Differential nested patterns of Anaplasma marginale and Coxiella-like endosymbiont across Rhipicephalus microplus ontogeny.

Understanding the intricate ecological interactions within the microbiome of arthropod vectors is crucial for elucidating disease transmission dynamics and developing effective control strategies. In this study, we investigated the ecological roles of Coxiella-like endosymbiont (CLE) and Anaplasma marginale across larval, nymphal, and adult stages of Rhipicephalus microplus. We hypothesized that CLE would show a stable, nested pattern reflecting co-evolution with the tick host, while A. marginale would exhibit a more dynamic, non-nested pattern influenced by environmental factors and host immune responses. Our findings revealed a stable, nested pattern characteristic of co-evolutionary mutualism for CLE, occurring in all developmental stages of the tick. Conversely, A. marginale exhibited variable occurrence but exerted significant influence on microbial community structure, challenging our initial hypotheses of its non-nested dynamics. Furthermore, in silico removal of both microbes from the co-occurrence networks altered network topology, underscoring their central roles in the R. microplus microbiome. Notably, competitive interactions between CLE and A. marginale were observed in nymphal network, potentially reflecting the impact of CLE on the pathogen transstadial-transmission. These findings shed light on the complex ecological dynamics within tick microbiomes and have implications for disease management strategies.

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.

Efficacy of the Vaccine Candidate Based on the P0 Peptide against Dermacentor nitens and Ixodes ricinus Ticks.

The control of ticks through vaccination offers a sustainable alternative to the use of chemicals that cause contamination and the selection of resistant tick strains. However, only a limited number of anti-tick vaccines have reached commercial realization. In this sense, an antigen effective against different tick species is a desirable target for developing such vaccines. A peptide derived from the tick P0 protein (pP0) conjugated to a carrier protein has been demonstrated to be effective against the Rhipicephalus microplus, Rhipicephalus sanguineus, and Amblyomma mixtum tick species. The aim of this work was to assess the efficacy of this peptide when conjugated to the Bm86 protein against Dermacentor nitens and Ixodes ricinus ticks. An RNAi experiment using P0 dsRNA from I. ricinus showed a dramatic reduction in the feeding of injected female ticks on guinea pigs. In the follow-up vaccination experiments, rabbits were immunized with the pP0-Bm86 conjugate and challenged simultaneously with larvae, nymphs, and the adults of I. ricinus ticks. In the same way, horses were immunized with the pP0-Bm86 conjugate and challenged with D. nitens larva. The pP0-Bm86 conjugate showed efficacies of 63% and 55% against I. ricinus and D. nitens ticks, respectively. These results, combined with previous reports of efficacy for this conjugate, show the promising potential for its development as a broad-spectrum anti-tick vaccine.

The Bm86 Discovery: A Revolution in the Development of Anti-Tick Vaccines.

The presence in nature of species with genetic resistance to ticks, or with acquired resistance after repeated tick infestations, has encouraged the scientific community to consider vaccination as an alternative to the unsustainable chemical control of ticks. After numerous attempts to artificially immunize hosts with tick extracts, the purification and characterization of the Bm86 antigen by Willadsen et al. in 1989 constituted a revolutionary step forward in the development of vaccines against ticks. Previously, innovative studies that had used tick gut extracts for the immunization of cattle against Rhipicepahalus microplus (previously named Boophilus microplus) ticks, with amazingly successful results, demonstrated the feasibility of using antigens other than salivary-gland-derived molecules to induce a strong anti-tick immunity. However, the practical application of an anti-tick vaccine required the isolation, identification, and purification of the responsible antigen, which was finally defined as the Bm86 protein. More than thirty years later, the only commercially available anti-tick vaccines are still based on this antigen, and all our current knowledge about the field application of immunological control based on vaccination against ticks has been obtained through the use of these vaccines.

Mammalian Cell Culture as a Platform for Veterinary Vaccines.

For more than three decades, mammalian cells have been the host par excellence for the recombinant protein production for therapeutic purposes in humans. Due to the high cost of media and other supplies used for cell growth, initially this expression platform was only used for the production of proteins of pharmaceutical importance including antibodies. However, large biotechnological companies that used this platform continued research to improve its technical and economic feasibility. The main qualitative improvement was obtained when individual cells could be cultured in a liquid medium similar to bacteria and yeast cultures. Another important innovation for growing cells in suspension was the improvement in chemically defined media that does not contain macromolecules; they were cheaper to culture as any other microbial media. These scientific milestones have reduced the cost of mammalian cell culture and their use in obtaining proteins for veterinary use. The ease of working with mammalian cell culture has permitted the use of this expression platform to produce active pharmaceutic ingredients for veterinary vaccines. In this chapter, the protocol to obtain recombinant mammalian cell lines will be described.

Challenges in Veterinary Vaccine Development.

Animals provide food and clothing in addition to other value-added products. Changes in diet and lifestyle have increased the consumption and the use of animal products. Infectious diseases in animals are a major threat to global animal health and its welfare; their effective control is crucial for agronomic health, for safeguarding food security and also alleviating rural poverty. Development of vaccines has led to increased production of healthy poultry, livestock, and fish. Animal production increases have alleviated food insecurity. In addition, development of effective vaccines has led to healthier companion animals. However, challenges remain including climate change that has led to enhancement in vectors and pathogens that may lead to emergent diseases in animals. Preventing transmission of emerging infectious diseases at the animal-human interface is critically important for protecting the world population from epizootics and pandemics. Hence, there is a need to develop new vaccines to prevent diseases in animals. This review describes the broad challenges to be considered in the development of vaccines for animals.

Characterization of two Cuban colonies of Rhipicephalus microplus ticks.

Rhipicephalus microplus (Canestrini, 1888) is one of the species with medical and economic relevance that has been reported in the list of Cuban tick species. Some morphological characterizations about the R. microplus species in Cuba have been published; however, molecular studies are lacking. Molecular phylogenetic analyses have grouped R. annulatus, R. australis and three clades of R. microplus in a complex named R. microplus. The present study aimed to characterize two R. microplus tick isolates, established as colonies at the Cuban National Laboratory of Parasitology. Morphological characterization of adult specimens was carried out by using Scanning Electron Microscopy. The sequences of mitochondrial genes: 12S rRNA, 16S rRNA and the subunit I of cytochrome c oxidase (coxI) and one nuclear sequence: internal transcribed spacer 2 (its2) were used for phylogenetic analyses. The life cycle under laboratory conditions for both isolates was also characterized. Tick specimens of both colonies showed morphological characteristics comparable with those distinctive for the R. microplus species. Phylogenies based on mitochondrial gene sequences identified congruently the Cuban tick colonies within the clade A of R. microplus. The life cycle of both isolates under laboratory conditions lasted 65 ± 5 days and the reproductive performance of female ticks of each colony also were similar with approximately 2500 larvae obtained from fully engorged female ticks. This study constitutes the first molecular characterization of ticks from the R. microplus species in Cuba.

Synthesis, LC-MS/MS analysis, and biological evaluation of two vaccine candidates against ticks based on the antigenic P0 peptide from R. sanguineus linked to the p64K carrier protein from Neisseria meningitidis.

A peptide from the P0 acidic ribosomal protein (pP0) of ticks conjugated to keyhole limpet hemocyanin from Megathura crenulata has shown to be effective against different tick species when used in host vaccination. Turning this peptide into a commercial anti-tick vaccine will depend on finding the appropriate, technically and economically feasible way to present it to the host immune system. Two conjugates (p64K-Cys1pP0 and p64K-ßAla1pP0) were synthesized using the p64K carrier protein from Neisseria meningitidis produced in Escherichia coli, the same cross-linking reagent, and two analogues of pP0. The SDS-PAGE analysis of p64K-Cys1pP0 showed a heterogeneous conjugate compared to p64K-ßAla1pP0 that was detected as a protein band at 91kDa. The pP0/p64K ratio determined by MALDI-MS for p64K-Cys1pP0 ranged from 1 to 8, being 3-5 the predominant ratio, while in the case of p64K-ßAla1pP0 this ratio was 5-7. Cys1pP0 was partially linked to 35 out of 39 Lys residues and the N-terminal end, while ßAla1pP0 was mostly linked to the six free cysteine residues, to the N-terminal end, and, in a lesser extent, to Lys residues. The assignment of the conjugation sites and side reactions were based on the identification of type 2 peptides. Rabbit immunizations showed the best anti-pP0 titers and the highest efficacy against Rhipicephalus sanguineus ticks when the p64K-Cys1pP0 was used as vaccine antigen. The presence of high molecular mass aggregates observed in the SDS-PAGE analysis of p64K-Cys1pP0 could be responsible for a better immune response against pP0 and consequently for its better efficacy as an anti-tick vaccine. Graphical abstract.

Morphological and molecular characterization supporting Amblyomma mixtum presence in Cuba.

Amblyomma cajennense Fabricius, 1787 (Acari: Ixodidae) is a widely distributed tick taxon. Recent studies have reassessed this taxon as a complex of six species. Amblyomma mixtum Koch, 1844 has been suggested by some authors as the only species of this complex that is present in Cuba. Other authors have pointed a niche overlapping for A. mixtum and A. cajennense s.s. in the country. Detailed taxonomic studies on the Cuban species belonging to this complex are needed in order to evaluate their current distribution according to the recent classification. This study aimed to characterize Cuban populations from the A. cajennense complex by using tick samples obtained from 3 occidental provinces and 1 central province of the country. Morphological identification and measurements of the main relevant taxonomic structures were conducted by using Scanning Electron Microscopy. Phylogenetic analyzes were carried out with 16S ribosomal RNA, internal transcribed spacer 2 and the subunit I of mitochondrial cytochrome c oxidase gene sequences. The results of these studies demonstrated that all samples belonged to the species A. mixtum (Koch, 1844). This study constitutes the first molecular characterization of this Amblyomma species in Cuba. Further studies will be necessary in order to corroborate if A. cajennense s.s. is also present in the island.

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.

Functional and Mass Spectrometric Evaluation of an Anti-Tick Antigen Based on the P0 Peptide Conjugated to Bm86 Protein.

A synthetic 20 amino acid peptide of the ribosomal protein P0 from ticks, when conjugated to keyhole limpet hemocyanin from Megathura crenulata and used as an immunogen against Rhipicephalus microplus and Rhipicephalus sanguineus s.l. species, has shown efficacies of around 90%. There is also experimental evidence of a high efficacy of this conjugate against Amblyomma mixtum and Ixodes ricinus species, which suggest that this antigen could be a good broad-spectrum anti-tick vaccine candidate. In this study, the P0 peptide (pP0) was chemically conjugated to Bm86 as a carrier protein. SDS-PAGE analysis of this conjugate demonstrated that it is highly heterogeneous in size, carrying from 1 to 18 molecules of pP0 per molecule of Bm86. Forty-nine out of the 54 lysine residues and the N-terminal end of Bm86 were found partially linked to pP0 by using LC-MS/MS analysis and the combination of four different softwares. Several post-translational modifications of Bm86 protein were also identified by mass spectrometry. High immunogenicity and efficacy were achieved when dogs and cattle were vaccinated with the pP0-Bm86 conjugate and challenged with R. sanguineus s.l. and R. microplus, respectively. These results encourage the development of this antigen with promising possibilities as an anti-tick vaccine.

A chemical conjugate of the tick P0 peptide is efficacious against Amblyomma mixtum.

After Rhipicephalus microplus, the most important tick species affecting livestock industry in Cuba belong to the Amblyomma genus. There are few reports of effective vaccine antigens for these species. Recently, vaccination and challenge trials using a peptide from the P0 acidic ribosomal protein of R. microplus ticks (pP0) as antigen have shown an efficacy around 90% against tick species from the Rhipicephalus genus. Given the high degree of sequence conservation among tick species, pP0 could be an antigen of versatile use in anti-tick vaccine formulations. In this paper, seven rabbits were immunized with a chemical conjugate of pP0 to keyhole limpet haemocyanin. Rabbits were challenged with an average of 1,900 Amblyomma mixtum larvae from a Cuban tick strain. The average number of recovered fed larvae and the viability of larvae in the moulting process were significantly lower in vaccinated animals compared with the control group. The overall vaccine efficacy of the P0 peptide antigen is 54% according to the calculated parameters.

Plasticity of the HEK-293 cells, related to the culture media, as platform to produce a subunit vaccine against classical swine fever virus.

Classical swine fever (CSF) is a contagious disease that causes a high mortality to domestic and wild pigs. Its causative agent is an enveloped Pestivirus named Classical Swine Fever Virus (CSFV). Due to the huge economic affectations produced by this disease to porcine industry, several vaccines have been developed using principally the CSFV E2 glycoprotein. Recently, a subunit vaccine based on this structural protein of the CSFV fused to the porcine CD154 molecule as immunomodulator named E2-CD154 was assayed by us. This chimeric protein was produced in the Human Embryonic Kidney (HEK-293) cell line. In this work, the growth and the expression profiles of HEK-293 E2-CD154 cells in four commercially available culture media were studied. The oligosaccharide structures in the N-glycosylation patterns of the E2-CD154 protein produced by this cell line in 10 L fermenters with two different culture media were also analyzed. In addition, the neutralizing antibody response generated in mice vaccinated with these antigens was assayed. Our results suggest that the culture media CDM4HEK293 and SFM4HEK293 which are recommended for HEK-293 growth are the best choice to growth the cell clone expressing the E2-CD154 protein. The glycosylation pattern and the neutralizing antibody response generated by the E2-CD154 protein were independent of the culture medium used which demonstrates the high reproducibility and consistency among protein batches produced by HEK-293 cells even in different culture conditions.

CYTED Network to develop an immunogen compatible with integrated management strategies for tick control in cattle.

INCOGARR is a thematic network recently approved to be financially supported by the Ibero-American Program of Science and Technology for Development (CYTED). The objectives of this Network are the design and evaluation of an efficient and feasible anti-tick vaccine candidate from the technical and economical points of view and also sharing experiences in the immunological control of ticks as part of an Integrated Control Program. The Network consists of seven laboratories and one company from six countries. The first meeting of the Network took place with the representation of each laboratory involved. In the meeting, general and specific objectives and activities of the Network were discussed and it was a very nice example of international collaboration to address an unsolved worldwide topic on tick control in which laboratories with different competencies and expertise join their efforts in a common goal.

Growth hormone releasing peptide-6 enhanced antibody titers against subunit antigens in mice (BALB/c), tilapia (Oreochromis niloticus) and African catfish (Clarias gariepinus).

Modern subunit vaccines have excellent safety profiles and improved tolerability, but do not elicit strong immune responses without the addition of adjuvants. Developing a safe and affective adjuvant remains a challenge for peptide-based vaccine design. Growth Hormone Releasing Peptide-6 (GHRP-6) is one of the earliest-developed, synthetic, peptidyl growth hormone secretagogue receptor agonists. These compounds mimic the effect of the endogenous ligand, ghrelin. In the present study, we evaluated the ability of GHRP-6 to enhance the humoral immune response against co-injected antigens in mice, tilapia and African catfish. This peptide was able to increase the antigen-specific antibody response using heterologous proteins and peptides as antigens, which were also formulated in "water in oil" emulsions (Freund and Montanide). As long as we know there is no previous report describing any ghrelin analogous as molecular immunomodulator stimulating a humoral immune response. Further studies will be conducted to evaluate the functionality of this humoral immune response in challenge trials.

A single dose of the novel chimeric subunit vaccine E2-CD154 confers early full protection against classical swine fever virus.

Classical swine fever is an economically important, highly contagious disease of swine worldwide. Subunit vaccines are a suitable alternative for the control of classical swine fever. However, such vaccines have as the main drawback the relatively long period of time required to induce a protective response, which hampers their use under outbreak conditions. In this work, a lentivirus-based gene delivery system is used to obtain a stable recombinant HEK 293 cell line for the expression of E2-CSFV antigen fused to porcine CD154 as immunostimulant molecule. The E2-CD154 chimeric protein was secreted into the medium by HEK293 cells in a concentration around 50mg/L in suspension culture conditions using spinner bottles. The E2-CD154 immunized animals were able to overcome the challenge with a high virulent CSF virus strain performed 7days after a unique dose of the vaccine without clinical manifestations of the disease. Specific anti-CSFV neutralizing antibodies and IFN-γ were induced 8days after challenge equivalent to 14days post-vaccination. The present work constitutes the first report of a subunit vaccine able to confer complete protection by the end of the first week after a single vaccination. These results suggest that the E2-CD154 antigen could be potentially used under outbreak conditions to stop CSFV spread and for eradication programs in CSF enzootic areas.

Developing Anti-tick Vaccines.

Ticks are responsible for the transmission of viral, bacterial, and protozoal diseases of man and animals and also produce significant economic losses to cattle industry. The use of acaricides constitutes a major component of integrated tick control strategies. However, this is accompanied by the selection of acaricide-resistant ticks and contamination of environment and milk and meat products with drug residues. These issues highlight the need for alternative approaches to control tick infestations and have triggered the search for tick protective antigens for vaccine development. Vaccination as a tick control method has been practiced since the introduction of TickGARD and Gavac that were developed using the midgut glycoprotein Bm86 as antigen. Gavac within integrated tick management systems has proven to reduce the number of acaricidal applications per year that are required to control some strains of R. microplus ticks in different geographical regions. Nevertheless, it has limited or no efficacy against other tick species. These issues have stimulated research for additional tick protective antigens with critical functions in the tick. This chapter presents methodologies for the design and test of molecules as antigens against ticks. Considerations about different methods for the tick control compared to the immunological methods, the desirable characteristics for an anti-tick vaccine and the obstacles encountered for developing this kind of vaccines are discussed. Detailed methodologies for the establishment of a biological model to test new molecules as immunogens against ticks and to perform challenge trials with this model are presented. General considerations in the efficacy calculation for any anti-tick vaccine are also discussed.

Molecular, biological, and morphometric comparisons between different geographical populations of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae).

In this study, different geographical populations of Rhipicephalus sanguineus sensu lato were compared by molecular, biological, and morphometric methods. Phylogenetic trees were constructed using 12S and 16S rDNA sequences and showed two distinct clades: one composed of ticks from Brazil (Jaboticabal, SP), Cuba (Havana) Thailand (Bangkok) and the so-called "tropical strain" ticks. The second clade was composed of ticks from Spain (Zaragoza), Argentina (Rafaela, Santa Fe) and the so-called "temperate strain" ticks. Morphometric analysis showed good separation between females of the two clades and within the temperate clade. Males also exhibited separation between the two clades, but with some overlap. Multiple biological parameters revealed differences between the two clades, especially the weight of the engorged female. These results confirm the existence of at least two species under the name "R. sanguineus".

High efficacy of a 20 amino acid peptide of the acidic ribosomal protein P0 against the cattle tick, Rhipicephalus microplus.

Current strategies to control cattle ticks use integrated control programs (ICP) that include vaccination. Reduction in the use of chemicals and in the cost of tick control, the delay or elimination of acaricide resistance and the decreasing of environmental pollution are the advantages of using these programs. This integrated program is potentially applicable to all genotypes of chemical resistant ticks. However, the problem here is to improve the efficacy of anti-tick vaccines. The P0 protein is a structural component of the ribosome of all organisms. We have identified an immunogenic region of ribosomal protein P0 from Rhipicephalus spp. ticks that is not very conserved compared to the orthologous protein in their hosts. A synthetic 20 amino acid peptide from this sequence was effective as a vaccine against Rhipicephalus sanguineus infestations in an immunization and challenge experiment using rabbits. In this paper, the same peptide used as vaccine against the cattle tick Rhipicephalus Boophilus microplus shows a significant diminution in the number of engorged females recovered, in the weight of females and the weight of egg masses. The number of eggs hatched was also significantly reduced for the vaccinated group, with an overall effectivity for the antigen pP0 of 96%. These results, together with the conserved sequence of the P0 peptide among ticks, suggest that this antigen could be a good broad spectrum vaccine candidate. It would be expected to be active against many species of ticks and thus has promise in an ICP for effective control of ticks and thereby to improve the efficiency and productivity of the livestock industry.