Immunization with a Multivalent Listeria monocytogenes Vaccine Leads to a Strong Reduction in Vertical Transmission and Cerebral Parasite Burden in Pregnant and Non-Pregnant Mice Infected with Neospora caninum.

Neospora caninum is an apicomplexan parasite that causes abortion and stillbirth in cattle. We employed the pregnant neosporosis mouse model to investigate the efficacy of a modified version of the attenuated Listeria monocytogenes vaccine vector Lm3Dx_NcSAG1, which expresses the major N. caninum surface antigen SAG1. Multivalent vaccines were generated by the insertion of gra7 and/or rop2 genes into Lm3Dx_NcSAG1, resulting in the double mutants, Lm3Dx_NcSAG1_NcGRA7 and Lm3Dx_NcSAG1_NcROP2, and the triple mutant, Lm3Dx_NcSAG1_NcGRA7_NcROP2. Six experimental groups of female BALB/c mice were inoculated intramuscularly three times at two-week intervals with 1 × 107 CFU of the respective vaccine strains. Seven days post-mating, mice were challenged by the subcutaneous injection of 1 × 105N. caninum NcSpain-7 tachyzoites. Non-pregnant mice, dams and their offspring were observed daily until day 25 post-partum. Immunization with Lm3Dx_NcSAG1 and Lm3Dx_NcSAG1_NcGRA7_NcROP2 resulted in 70% postnatal pup survival, whereas only 50% and 58% of pups survived in the double mutant-vaccinated groups. Almost all pups had died at the end of the experiment in the infection control. The triple mutant was the most promising vaccine candidate, providing the highest rate of protection against vertical transmission (65%) and CNS infection. Overall, integrating multiple antigens into Lm3Dx_SAG1 resulted in lower vertical transmission and enhanced protection against cerebral infection in dams and in non-pregnant mice.

Bovine neutrophil chemotaxis to Listeria monocytogenes in neurolisteriosis depends on microglia-released rather than bacterial factors.

BACKGROUND: Listeria monocytogenes (Lm) is a bacterial pathogen of major concern for humans and ruminants due to its neuroinvasive potential and its ability to cause deadly encephalitis (neurolisteriosis). On one hand, polymorphonuclear neutrophils (PMN) are key players in the defense against Lm, but on the other hand intracerebral infiltration with PMN is associated with significant neural tissue damage. Lm-PMN interactions in neurolisteriosis are poorly investigated, and factors inducing PMN chemotaxis to infectious foci containing Lm in the central nervous system (CNS) remain unidentified. METHODS: In this study, we assessed bovine PMN chemotaxis towards Lm and supernatants of infected endogenous brain cell populations in ex vivo chemotaxis assays, to identify chemotactic stimuli for PMN chemotaxis towards Lm in the brain. In addition, microglial secretion of IL-8 was assessed both ex vivo and in situ. RESULTS: Our data show that neither Lm cell wall components nor intact bacteria elicit chemotaxis of bovine PMN ex vivo. Moreover, astrocytes and neural cells fail to induce bovine PMN chemotaxis upon infection. In contrast, supernatant from Lm infected microglia readily induced chemotaxis of bovine PMN. Microglial expression and secretion of IL-8 was identified during early Lm infection in vitro and in situ, although IL-8 blocking with a specific antibody could not abrogate PMN chemotaxis towards Lm infected microglial supernatant. CONCLUSIONS: These data provide evidence that host-derived rather than bacterial factors trigger PMN chemotaxis to bacterial foci in the CNS, that microglia have a primary role as initiators of bovine PMN chemotaxis into the brain during neurolisteriosis and that blockade of these factors could be a therapeutic target to limit intrathecal PMN chemotaxis and PMN associated damage in neurolisteriosis.

Vaccine-Linked Chemotherapy Approach: Additive Effects of Combining the Listeria monocytogenes-Based Vaccine Lm3Dx_NcSAG1 With the Bumped Kinase Inhibitor BKI-1748 Against Neospora caninum Infection in Mice.

The apicomplexan parasite Neospora (N.) caninum causes neosporosis in numerous host species. There is no marketed vaccine and no licensed drug for the prevention and/or treatment of neosporosis. Vaccine development against this parasite has encountered significant obstacles, probably due to pregnancy-induced immunomodulation hampering efficacy, which has stimulated the search for potential drug therapies that could be applied to limit the effects of neosporosis in dams as well as in offspring. We here investigated, in a pregnant neosporosis mouse model, the safety and efficacy of a combined vaccination-drug treatment approach. Mice were vaccinated intramuscularly with 1 × 107 CFU of our recently generated Listeria (L.) monocytogenes vaccine vector expressing the major N. caninum tachyzoite surface antigen NcSAG1 (Lm3Dx_SAG1). Following mating and experimental subcutaneous infection with 1 × 105 N. caninum (NcSpain-7) tachyzoites on day 7 of pregnancy, drug treatments were initiated using the bumped kinase inhibitor BKI-1748 at 20 mg/kg/day for 5 days. In parallel, other experimental groups were either just vaccinated or only treated. Dams and offspring were followed-up until day 25 post-partum, after which all mice were euthanized. None of the treatments induced adverse effects and neither of the treatments affected fertility or litter sizes. Cerebral infection in dams as assessed by real-time PCR was significantly reduced in the vaccinated and BKI-1748 treated groups, but was not reduced significantly in the group receiving the combination. However, in non-pregnant mice, all three treatment groups exhibited significantly reduced parasite burdens. Both, vaccination as well BKI-1748 as single treatment increased pup survival to 44 and 48%, respectively, while the combination treatment led to survival of 86% of all pups. Vertical transmission in the combination group was 23% compared to 46 and 50% in the groups receiving only BKI-treatment or the vaccine, respectively. In the dams, IgG titers were significantly reduced in all treatment groups compared to the untreated control, while in non-pregnant mice, IgG titers were reduced only in the group receiving the vaccine. Overall, vaccine-linked chemotherapy was more efficacious than vaccination or drug treatment alone and should be considered for further evaluation in a more relevant experimental model.

A Listeria monocytogenes-Based Vaccine Formulation Reduces Vertical Transmission and Leads to Enhanced Pup Survival in a Pregnant Neosporosis Mouse Model.

The apicomplexan parasite Neospora caninum is the worldwide leading cause of abortion and stillbirth in cattle. An attenuated mutant Listeria monocytogenes strain (Lm3Dx) was engineered by deleting the virulence genes actA, inlA, and inlB in order to avoid systemic infection and to target the vector to antigen-presenting cells (APCs). Insertion of sag1, coding for the major surface protein NcSAG1 of N. caninum, yielded the vaccine strain Lm3Dx_NcSAG1. The efficacy of Lm3Dx_NcSAG1 was assessed by inoculating 1 × 105, 1 × 106, or 1 × 107 CFU of Lm3Dx_NcSAG1 into female BALB/c mice by intramuscular injection three times at two-week intervals, and subsequent challenge with 1 × 105N. caninum tachyzoites of the highly virulent NcSpain-7 strain on day 7 of pregnancy. Dose-dependent protective effects were seen, with a postnatal offspring survival rate of 67% in the group treated with 1 × 107 CFU of Lm3Dx_NcSAG1 compared to 5% survival in the non-vaccinated control group. At euthanasia (25 days post-partum), IgG antibody titers were significantly decreased in the groups receiving the two higher doses and cytokines recall responses in splenocyte culture supernatants (IFN-γ, IL-4, and IL-10) were increased in the vaccinated groups. Thus, Lm3Dx_NcSAG1 induces immune-protective effects associated with a balanced Th1/Th2 response in a pregnant neosporosis mouse model and should be further assessed in ruminant models.

Safety of a Novel Listeria monocytogenes-Based Vaccine Vector Expressing NcSAG1 (Neospora caninum Surface Antigen 1).

Listeria monocytogenes (LM) has been proposed as vaccine vector in various cancers and infectious diseases since LM induces a strong immune response. In this study, we developed a novel and safe LM-based vaccine vector platform, by engineering a triple attenuated mutant (Lm3Dx) (ΔactA, ΔinlA, ΔinlB) of the wild-type LM strain JF5203 (CC 1, phylogenetic lineage I). We demonstrated the strong attenuation of Lm3Dx while maintaining its capacity to selectively infect antigen-presenting cells (APCs) in vitro. Furthermore, as proof of concept, we introduced the immunodominant Neospora caninum (Nc) surface antigen NcSAG1 into Lm3Dx. The NcSAG1 protein was expressed by Lm3Dx_SAG1 during cellular infection. To demonstrate safety of Lm3Dx_SAG1 in vivo, we vaccinated BALB/C mice by intramuscular injection. Following vaccination, mice did not suffer any adverse effects and only sporadically shed bacteria at very low levels in the feces (<100 CFU/g). Additionally, bacterial load in internal organs was very low to absent at day 1.5 and 4 following the 1st vaccination and at 2 and 4 weeks after the second boost, independently of the physiological status of the mice. Additionally, vaccination of mice prior and during pregnancy did not interfere with pregnancy outcome. However, Lm3Dx_SAG1 was shed into the milk when inoculated during lactation, although it did not cause any clinical adverse effects in either dams or pups. Also, we have indications that the vector persists more days in the injected muscle of lactating mice. Therefore, impact of physiological status on vector dynamics in the host and mechanisms of milk shedding requires further investigation. In conclusion, we provide strong evidence that Lm3Dx is a safe vaccine vector in non-lactating animals. Additionally, we provide first indications that mice vaccinated with Lm3Dx_SAG1 develop a strong and Th1-biased immune response against the Lm3Dx-expressed neospora antigen. These results encourage to further investigate the efficiency of Lm3Dx_SAG1 to prevent and treat clinical neosporosis.

Transcriptome of microglia reveals a species-specific expression profile in bovines with conserved and new signature genes.

Evidence is growing that microglia adopt different roles than monocyte-derived macrophages (MDM) during CNS injury. However, knowledge about their function in the pathogenesis of neuroinfections is only rudimentary. Cattle are frequently affected by neuroinfections that are either zoonotic or related to diseases in humans, and, hence, studies of bovine neuroinfections as a natural disease model may generate fundamental data on their pathogenesis potentially translatable to humans. We investigated the transcriptomic landscape and lineage markers of bovine microglia and MDM. Although bovine microglia expressed most microglial signature genes known from humans and mice, they exhibited a species-specific transcriptomic profile, including strikingly low expression of TMEM119 and enrichment of the two scavenger receptors MEGF10 and LY75. P2RY12 was amongst the most enriched genes in bovine microglia, and antibodies against P2RY12 labeled specifically resting microglia, but also reactive microglia within neuroinfection foci in-situ. On the other hand, F13A1 was amongst the most enriched genes in bovine monocytes and MDM and, additionally, the encoded protein was expressed in-situ in monocytes and MDM in the inflamed brain but not in microglia, making it a promising marker for infiltrating MDM in the brain. In culture, primary bovine microglia downregulated signature genes, expressed markers of activation, and converged their transcriptome to MDM. However, they retained several microglia signature genes that clearly distinguished them from bovine MDM, making them a promising in-vitro tool to study mechanisms of microglia-pathogen interactions.

Influence of nutrient availability on in vitro growth of major bovine mastitis pathogens.

The aim of the present study was to investigate the effects of milk composition changes on the in vitro growth of bovine mastitis pathogens. Nutritional requirements of three major bovine mastitis pathogens Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Streptococcus uberis (S. uberis) were investigated in vitro. We used ultra-high temperature (UHT) treated milk with different contents of fat, protein, and carbohydrates to test the influence of the availability of various milk constituents on pathogen growth characteristics. Additionally, the bacterial growth was investigated under experimentally modified nutrient availability by dilution and subsequent supplementation with individual nutrients (carbohydrates, different nitrogen sources, minerals, and different types of B vitamins) either to milk or to a conventional medium (thioglycolate broth, TB). Varying contents of fat, protein or lactose did not affect bacterial growth with the exception of growth of S. uberis being promoted in protein-enriched milk. The addition of nutrients to diluted whole milk and TB partly revealed different effects, indicating that there are media-specific growth limiting factors after dilution. Supplementation of minerals to diluted milk did not affect growth rates of all studied bacteria. Bacterial growth in diluted whole milk was decreased by the addition of high concentrations of amino acids in S. aureus, and by urea and additional B vitamins in E. coli and S. aureus. The growth rate of S. uberis was increased by the addition of B vitamins to diluted whole milk. The present results demonstrate that growth-limiting nutrients differ among pathogen types. Because reduced bacterial growth was only shown in diluted milk or TB, it is unlikely that alterations in nutrient availability occurring as a consequence of physiological changes of milk composition in the cow's udder would directly affect the susceptibility or course of bovine mastitis.

Hyperinvasiveness of Listeria monocytogenes sequence type 1 is independent of lineage I-specific genes encoding internalin-like proteins.

Listeriosis is a severe disease caused by the opportunistic bacterial pathogen Listeria monocytogenes (L. monocytogenes). Previous studies indicate that of the four phylogenetical lineages known, lineage I strains are significantly more prevalent in clinical infections than in the environment. Among lineage 1, sequence type (ST1) belongs to the most frequent genotypes in clinical infections and behaves hyperinvasive in experimental in vitro infections compared to lineage II strains suggesting that yet uncharacterized virulence genes contribute to high virulence of certain lineage I strains. This study investigated the effect of four specific lineage I genes encoding surface proteins with internalin-like structures on cellular infection. CNS derived cell lines (fetal bovine brain cells, human microglia cells) and non-CNS derived cell lines (bovine macrophage cells, human adenocarcinoma cells) that represent the various target cells of L. monocytogenes were infected with the parental ST1 strain and deletion mutants of the four genes. Despite their association with lineage I, deletion of the four genes investigated did not dampen the hyperinvasiveness of the ST1 strain. Similarly, these genes did not contribute to the intracellular survival and intercellular spread of L. monocytogenes ST1, indicating that these genes may have other functions, either during the infection process or outside the host.