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.

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.

Objective: To evaluate the prevalence of chronic postsurgical pain (CPSP) after tibial plateau leveling osteotomy (TPLO) in dogs and to determine the influence of preemptive locoregional analgesia on CPSP. Study design: Retrospective study. Animals: One hundred twenty client-owned dogs. Methods: Medical records of dogs that underwent TPLO between 2012 and 2016 were reviewed for demographic information and type of preemptive analgesia. Owners were contacted to retrospectively assess the quality of life of their dogs by using the Helsinki Chronic Pain Index (HCPI) before and 6 months after surgery and at the time of questioning. An HCPI score > 12 was considered indicative of CPSP. Medical records were reviewed for demographic information and type of preemptive analgesia. A cumulative logit model was used to assess correlation of type of perioperative analgesia, HCPI, and demographic data. Results: The HCPI score was consistent with CPSP in 41 of 101 dogs with long-term follow-up (2.9 ± 1.5 years after surgery). Chronic postsurgical pain was documented in 11 of 32 and 13 of 28 dogs that received a spinal or epidural injection, respectively, or in 28 of 80 and 25 of 67 dogs with sciatic-femoral block at 6 months or with long-term follow-up after TPLO, respectively (P > .05). A negative correlation was found between HCPI and both weight and age 6 months after surgery. Only weight remained negatively correlated 2.9 years after surgery. Conclusion: Forty-one percent of dogs that were evaluated exhibited HCPI values compatible with CPSP long-term after TPLO, regardless of the type of preemptive analgesia. Increased body weight was a negative prognostic factor for CPSP development. Clinical significance: Additional studies are required to evaluate CPSP development after TPLO.

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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.