Inclusion keratoconjunctivitis ('pink eye') in sheep. A proposal for a new name for chlamydial keratoconjunctivitis in sheep and comment on recent clinical trials

The cytoplasmatic inclusion bodies, which, in 1931, Coles discovered in the corneal cells of sheep suffering from contagious keratoconjunctivitis are now considered to be the reticulate bodies of a chlamydia, Colesiota conjunctivae (synonym: Chlamydia psittaci ovis). According to the postulates of Koch Colesiota conjunctivae is a primary cause of contagious keratoconjunctivitis in sheep, but the clinical picture is complex and is a result of the interaction between the infecting chlamydiae, host resistance factors, and secondary infections caused by opportunistic bacterial ocular pathogens. The clinical syndrome might also be caused by other micro-organisms, such as Mycoplasma conjunctivae or environmental factors, such as dust. However, in these cases, cytoplasmatic inclusion bodies cannot be found in the corneal cells of diseased eyes. To differentiate chlamydial keratoconjunctivitis from keratoconjunctivitis due to other causes, it is proposed to include in the name the laboratory findings typical for this disease: Sheep Inclusion Keratoconjunctivitis. Chlamydia are Gram-negative bacteria, which are obligate intracellular parasites. Prolonged treatment seems to be required to eradicate chlamydiae from a host and antibiotics must reach intracellular levels that are higher than their minimum inhibitory concentration for chlamydiae. Tetracyclines are the drugs of choice. This means that for a microbiological cure, diseased sheep must be injected several times a day for a week or more. Because the disease is usually self-limiting and economic losses are considered low, this seems unnecessary and control of the disease by local treatment of secondary infections seems sufficient. However, this will not prevent spreading of the disease in a herd and relapses may occur.     

Review papers

The occurrence of keratoconjunctivitis infectiosa ovis (KIO) in the Netherlands was reported by Holland et al. in 1969 for the first time. At present the disease is well known in the Netherlands. Nevertheless there are still questions about the causative agent and the most effective and easiest therapy.

Most authors suppose that the disease is caused by Colesiota conjunctivae, although others mention infections by other chlamydia, Mycoplasma conjunctivae, Mycoplasma ovipneumoniae, Acholeplasma oculi and a wide variety of bacteria.

The diagnosis can be made on the basis of the symtoms and the detection of the agent in Conjunctival scrapings. The bacilliform bodies can be found in conjunctival smears in the cytoplasm (Giemsa, Stamp).

Many therapies are used topically, parenterally or orally. Locally used eye‐ointments must be effective against Colesiota; antimicrobial drugs administered by injection must be effective against the latter and also provide a sufficient cell tissue penetration with excretion into the lacrimal fluid. Injections have proved to be easier to administer, especially in serious outbreaks, but such outbreaks are exceptional (18). Some therapies have been evaluated in small scale experiments (28) as well as in field trials (17).     

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.     

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.     

A Survey of the Conjunctival Flora of Clinically Normal Cats and Cats with Conjunctivitis

Conjunctival swabs obtained from 39 cats with conjunctivitis and from 50 cats with clinically normal conjunctivae were cultured for bacteria, mycoplasmas, viruses and chlamydiae. Non hemolytic streptococci and Staphylococcus epidermidis were isolated from both groups, but B hemolytic streptococci, rhinotracheitis (feline herpes I) virus, Mycoplasma felis and Chlamydia psittaci were recovered only from cases of conjunctivitis. The isolation rate of microorganisms was low; only two of 50 normal and 14 of 39 diseased cats yielded positive cultures.     

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.     

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.     

Detection of specific Mycoplasma conjunctivae antibodies in the sera of sheep with infectious keratoconjunctivitis

The serological cross reactions between Mycoplasma conjunctivae, the etiological agent of infectious keratoconjunctivitis (IKC), and the antigenetically and phylogenetically closely related Mycoplasma ovipneumoniae, which is often found in sheep, were analysed. Cross reacting antigens were identified using sera from sheep with IKC and from sheep of herds known to be free of IKC, as well as rabbit hyperimmune serum specific to the two Mycoplasma species. Cross reactions were predominantly due to the strongly antigenic proteins of 42 kDa and 83 kDa. Serospecific antigens of M. conjunctivae could be separated from cross-reacting antigens by the extraction of Tween 20-soluble membrane proteins. The Tween 20-extracted proteins of the M. conjunctivae strain HRC/581T were used for the development of an indirect ELISA test. This ELISA test was shown to be a useful serological method for the diagnosis of M. conjunctivae infections and to identify infected sheep herds.     

Experimental conjunctival infection of lambs with a strain of Chlamydia psittaci isolated from the eyes of a sheep naturally affected with keratoconjunctivitis

Five ram-lambs were inoculated into the left conjunctival sac with the 15R isolate of Chlamydia psittaci, recovered from a sheep with keratoconjunctivitis. A sixth ram-lamb was kept in contact with them. The five lambs developed varying degrees of acute conjunctivitis and 14 days later C psittaci could be recovered from the inoculated eyes, from which Branhamella ovis was also isolated. The eyes were examined regularly for four months; C psittaci could not be re-isolated but the eyes developed varying degrees of follicular conjunctivitis. After four months the sheep were treated with corticosteroids in an attempt to reactivate a latent chlamydial infection but no chlamydiae could be isolated. Five months after the start of the experiment the six lambs were inoculated with 15R into the left conjunctival sacs. Acute conjunctivitis developed which was not as severe as after the first inoculation, but C psittaci could only be recovered from the left eyes of three sheep three days after inoculation. The eyes remained chronically affected by follicular conjunctivitis. Six months after the start of the experiment the left eyes were again inoculated with 15R; on this occasion acute conjunctivitis did not develop and chlamydiae could not be isolated. Chronic follicular conjunctivitis persisted until the experiment was terminated three months later.     

Animal spongiform encephalopathies ‐ an update part 1. Scrapie and lesser known animal spongiform encephalopathies

Summary

The present article (part I) reviews recent developments in animal spongiform encephalopathies (SEs), with the exception of bovine spongiform encephalopathy (BSE), which is dealt with in part II.

The article focuses on scrapie and describes epidemiological aspects and the prospects for a preclinical diagnosis. Up to now, confirmatory diagnosis of scrapie depended on histological examination of the brain, collected during post‐mortem examination from sheep with clinical signs of the disease. An altered protein, PrP^Sc, can be detected in the brain of diseased animals. The demonstration of the same protein in the spleen and in peripheral lymph nodes of infected animals seems to offer interesting possibilities of arriving at a method for a preclinical diagnosis, and thus a diagnosis in the live animal. Progress has also been made in our understanding of the relationship between the genetic constitution and susceptibility of the host. Susceptibility is expressed as the survival time of sheep inoculated with scrapie. This was thought to be determined by a single genetic locus designated the Sip gene (scrapie incubation period gene). Putative markers for the two alleles of the Sip gene, sA and pA, have been discovered, consisting of restriction fragment length polymorphisms (RFLPs). In field tests, however, the link between these markers and the length of incubation time was far from consistent. These RFLPs were found to be situated outside the prion‐protein‐co‐ding region of the ovine gene. In later studies, RFLPs were detected inside this region. These markers appear to be more informative, i.e. they correspond with a difference in the length of the scrapie incubation period.

Finally, the article briefly describes recent developments in other, lesser known, animal spongiform encephalopathies: chronic wasting disease and other spongiform encephalopathies in exotic ungulates, transmissible mink encephalopathy, and feline spongiform encephalopathy, focusing on their possible links with scrapie or bovine spongiform encephalopathy.     

Efficacy against ovine enzootic abortion of an experimental vaccine containing purified elementary bodies of Chlamydia psittaci

A vaccine prepared from purified, inactivated elementary bodies of Chlamydia psittaci protected sheep against abortion after subcutaneous challenge with live chlamydiae. Immunoblot analysis of serum samples revealed a consistently dominant antibody response against the chlamydial major outer membrane protein in all vaccinated sheep. Reactions to other chlamydial antigens were also detected but were less pronounced or inconsistent. Serological responses detected by complement fixation were variable and did not correlate with immunity.     

Molecular evidence for chlamydial infections in the eyes of sheep

Ocular infections by chlamydiae are associated with ocular disease manifestations such as conjunctivitis and keratitis in humans and animals. Limited evidence exists that members of the order Chlamydiales can also cause ocular disease in sheep. In the current study, the prevalence of chlamydiae in the eyes of sheep was investigated by using PCR methods. Data obtained in sheep by broad-range 16S rRNA order Chlamydiales-specific PCR were compared to the prevalence of antibodies against chlamydiae detected by a competitive enzyme-linked immunosorbent assay (cELISA). Flocks tested included a clinically healthy flock and two flocks suffering from ocular disease and with histories of Ovine Enzootic Abortion (OEA). PCR detected DNA of Chlamydophila (Cp.) abortus and Cp. pecorum in the eyes of both healthy and sick animals but also identified Chlamydia (C.) suis and a variety of uncultured chlamydia-like organisms. Good correlation was found between the presence of Cp. abortus DNA in sheep conjunctival samples and seropositivity detected by cELISA. Despite these findings, no association was found between the presence of chlamydial DNA in the sheep conjunctival samples and the onset of clinical disease. These results suggest that the biodiversity of chlamydiae in the eyes of sheep is greater than that previously thought. Further investigations are needed to determine whether a causal relationship between infection by chlamydiae and ocular disease exists in these animals.     

Keratoprosthesis with retrocorneal fixation: preliminary results in dogs with corneal blindness

Objective To evaluate the use and complications of a penetrating keratoprosthesis implantation in the management of corneal opacification in dogs. Methods A retrospective clinical study describes the indications for the surgical technique utilized and the outcomes of this procedure in 20 eyes of 19 dogs with blindness of corneal origin. A successful surgical outcome was defined as a clear keratoprosthesis optic and improvement or restoration of functional vision over a follow‐up period ranging from at least 8 months to a maximum of 7 years. Results Eyes with total corneal opacification resulting from chronic superficial keratitis (n = 11), keratoconjunctivitis sicca (n = 5), endothelial dystrophy (n = 3) and chemical burn (n = 1) were treated by unilateral (n = 18) or bilateral (n = 1) full‐thickness implantation of a keratoprosthesis. Keratoprostheses were retained in 15 eyes (75%) which regained vision to the date of reporting. Among these eyes, six had uncomplicated postoperative course, five developed retroprosthetic membranes and four developed granulation tissue over the optic of the keratoprostheses. These complications were successfully removed surgically in the nine eyes. The five remaining eyes (25%) developed serious early postoperative complications, for which enucleation had to be performed. Conclusion In keratopathies in which the corneal opacification could not be treated by standard medical or surgical procedures, this keratoprosthesis appears to be promising to restore vision in chronic superficial keratitis and deep corneal dystrophy. It appears to have a poor prognosis in keratoconjunctivitis sicca in brachycephalic dogs. The post operative complications retro‐prosthetic membranes and granulomatous overgrowth could be treated well.     

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.     

Detection of enzootic nasal tumor virus (ENTV) in a sheep flock in southern Brazil

Enzootic nasal tumor (ENT) is a contagious neoplasm associated with enzootic nasal tumor virus (ENTV), which may induce disease in sheep (ENTV-1) and goats (ENTV-2). This study aimed to describe the occurrence of ENT in two Texel sheep (Ovis aries) from a 75-sheep flock, located in the city of Gravataí, southern Brazil. Animals used to be purchased from different origins, and no specific tests for disease monitoring or quarantine procedure were performed. Affected animals presented respiratory distress, anorexia with severe weight loss, and mucopurulent unilateral nasal discharge. Necropsy was performed in both animals and nasal cavity masses were observed. Histopathological analysis demonstrated an epithelial neoplasm compatible with nasal adenocarcinoma. PCR using a protocol that amplifies a 591 bp sequence of 5’LTR-gag region of ENTV1 was performed followed by DNA sequencing. Both samples were positive, and the sequences obtained presented highest identity (97%) with ENTV strain TN28 (GenBank accession number MH899613) detected in a Texel sheep from Scotland. This is the first report of ENTV-1 leading to enzootic nasal tumor in sheep in Latin America, which confirms the presence of the retrovirus in sheep flocks in the Brazilian territory.

     

Control of Peste des Petits Ruminants and Poverty Alleviation?

Peste des petits ruminants (PPR) is a contagious and often fatal viral disease of sheep and goats and also wild small ruminants. The PPR virus is distinct from but closely related to rinderpest virus and both belong to the morbivillivirus genus within the family Paramyxoviridae. PPR is a contagious transboundary disease with a significant impact on rural poor farmers. Its control should therefore be considered in programs that aim at alleviating poverty in developing countries.     

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.