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.

Reservoir targeted vaccine against Borrelia burgdorferi: a new strategy to prevent Lyme disease transmission.

A high prevalence of infection with Borrelia burgdorferi in ixodid ticks is correlated with a high incidence of Lyme disease. The transmission of B. burgdorferi to humans can be disrupted by targeting 2 key elements in its enzootic cycle: the reservoir host and the tick vector. In a prospective 5-year field trial, we show that oral vaccination of wild white-footed mice resulted in outer surface protein A-specific seropositivity that led to reductions of 23% and 76% in the nymphal infection prevalence in a cumulative, time-dependent manner (2 and 5 years, respectively), whereas the proportion of infected ticks recovered from control plots varied randomly over time. Significant decreases in tick infection prevalence were observed within 3 years of vaccine deployment. Implementation of such a long-term public health measure could substantially reduce the risk of human exposure to Lyme disease.

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.

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.

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.

Experimental infection of Swiss webster mice with four rat bartonella strains: host specificity, bacteremia kinetics, dose dependent response, and histopathology.

Groups of Swiss Webster outbred mice were each inoculated with one of four bartonella strains originally isolated from Rattus spp. at doses ranging from 10(1) to 10(7) bacteria per mouse. One strain, Rn1691yn (Bartonella coopersplainensis-like), infected mice and produced bacteremias at levels up to 10(5)bacteria/ml of blood and from 3 to 8 weeks duration. A dose dependent response was also observed with differing proportions of mice bacteremic following inoculation at different doses. In addition weeks-to-months long lags in bacteremia manifestation occurred following lower dose exposures. The possibility of bacterial transmission from bacteremic mice to uninfected cagemates was assessed and no naïve mice became infected from contacts with infected mice. Finally, a subset of bacteremic mice inoculated with high doses of Rn1691yn were examined histopathologically and multifocal, granulomatous lesions were detected in both liver and kidneys. The host specificity and infectivity of the strains is discussed in relation to their potential for zoonotic transmission to incidental hosts.

Human seroreactivity against Bartonella species in the Democratic Republic of Congo.

OBJECTIVE: To assess the presence and identity of Bartonella species in a pool of human blood samples from DRC Congo. METHODS: Blood (±120µL) was collected anonymously from Congolese patients and placed on calibrated filter papers. Bartonella serology determination was performed using an indirect immunofluorescence assay (IFA) against six specific Bartonella antigens and Coxiella burnetii (C. burnetii) antigen. The end cut-off value for Bartonella sp. was a titre greater than 1:200. RESULTS: None of the patients was positive for Bartonella elizabethae, Bartonella vinsonii subsp. vinsonii or Bartonella vinsonii subsp. arupensis nor for C. burnetti, but 4.5% of the 155 samples were positive for either Bartonella henselae, Bartonella quintana, or Bartonella clarridgeiae. CONCLUSIONS: This preliminary study presents the first report of Bartonella species in the DR Congo and the first report of antibodies to Bartonella clarridgeiae in an African human population. Although few experimental trials have established the link between fleas and Bartonella transmission, the repeated detection of similar Bartonella species in fleas and humans in several countries suggests that Bartonellosis could be another flea-borne disease which specific reservoirs are still unknown.

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.

The first reported case of Bartonella endocarditis in Thailand.

Bartonella species have been shown to cause acute, undifferentiated fever in Thailand. A study to identify causes of endocarditis that were blood culture-negative using routine methods led to the first reported case in Thailand of Bartonella endocarditis A 57 year-old male with underlying rheumatic heart disease presented with severe congestive heart failure and suspected infective endocarditis. The patient underwent aortic and mitral valve replacement. Routine hospital blood cultures were negative but B. henselae was identified by serology, PCR, immunohistochemistry and specific culture techniques.

Human isolates of Bartonella tamiae induce pathology in experimentally inoculated immunocompetent mice.

BACKGROUND: Bartonella tamiae, a newly described bacterial species, was isolated from the blood of three hospitalized patients in Thailand. These patients presented with headache, myalgia, anemia, and mild liver function abnormalities. Since B. tamiae was presumed to be the cause of their illness, these isolates were inoculated into immunocompetent mice to determine their relative pathogenicity in inducing manifestations of disease and pathology similar to that observed in humans. METHODS: Three groups of four Swiss Webster female mice aged 15-18 months were each inoculated with 10(6-7) colony forming units of one of three B. tamiae isolates [Th239, Th307, and Th339]. A mouse from each experimental group was sampled at 3, 4, 5 and 6 weeks post-inoculation. Two saline inoculated age-matched controls were included in the study. Samples collected at necropsy were evaluated for the presence of B. tamiae DNA, and tissues were formalin-fixed, stained with hematoxylin and eosin, and examined for histopathology. RESULTS: Following inoculation with B. tamiae, mice developed ulcerative skin lesions and subcutaneous masses on the lateral thorax, as well as axillary and inguinal lymphadenopathy. B. tamiae DNA was found in subcutaneous masses, lymph node, and liver of inoculated mice. Histopathological changes were observed in tissues of inoculated mice, and severity of lesions correlated with the isolate inoculated, with the most severe pathology induced by B. tamiae Th239. Mice inoculated with Th239 and Th339 demonstrated myocarditis, lymphadenitis with associated vascular necrosis, and granulomatous hepatitis and nephritis with associated hepatocellular and renal necrosis. Mice inoculated with Th307 developed a deep dermatitis and granulomas within the kidneys. CONCLUSIONS: The three isolates of B. tamiae evaluated in this study induce disease in immunocompetent Swiss Webster mice up to 6 weeks after inoculation. The human patients from whom these isolates were obtained had clinical presentations consistent with the multi-organ pathology observed in mice in this study. This mouse model for B. tamiae induced disease not only strengthens the causal link between this pathogen and clinical illness in humans, but provides a model to further study the pathological processes induced by these bacteria.

Epidemiologic and clinical aspects of a Rift Valley fever outbreak in humans in Tanzania, 2007.

In January 2007, an outbreak of Rift Valley fever (RVF) was detected among humans in northern Tanzania districts. By the end of the outbreak in June, 2007, 511 suspect RVF cases had been recorded from 10 of the 21 regions of Tanzania, with laboratory confirmation of 186 cases and another 123 probable cases. All confirmed RVF cases were located in the north-central and southern regions of the country, with an eventual fatality rate of 28.2% (N = 144). All suspected cases had fever; 89% had encephalopathy, 10% hemorrhage, and 3% retinopathy. A total of 169 (55%) of the 309 confirmed or probable cases were also positive for malaria as detected by peripheral blood smear. In a cohort of 20 RVF cases with known outcome that were also positive for human immunodeficiency virus, 15 (75%) died. Contact with sick animals and animal products, including blood, meat, and milk, were identified as major risk factors of acquiring RVF.

Molecular characterization of a new isolate of Borrelia lusitaniae derived from Apodemus sylvaticus in Portugal.

A total of 196 small mammals were collected in Portugal and tested for Borrelia burgdorferi sensu lato. Tissue samples were taken from each animal and cultured in Barbour-Stoenner-Kelly (BSK)-II medium. The single strain of spirochete isolated was confirmed as Borrelia lusitaniae by genetic analyses. This is the first report of B. lusitaniae isolated from Apodemus sylvaticus.

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.

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.

Identification of flea blood meals using multiplexed real-time polymerase chain reaction targeting mitochondrial gene fragments.

Human plague is found in the West Nile region of Uganda and Democratic Republic of the Congo where flea vectors are often found inhabiting homes. We have developed a multiplexed, real-time polymerase chain reaction assay targeting mitochondrial genes that is capable of detecting blood meal sources in fleas collected off-host in East Africa. Laboratory tests showed that the assay is specific for the intended targets and has a detection limit below one picogram of DNA. Testing of wild-caught fleas from the Democratic Republic of Congo suggests that humans are at significant risk from flea-borne disease and implicates domestic animals including cats, chickens, and the black rat as potential sources of human exposure to fleas and flea-borne diseases. Future application of the assay will help us better define the ecology of plague in East Africa to implement effective control measures to combat the spread of disease.

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.

A borreliacidal factor in Amblyomma americanum saliva is associated with phospholipase A2 activity.

Previous work in our laboratory described the in vitro killing of Borrelia burgdorferi when co-cultured with saliva from adult Amblyomma americanum. Borreliacidal activity was not evident using Ixodes scapularis saliva. Mixing trypsin with saliva eliminated the borreliacidal activity of A. americanum saliva, while incorporating a trypsin inhibitor restored all borreliacidal activity, indicating this factor was of protein or peptide origin. One-dimensional PAGE indicated at least 7 major protein differences between I. scapularis and A. americanum saliva. To determine the borreliacidal factor, A. americanum saliva was fractionated by gel filtration and subsequent killing of B. burgdorferi was associated with a single fraction. Two-dimensional gel analysis indicated protein and/or peptide(s) in borreliacidal fractions running between 38 and 64 kDa. Finally, admixing saliva with the phospholipase A2 inhibitor oleyloxyethyl phosphorylcholine completely eliminated the ability of A. americanum saliva to kill B. burgdorferi. These studies indicate the borreliacidal activity found in A. americanum saliva is likely due to phospholipase A2 enzymatic activity.

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.

Development of a real-time quantitative PCR assay to enumerate Yersinia pestis in fleas.

A real-time quantitative polymerase chain reaction (qPCR) assay was developed for Yersina pestis. The qPCR assay was developed utilizing a conserved region of the Y. pestis ferric iron uptake regulator gene (fur) to design primers and a fluorescent (FAM-labeled) TaqMan probe. The assay was optimized using cultured Y. pestis (UG05-0454) and was confirmed to work with strains from 3 Y. pestis biovars. The optimized assay was capable of detecting a single organism of cultured Y. pestis and as little as 300 bacteria in infected flea triturates. This qPCR assay enables rapid enumeration of Y. pestis bacterium in laboratory-infected fleas when compared with conventional serial dilution plating.