasing order) for the serovars saxkoebing australis, and hardjo. The percentage of serologically positive animals ranged from 34.4% in the house mouse, 13,9% in the black-striped field mouse, 10.8% in the yellow-necked fieldmouse, 9.4% in the bank vole (Clethrionomys glareolus), to 6.5% in the long-tailed field mouse (Apodemus sylvaticus). The research results suggest that pedunculate oak forests can be considered as an integral part of a vast natural focus of leptospirosis. The specificity and role of this biotope in maintaining leptospira should be investigated to a greater extent in the future.U šumama hrasta lužnjaka, na 11 lokaliteta koji se prostiru u širem području rijeke Save, poznatom kao staro prirodno žarište leptospiroza, tijekom 2000. godine ulovljeno je 381 životinja. Na kliconoštvo leptospira pretraženo je 227 životinja i 381 životinja na prisutnost specifičnih protutijela za leptospire. Kultivacijom komadića bubrega u Korthof-ovom hranilištu, izdvojeno je 17 sojeva leptospira, u 3 vrste mišolikih sisavaca, od čega u kućnog miša (Mus musculus) serološka varijanta sejroe u 10 ili 71,4% životinja, u poljskog miša (Apodemus agrarius) serološka varijanta pomona u 4 ili 5,4% i žutogrlog miša (Apodemus flavicollis) serološka varijanta australis, serološka varijanta pomona i jedan nedeterminirani izolat u tri ili 4,5% životinja, što predstavlja ukupno 7,5% životinja. Reakcijom mikroskopske aglutinacije u 47 ili 12,3% životinja ustanovljena su protutijela za 8 seroloških varijanti leptospira, od čega najviše za serološku varijantu pomona, a zatim redom serološku varijantu saxkoebing, australis i hardjo. Postotak serološki pozitivnih životinja kretao se od 34,4% u kućnog miša, 13,9% u poljskog miša, 10,8% žutogrlog miša, 9,4% u riđe voluharice (Clethrionomys glareolus) do 6,5% u šumskog miša (Apodemus sylvaticus). Rezultati istraživanja upućuju na zaključak da šume hrasta lužnjaka možemo smatrati sastavnim dijelom velikog prirodnog žarišta leptospiroze. Specifičnosti i ulogu ovog biotopa u održanju leptospira trebalo bi u budućnosti opsežnije istražiti

Identification and Typing of Leptospira spp. by Restriction Fragment Length Polymorphism (RFLP) of 16S Gene rDNA and Pulsed-field Gel Electrophoresis (PFGE)

Ukupno 13 izolata bakterije roda Leptospira izdvojenih iz domaćih i divljih životinja iz kontinentalnog dijela Republike Hrvatske identificirano je najprije serološki metodom mikroskopske aglutinacije (MAT), a zatim genetski uporabom molekularnih metoda; raznolikosti dužine restrikcijskih fragmenata rDNK za gen 16S (RFLP) te gel elektroforeze u pulsirajućem polju (PFGE). Mikroskopskom aglutinacijom s 23 hiperimuna seruma seroloških skupina ustanovljeno je da pet izolata pripada serološkoj skupini Pomona, po dva izolata serološkoj skupini Bataviae, Canicola i Icterohaemorrhagiae i po jedan serološkoj skupini Australis i Grippotyphosa. Umnažanjem početnih 330 bp rDNK za gen 16S analiziranih izolata leptospira i mikrorestrikcije umnoženih fragmenata DNK s restrikcijskim enzimima MnlI i DdeI ustanovljeno je da svi izolati pripadaju genomskoj vrsti L. interrogans sensu stricto. Makrorestrikcijom čitave genomske DNK pretraživanih izolata leptospira s restrikcijskim enzimima NotI i SgrAI i gel elektroforezom u pulsirajućem polju ustanovljeno je osam različitih serovara: Copenhageni (1), Icterohaemorrhagiae (1), Bataviae (2), Pomona (4), Pomona Kennewicki (1), Canicola (2), Lora (1) i Grippotyphosa (1). Budući smo od 13 izolata leptospira na osnovi analize razlika u restrikcijskim profilima ustanovili osam različitih serovara, smatramo kako u Republici Hrvatskoj, osobito u nizinama Save i Drave postoji velika genetska raznolikost leptospira unutar jedne genomske vrste, Leptospira interrogans sensu stricto.Atotal of 13 Leptospira spp. strains isolated from domestic and wild mammals in different regions of inland Croatia were identified and characterized serologically by microscopic agglutination (MAT) and by modern molecular biology methods; polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) and pulsed field gel electrophoresis (PFGE). After the first typing according to serogroup affinities the isolates appeared to belong to serogroups Pomona (five isolates), Bataviae (two isolates), Canicola (two isolates), Icterohaemorrhagiae (two isolates), Australis (one isolate) and Grippotyphosa (one isolate). RFLP were examined in PCR products from first 330 bp long portion of rrs (16S rDNA gene) digested with endonucleases MnlI and DdeI revealed that all strains belonged to Leptospira interrogans sensu stricto genomic species. Intact genomic DNA from 13 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analyzed by PFGE. The results showed eight different serovars: Copenhageni (1), Icterohaemorrhagiae (1), Bataviae (2), Pomona (4), Pomona Kennewicki (1), Canicola (2), Lora (1) and Grippotyphosa (1). By revealing the degress of relatedness among 13 leptospiral restriction fingerprints in Croatia we demonstrated the existence of great genetic diversity within one genomic species, Leptospira interrogans sensu stricto especially in the Sava and Drava river valleys

Leptospiral antibodies in red foxes (Vulpes vulpes) in northwest Croatia

In 2000 sera from 59 red foxes (Vulpes vulpes) from northwest Croatia were tested for antibodies against 12 Leptospira interrogans serovars: grippotyphosa, sejroe, australis, pomona, canicola, icterohaemorrhagiae, tarassovi, saxkoebing, ballum, bataviae, poi and hardjo. Using the microscopic agglutination test in 34 (57.6%) of the 59 sera, leptospira antibodies were found for 11 serovars, mainly for the serovar australis, then (in decreasing order) for the serovars sejroe, icterohaemorrhagiae, grippotyphosa and saxkoebing. The results showed a strong correlation with previous results of seroepidemiology and isolation of leptospira from small mammals in northwest Croatia. The role of the red fox as a maintaining leptospira reservoir in the natural foci of leptospirosis is still a matter of question and should be further investigated. Key words: red fox, Vulpes vulpes, leptospira, natural foci, Croati