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According to Greig (5), bacteria, particularly Branhamella ovis, may monopolize on the primary damage caused by the smaller microorganisms and may help produce a more serious form of the disease. The outbreaks of IKC have been reported in many countries, recently including Croatia (6). Although not being fatal, IKC is a serious ocular infection which requires antimicrobial treatment in order to prevent frequent relapses or total blindness (7). The most commonly used antimicrobial agents in therapy are tetracycline antibiotics oxytetracycline and chlortetracycline, followed by some other antimicrobials. Antibiotics are used locally and/or systematically, and the therapy is generally effective if animals were treated early enough in the disease process before major eye damage has occurred (5). Since there are only a few reports on in vitro sensitivity of M. conjunctivae, this study was initiated to examine the inhibitory and mycoplasmacidal concentrations of nine antimicrobials against our isolates. These are generally used locally or systematically in the treatment of ocular disease in small ruminants. MATERIAL AND METHODS Test organisms. Six of seven strains of M. conjunctivae selected for susceptibility testing were isolated from affected ovine and one (strain My-7) from caprine eyes during the first outbreak of infectious keratoconjunctivitis in Croatia in 1995. The strains were characterized both by cultural and biochemical properties. Molecular characterization was performed in Berne, Switzerland, by a PCR method which amplifies the specific parts of the rrs gene of M. conjunctivae (6, 8). All tested strains were cloned once from a solid medium, freeze-dried and stored at -20 0C until use. There are not reliable data on the number of passages of strains on PPLO media prior to testing their sensitivity. Media. Cultivation of mycoplasma and sensitivity testing were carried out in Hayflick's PPLO agar and broth medium (9), enriched with horse serum, fresh yeast extract, DNA and glucose (0,1% wt/vol.). For the sensitivity tests liquid medium (pH 7.8) without penicillin and thallium acetate was prepared, and sterilized by filtration through Seitz EKS membrane filter. Thereafter, a sterile phenol red solution (0,005%) was added to the medium. Antimicrobials. The following nine antimicrobial agents were used for the test: oxytetracycline, chlortetracycline, chloramphenicol, streptomycin, gentamicin, neomycin, tylosin, tiamulin, and enrofloxacin. Stock solutions (1024 (g/ml) were purchased from Pharmaceutical firm PLIVA Zagreb, and stored at - 20 0C until used in test. Test procedure. Minimal inhibitory concentrations (MICs) and minimal mycoplasmacidal concentrations (MMCs) basically were studied according to the method described by Aarestrup and Friis (10). For use in the test the stock solutions were diluted in liquid mycoplasma medium to double the final concentrations required. Based on previous experiences the following two-fold dilutions in liquid medium with phenol red, but without penicillin and thallium acetate, were used for determination of MICs and MMCs, from 0,25 to 128 (g/ml for streptomycin, neomycin, chloramphenicol and gentamicin, from 0,0075 to 16 (g/ml for oxytetracycline, chlortetracycline, and tylosin, and from 0,0037 to 1 (g/ml for tiamulin and enrofloxacin. For sensitivity determination a single tube containing 1,8 ml of liquid medium for each concentration was inoculated with 0,2 ml of three to five days incubated broth culture of tested strain. Inoculated tubes together with positive control (inoculated medium w/o antibiotics) and negative (uninoculated medium w/o antibiotics) control were incubated at 37 0C. Parallel with sensitivity testing, culture of used strain was serially diluted 10-fold in tubes with broth medium to determinate color-changing units (CCU). If titer of used mycoplasma culture was not from 104 to 106 CCU/ml, sensitivity testing procedure was repeated. The incubated broth were controlled daily, and the initial reading was taken as soon as the inoculum controls without antimicrobial agent changed color from the red to yellow. The MIC was the lowest drug concentration to show no color change in the medium. The final reading representing mycoplasmacidal activity (MMC) was made after incubation of 10 days. RESULTS The results of sensitivity testing for each M. conjunctivae strains separately are presented in table 1 as MICs and MMCs. Table 1. Antimicrobial agents sensitivity of 7 isolates of Mycoplasma conjunctivae Strain A n t i m i c r o b i a l s MICs/MMCs ((g/ml) OTCCTCCSGM***NETY***TMENO My-7*  0,125 (0,25) 0,125 (0,125) 2 (4) 8 (16) 0,25 (0,25) 1 (2) 0,125 (0,5) 0,06 (0,06) 0,06 (0,06) My-72** 0,125 (0,25)  0,06 (0,06) 2 (2) 4 (64) 0,125 (0,5) 1 (4) 0,25 (1) 0,125 (0,125) 0,015 (0,03)My-83 0,125 (0,125)0,06 (0,06)1 (4)4 (16)0,125 (0,5)0,5 (4)0,25 (0,5)0,03 (0,06)0,03 (0,06)My-87 0,25 (0,5)0,125 (0,125)0,5 (2)4 (16)0,125 (0,5)2 (8)1 (4)0,06 (0,25)0,06 (0,125)My-88 0,125 (0,125) 0,06 (0,06)1 (2)2 (64)0,125 (1)4 (8)0,5 (0,5)0,03 (0,03)0,03 (0,03)My-93 0,125 (0,25)0,06 (0,125)2 (2)64 ((128)1 (1)8 (8)1 (2)0,06 (0,06)0,06 (0,25)My-94a 0,125 (0,125)0,125 (0,125)2 (4)4 (16)0,25 (1)2 (4)0,5 (0,5)0,06 (0,06)0,06 (0,06) ( ) = mycoplasmacidal concentrations; * = CCU 102; **= CCU 103;*** = variable values in repeated tests; oxytetracycline (OTC), chlortetracycline (CTC), chloramphenicol (C), streptomycin (S), gentamicin (GM), neomycin (NE), tylosin (TY), tiamulin (TM), enrofloxacin (ENO). Susceptibility of tested mycoplasma strains as MICs and MMCs ranges are shown in the table 2. Table 2. MICs and MMCs ranges of 9 antimicrobials to 7 isolates of M. conjunctivae AntimicrobialMICs range ((g/ml)MMCs range ((g/ml)Oxytetracycline0,125 - 0,250,125 - 0,5Chlortetracycline0,06 - 0,1250,06 - 0,125Chloramphenicol0,5 - 22 - 4Streptomycin2 - 6416 - >128Gentamicin0,125 - 10,25 - 1Neomycin0,5 - 82 - 8Tylosin0,125 - 10,5 - 4Tiamulin0,03 - 0,1250,03 - 0,25Enrofloxacin0,015 - 0,060,03 - 0,25 DISCUSSION AND CONCLUSIONS In our study the most of 9 antimicrobial agents showed considerable in vitro effectiveness against tested M. conjunctivae isolates. The most effective were already well known antimycoplasmal agents enrofloxacin, tiamulin, chlortetracycline and oxytetracycline, followed by tylosin and gentamicin. The highest resistance was recorded with chloramphenicol, neomycin, and streptomycin. For tests that were repeated, because an M. conjunctivae titer was not within the desired range, similar results were obtained. These results varied by one or two dilutions from the results obtained when CCU's were within desired range. In spite of many attempts, we could not obtain the higher number of CCU in medium used for caprine isolate My-7 and ovine My-72. In repeated tests with the strain My-7, sensitivity to tylosin and gentamicin varied, which is noted in the table. The results presented in this study for the most effective agents enrofloxacin, tiamulin, chlortetracycline and oxytetracycline, including tylosin, are similar to those recorded by Hannan et al. (11), however, the strains used by these author included type strains and field strains isolated from poultry, swine and ruminants from different geographical location. Until now only a limited number of reports on sensitivity testing of M. conjunctivae isolates are available. There is a good correlation between our results and those obtained by Eqwu (7) for the sensitivity of M. conjunctivae field strains against tetracyclines and tylosin. These therapeutics have been of primary importance in treating animal mycoplasmal infections for a long time. Remarkable in vitro sensitivity of our M. conjunctivae isolates to tetracyclines was verified by good results obtained by topic and systemic administration of these drugs during the first outbreak of ovine infectious keratoconjunctivitis in Croatia (6). However, the strains tested by this author were more sensitive against streptomycin, but markedly more resistant against chlortetracycline, gentamicin, neomycin and chloramphenicol. There are a number of possible explanations for this phenomena, such as differences in procedures, a long time persistence of IKC in England and possible acquired resistance of causative agent to commonly used antibiotics. In testing the sensitivity we have used classical procedures, and the tested strains were isolated during the first outbreak of IKC in Croatia. As an ophthalmic ointment chloramphenicol and gentamicin are frequently used in the therapy of bacterial infections. A clinical verification of the use of gentamicin ointments in treating mycoplasmal eye infections has not been done, and the contrary results of the effectiveness of cloramphenicol use in treating IKC are published (7). It can be concluded from our study that enrofloxacin, tiamulin, tetracyclines and tylosin are promising for the local and/or systemic treatment of IKC in small ruminants. Taking into account the broader antibacterial activity and possibilities of incorporation in ophthalmic preparations, tetracyclines may be preferred. REFERENCES 1. Jones, G.E., and A. Foggie. 1976. Mycoplasmas and ovine keratoconjunctivitis. Vet. Record 99: 137-141. 2. Terlaak, E.A., B.E.C. Schreuder, T.G. Kinmman, D.J. Houwers. 1987. Keratoconjunctivitis in sheep experimentally produced by Mycoplasma conjunctivae. Israel J. Med. Sci. 23:533. 3. Egwu, G.O., W.B. Faull, J.M. Bradbury, M.J. Clarkson. 1989. Ovine keratoconjunctivitis: a microbiological study of clinically unaffected and affected sheep's eyes with special reference to Mycoplasma conjunctivae. Vet. Record 125: 253-256. 4. Mayer, D., J. Nicolet, M. Giacometti, M. Schmitt, T. Wahli, W. Meier. 1996. Isolation of Mycoplasma conjunctivae from conjunctival swabs of alpine ibex (Capra ibex ibex) affected with infectious keratoconjunctivitis. J. Vet. Med. B 43: 155-161. 5. Greig, A. 1989. Ovine keratoconjunctivitis. In Practise 11: 110-113. 6. Naglic, T., J. Frey, D. Hajsig, K. Busch, B. Seol.1998. Epizootiological and microbiological study of infectious keratoconjunctivitis in sheep. In: Mycoplasmas of ruminants: pathogenicity, diagnostics, epidemiology and molecular genetics, Volume 2. Ed. G. Leori, F. Santini, E. Scanziani & J. Frey, (Eds.). European Commission EUR 18018, pp. 168-171. 7. Egwu, G.O. 1991. Ovine infectious keratoconjunctivitis: an update. Vet. Bull. 61: 547-558. 8. Giacometti, M., J. Nicolet, K.-E. Johansson, T. Naglic, M.-P. Degiorgis, J. Frey. 1999. Detection and identification of Mycoplasma conjunctivae in infectious keratoconjunctivitis by PCR based on the 16S rRNA gene. J. Vet. Medicine B 46, 173-180. 9. Hayflick, L. 1965: Tissue cultures and mycoplasmas. Tex. Rep. Biol. Med. 23, Suppl. 1, 285-303. 10. Aarestrup, F.M., N.F. Friis. 1998. Antimicrobial susceptibility testing of Mycoplasma hyosinoviae isolated from pigs during 1968 to 1971 and during 1995 and 1996. Vet. Microbiol. 61: 33-33. 11. Hannan, P.C.T., G.D. Windsor, A. Jong, N. Schmeer, M. Stegemann. 1997. Comparative susceptibilities of various animal pathogenic mycoplasmas to fluoroquinolones. Antimicrob. Agents Chem. 41: 2037-2040. Zavod za mikrobiologiju i zarazne bolesti Veterinarskog fakulteta Sveu~ili{ta u Zagrebu Zagreb, 8. lipnja 1999. Predmet: prof. dr. sc. T. Nagli} - izvje{}e sa sudjelovanja u radu me|unarodnog simpozija MINISTARSTVO ZNANOSTI I TEHNOLOGIJE R. HRVATSKE STROSSMAYEROV TRG 4, 10000 ZAGREB prof. Jeli~i} Kao koordinator za Hrvatsku me|unarodnog projekta COST-826 sudjelovao sam u radu "International Symposium Mycoplasmas of ruminants", koji je odr`an u Toulouse u Francuskoj, u razdoblju od 2-4. lipnja, 1999. U tijeku simpozija upoznao sam ostale sudionike sa epizootiolo{kim podacima o mikoplazmozama u Hrvatskoj i radom na istra`ivanju osjetljivosti na{ih izolata vrste M. conjunctivae prema razli~itim antibioticima (izvje{}e: T. Nagli}, B. [eol, @. Kelneri}, K. Busch: In vitro susceptibility of field isolates of Mycoplasma conjunctivae to some antimicrobials). Izvje{}e je u obliku sa`etka otisnuto u Knjizi sa`etaka na str. 69., a naknadno }e biti u potpunosti objavljeno u Izvje{}ima sa simpozija. Na sastanku Upravnog odbora, kojeg sam ~lan, predlo`eno je da se petogodi{nji projekt istra`ivanja mikoplazmoza u pre`iva~a produ`i za 6 mjeseci, a da se sljede}i sastanak stru~njaka koji sudjeluju u projektu EU COST 826 odr`i na Kanarskim otocima. Svi sudionici simpozija naglasili su potrebu organiziranog suzbijanja mikoplazmoza pre`iva~a, koje su se pro{irene u mnogim europskim zemljama. To se posebice odnosi na zaraznu pleuropneumoniju goveda, infekcije vimena, di{nog i genitalnog sustava goveda vrstom M. bovis, zaraznu agalakciju ovaca i koza, infekcije malih pre`iva~a pripadnicima skupine M. mycoides, a po prvi puta se velika pozornost pridala i zaraznom keratokonjunktivitisu ovaca i koza. Sudionici projekta COST 826 iz Ma|arske pokrenuli su inicijativu za novi projekt, kojim bi se pod patronatom Italije od Europske unije osigurala sredstva za istra`ivanja mikoplazmoza u pre`iva~a u Ma|arskoj, Poljskoj, ^e{koj i Hrvatskoj. Osnovna je svrha tog projekta da se ustanovi prisutnost, pro{irenost i gospodarske {tete od mikoplazmoza u tim zemljama, posebice uzrokovanih pripadnicima skupine M. mycoides i da se uvede etiolo{ka dijagnostika. S obzirom na nepovoljnu situaciju u vezi s najva`nijim mikoplazmozama u ve}ini zapadnoeuropskih zemalja, na osnovi tih istra`ivanja moglo bi se objektivno utvrditi ili isklju~iti prisutnost tih bolesti u nas i sprije~iti njihovo uno{enje uvozom `ivotinja iz inozemstva. prof. dr. sc. T. Nagli} Zavod za mikrobiologiju i zarazne bolesti Veterinarskog fakulteta Sveu~ili{ta u Zagrebu Zagreb, 9. lipnja 1999. FAKULTETSKO VIJE]E Ovdje Izvje{}ujem Vije}e da sam od 1-6 lipnja 1999. sklopu projekta Europske unije COST 826 sudjelova o radu: International Symposium Mycoplasmas of ruminants, koji je odr`an u Toulouse u Francuskoj, sa izvje{}em: T. Nagli}, B. [eol, @. Kelneri}, K. Busch: IN VITRO SUSCEPTIBILITY OF FIELD ISOLATES OF MYCOPLASMA CONJUNCTIVAE TO SOME ANTIMICROBIAL AGENTS. Prof. dr. sc. T. Nagli} Protutijela za virus imunodeficijencije i virus leukemije u ma~aka u Hrvatskoj Seroepidemiolo{ka istra`ivanja ma~je imunodeficijencije i leukemije u Hrvatskoj Єx.ЅТAІŠЇŠЈŠЉŠŒаЯрЁБс;ўџ ўџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџSummaryInformation(џџџџџџџџџџџџ@џџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџ......@†'ЈgvЇО@†п㇕БО@†щЧ6БО@ъљлMicrosoft Word 6.018џџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџ&/Tk‰•—ЄЅЖЗЧЪЬXZfяљћќџ ОЮир№џя   2 A Ч Ъ г у C D Ц в ‡špqРСзцфх89ˆ‰цчэюŠ‘РЯKcdГДМНќіёіёќьќьќьќьщчщчќёќтќтќтќтќтќтќёќтќтќтќтќьќтќтќмќьќтќмќмќмќьќьќьќёќтќтзгЭгзгЧ J>]c Jm]c]cV]c Jm]cV]c]]h]chU]c UV]c]cNЎопыьР!du‚ƒ•–ЂЃЈЊЙКЦЧвдхцђѓџ ,-34>@JKTU]_ghopuw~ˆ‰’š›ЇЈГЕСТЮЯкмољ@HfuЄГ##œ#Ћ#V$^$r$ќњїњїњѓќюќюќшќюќшќюќюќюќюќюќюќюќюќюќюќюќюќюќюќюќюќюќюќюќюќюќюќюќюќюќюќюќюѓуѓоѓоѓоѓоѓоѓоѓV]cU]c Jm]cV]c]c]h]]cXr$$**l*n*љ*+&+(+ю+,,,~,•,О,Ш,Щ,Э, --V-X-і-ј-//Ÿ/Ж/00k0m0г0н0о0щ0;1=1222З4Ц45%5J5a588`8k8Y:d:`<f<b=j==І=р=с=т=‡>Ё>ћїђющюфющюфющюфюфюфющющюфющюфющющюфюфющющюрлрлрлрлрлрлржргбгбЯбрЭu]]V]]^cV]c]cV]cU]c]cU]c]cV]cA&'‰ŠЩЪXYZghю я   Ц ‡з#‰Š’“cdeМНОТЦШЪагйм§њњd#њd#њd#њd#њd#фњd#фњd#§§d#§d#§џd#r§d#§d#яя§§џd#w§џd#‰§d#§d#§d#§d#§џd#*§d#ээd#ўщd# фэррўрўр^ўр|ўруўр.ўр—ўрў& &  7рp(мрстшщъ№їјў  "#%)*067<CDIќБЎЎvўЎvўќќўќўќўќўќўќ^ўќ^ўќ^ўќўќўќўќўќўќўќўќўќўќ^ўќ^ўќ^ўќ5ўќ5ўќ5ўќўќўJИlЛ О| ”џсСЁ и +цыY9!5&џџ IPQRZ[ahijovwy}~€…†Œ’“•™šŸЃЄЊВГЙРСЧШЩЯзмухќщццvўќќўќўќўќўќўќўќ^ўќ^ўќ^ўќўќўќўќўќўќўќўќўќўќ^ўќ^ўќ^ўќ5ўќ5ўќ5ўќўќўќўщўццvўцvўќќўќўќўќ^ўИlО ”џсСЁ и >7D!5&)хщы№іќ  '(./05;AIMQSX^dfjlpv}‚Š‹‘’˜ Ёќќўќўќўќўќўќўќ^ўќ^ўќ5ўќ5ўќўќўщўццvўцvўќќўќўќўќ^ўќ^ўќўќўќўќўќўќўќ^ўќ^ўќ5ўќ5ўќўќўщўццvўќќўќўИlО ”џсСЁ и >7D!5&)ЁІ­ЏГЕКРФЦЪЮдйрхьэѓєѕћ!%'+-16=BIJQRќќўќ^ўќ^ўќўќўќўќўќўќўќ^ўќ^ўќ5ўќ5ўќўќўщўццvўцvўќќўќўќўќ^ўќ^ўќўќ ќўќўќўќўќ^ўќ^ўќ5ўќ5ўќўќўщўццvўИlО ”џсСЁ и >7D!5&)RX`fnptv{€„†ŠŽ”™ ЅЌ­ПР !tuƒ–Љќќўќўќўќ^ўќ^ўќўќўќўќўќўќўќ^ўќ^ўќ5ўќ5ўќўќўЗўДДd#фВВd#Дd#ўДd#ўЎЋЋ; DИlО| ”џсСЁ и >7D!5& ЉЊКЧгдцѓ -4?@KU^_hpvw‰‘’›ЈДЕТЯлх; соо; ўв; ўсоо; ўв; ўсоо; ўв; ўсоо; ўв; ўсоо; ўв; ўсоо; ўв; ўсоо; ўв; ўсоо; ўв; ўсоо; ў ИlО ”џЈ Ма#ИlО(”џЈ Ма# $лмнољћn'****z*.+",-c-Х.$/0„0F12@2n2o2‡2ˆ2т2у2ф2363D3E37š;›;œ;Д;Ж;х; ўууd#рd#рd#рџd#i р d#рd#рd#рd#рџd#рџd#рџd#рџd#рd#рd#ўрd#рџd#рџd#рџd#рџd#ррd#њрd#њрd#њрd#њрd#њрd#њрd#њннd#њнd#њнd#њррd#њрd#њрd#њрd#њрИlО(”џЈ Ма# 'Ж;З;И;Й;К;ф;<<+<,<-<@<A<f<g<Ц=Ч=Ш=Щ=Ъ=т=у=ф=х=ц=6>†>‡>§§d#њ§d#њ§d#њ§d#њ§d#њ§d#њ§d#њ§d#њ§d#њњњd#њњd#њ§§џd#З§d#ф§d#ф§d#ф§d#ф§d#ф§d#њ§d#њ§d#њ§d#њ§§d#њ§d#њK@ёџNormala "A@ђџЁ"Default Paragraph Font‡; ‡> џџџџ!џџ€!џџ„!џџ!џџ!џџ џџ џџ‡ /0Š‹u!/Е8ч:‡;P[*Or$Ё> !"мIхЁRЉлЖ;‡>#$%&'()*+z...'C:\DOCUMENT\PROJEKTI\STRANI\TOUPOST.DOC...A:\TOUPOST.DOC...A:\TOUPOST.DOC...A:\TOUPOST.DOC...A:\TOUPOST.DOCџ@HP LaserJet 5LLPT1:HPWHP LaserJet 5L PDиoъ odXџџXLPT1:22ћЌџSP51HP LaserJet 5L PDиoъ odXџџXLPT1:22ћЌџSP51€ч:ч:€ч:ч:A&Times New Roman Symbol &Arial&Arial CRO"‰h3г5fЖB6F{B6F‡ƒќ"{aIN VITRO SUSCEPTIBILITY OF FIELD ISOLATES OF MYCOPLASMA CONJUNCTIVAE TO SOME ANTIMICROBIAL AGENTS......аЯрЁБс;ўџ ўџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџ