A new bovine conjunctiva model shows that Listeria monocytogenes invasion is associated with lysozyme resistance

Abstract

Listerial keratoconjunctivitis ('silage eye') is a wide spread problem in ruminants causing economic losses to farmers and impacts negatively on animal welfare. It results from direct entry of Listeria monocytogenes into the eye, often following consumption of contaminated silage. An isolation protocol for bovine conjunctival swabbing was developed and used to sample both infected and healthy eyes bovine eyes (n=46). L. monocytogenes was only isolated from one healthy eye sample, and suggests that this organism can be present without causing disease. To initiate a study of this disease, an infection model was developed using isolated conjunctiva explants obtained from cattle eyes post slaughter. Conjunctiva were cultured and infected for 20 h with a range of L. monocytogenes isolates (n=11), including the healthy bovine eye isolate and also strains isolated from other bovine sources, such as milk or clinical infections. Two L. monocytogenes isolates (one from a healthy eye and one from a cattle abortion) were markedly less able to invade conjunctiva explants, but one of those was able to efficiently infect Caco2 cells indicating that it was fully virulent. These two isolates were also significantly more sensitive to lysozyme compared to most other isolates tested, suggesting that lysozyme resistance is an important factor when infecting bovine conjunctiva. In conclusion, we present the first bovine conjunctiva explant model for infection studies and demonstrate that clinical L. monocytogenes isolates from cases of bovine keratoconjunctivitis are able to infect these tissues.

Stimulation of PgdA‐dependent peptidoglycan N‐deacetylation by GpsB‐PBP A1 in Listeria monocytogenes

“…Intracellular passages not only facilitate escape from the immune system but also impair antibiotic therapy, as antibiotics have to penetrate the host cells in order to exert their anti‐listerial effects (Hof, ). Another mechanism, which impairs efficient eradication of L. monocytogenes by the innate immune system is their high intrinsic resistance against the muramidase lysozyme (Rae et al ., ; Warren et al ., ), which is part of the unspecific humoral host defense against bacterial pathogens.…”

Sensitivity ofListeria monocytogenesto lysozyme predicts ability to proliferate in bovine caruncular epithelial cells

“…Interestingly, the number of recovered bacteria from BCEC cells after 24h of infection correlated strongly with levels of lysozyme resistance (MIC [r=0.82, p=0.004]; bacterial survival in 580 µg lysozyme/ml [r=0.72, p=0.02]). However, there was no correlation with growth rate (r=0.49, p>0.05) or with intracellular bacteria recovered from conjunctiva explant infections (r=0.55, p>0.05) (Table 3, MIC & survival in lysozyme were previously reported) [22].…”

“…Lysozyme sensitivity is an important factor in determining the ability of most L. monocytogenes strains to infect a bovine conjunctiva explant model [22]. Interestingly, the number of recovered bacteria from BCEC cells after 24h of infection correlated strongly with levels of lysozyme resistance (MIC [r=0.82, p=0.004]; bacterial survival in 580 µg lysozyme/ml [r=0.72, p=0.02]).…”

“…However, loss of infectivity may also be due to mutations accumulated during long term culture of the bacteria in a laboratory environment. In our previous study, both LM4 and 7644 were attenuated in a Caco2 infection model, whereas AR008 was able to infect Caco2 cells at similar levels to the control strain 10403S [22]. The fact that AR008 was attenuated in the BCEC model suggest that there are specific factors in these bovine placental cells involved in the interactions with L. monocytogenes .…”

“…Previously, we have shown that resistance to lysozyme was a positive predictive factor for infection of bovine conjunctiva explant model [22] but genome analysis performed in this study did not reveal any differences between the isolates used in the genes suspected to be involved in lysozyme resistance ( pdgA , oatA , degU ) [22, 34, 35]. Interestingly, there was also a strong correlation between L. monocytogenes replication in BCECs 24h post-infection and their level of lysozyme resistance.…”