Prevalence and molecular characterization of Anaplasmataceae agents in free-ranging Brazilian marsh deer (Blastocerus dichotomus)

Anaplasmataceae organisms comprise a group of obligate intracellular gram-negative, tick-borne bacteria that can infect both animals and humans. In the present work we investigate the presence of Ehrlichia, Anaplasma, and Neorickettsia species in blood samples from Brazilian marsh deer (Blastocerus dichotomus), using both molecular and serologic techniques. Blood was collected from 143 deer captured along floodplains of the Paraná River, near the Porto Primavera hydroelectric power plant. Before and after flooding, marsh deer were captured for a wide range research program under the financial support of S?o Paulo State Energy Company (CESP), between 1998 and 2001. Samples were divided into four groups according to time and location of capture and named MS01 (n=99), MS02 (n=18) (Mato Grosso do Sul, before and after flooding, respectively), PX (n=9; Peixe River, after flooding), and AGUA (n=17; Aguapeí River, after flooding). The seroprevalences for Ehrlichia chaffeensis and Anaplasma phagocytophilum were 76.76% and 20.2% in MS01, 88.88% and 5.55% in MS02, 88.88% and 22.22% in PX, and 94.12% and 5.88% in AGUA, respectively. Sixty-one animals (42.65% of the total population) were PCR-positive for E. chaffeensis PCR (100.0% identity based on 16S rRNA, dsb, and groESL genes). Seventy deer (48.95% of the total population) were PCR-positive for Anaplasma spp. (99.0% of identity with A. platys, and in the same clade as A. phagocytophilum, A. bovis, and A. platys based on 16S rRNA phylogenetic analysis). Our results demonstrate that Brazilian marsh deer are exposed to E. chaffeensis and Anaplasma spp. and may act as reservoirs for these rickettsial agents, playing a role in disease transmission to humans and other animals.     

Tuberculosis survey of free-ranging marsh deer (Blastocerus dichotomus) in Brazil.

Esophageal-pharyngeal fluids from 53 free-ranging marsh deer (Blastocerus dichotomus) captured for a research program in the state of Mato Grosso do Sul, Brazil, were assayed for tuberculosis. Total DNA was extracted. amplified by polymerase chain reaction using specific primers for Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, M. microti, and M. africanum), and observed by agarose gel electrophoresis stained with ethidium bromide. All samples were negative. This, along with necropsy and histopathology data, suggests that these animals are not shedding and probably do not have active disease.     

Hematology of free-living marsh deer (Blastocerus dichotomus) from southeast Brazil.

This work reports basic hematologic values of a sample of a population of free-living marsh deer (Blastocerus dichotomus) living by the Paraná River in Southeast Brazil. Hematologic values are presented separately for male, female, and young animals as well as for anesthetized and nonanesthetized cervids. Nonanesthetized deer restrained by physical means had significantly higher erythrocyte indices and total leukocyte counts. Comparisons of blood parameters of anesthetized animals of different ages and gender differed slightly, with only two significant differences observed: young animals had significantly higher red blood cell counts than adult males and a lower blood total protein content when compared to adult females. Results indicate that two main reference ranges for blood values should be considered for marsh deer, for blood obtained from anesthetized or physically restrained individuals.     

Cat flea (Ctenocephalides felis) infestation in quarantined marsh deer (Blastocerus dichotomus) populations.

Marsh deer (Blastocerus dichotomus) were captured for a research program in Brazil and maintained in quarantine stations. After 60 days, fleas were detected on animals and identified as Ctenocephalides felis felis. Elimination of the infestation was difficult. Animal treatment with a fipronil-based compound was effective, and subsequently captured animals were treated prophylactically. Some animals remained infested, and some died from the infestation.     

Detection of Theileria and Babesia in brown brocket deer (Mazama gouazoubira) and marsh deer (Blastocerus dichotomus) in the State of Minas Gerais, Brazil

Intraerythrocytic protozoan species of the genera Theileria and Babesia are known to infect both wild and domestic animals, and both are transmitted by hard-ticks of the family Ixodidae. The prevalences of hemoprotozoa and ectoparasites in 15 free-living Mazama gouazoubira, two captive M. gouazoubira and four captive Blastocerus dichotomus from the State of Minas Gerais, Brazil, have been determined through the examination of blood smears and the use of nested polymerase chain reaction (nPCR). The cervid population was inspected for the presence of ticks and any specimens encountered were identified alive under the stereomicroscope. Blood samples were collected from all 21 animals, following which blood smears were prepared, subjected to quick Romanowsky staining and examined under the optical microscope. DNA was extracted with the aid of commercial kits from cervid blood samples and from tick salivary glands. The nPCR assay comprised two amplification reactions: the first was conducted using primers specific for a 1700 bp segment of the 18S rRNA gene of Babesia and Theileria species, whilst the second employed primers designed to amplify a common 420 bp Babesia 18S rRNA fragment identified by aligning sequences from Babesia spp. available at GenBank. The ticks Amblyomma cajennense, Rhipicephalus microplus and Dermacentor nitens were identified in various of the cervids examined. Of the animals investigated, 71.4% (15/21) were infected with hemoprotozoa, including Theileria cervi (47.6%), Theileria sp. (14.3%), Babesia bovis (4.8%) and Babesia bigemina (4.8%). However, only one of the infected wild cervids exhibited accentuated anaemia (PCV=17%). This is first report concerning the occurrence of Theileria spp. in Brazilian cervids.     

Species diversity and seasonality of free-living ticks (Acari: Ixodidae) in the natural habitat of wild Marsh deer (Blastocerus dichotomus) in Southeastern Brazil

This study evaluated the presence and seasonal activity of free-living ticks in remaining marsh areas by the Paraná river, in Brazil. Eight field trips (once per season) for collection of ticks were performed during 2 years. Using CO2 traps, dragging, and visual inspection of vegetation, five free-living tick species were collected, in the following order of abundance: Amblyomma cajennense, Amblyomma dubitatum, Amblyomma triste, Amblyomma coelebs, and Amblyomma nodosum. The seasonal pattern of A. cajennense was characterized by the highest peaks for adult ticks in the summer/spring months, for nymphs in the winter and for larvae in the autumn and winter. A. dubitatum and A. triste presented similar seasonal patterns characterized by peaks of adult ticks in the autumn. Nymphs of A. dubitatum peaked in the winter of the first year and in the winter/spring of the second year. A. triste was the only species to be collected in significantly higher numbers in the marsh than in surrounding drier areas such as forest patches. Among domestic animals living close the marsh areas, horses were infested by Anocentor nitens, A. cajennense, and Boophilus microplus, bovines were infested solely by B. microplus, and dogs were infested by Rhipicephalus sanguineus. Adults of A. triste showed to be well adapted to the marsh environment. This result, at least partially, explains local previous observations on the association of A. triste with marsh deer, as this vertebrate host inhabits mainly the marsh area.     

Detection of Ehrlichia muris DNA from sika deer (Cervus nippon yesoensis) in Hokkaido, Japan

Ehrlichia muris DNA was detected in the blood of sika deer (Cervus nippon yesoensis) by species-specific PCR based on the citrate synthase gene, which was shown to be more sensitive than species-specific PCR based on the 16S rRNA gene. Among 102 deer examined, one deer was positive. Deer may be a possible mammalian reservoir of E. muris.     

Cultivation and molecular identification of Ehrlichia canis and Ehrlichia chaffeensis from a naturally co-infected dog in Venezuela

Background: Diagnosis of canine ehrlichiosis in Venezuela is normally performed by examination of buffy coat smears (BCS). Characteristic inclusion bodies are frequently observed in leukocytes and platelets from dogs with clinical signs of the disease. Objective: The purpose of this study was to investigate the co-infection of a dog with Ehrlichia canis and E hrlichia chaffeensis using microbiological and molecular techniques. Methods: Primary cultures of monocytes from a dog showing signs of ehrlichiosis were performed. Ehrlichial inclusions in blood cells were demonstrated by BCS and in cultured cell smears with direct immunofluorescence and Dip Quick staining. Nested PCR analysis was performed with DNA from blood samples and cultures, using primers specific for E. canis and E. chaffeensis. The amplified DNA fragments were sequenced to confirm the specificity of the amplifications. Results: The BCS of the naturally infected dog contained intracellular morulae. Ehrlichial inclusions were observed 9 days after inoculation of the primary cultures. After 3 passages with monocytes from a healthy dog, 65% of infected cells, and cells with >60 morulae were observed. A healthy female German Shepherd dog, seronegative for E. canis and E. chaffeensis antigens and without contact to ticks, was inoculated with an infected culture. The animal developed signs of canine monocytic ehrlichiosis and became seropositive. Nested PCR results and sequencing of amplified DNA fragments demonstrated the simultaneous presence of E. canis and E. chaffeensis in both dogs. Conclusions: This is the first report of E. chaffeensis in dogs in South America. This organism was previously identified in dogs by PCR only in the United States.     

Molecular events involved in cellular invasion by Ehrlichia chaffeensis and Anaplasma phagocytophilum

Ehrlichia chaffeensis and Anaplasma phagocytophilum are obligatory intracellular bacteria that preferentially replicate inside leukocytes by utilizing biological compounds and processes of these primary host defensive cells. These bacteria incorporate cholesterol from the host for their survival. Upon interaction with host monocytes and granulocytes, respectively, these bacteria usurp the lipid raft domain containing GPI-anchored protein to induce a series of signaling events that result in internalization of the bacteria. Monocytes and neutrophils usually kill invading microorganisms by fusion of the phagosomes containing the bacteria with granules containing both antimicrobial peptides and lysosomal hydrolytic enzymes and/or through sequestering vital nutrients. However, E. chaffeensis and A. phagocytophilum alter vesicular traffic to create a unique intracellular membrane-bound compartment that allows their replication in seclusion from lysosomal killing. These bacteria are quite sensitive to reactive oxygen species (ROS), so in order to survive in host cells that are primary mediators of ROS-induced killing, they inhibit activation of NADPH oxidase and assembly of this enzyme in their inclusion compartments. Moreover, host phagocyte activation and differentiation, apoptosis, and IFN-γ signaling pathways are inhibited by these bacteria. Through reductive evolution, lipopolysaccharide and peptidoglycan that activate the innate immune response, have been eliminated from these gram-negative bacteria at the genomic level. Upon interaction with new host cells, bacterial genes encoding the Type IV secretion apparatus and the two-component regulatory system are up-regulated to sense and adapt to the host environment. Thus dynamic signal transduction events concurrently proceed both in the host cells and in the invading E. chaffeensis and A. phagocytophilum bacteria for successful establishment of intracellular infection. Several bacterial surface-exposed proteins and porins are recently identified. Further functional studies on Ehrlichia and Anaplasma effector or ligand molecules and cognate host cell receptors will undoubtedly advance our understanding of the complex interplay between obligatory intracellular pathogens and their hosts. Such data can be applied towards treatment, diagnosis, and control of ehrlichiosis and anaplasmosis.     

Susceptibility of dogs to infection with Ehrlichia chaffeensis, causative agent of human ehrlichiosis.

Ehrlichia chaffeensis, the newly recognized agent of human ehrlichiosis, is closely related to E canis, the causative agent of canine ehrlichiosis. Eight pups were inoculated IV with E chaffeensis-, or with E canis-infected DH82 cells, or organisms released from these host cells. Two additional pups served as nonexposed controls. Marked thrombocytopenia was observed in the E canis-infected pups, but not in those infected with E chaffeensis. Homologous serologic response was observed in the E chaffeensis-exposed pups by postinoculation day (PID) 14 and in the E canis-exposed pups by PID 21. Ehrlichia chaffeensis and E canis were reisolated from the respective inoculated pups on each of 8 attempts from PID 7 to 26. One E chaffeensis-exposed pup that was challenge exposed with E canis via blood transfusion, developed fever, anorexia, and thrombocytopenia, suggesting lack of cross protection against E canis.     

Detection of Ehrlichia platys in dogs in Australia

OBJECTIVE: To describe the detection of Ehrlichia platys in free-roaming dogs in Central Australia. PROCEDURE: Blood samples were collected from four dogs and examined for bacterial 16S ribosomal DNA using Polymerase Chain Reaction (PCR)-based assays. The three positive samples obtained were then sequenced and identification of the PCR product carried out. As a result of all three samples being identical to or closely related to part of the 16S rRNA gene of E. platys, blood samples were subsequently obtained from a further 24 dogs. These samples were screened using a PCR-assay to determine the presence of Ehrlichia DNA using genus-specific primers. The positive samples obtained from the screening process were then subjected to a further PCR-assay using E. platys specific primers. RESULTS: Of 28 dogs sampled, Ehrlichia DNA was detected in the blood of 13 dogs. Sequencing of the amplicons obtained indicated a high homology with the 16S rRNA gene for E. platys. When the E. platys-specific PCR was performed for 10 of those dogs, the 678 bp product obtained from the PCR amplification confirmed the identification as part of the 16S rRNA gene of E. platys in all 10 dogs. CONCLUSION: This study reports for the first time Ehrlichia carriage by dogs in Australia. It also indicates the usefulness of the PCR technique in rapidly and accurately identifying diseases that are otherwise difficult to detect. By using universal primers directed against bacterial 16S ribosomal DNA and sequencing analysis, the detection of potentially pathogenic Ehrlichia organisms that had not previously been found in Australia has been made possible.     

Detection of specific antibodies anti-Neospora caninum in the fallow deer (Dama dama)

Sera from 335 farmed fallow deer (Dama dama) at the breeding station in Kosewo Górne in the Mazurian Lake District, North-East Poland, were investigated for the presence of antibodies against Neospora caninum. The distribution of age groups was as follow: >4 years - 154 animals; 2 years - 76 animals; 1 year - 105 animals. Ten sera with the optical density exceeding 0.159 absorbance units (i.e., cut-off value) in ELISA test were also analyzed by Western blot. Western blot analysis revealed seroreactivity against immunodominant N. caninum antigens of 37, 25, and 16kDa; however, in some sera additional bands were also visible. This is the first screening studies for antibodies against N. caninum in farmed fallow deer in Poland, in the region where neosporosis was confirmed in cattle and in farmed and free-ranging European red deer (Cervus elaphus).     

Detection of paramyxoviruses in Magellanic penguins (Spheniscus magellanicus) on the Brazilian tropical coast

Aquatic migratory birds are a major vectors by which influenza viruses and paramyxoviruses are spread in nature. Magellanic penguins (Spheniscus magellanicus) are usually present on the southern shores of South America and can swim as far as the southern coast of Brazil in winter. In 2008, however, several Magellanic penguins were observed on the northeastern coast of Brazil. Paramyxoviruses were isolated from Magellanic penguins on the Espírito Santo state coast, approximately 4000 km from their breeding colonies, although influenza viruses were not detected. Among the paramyxoviruses, five Avulavirus isolates belonging to serotype APMV-2 and the serotype APMV-10, which was proposed by Miller et al. (2010), were identified. These results highlight the risks associated with the spread of paramyxoviruses between natural to non-natural habitats by birds exhibiting unusual migration patterns, and they document for the first time the presence of the APMV-2 and APMV-10 serotypes on penguins in Brazil. The local avifauna may become infected with these viruses through close contact between migratory and resident birds. Continued surveillance of virus incidence in these migratory populations of penguins is necessary to detect and prevent the potential risks associated with these unusual migration patterns.     

Detection of an Ehrlichia bovis-like Organism in Cultured Buffalo Monocytes

Monocytes from a buffalo were cultured in RPMI 1640 medium following separation of plasma by the erythrocyte sedimentation technique and subsequent separation of mononuclear cells by density gradient centrifugation. Growth of an organism considered to be Ehrlichia bovis was noticed in the cultured monocytes after 10 days. The inclusions were considered to be those of E. bovis from their morphology, staining characteristics and growth characteristics in culture, and by indirect immunofluorescence examination with an anti-E. canis serum. The utility of peripheral blood monocyte cultures opens the possibility of diagnosing the carrier status of ehrlichiosis in animals.     

Detection of Neorickettsia (Ehrlichia) risticii in tissues of mice experimentally infected with cercariae of trematodes by in situ hybridization

Neorickettsia (Ehrlichia) risticii was demonstrated to occur in cercariae developing in Juga yrekaensis snails by experimental transmission, genetic detection and histopathology. Cercariae were isolated from the digestive glands of snails collected in a fresh stream water area of Siskiyou County, CA, and inoculated into CF1 mice. Mice developed clinical signs, splenomegaly and histopathologic abnormalities. The agent was maintained by serial passages of whole blood in CF1 mice. A 527-bp product of the 16S rRNA gene of N. risticii was serially detected by nested PCR in blood, feces, salivary gland, suprarenal gland, spleen, intestine and bone marrow of inoculated mice. N. risticii DNA was detected by in situ hybridization with DIG-labeled probe in PCR-positive salivary gland, intestine and spleen tissue sections of experimental mice on day 30 after inoculation. Infection in mice was established when cercariae were inoculated by either IP or SC routes but not established following intraoral route. N. risticii was detected by PCR in spleen, intestine and bone marrow even after 73 days post-inoculation whereas blood from the same animals became negative at 58 days. N. risticii was observed by in situ hybridization in salivary gland, spleen and intestine of mice infected by IP or SC inoculation. This ISH protocol should aid investigations on the host range of the Neorickettsiosis and pathogenesis of neorickettiosis in vector, animal or human.     

Genetic characterization of Anaplasma (Ehrlichia) platys in dogs in Spain

This paper reports the first genetic characterization of Anaplasma (Ehrlichia) platys in Spain from a naturally infected dog. The dog presented clinical signs compatible with canine ehrlichiosis. After DNA extraction and PCR amplification, 16S rRNA gene and citrate synthase gene ( gltA) of this agent were amplified. The GenBank accession number for the nucleotide sequence of the 16S rRNA gene of this strain is AY530806. The A. platys strains registered in France and Japan showed the highest similarity to the 16S rRNA gene sequence obtained from the Spanish strain. In the amplification of the gltA gene, a 1443 bp fragment was obtained, and three nucleotide differences were detected in comparison with other strains sequences. These data confirm the presence of A. platys in a dog showing clinical signs compatible with ehrlichiosis in Spain.     

Detection of Ehrlichia risticii using an avidin-biotin-immunoperoxidase staining system

An indirect immunoperoxidase procedure using a specific anti-Ehrlichia risticii monoclonal antibody and an avidin-biotin-peroxidase staining method was used to detect E. risticii antigen in infected P388D1 murine monocytes. Several different methods of cytological fixation were used, including acetone (15 min), 95% ethanol (15 min), Bouin's fixative (5 hr), and 10% buffered neutral formalin (24 hr). The E. risticii organisms were labeled effectively and identified in cells fixed with acetone and ethanol. However, infected P388D1 cells fixed in 10% formalin or Bouin's fixative required enzymatic digestion with 1.0% trypsin for 15 min at 37 C before positive results were evident. This indirect immunoperoxidase avidin-biotin staining procedure proved to be a sensitive assay for the detection of intracellular E. risticii and may be an effective diagnostic procedure for formalin-fixed paraffin-embedded tissue.