A. O. Vet. Fak. Derg.
32 (1): 172--177, 1985
ISOLATION A?\D CHARACTERIZATlON OF :'JEISSERIA OVIS fROM OVI?\E KERATOCO:'\JU:\CTIVITIS
K.Serdar Diker* Zeki Alkan * *
Keratokonjuktivitisli koyunlardan Neisseria ovis izolasyonu
Özet: lnfiksiyö'z keratokonjuktivitisli kuzulardan bir Neisseria ovis suşu izole edildi. Hasta ha)'vanların konjuktivalarından alınan ekü1!Y0niar kanlı agar besiyri üzerine ekiidiler. Gram negatif, hemolitik koklar saf olarak izole edildiler. Koloniler, gri renkli, yarı saydam, "S" tipinde, düzgün ke-narlı konveks ı'e belirgin olarak hemolitik idiler. Hareketsiz ve spor oluştur-mayan bakteri çoğunlukla diploid şekilde görüldü. Penisilin varlığında, hüc-relerde uzama görülmedi. Organizma, buyyonda parçalanabilir bir pelikül, biraz bulanıklık ve granüllü bir çüküntü oluşturdu. hole edilen organizma ok-sidaz ve katalaz pozitif bulundu. Karbonhidratların hiçbirisinden asit üretimi gözlenmedi. Nitrat, nitrite indirgendi. holat indol ve hidrojen sülfit oluştur-madı. Ureaz, DNAaz, Foifata;::, MR ve VP testleri negatif bulundu. Eriye-bilir bir hemolizin belirlenmedi. Organizma, denenen tüm antibiyotiklere duyarlı bulundu. Parenteral ve konjuktival yolla N. ovis verilen fareler hiçbir semptom göstermediler.
Sunınıary: A Neisseria ovis strain was isolated from lambs with infectious keratoconjunctivitis. Swabs taken from the conjunctival sac of dise-ased animals were inoculated onto blood agar plates. The pure czıltures of gram-negative hemolytic cocci were isolated. The colonies were gre.J'ish, translucent, smooth, convex wıth regular ed,ges and hemolytic with a dear zoııe. The bacteriae were non-motile, non-spor forming which often occured iıı diploid arrangement. Elongation was not seen in the presence of penicilline. In broth culture, organism produce a fragile pellide, some turbidity and a gramdar deposit. The isolated organism was oxidase and catalase positive. No acid production lOas obser-ved from mry of the carbohydrates tested. Nitrate was reduced to nitrite .
•• Dr., A.Ü.Veteriner Fakültesi, Mikrobiyoloji Anabilim Dalı, Ankara, Turkiye . ••" Araş.Cör.,A.Ü.Vcıcriner Fak~ilıesi, Cerrahi Anabilim Dalı, Ankara, Turkiyc.
The isolate did not produce indole and hydrogen suljideo Urease, DNAase, phosphatase, /1;/ R, and VP tests were negative. A filtraUe hemolysin was not
demonstrated. The organism was sensitive to all antibiotics tested. Mice exposed to parenteral and conjunctival administratioll of N. ovis did not show any symptom.
Introduction
lnfectious keratoconjunctivitis (pink-eye) is an acute conta-gious disease of ruminants. The etiological agent of ovine keratocon-junctivitis has not been definetely demonstrated; some investigators (4, 7) have deseribed Rickettsia, Chylamidia and various Gram-negative bacteria as being associated with the disease. In 1960, Ne-isseria ovis, as a new species of the genus Neisseria has been isolated from sheep with keratoconjunctivitis (6). Since then, a limited number of investigators have reported the isolation of N. ovis from both heal-thyand diseased eyes of sheep (I, 5, 7, 13, 14,
ı
6). This organism has also been isolated from cattle with and without keratoconjunctivitis (8, iO, 12, i8) and frcm a:' alonrted boyine fetus (I ı).This paper describes the isolation and characterization of a Ne- o isseria ovis strain from the eye s of lambs with keratoeonjunctivitis in Turkey.
Materials and Methods
Two lambs with the symptoms of keratoconjunctivitis were brought to the clinic. There were lachrymation, s,velling eye-lids, and corneal opacity in h, ıt!: cyes of effected lambs. Samples were collected from both eyes of two lambs, using a cotton wool swab. The swabs were inoculated onto iO per cent slıeep blood agar plates, wlıich were incubated aerobically at 37°C for 24-48 hours.
After incubation period, isolateci colonies.were stained by Gram's method. Diftcrentiation betwccn cocci and rods was alsa carricd out by observing cellular elongation in the prescnce of renicilline. A disc of penicilline G was placed on the surface of blood agar inoculated with suspected organism. After 18 lı incubation at 37oC, a film was made from the edge of the zone of inhibition and examined by phase contrast microscopy for the cvidenee of elongation. Motility was
174 K. SERDAR DIKER - Z. ALKAN
determined by phase-contrast iııumination and in semi-solid broth. Pigment produetion was obseved on egg-yolk agar. The ability of growth at 20 oC and 22°C, on MeConkey agar and in the presenee of2% and 5 % NaCl was also investigated.
The oxidase and eatalase tests were earried out using Baet~ ident Oxidase (Merek) and 5 per cent Hı02ı respectivcly.
Carbohy-rat c fermentation studies wc re performed in Trypticase Soy Broth (TSB) with i % of the sugar under test addcd. The sugars tested were adonitol, arabinose, dulcital, fruetose, galactose, glueose, lactose, mal-tosc, mannitol, mannose, rhamnose, sorbitol, suerose and xylose. Nit-rat c reduction was test ed in a medium eontaining O.i
%
Kalium nitrate in a TSB basc. Tests for urease, DNAase, phosphatase, indol and hydrogen sulfidc production and MR and VP tests were also per-formed.The filtrate of broth eulture was tested for hemolytie aetivity. Sensitivity to antibioties was determined by di sc difIusian method. Foııowing antibioties were used; ampicillin, earbeııicillin, ehloramp-henieol, ehlortetraeyclin, eoiistin sulfate, erythromyein, gentamicin, neomyein, nitrofurontoin, oxytetraeyelin, rifamycin, streptomyein, tetraeyelin, trimethoprim and penieillin.
To test the pathogenieity of organism, an inoeulum was pre-pared from IS h broth culture. This inoeulum was injeeted iv and ip the groups of 3 miee (O.i ml eaeh) and inoeulated to the con-junetivae of miee b
f
swabbing.Results
. The pure cultures of gram-negative, Iıemolytie eoeci were obta-ined from eaeh of blood agar platcs. The isolated organism was named as Neisseria ovis according to the basis of foııowing criteria.
The eolonies were greyish, translueent, smotlı, convex with rc-gular edges and hemolytic with a elear zone. The colonies ,;,,"eredif-fieult to emulsify and cl suspension in saline was usuaııy granular.
Theisolated organism eonsisted cxclusively of gram-nega-tive coed of irregular size with a tendeney to retain variably the gram stain. The baeteriac were non-motile, non-spor forming whiehoften oeeurcd in diploid arrangament. Elongation was not seen in the' pre-sence of penieiııine.
Organism grew well on solid media with or without blood. No growth on McConkey agar and no pigmentation on egg yolk meditim wc re observed. In broth culture, organism produce a fragile pellicle, some turbidity and a graular deposit. Very scant growth was detec-ted at 20°C and
noc.
The organism had the ability to grow in the presence of 2 percent NaCl but not in 5%
NaCL.The isolated organism was oxidase and catalase posltıve. No acid production was observed from any of the carbohydrates testcd. Nitrate was reduced to nitritc. The isoIate did not produce indol and hydrogen sulfite. Urease, DNA ase, phosphatase, MR and VP tests wc re negative. A filtrablc hemolysin was not dctermined. The organism was sensitiye to all antibiotics tested.
The mice exposed to parenteral and conjunctival administra-tion of N. ovis did not show any symptom.
Discussion and Condusion
In 1960, Lindqvist (6) described a Neisseria strain associated with infectious keratoconjunctivitis in Norwegian sheep and propo-sed the species name of Neisseria ovis. In this study, results obtained from the characterization of isolated strain from ovine keratoconjunc-tivitis justify the placing of this organism within the Novis. The morp-hological, cultural and biochemical characteristics of N. ovis isolated in this case were also similar to the N. ovis strains İsolated by Fraser and Gilmour (3) and Wilcox (18). Differentiation of Novisfromother Neisseria sp. and kloraxella sp. was readily performed by means of c'ar-bohdydrate fermentation, catalasc ad nitrate tests and elongation İn the presence of peniciIlin. Original N. ovis strain was sensitive .to all antibiotics tested. Wilcox (ı 8) has also found that Novis strains were sensitiye to same antibiotics tested İn the present study.
Madrid and Terzolo (7) and Buczek et al (I) have reported the outbreaks of infectious keratoconjunctivitis associated with N. ovis and showed some unique features in that the disease occured only in lambs and started in he first few hours of life. In the present study, N. ovis was similady isoalted from lambs. However, other wor-kers(6,9, 15) have not pointed out the specific sensitivity of lambs to this infection.
176 K. SERDAR DIKER - Z. ALKAN
The pathogenieity of N. oüis for cxperimental animals was in-vestigated by several workers. Lindqvist (6), Moreno ct al (9), Peder-sen (I 2) and Spradbrow (I 5) have found that Novis in large doses was lethal to miee and eould experimentall)' eause keratoeonjunet.i-vitis in sheep. However, in this study, attempts to produee an expe-rimental infeetion in miee did not give positive result. Similarly, Fairlie (2) and Spradbrow and Smith (I 6) have reported that no sym-ptoms were reeorded in the experimental animals inoeulated with N. ovis. PossibiIity of the strain of N. ovis became attenued after three or four passages on artifieial medium, the minimum number neces-eary to prepare a purc eulture and inoeulum, may be causc the lass of pathogenicity.
it is morc probablc that N. ovls ıs a commcnsal organism well adapted to survival in the ovine conjunctival sac and a predispo-sing factor, either iııfective or traumatic, is rcquired to initially lower the resistance of conjunctivae, bdüre the establisment of N.
ovis infCction. The precise role of N. oıis in eye infections of sh cep in this country requires further investigations.
References
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