Thermostable Keystone bacteria maintain the functional diversity of the Ixodes scapularis microbiome under heat stress

Variations in the composition and diversity of tick microbiome due to high temperatures may influence the hierarchy of community members as a response to environmental change. Modifications in the community structure are hypothesized to drive alterations in the presence and/or abundance of functional pathways in the bacterial metagenome. In this study, this hypothesis was tested by using published 16S rRNA datasets of Ixodes scapularis males incubated at different temperatures (i.e., 4, 20, 30, and 37 degrees C) in a laboratory setting. Changes in community structure and functional profiles in response to temperature shifts were measured using co-occurrence networks and metagenome inference. Results from laboratory-reared ticks were then compared with those of field-collected ticks. The results from laboratory-reared ticks showed that high temperature altered the structure of the microbial community and decreased the number of keystone taxa. Notably, four taxa were identified as keystone in all the temperatures, and the functional diversity of the tick microbiome was contained in the four thermostable keystone their associated bacterial taxa. Three of the thermostable keystone taxa were also found in free-living ticks collected in Massachusetts. Moreover, the comparison of functional profiles of laboratory-reared and field-collected ticks revealed the existence of an important set of metabolic pathways that were common among the different datasets. Similar to the laboratory-reared ticks, the keystone taxa identified in field-collected ticks alongside their consortia (co-occurring taxa) were sufficient to retain the majority of the metabolic pathways in the functional profile. These results suggest that keystone taxa are essential in the stability and the functional resiliency of the tick microbiome under heat stress

Rapid evolution of a novel protective symbiont into keystone taxon in Caenorhabditis elegans microbiota

Protective microbes have a major role in shaping host–pathogen interactions, but their relative importance in the structure of the host microbiota remains unclear. Here, we used a network approach to characterize the impact of a novel, experimentally evolved ‘protective microbial symbiont’ (Enterococcus faecalis) on the structure and predicted function of the natural microbiota of the model organism Caenorhabditis elegans. We used microbial network analysis to identify keystone taxa and describe the hierarchical placement of protective and non-protective symbionts in the microbiota. We found that early colonization with symbionts produce statistically significant changes in the structure of the community. Notably, only the protective E. faecalis became a keystone taxon in the nematode microbiota. Non-protective lineages of the same bacterial species remained comparatively unimportant to the community. Prediction of functional profiles in bacterial communities using PICRUSt2 showed that the presence of highly protective E. faecalis decreased the abundance of ergothioneine (EGT) biosynthesis pathway involved in the synthesis of the antioxidant molecule EGT, a potential public good. These data show that in addition to direct antagonism with virulent pathogens, keystone protective symbionts are linked to modified bacterial community structure and possible reductions in public goods, potentially driving decreased antioxidant defense. We suggest that this response could suppress infection via wholesale microbial community changes to further benefit the host. These findings extend the concept of protective symbionts beyond bodyguards to ecosystem engineers

Bovine ticks harbour a diverse array of microorganisms in Pakistan

International audienceBackground: Ticks and tick-borne pathogens (TTBP) are a major constraint to livestock production in Pakistan; despite a high prevalence of TTBPs, knowledge on the capacity of Pakistani ticks to carry pathogens and endosymbionts is limited. Furthermore, mixed infections with multiple microorganisms further complicate and limit the detection potential of traditional diagnostic methods. The present study investigated the tick-borne microorganisms in bovine ticks in Pakistan, employing a high-throughput microfluidic real-time PCR based technique. Methods: Ticks were collected from clinically healthy cattle (n = 116) and water buffaloes (n = 88) from 30 villages across six districts located in five agro-ecological zones (AEZs) of Pakistan from September to November 2017. The microfluidic real-time PCR was used to test the genomic DNA of individual ticks for the presence of 27 bacterial and eight parasitic microorganisms. Phylogenetic methods were used to assess the genetic relationship of DNA sequences determined herein. Results: PCR detected DNA of at least one microorganism in each of 221 ticks tested (94.4%, 221/234). DNA-based detection inferred that single pathogens/endosymbionts were the most common (43.4%, 96/221) followed by double (38.9%, 86/221), triple (14.5%, 32/221), quadruple (2.3%, 5/221) and quintuple (0.9%, 2/221) mixed infections. Piroplasms (Babesia/Theileria spp.) were the most prevalent (31.6%, 74/234), followed by Ehrlichia spp. (20%, 47/234) and Anaplasma marginale (7.7%, 18/234). Anaplasma phagocytophilum, A. ovis, A. centrale, Babesia ovis, Borrelia spp., Rickettsia spp., R. massiliae, Bartonella spp. and Hepatozoon spp. were also detected. Endosymbionts such as Francisella-like (91.5%, 214/234) and Coxiella-like (1.3%, 3/234) organisms were also detected in ticks. The highest diversity of microorganisms was detected in Hyalomma anatolicum ticks (test-positive for 14/14 microorganisms), followed by Rhipicephalus microplus (4/14), Hy. hussaini (3/14) and Rh. annulatus (2/14). Ticks collected from cattle carried significantly more frequently piroplasms (41.2%, 54/131; P < 0.05) than those from buffaloes (19.4%, 20/103). However, the overall prevalence of microorganisms did not vary significantly among ticks from the two host species as well as across different AEZs. Conclusions: To our knowledge, this is the first study to investigate a wide range of tick-borne microorganisms in bovine ticks using a high-throughput diagnostic method from different AEZs in Pakistan. These findings will aid in establishing the distribution patterns and the control of tick-borne pathogens of bovines in Pakistan

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

International audienceBovine 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

Exploring the ecological implications of microbiota diversity in birds: Natural barriers against avian malaria

Natural antibodies (Abs), produced in response to bacterial gut microbiota, drive resistance to infection in vertebrates. In natural systems, gut microbiota diversity is expected to shape the spectrum of natural Abs and resistance to parasites. This hypothesis has not been empirically tested. In this ‘Hypothesis and Theory’ paper, we propose that enteric microbiota diversity shapes the immune response to the carbohydrate α-Gal and resistance to avian malaria. We further propose that anti-α-Gal Abs are transmitted from mother to eggs for early malaria protection in chicks. Microbiota modulation by anti-α-Gal Abs is also proposed as a mechanism favoring the early colonization of bacterial taxa with α1,3-galactosyltransferase (α1,3GT) activity in the bird gut. Our preliminary data shows that bacterial α1,3GT genes are widely distributed in the gut microbiome of wild and domestic birds. We also showed that experimental infection with the avian malaria parasite P. relictum induces anti-α-Gal Abs in bird sera. The bird-malaria-microbiota system allows combining field studies with infection and transmission experiments in laboratory animals to test the association between microbiota composition, anti-α-Gal Abs, and malaria infection in natural populations of wild birds. Understanding how the gut microbiome influences resistance to malaria can bring insights on how these mechanisms influence the prevalence of malaria parasites in juvenile birds and shape the host population dynamics

Anti-microbiota vaccine reduces avian malaria infection within mosquito vectors

Animal and human pathogens that are transmitted by arthropods are a global concern, particularly those vectored by mosquitoes (e.g., Plasmodium spp. and dengue virus). Vector microbiota may hold the key to vector-borne pathogen control, as mounting evidence suggests that the contributions of the vector microbiota to vector physiology and pathogen life cycle are so relevant that vectorial capacity cannot be understood without considering microbial communities within the vectors. Anti-tick microbiota vaccines targeting commensal bacteria of the vector microbiota alter vector feeding and modulate the taxonomic and functional profiles of vector microbiome, but their impact on vector-borne pathogen development within the vector has not been tested. In this study, we tested whether anti-microbiota vaccination in birds targeting Enterobacteriaceae within mosquito midguts modulates the mosquito microbiota and disrupt Plasmodium relictum development in its natural vector Culex quinquefasciatus. Domestic canaries (Serinus canaria domestica) were experimentally infected with P. relictum and/or immunized with live vaccines containing different strains of Escherichia coli. Immunization of birds induced E. coli-specific antibodies. The midgut microbial communities of mosquitoes fed on Plasmodium-infected and/or E. coli-immunized birds were different from those of mosquitoes fed on control birds. Notably, mosquito midgut microbiota modulation was associated with a significant decrease in the occurrence of P. relictum oocysts and sporozoites in the midguts and salivary glands of C. quinquefasciatus, respectively. A significant reduction in the number of oocysts was also observed. These findings suggest that anti-microbiota vaccines can be used as a novel tool to control malaria transmission and potentially other vector-borne pathogens

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

International audienceAnaplasmosis 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 5ʹUTR 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

First molecular evidence of bovine hemoplasma species (Mycoplasma spp.) in water buffalo and dairy cattle herds in Cuba

Background: Hemotropic mycoplasmas (aka hemoplasmas) are small bacteria which cause infectious anemia in several mammalian species including humans. Information on hemoplasma infections in Cuban bovines remains scarce and no studies applying molecular methods have been performed so far. The aim of the present study was to utilize real-time PCR and sequence analysis to investigate dairy cattle and buffalo from Cuba for the presence of bovine hemoplasma species. Results: A total of 80 blood samples from 39 buffalo and 41 dairy cattle were investigated for the presence of Mycoplasma wenyonii and Candidatus Mycoplasma haemobos using two species-specific real-time TaqMan PCR assays. PCR results revealed overall 53 (66.2%; 95% CI: 55.3-75.7%) positive animals for M. wenyonii and 33 (41.2%; 95% CI: 31.1-52.2%) for Ca. M. haemobos; the latter were all co-infections with M. wenyonii. The sample prevalences were similar in cattle and buffalo. Based on the sequence analysis of the nearly full-length 16S rRNA gene from two cattle and two buffalo, the presence of M. wenyonii and Ca. M. haemobos was confirmed. Statistical analysis revealed that buffalo and cattle one year of age or older were more frequently infected with M. wenyonii or Ca. M. haemobos than younger animals. PCR-positivity was not associated with anemia; however, the infection stage was unknown (acute infection versus chronic carriers). Conclusions: The high occurrence of bovine hemoplasma infections in buffalo and dairy cattle may have a significant impact on Cuban livestock production. To the best of our knowledge, this is the first molecular evidence of bovine hemoplasma species infection in dairy cattle and buffalo from Cuba and the Caribbean

High co-infection rates of babesia bovis, <em>babesia bigemina</em>, and anaplasma marginale in water buffalo in western cuba

International audienceWater buffalo is important livestock in several countries in the Latin American and Caribbean regions. This buffalo species can be infected by tick-borne hemoparasites and remains a carrier of these pathogens which represent a risk of infection for more susceptible species like cattle. Therefore, studies on the epidemiology of tick-borne hemoparasites in buffaloes are required. In this study, the prevalence of Babesia bovis, Babesia bigemina, and Anaplasma marginale were determined in water buffalo herds of western Cuba. To this aim, a cross-sectional study covering farms with large buffalo populations in the region was performed. Eight buffalo herds were randomly selected, and blood samples were collected from 328 animals, including 63 calves (3-14months), 75 young animals (3-5years), and 190 adult animals (>5years). Species-specific nested PCR and indirect ELISA assays were used to determine the molecular and serological prevalences of each hemoparasite, respectively. The molecular and serological prevalence was greater than 50% for the three hemoparasites. Differences were found in infection prevalence among buffalo herds, suggesting that local epidemiological factors may influence infection risk. Animals of all age groups were infected, with a higher molecular prevalence of B. bigemina and A. marginale in young buffalo and calves, respectively, while a stepwise increase in seroprevalence of B. bovis and B. bigemina from calves to adult buffaloes was found. The co-infection by the three pathogens was found in 12% of animals, and when analyzed by pair, the co-infections of B. bovis and B. bigemina, B. bigemina and A. marginale, and B. bovis and A. marginale were found in 20%, 24%, and 26%, respectively, underlying the positive interaction between these pathogens infecting buffaloes. These results provide evidence that tick-borne pathogen infections can be widespread among water buffalo populations in tropical livestock ecosystems. Further studies should evaluate whether these pathogens affect the health status and productive performance of water buffalo and infection risk of these pathogens in cattle cohabiting with buffalo