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 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 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.     

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).     

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).     

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.     

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.     

Serotypes and antibiotic sensitivity of Moraxella bovis isolated from an outbreak of infectious bovine keratoconjunctivitis.

Samples of conjunctival fluid of four calves, from a herd with infectious bovine keratoconjunctivitis, were collected five times at monthly intervals and from another 16 calves in the initial stages of the disease, were collected only once. Moraxella bovis was recovered from 82.3% of the samples. Twenty-six isolates were typed with polyvalent sera and 25 with monovalent sera. The antibiotic sensitivity of 13 isolates was also studied. Twenty of the 26 isolates reacted with polyvalent sera and 16 of 25 tested isolates, reacted with monovalent sera. The isolates recovered from the same animal in a different collection or from a different animal in the same collection did not always belong to the same serogroup. Different serotypes were prevalent in each collection. Differences in the antibiotic sensitivity of the isolates were also detected.     

Prevalence and serum protein values of strangles (Streptococcus equi) affected mules at Remount Depot,Sargodha (Pakistan)

The prevalence of Streptococcus equi serovar equi (S.equi) in nasal discharge and pus samples from sub‐mandibular lymph nodes in mules at the Remount Depot, Sargodha was examined and total serum proteins, serum albumin, serum globulin and fibrinogen measured. A total of 250 nasal swabs and pus samples were collected from mules and examined microbiologically: 99 (39.6%) were positive for S. equi. A higher occurrence of S. equi was recorded in foals as compared to adults. The concentrations of total serum protein, serum globulin and fibrinogen were significantly increased (P<0.05), while the concentration of serum albumin significantly decreased (P<0.05) in strangles‐affected mules. It was concluded that increased total serum proteins, serum globulin and fibrinogen along with decreased serum albumin were important indicators of infection by S. equi in mules.     

A national survey of the clinical features, treatment and importance of infectious bovine keratoconjunctivitis

SUMMARY A postal survey of cattle producers throughout Australia was conducted to obtain information concerning the occurrence, signs and treatment of infectious bovine keratocojunctivitis, animal breeds, animal numbers and types, environmental conditions under which the animals were kept and management routines and systems of the animals (Slatter et al 1982). The most common clinical signs reported were ocular discharge (43.9%), corneal opacity (9.9%), or both (46.1%). The majority of respondents (54.8%) indicated duration of infections of at least 3 weeks. The condition was predominantly unilateral (74.7%) but 22.3% of respondents reported an equal occurrence of the condition unilaterally and bilaterally. The most frequently used drugs were homidium bromide (26.7%), oxytetracycline hydrochloride (22.8%), chloramphenicol derivatives (13.7%) and penicillin derivatives (13.5%). However, described treatment regimes indicated that therapeutic levels of antibiotics would not be maintained in the eyes of treated animals. Producers considered that 75% of affected animals showed reduced rates of weight gain, and 64% indicated they were more difficult to handle. An approximate figure of $22,000,000 was determined for loss of national production due to the disease, based on producers' estimates. In addition, a further cost of $1,566,500 was estimated for the labour involved in current treatment regimes. Beef and dairy producers spent different amounts on medications and treated for different durations. The economic significance of the disease justifies further studies on production losses due to the disease and cost effective methods of treatment.     

The in vitro effect of six antimicrobials against Mycoplasma putrefaciens, Mycoplasma mycoides subsp. mycoides LC and Mycoplasma capricolum subsp. capricolum isolated from sheep and goats in jordan

Respiratory disease in sheep and goats is a major problem in Jordan and is often associated with Mycoplasma species. Without effective vaccines, control is mainly by chemotherapy, but the uncontrolled use of antimicrobials has led to concerns about the potential development of antimicrobial resistance. The in vitro effect of chloramphenicol, florfenicol, enrofloxacin, tylosin, erythromycin and oxytetracycline was determined against 32 isolates of Mycoplasma species-M. mycoides subsp. mycoides LC (6), M. capricolum subsp. capricolum (8) and M. putrefaciens (18), all isolated from either nasal swabs or milk, from sheep and goats in different regions of Jordan. The antimicrobial susceptibility showed some Mycoplasma species-specific differences, with M. capricolum subsp. capricolum being more susceptible to tylosin and erythromycin. Chloramphenicol and florfenicol were the least effective for all three Mycoplasma species. No trends or significant differences in antimicrobial susceptibilities were observed between sheep and goat isolates, between milk or nasal swab isolates, or between isolates from different regions of Jordan. Some isolates of M. capricolum subsp. capricolum and M. putrefaciens showed higher MIC levels with oxytetracycline, as did two isolates of M. mycoides subsp. mycoides LC with tylosin, possibly indicating signs of development of antimicrobial resistance.     

Pathogenicity and molecular analysis of an infectious bursal disease virus isolated from Malaysian village chickens

The characteristics of the pathogenic infectious bursal disease virus (IBDV) that infected avian species other than commercial chickens were largely unknown. In this study, by using in vivo and molecular methods, we had characterized an IBDV isolate (named 94268) isolated from an infectious bursal disease (IBD) outbreak in Malaysian village chickens--the adulterated descendant of the Southeast Asian jungle fowl (Gallus bankiva) that were commonly reared in the backyard. The 94268 isolate was grouped as the very virulent IBDV (vvIBDV) strain because it caused severe lesions and a high mortality rate in village chickens (>88%) and experimentally infected specific-pathogen-free chickens (>66%). In addition, it possessed all of the vvIBDV molecular markers in its VP2 gene. Phylogenetic analysis using distance, maximum parsimony, and maximum likelihood methods revealed that 94268 was monophyletic with other vvIBDV isolates and closely related to the Malaysian vvIBDV isolates. Given that the VP2 gene of 94268 isolate was almost identical and evolutionarily closely related to other field IBDV isolates that affected the commercial chickens, we therefore concluded that IBD infections had spread across the farm boundary. IBD infection in the village chicken may represent an important part of the IBD epidemiology because these birds could harbor the vvIBDV strain and should not be overlooked in the control and prevention of the disease.     

Prevalence and characteristics of attaching and effacing strains of Escherichia coil isolated from diarrheic and healthy sheep and goats

OBJECTIVE: To determine the prevalence and characteristics of attaching and effacing Escherichia coli (AEEC) in diarrheic and healthy small ruminants. ANIMALS: 502 lambs and kids with diarrhea and 511 healthy sheep and goats. PROCEDURE: Fecal samples from diarrheic and healthy sheep and goats were screened for the eae gene. In addition, E coli isolates with positive results for the eae gene (E coli eae+) were analyzed for the espB gene, production of verotoxins (VT), and serogroup. RESULTS: A significantly higher prevalence of healthy lambs and kids were infected with AEEC, compared with diarrheic lambs and kids and healthy adult sheep and goats. Some differences in the characteristics of E coli eae strains isolated from diarrheic and healthy animals were detected. Thus, the espB gene was detected more frequently among E coli eae+ strains isolated from healthy animals than in those isolated from diarrheic animals, and VT production was only detected in E coli eae+ strains isolated from healthy lambs and kids. The E coli eae+ isolates belonged to several O serogroups. However, 17 of 40 (42.5%) isolates from diarrheic lambs and only 4 of 168 (2.4%) isolates from healthy sheep belonged to serogroup 026. CONCLUSIONS AND CLINICAL RELEVANCE: Our results suggest that E coli eae+ 026 strains may play a role in diarrheal disease in lambs, whereas E coli eae+ strains that also had VT production and eae+ strains that had positive results for the espB gene did not appear to be associated with diarrhea in small ruminants.     

Presence of Mycoplasma haemofelis,Mycoplasma haemominutum and piroplasmids in cats from southern Europe: a molecular study

Clinical symptoms produced by Mycoplasma spp. and piroplasmids in cats are sometimes similar. Diagnosis of these pathogens is difficult by microscopic procedures and molecular methods have been used as an alternative. We present in this work, the development of new molecular procedures for diagnosis of the aforementioned organisms, together with a molecular characterization of isolates found in southern European cats.A single PCR-RFLP procedure was designed for diagnosis of Mycoplasma spp. and a seminested PCR-RFLP was designed for diagnosis of piroplasmids. The 16S or 18S rRNA genes of isolates found in clinical samples were partially sequenced in all positive cases.Mycoplasma spp. was detected in 9 (30%) out of 30 symptomatic cats from Spain. Sequencing indicated that 66.6% of these isolates can be ascribed to Mycoplasma haemofelis and only 33.3% to Mycoplasma haemominutum. Partial 16S rRNA sequences obtained in Spanish isolates were very similar to those previously published from the UK and the USA.The presence of piroplasmids (Babesia and Theileria spp.) was studied in 16 cats from Spain (n=13) and Portugal (n=3). Animals analyzed were 10 cats with immunosuppressive viral infection (either FeLV or FIV), 5 asymptomatic cats and 1 cat with Babesia-compatible symptoms. Asymptomatic cats were all PCR-negative. Partial sequencing of 18S rRNA gene demonstrated that the Babesia-symptomatic cat was infected with Babesia canis canis whereas 3 (30%) out of the 10 cats with immunosuppressive viral infection were coinfected with piroplasmids (1 with B. canis canis, 1 with Theileria annae, and 1 with B. canis canis and T. annae both).     

Development and survival of Haemonchus contortus isolated from sheep on a pasture at Bunia (Itiri, Zaire)]

Development and survival of Haemonchus contortus larvae were studied from December 1987 to November 1988 during three different periods (dry season, first and second rainy seasons) on an experimentally infected pasture at Bunia (Ituri, Za?re). Whatever the season, eggs developed into infective larvae within six days and the largest number of larvae on the herbage occurred between the 12th and the 18th day post deposition. However, the two rainy seasons were the most favourable for transmission because of the high number of larvae on the pasture and the increased survival of these larvae after 4 weeks.     

Prevalence and molecular mechanism of macrolide and lincosamide resistance in staphylococci isolated from subclinical bovine mastitis in Turkey

Macrolide and lincosamide (ML) resistance and the related resistance genes of staphylococci were assessed from cases of bovine subclinical mastitis. Of the 104 Staphylococcus aureus and 62 coagulase negative staphylococcus (CoNS) isolates, 26 (25%) and 12 (19.4%) were resistant to ML, respectively. While constitutive ML resistance phenotype accounted for 15.4% (16/104) of S. aureus and 8.1% (5/62) of CoNS, inducible ML resistance phenotype accounted for 2.9% (3/104) of S. aureus and 3.2% (2/62) of CoNS. Among erythromycin-resistant isolates, single or various combination of different resistance genes were detected. The results of this study showed that ML resistance was prevalent among staphylococci from subclinical bovine mastitis cases in Hatay, Turkey. Therefore, a continuous surveillance is necessary to minimise the spread of antimicrobial-resistant staphylococci.     

Prevalence of and carcass condemnation from maedi-visna,paratuberculosis and caseous lymphadenitis in culled sheep from Quebec,Canada

We determined the prevalence of lung and mammary gland lesions associated with maedi-visna (MV) infection, the prevalence of paratuberculosis (PTB), and the prevalence and lesions distribution of caseous lymphadenitis (CL) in culled sheep. Total of 451 ewes and 34 rams were selected randomly from two slaughterhouses in Quebec, Canada. MV serostatus was determined by recombinant ELISA test. PTB diagnosis was based on characteristic histological lesions in the terminal ileum, ileocecal lymph node and/or ileocecal valve and CL by gross detection of abscesses and isolation of Corynebacterium pseudotuberculosis. Seroprevalence of MV was 44% (95% CI: 40, 48). Seropositivity increased with age and was higher in ewes than in rams. The percentages of lung and mammary gland lesions in seropositive sheep were 14 and 40%, respectively, but mammary gland lesions lack specificity. The prevalence of PTB was 3% (95% CI: 2, 5). PTB increased with age and was lower among sheep with abscesses. The prevalence of CL was >/=21% (95% CI: 17, 24). The most-prevalent site of caseous lymphadenitis lesions was the thoracic cavity. The risk of carcass condemnation was significantly associated with region, body score and abscesses. Only the presence of abscesses was associated with an increase in trimming of carcasses.