Mycoplasma conjunctivae in domestic small ruminants from high mountain habitats in Northern Spain

Background Infectious keratoconjunctivitis (IKC) is a clinical condition affecting eyes of domestic and wild Caprinae worldwide, and Mycoplasma conjunctivae is considered the primary causative agent of IKC in sheep, goats and wild Caprinae. Domestic ruminants from high mountain habitats share grazing areas with wild mountain ungulates, such as chamois (Rupicapra spp.), Alpine ibex (Capra ibex) and European mouflon (Ovis aries musimon), and domestic sheep seem to act as M. conjunctivae reservoir. In this study, the presence of M. conjunctivae in domestic sheep and goats from the two main mountain ranges of Northern Spain, the Pyrenees and the Cantabrian Mountains, has been investigated. Results Eye swabs were obtained from 439 domestic small ruminants selected from flocks that seasonally graze in alpine meadows during three consecutive years (2011-2012-2013). Seventy-nine out of the 378 domestic sheep (20.9%) tested positive to a M. conjunctivae specific real time-PCR (rt-PCR) in at least one eye, whereas all the 61 sampled domestic goats were negative. Statistically significant higher prevalence and higher proportion of infected flocks (P < 0.001) was observed in the Pyrenees (25.7%; 12 flocks out of 13), where M. conjunctivae is widespread and probably endemic in domestic sheep, than in the Cantabrian Mountains (7.8%; one flock out of six). Twenty-five sheep (three from the Pyrenees and 22 from the Cantabrian Mountains) which showed clinical signs consistent with infectious keratoconjunctivitis (IKC) were negative by rt-PCR. In contrast, 62 out of the 71 (87.3%) M. conjunctivae-positive sheep from the Pyrenees and the eight positive sheep from the Cantabrian Mountains were asymptomatic. Conclusions This study provides rt-PCR-based evidences of M. conjunctivae maintenance in domestic sheep, as well as a relationship between prevalence in domestic sheep and previously reported M. conjunctivae and IKC in wild ruminants. Domestic goats do not seem to play an important role in the epidemiology of M. conjunctivae in alpine habitats from Northern Spain.


Background
Infectious keratoconjunctivitis (IKC) is a clinical condition affecting eyes of domestic and wild Caprinae worldwide. Several infectious agents such as Mycoplasma conjunctivae, Chlamydophila psittaci or Moraxella ovis (formerly Branhamella ovis) have been isolated from eyes of small ruminants affected by IKC [1]. However, M. conjunctivae is considered the primary causative agent of IKC in sheep, goats and wild Caprinae [1][2][3][4]. Susceptibility to M. conjunctivae infection differs among host species. While in sheep and goats IKC usually appears in form of transitory blindness causing little concern and economic consequences, pathogenicity to wild species is generally high though variable, causing outbreaks with morbidity and mortality up to 30% [1].
The epidemiology of M. conjunctivae is particularly worth investigating in mountain habitats, where domestic ruminants share grazing areas with susceptible wild mountain ungulates during late spring (May-June) to early fall (September-October), such as chamois (Rupicapra spp.), Alpine ibex (Capra ibex) and European mouflon (Ovis aries musimon) [1,5]. Interspecific transmission may occur [6], and domestic sheep seem to play a key role as a reservoir host for M. conjunctivae in such a complex scenario of host interaction [7,8]. Several outbreaks of IKC have been described in domestic sheep and goats worldwide [3,[9][10][11], but few active surveillance studies have been conducted in small domestic ruminants, particularly in high mountain habitats.
In this study, the presence of M. conjunctivae in domestic sheep and goats from the two main mountain ranges of Northern Spain, the Pyrenees and the Cantabrian Mountains, has been investigated ( Figure 1). M. conjunctivae and IKC outbreaks have been reported in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in the Pyrenees [5,12,13], but not in Cantabrian chamois (Rupicapra pyrenaica parva) from the Cantabrian mountains. Therefore, both study areas represent two different epidemiological scenarios.

Methods
Conjunctival swabs were obtained from below the nictitating membrane from both eyes in 439 physically restrained small ruminants (19 flocks; Table 1). Two flocks in each region were sampled in at least two periods. All the goats were sampled in mixed goat and sheep flocks, except for 20 goats from a goat flock in the Cantabrian Mountains. Clinical signs compatible with IKC were recorded at sampling and swabs were stored at -20ºC until analyzed. During the study period, no outbreak of IKC was detected neither in domestic ruminants or wild mountain ungulates in the study area, though IKC cases in Pyrenean chamois are observed every year (Fernández-Aguilar et al., in prep.).
At the laboratory, swabs were thawed and mixed with 0.5 ml of lysis buffer (100 mM Tris-HCl, pH 8.5, 0.05% Tween 20, 0.24 mg/ml proteinase K) in microcentrifuge tubes and cells were lysed for 60 minutes at 60°C and 15 minutes at 97°C. The presence of M. conjunctivae was determined by a TaqMan real-time PCR (rt-PCR) with an exogenous external positive control in each reaction, as previously described [15]. For analysis of Mycoplasma agalactiae as a potential cause of IKC, the real time PCR method based on the cytadhesin P40 [16] was used as described [17].
Chi-square tests were performed in order to detect statistically significant differences in M. conjunctivae prevalence both at flock and individual level according to area and clinical status, using the PROC FREQ of the SAS® 9.1.3 System for Windows (SAS Institute, Cary, NC, USA). As Mycoplasma agalactiae has been reported to be a mycoplasmal cause of IKC in domestic ruminants [18], samples of these sheep were also analyzed by real time PCR for the presence of this pathogen. This analysis did not detect M. agalactiae in the eye swabs of these animals. In contrast, 62 out of the 71 (87.3%) M. conjunctivae-positive sheep from the Pyrenees and the eight positive sheep from the Cantabrian Mountains were asymptomatic. In most flocks, IKC was unnoticed by the owners, as clinical cases were found to be occasional and mild. The clinical signs of the two sheep from the Pyrenees which were positive to M. conjunctivae in two samplings periods evolved from lachrymation and bilateral conjunctivitis in the first sampling to apparently healthy eyes in the second sampling in one sheep, and conversely for the other sheep.

Results
M. conjunctivae was not detected in any of the goats sampled in both study areas (Table 1), although three goats from one flock in the Cantabrian Mountains showed mild clinical ocular signs.

Discussion
The high prevalence of M. conjunctivae in domestic sheep from the Pyrenees and its maintenance over Table 1 Results of Mycoplasma conjunctivae prevalence assessed by rt-PCR (as described in [15])   [4,7]. In contrast, the lower prevalence found in the Cantabrian Mountains and the fact that M. conjunctivae was detected in only one flock in this region suggest that M. conjunctivae is currently less common in this area. Moreover, the only M. conjunctivaepositive flock in the Cantabrian Mountains seasonally migrates to Caceres (South-Western Spain) in winter. Hence, the origin of M. conjunctivae in this flock is unclear and could lead to an overestimation of the endemic situation of M. conjunctivae in this region. Furthermore, the occurrence of M. conjunctivae relative to IKC cases in the two study areas strongly differed. Whereas there is a good agreement between IKC cases and the presence of M. conjunctivae in the Pyrenees, this is not the case in the Cantabrian Mountains. The prevalence of M. conjunctivae among asymptomatic sheep was higher than previous data obtained by traditional culture methods [19]. However, strain pathogenicity or individual host factors such as immunity may influence the outcome of clinical disease. Spontaneous clinical recoveries and relapses are a common feature of IKC in sheep [4], which agrees with the clinical evolution of the two positive sheep from the Pyrenees sampled twice, further suggesting the role of sheep as a maintenance host of M. conjunctivae in this area, as previously suggested in Switzerland assessed by serology [7] and Central Pyrenees [12].
Several IKC outbreaks caused by M. conjunctivae have been reported in domestic goats [3,9,10], and IKC has been experimentally reproduced in goats by inoculating M. conjunctivae previously isolated from a goat with IKC [2]. However, the absence of M. conjunctivae occurrence in goats in this study, even in mixed flocks with high prevalence of M. conjunctivae in sheep, suggests a lower susceptibility to M. conjunctivae infection or a host specificity of the strains circulating in the area among domestic sheep. Therefore, domestic goats do not seem to contribute to M. conjunctive epidemiology in nonepidemic IKC in mountain habitats from Northern Spain. The finding of sheep and goats negative for M. conjunctivae and M. agalactiae but showing clinical signs consistent with IKC suggests a possible implication of other pathogens, such as Chlamydophila psittaci, Moraxella ovis, or Listeria monocytogenes previously detected in domestic small ruminants with IKC [1,19]. The higher occurrence of M. conjunctivaenegative sheep and goats with clinical signs of keratoconjunctivitis in the Cantabrian Mountains suggests that other pathogens may be more relevant than M. conjunctivae for keratoconjunctivitis in this region. Although M. conjunctivae is considered the main etiological agent of IKC outbreaks in both domestic small ruminants and wild mountain ruminants [1,2,11], pathogens or conditions associated with nonepidemic IKC warrant further research, as previously suggested in wild mountain ungulates [20].
The widespread and consistent presence of M. conjunctivae in domestic sheep from the Pyrenees and the less relevant role of this pathogen in the Cantabrian Mountains seem to correspond to previous reports of IKC in wild sympatric susceptible hosts, such as European mouflon and Pyrenean chamois in the Pyrenees [1,5,12,13], but not in Cantabrian chamois from the Cantabrian Mountains. Phylogenetical analyses of M. conjunctivae strains circulating in domestic sheep and wild mountain ruminants would help clarifying the specific role of different host species in the epidemiology of IKC from the studied areas, particularly in the Pyrenees.

Conclusions
This study provides rt-PCR-based evidence of M. conjunctivae maintenance in domestic sheep, as well as a relationship between prevalence in domestic sheep and previously reported M. conjunctivae and IKC in wild ruminants. This finding adds new relevant information into the epidemiology of M. conjunctivae in the domestic-wildlife interface. Domestic goats do not seem to play an important role in the epidemiology of M. conjunctivae in alpine habitats from Northern Spain.