Detection of Mycoplasma conjunctivae in sheep affected with conjunctivitis and infectious keratoconjunctivitis

AIM: To determine the aetiolog y of a recurring and severe form of infectious keratoconjunctivitis (IKC) in sheep.

METHODS: Five sheep flocks that had experienced a severe form of IKC were examined. Clinical history, conjunctival swabs and blood samples were collected from affected animals. Culture for bacteria, and also specifically for Mycoplasma and Chlamydophila spp, and detection of Mycoplasma conjunctivae DNA by polymerase chain reaction (PCR) were attempted. Serum samples were tested for antibodies to M. agalactiae, M. capricolum, M. conjunctivae and Chlamydophila spp.

RESULTS: Mycoplasma conjunctivae DNA was detected using PCR in 3/5 flocks, and in all flocks antibodies to M. conjunctivae were detected in sera. A pure growth of Branhamella ovis was cultured from conjunctival swabs from a small proportion of sheep in two flocks. No other pathogens were detected.

CONCLUSIONS: This investigation demonstrated that M. conjunctivae was a primary pathogen causing severe IKC in sheep, and is the first report of detection of this organism in sheep in New Zealand. Introduction of clinically normal carrier sheep appeared to have caused the outbreaks.     

Detection of Mycoplasma conjunctivae in sheep affected with conjunctivitis and infectious keratoconjunctivitis

AIM: To determine the aetiology of a recurring and severe form of infectious keratoconjunctivitis (IKC) in sheep. METHODS: Five sheep flocks that had experienced a severe form of IKC were examined. Clinical history, conjunctival swabs and blood samples were collected from affected animals. Culture for bacteria, and also specifically for Mycoplasma and Chlamydophila spp, and detection of Mycoplasma conjunctivae DNA by polymerase chain reaction (PCR) were attempted. Serum samples were tested for antibodies to M. agalactiae, M. capricolum, M. conjunctivae and Chlamydophila spp. RESULTS: Mycoplasma conjunctivae DNA was detected using PCR in 3/5 flocks, and in all flocks antibodies to M. conjunctivae were detected in sera. A pure growth of Branhamella ovis was cultured from conjunctival swabs from a small proportion of sheep in two flocks. No other pathogens were detected. CONCLUSIONS: This investigation demonstrated that M. conjunctivae was a primary pathogen causing severe IKC in sheep, and is the first report of detection of this organism in sheep in New Zealand. Introduction of clinically normal carrier sheep appeared to have caused the outbreaks. KEYWORDS: Infectious keratoconjunctivitis, Mycoplasma conjunctivae, Chlamydophila pecorum, Branhamella ovis, polymerase chain reaction, ELISA, complement fixation test.     

Detection and identification of Mycoplasma conjunctivae in infectious keratoconjunctivitis by PCR based on the 16S rRNA gene.

A specific PCR assay based on unique sequences of the rrs genes (16S rRNA) of Mycoplasma conjunctivae was developed for direct detection and identification of this pathogen from clinical material. DNA from eye swabs was amplified after a simple lysis step by either a single PCR with the M. conjunctivae specific primer pair McoR1 and McoF1, or by a nested PCR with the Mycoplasma genus specific primer pair MOLIGEN1-L and 16UNI-R in the first step and McoR1 and McoF1 in the second step. The specificity of the primer pair McoR1 and McoF1 was verified with purified DNA from the type strain, from 17 field isolates of M. conjunctivae and from several Mollicutes which are phylogenetically related to M. conjunctivae or which can be isolated from the same host animals. This method identified mycoplasma isolates from goat, sheep, ibex and chamois originating from different countries as M. conjunctivae. No cross amplifications with other mycoplasmas which are related to M. conjunctivae were observed. Eye swab samples containing known numbers of M. conjunctivae cells were analysed after direct lysis of the material. The detection level was estimated to be 20 cells per swab when the nested PCR procedure was used and 2 x 10(5) by the single PCR method. In an experimental infection model of sheep, the nested PCR method for detection of M. conjunctivae gave results which were comparable to mycoplasmal culture. These are the implications for diagnostic purposes: M. conjunctivae isolates can be identified by the one-step PCR method, whereas for detection and identification of M. conjunctivae in clinical material the two-step method should be used (higher sensitivity).     

Ovine keratoconjunctivitis experimentally induced by instillation of Mycoplasma conjunctivae

Five sheep, free from Mycoplasma conjunctivae and ocular Chlamydia infection, were experimentally inoculated with M. conjunctivae and five more sheep were exposed to the infection by contact. Keratoconjunctivitis developed in all ten sheep. As in natural outbreaks of infectious keratoconjunctivitis (IKC), clinical signs were generally moderate and transient, and recurred in some sheep. M. conjunctivae was detected throughout the 53-day observation period. Clinical diagnosis was confirmed by histopathologic examination of three sheep. Moraxella ovis, found in six of the ten sheep before the start of the experiment, appeared to play no etiologic role in the development of IKC.     

Ovine keratoconjunctivitis experimentally induced by instillation of Mycoplasma conjunctivae

Summary

Five sheep, free from Mycoplasma conjunctivae and ocular Chlamydia infection, were experimentally inoculated with M. conjunctivae and five more sheep were exposed to the infection by contact. Keratoconjunctivitis developed in all ten sheep. As in natural outbreaks of infectious keratoconjunctivitis (IKC), clinical signs were generally moderate and transient, and recurred in some sheep. M. conjunctivae was detected throughout the 53‐day observation period. Clinical diagnosis was confirmed by histopathologic examination of three sheep. Moraxella ovis, found in six of the ten sheep before the start of the experiment, appeared to play no etiologic role in the development of IKC.     

Ovine infectious keratoconjunctivitis: a microbiological study of clinically unaffected and affected sheep's eyes with special reference to Mycoplasma conjunctivae

In a field survey of ovine infectious keratoconjunctivitis, the microbiological flora of 240 clinically unaffected eyes from sheep in 10 flocks was compared with the flora of an equivalent number of clinically affected eyes from 12 natural outbreaks of the disease. Totals of 16 and 17 genera of bacteria were recovered from unaffected and affected eyes, respectively. Staphylococcus, bacillus and branhamella were isolated significantly more often than the other genera of bacteria, in both the unaffected and affected eyes (P less than 0.05). Branhamella ovis and Escherichia coli occurred more frequently in affected eyes, and Staphylococcus aureus occurred more frequently in severely than mildly affected eyes. The genera Mycoplasma and Acholeplasma were isolated from both groups, and Mycoplasma conjunctivae occurred in 92 affected eyes (38.3 per cent), and 27 unaffected eyes (11.3 per cent).     

Mycoplasma conjunctivae infection is self-maintained in the Swiss domestic sheep population

Bacteriological and serological investigations were performed to assess whether the domestic sheep population is a reservoir of Mycoplasma conjunctivae in Switzerland. Among a sample of 69 sheep showing clinical signs of infectious keratoconjunctivitis (IKC) in three Swiss cantons, M. conjunctivae was identified 53 times (76.8%). A commercially prepared indirect ELISA was used to detect M. conjunctivae antibodies in 674 sera of adult sheep. We analysed a stratified random sample of 123 sheep herds from 25 out of the 26 Swiss cantons. At least one positive animal was detected in 89.4% of the herds. In positive herds (n=110), 57.1% of the individual animals tested positive. To assess the importance of sheep's age in the spread of M. conjunctivae, 209 sera of adult sheep and 93 lamb sera among eight sheep herds were analysed using the indirect ELISA. Seroprevalence in 2-6-month-old lambs was 50.5%, indicating that the IKC agent is spread in sheep flocks during raising. Lambs experimentally infected with M. conjunctivae carried the agent for 8 and 23 weeks, respectively, depending on the strain used for challenge. We conclude that the M. conjunctivae-infection is endemic and self-maintained in the domestic sheep population in Switzerland.     

Prevalence, molecular diagnosis and treatment of Mycoplasma conjunctivae isolated from infectious keratoconjunctivitis affected Lohi sheep maintained at Livestock Experiment Station, Bahadurnagar, Okara, Pakistan

Mycoplasma conjunctivae are etiological agents of infectious keratoconjunctivitis (IKC), commonly known as pink-eye in domestic sheep, goats and other wild animals in many parts of the world. A few young Lohi lambs maintained at Livestock Experiment Station (LES), Bahadurnagar, Okara, Pakistan showed clinical signs and symptoms of conjunctivitis, keratitis, severe lacrimation and varying degree of blindness. During January to March, 2011, a total of 36 ocular swabs were collected from IKC affected animals and were processed for isolation, identification, and characterization of M. conjunctivae. Sixteen (44.44 %) out of 36 samples showed turbidity in PPLO broth. Twelve (75 %) out of 16 broth samples showed colony growth on PPLO agar. All 16 (44.44 %) out of 36 turbid broth samples, 12 (75 %) out of 16 cultured on agar plate samples, and 21 (59 %) out of 36 sheep ocular direct swab samples were found positive for M. conjunctivae through polymerase chain reaction test by using M. conjunctivae-specific primer pair McoF1 and McoR1 and detecting a 750 base pair fragment on agarose gel. Topical application of 0.5 % sterile solution of gentamycin (100 mg/ml) (Gentafar 10 %, FARVET, Netherlands) proved suitable for the treatment of IKC in Lohi lambs as all clinical signs of IKC disappeared after 5 days of treatment with this antibiotic. This is the first report about the prevalence, molecular diagnosis, and treatment of M. conjunctivae in Lohi sheep affected with infectious keratoconjunctivitis at LES, Bahadurnagar, Okara, Pakistan.     

Bacteriological investigation of infectious keratoconjunctivitis in Norwegian sheep

Contagious keratoconjunctivitis is a rather common disease in Norwegian sheep. Since the knowledge of its aetiology is limited, the present study was performed to determine the microorganisms involved. Local veterinarians throughout the country collected conjunctival swabs from both sick (n = 43) and healthy (n = 42) sheep on 15 farms with outbreaks of ovine keratoconjunctivitis, and further from healthy sheep (n = 50) on 17 farms not showing any signs of conjunctival disease. All samples were cultivated for bacteria and mycoplasma. Listeria monocytogenes was isolated from 3 cases (1%) in one single herd. Staphylococcus aureus (5%), Corynebacterium spp. (2%) and Escherichia coli (4%) were isolated only in herds with keratoconjunctivitis, but from both sick and healthy animals. Moraxella (Branhamella) ovis was isolated from 28% of sampled animals in affected herds and from 10% of sampled animals in healthy herds. The corresponding numbers for Moraxella spp. were 9%/12%, for Pseudomonas spp. 7%/8%, for Staphylococcus spp. 22//22%, for Bacillus spp. 12%/14%, for Micrococcus spp. 6%/2% and for Streptococcus/Enterococcus spp. 2%/2%. Mycoplasma conjunctivae was isolated from 16 animals with keratoconjunctivitis (37%) and from 3 animals without clinical signs (7%) in farms with keratoconjunctivitis. In farms without clinical signs of keratoconjunctivitis, M. conjunctivae was isolated in 4 animals (8%). To our knowledge, this is the first time M. conjunctivae has been isolated in Norway. Other predisposing agents found were Moraxella (Branhamella) ovis and Listeria monocytogenes. The etiological importance of different microorganisms in ovine keratoconjunctivitis seems to vary; some are probably only present as secondary invaders. Other possible causes of ovine keratoconjunctivitis in Norway, such as Chlamydia psittaci, remain to be investigated.     

Detection of Mycoplasma conjunctivae in the eyes of healthy, free-ranging Alpine ibex: possible involvement of Alpine ibex as carriers for the main causing agent of infectious keratoconjunctivitis in wild Caprinae

Mycoplasma conjunctivae is considered the major cause of infectious keratoconjunctivitis (IKC) in Alpine ibex (Capra i. ibex) and chamois (Rupicapra r. rupicapra). While it is known that domestic sheep can act as healthy carriers for M. conjunctivae, this question has not been addressed in wild ungulates so far. In this study, bacteriological investigations and field observations were performed to assess whether free-ranging Alpine ibex can be healthy carriers of M. conjunctivae. Among 136 ibex without clinical signs of IKC, M. conjunctivae was identified 26 times (19.1%) by TaqMan PCR. To assess the potential pathogenicity of M. conjunctivae strains isolated from asymptomatic eyes, strains from three healthy ibex and from 15 IKC-ibex and IKC-chamois were analysed genetically by DNA sequence analysis of the variable part of the lppS gene. No significant differences were observed between strains from asymptomatic and clinically affected animals, reflecting the assumption that healthy ibex may act as carriers for M. conjunctivae strains that may be pathogenic for other individuals. Our results further indicate that development of IKC is associated with M. conjunctivae load in the eyes. In addition, a questionnaire survey revealed that IKC is generally less common in ibex than chamois and that infection in wild ungulates is not necessarily linked to the presence of sheep. These data support the hypothesis that apparently healthy ibex may be important in the epizootiology of IKC and indicate that host predilection may play a role in IKC development.     

感染绵羊肺炎支原体前后绵羊血清中相关细胞因子的动态检测

为研究绵羊感染绵羊肺炎支原体(Mycoplasma ovipneumoniae,MO)前后体内细胞因子含量的变化,将6只盘羊杂交羊和6只巴什拜羊人工感染MO,在感染前后采集静脉血,分离血清,用ELISA方法检测IL-5、IL-9、IL-12及IL-13含量。结果显示:2组羊在感染后IL-5浓度均升高,第7(P0.05)、14(P0.01)和21天(P0.01)盘羊杂交羊IL-5含量显著和极显著高于巴什拜羊;感染后第7(P0.01)、14(P0.01)和21天(P0.05)盘羊杂交羊的IL-9浓度极显著和显著地高于巴什拜羊;在感染后的第7(P0.05)、14(P0.01)和第21天(P0.01),盘羊杂交羊的IL-12浓度显著和极显著地高于巴什拜羊;在感染后的第5(P0.05)、7(P0.05)、14(P0.01)和21天(P0.01),IL-13浓度巴什拜羊显著和极显著低于盘羊杂交羊。结果表明:绵羊感染MO前后血清中IL-5、IL-9、IL-12及IL-13的浓度有明显变化,巴什拜羊和盘羊杂交羊的上述细胞因子变化也有显著差异,这对研究支原体肺炎发病机理及临床诊断有指导意义。     

Isolation of Mycoplasma bovoculi from cases of infectious bovine keratoconjunctivitis.

Five outbreaks of infectious bovine keratoconjunctivitis were examined for bacteria and mycoplasmas. Mycoplasma bovoculi was demonstrated in four of the five outbreaks. Other mycoplasmatales were represented by Ureaplasma in one sample. Moraxella bovis and Neisseria ovis were found in all the outbreaks, the former being present in the vast majority of the animals. Transmission experiments with Mycoplasma bovoculi and Moraxella bovis in combination were carried out on four young, colostrumdeprived calves. Mycoplasma bovoculi appeared to have an enhancing effect on the pathogenicity of Moraxella bovis.     

Epidemiological and microbiological study of an outbreak of infectious keratoconjunctivitis in sheep

After several thousand sheep had been imported from Australia and New Zealand to Croatia during 1995, many native sheep that had been in contact with the imported animals acquired a severe ocular disease closely resembling infectious keratoconjunctivitis. In affected flocks glucose-fermenting mycoplasma were isolated from 48 per cent of conjunctival swabs and Branhamella ovis from 58 per cent. Twelve of 42 culturally and biochemically identical isolates were identified as Mycoplasma conjunctivae by polymerase chain reaction. From the conjunctivae of two animals M conjunctivae and M arginini were isolated in mixed culture. For many reasons most farmers removed the imported animals from their flocks and only sporadic cases of the disease were recognised in 1996. At the end of 1997, six flocks which were clinically free of the disease but had been affected during 1995, and five flocks with no history of the severe ocular disease were examined clinically and microbiologically, and were found to be free of M conjunctivae infection. At the time, B ovis was cultured almost exclusively from sheep originating from flocks which had been affected during 1995 and/or 1996. It was usually isolated in pure culture or as the predominant bacterial species, and was often accompanied by mild conjunctivitis. There were no microbiologically confirmed new cases of infectious keratoconjunctivitis during 1998 and 1999.     

Occurrence of antibodies to group specific chlamydia antigen in Finnish sheep, cattle and horse sera

A serological survey on the occurrence of group-specific chlamydial antibodies in random sera of Finnish sheep, cattle and horses was performed. The whole material consisted of 1347 serum samples, including 432 ovine, 454 bovine and 461 equine sera. The sera were sent to the laboratory for various serological tests during 1968–1972. Of the ovine sera 9.5%, bovine 12.8 % and equine 7.1 % showed a titer ≥ 1:16 in the complement fixation test.No definite geographic differences could be found in the distribution of the herds which showed positive results. The ubiquity of chlamydial infections in domestic mammals and their role as a cause of clinical diseases is discussed.     

Mycoplasma infections in farm animals: specific prevention of infectious (contagious) agalactia in the sheep and goat

The authors of this paper have produced live vaccine to cope with infectious agalactia in goat. The vaccine (culture) was used on 360, 924 goats kept on ajmaks in Bajanchongor and Gobi-Altaj, in 1986. The number of pathologically affected goats went down by a factor of 4.5, as compared to 1985. Mortality was reduced to one third. Abortions declined by a factor of 2.6 and infertility among goats by 1.6. The conclusion is that sheep of the Mongolian breed are not susceptible to infectious agalactia. Large-scale production tests of live cultures supported hopes for successful action on this infectious disease under Mongolian conditions.