Transgene expression in tick cells using Agrobacterium tumefaciens.

Ticks transmit infectious agents to humans and other animals. Genetic manipulation of vectors like ticks could enhance the development of alternative disease control strategies. Transgene expression using the phytopathogen Agrobacterium tumefaciens has been shown to promote the genetic modification of non-plant cells. In the present work we developed T-DNA constructs for A. tumefaciens to mediate transgene expression in HeLa cells as well as Rhipicephalus microplus tick cells. Translational fusions eGfp:eGfp or Salp15:eGfp, including the enhanced-green fluorescent protein and the Ixodes scapularis salivary factor SALP15 genes, were constructed using the CaMV 35S (cauliflower mosaic virus) promoter, "PBm" tick promoter (R. microplus pyrethroid metabolizing esterase gene) or the Simian Virus SV40 promoter. Confocal microscopy, RT-PCR and Western-blot assays demonstrated transgene(s) expression in both cell lines. Transgene expression was also achieved in vivo, in both R. microplus and I. scapularis larvae utilizing a soaking method including the A. tumefaciens donor cells and confirmed by nested-RT-PCR showing eGfp or Salp15 poly-A-mRNA(s). This strategy opens up a new avenue to express exogenous genes in ticks and represents a potential breakthrough for the study of tick-host pathophysiology.

A prevalent alpha-proteobacterium Paracoccus sp. in a population of the Cayenne ticks (Amblyomma cajennense) from Rio de Janeiro, Brazil.

As Rocky Mountain Spotted Fever is the most common tick-borne disease in South America, the presence of Rickettsia sp. in Amblyomma ticks is a possible indication of its endemicity in certain geographic regions. In the present work, bacterial DNA sequences related to Rickettsia amblyommii genes in A. dubitatum ticks, collected in the Brazilian state of Mato Grosso, were discovered. Simultaneously, Paracoccus sp. was detected in aproximately 77% of A. cajennense specimens collected in Rio de Janeiro, Brazil. This is the first report of Paracoccus sp. infection in a specific tick population, and raises the possibility of these bacteria being maintained and/or transmitted by ticks. Whether Paracoccus sp. represents another group of pathogenic Rhodobacteraceae or simply plays a role in A. cajennense physiology, is unknown. The data also demonstrate that the rickettsial 16S rRNA specific primers used forRickettsia spp. screening can also detect Paracoccus alpha-proteobacteria infection in biological samples. Hence, a PCR-RFLP strategy is presented to distinguish between these two groups of bacteria.

Coxiella symbionts in the Cayenne tick Amblyomma cajennense.

Members of the Coxiella genus are intracellular bacteria that can infect a variety of animals including humans. A symbiotic Coxiella was recently described in Amblyomma americanum ticks in the Northern Hemisphere with no further investigations of other Amblyomma species in other geographic regions. These ixodid ticks represent a group of important vectors for human infectious agents. In the present work, we have demonstrated that symbiotic Coxiella (SCox) are widespread, occurring in South America and infecting 100% of all life stages and eggs of the Cayenne ticks Amblyomma cajennense from Brazil and the USA. Using light microscopy, in situ hybridization, and PCR, we demonstrated SCox in salivary glands, ovaries, and the intestines of A. cajennense. These symbionts are vertically and transtadially transmitted in laboratory reared A. cajennense, and quantitative PCR analyses indicate that SCox are more abundant in adult female ticks, reaching values corresponding to an 11×, 38×, and 200× increase in SCox 16S rRNA gene copy number in unfed females, compared to unfed nymphs, larvae, and eggs, respectively. Phylogenetic analyses showed distinct SCox subpopulations in the USA and Brazil and demonstrated that SCox bacteria do not group with pathogenic Coxiella burnetii.

Molecular identification of Salp15, a key salivary gland protein in the transmission of lyme disease spirochetes, from Ixodes persulcatus and Ixodes pacificus (Acari: Ixodidae).

Salp15 is a multifunctional protein, vital to the tick in its need to obtain vertebrate host blood without stimulating a host inflammatory and immune response. The Salpl5 protein from both Ixodes scapularis Say and Ixodes ricinus (L.), the principal vectors of the Lyme disease spirochete in eastern North America and Europe, respectively, have been well characterized and found to bind the murine CD4 receptor, DC-SIGN, and the OspC protein of Borrelia burgdorferi. In the current study, we characterized the full salp15 gene in Ixodes pacificus Cooley & Kohls and Ixodes persulcatus Schulze, the principal vectors of Lyme disease spirochetes in western North America and Asia, respectively. In comparing the Salp15 protein of all four principal vector ticks of public health importance for the transmission of Lyme disease spirochetes, we find the 53 C-terminal amino acids to have a high degree of similarity. There are at least three clades in the tree of Salp15 and its homologues, probably representing a multigene family.

Francisella-like endosymbiont DNA and Francisella tularensis virulence-related genes in Brazilian ticks (Acari: Ixodidae).

Ticks are vectors of a variety of pathogens, including Francisella tularensis. Bacteria in the genus Francisella have been identified mostly in the Northern Hemisphere and include tick endosymbionts. Francisella has never been described in Brazil, where Amblyomma spp. ticks are known as the vector of many bacterial zoonotic pathogens. In the present work, we have identified bacterial DNA sequences with identity to Francisella genes in Amblyomma dubitatum Neumann Dermacentor nitens (Neumann), and Rhipicephalus microplus (Canestrini) in Brazil. DNA fragments with homology to Francisella spp. 16S rDNA and the tul4 gene were polymerase chain reaction amplified from tick DNA samples collected in Minas Gerais and Mato Grosso states. These sequences were 96-99% identical to the reported sequences for Francisella-like tick endosymbionts (FLEs). Sequences similar to the tularemia agent F. tularensis pathogenicity island gene iglC and its regulatory gene mglA also were identified in FLEs.

Tularemia type A in captive Bornean orangutans (Pongo pygmaeus pygmaeus).

In 2003, tularemia was suspected to be the cause of severe illness in two orangutans (Pongo pygmaeus pygmaeus) and the cause of death in a third orangutan at an urban zoo. The two sick orangutans were treated two times under chemical immobilization with i.v. doxycycline, fluids, and antipyretic drugs, followed by a sustained course of oral doxycycline. The rest of the orangutan group was treated prophylactically with oral doxycycline. Postmortem diagnosis was obtained via immunohistochemistry and bacterial culture that revealed Francisella tularensis type A. Tularemia was also confirmed in the two surviving orangutans via paired serology testing. In addition, F. tularensis was identified in two wild rabbit carcasses submitted during a die-off, several weeks prior to the tularemia outbreak in the apes, indicating that rabbits were possibly a reservoir for tularemia within the zoo premises.

A sustained-release formulation of doxycycline hyclate (Atridox) prevents simultaneous infection of Anaplasma phagocytophilum and Borrelia burgdorferi transmitted by tick bite.

Current prophylaxis for infected tick bites consists of personal protective measures directed towards ticks. This study compared the efficacy of a single oral dose of doxycycline with that of a single injection of sustained-release doxycycline in a model of Lyme borreliosis and Anaplasma phagocytophilum infection. Dosages of doxycycline were equilibrated based on previously determined peak plasma levels in mice [oral, 2.4 microg (ml plasma)(-1); sustained release, 1.9 microg (ml plasma)(-1)] determined 8 h after inoculation. In challenge experiments where five Borrelia burgdorferi-infected and five A. phagocytophilum-infected nymphs were used per mouse, only 20 and 30 % of mice were protected from B. burgdorferi and A. phagocytophilum infection, respectively, using oral doxycycline. In contrast, 100 % of mice receiving sustained-release doxycycline were protected from A. phagocytophilum infection, as indicated by real-time PCR of blood samples, quantitative PCR and culture isolation of spleen samples, and protected against B. burgdorferi infection as demonstrated by culture of ear, heart and bladder. Although 15-40 copies of A. phagocytophilum could be amplified from the spleens of mice treated with sustained-release doxycycline, no viable A. phagocytophilum from these spleens could be cultured in HL-60 cells. In contrast, 7/10 mice receiving oral doxycycline were PCR- and culture-positive for A. phagocytophilum, with copy numbers ranging from 800 to 10 000 within the spleen, as determined by quantitative PCR. Other correlates with A. phagocytophilum infection included a significant difference in spleen mass (mean of 110 mg for sustained-release treatment versus a mean of 230 mg for oral treatment) and the number of splenic lymphoid nodules (mean of 8 for sustained-release treatment versus mean of 12.5 for oral doxycycline) as determined by histopathology. These studies indicate that a single injection of a sustained-release formulation antibiotic may offer a viable prophylactic treatment option for multiple infectious agents in patients presenting with tick bites.

Suppression of Th2 cytokines reduces tick-transmitted Borrelia burgdorferi load in mice.

Previous work has indicated that both Borrelia burgdorferi and the process of tick feeding (saliva) modulate the host immune response. Molecules have been identified in tick saliva that effect T cell proliferation by binding to specific cytokines, thereby promoting a Th2 cytokine response that does not afford protection against tick-transmitted B. burgdorferi in mice. Moreover, reconstitution of a Th1-biased T cell response prior to spirochete challenge effectively neutralizes tick modulation of host immunity and affords protection against tick transmission of spirochetes. The current studies were undertaken to determine the effect of neutralizing specific Th2 cytokines prior to tick feeding and subsequent transmission of B. burgdorferi. The results indicate that suppression of both IL-4 and IL-5 prior to the feeding of B. burgdorferi-infected ticks significantly decreased spirochete load in target organs such as joint, bladder, heart, and skin of the Lyme disease-susceptible host.

A hard tick relapsing fever group spirochete in a Brazilian Rhipicephalus (Boophilus) microplus.

Tick-borne diseases usually comprise a complex epidemiological and ecological network connecting the vector, pathogen, and a group of host species. Symptoms associated with Lyme disease have been reported in Brazil, but no Borrelia sp. has been definitively related to these events. Here we have identified a B. lonestari/B. theileri-related spirochete DNA in the cattle tick Rhipicephalus (Boophilus) microplus from Brazil. Four hundred R. microplus and 80 Amblyomma cajennense ticks were screened, and only 1 horse-fed R. microplus was infected. A Borrelia sp. 16S rDNA sequence was amplified by polymerase chain reaction (PCR) from the total tick DNA with 99% similarity to B. theileri and B. lonestari. Partial flaB sequence was also obtained, demonstrating 96% similarity to the B. lonestari flagellin gene, and the resultant putative amino acid sequence demonstrated 97% identity to B. lonestari flagellin. Moreover, partial glpQ sequence demonstrated 92% similarity to the B. lonestari gene, with a putative amino acid sequence 90% identical to the B. lonestari glycerophosphodiester phosphodiesterase. Phylogenetic analyses clearly include this Brazilian Borrelia sp., denoted "Borrelia," sp-BR in a group of spirochetes aligned with B. theileri and B. lonestari. Thus, hard tick relapsing fever group spirochetes represent a clade of widespread bacteria and herein we describe the first molecular identification of a Borrelia sp. in South America.

Transfer of Borrelia burgdorferi s.s. infection via blood transfusion in a murine model.

Without antibiotic treatment, the Lyme-disease-causing bacterium, Borrelia burgdorferi can be cultured from the peripheral blood of human patients nearly 6 wk post-tick bite. To determine if Lyme disease spirochetes can be transmitted from a spirochetemic donor mouse to a naive recipient during blood transfusion, blood taken from immunocompetent infected mice was transfused into either immunodeficient (SCID) mice, inbred immunocompetent animals (C3H/HeJ), or outbred mice. Nine of 19 (47.7%) immunodeficient mice, 7 of 15 (46.8%) inbred immunocompetent mice, and 6 of 10 (60.0%) outbred mice became infected with B. burgdorferi after transfusion. Our results indicate that it is possible to acquire B. burgdoferi infection via transfused blood in a mouse model of Lyme borreliosis.

Prophylactic use of sustained-release doxycycline blocks tick-transmitted infection by Anaplasma phagocytophilum in a murine model.

A sustained-release formulation of doxycycline hyclate was tested for its ability to block Anaplasma phagocytophilum infection in mice. Mice treated with sustained-release doxycycline showed no splenomegaly and their blood samples were negative by PCR on days 7, 14, and 21. Control mice treated with either oral doxycycline or water had significant splenomegaly and were PCR positive at multiple time points. The sustained-release doxycycline formulation was shown to be efficacious for preventing tick-transmitted A. phagocytophilum infection in a mouse model.

Aedes aegypti salivary gland extracts modulate anti-viral and TH1/TH2 cytokine responses to sindbis virus infection.

Vector-borne viruses are naturally transmitted when a vector salivates during feeding on a vertebrate host. Most laboratory studies of infection disregard the role that the vector plays in the pathogenesis of the virus. In this study, intradermal inoculations of Aedes aegypti salivary gland extract (SGE) and Sindbis virus (SINV) were used to investigate the effect of mosquito feeding on the vertebrate immune response to infection with an arthropod-borne virus. Murine cytokine expression in the skin was quantified by means of real-time RT-PCR. In response to co-inoculation of SINV with SGE, interferon (IFN)-beta expression at 24 and 72 h post inoculation was significantly reduced by 2.2- and 2.3-fold, respectively, when compared to injection of virus alone. IFN-gamma expression in response to SINV infection was significantly decreased by 1.6-fold at 24 h post inoculation when SGE was co-inoculated. In contrast, interleukin (IL)-4 expression was significantly up regulated when SGE was co-inoculated at 24 h post inoculation becoming a 3.3-fold increase by 72 h post inoculation. Compared to expression with SINV alone, IL-10 expression showed a 7.6-fold increase by 72 h post inoculation in mice receiving SGE concurrently with virus. This study suggests that the response to virus is significantly different when an infection is initiated in the presence of mosquito salivary factors, and we identify a possible mechanism for potentiation of viral infections initiated by the natural mosquito vector or in the presence of mosquito saliva.

A portuguese isolate of Borrelia lusitaniae induces disease in C3H/HeN mice.

A low-passage, Portuguese isolate of Borrelia lusitaniae, strain PotiB2, was inoculated into C3H/HeN mice and disease was monitored by histopathology at 8 weeks after spirochaete challenge. Ear, heart, bladder, femoro-tibial joint, brain and spinal cord were examined. B. lusitaniae strain PotiB2 (6 of 10 mice) and B. burgdorferi sensu stricto strain N40 (9 of 10 mice) induced similar lesions in the bladder of infected mice characterised as a multifocal, lymphoid, interstitial cystitis. Moreover, both B. lusitaniae PotiB2 and B. burdorferi N40 induced lesions in the heart of infected mice. The lesions induced by B. lusitaniae PotiB2 (2 of 10 mice) were characterised as a severe, necrotising endarteritis of the aorta, with a minimal, mixed inflammatory infiltrate (neutrophils, macrophages and lymphoid cells) extending into the adjacent myocardium. In contrast, B. burgdorferi N40 induced a periarteritis of the pulmonary artery (7 of 10 mice), with no involvement of the endothelium and more extensive inflammation and subsequent necrosis of the adjacent myocardium. This infiltrate was composed entirely of mononuclear cells, predominantly mature lymphocytes and plasma cells. No lesions were noted in the joints or central nervous system with inoculation of strains N40 or PotiB2, and co-inoculation of either strain with Ixodes ricinus salivary gland lysate did not affect the resulting pathology. Serology, examined 8 weeks after inoculation, indicated a different reactivity in mice infected with B. lusitaniae PotiB2 compared with B. burgdorferi N40. Immunoblot analysis demonstrated that mice with lesions resulting from infection with B. lusitaniae PotiB2 reacted only to the flagellin protein (41 kDa) or to flagellin and OspC, whereas mice infected with B. burgdorferi N40 reacted with multiple high and low mol. wt proteins, including flagellin, p93, p39, OspA, OspB and OspC. These results indicate that B. lusitaniae PotiB2 induced pathology similar to B. burgdorferi N40 when inoculated into susceptible mice. Moreover, these results establish the first animal model of disease with B. lusitaniae. This mouse model can be used to characterise the immunopathogenesis of B. lusitaniae infection and to delineate the proteins responsible for disease induction in susceptible mice.

Dynamic changes in Borrelia burgdorferi populations in Ixodes scapularis (Acari: Ixodidae) during transmission: studies at the mRNA level.

Many B. burgdorferi genes are regulated at the level of transcription during B. burgdorferi passage from ticks to mammals. Particular spirochete outer surface proteins of interest are OspA, OspC, and vlsE. The messenger RNA (mRNA) levels produced by these three genes were determined by a quantitative reverse transcription PCR (q-RT-PCR) procedure for spirochete populations in nymphal I. scapularis midguts and salivary glands at specific intervals during the feeding process. The mRNA values were compared to that of a standard, the mRNA levels of the constitutively expressed Flagellin (fla) gene. The levels of OspA and vlsE did not increase markedly in the midgut during feeding, but the mRNA levels of OspC increased significantly during feeding. In tick salivary glands, OspA mRNA levels actually decreased during feeding, while OspC levels increased six orders of magnitude. The mRNA levels of vlsE in tick salivary glands increased significantly only during the last 2 days of tick feeding. Overall, OspA mRNA was more abundant in tick midguts, whereas OspC and vlsE mRNA was more abundant in tick salivary glands. Further studies on the regulation of B. burgdorferi transcription activity during the act of transmission will lead to a better understanding of spirochete transmission dynamics, and hopefully facilitate the development of novel ways of interrupting the spread of Lyme disease.

Control of immature Ixodes scapularis (Acari: Ixodidae) on rodent reservoirs of Borrelia burgdorferi in a residential community of southeastern Connecticut.

A 3-yr community-based study was conducted on residential properties on Mason's Island, Mystic, CT, to determine the efficacy of a rodent-targeted acaricide (fipronil) to control immature Ixodes scapularis (Say) on Peromyscus leucopus. Results indicated that modified commercial bait boxes were effective as an acaricide delivery method for reducing nymphal and larval tick infestations on white-footed mice by 68 and 84%, respectively. Passive application of fipronil significantly reduced the infection rate of Borrelia burgdorferi among white-footed mice by 53%. Moreover, the abundance of questing I. scapularis adults on treated properties was reduced by 77% and fewer were infected with spirochetes (31%) compared with untreated sites (47%) after 3 yr of treatment. Likewise, the abundance of host-seeking nymphs was significantly reduced on treated properties by >50%. Finally, infection rates in flagged nymphal ticks for both B. burgdorferi and Anaplasma phagocytophilum were reduced by 67 and 64%, respectively, after only 2 yr of treatment. Results from this 3-yr trial indicate that the use of fipronil passively applied to reservoir animals by bait boxes is an environmentally acceptable means to control ticks, interrupt the natural disease transmission cycle, and reduce the risk of Lyme disease for residents of treated properties.

An outbreak of Francisella tularensis in captive prairie dogs: an immunohistochemical analysis.

An immunohistochemical assay was developed and tested for detection of Francisella tularensis lipopolysaccaride antigen in tissues of captive prairie dogs (Cynomys ludovicianus). Tissues from 59 cases of F. tularensis were examined by this technique, which was corroborated by direct fluorescent antibody assay and direct isolation of the organism. In infected prairie dogs, studies indicated multiple, severe, necroprurulent foci occurring in the liver, lung, spleen, terminal ileum, and mandibular lymph node. Immunohistochemical analysis of the same formalin-fixed tissues indicated the presence of F. tularensis antigen in neutrophils and macrophages of these lesions and occurring extracellularly in areas of necrosis. This report demonstrates that immunohistochemical analysis is a rapid procedure that can be used to determine the pathogenesis of F. tularensis in rodent populations.

Novel potential reservoirs for Borrelia sp. and the agent of human granulocytic ehrlichiosis in Colorado.

Previous work demonstrated that Ixodes spinipalpis ticks maintained an enzootic cycle of Borrelia bissettii and the agent of human granulocytic ehrlichiosis (aoHGE) within woodrats (Neotoma mexicana) and deer mice (Peromyscus maniculatus) in northern Colorado (USA). Because I. spinipalpis is the only known vector of B. bissettii and aoHGE in Colorado, this study was designed to determine the reservoir status of other hosts of I. spinipalpis in five distinct ecological zones along the front range and foothills of Colorado. One hundred and twelve rodents of nine species were examined and 11 (10%) were polymerase chain reaction (PCR) positive for aoHGE; 37 (33%) were culture positive for B. bissettii, and five (4%) were coinfected with both organisms based on PCR and culture. Of these, three chipmunk species (Tamias minimus, T. quadrivittatus, and T. umbrinus) were culture positive for B. bissettii, with a single T. minimus coinfected with B. bissettii and aoHGE. In addition, one golden-mantled ground squirrel (Spermophilus lateralis) was positive for both B. bissettii and aoHGE. This is the first report of a golden-mantled ground squirrel harboring either B. bissettii or aoHGE and the initial observation that chipmunks may be a reservoir for B. bissettii in Colorado.

Immunization with Culex tarsalis mosquito salivary gland extract modulates West Nile virus infection and disease in mice.

Mosquito salivary proteins inoculated during blood feeding modulate the host immune response, which can contribute to the pathogenesis of viruses transmitted by mosquito bites. Previous studies with mosquito bite-naïve mice indicated that exposure to arthropod salivary proteins resulted in a shift toward a Th2-type immune response in flavivirus-susceptible mice but not flavivirus-resistant animals. In the study presented here, we tested the hypothesis that immunization with high doses of Culex tarsalis salivary gland extracts (SGE) with an adjuvant would prevent Th2 polarization after mosquito bite and enhance resistance to mosquito-transmitted West Nile virus (WNV). Our results indicate that mice immunized with Cx. tarsalis SGE produced increased levels of Th1-type cytokines (IFNγ and TNFα) after challenge with mosquito-transmitted WNV and exhibited both a delay in infection of the central nervous system (CNS) and significantly lower WNV brain titers compared to mock-immunized mice. Moreover, mortality was significantly reduced in the SGE-immunized mice, as none of these mice died, compared to mortality of 37.5% of mock-vaccinated mice by 8 days after infected mosquito bite. These results suggest that development of a mosquito salivary protein vaccine might be a strategy to control arthropod-borne viral pathogens such as WNV.

Elimination of Borrelia burgdorferi and Anaplasma phagocytophilum in rodent reservoirs and Ixodes scapularis ticks using a doxycycline hyclate-laden bait.

A field trial was conducted in a Lyme disease-endemic area of New Jersey to determine the efficacy of a doxycyline hyclate rodent bait to prophylactically protect and cure small-mammal reservoirs and reduce infection rates in questing Ixodes scapularis ticks for Borrelia burgdorferi and Anaplasma phagocytophilum. The doxycycline-laden bait was formulated at a concentration of 500 mg/kg and delivered during the immature tick feeding season in rodent-targeted bait boxes. The percentage of infected small mammals recovered from treated areas after 2 years of treatment was reduced by 86.9% for B. burgdorferi and 74% for A. phagocytophilum. Infection rates in questing nymphal ticks for both B. burgdorferi and A. phagocytophilum were reduced by 94.3% and 92%, respectively. Results from this study indicate that doxycycline-impregnated bait is an effective means of reducing infection rates for B. burgdorferi and A. phagocytophilum in both rodent reservoirs and questing I. scapularis ticks.