Further in vivo evidence implying DNA apurinic/apyrimidinic endonuclease activity in Trypanosoma cruzi oxidative stress survival.

Trypanosoma cruzi is under the attack of reactive species produced by its mammalian and insect hosts. To survive, it must repair its damaged DNA. We have shown that a base excision DNA repair (BER)-specific parasite TcAP1 endonuclease is involved in the resistance to H2 O2 . However, a putative TcAP1 negative dominant form impairing TcAP1 activity in vitro did not show any in vivo effect. Here, we show that a negative dominant form of the human APE1 apurinic/apyrimidinic (AP) endonuclease (hAPE1DN) induces a decrease in epimastigote and metacyclic trypomastigote viability when parasites were exposed to H2 O2 . Those results confirm that TcAP1 AP endonuclease activity plays an important role in epimastigote and in infective metacyclic trypomastigote oxidative DNA damage resistance leading to parasite persistence in the insect and mammalian hosts. All along its biological cycle and in its different cellular forms, T. cruzi, the etiological parasite agent of Chagas' disease, is under the attack of reactive species produced by its mammalian and insect hosts. To survive, T. cruzi must repair their oxidative damaged DNA. We have previously shown that a specific parasite TcAP1 AP endonuclease of the BER is involved in the T. cruzi resistance to oxidative DNA damage. We have also demonstrated that epimastigotes and cell-derived trypomastigotes parasite forms expressing a putative TcAP1 negative dominant form (that impairs the TcAP1 activity in vitro), did not show any in vivo effect in parasite viability when exposed to oxidative stress. In this work, we show the expression of a negative dominant form of the human APE1 AP endonuclease fused to a green fluorescent protein (GFP; hAPE1DN-GFP) in T. cruzi epimastigotes. The fusion protein is found both in the nucleus and cytoplasm of noninfective epimastigotes but only in the nucleus in metacyclic and cell-derived trypomastigote infective forms. Contrarily to the TcAP1 negative dominant form, the ectopic expression of hAPE1DN-GFP induces a decrease in epimastigote and metacyclic trypomastigote viability when parasites were exposed to increasing H2 O2 concentrations. No such effect was evident in expressing hAPE1DN-GFP cell-derived trypomastigotes. Although the viability of both wild-type infective trypomastigote forms diminishes when parasites are submitted to acute oxidative stress, the metacyclic forms are more resistant to H2 O2 exposure than cell-derived trypomastigotes.Those results confirm that the BER pathway and particularly the AP endonuclease activity play an important role in epimastigote and metacyclic trypomastigote oxidative DNA damage resistance leading to parasite survival and persistence inside the mammalian and insect host cells.

Mechanisms by which Factor H protects Trypanosoma cruzi from the alternative pathway of complement.

Chagas disease, a chronic disabling disease caused by the protozoan Trypanosoma cruzi, has no standardized treatment or preventative vaccine. The infective trypomastigote form of T. cruzi is highly resistant to killing by the complement immune system. Factor H (FH), a negative regulator of the alternative pathway (AP) of complement on cell surfaces and in blood, contains 20 short consensus repeat domains. The four N-terminal domains of FH inactivate the AP, while the other domains interact with C3b/d and glycan markers on cell surfaces. Various pathogens bind FH to inactivate the AP. T. cruzi uses its trans-sialidase enzyme to transfer host sialic acids to its own surface, which could be one of the approaches it uses to bind FH. Previous studies have shown that FH binds to complement-opsonized T. cruzi and parasite desialylation increases complement-mediated lysis of trypomastigotes. However, the molecular basis of FH binding to T. cruzi remain unknown. Only trypomastigotes, but not epimastigotes (non-infective, complement susceptible) bound FH directly, independent of C3 deposition, in a dose-dependent manner. Domain mapping experiments using 3-5 FH domain fragments showed that domains 5-8 competitively inhibited FH binding to the trypomastigotes by ~35% but did not decrease survival in complement. FH-Fc or mutant FH-Fc fusion proteins (3-11 contiguous FH domains fused to the IgG Fc) also did not kill trypomastigotes. FH-related protein-5, whose domains bear significant sequence identity to all known polyanion-binding FH domains (6-7, 10-14, 19-20), fully inhibited FH binding to trypomastigotes and reduced trypomastigote survival to < 24% in the presence of serum. In conclusion, we have elucidated the role of FH in complement resistance of trypomastigotes.

High Exposure to Livestock Pathogens in Southern Pudu (Pudu puda) from Chile.

A significant gap in exposure data for most livestock and zoonotic pathogens is common for several Latin America deer species. This study examined the seroprevalence against 13 pathogens in 164 wild and captive southern pudu from Chile between 2011 and 2023. Livestock and zoonotic pathogen antibodies were detected in 22 of 109 wild pudus (20.18%; 95% CI: 13.34-29.18) and 17 of 55 captive pudus (30.91%; 95% CI: 19.52-44.96), including five Leptospira interrogans serovars (15.38% and 10.71%), Toxoplasma gondii (8.57% and 37.50%), Chlamydia abortus (3.03% and 12.82%), Neospora caninum (0.00% and 9.52%), and Pestivirus (8.00% and 6.67%). Risk factors were detected for Leptospira spp., showing that fawn pudu have statistically significantly higher risk of positivity than adults. In the case of T. gondii, pudu living in "free-range" have a lower risk of being positive for this parasite. In under-human-care pudu, a Pestivirus outbreak is the most strongly suspected as the cause of abortions in a zoo in the past. This study presents the first evidence of Chlamydia abortus in wildlife in South America and exposure to T. gondii, L. interrogans, and N. caninum in wild ungulate species in Chile. High seroprevalence of livestock pathogens such as Pestivirus and Leptospira Hardjo in wild animals suggests a livestock transmission in Chilean template forest.

Zoonotic Cryptosporidium meleagridis in urban invasive monk parakeets.

Zoonotic Cryptosporidium meleagridis was identified in invasive monk parakeets (Myiopsitta monachus) from Santiago metropolis in Chile. Oocysts were isolated from nestlings' faeces, and a fragment of 18S ribosomal RNA gene was amplified and sequenced. This finding emphasizes the importance of permanent pathogen monitoring in synanthropic species with wide global distribution.

Epidemiological Characterization of Isolates of Salmonella enterica and Shiga Toxin-Producing Escherichia coli from Backyard Production System Animals in the Valparaíso and Metropolitana Regions.

Backyard production systems (BPS) are distributed worldwide, rearing animals recognized as reservoirs of Salmonella enterica and Shiga toxin-producing Escherichia coli (STEC), both zoonotic pathogens. The aim of this study was to characterize isolates of both pathogens obtained from animals raised in BPS from two central Chile regions. The presence of pathogens was determined by bacterial culture and confirmatory PCR for each sampled BPS, calculating positivity rates. Multivariate logistic regression was used to determine risk factors. Additionally, phenotypic antimicrobial resistance was determined. A positivity rate of 2.88% for S. enterica and 14.39% for STEC was determined for the complete study region (Valparaíso and Metropolitana regions). Risk factor analysis suggests that the presence of ruminants (OR = 1.03; 95% CI = 1.002-1.075) increases the risk of STEC-positive BPS, and the presence of ruminants (OR = 1.05; 95% CI = 1.002-1.075) and the animal handlers being exclusively women (OR = 3.54; 95% CI = 1.029-12.193) increase the risk for S. enterica/STEC positivity. Eighty percent of S. enterica isolates were multidrug resistant, and all STEC were resistant to Cephalexin. This study evidences the circulation of multidrug-resistant zoonotic bacterial strains in animals kept in BPS and the presence of factors that modify the risk of BPS positivity for both pathogens.

Identification and characterization of porcine Rotavirus A in Chilean swine population.

Rotavirus A (RVA) is a common cause of diarrhea in newborn pigs, leading to significant economic losses. RVA is considered a major public health concern due to genetic evolution, high prevalence, and pathogenicity in humans and animals. The objective of this study was to identify and characterize RVA in swine farms in Chile. A total of 154 samples (86 oral fluids and 68 fecal samples) were collected, from 22 swine farms. 58 (38%) samples belonging to 14 farms were found positive for RVA by real-time RT-PCR. The samples with low Ct values (21) and the two isolates were selected for whole genome sequencing. Nearly complete genomes were assembled from both isolates and partial genomes were assembled from five clinical samples. BLAST analysis confirmed that these sequences are related to human and swine-origin RVA. The genomic constellation was G5/G3-P[7]-I5-R1-C1-M1-A8-N1-T1-E1-H1. Phylogenetic analysis showed that VP4, VP1, VP2, NSP2, NSP3, NSP4, and NSP5 sequences were grouped in monophyletic clusters, suggesting a single introduction. The phylogenies for VP7, VP6, VP3, and NSP1 indicated two different origins of the Chilean sequences. The phylogenetic trees showed that most of the Chilean RVA sequences are closely related to human and swine-origin RVA detected across the world. The results highlight the potential zoonotic nature of RVA circulating in Chilean swine farms. Therefore, it is important to continue RVA whole genome sequencing globally to fully understand its complex epidemiology and early detection and characterization of zoonotic strains.

Molecular surveillance of potential SARS-CoV-2 reservoir hosts in wildlife rehabilitation centers.

BACKGROUND: The COVID-19 pandemic, caused by SARS-CoV-2 infection, has become the most devastating zoonotic event in recent times, with negative impacts on both human and animal welfare as well as on the global economy. Although SARS-CoV-2 is considered a human virus, it likely emerged from animals, and it can infect both domestic and wild animals. This constitutes a risk for human and animal health including wildlife with evidence of SARS-CoV-2 horizontal transmission back and forth between humans and wild animals. AIM: Molecular surveillance in different wildlife rehabilitation centers and wildlife associated institutions in Chile, which are critical points of animal-human interaction and wildlife conservation, especially since the aim of wildlife rehabilitation centers is to reintroduce animals to their original habitat. MATERIALS AND METHODS: The survey was conducted in six WRCs and three wildlife associated institutions. A total of 185 samples were obtained from 83 individuals belonging to 15 different species, including vulnerable and endangered species. Each specimen was sampled with two different swabs: one oropharyngeal or nasopharyngeal according to the nostril diameter, and/or a second rectal sample. RNA was extracted from the samples and two different molecular assays were performed: first, a conventional RT-PCR with pan-coronavirus primers and a second SARS-CoV-2 qPCR targeting the N and S genes. RESULTS: All 185 samples were negative for SARS-CoV-2. CLINICAL RELEVANCE: This study constitutes the first report on the surveillance of SARS-CoV-2 from wildlife treated in rehabilitation centers in Chile, and supports the biosafety procedures adopted in those centers.

Elucidating the Role of Innate and Adaptive Immune Responses in the Pathogenesis of Canine Chronic Inflammatory Enteropathy-A Search for Potential Biomarkers.

Canine chronic inflammatory enteropathy (CIE) is one of the most common chronic gastrointestinal diseases affecting dogs worldwide. Genetic and environmental factors, as well as intestinal microbiota and dysregulated host immune responses, participate in this multifactorial disease. Despite advances explaining the immunological and molecular mechanisms involved in CIE development, the exact pathogenesis is still unknown. This review compiles the latest reports and advances that describe the main molecular and cellular mechanisms of both the innate and adaptive immune responses involved in canine CIE pathogenesis. Future studies should focus research on the characterization of the immunopathogenesis of canine CIE in order to advance the establishment of biomarkers and molecular targets of diagnostic, prognostic, or therapeutic utility.

SEROSURVEY FOR SELECTED PARASITIC AND BACTERIAL PATHOGENS IN DARWIN'S FOX (LYCALOPEX FULVIPES): NOT ONLY DOG DISEASES ARE A THREAT.

The Darwin's fox (Lycalopex fulvipes) is one of the most endangered carnivores worldwide, with the risk of disease spillover from domestic dogs being a major conservation threat. However, lack of epidemiologic information about generalist, non-dog-transmission-dependent protozoal and bacterial pathogens may be a barrier for disease prevention and management. To determine the exposure of some of these agents in Darwin's fox populations, 54 serum samples were collected from 47 Darwin's foxes in Southern Chile during 2013-18 and assessed for the presence of antibodies against Brucella abortus, Brucella canis, Coxiella burnetii, pathogenic Leptospira (serovars Grippotyphosa, Pomona, Canicola, Hardjo, and Copehageni), Toxoplasma gondii, and Neospora caninum. The highest seroprevalence was detected for T. gondii (78%), followed by pathogenic Leptospira (14%). All the studied Leptospira serovars were confirmed in at least one animal. Two foxes seroconverted to Leptospira and one to T. gondii during the study period. No seroconversions were observed for the other pathogens. No risk factors, either intrinsic (sex, age) or extrinsic (season, year, and degree of landscape anthropization), were associated with the probability of being exposed to T. gondii. Our results indicate that T. gondii exposure is widespread in the Darwin's fox population, including in areas with minimal anthropization, and that T. gondii and pathogenic Leptospira might be neglected threats to the species. Further studies identifying the causes of morbidity and mortality in Darwin's fox are needed to determine if these or other pathogens are having individual or population-wide effects in this species.

Urban public squares as potential hotspots of dog-human contact: A spatial analysis of zoonotic parasites detection in Gran Santiago, Chile.

Zoonotic parasites are a major public health problem globally, representing a hazard to human health. The infections occur through contact with different parasite forms. Public squares are a common meeting place for people and dogs, becoming a risk area for transmission to other dogs and humans. A spatial analysis of zoonotic parasite distribution in urban public squares in Gran Santiago, Chile was performed to elucidate its importance, exploring spatial aggregation on positivity rates at commune level. A total of 170 stool samples were analysed; 54 (31.7%) were positive for gastrointestinal-zoonotic parasites. Positivity was detected in 27 (79.4%) squares. Diversity of parasite species was variable across sites. Toxocara canis was the most frequently detected (27 samples, 21/34 squares), followed by Toxascaris leonina (13 samples, 10/34 squares), cestode eggs (10 samples, 9/34 squares), Giardia sp. (6 samples, 5/34 squares), Ancylostomatidae-like eggs (4 samples, 4/34 squares), Cryptosporidium spp. (2 samples, 2/34 squares) and Entamoeba spp. (1 sample, 1/34 square). Spatial autocorrelation (SA) was observed at commune level. The local indicators of the spatial association test showed statistical significance for cestode eggs (p < 0.001) with a negative SA (Moran's I Index = -0.2929, CI-95% = -0.3869--0.1989) and Ancylostomatidae (p = 0.046) with a positive SA (Moran's I Index = 0.1404, CI-95% = 0.0499-0.2309). Toxocara canis is the most prevalent geo-helminth in public places of Santiago, Chile. Spatial autocorrelation was detected for cestode eggs and Ancylostomatidae. Diagnosis and detection of parasites is key for the establishment of control/eradication of environmental dissemination following One Health guidelines.