Background: 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.

Global population growth poses a threat to food security in an era of increased ecosystem degradation, climate change, soil erosion, and biodiversity loss. In this context, harnessing naturally-occurring processes such as those provided by soil and plant-associated microorganisms presents a promising strategy to reduce dependency on agrochemicals. Biofertilizers are living microbes that enhance plant nutrition by either by mobilizing or increasing nutrient availability in soils. Various microbial taxa including beneficial bacteria and fungi are currently used as biofertilizers, as they successfully colonize the rhizosphere, rhizoplane or root interior. Despite their great potential to improve soil fertility, biofertilizers have yet to replace conventional chemical fertilizers in commercial agriculture. In the last 10 years, multi-omics studies have made a significant step forward in understanding the drivers, roles, processes, and mechanisms in the plant microbiome. However, translating this knowledge on microbiome functions in order to capitalize on plant nutrition in agroecosystems still remains a challenge. Here, we address the key factors limiting successful field applications of biofertilizers and suggest potential solutions based on emerging strategies for product development. Finally, we discuss the importance of biosafety guidelines and propose new avenues of research for biofertilizer development.

The main importance of ticks resides in their ability to harbor pathogens that can be transmitted to terrestrial vertebrates including humans. Recently, studies have focused on the taxonomic and functional composition of the tick microbiome, its microbial diversity and variation under different factors including tick species, sex, and environment among others. Of special interest are the interactions between the tick, the microbiome and pathogens since tick microbiome can influence pathogen colonization within the tick vector, and potentially, transmission to the vertebrate host. In this review, we tackled a synthesis on the growing field of tick microbiomes. We focus on the current state of tick microbiome research, addressing controversial and hotly debated topics and advances in the precise manipulation of tick microbiome. Furthermore, we discuss the innovative anti-tick microbiota vaccines as a possible tool for microbiome modulation and thus, control of tick-borne diseases. Deciphering tick-microbiome pathogen interactions can spur new strategies to control tick-borne diseases via modulation of tick microbiome.

The tick microbiota is a highly complex ensemble of interacting microorganisms. Keystone taxa, with a central role in the microbial networks, support the stability and fitness of the microbial communities. The keystoneness of taxa in the tick microbiota can be inferred from microbial co-occurrence networks. Microbes with high centrality indexes are highly connected with other taxa of the microbiota and are expected to provide important resources to the microbial community and/or the tick. We reasoned that disturbance of vector microbiota by removal of ubiquitous and abundant keystone bacteria may disrupt the tick-microbiota homeostasis causing harm to the tick host. These observations and reasoning prompted us to test the hypothesis that antibodies targeting keystone bacteria may harm the ticks during feeding on immunized hosts. To this aim, in silico analyses were conducted to identify keystone bacteria in the microbiota of Ixodes nymphs. The family Enterobacteriaceae was among the top keystone taxa identified in Ixodes microbiota. Immunization of α-1,3-galactosyltransferase-deficient-C57BL/6 (α1,3GT KO) mice with a live vaccine containing the Enterobacteriaceae bacterium Escherichia coli strain BL21 revealed that the production of anti-E. coli and anti-α-Gal IgM and IgG was associated with high mortality of I. ricinus nymphs during feeding. However, this effect was absent in two different strains of wild type mice, BALB/c and C57BL/6. This result concurred with a wide distribution of α-1,3-galactosyltransferase genes, and possibly α-Gal, in Enterobacteriaceae and other bacteria of tick microbiota. Interestingly, the weight of I. ricinus nymphs that fed on E. coli-immunized C57BL/6 was significantly higher than the weight of ticks that fed on C57BL/6 immunized with a mock vaccine. Our results suggest that anti-tick microbiota vaccines are a promising tool for the experimental manipulation of vector microbiota, and potentially the control of ticks and tick-borne pathogens.

The lack of tools for the precise manipulation of the tick microbiome is currently a major limitation to achieve mechanistic insights into the tick microbiome. Anti-tick microbiota vaccines targeting keystone bacteria of the tick microbiota alter tick feeding, but their impact on the taxonomic and functional profiles of the tick microbiome has not been tested. In this study, we immunized a vertebrate host model (Mus musculus) with live bacteria vaccines targeting keystone (i.e., Escherichia-Shigella) or non-keystone (i.e., Leuconostoc) taxa of tick microbiota and tested the impact of bacterial-specific antibodies (Abs) on the structure and function of tick microbiota. We also investigated the effect of these anti-microbiota vaccines on mice gut microbiota composition. Our results showed that the tick microbiota of ticks fed on Escherichia coli-immunized mice had reduced Escherichia-Shigella abundance and lower species diversity compared to ticks fed on control mice immunized with a mock vaccine. Immunization against keystone bacteria restructured the hierarchy of nodes in co-occurrence networks and reduced the resistance of the bacterial network to taxa removal. High levels of E. coli-specific IgM and IgG were negatively correlated with the abundance of Escherichia-Shigella in tick microbiota. These effects were not observed when Leuconostoc was targeted with vaccination against Leuconostoc mesenteroides. Prediction of functional pathways in the tick microbiome using PICRUSt2 revealed that E. coli vaccination reduced the abundance of lysine degradation pathway in tick microbiome, a result validated by qPCR. In contrast, the gut microbiome of immunized mice showed no significant alterations in the diversity, composition and abundance of bacterial taxa. Our results demonstrated that anti-tick microbiota vaccines are a safe, specific and an easy-to-use tool for manipulation of vector microbiome. These results guide interventions for the control of tick infestations and pathogen infection/transmission.

The taxonomic composition and diversity of tick midgut microbiota have been extensively studied in different species of the genera Rhipicephalus, Ixodes, Amblyomma, Haemaphysalis, Hyalomma, Dermacentor, Argas and Ornithodoros, while the functional significance of bacterial diversity has been proportionally less explored. In this study, we used previously published 16S amplicon sequence data sets from three Ixodes scapularis cohorts, two of uninfected nymphs, and one of larvae experimentally infected with Borrelia burgdorferi, to test the functional redundancy of the tick microbiome. We predicted the metabolic profiling of each sample using the state-of-the-art metagenomics tool PICRUSt2. The results showed that the microbiomes of all I. scapularis samples share only 80 taxa (24.6%, total 324), while out of the 342 metabolic pathways predicted, 82.7%, were shared by all the ticks. Borrelia-infected larvae lack 15.4% of pathways found in the microbiome of uninfected nymphs. Taxa contribution analysis showed that the functional microbiome of uninfected ticks was highly redundant, with, in some cases, up to 198 bacterial taxa contributing to a single pathway. However, Borrelia-infected larvae had a smaller redundancy with 6.7% of pathways provided by more than 100 genera, while 15.7–19.2% of pathways were provided by more than 100 genera in the two cohorts of uninfected ticks. In addition, we compared the functional profiles of three microbial communities from each data set, identified through a network-based approach, and we observed functional similarity between them. Based on the functional redundancy and functional similarity of the microbiome of ticks in different developmental stages and infection status, we concluded that the tick gut microbiota is a self-regulating community of very diverse bacteria contributing to a defined set of metabolic pathways and functions with yet unexplored relevance for tick fitness and/or bacterial community stability. We propose a change of focus in which the tick microbiome must be analyzed in all dimensions, highlighting their functional traits, instead of the conventional taxonomic profiling.