Mammalian Reservoir Hosts Research Articles

Tick-Borne Transmission of Two Genetically DistinctAnaplasma marginaleStrains following Superinfection of the Mammalian Reservoir Host

Strain superinfection affects the dynamics of epidemiological spread of pathogens through a host population. Superinfection has recently been shown to occur for two genetically distinct strains of the tick-borne pathogen Anaplasma marginale that encode distinctly different surface protein variants. Superinfected animals could serve as a reservoir for onward transmission of both strains if the tick vector is capable of acquiring and transmitting both strains. Whether competition among strains during development within the tick vector, which requires sequential invasion and replication events, limits colonization and subsequent transmission to a single strain is unknown. We tested this possibility by acquisition feeding Dermacentor andersoni ticks on a reservoir host superinfected with the genetically distinct St. Maries and EMPhi strains. Although the St. Maries strain consistently maintained higher bacteremia levels in the mammalian host and the EMPhi strain had an early advantage in colonization of the tick salivary glands, individual ticks were coinfected, and there was successful transmission of both strains. These results indicate that a genetically distinct A. marginale strain capable of superinfecting the mammalian host can subsequently be cotransmitted and become established within the host population despite the presence of an existing established strain.

Infection-associated type IV secretion systems ofBartonellaand their diverse roles in host cell interaction

Type IV secretion systems (T4SSs) are transporters of Gram-negative bacteria that mediate interbacterial DNA transfer, and translocation of virulence factors into eukaryotic host cells. The α-proteobacterial genus Bartonella comprises arthropod-borne pathogens that colonize endothelial cells and erythrocytes of their mammalian reservoir hosts, thereby causing long-lasting intraerythrocytic infections. The deadly human pathogen Bartonella bacilliformis holds an isolated position in the Bartonella phylogeny as a sole representative of an ancestral lineage. All other species evolved in a separate ‘modern’ lineage by radial speciation and represent highly host-adapted pathogens of limited virulence potential. Unlike B. bacilliformis, the species of the modern lineage encode at least one of the closely related T4SSs, VirB/VirD4 or Vbh. These VirB-like T4SSs represent major host adaptability factors that contributed to the remarkable evolutionary success of the modern lineage. At the molecular level, the VirB/VirD4 T4SS was shown to translocate several effector proteins into endothelial cells that subvert cellular functions critical for establishing chronic infection. A third T4SS, Trw, is present in a sub-branch of the modern lineage. Trw does not translocate any known effectors, but produces multiple variant pilus subunits critically involved in the invasion of erythrocytes. The T4SSs laterally acquired by the bartonellae have thus adopted highly diverse functions during infection, highlighting their versatility as pathogenicity factors.

Recognizing and Reducing the Risks of Chagas Disease (American Trypanosomiasis) in Travelers

Chagas disease, or American trypanosomiasis, is an arthropod‐borne protozoan infectious disease endemic throughout most of the Americas, caused by the trypanosome, Trypanosoma cruzi , and transmitted to humans by reduviid, or kissing, bugs. Reduviid bugs (phylum: Insecta, order: Hemiptera, family: Reduviidae, subfamily: Triatominae) transmit several zoonotic strains of T cruzi among many mammalian reservoir hosts throughout the Americas. Chagas disease is most commonly transmitted to humans via T cruzi –infected reduviid bug defecations near bite wounds or exposed mucosal surfaces. Chagas disease may also be transmitted congenitally, by ingestion of T cruzi –infected reduviids or their feces, by blood product transfusions, and by organ transplants. Indeterminate and chronic infections may be reactivated by immunosuppression, particularly human immunodeficiency virus (HIV) infection or acquired immunodeficiency syndrome (AIDS), and by pregnancy. Characterized by an influenza‐like illness acutely in adults, Chagas disease may result in chronic heart disease or gastrointestinal megasyndromes following a prolonged, indeterminate stage of subclinical infection. In its 2003 World Health Report, the World Health Organization (WHO) noted that Chagas disease caused more deaths from parasitic disorders than from any other parasitic disease in Latin America and that T cruzi was responsible for the third highest number of parasitic infections in the world following malaria and schistosomiasis.1 Chagasic heart disease has become an increasingly common indication for heart transplantation in Latin America and the United States.2,3 Chagas disease has now occurred in three US recipients of heart, kidney, liver, and pancreas transplants from a single Central American immigrant donor and in two US recipients of heart transplants from T cruzi –infected donors (Figure 1).3–5 Chagas disease may pose increasing communicable disease risks to travelers in endemic regions of the Americas, including beachside resort regions and interior rural regions. Figure 1 Trypanosoma cruzi metacyclic trypomastigote on a peripheral blood smear … Corresponding Author: James H. Diaz, MD, MPH&TM, DrPH, Program in Environmental and Occupational Health Sciences, School of Public Health, Louisiana State University Health Sciences Center, 2021 Lakeshore Drive, Suite 200, New Orleans, LA 70122, USA. E‐mail: jdiaz{at}lsuhsc.edu.

Development of a Molecular Tool for the Identification of Leishmania Reservoir Hosts by Blood Meal Analysis in the Insect Vectors

The transmission of parasites of the genus Leishmania involves a large diversity of mammalian reservoir hosts. However, many of these are yet to be identified, mainly in isolated biotopes such as the Amazonian rain forest. Furthermore, the trophic preferences of insect vectors have major epidemiologic implications. In this study, we developed a molecular tool for the identification of blood meals of phlebotomine sand flies. This assay is based on specific amplification and sequencing of the blood meal-derived single copy prepronociceptin (PNOC) gene, which is used as a target in phylogenetic studies of mammals. Sand flies were identified simultaneously with the blood-meal identification, using molecular analysis of a ribosomal locus. After a systematic assessment of the sensitivity and specificity of polymerase chain reaction amplification of the PNOC gene using human fed sand flies, the assay was tested on wild-caught sand flies. This work has important implications for the discovery of new Leishmania reservoir hosts and for a better understanding of complex parasite life cycles.

First report of genetic hybrids between two very divergent Leishmania species: Leishmania infantum and Leishmania major

The genus Leishmania includes many pathogenic species which are genetically very distant. The possibility of genetic exchange between different strains is still an important and debated question. Very few genetic hybrids (i.e., offspring of genetically dissimilar species) have been described in Leishmania. In this study, we report the first example of genetic hybrids occurring between two divergent Leishmania species, Leishmania infantum and Leishmania major. These two species have distinct geographical distributions and are transmitted by different vector species to different mammalian reservoir hosts. These hybrid strains were isolated in Portugal from immunocompromised patients and characterized by molecular and isoenzymatic techniques. These approaches showed that these chimeric strains probably contained the complete genome of both L. major and L. infantum. We believe this is the first report of genetic hybrids between such phylogenetically and epidemiologically distant species of Leishmania. This raises questions about the frequency of such cross-species genetic exchange in natural conditions, modalities of hybrid transmission, their long term maintenance as well as the consequences of these transfers on phenotypes such as drug resistance or pathogenicity.

Borrelia garinii in Seabird Ticks (Ixodes uriae), Atlantic Coast, North America

Borrelia garinii is the most neurotropic of the genospecies of B. burgdorferi sensu lato that cause Lyme disease in Europe, where it is transmitted to avian and mammalian reservoir hosts and to humans by Ixodes ricinus. B. garinii is also maintained in an enzootic cycle in seabirds by I. uriae, a tick found at high latitudes in both the Northern and Southern Hemispheres. To determine whether B. garinii is present in seabird ticks on the Atlantic Coast of North America, we examined 261 I. uriae ticks by polyclonal antiborrelial fluorescent antibody. Ten of 61 ticks from Gull Island, Newfoundland, were positive for borreliae by this screen. Amplicons of DNA obtained by PCR that targeted the B. garinii rrs-rrla intergenic spacer were sequenced and matched to GenBank sequences for B. garinii. The potential for introduction of this agent into the North American Lyme disease enzootic is unknown.

ECOLOGY OF THE LEISHMANIA SPECIES IN THE GUIANAN ECOREGION COMPLEX

In the Guianan ecoregion complex, at least seven Leishmania species belonging to both the L. (Leishmania) Saf' janova, 1982 and L. (Viannia) Lainson and Shaw, 1987 sub-genera are causative agents of American visceral leishmaniasis, mucocutaneous leishmaniasis, and cutaneous leishmaniases, including localized and diffuse forms. They are mostly sylvatic species subjugated to their own specific Lutzomyia sand fly vectors and to particular wild mammalian reservoir hosts of the dense rain forest. Originally implicated strictly in wild zoonoses and developing in forested ecotopes, Leishmania show a particular adaptive plasticity to face environmental modifications following deforestation and anthropization. This review presents the different pathogenic complexes of these parasites, emphasizes their implication in human diseases, and considers the influence of humans on their ecology.

Expression of multiple outer membrane protein sequence variants from a single genomic locus of Anaplasma phagocytophilum.

Anaplasma phagocytophilum is the causative agent of an emerging tick-borne zoonosis in the United States and Europe. The organism causes a febrile illness accompanied by other nonspecific symptoms and can be fatal, especially if treatment is delayed. Persistence of A. phagocytophilum within mammalian reservoir hosts is important for ensuring continued disease transmission. In the related organism Anaplasma marginale, persistence is associated with antigenic variation of the immunoprotective outer membrane protein MSP2. Extensive diversity of MSP2 is achieved by combinatorial gene conversion of a genomic expression site by truncated pseudogenes. The major outer membrane protein of A. phagocytophilum, MSP2(P44), is homologous to MSP2 of A. marginale, has a similar organization of conserved and variable regions, and is also encoded by a multigene family containing some truncated gene copies. This suggests that the two organisms could use similar mechanisms to generate diversity in outer membrane proteins from their small genomes. We define here a genomic expression site for MSP2(P44) in A. phagocytophilum. As in A. marginale, the msp2(p44) gene in this expression site is polymorphic in all populations of organisms we have examined, whether organisms are obtained from in vitro culture in human HL-60 cells, from culture in the tick cell line ISE6, or from infected human blood. Changes in culture conditions were found to favor the growth and predominance of certain msp2(p44) variants. Insertions, deletions, and substitutions in the region of the genomic expression site encoding the central hypervariable region matched sequence polymorphisms in msp2(p44) mRNA. These data suggest that, similarly to A. marginale, A. phagocytophilum uses combinatorial mechanisms to generate a large array of outer membrane protein variants. Such gene polymorphism has profound implications for the design of vaccines, diagnostic tests, and therapy.

Molecular epidemiology of cutaneous leishmaniasis in Venezuela

This paper discusses the utility of a set of primers (3J1, 3J2) designed from a repetitive nuclear deoxyribonucleic acid sequence for the diagnosis of Leishmania braziliensis infection in samples obtained from humans, insect vectors and mammalian reservoir hosts from different endemic areas in Venezuela. A high incidence of Leishmania (Viannia) braziliensis infection was found in the endemic areas studied. The sensitivity and specificity of the primers used were adequate for the identification of the natural vectors and reservoir hosts of L. (V.) braziliensis. The polymerase chain reaction was more sensitive than culture and stained smear examination in the diagnosis of cutaneous leishmaniasis, detecting 80% of cases compared to 42% and 72%, respectively.

Antigenic variation of Anaplasma marginale msp2 occurs by combinatorial gene conversion

The rickettsial pathogen Anaplasma marginale establishes lifelong persistent infection in the mammalian reservoir host, during which time immune escape variants continually arise in part because of variation in the expressed copy of the immunodominant outer membrane protein MSP2. A key question is how the small 1.2 Mb A. marginale genome generates sufficient variants to allow long-term persistence in an immunocompetent reservoir host. The recombination of whole pseudogenes into the single msp2 expression site has been previously identified as one method of generating variants, but is inadequate to generate the number of variants required for persistent infection. In the present study, we demonstrate that recombination of a whole pseudogene is followed by a second level of variation in which small segments of pseudogenes recombine into the expression site by gene conversion. Evidence for four short sequential changes in the hypervariable region of msp2 coupled with the identification of nine pseudogenes from a single strain of A. marginale provides for a combinatorial number of possible expressed MSP2 variants sufficient for lifelong persistence.

Leptospirosis

Leptospirosis is a globally important zoonotic disease that affects humans on all continents, in both urban and rural contexts, and in temperate and tropical climes. Leptospirosis is a disease of the environment; transmission depends on interactions between humans and mammalian reservoir hosts. A variety of infectious diseases that present as undifferentiated febrile syndromes, such as malaria, dengue and influenza, as well as viral hemorrhagic fevers can mimic leptospirosis. The importance of pulmonary hemorrhage as a lethal complication of leptospirosis has become more widely recognized. In contrast to textbook dogma, population-based studies indicate that there is a poor correlation between infecting leptospiral strain and clinical expression of disease. Genetic transformation of a Leptospira sp. has now been reported, which should allow for detailed analysis of a variety of leptospiral genes. Publication of the whole Leptospira genome is eagerly awaited. Following recent reports of a new, highly effective conjugate typhoid vaccine, new efforts to find leptospirosis vaccines should include the manufacture and testing of conjugate leptospiral lipopolysaccharide vaccines. Recent advances, particularly in epidemiology, molecular genetics and pathogenesis, are placing leptospirosis at the cutting edge of biomedical science.

Analysis of Borrelia burgdorferi gene expression during life cycle phases of the tick vector Ixodes scapularis

Borrelia burgdorferi exists in nature via an enzootic cycle whereby the organism must adapt to the diverse environmental conditions provided inside the arthropod transmission vector and the mammalian reservoir hosts. B. burgdorferi genes shown to be regulated by temperature, pH and/or cell density during the organism’s growth in culture medium were assayed for expression during various stages of the tick feeding cycle by reverse transcription-polymerase chain reaction (RT-PCR). ospA, ospC, flaB, erpA/I/N, erpB/J/O, rev and mlpA, were transcriptionally active following the larval and nymphal stages of feeding as determined by qualitative RT-PCR. During tick resting periods between feedings, ospC, mlpA and rev transcription were undetectable, in contrast to ospA, flaB, erpA/I/N and erpB/J/O. bba64, a gene induced by environmental changes in culture and expressed during mammalian infection, was not detectable during any of the tick life cycle phases. Quantitative PCR to determine B. burgdorferi genome equivalents in these tick samples using DNA co-purified with the RNA allowed an estimation of gene expression relative to the numbers of B. burgdorferi present in the ticks. Although the spirochete totals varied widely between individual tick pools of fed, replete nymphs, the relative expression ratios between individual target genes following a nymphal feed were comparable. Similarly, borrelial gene transcription from the larval feeding and the nymphal feeding were observed and compared. These findings analogize B. burgdorferi gene expression observed by environmental stimuli in vitro with the transcriptional activity occurring during the organism’s infectious cycle within the tick.

Cutting edge: antibody-mediated cessation of hemotropic infection by the intraerythrocytic mouse pathogen Bartonella grahamii.

The genus Bartonella includes important human-specific and zoonotic pathogens which cause intraerythrocytic bacteremia in their mammalian reservoir host(s). It is accepted that cellular immunity plays a decisive role in the host's defense against most intracellular bacteria. Bartonella sp. infection in the immunocompetent host typically leads to immunity against homologous challenge. The basis of this immunity, be it cellular or humoral, is unclear. In this study, the course of Bartonella grahamii bacteremia in immunocompetent and immunocompromised mice was compared. In immunocompetent hosts, the bacteremia is transient and induces a strong humoral immune response. In contrast, bacteremia persists in immunocompromised B and T cell-deficient mice. Immune serum transfer beginning with day 6 postinfection to B cell-deficient mice unable to produce Igs converted the persistent bacteremia to a transient course indistinguishable from that of immunocompetent animals. These data demonstrate an essential role for specific Abs in abrogating the intraerythrocytic bacteremia of B. grahamii in mice.

Bacterial persistence within erythrocytes: A unique pathogenic strategy of Bartonella spp.

Abstract The genus Bartonella comprises human-specific and zoonotic pathogens responsible for a wide range of clinical manifestations, including Carrion's disease, trench fever, cat scratch disease, bacillary angiomatosis and peliosis, endocarditis and bacteremia. These arthropod-borne pathogens typically parasitise erythrocytes in their mammalian reservoir host(s), resulting in a long-lasting haemotropic infection. We have studied the process of Bartonella erythrocyte parasitism by tracking green fluorescent protein-expressing bacteria in the blood of experimentally infected animals. Following intravenous infection, bacteria colonise a yet enigmatic primary niche, from where they are seeded into the blood stream in regular intervals of ∼five days. Bacteria invade mature erythrocytes, replicate temporarily and persist in this unique intracellular niche for the remaining life span of the infected erythrocytes. A triggered antibody response typically results in an abrogation of bacteremia within 3 months of infection, likely by blocking new waves of bacterial invasion into erythrocytes. The recent establishment of genetic tools for Bartonella spp. permitted us to identify several putative pathogenicity determinants. Application of differential fluorescence induction technology resulted in the isolation of bacterial genes differentially expressed during infection in vitro and in vivo, including an unknown family of autotransporter proteins as well as a novel type IV secretion system homologous to the conjugation system of E. coli plasmid R388. Mutational analysis of a previously described type IV secretion system displaying homology to the virB locus of Agrobacterium tumefaciens provided the first example of an essential pathogenicity locus in Bartonella. Though required for establishing haemotropic infection, it remains to be demonstrated if this type IV secretion system is necessary for colonisation of the primary niche or for the subsequent colonisation of erythrocytes.

Antigenic variation of Anaplasma marginale by expression of MSP2 mosaics.

Anaplasma marginale is a tick-borne pathogen, one of several closely related ehrlichial organisms that cause disease in animals and humans. These Ehrlichia species have complex life cycles that require, in addition to replication and development within the tick vector, evasion of the immune system in order to persist in the mammalian reservoir host. This complexity requires efficient use of the small ehrlichial genome. A. marginale and related ehrlichiae express immunoprotective, variable outer membrane proteins that have similar structures and are encoded by polymorphic multigene families. We show here that the major outer membrane protein of A. marginale, MSP2, is encoded on a polycistronic mRNA. The genomic expression site for this mRNA is polymorphic and encodes numerous amino acid sequence variants in bloodstream populations of A. marginale. A potential mechanism for persistence is segmental gene conversion of the expression site to link hypervariable msp2 sequences to the promoter and polycistron.

Prevalence of Ehrlichia chaffeensis (Rickettsiales: Rickettsiaceae) in Amblyomma americanum (Acari: Ixodidae) from the Georgia coast and Barrier Islands.

Human monocytic ehrlichiosis is an emerging zoonosis caused by infection with Ehrlichia chaffeensis Anderson, Dawson, Jones & Wilson, which is transmitted to mammals by ixodid ticks. Prevalence of infected ticks and distribution of infection foci indicate relative risk of human exposure to ehrlichiosis and may be influenced by factors such as geographic isolation, human disturbance, and the availability of suitable mammalian reservoir hosts. To test, this, individual and pooled lone star ticks, Amblyomma americanum (L.), were collected from three populations from the Georgia coast (1 mainland site and two barrier islands) and screened for E. chaffeensis. A species-specific, nested polymerase chain reaction (PCR) assay was used to amplify a 572 bp fragment of the E. chaffeensis 16S rRNA gene from infected ticks. PCR product specificity was confirmed by nucleotide sequencing. Our results showed the prevalence of infected ticks to be 0.0% (n = 151), 0.9% (n = 111), and 9.3% (n = 129) for Sapelo Island, St. Catherine's Island, and Fort McAllister, respectively.

Comparative seroreactivity to Bartonella henselae and Bartonella quintana among cats from Israel and North Carolina

Bartonella henselae, the predominant cause of cat scratch disease, and Bartonella quintana, the cause of trench fever, are closely related Bartonella species that induce cross-reactivity when cat or human sera are tested using an indirect immunofluorescence antibody (IFA) test. Cats are the natural reservoir for B. henselae, whereas a mammalian reservoir host for B. quintana has not been identified. Serum samples from 114 cats from Israel and 114 cats from North Carolina were tested by IFA for seroreactivity to B. henselae and B. quintana antigens. Similar numbers of cats from Israel [45 (39.5%)] and from North Carolina [46 (40.4%)] were seroreactive to both antigens, however, as compared to cats from North Carolina [8 (7%)], a significantly ( P = 0.001) larger number of cats from Israel were seroreactive to B. quintana antigen only [23 (20.2%)]. In addition, mean antibody titers were lower to B. henselae than to B. quintana ( P = 0.0001) in the cats from Israel, whereas similar mean titers to both antigens were identified in cats from North Carolina. Absorption of serum using whole B. henselae organisms resulted in a significantly greater ( P = 0.0001) decreacse in antibody titer to B. henselae between absorbed and non-absorbed sera, as compared to the decrease in antibody titer following absorption with whole B. quintana organisms. There was a similar decrease in antibody titer in sera from cats experimentally infected with B. henselae and in cats naturally exposed to Bartonella species from Israel and North Carolina. Our results indicate that absorption of serum will, in most instances, distinguish species-specific reactivity by IFA to B. henselae from cross-reactivity to B. quintana in cats experimentally infected with B. henselae. The data support the conclusion that B. henselae is the principal Bartonella species responsible for seroreactivity against B. henselae and B. quintana in naturally exposed cats from Israel or North Carolina. It also suggests that in Israel, cats are exposed to one or more antigenically different Bartonella species, sub-species or strains, that seroreact by IFA more intensely with B. quintana antigen.

Inter-relation of sylvatic and domestic transmission of Trypanosoma cruzi in areas with and without domestic vectorial transmission in Minas Gerais, Brazil.

During the period 1980-1986, we captured triatomine bugs and mammalian reservoir hosts from sylvatic and domestic situations in different municipalities of the State of Minas Gerais. Trypanosoma cruzi was isolated from captured bugs, mammals and patients. After cultivation in LIT medium, the electrophoretic enzyme profiles were determined. We obtained a total of 32 parasite isolates from regions with active domestic transmission, and 24 isolates from areas under control. For the first areas the results suggest introduction of T. cruzi from sylvatic habitats, through incursion of infected opossums and/or sylvatic T. sordida, which appears to have given rise to at least one acute human infection. Of particular interest is the finding of sylvatic opossums and a T. sordida nymph infected with ZB, that could indicate return of parasites from chronic human infections to sylvatic transmission cycles. For the areas under control we also interpret the results as interaction between sylvatic and domestic cycles of transmission, here through the invasion of houses by bugs carrying the Z1 zymodeme from the sylvatic environment. The Multivariate Correspondence Analysis gives a spatial description between the different parasite isolates and confirms the existence of a bridge in the opposite direction in the region with active vectorial transmission including the exporting of Z2 through the peridomestic environment into the sylvatic cycle. For the other areas this bridge corresponds especially to Panstrongylus megistus, importing Z1 into the domestic environment.

Apparent Incompetence of Dermacentor variabilis (Acari: Ixodidae) and Fleas (Insecta: Siphonaptera) as Vectors of Borrelia burgdorferi in an Ixodes dammini Endemic Area of Ontario, Canada

From April to October 1990, white-footed mice, Peromyscus leucopus (Rafinesque), were examined for ectoparasites on Long Point, Ontario, the only endemic area for Ixodes dammini Clifford, Spielman, Piesman & Corwin and Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner known in Canada. Larval and nymphal I. dammini and Dermacentor variabilis (Packard), and adult fleas Orchopeas leucopus (Baker), Epitedia wenmanni (Rothschild), and Ctenophthalamus pseudagrytes Baker were common on trapped mice. Questing ticks were collected by dragging, near the sites of mouse trapping, from April to November 1990. Indirect immunofluorescent assay established that 58.3% of adult, 17.3% of nymphal, and 0.15% of larval I. dammini questing at Long Point were infected with B. burgdorferi, indicating that infected mammalian reservoir hosts are common. None of 593 adult, 2 nymphal, and 4 larval D. variabilis collected while questing were infected; and only 1 of 322 fleas (O. leucopus) removed from white-footed mice was infected. The fact that no unfed adult D. variabilis and only one flea were infected, in a situation where the probability of exposure of hematophagous ectoparasites is moderately high, suggests that this species of tick and the fleas examined are poor vectors for the Lyme disease spirochete.