Caracterização das propriedades funcionais e probióticas de bactérias láticas isoladas de cães para uso em vacinas orais contra leptospirose como adjuvante imune ou carreador antigênico

Abstract

Leptospirosis is an important reemerging infectious disease that affects populations worldwide. Caused by pathogenic spirochaetes of the genus Leptospira, the disease presents higher incidence in tropical and subtropical regions. Aimming new live vehicles or immune adjuvants for a new leptospirosis vaccine, Lactic Acid Bacteria were isolated and identified in the faeces of Chinese Crested and Yorkshire terrier pups and their probiotic features were investigated in vitro. Thirty-seven isolates were identified as Lactobacillus or Enterococcus. Out of these isolates, 31 were lactic acid bacteria (LAB) and belonged to the species Lactobacillus reuteri (16/37; 43.3%), Lactobacillus animalis (7/37; 18.9%), Lactobacillus acidophilus (3/37; 8.1%), Lactobacillus sanfranciscensis (2/37; 5.4%), Lactobacillus murinus (2/37; 5.4%), and Lactobacillus paraplantarum (1/37; 2.7%), while six other LAB isolates were Enterococcus spp. (6/37; 16.2%). Based on these results, two Lactobacillus strains L. paraplantarum (15) and L. reuteri (2) showed promising probiotic-related features and merit investigation as probiotics for dogs. Golden Syrian Hamsters fed with both strains had immunomodulatory properties assessed by RT-qPCR for selected cytokines and they stimulated proinflammatory (INF-, IL-12, TNF- ) and regulatory cytokines (IL-10, TGF-). The genes of LipL32 and Loa22 antigens of three Leptospira serovars were amplified by PCR, sequenced and then aligned with each other, confirming the high degree of evolutionary conservation of these genes. Recombinant LipL32 (rLipL32) was expressed in an E. coli heterologous system and purified as soluble fraction, yielding 7.96 mg of protein per liter of IPTG-induced culture. Antisera against Leptospira, were produced in BALB/c mice by inoculation of a commercial vaccine produced with five serovars of L. interrogans. The molecular mass of rLipL32 was determined by Western blot to be 32 kDa. Polyclonal antisera anti-rLipL32 was also produced in mice. Recombinat Loa antigen was not produced at this time.