Protective response mediated by immunization with recombinant proteins in a murine model of toxocariasis and canine infection by Toxocara canis.

Toxocariasis is a neglected parasitic zoonosis of global importance. The development of a formulation that can be used as a vaccine would help the definitive control of the infection. Preclinical studies selected two recombinant T. canis proteins (rTcVcan and rTcCad) which significantly protected mice against larval migration. In the present work, these proteins plus three adjuvants (Alhydrogel®, PAM3CSK4®, and Quil-A®) were used to immunize mice against toxocariasis; blood samples were collected three times to measure IgG (total, IgG1, IgG2a), IgA, and IgE via indirect ELISA. Cytokines (IL-5, TNF-α, and IL-10) were measured in splenocytes supernatant, and T. canis larvae were quantified in tissues. The best protein + adjuvant pair found (rTVcan + QuialA®) was then used to immunize T. canis-free puppies (n = 18) that were experimentally infected with T. canis and T. canis naturally-infected puppies (n = 6). Immunoglobulin (IgA, IgE, IgG, IgG1, and IgG2a), parasite load (eggs in feces), number of expelled adults and eggs extracted from the female uterus, and their fertility percentages were analyzed. In mice, it was observed a highly significant reduction (73%) of tissue larvae, a mixed cytokine profile (Th1/Th2), and anti-T. canis antibody titers (IgG, IgG1, IgG2a) using rTVcan + QuialA® mix. In canines, rTVcan + QuialA® promoted reduction in the parasite eggs in feces (95%) and eggs reduction obtained from the uteri of pharmacologically expelled adult females (58.38%). In our knowledge this is the first canine clinical trial of a vaccine with T. canis recombinant proteins. The formulation used has been shown to efficiently stimulate the production of antibodies against infection by T. canis. In the canine, a significant reduction in the number of eggs expelled by the experimental animals that received the formulation prophylactically was evidenced. Future tests should be developed to evaluate the duration of the protective effect and analyze other immune pathways that could be stimulated by the formulation used.

Seroprevalence of exposure to SARS-CoV-2 in domestic dogs and cats and its relationship with COVID-19 cases in the city of Villavicencio, Colombia.

Background: Since the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak, different animal species have been implicated as possible intermediate hosts that could facilitate the transmission of the virus between species. The detection of these hosts has intensified, reporting wild, zoo, farm, and pet animals. The goal of this study was to determine the seroprevalence of anti-SARS-CoV-2 immunoglobulins (IgG) in domestic dogs and cats and its epidemiological association with the frequency of coronavirus disease 2019 (COVID-19) patients in Villavicencio, Colombia. Methods: 300 dogs and 135 cats were randomly selected in a two-stage distribution by clusters according to COVID-19 cases (positive RT-qPCR for SARS-CoV-2) within the human population distributed within the eight communes of Villavicencio. Indirect enzyme-linked immunosorbent assay (ELISA) technique was applied in order to determine anti-SARS-CoV-2 IgG in sera samples. Kernel density estimation was used to compare the prevalence of COVID-19 cases with the seropositivity of dogs and cats. Results: The overall seroprevalence of anti-SARS-CoV-2 IgG was 4.6% (95% CI=3.2-7.4). In canines, 3.67% (95% CI=2.1-6.4) and felines 6.67% (95% CI=3.6-12.18). Kernel density estimation indicated that seropositive cases were concentrated in the southwest region of the city. There was a positive association between SARS-CoV-2 seropositivity in pet animals and their habitat in Commune 2 (adjusted OR=5.84; 95% CI=1.1-30.88). Spearman's correlation coefficients were weakly positive ( p=0.32) between the ratio of COVID-19 cases in November 2020 and the results for domestic dogs and cats from the eight communes of Villavicencio. Conclusions: In the present research cats were more susceptible to SARS-CoV-2 infection than dogs. This study provides the first positive results of anti-SARS-CoV-2 ELISA serological tests in domestic dogs and cats in Colombia with information about the virus transmission dynamics in Latin America during the COVID-19 pandemic.

Immunogenicity and protection induced by recombinant Toxocara canis proteins in a murine model of toxocariasis.

Toxocariasis, a natural helminth infection of dogs and cats caused by Toxocara canis and T. cati, respectively, that are transmitted to mammals, including humans. Infection control is based currently on periodic antihelmintic treatment and there is a need for the development of vaccines to prevent this infection. MATERIALS AND METHODS: Eight potential vaccine candidate T. canis recombinant proteins were identified by in silico (rTcGPRs, rTcCad, rTcVcan, rTcCyst) and larval proteomics (rTES26, rTES32, rMUC-3 and rCTL-4) analyses. Immunogenicity and protection against infectious challenge for seven of these antigens were determined in a murine model of toxocariasis. C57BL/6 female mice were immunized with each of or combinations of recombinant antigens prior to challenge with 500 T. canis embryonated eggs. Levels of specific antibodies (IgG, IgG1, IgG2a and IgE) in sera and cytokines (IL-5, INF-É£ and IL-10) produced by antigens-stimulated splenocytes, were measured. Presence of specific antibodies to the molecules was measured in sera of T. canis-seropositive dogs and humans. RESULTS: All seven molecules were immunogenic in immunized mice; all stimulated significantly elevated levels of specific IgG, IgG1 or IgG2a and six were associated with elevated levels of specific IgE; all induced elevated production of IFN- É£ and IL-10 by splenocytes, but only the in silico-identified membrane-associated recombinants (rTcCad, rTcVcan, and rTcCyst) induced significantly increased IL-5 production. Vaccination with two of the latter (rTcCad and rTcVcan) reduced larval loads in the T. canis challenged mice by 54.3% and 53.9% (P < 0.0001), respectively, compared to unimmunized controls. All seven recombinants were recognized by T. canis-seropositive dog and human sera. CONCLUSION: The identification of vaccine targets by in silico analysis was an effective strategy to identify immunogenic T. canis proteins capable of reducing larval burdens following challenge with the parasite. Two recombinant proteins, rTcCad and rTcVcan, were identified as promising vaccine candidates for canine toxocariasis.