Detection and identification of species-specific bacteria associated with synanthropic mites

Internal bacterial communities of synanthropic mites Acarus siro, Dermatophagoides farinae, Lepidoglyphus destructor, and Tyrophagus putrescentiae (Acari: Astigmata) were analyzed by culturing and culture-independent approaches from specimens obtained from laboratory colonies. Homogenates of surface-sterilized mites were used for cultivation on non-selective agar and DNA extraction. Isolated bacteria were identified by sequencing of the 16S rRNA gene. PCR amplified 16S rRNA genes were analyzed by terminal restriction fragment length polymorphism analysis (T-RFLP) and cloning sequencing. Fluorescence in situ hybridization using universal bacterial probes was used for direct bacterial localization. T-RFLP analysis of 16S rRNA gene revealed distinct species-specific bacterial communities. The results were further confirmed by cloning and sequencing (284 clones). L. destructor and D. farinae showed more diverse communities then A. siro and T. putrescentiae. In the cultivated part of the community, the mean CFUs from four mite species ranged from 5.2 × 102 to 1.4 × 103 per mite. D. farinae had significantly higher CFUs than the other species. Bacteria were located in the digestive and reproductive tract, parenchymatical tissue, and in bacteriocytes. Among the clones, Bartonella-like bacteria occurring in A. siro and T. putresecentiae represented a distinct group related to Bartonellaceae and to Bartonella-like symbionts of ants. The clones of high similarity to Xenorhabdus cabanillasii were found in L. destructor and D. farinae, and one clone related to Photorhabdus temperata in A. siro. Members of Sphingobacteriales cloned from D. farinae and A. siro clustered with the sequences of “Candidatus Cardinium hertigii” and as a separate novel cluster.

Surveillance for zoonotic vector-borne infections using sick dogs from southeastern Brazil

For many vector-borne organisms, dogs can be used as sentinels to estimate the risk of human infection. The objective of this study was to use dogs as sentinels for multiple vector-borne organisms in order to evaluate the potential for human infection with these agents in southeastern Brazil. Blood from 198 sick dogs with clinicopathological abnormalities consistent with tick-borne infections were selected at the São Paulo State University Veterinary Teaching Hospital in Botucatu and tested for DNA and/or antibodies against specific vector-borne pathogens. At least one organism was detected in 88% of the dogs, and Ehrlichia canis DNA was amplified from 78% of the blood samples. Bartonella spp. seroreactivity was found in 3.6%. Leishmania chagasi antibodies were detected in 1% of the dogs. There was no serological or polymerase chain reaction evidence of infection with Anaplasma phagocytophilum, Borrelia burgdorferi, Ehrlichia chaffeensis, Ehrlichia ewingii, and Rickettsia rickettsii. The full E. canis 16S rRNA gene sequence of one of the Brazilian strains obtained in this study was identical to the causative agent of human ehrlichiosis in Venezuela. Ehrlichia canis may pose a human health hazard and may be undiagnosed in southeastern Brazil, whereas exposure to the other organisms examined in this study is presumably infrequent.

Arthropod-borne pathogens circulating in free-roaming domestic cats in a zoo environment in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Diagnóstico sorológico e molecular de agentes transmitidos por artrópodes em aves carnívoras

Pós-graduação em Medicina Veterinária - FCAV

Surveillance using serological and molecular methods for the detection of infectious agents in captive Brazilian neotropic and exotic felids

The aim of the current study was to investigate the exposure of captive wild felids to various infectious pathogens using serological and molecular methods. One hundred and fifty-nine neotropic felids and 51 exotic felids from 28 captive settings in Brazil were tested. While antibodies against Feline parvovirus and Feline coronavirus (FCoV), Feline calicivirus and Bartonella spp. were frequently detected by serologic tests, antibodies against Felid herpesvirus 1 or infection with hemotropic mycoplasmas were less prevalent. Serologic evidence of exposure to Ehrlichia spp., Feline immunodeficiency virus, and Feline leukemia virus (FeLV) was detected rarely, and infections with FeLV, Ehrlichia spp., and Cytauxzoon spp. were found infrequently. The detected Bartonella sequence was molecularly similar to B. koehlerae and B. henselae; for Cytauxzoon, the sequence resembled those from domestic cats. No Anaplasma phagocytophilum and Theileria spp. infections were detected. The positive test results varied significantly among different facilities and species. Additionally, FCoV seropositivity was more prevalent in captivity than in free-ranging populations. Results suggest that testing is appropriate prior to relocation of felids.

Fic domain-catalyzed adenylylation: insight provided by the structural analysis of the type IV secretion system effector BepA

Numerous bacterial pathogens subvert cellular functions of eukaryotic host cells by the injection of effector proteins via dedicated secretion systems. The type IV secretion system (T4SS) effector protein BepA from Bartonella henselae is composed of an N-terminal Fic domain and a C-terminal Bartonella intracellular delivery domain, the latter being responsible for T4SS-mediated translocation into host cells. A proteolysis resistant fragment (residues 10-302) that includes the Fic domain shows autoadenylylation activity and adenylyl transfer onto Hela cell extract proteins as demonstrated by autoradiography on incubation with α-[(32)P]-ATP. Its crystal structure, determined to 2.9-Å resolution by the SeMet-SAD method, exhibits the canonical Fic fold including the HPFxxGNGRxxR signature motif with several elaborations in loop regions and an additional β-rich domain at the C-terminus. On crystal soaking with ATP/Mg(2+), additional electron density indicated the presence of a PP(i) /Mg(2+) moiety, the side product of the adenylylation reaction, in the anion binding nest of the signature motif. On the basis of this information and that of the recent structure of IbpA(Fic2) in complex with the eukaryotic target protein Cdc42, we present a detailed model for the ternary complex of Fic with the two substrates, ATP/Mg(2+) and target tyrosine. The model is consistent with an in-line nucleophilic attack of the deprotonated side-chain hydroxyl group onto the α-phosphorus of the nucleotide to accomplish AMP transfer. Furthermore, a general, sequence-independent mechanism of target positioning through antiparallel β-strand interactions between enzyme and target is suggested.

Survey of selected tick-borne diseases in dogs in Finland

BACKGROUND: Due to climate changes during the last decades, ticks have progressively spread into higher latitudes in northern Europe. Although some tick borne diseases are known to be endemic in Finland, to date there is limited information with regard to the prevalence of these infections in companion animals. We determined the antibody and DNA prevalence of the following organisms in randomly selected client-owned and clinically healthy hunting dogs living in Finland: Ehrlichia canis (Ec), Anaplasma phagocytophilum (Ap), Borrelia burgdorferi (Bb) and Bartonella. METHODS: Anti-Ap, -Bb and -Ec antibodies were determined in 340 Finnish pet dogs and 50 healthy hunting dogs using the 4DX Snap(R)Test (IDEXX Laboratories). In addition, PCRs for the detection of Ap and Bartonella DNA were performed. Univariate and multivariate logistic regression analyses were used to identify risk factors associated with seropositivity to a vector borne agent. RESULTS: The overall seroprevalence was highest for Ap (5.3%), followed by Bb (2.9%), and Ec (0.3%). Seropositivities to Ap and Bb were significantly higher in the Aland Islands (p <0.001), with prevalence of Ap and Bb antibodies of 45 and 20%, respectively. In healthy hunting dogs, seropositivity rates of 4% (2/50) and 2% (1/50) were recorded for Ap and Bb, respectively. One client-owned dog and one hunting dog, both healthy, were infected with Ap as determined by PCR, while being seronegative. For Bartonella spp., none of the dogs tested was positive by PCR. CONCLUSIONS: This study represents the first data of seroprevalence to tick borne diseases in the Finnish dog population. Our results indicate that dogs in Finland are exposed to vector borne diseases, with Ap being the most seroprevalent of the diseases tested, followed by Bb. Almost 50% of dogs living in Aland Islands were Ap seropositive. This finding suggests the possibility of a high incidence of Ap infection in humans in this region. Knowing the distribution of seroprevalence in dogs may help predict the pattern of a tick borne disease and may aid in diagnostic and prevention efforts.

Multiple infections of rodents with zoonotic pathogens in Austria

Rodents are important reservoirs for a large number of zoonotic pathogens. We examined the occurrence of 11 viral, bacterial, and parasitic agents in rodent populations in Austria, including three different hantaviruses, lymphocytic choriomeningitis virus, orthopox virus, Leptospira spp., Borrelia spp., Rickettsia spp., Bartonella spp., Coxiella burnetii, and Toxoplasma gondii. In 2008, 110 rodents of four species (40 Clethrionomys glareolus, 29 Apodemus flavicollis, 26 Apodemus sylvaticus, and 15 Microtus arvalis) were trapped at two rural sites in Lower Austria. Chest cavity fluid and samples of lung, spleen, kidney, liver, brain, and ear pinna skin were collected. We screened selected tissue samples for hantaviruses, lymphocytic choriomeningitis virus, orthopox viruses, Leptospira, Borrelia, Rickettsia, Bartonella spp., C. burnetii, and T. gondii by RT-PCR/PCR and detected nucleic acids of Tula hantavirus, Leptospira spp., Borrelia afzelii, Rickettsia spp., and different Bartonella species. Serological investigations were performed for hantaviruses, lymphocytic choriomeningitis virus, orthopox viruses, and Rickettsia spp. Here, Dobrava-Belgrade hantavirus-, Tula hantavirus-, lymphocytic choriomeningitis virus-, orthopox virus-, and rickettsia-specific antibodies were demonstrated. Puumala hantavirus, C. burnetii, and T. gondii were neither detected by RT-PCR/PCR nor by serological methods. In addition, multiple infections with up to three pathogens were shown in nine animals of three rodent species from different trapping sites. In conclusion, these results show that rodents in Austria may host multiple zoonotic pathogens. Our observation raises important questions regarding the interactions of different pathogens in the host, the countermeasures of the host's immune system, the impact of the host–pathogen interaction on the fitness of the host, and the spread of infectious agents among wild rodents and from those to other animals or humans.

Excessive tumor-elaborated VEGF and its neutralization define a lethal paraneoplastic syndrome

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and key regulator of both physiologic and pathologic (e.g., tumor) angiogenesis. In the course of studies designed to assess the ability of constitutive VEGF to block tumor regression in an inducible RAS melanoma model, mice implanted with VEGF-expressing tumors sustained high morbidity and mortality that were out of proportion to the tumor burden. Documented elevated serum levels of VEGF were associated with a lethal hepatic syndrome characterized by massive sinusoidal dilation and endothelial cell proliferation and apoptosis. Systemic levels of VEGF correlated with the severity of liver pathology and overall clinical compromise. A striking reversal of VEGF-induced liver pathology and prolonged survival were achieved by surgical excision of VEGF-secreting tumor or by systemic administration of a potent VEGF antagonist (VEGF-TRAPR1R2), thus defining a paraneoplastic syndrome caused by excessive VEGF activity. Moreover, this VEGF-induced syndrome resembles peliosis hepatis, a rare human condition that is encountered in the setting of advanced malignancies, high-dose androgen therapy, and Bartonella henselae infection. Thus, our findings in the mouse have suggested an etiologic role for VEGF in this disease and may lead to diagnostic and therapeutic options for this debilitating condition in humans.

Bartonelosis sistémica con lesiones hepatoesplénicas en un paciente inmunocompetente: Presentación de un caso y revisión de la bibliografía

Resumen La enfermedad por arañazo de gato (EAG) es una enfermedad infecciosa causada por Bartonella henselae. Esta enfermedad es de evolución habitualmente benigna, el beneficio del tratamiento antibiótico no está claramente demostrado, sin embargo algunos pacientes desarrollan formas atípicas y más graves con compromiso sistémico como la formación de granulomas hepáticos y esplénicos. Se presenta el caso clínico de una niña de 10 años con granulomas hepáticos que fue asistida en el Centro Hospitalario Pereira Rossell por fiebre prolongada, astenia, dolor abdominal y adelgazamiento. Lo que obliga a investigar la etiología y descartar diagnósticos diferenciales y proponer alternativas terapéuticas. En ese momento no disponíamos de estudios serológicos para la búsqueda etiológica en el país lo cual generó dificultades diagnósticas en esta paciente. Finalmente se pudo obtener la confirmación serológica de EAG de la paciente al enviar la muestra al Hospital de Niños Ricardo Gutiérrez en Buenos Aires prácticamente un mes después de iniciado los síntomas. Fue tratada inicialmente con azitromicina vía oral 10 mg/kg/día por 10 días y dada la tórpida evolución se utiliza un segundo plan antibiótico que incluyó claritromicina 15 mg/kg/día más rifampicina 20 mg/kg/día por 6 semanas. La paciente evolucionó favorablemente, presentando disminución progresiva del tamaño de los granulomas hepáticos y esplénicos. Discusión: Los granulomas hepatoesplénicos constituyen una forma de presentación poco frecuente pero orientadora de la EAG. La evidencia en relación a su tratamiento es escasa y deriva de series de casos. Es posible que los pacientes con manifestaciones sistémicas requieran tratamientos combinados y prolongados.