Chagas disease (ChD), a complex and persistent parasitosis caused by Trypanosoma cruzi , represents a natural model of chronic infection, in which some people exhibit cardiac or digestive complications that can result in death 20–40 years after the initial infection. Nonetheless, due to unknown mechanisms, some T. cruzi -infected individuals remain asymptomatic throughout their lives. Actually, no vaccine is available to prevent ChD, and treatments for chronic ChD patients are controversial. Chronically T. cruzi -infected individuals exhibit a deterioration of T cell function, an exhaustion state characterized by poor cytokine production and increased inhibitory receptor co-expression, suggesting that these changes are potentially related to ChD progression. Moreover, an effective anti-parasitic treatment appears to reverse this state and improve the T cell response. Taking into account these findings, the functionality state of T cells might provide a potential correlate of protection to detect individuals who will or will not develop the severe forms of ChD. Consequently, we investigated the T cell response, analyzed by flow cytometry with two multicolor immunofluorescence panels, to assess cytokines/cytotoxic molecules and the expression of inhibitory receptors, in a murine model of acute (10 and 30 days) and chronic (100 and 260 days) ChD, characterized by parasite persistence for up to 260 days post-infection and moderate inflammation of the colon and liver of T. cruzi -infected mice. Acute ChD induced a high antigen-specific multifunctional T cell response by producing IFN-γ, TNF-α, IL-2, granzyme B, and perforin; and a high frequency of T cells co-expressed 2B4, CD160, CTLA-4, and PD-1. In contrast, chronically infected mice with moderate inflammatory infiltrate in liver tissue exhibited monofunctional antigen-specific cells, high cytotoxic activity (granzyme B and perforin), and elevated levels of inhibitory receptors (predominantly CTLA-4 and PD-1) co-expressed on T cells. Taken together, these data support our previous results showing that similar to humans, the T. cruzi persistence in mice promotes the dysfunctionality of T cells, and these changes might correlate with ChD progression. Thus, these results constitute a model that will facilitate an in-depth search for immune markers and correlates of protection, as well as long-term studies of new immunotherapy strategies for ChD.

In this work, we statistically improved culture media for rPOXA 1B laccase production, expressed in Pichia pastoris containing pGAPZαA-LaccPost-Stop construct and assayed at 10 L bioreactor production scale (6 L effective work volume). The concentrated enzyme was evaluated for temperature and pH stability and kinetic parameter, characterized by monitoring oxidation of different ABTS [2,] substrate concentrations. Plackett-Burman experimental design (PBED) implementation improved previous work results by 3.05-fold, obtaining a laccase activity of 1373.72 ± 0.37 U L −1 at 168 h of culture in a 500 mL shake flask. In contrast, one factor experimental design (OFED) applied after PBED improved by threefold the previous study, additionally increasing the C/N ratio. Employing OFED media at 10 L bioreactor scale was capable of producing 3159.93 ± 498.90 U L −1 at 192 h, representing a 2.4-fold increase. rPOXA 1B concentrate remained stable between 10 and 50 °C and retained over 70% residual enzymatic activity at 60 °C and 50% at 70 °C. Concerning pH stability, the enzyme was stable at pH 4.0 ± 0.2 with a residual activity greater than 90%. The lowest residual activity (60%) was obtained at pH 10.0 ± 0.2. Furthermore, the apparent kinetic parameters were V max of 3.163 × 10 −2 mM min −1 and K m of 1.716 mM. Collectively, regarding enzyme stability our data provide possibilities for applications involving a wide range of pH and temperatures. Keywords Plackett-Burman experimental design • One-factor experimental design • Pichia pastoris • Recombinant laccase • Enzyme stability • Enzyme kinetics Abbreviations MCOs Multicopper oxidases ABTS 2, 20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) PBL Pulping black liquor LDPE Low density polyethylene pAOX1 Alcohol oxidase promoter pGAP Glyceraldehyde-3-phosphate dehydrogenase promoter POXA 1B Laccase from P. ostreatus rPOXA 1B Recombinant laccase from P. ostreatus V max Maximum reaction rate K M Michaelis constant YPG Yeast extract, peptone and glucose culture media MCB Master cell bank YPG-Z Yeast extract, peptone, glucose-zeocin culture media EWV Effective work volume PBED Plackett-Burman experimental design OFED One-factor experimental design v.v.m. Air volume per media volume µ x Specific growth rate T d Duplication time r.p.m. Revolutions per minute Y (x/s) Biomass/substrate yield Y (p/s) Enzyme/substrate yield P (x) Biomass productivity P (p) Enzyme productivity SD Standard deviation GFP Green fluorescent protein

Bats have been implicated as reservoirs of relapsing fever group spirochaetes since the beginning of the last century. Recently, bat‐associated spirochaetes have been reported as human pathogens. In 1968, a spirochaete was detected in blood of the bat Natalus tumidirostris captured inside the Macaregua cave, Colombia. Data on this microorganism were never published again. The aim of this study was to evaluate the presence of Borrelia DNA in blood from bats of Macaregua cave. We performed molecular analyses using a genus‐specific real‐time PCR targeting the 16S rRNA to detect DNA of Borrelia in blood samples from 46 bats captured in the Macaregua cave. Positive samples were submitted to a battery of PCRs aiming to amply Borrelia 16S rRNA, flaB, glpQ, p66, ospC, clpA, clpX, nifS, pepX, pyrG, recG, rplB and uvrA genes. Seventeen samples were positive for Borrelia after real‐time PCR. With the exception of flaB gene, attempts to amplify further loci were unsuccessful. Nucleotide and amino acid divergences of four flaB haplotypes characterized from blood of Carollia perspicillata showed Borrelia burgdorferi sensu lato (Bbsl) as the most closely related group. A phylogenetic tree including 74 sequences of the genus confirmed this trend, since Borrelia genotypes detected in bats from Macaregua formed a monophyletic group basally positioned to Bbsl. Our results suggest that Borrelia genotypes characterized from bats roosting in the Macaregua cave might constitute a new taxon within the genus. This is the first molecular characterization of a Borrelia sp. in Colombia.

In 2015, we investigated Bartonella quintana and typhus group rickettsiae in body lice from homeless persons in Bogotá, Colombia. We found B. quintana–infected body lice and seroprevalence of this microorganism in 19% of homeless persons and typhus group rickettsiae in 56%. Public health professionals should start preemptive measures and active vector control.

Introduction: Bats have become an epidemiologically significant source of pathogenic microorganisms, such as leptospires, the causative agents of leptospirosis. However, little information exists about bats and their potential role as a reservoir of pathogenic Leptospira spp. in Colombia. The aim of this study was to evaluate the presence of pathogenic Leptospira spp. in the kidneys of bats from the Caribbean region of Colombia deposited in the collection of mammals of the Museo Javeriano de Historia Natural (MPUJ-MAMM). Methodology: DNA was extracted from twenty-six kidney samples from a total of 13 species of bats captured in Colombia. First, 16S ribosomal RNA conventional PCR was performed to detect the presence of Leptospira spp. Then, in samples that tested positive, LipL32 PCR was performed to detect pathogenic Leptospira spp. by sequencing and phylogenetic analysis. Results: The presence of Leptospira spp. was observed in 7/26 (26.9%) bats from the following 6 species: Carollia perspicillata, Glossophaga soricina, Dermanura phaeotis, Uroderma bilobatum, Desmodus rotundus, and Lophostoma silvicolum, and pathogenic Leptospira spp. were detected in 4/26 samples (15.4%). Conclusions: This study suggests that bats present in the Caribbean region of Colombia could be potential reservoirs of pathogenic Leptospira spp.

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Trypanosomatids are early divergent parasites which include several species of medical interest. Trypanosoma rangeli is not pathogenic for humans but shows a high immunological cross-reactivity with Trypanosoma cruzi, the causative agent of Chagas' disease that affects more than 17 million people throughout the world. Recent studies have suggested that T. cruzi KMP-11 antigen could be a good candidate for the induction of immunoprotective cytotoxic responses against T. cruzi natural infection. In the present paper the genes coding for the T. rangeli kinetoplastid membrane protein-11 have been characterized. The results show that the locus encoding this protein is formed by 4 gene units measuring 550 nucleotides in length, organized in tandem, and located in different chromosomes in KP1(+) and KP1(-) strains. The gene units are transcribed as a single mRNA of 530 nucleotides in length. Alignment of the T. rangeli KMP-11 deduced amino acid sequence with the homologous KMP-11 protein from T. cruzi revealed an identity of 97%. Interestingly, the T and B cell epitopes of the T. cruzi KMP-11 protein are conserved in the T. rangeli KMP-11 amino acid sequence.

Laccases are multicopper oxidases that catalyze aromatic and nonaromatic compounds with concomitant reduction of molecular oxygen to water. They are of great interest due to their potential biotechnological applications. In this work we statistically improved culture media for recombinant GILCC1 (rGILCC1) laccase production at low scale from Ganoderma lucidum containing the construct pGAPZαA-GlucPost-Stop in Pichia pastoris. Temperature, pH stability, and kinetic parameter characterizations were determined by monitoring concentrate enzyme oxidation at different ABTS substrate concentrations. Plackett-Burman Design allowed improving enzyme activity from previous work 36.08-fold, with a laccase activity of 4.69 ± 0.39 UL−1 at 168 h of culture in a 500 mL shake-flask. Concentrated rGILCC1 remained stable between 10 and 50°C and retained a residual enzymatic activity greater than 70% at 60°C and 50% at 70°C. In regard to pH stability, concentrated enzyme was more stable at pH 4.0 ± 0.2 with a residual activity greater than 90%. The lowest residual activity greater than 55% was obtained at pH 10.0 ± 0.2. Furthermore, calculated apparent enzyme kinetic parameters were a Vmax of 6.87 × 10−5 mM s−1, with an apparent Km of 5.36 × 10−2 mM. Collectively, these important stability findings open possibilities for applications involving a wide pH and temperature ranges.