Exploring the impact of Anaplasma phagocytophilum on colonization resistance of Ixodes scapularis microbiota using network node manipulation.

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.

Importance of Probiotics in Fish Aquaculture: Towards the Identification and Design of Novel Probiotics.

Aquaculture is a growing industry worldwide, but it faces challenges related to animal health. These challenges include infections by parasites, bacteria, and viral pathogens. These harmful pathogens have devastating effects on the industry, despite efforts to control them through vaccination and antimicrobial treatments. Unfortunately, these measures have proven insufficient to address the sanitary problems, resulting in greater environmental impact due to the excessive use of antimicrobials. In recent years, probiotics have emerged as a promising solution to enhance the performance of the immune system against parasitic, bacterial, and viral pathogens in various species, including mammals, birds, and fish. Some probiotics have been genetically engineered to express and deliver immunomodulatory molecules. These promote selective therapeutic effects and specific immunization against specific pathogens. This review aims to summarize recent research on the use of probiotics in fish aquaculture, with a particular emphasis on genetically modified probiotics. In particular, we focus on the advantages of using these microorganisms and highlight the main barriers hindering their widespread application in the aquaculture industry.

Tick-borne viruses and their risk to public health in the Caribbean: Spotlight on bats as reservoirs in Cuba.

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.

Microfluidic PCR and network analysis reveals complex tick-borne pathogen interactions in the tropics.

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.

Dissecting the impact of Anaplasma phagocytophilum infection on functional networks and community stability of the tick microbiome.

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.

First molecular characterization of Dirofilaria Immitis in Cuba.

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.

Transovarial Transmission of Anaplasma marginale in Rhipicephalus (Boophilus) microplus Ticks Results in a Bottleneck for Strain Diversity.

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.

Morphological and molecular identification of the brown dog tick in Mexico.

Ticks of the Rhipicephalus sanguineus complex are known as the brown dog ticks. This complex groups at least 12 species of ticks that are distributed worldwide. On the American continents, R. sanguineus sensu stricto (s.s.), is distributed in temperate areas, while Rhipicephalus sanguineus sensu lato (s.l.), also called "tropical lineage" is distributed in tropical regions. Previous analyses of brown dog ticks from Mexico have identified the so-called tropical lineage and the country generally has a climate more favorable for these ticks (> 20o C in average). In addition, some pathogens thought to be transmitted by this lineage (such as Ehrlichia canis, and Rickettsia rickettsii) are prevalent in Mexico. Herein we aim to contribute to the study of brown dog ticks by providing morphological identification and molecular analysis of mt 12S rDNA and 16S rDNA sequences from ticks collected from 12 states in Mexico. Our results indicate that the tropical lineage of R. sanguineus s.l., recently redescribed as R. linnaei is widely distributed in Mexico.

Comparison of salivary gland and midgut microbiome in the soft ticks Ornithodoros erraticus and Ornithodoros moubata.

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.

Differential Expression of Immune Genes in the Rhipicephalus microplus Gut in Response to Theileria equi Infection.

Rhipicephalus microplus is the only tick species known to serve as a biological vector of Theileria equi for horses and other equids in Brazil. The protozoan T. equi is one of the causal agents of equine piroplasmosis, a major threat in horse breeding systems. Vector competence is closely linked to the pathogens' ability to evade tick defense mechanisms. However, knowledge of tick immune response against infections by hemoparasites of the Theileria genus is scarce. In the present study, the expression of genes involved in immune signaling pathways of R. microplus adults' guts when challenged with a high or low parasitic load of T. equi was evaluated. This research demonstrates divergences in the immune gene expression pattern linked to T. equi infection in R. microplus since the Toll, IMD, and JNK signaling pathways were transcriptionally repressed in the guts of adult ticks infected with T. equi. Moreover, the results showed that different infectious doses of T. equi induce differential gene expression of key components of immune signaling cascades in R. microplus gut, suggesting a link between the intensity of infection and the activation of tick immunity response. The present study adds knowledge to elucidate the gut immune signaling response of R. microplus to T. equi infection. In addition, the generated data can serve as a basis for further investigations to develop strategies for controlling and preventing equine piroplasmosis.

Novel Ehrlichia canis genogroup in dogs with canine ehrlichiosis in Cuba.

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.

Murine Mammary Carcinoma Induces Chronic Systemic Inflammation and Immunosuppression in BALB/c Mice.

PURPOSE: The F3II cell line is a highly invasive variant of mammary carcinoma. Although it is frequently used as a model to evaluate the efficacy of immunotherapy, its impact on the immune system remains poorly understood. The main objectives of this study were to evaluate the effects of F3II tumors on the development of chronic inflammation and to characterize tumor-associated immunosuppression. METHODS: Following the experimental implantation of F3II tumors in BALB/c mice, alterations in the liver and spleen anatomy and the numbers of circulating leukocytes, myeloid-derived suppressor cells (MDSCs), and regulatory T cells were measured using hematological techniques, histopathological analysis, and flow cytometry. The capacity of the F3II tumor-bearing mice to reject MB16F10 allogeneic tumor transplantation was also evaluated. In addition, the restoration of immune parameters in tumor-bearing mice was evaluated after standard breast cancer chemotherapy and surgical tumor excision. RESULTS: F3II tumor implantation increased the levels of chronic inflammatory markers, such as the neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios, and caused myeloid alterations, including extramedullary granulopoiesis and megakaryopoiesis, along with the recruitment of MDSCs to the spleen. Chemotherapy or surgical F3II tumor removal completely rescued the tumor-associated extramedullary granulopoiesis and megakaryopoiesis. Notably, the presence of F3II tumors reduced the capacity of BALB/c mice to reject MB16F10 allogeneic tumor transplantation. CONCLUSION: These results support the occurrence of F3II tumor-mediated immune cell dysfunction, which mimics the immune alterations characterized by chronic systemic inflammation and immunosuppression observed in breast cancer in clinical settings. Thus, the F3II tumor model is relevant for evaluating novel breast cancer immunotherapies and combinations in preclinical studies. This model could also be useful for identifying appropriate therapeutic targets and developing proof-of-concept experiments in the future.

Ticks and Tick-Borne Diseases in Central America and the Caribbean: A One Health Perspective.

Ticks have complex life cycles which involve blood-feeding stages found on wild and domestic animals, with humans as accidental hosts. At each blood-feeding stage, ticks can transmit and/or acquire pathogens from their hosts. Therefore, the circulation of tick-borne pathogens (TBPs), especially the zoonotic ones, should be studied in a multi-layered manner, including all components of the chain of infections, following the 'One Health' tenets. The implementation of such an approach requires coordination among major stakeholders (such as veterinarians, physicians, acarologists, and researchers) for the identification of exposure and infection risks and application of effective prevention measures. In this review, we summarize our current knowledge on the epidemiology of tick-borne diseases in Central America and the Caribbean and the challenges associated with the implementation of 'One Health' surveillance and control programs in the region.

Serological evidence of Ehrlichia minasensis infection in Brazilian dogs.

Ehrlichia spp. are important tick-borne pathogens of animals in Brazil, and Ehrlichia canis is the most prevalent species infecting dogs. Moreover, Ehrlichia minasensis has also recently been identified as a novel ehrlichial agent that infects cattle in Brazil. The objective of this study was to determine whether dogs could be infected by E. minasensis. To investigate this possibility, sera (n = 429) collected from dogs in the Pantanal region were retrospectively analyzed for the presence of antibodies against E. canis and E. minasensis. Canine sera were screened by two isolates of E. canis in indirect immunofluorescence assay (IFA) and the majority (n = 298; 69.4%) had antibodies with endpoint titers ranging from 80 to 327,680. In order to further confirm E. canis-specific antibodies, IFA positive sera were analyzed by ELISA using E. canis-specific peptides (i.e. TRP19 and TRP36 US/BR/CR), which detected E. canis antibodies in 80.2% (239/298) of the dog sera. Fifty-nine (13.7%) samples had detectable antibodies to E. canis by IFA but were negative by E. canis peptide ELISA. These sera were then tested by E. minasensis IFA (Cuiaba strain) as antigen and 67.8% (40/59) were positive (titers ranging from 80 to 20,480). Eleven sera had antibody titers against E. minasensis at least two-fold higher than observed for E. canis and suggests that these dogs were previously infected with E. minasensis. The results of the present study suggest that multiple ehrlichial agents infect dogs in Brazil, which highlights the need to consider different Ehrlichia spp. in Brazilian dogs, particularly in areas where dogs are frequently exposed to multiple tick species. This investigation is the first to provide serologic evidence of E. minasensis infection in dogs from Brazil.

First Evidence of Ehrlichia minasensis Infection in Horses from Brazil.

The genus Ehrlichia includes tick-borne bacterial pathogens affecting humans, domestic and wild mammals. Ehrlichia minasensis has been identified in different animal species and geographical locations, suggesting that this is a widely distributed and generalist Ehrlichia. In the present study, we evaluated Ehrlichial infection in 148 Equidae presented to the Medical Clinic Department of a Veterinary Hospital from a midwestern region of Brazil. Blood samples and ticks collected from the animals were tested by Polymerase Chain Reaction (PCR) for the presence of Ehrlichia spp. A multigenic approach including Anaplasmataceae-specific (i.e., 16S rRNA, groEL, gltA) and Ehrlichia-specific (i.e., dsb and trp36) genes was used for accurate bacteria identification. Sera samples were also collected and evaluated for the detection of anti-Ehrlichia antibodies by indirect fluorescent antibody test (IFA). Possible associations between molecular and serological diagnostics and clinical and hematological manifestations were tested using chi-squared or Fisher's exact tests. Sequence analysis of the dsb fragment revealed that three horses (2.03%) were exposed to E. minasensis. Sixty-one (41.2%) Equidae (58 equines and three mules), were seropositive for Ehrlichia spp., with antibody titers ranging between 40 and 2560. Seropositivity to ehrlichial antigens was statistically associated with tick infestation, rural origin, hypoalbuminemia and hyperproteinemia (p ≤ 0.05). The present study reports the first evidence of natural infection by E. minasensis in horses from Brazil.

Ticks and Tick-Borne Diseases in Cuba, Half a Century of Scientific Research.

Ticks and the vast array of pathogens they transmit, including bacteria, viruses, protozoa, and helminths, constitute a growing burden for human and animal health worldwide. In Cuba, the major tropical island in the Caribbean, ticks are an important cause of vector-borne diseases affecting livestock production, pet animal health and, to a lesser extent, human health. The higher number of tick species in the country belong to the Argasidae family and, probably less known, is the presence of an autochthonous tick species in the island, Ixodes capromydis. Herein, we provide a comprehensive review of the ticks and tick-borne pathogens (TBPs) affecting animal and human health in Cuba. The review covers research results including ecophysiology of ticks, the epidemiology of TBPs, and the diagnostic tools used currently in the country for the surveillance of TBPs. We also introduce the programs implemented in the country for tick control and the biotechnology research applied to the development of anti-tick vaccines.

Epidemiology and genetic diversity of Anaplasma marginale in Zamora-Chinchipe, Ecuador.

Bovine anaplasmosis, caused by the tick-borne pathogen Anaplasma marginale, is a hemolytic disease that constitutes a major constraint to cattle production in tropical and subtropical regions including Ecuador. However, the epidemiological situation of this hemoparasitosis in Ecuador is poorly characterized. The present study was aimed to determine the prevalence and genetic diversity of A. marginale in cattle of Ecuador. A cross-sectional study was carried out covering several farms from six out nine cantons of the Zamora-Chinchipe province. A total of 185 cattle were randomly selected and blood samples were collected from the animals. The studied group of animals included six breeds, three age groups, and both sexes. The molecular diagnostic was performed based on a nPCR assay targeting the A. marginale msp5 gene. Anaplasma marginale prevalence was 63.8 % and the bacteria were detected in all the cantons studied. Thirteen representative strains were selected and genetically characterized based on the msp1α gene. Genetic diversity analysis revealed that different strains circulate in the bovine herds studied. The results suggest that cattle movement may contribute to the circulation of common strains in the area. The results demonstrate a high prevalence of A. marginale in the region which should be considered by the sanitary authorities. The epidemiological surveillance for this disease should increase to anticipate acute disease outbreaks with high mortality. Bovine anaplasmosis outbreaks can cause economic losses and the death of several animals; therefore, measures for the prevention and control of this disease are required.

Development and application of a multiplex TaqMan® real-time qPCR assay for the simultaneous detection of Anaplasma marginale and Theileria annulata and molecular characterization of Anaplasma marginale from cattle in Western Cuba.

Anaplasmosis and theileriosis are considered the most important tick-borne diseases for livestock production worldwide, causing significant economic losses in tropical and subtropical regions. The present study was aimed to develop a multiplex TaqMan® qPCR assay to simultaneously detect Anaplasma marginale and Theileria annulata and to applied it to investigate naturally infected cattle in Cuba. The assay was highly specific, sensible, and efficient; it was more sensitive than a well-established nested PCR and detected 1 DNA copy of each target. Consistent repeatability and reproducibility within and between multiplex qPCR runs was shown. A total of 223 blood samples collected in western Cuba were analyzed for haemoparasites infection in cattle. The multiplex qPCR assay detected A. marginale in 213 samples (95.5%; CI: 95%; 91.9%-97.5%), but all samples were negative for T. annulata. Additionally, the genetic diversity of A. marginale was assessed using 16S rRNA, MSP1a and MSP4 nucleotide and protein sequences. The MSP1a tandem repeats ranged from three to five, and twelve different MSP1a tandem repeats of A. marginale were found, which presented genotypes C, E, and G in the 5'UTR microsatellite region. Phylogenetic analysis using the msp4 gene showed that Cuban strains were closely related to others previously reported in Mexico, Brazil and Asian countries. The multiplex qPCR described here proved to be a rapid, specific and cost-effective mean for the simultaneous detection of A. marginale and T. annulata. Further epidemiological studies using this assay will improve the surveillance of the associated diseases in regions where they are endemic.

Isolation and Characterization of a Novel Pathogenic Strain of Ehrlichia minasensis.

The genus Ehrlichia is composed of tick-borne obligate intracellular gram-negative alphaproteobacteria of the family Anaplasmataceae. Ehrlichia includes important pathogens affecting canids (E. canis, E. chaffeensis, and E. ewingii), rodents (E. muris), and ruminants (E. ruminantium). Ehrlichia minasensis, an Ehrlichia closely related to E. canis, was initially reported in Canada and Brazil. This bacterium has now been reported in Pakistan, Malaysia, China, Ethiopia, South Africa, and the Mediterranean island of Corsica, suggesting that E. minasensis has a wide geographical distribution. Previously, E. minasensis was found to cause clinical ehrlichiosis in an experimentally infected calf. The type strain E. minasensis UFMG-EV was successfully isolated from Rhipicephalus microplus ticks and propagated in the tick embryonic cell line of Ixodes scapularis (IDE8). However, the isolation and propagation of E. minasensis strains from cattle has remained elusive. In this study, the E. minasensis strain Cuiabá was isolated from an eight-month-old male calf of Holstein breed that was naturally infected with the bacterium. The calf presented clinical signs and hematological parameters of bovine ehrlichiosis. The in vitro culture of the agent was established in the canine cell line DH82. Ehrlichial morulae were observed using light and electron microscopy within DH82 cells. Total DNA was extracted, and the full genome of the E. minasensis strain Cuiabá was sequenced. A core-genome-based phylogenetic tree of Ehrlichia spp. and Anaplasma spp. confirmed that E. minasensis is a sister taxa of E. canis. A comparison of functional categories among Ehrlichia showed that E. minasensis has significantly less genes in the 'clustering-based subsystems' category, which includes functionally coupled genes for which the functional attributes are not well understood. Results strongly suggest that E. minasensis is a novel pathogen infecting cattle. The epidemiology of this Ehrlichia deserves further attention because these bacteria could be an overlooked cause of tick-borne bovine ehrlichiosis, with a wide distribution.

Sex-Specific Linkages Between Taxonomic and Functional Profiles of Tick Gut Microbiomes.

Ticks transmit the most diverse array of disease agents and harbor one of the most diverse microbial communities. Major progress has been made in the characterization of the taxonomic profiles of tick microbiota. However, the functional profiles of tick microbiome have been comparatively less studied. In this proof of concept we used state-of-the-art functional metagenomics analytical tools to explore previously reported datasets of bacteria found in male and female Ixodes ovatus, Ixodes persulcatus, and Amblyomma variegatum. Results showed that both taxonomic and functional profiles have differences between sexes of the same species. KEGG pathway analysis revealed that male and female of the same species had major differences in the abundance of genes involved in different metabolic pathways including vitamin B, amino acids, carbohydrates, nucleotides, and antibiotics among others. Partial reconstruction of metabolic pathways using KEGG enzymes suggests that tick microbiome form a complex metabolic network that may increase microbial community resilience and adaptability. Linkage analysis between taxonomic and functional profiles showed that among the KEGG enzymes with differential abundance in male and female ticks only 12% were present in single bacterial genera. The rest of these enzymes were found in more than two bacterial genera, and 27% of them were found in five up to ten bacterial genera. Comparison of bacterial genera contributing to the differences in the taxonomic and functional profiles of males and females revealed that while a small group of bacteria has a dual-role, most of the bacteria contribute only to functional or taxonomic differentiation between sexes. Results suggest that the different life styles of male and female ticks exert sex-specific evolutionary pressures that act independently on the phenomes (set of phenotypes) and genomes of bacteria in tick gut microbiota. We conclude that functional redundancy is a fundamental property of male and female tick microbiota and propose that functional metagenomics should be combined with taxonomic profiling of microbiota because both analyses are complementary.