Molecular Epidemiology of Mycoplasma conjunctivae in Caprinae: Transmission across Species in Natural Outbreaks

Mycoplasma conjunctivae is the etiological agent of infectious keratoconjunctivitis, a highly contagious ocular infection that affects both domestic and wild Caprinae species in the European Alps. In order to study the transmission and spread of M. conjunctivae across domestic and wild Caprinae populations, we developed a molecular method for subtyping and identifying strains of M. conjunctivae. This method is based on DNA sequence determination of a variable domain within the gene lppS, a gene that encodes an antigenic lipoprotein of M. conjunctivae. This domain of lppS shows variations among different strains but remains constant upon generations of individual strains on growth medium and thus allows identification of individual strains and estimation of their phylogenetic intercorrelations. The variable domain of lppS is amplified by PCR using primers that match conserved sequences of lppS flanking it. Sequence analysis of the amplified fragment enables fine subtyping of M. conjunctivae strains. The method is applicable both to isolated strains and to clinical samples directly without requiring the cultivation of the strain. Using this method, we show that M. conjunctivae was transmitted between domestic and wild animals that were grazing in proximate pastures. Certain animals also presented infections with two different strains simultaneously

Spatial distribution of Melissococcus plutonius in adult honey bees collected from apiaries and colonies with and without symptoms of European foulbrood

In Switzerland, the incidence of European foulbrood (EFB), caused by the Gram-positive bacterium Melissococcus plutonius, has increased dramatically between 1997 and 2005 but the epidemiology, including the transmission of M. plutonius, remains poorly understood. In this study, the distribution of M. plutonius among bees originating from apiaries and colonies with and without symptoms of EFB was evaluated using a specific and sensitive hemi-nested PCR. In more than 90% of colonies without EFB symptoms located in apiaries with EFB symptoms, the bees were carriers of M. plutonius. In apiaries without EFB symptoms, but near apiaries with EFB symptoms, bees carrying M. plutonius were detected in about 30% of the colonies. In regions without European foulbrood history, all bee samples were negative. The proportion of adult bees carrying M. plutonius in colonies without symptoms appeared to increase when the distance to apiaries with clinical EFB symptoms decreased

Investigating the role of free-ranging wild boar (Sus scrofa) in the re-emergence of enzootic pneumonia in domestic pig herds: a pathological, prevalence and risk-factor study

Enzootic pneumonia (EP) caused by Mycoplasma hyopneumoniae has a significant economic impact on domestic pig production. A control program carried out from 1999 to 2003 successfully reduced disease occurrence in domestic pigs in Switzerland, but recurrent outbreaks suggested a potential role of free-ranging wild boar (Sus scrofa) as a source of re-infection. Since little is known on the epidemiology of EP in wild boar populations, our aims were: (1) to estimate the prevalence of M. hyopneumoniae infections in wild boar in Switzerland; (2) to identify risk factors for infection in wild boar; and (3) to assess whether infection in wild boar is associated with the same gross and microscopic lesions typical of EP in domestic pigs. Nasal swabs, bronchial swabs and lung samples were collected from 978 wild boar from five study areas in Switzerland between October 2011 and May 2013. Swabs were analyzed by qualitative real time PCR and a histopathological study was conducted on lung tissues. Risk factor analysis was performed using multivariable logistic regression modeling. Overall prevalence in nasal swabs was 26.2% (95% CI 23.3-29.3%) but significant geographical differences were observed. Wild boar density, occurrence of EP outbreaks in domestic pigs and young age were identified as risk factors for infection. There was a significant association between infection and lesions consistent with EP in domestic pigs. We have concluded that M. hyopneumoniae is widespread in the Swiss wild boar population, that the same risk factors for infection of domestic pigs also act as risk factors for infection of wild boar, and that infected wild boar develop lesions similar to those found in domestic pigs. However, based on our data and the outbreak pattern in domestic pigs, we propose that spillover from domestic pigs to wild boar is more likely than transmission from wild boar to pigs

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.Détection d'anticorps spécifiques de Mycoplasma conjunctivae dans les sérums de moutons atteints de kérato-conjonctivite infectieuse. Les réactions sérologiques croisées entre Mycoplasma conjunctivae, l'agent étiologique de la kérato-conjonctivite infectieuse (KCI), et Mycoplasma ovipneumoniae, qui lui est antigéniquement et phylogénétiquement le plus proche et qui est souvent présent chez le mouton, ont été analysées. Des sérums provenant de moutons atteints de KCI et de moutons issus de troupeaux exempts de KCI, ainsi que des sérums hyperimmuns de lapin spécifiques pour les deux espèces de mycoplasmes, ont permis l'identification des antigènes impliqués dans les réactions croisées. Les réactions croisées sont principalement dues aux protéines fortement antigéniques de 42 kDa et 83 kDa. Les antigènes sérospécifiques de Mycoplasma conjunctivae peuvent être séparés des antigènes provoquant une réaction croisée par une extraction des protéines membranaires au Tween 20. Les protéines de Mycoplasma conjunctivae HRC/581T extraites par le détergent Tween 20 ont été utilisées dans le développement d'un ELISA indirect. Cet ELISA s'est avéré être une méthode sérologique utile pour le diagnostique des infections dues à Mycoplasma conjunctivae ainsi que pour identifier les troupeaux de moutons infectés

Map of Switzerland depicting the location of the study areas.

<p>The five study areas (units A-E) are indicated by shades of grey. Black lines correspond to canton borders, and blue areas are main lakes. The origin of wild boar (<i>Sus scrofa</i>) samples and their real-time PCR results are indicated by colored dots: Samples negative for <i>Mycoplasma hyopneumoniae</i> are green and positive samples are red.</p

Histological lesions associated with <i>Mycoplasma hyopneumoniae</i> infection and their distribution in wild boar lungs.

<p>A: Lung without macroscopic enzootic pneumonia-like lesions (MaEPL) with moderate multifocal BALT hyperplasia along a bronchiole (measurement bar = 500nm). B: Lung with early type MaEPL and mild to moderate multifocal BALT hyperplasia (black arrow) and diffuse moderate thickening of the interalveolar septa (broncho-interstitial pneumonia). Marginal subpleural atelectasis is considered an artefact (measurement bar = 50mm). C: Lung with early type MaEPL and severe infiltration of mononuclear cells expanding the interlobular septum (thick black arrow) and compressing the regional airways. A discrete hyperplastic lymphoid follicle is also present (thin black arrow, measurement bar = 2mm). D: Lung with late type MaEPL and moderate to severe multifocal BALT hyperplasia (thick arrows), moderate infiltration of the submucosa of the airways (thin black arrow) and mild to moderate multifocal thickening of the interalveolar septa by infiltrating inflammatory cells (measurement bar = 500nm). E: Magnification (20x) of the framed area in D (inset). Intraluminal collection of neutrophils (thin black arrows, measurement bar = 20μm). F: Lung with late type MaEPL and diffuse intraalveolar collection of amorphous eosinophilic material (edema, black arrows) with mild to moderate multifocal lympho-plasmacytic clusters in the interstitium (black circles). Few numbers of mononuclear cells are also observed in the alveolar spaces along with free erythrocytes (hemorrhages, gunshot-related, measurement bar = 20μm).</p