ࡱ; 23  !"#$%&'()*+,-./01456789:;<Root Entry F8˾CompObjbWordDocumentEmObjectPool8˾8˾  FMicrosoft Word 6.0 Document MSWordDocWord.Document.6;  Oh+'0 D h   @d (C:\MSOFFICE\WINWORD\TEMPLATE\NORMAL.DOT-FURTHER EPIZOOTIOLOGICAL AND MICROBIOLOGICAL......ܥe3 eZEmWhhhhhhhpipipipipiiipijlAii"iiiiiiiiiij0kLllTlFjlhi.1iiiijlihhiiiiiihihiihDhrhhhhiii+i EPIDEMIOLOGICAL AND MICROBIOLOGICAL STUDY OF AN OUTBREAK OF INFECTIOUS KERATOCONJUNCTIVITIS IN SHEEP Tomo Nagli}1, Danko Hajsig2, Joachim Frey3, Branka [eol1, Kristina Busch2, Mirko Lojki}4 1Department of Microbiology and Infectious Diseases, Veterinary Faculty University of Zagreb, 10000 Zagreb, Heinzelova 55, Croatia 2PLIVA d.d. Zagreb, Ulica grada Vukovara 49, 10000 Zagreb, Croatia 3Institute for Veterinary Bacteriology, Laenggasstrasse 122, CH-3012 Berne, Switzerland 4Croatian Veterinary Institute, Savska 143, 10000 Zagreb, Croatia Summary Connected with the import of several thousand sheep from Australia and New Zealand in Croatia during 1995, a great number of native sheep that have been in contact with imported animals acquired a severe ocular disease closely resembling ovine infectious keratoconjunctivitis (OIKC). In affected flocks glucose-fermenting mycoplasma were isolated from 48% and Branhamella ovis from 58% of microbiologically examined conjunctival swabs. As representatives, 12 of 42 culturally and biochemically identical isolates were identified as Mycoplasma conjunctivae by PCR. From the conjunctivas of two animals M. conjunctivae and M. arginini were isolated in mixed culture. For many reasons most of the farmers eliminated imported animals from their flocks and only sporadic cases of OIKC were recognized in 1996. By the end of 1997 six flocks clinically free from OIKC but affected with this disease in the course of 1995 and five flocks with no history of the severe ocular disease at all underwent clinical and microbiological examinations and all were found free of M. conjunctivae infection. At the time Branhamella ovis was almost exclusively cultured from the sheep originated from the flocks affected with OIKC during 1995 and/or 1996. It was customarily found in pure culture or as predominant bacterial species, and frequently was accompanied with mild conjunctivitis. Its implication in pathogenesis of OIKC or conjunctivitis, respectively, requires further investigation. There was not microbiologically evidenced a new case of OIKC during 1998. Related to costs of medication and need of special nursing, anxiety of the farmers caused by severity of clinical signs, and other inconveniences, there is a strong suggestion that the import of OIKC in the countries free of infection should be controlled. Key words: Mycoplasma conjunctivae, Mycoplasma arginini, Branhamella ovis, ovine infectious keratoconjunctivitis Introduction Ovine infectious keratoconjunctivitis (OIKC), also described as "pink eye" (Hopkirk, 1934), is an acute contagious disease occurring throughout the world (Egwu, 1991; Jones, 1991). It is characterized by inflammation of the conjunctiva and cornea, often resulting in a period of temporal or even persistent blindness (Egwu et al., 1989; Greig, 1989). Although lambs as young as 5-10 days can become ill (Jones et al., 1976), adult animals experience more severe symptoms (Greig, 1989). In many cases ocular changes regress spontaneously, but relapses are not unusual. An outbreak of OIKC can be expected in all seasons of the year, particularly during the pasture season (Nicolet and Freundt, 1975). Concerning frequent relapses, regardless of treatment, and need of special nursing during the period of reduced vision or total blindness, the disease is of equal nuisance to veterinarians and farmers (Egwu et al., 1989; Egwu, 1991). Although being clinically well defined and easily recognized, the etiology of OIKC still remains uncertain (Jones et al., 1976; Mayer et al., 1996). Numerous microorganisms have been suggested as etiologic agents, but the cause and possible predisposing factors of the disease are still disputable (Egwu, 1991). Among a great variety of microorganisms that have been incriminated as causative agents special attention has been focused on Branhamella (B.) ovis, Chlamydia psittaci and Mycoplasma (M.) conjunctivae. Recent investigations indicate that M. conjunctivae could be the primary etiologic agent of OIKC (Egwu, 1991; Giacometti et al., 1998). This paper reports our microbiological and epidemiological observations during the first occurrence of OIKC in Croatia. Case report In early 1995, approximately 6,000 sheep (Corriedale, Comeback, Crossbreed I., Poll Dorset, Border Leicester) from Australia and New Zealand were donated to Croatia. After quarantine they were distributed to farmers all over the country. Native and imported animals were kept either together or in separate flocks, about 20 to 40 animals in each. During spring and summer of the same year a great number of native sheep that have been in contact with the imported animals developed signs of a severe ocular disease closely resembling infectious keratoconjunctivitis. At the time of the first monitoring, 20% to 80% of native animals had ocular changes involving one or both eyes. The signs of the disease varied from mild conjunctivitis with hyperemia of palpebral conjunctiva and clear ocular discharge observed in some cases to marked keratoconjunctivitis in most of them. Severely diseased animals showed the signs of blepharospasm and marked photophobia. They frequently stood with their eyes closed or bumped into barriers when in motion. In more advanced cases mucopurulent keratitis with opacity and blood vessels migrating into cornea and corneal ulceration were seen. There was a history of an ocular disease in imported sheep during the prolonged transportation or quarantine. At the time of our observation, a majority of these animals appeared healthy, or showed the signs of mild conjunctivitis. Keratoconjunctivitis with copious ocular discharge and marked conjunctival hyperemia in imported sheep occurred sporadically. The reported outbreaks of the disease in native sheep were strongly related to their mixing with the imported animals. Those unexposed directly to the imported animals remained healthy even if housed in neighbouring pens. In some cases the animals were probably infected during common pasture. In the beginning of the epizootic only topical or systemic treatment of affected sheep was predominately applied using oxytetracycline eye ointment and oxytetracycline dehydrate (Geomycin Retard, PLIVA Zagreb), respectively. The topical treatment was predominately effective only for mildly affected animals and if oxytetracycline ointment was used two or three times daily during one week. Five or six days after the initial amelioration, clinical signs usually relapsed requiring further treatment. The similar effects were achieved with single or triple parenteral administration of only long acting oxytetracycline. The most effective was a combination of daily topical application of oxytetracycline eye ointment and triple (on days 0, 3 and 6) intramuscular injection of oxytetracycline dehydrate. In this manner treated animals usually recovered in three to six days and relapsed more rarely. For many reasons, in the course of 1996 most of the farmers completely eliminated imported sheep from their flocks and only sporadic outbreaks of OIKC occurred among native sheep during this year. At the time of our second monitoring by the end of 1997, all 11 inspected flocks located in different regions of Croatia were clinically free from OIKC. No new cases of OIKC were diagnosed officially during 1998. Material and methods First monitoring (1995). From May to December 1995 conjunctival swabs from 101 native and imported sheep, including two lambs and two rams, of different ages and clinical status, were taken for microbiological examinations. The animals originated from 14 flocks located in different parts of Croatia, 88 of them were from 12 OIKC affected flocks and 13 sheep were from 2 healthy flocks which had no contact with infected animals. Most of the affected flocks had been treated with antibiotics before sampling. Second monitoring (1997). During the last three months of 1997, six currently healthy flocks which were affected with OIKC in the course of 1995, and five flocks with no history of the severe ocular disease underwent clinical and microbiological examinations. The flocks, with 20 to 116 animals in each, are located in central, northern and western parts of Croatia. The sheep in the flocks are of different native breeds (Pramenka, Cigaja-Suffolk, Solcavka, Istrian Sheep, Wuertemberg cross-breed). Imported animals were completely eliminated from all but one previously affected flock, in which eleven imported animals were kept together with 105 of native sheep. In each of the previously affected flocks examined, farmers could identify at least some animals which showed typical ocular changes during 1995 or 1996. The animals from the flocks with no history of OIKC had never been in contact with animals imported from abroad. At the time of monitoring all inspected sheep were clinically free of OIKC but about 10% in each flock showed the signs of mild conjunctivitis. The highest number (about 30%) of animals with conjunctivitis was found in a flock with native and imported sheep kept together. At that time, conjunctival swabs were taken for microbiological examination from 67 animals that originated from 6 previously infected flocks, and from 38 animals derived from the 5 flocks with no history of OIKC. The age of animal varied from several months to five years. Methods. Sheep blood agar plates and PPLO agar and broth (Hayflick, 1965) were inoculated with ocular swabs immediately after sampling. Cultivation and identification of isolated bacteria and mycoplasma were performed according to the established diagnostic techniques (Razin and Tully, 1983; Holt et al.,1994). In the identification of bacteria, special attention was paid to branhamella, Escherichia coli, staphylococci, streptococci and pseudomonas. Other opportunistic and saprophytic microflora were only provisionally identified if more than 5 colonies were grown up. At the first and second monitoring, the smears prepared from ocular swabs from 20 sheep were stained by Giemsa and modified Ziehl-Neelsen methods for microscopic detection of chlamydia (Stephenson et al., 1974; Greig, 1989). Additionally, at the time of first monitoring, the ocular materials from 12 diseased sheep were also inoculated into 7-days old chicken embryos and yolk sac impression smears were stained by the above mentioned methods. Molecular identification of Mycoplasma conjunctivae was made by a PCR method which amplifies the specific parts of the rrs gene of M. conjunctivae (Giacommetti et al., 1999). Results and Discussion At the first monitoring, glucose-fermenting mycoplasmas were cultivated from 42 (48%) out of 88 examined conjunctival swabs from affected flocks. Two of these isolates were contaminated with arginine-hydrolyzing strains. Where ocular swabbing had not preceded the therapy, the rate of infected animals in individual flocks was 77%. Although rarely with clear-cut clinical changes, the imported animals were infected with mycoplasmas in higher percentage (60%) than the native (38%) ones. (Table 1) In affected flocks, mycoplasmas were isolated from clinically affected and unaffected animals. They were, however, never found in the eyes of the animals originating from native flocks with no history of OIKC. Mycoplasma isolated from 42 animals grew in brownish colonies. They were glucose-positive and arginine-negative, digitonin-sensitive, film and spots- and phosphatase-negative. Two strains separated from mixed cultures were also digitonin-sensitive, phosphatase-negative, but hydrolyzed arginine and did not ferment glucose, and their colonies were not brownish. From the glucose-fermenting mycoplasma isolates, 12 were further analyzed by PCR amplification. This included amplification of the entire rrs gene with universal prokaryotic primers, amplification of parts of rrs gene specific to M. conjunctivae and a PCR method for the identification of M. agalactiae (Gonzales et al., 1995; Giacometti et al., 1999). These isolates were shown to be M. conjunctivae by the amplification of the specific 748 bp PCR fragment (Fig. 1). It therefore can be assumed that the majority of the other isolates with the same cultural and physiological characteristic also belonged to this species. Two arginine-positive isolates were identified as M. arginini. Branhamella ovis was recovered from 51 (58%) conjunctival swabs out of 88 examined from effected flocks. It occurred in higher percentage in imported (85%) than in native sheep (35%). Thirteen (33%) of 39 examined and clinically affected native sheep and 4 (33%) of 12 examined and apparently healthy were infected with B. ovis. The bacteria was also found in 8 (89%) of 9 examined and clinically affected and in 20 (61%) of 33 examined and apparently healthy imported animals. It was not found in any case in the native sheep from two uninfected flocks. All isolates were glucose nonfermenting gram-negative cocci, growing in "S" form, catalase- and oxidase-positive, urease-negative and (-hemolytic (sheep erythrocytes). Other bacteria belonging to genera Staphylococcus, Streptococcus, Bacillus or Escherichia were only occasionally found in affected or unaffected ovine eyes, predominately in mixed cultures. Chlamydias were absent in all cases. At the second monitoring by the end of 1997, colonies suspected to be Mycoplasma sp. grew up from conjunctival swabs of four native sheep that originated from a flock affected with OIKC during 1995. Two isolates were slow growing and two were fast growing glucose-positive and arginine-negative strains. The isolates did not require serum for growth and grew at room temperature. Slow growing strains were isolated from the eyes with the signs of mild conjunctivitis in pure culture or together with B. ovis. One fast-growing strain was isolated in pure culture from an animal with mild conjunctivitis, and the other one with coliforms from apparently healthy eyes. The isolates were not serologically identified, but regarding the cultural and biochemical characteristics they obviously did not belong to M. conjunctivae. The overall results of bacteriological examinations are presented in table 2. (Table 2) In the flocks affected with OIKC during 1995, B. ovis was customarily found in pure culture or as predominant bacterial species, frequently accompanied with mild conjunctivits. Branhamellas were isolated more often from animals less than one year old (44%) than from those aging 1-3 years (33%) and over 3 years (22%). The conjunctivitis was characterized with serous lachrymation and mild hyperemia of palpebral and bulbar vessels. Using microscopical evidence, chlamydia were not detected in any examined specimen. Our microbiological and epidemiological investigations and clinical observations during the first epizootic of OIKC in Croatia support the thesis that OIKC is primarily a mycoplasmal disease caused by M. conjunctivae as described previously (Nicolet and Freundt, 1975; Egwu et al., 1989). This microorganism was found in diseased as well as in apparently healthy animals, but always in association with occurrence of OIKC in the flock. Mycoplasmal infections were commonly accompanied with B. ovis isolated almost exclusively from sheep affected with OIKC or which originated from affected flocks. Its implication in pathogenesis of OIKC as an associated causative agent or as a secondary pathogen requires further investigation. Clinical and microbiological observations indicate that B. ovis might be the only microbial agent sporadically causing mild conjunctivitis. All investigations clearly indicated that the epizootic of OIKC in native sheep was related to the import of infected animals. Introduction of imported sheep and rams into the flocks and direct contact with them were mostly responsible for the appearance of the disease in native sheep. The first observations raise expectations that prompt elimination of imported animals and acquired resistance of native sheep resulted with eradication of OIKC in Croatia. Our negative results at the second monitoring, however, can not exclude the possibilities of new cases of OIKC after diminished flock immunity. Therefore, methods more sensitive than isolation of mycoplasmas by cultivation will be needed to exclude animals carrying M. conjunctivae and to prevent from reinfection of flocks with the agent (Giacometti et al., 1999). Related to costs of medication and need of special nursing during the period of reduced vision or total blindness, anxiety of the farmers caused by severity of clinical signs, and other inconveniences, we suggest that in countries free of infection small ruminants should be controlled by strict veterinary regulations in order to prevent from outbreaks of OIKC. . References Egwu, G. O., W. B. Faull, J. M. Bradbury, M. J. Clarkson (1989): Ovine infectious keratoconjunctivitis: a microbiological study of clinically unaffected and affected sheep's eye with special reference to Mycoplasma conjunctivae. Vet. Rec. 125, 253-256. Egwu, G. O. (1991): Ovine infectious keratoconjunctivitis: an update. Vet. Bull. 61, 547-559. Giacometti, M., J. Nicolet, J. Frey, M. Krawinkler, W. Meier, M. Welle, K-E. Johansson, M. P. Degiorgis (1998): Susceptibility of alpine ibex to conjunctivitis caused by inoculation of a sheep-strain of Mycoplasma conjunctivae. Vet. Microbiol. 61, 279-288. Giacometti, M., J. Nicolet, K-E. Johansson, T. Nagli}, 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. Med. B 46, 173-180. Gonzales, Y. R. C., C. R. Bascunana, G. Bolske, J. G. Mattsson, C. F. Molina, K-E. Johansson (1995): In vitro amplification of the 16s rRNA genes from Mycoplasma bovis and Mycoplasma agalactiae by PCR. Vet. Microbiol. 47, 183-190. Greig, A. (1989): Ovine keratokonjunctivitis. In Practice 11, 110-113. Hayflick, L: (1965): Tissue cultures and mycoplasmas. Tex. Rep. Biol. Med. 23, Suppl. 1, 285-303. Holt, J. G., N. R. Krieg, P. H. A. Sneath, J. T. Staley, S. T. Williams: Bergey's Manual of Determinative Bacteriology. 9th ed. Williams & Wilkins. Baltimore, Philadelphia, Hong Kong, London, Munich, Sydney, Tokyo, 1994. Hopkirk, C. S. M. (1934): Pink eye in sheep. N. Z. J. Agriculture 48, 224-225. Jones, G. E., A. Foggie, A. Sutherland, D. B. Harker (1976): Mycoplasmas and ovine keratoconjunctivitis. Vet. Rec. 99, 137-141. Jones, G. E.: Infectious keratoconjunctivitis. 1991. In: W. B. Martin and I. D. Aitken (ed); Diseases of sheep. Blackwell Scientific Publications, London, pp. 280-283. 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. Nicolet, J., E. A. Freundt (1975): Isolations of Mycoplasma conjunctivae from chamois and sheep affected with keratoconjunctivitis. Zbl. Vet. Med. B. 22, 302-307. Razin, S., J. G. Tully: Methods in Mycoplasmology. Vol.1. Mycoplasma characterization. Academic Press. Inc., New York, London, Paris, San Diego, San Francisco, Sao Paulo, Sydney, Tokyo, Toronto,1983. Stephenson, E. H., J. Storz, J. B. Hopkins (1974): Properties and frequency of isolation of chlamydiae from eyes of lambs with conjunctivitis and polyarthritis. Am. J. Vet. Res. 35, 177-180. Table 1. Occurrence of bacteria and mycoplasma in ovine eyes from infected flocks --------------------------------------------------------------------------------------------------------------- Microorganism Native sheep (48*) Imported sheep (40*) Total (88*) ___________________________________________________________________ Mycoplasma sp. 18 (38%) 24 (60%) 42 (48%) Branhamella ovis 17 (35%) 34 (85%) 51 (58%) Other bacteria 10 (21%) 19 (48%) 29 (33%) _______________________________________________________________ * Number of examined animals Table 2. Bacteria and mycoplasma in ovine eyes at the second monitoring --------------------------------------------------------------------------------------------------------------- Microorganism Flocks with history of Flocks without history Total OIKC OIKC (67 examined animals) (38 examined animals) (105) --------------------------------------------------------------------------------------------------------------- Mycoplasma sp. 0 0 0 Acholeplasma sp. 4 (6%) 0 4 (4%) B. ovis 27 (40%) 1 (3%) 28 (27%) E. coli and coliforms 3 (4%) 5 (13%) 8 (8%) Bacillus sp. 10 (15%) 24 (63%) 34 (32%) Staphylococcus sp. 17 (25%) 21 (55%) 38 (36%) S. aureus 5 (7%) 1 (3%) 6 (6%) Streptococcus sp. 11 (16%) 5 (13%) 16 (15%) Negative 22 (33%) 8 (21%) 30 (29%) --------------------------------------------------------------------------------------------------------------- Figure 1. Confirmation of M. conjunctivae by PCR. Colonies suspected to be M. conjunctivae were lysed and submitted to specific PCR analysis as described (Giacometti et al., 1999). The photograph shows the analysis of the PCR products from 7 strains on a 1% agarose gel, stained with ethidium bromide and photographed upon exposure to UV light. + indicates the positive control M. conjunctivae type strain HRC/581T , ( is the negative control. Std is the molecular mass standard phage ( digested by Hind III. The arrow indicates the specific band of DNA of 748 bp. 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