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Upon ingestion from an infected host, tick-borne pathogens (TBPs) have to overcome colonization resistance, a defense mechanism by which tick microbiota prevent microbial invasions. Previous studies have shown that the pathogen Anaplasma phagocytophilum alters the microbiota composition of the nymphs of Ixodes scapularis, but its impact on tick colonization resistance remains unclear. We analyzed tick microbiome genetic data using published Illumina 16S rRNA sequences, assessing microbial diversity within ticks (alpha diversity) through species richness, evenness, and phylogenetic diversity. We compared microbial communities in ticks with and without infection with A. phagocytophilum (beta diversity) using the Bray-Curtis index. We also built co-occurrence networks and used node manipulation to study the impact of A. phagocytophilum on microbial assembly and network robustness, crucial for colonization resistance. We examined network robustness by altering its connectivity, observing changes in the largest connected component (LCC) and the average path length (APL). Our findings revealed that infection with A. phagocytophilum does not significantly alter the overall microbial diversity in ticks. Despite a decrease in the number of nodes and connections within the microbial networks of infected ticks, certain core microbes remained consistently interconnected, suggesting a functional role. The network of infected ticks showed a heightened vulnerability to node removal, with smaller LCC and longer APL, indicating reduced resilience compared to the network of uninfected ticks. Interestingly, adding nodes to the network of infected ticks led to an increase in LCC and a decrease in APL, suggesting a recovery in network robustness, a trend not observed in networks of uninfected ticks. This improvement in network robustness upon node addition hints that infection with A. phagocytophilum might lower ticks' resistance to colonization, potentially facilitating further microbial invasions. We conclude that the compromised colonization resistance observed in tick microbiota following infection with A. phagocytophilum may facilitate co-infection in natural tick populations.

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In recent decades, tick-borne diseases (TBDs) have surged and expanded globally due to factors like changes in human activities, land use patterns, and climate change, and it have been associated with the emergence of zoonotic diseases. Cuba faces the impact of ticks on human health and the economy. Although Cuba has studied TBDs extensively for the past 50 years, focus on tick-borne viral pathogens affecting humans remains scant. Despite TBDs not currently being a major health concern in Cuba, factors like inadequate clinician awareness, climate conditions, global tick emergence, and evidence of zoonotic pathogens in ticks underscore the importance of enhanced TBD surveillance in the country. Here we revised the available information on ticks as vectors of pathogenic viruses to humans, spotlighting bats as potential reservoirs of tick-borne viruses (TBVs). Ticks on bats have gained interest as potential reservoirs of pathogenic viruses to humans in Cuba and worldwide. Understanding their role in maintaining viruses and their potential transmission to humans is crucial for the implementation of surveillance and control programs to reduce the risk of tick-borne viral diseases and public health management.

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BACKGROUND: Ixodid ticks, particularly Rhipicephalus sanguineus s.l., are important vectors of various disease-causing agents in dogs and humans in Cuba. However, our understading of interactions among tick-borne pathogens (TBPs) in infected dogs or the vector R. sanguineus s.l. remains limited. This study integrates microfluidic-based high-throughput real-time PCR data, Yule's Q statistic, and network analysis to elucidate pathogen-pathogen interactions in dogs and ticks in tropical western Cuba. METHODS: A cross-sectional study involving 46 client-owned dogs was conducted. Blood samples were collected from these dogs, and ticks infesting the same dogs were morphologically and molecularly identified. Nucleic acids were extracted from both canine blood and tick samples. Microfluidic-based high-throughput real-time PCR was employed to detect 25 bacterial species, 10 parasite species, 6 bacterial genera, and 4 parasite taxa, as well as to confirm the identity of the collected ticks. Validation was performed through end-point PCR assays and DNA sequencing analysis. Yule's Q statistic and network analysis were used to analyse the associations between different TBP species based on binary presence-absence data. RESULTS: The study revealed a high prevalence of TBPs in both dogs and R. sanguineus s.l., the only tick species found on the dogs. Hepatozoon canis and Ehrlichia canis were among the most common pathogens detected. Co-infections were observed, notably between E. canis and H. canis. Significant correlations were found between the presence of Anaplasma platys and H. canis in both dogs and ticks. A complex co-occurrence network among haemoparasite species was identified, highlighting potential facilitative and inhibitory roles. Notably, H. canis was found as a highly interconnected node, exhibiting significant positive associations with various taxa, including A. platys, and E. canis, suggesting facilitative interactions among these pathogens. Phylogenetic analysis showed genetic diversity in the detected TBPs. CONCLUSIONS: Overall, this research enhances our understanding of TBPs in Cuba, providing insights into their prevalence, associations, and genetic diversity, with implications for disease surveillance and management.

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CONTEXT: Pathogens can manipulate microbial interactions to ensure survival, potentially altering the functional patterns and microbiome assembly. The present study investigates how Anaplasma phagocytophilum infection affects the functional diversity, composition, and assembly of the Ixodes scapularis microbiome, with a focus on high central pathways-those characterized by elevated values in centrality metrics such as eigenvector, betweenness, and degree measures, in the microbial community. METHODS: Using previously published data from nymphs' gut V4 region's amplicons of bacterial 16S rRNA, we predicted the functional diversity and composition in control and A. phagocytophilum-infected ticks and inferred co-occurrence networks of taxa and ubiquitous pathways in each condition to associate the high central pathways to the microbial community assembly. RESULTS: Although no differences were observed concerning pathways richness and diversity, there was a significant impact on taxa and functional assembly when ubiquitous pathways in each condition were filtered. Moreover, a notable shift was observed in the microbiome's high central functions. Specifically, pathways related to the degradation of nucleosides and nucleotides emerged as the most central functions in response to A. phagocytophilum infection. This finding suggests a reconfiguration of functional relationships within the microbial community, potentially influenced by the pathogen's limited metabolic capacity. This limitation implies that the tick microbiome may provide additional metabolic resources to support the pathogen's functional needs. CONCLUSIONS: Understanding the metabolic interactions within the tick microbiome can enhance our knowledge of pathogen colonization mechanisms and uncover new disease control and prevention strategies. For example, certain pathways that were more abundant or highly central during infection may represent potential targets for microbiota-based vaccines.

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BACKGROUND: Dirofilarioses are widespread diseases caused by mosquito-borne nematodes of the family Onchocercidae, genus Dirofilaria. The major etiologic agent of canine dirofilariosis in the American continent is the zoonotic parasite Dirofilaria immitis. Existing reports of filarioid nematodes in Cuba are based solely on morphological and immunological analysis which do not allow unambiguous identification and/or direct detection of causal agents. RESULTS: Here we present the molecular characterization of filarioid nematodes found in a dog in Cuba. Based on the molecular and phylogenetic analysis of the 5.8S-ITS2-28S region and cox1 gene fragments, the worms were unambiguously classified as D. immitis. Sequence analysis showed high identity of the gene fragments in this study with others previously obtained from D. immitis found in dogs, wolfs and jackals but also from mosquito vectors of D. immitis. CONCLUSIONS: Further studies are guarantee to better understand the epidemiological impact of canine dirofilariosis in Cuba as well as the competence of different species of culicid mosquitoes as vectors of Dirofilaria in the country.

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Anaplasma marginale is an obligate intraerythrocytic bacterium of bovines, responsible for large economic losses worldwide. It is mainly transmitted by Rhipicephalus (Boophilus) microplus ticks and, despite mounting evidence suggesting transovarial transmission, the occurrence of this phenomenon remains controversial. We evaluated the vector competence of R. microplus larvae vertically infected with A. marginale to transmit the bacterium to a naïve bovine. A subgroup of engorged female ticks collected from an A. marginale-positive animal was dissected and the presence of the pathogen in its tissues was confirmed. A second subgroup of ticks was placed under controlled conditions for oviposition. After confirming the presence of A. marginale in the hatched larvae, an experimental infestation assay was conducted. Larvae were placed on an A. marginale-free splenectomized calf. The bacterium was detected in the experimentally infested bovine 22 days post-infestation. We analyzed the A. marginale diversity throughout the transmission cycle using the molecular marker MSP1a. Different genotypes were detected in the mammalian and arthropod hosts showing a reduction of strain diversity along the transmission process. Our results demonstrate the vertical transmission of A. marginale from R. microplus females to its larvae, their vector competence to transmit the pathogen, and a bottleneck in A. marginale strain diversity.

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Introduction: Ornithodoros erraticus and Ornithodoros moubata are the main vectors of African swine fever virus (ASFV) and the human relapsing fever spirochetes Borrelia hispanica and Borrelia crocidurae in the Mediterranean region and Borrelia duttoni in continental Africa. Manipulation of the tick microbiome has been shown to reduce vector fitness and competence in tick vectors, suggesting that the identification of key microbial players associated with tick tissues can inform interventions such as anti-microbiota vaccines to block pathogen development in the midgut and/or salivary glands. Methods: In this study, we analyzed and compared the microbiome of the salivary glands and midgut of O. erraticus and O. moubata. For the taxonomic and functional characterization of the tissue-specific microbiome, we used 16S rRNA amplicon sequencing and prediction of metabolic profiles using PICRUSt2. Co-occurrence networks were built to characterize the community assembly and identify keystone taxa in each tick species. Results: Our results revealed differences in the composition, diversity, and assembly of the bacterial microbiome of salivary glands and midgut within each tick species, but differences were more noticeable in O. moubata. Differences were also found in the microbiome of each tissue, salivary gland and midgut, between species. However, the 'Core Association Networks (CAN)' analysis revealed conserved patterns of interacting taxa in tissues within and between tick species. Different keystone taxa were identified in O. erraticus and O. moubata tissues, but Muribaculaceae and Alistipes were found as keystone taxa in the salivary glands of both tick species which justifies their use as anti-microbiota vaccine candidates to alter the microbiome and reduce tick fitness and/or block pathogen transmission. The high similarity of predicted metabolic pathways profiles between tissues of the two tick species suggests that taxonomic variability of the microbiome is not associated with significant changes in microbial functional profiles. Conclusion: We conclude that the taxonomic structure of the microbiome in O. erraticus and O. moubata is tissue-specific, suggesting niche partitioning of bacterial communities associated to these soft ticks. However, shared keystone taxa and conserved patterns of interacting taxa between tissues and tick species suggest the presence of key microbial players that could be used as anti-microbiota vaccine candidates to affect tick physiology and/or pathogen colonization.

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Riparian buffer systems (RBS) are a common agroforestry practice that involves maintaining a forested boundary adjacent to water bodies to protect the aquatic ecosystems in agricultural landscapes. While RBS have potential for carbon sequestration, they also can be sources of methane emissions. Our study site at Washington Creek in Southern Ontario, includes a rehabilitated tree buffer (RH), a grassed buffer (GRB), an undisturbed deciduous forest (UNF), an undisturbed coniferous forest (CF), and an adjacent agricultural field (AGR). The objective of this study was to assess the diversity and activity of CH4 cycling microbial communities in soils sampled during hot moments of methane fluxes (July 04 and August 15). We used qPCR and high-throughput amplicon sequencing from both DNA and cDNA to target methanogen and methanotroph communities. Methanogens, including the archaeal genera Methanosaeta, Methanosarcina, Methanomassiliicoccus, and Methanoreggula, were abundant in all RBSs, but they were significantly more active in UNF soils, where CH4 emissions were highest. Methylocystis was the most prevalent taxon among methanotrophs in all the riparian sites, except for AGR soils where the methanotrophs community was composed primarily of members of rice paddy clusters (RPCs and RPC-1) and upland soil clusters (TUSC and USCα). The main factors influencing the composition and assembly of methane-cycling microbiomes were soil carbon and moisture content. We concluded that the differences in CH4 fluxes observed between RBSs were primarily caused by differences in the presence and activity of methanogens, which were influenced by total soil carbon and water content. Overall, this study emphasizes the importance of understanding the microbial drivers of CH4 fluxes in RBSs in order to maximize RBS environmental benefits.

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BACKGROUND: Canine monocytic ehrlichiosis (CME) is caused by the tick-borne pathogen Ehrlichia canis, an obligate intracellular Gram-negative bacterium of the family Anaplasmataceae with tropism for canine monocytes and macrophages. The trp36 gene, which encodes for the major immunoreactive protein TRP36 in E. canis, has been successfully used to characterize the genetic diversity of this pathogen in different regions of the world. Based on trp36 sequence analysis, four E. canis genogroups, United States (US), Taiwan (TWN), Brazil (BR) and Costa Rica (CR), have been identified. The aim of this study was to characterize the genetic diversity of E. canis in Cuba based on the trp36 gene. METHODS: Whole blood samples (n = 8) were collected from dogs found to be infested with the tick vector Rhipicephalus sanguineus sensu lato (s.l.) and/or presenting clinical signs and symptoms of CME. Total DNA was extracted from the blood samples and trp36 fragments were amplified by PCR. Nucleotide and protein sequences were compared using alignments and phylogenetic analysis. RESULTS: Four of the trp36 sequences obtained (n = 8) fall within the phylogenetic cluster grouping the US genogroup E. canis strains. The other E. canis trp36 sequences formed a separate and well-supported clade (94% bootstrap value) that is phylogenetically distant from the other major groups and thus represents a new genogroup, herein designated as the 'Cuba (CUB) genogroup'. Notably, dogs infected with the CUB genogroup presented frequent hemorrhagic lesions. CONCLUSIONS: The results of this study suggest that genetic diversification of E. canis in Cuba is associated with the emergence of E. canis strains with increased virulence.

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A total of 434 poultry cloacal samples were collected from seven different farms in different years (2013-2015) in the Cuban province of Mayabeque and analyzed for the presence of third-generation cephalosporin-resistant Escherichia coli (3GC-R-Ec). Sixty-two 3GC-R-Ec isolates were recovered in total from the farms, with detection rates of 2.9% in 2013, 10.3% in 2014, and 28.7% in 2015. Characterization of 32 3GC-R-Ec isolates revealed the presence of the extended-spectrum ß-lactamase (ESBL) genes blaCTX-M-1 (n = 27), blaCTX-M-15 (n = 4), and blaCTX-M-1 together with blaLAP-2 (n = 1). The isolates also contained different proportions of genes conferring decreased susceptibility to sulfonamides (sul1, sul2, sul3), trimethoprim (dfrA1, dfrA7, dfrA12, dfrA14, dfrA17), tetracyclines (tet(A), tet(B)), aminoglycosides (aac(6')-Ib-cr, strA, strB), chloramphenicol (cmlA1, floR), macrolides (mph(A), mph(D)), and quinolones (qnrS, qnrB, aac(6')-Ib-cr) as well as mutations in the fluoroquinolone-resistance determining regions of GyrA (S83L, D87N, D87Y) and ParC (S80I, E84G). The isolates belonged to 23 different sequence types and to phylogroups A (n = 25), B1 (n = 5), and D (n = 2), and they contained plasmid-associated incompatibility groups FII, X1, HI1, HI2, N, FIA, and FIB. These findings reveal a genetically diverse population of multiresistant ESBL-producing E. coli in poultry farms in Cuba, which suggests multiple sources of contamination and the acquisition of antibiotic resistance genes.