P26-based serodiagnosis for Bartonella spp. infection in cats

Bartonella henselae P26 has been identified as an immunodominant antigen expressed during feline infection. We used antisera from cats experimentally infected with B. henselae (n = 6), B. clarridgeiae (n = 4), or B. koehlerae (n = 2) and from a sample of naturally infected cats (B. henselae, n = 34; B. clarridgeiae, n = 1) to evaluate recombinant P26 (rP26) as a serodiagnostic antigen. Immunoblots using antisera from cats infected with B. henselae and B. clarridgeiae reacted strongly with rP26, whereas B. koehlerae antisera did not. A capture ELISA was designed to evaluate the kinetics of rP26 IgG in sera from experimentally infected cats. For B. henselae and B. clarridgeiae antisera, the kinetic profiles of reactivity were similar for rP26 capture ELISA and Bartonella spp. indirect fluorescence assay. However, for B. koehlerae antisera, reactivity in rP26 capture ELISA was consistently low. The serodiagnostic potential of rP26 capture ELISA was evaluated using sera from cats with known Bartonella sp. exposure histories. All 24 (100%) uninfected cats were seronegative, and 33 of 35 (94.3%) cats bacteremic for Bartonella spp. were seropositive. We propose that rP26-based serology can serve as a useful adjunct tool for the diagnosis of feline infection with B. henselae and B. clarridgeiae, but it may not be useful for feline infection with B. koehlerae.     

Role of Hippoboscidae flies as potential vectors of Bartonella spp. infecting wild and domestic ruminants

The putative role of biting flies in Bartonella transmission among ruminants was investigated. Amplification of the Bartonella citrate synthase gene from 83 Hippoboscidae was detected in 94% of 48 adult Lipoptena cervi flies, 71% of 17 adult Hippobosca equina flies, 100% of 20 adult Melophagus ovinus flies, and 100% of 10 M. ovinus pupae. Our findings suggest that Hippoboscidae play a role in the transmission of Bartonella among ruminants. The vertical transmission of Bartonella in M. ovinus and the presence of Bartonella DNA in all samples suggest a symbiotic association between Bartonella and M. ovinus.     

Role of Hippoboscidae Flies as Potential Vectors of Bartonella spp. Infecting Wild and Domestic Ruminants

The putative role of biting flies in Bartonella transmission among ruminants was investigated. Amplification of the Bartonella citrate synthase gene from 83 Hippoboscidae was detected in 94% of 48 adult Lipoptena cervi flies, 71% of 17 adult Hippobosca equina flies, 100% of 20 adult Melophagus ovinus flies, and 100% of 10 M. ovinus pupae. Our findings suggest that Hippoboscidae play a role in the transmission of Bartonella among ruminants. The vertical transmission of Bartonella in M. ovinus and the presence of Bartonella DNA in all samples suggest a symbiotic association between Bartonella and M. ovinus.     

Phagocytosis in the spleen of the sunfish Lepomis spp.

A histological investigation of the filtering function of the spleen of the sunfish Lepomis spp. was conducted by light, scanning, and transmission electron microscopy. The parenchyma of the organ is predominantly red pulp, a system of splenic cords and sinuses. The white pulp consists of loose lymphoid tissue which forms a cuff around the pulp arteries. Filtering of particulate matter from the blood occurs in the red pulp by phagocytes of the pulp cords and ellipsoids (periarterial macrophage sheaths). The ellipsoids are pale-staining cuffs of macrophages and reticular cells in a framework of reticular fibres surrounding the arterial capillaries. Destruction of effete blood cells (especially erythrocytes) is confined to the pigment nodules; particulate matter is not taken up by the nodules. These yellow-brown bodies are dispersed throughout the red pulp and are bounded by a reticular capsule. They contain masses of phagocytes and have the appearance of a morula. They are associated with blood vessels and are surrounded by sinusoids. Prussian Blue stain shows the presence of haemosiderin within their phagocytes. The phagocytes of the pigment nodules are filled with inclusions such as residual bodies, siderosomes, and fragments of erythrocytes. The early filtering of particulate matter by the phagocytes of the pulp cords and ellipsoids may allow for a more efficient phagocytosis of erythrocytes by the pigment nodules, followed by storage and reutilization of iron-containing compounds uncontaminated by other phagocytosed material.     

Prevalence and Genetic Diversity of Bartonella spp. in Small Mammals from Southeastern Asia

Among 1,341 blood samples from rodents that were trapped in Southeast Asia between 2008 and 2010, we found a prevalence of Bartonella infection ranging from 9.6 to 11.9%. Bartonella species identified (143 isolates) included B. elizabethae, B. coopersplainsensis, B. phoceensis, B. queenslandensis, B. rattimassiliensis, B. tribocorum, and three new putative Bartonella species.     

Bartonella spp. in Fruit Bats and Blood-Feeding Ectoparasites in Madagascar

We captured, ectoparasite-combed, and blood-sampled cave-roosting Madagascan fruit bats (Eidolon dupreanum) and tree-roosting Madagascan flying foxes (Pteropus rufus) in four single-species roosts within a sympatric geographic foraging range for these species in central Madagascar. We describe infection with novel Bartonella spp. in sampled Eidolon dupreanum and associated bat flies (Cyclopodia dubia), which nest close to or within major known Bartonella lineages; simultaneously, we report the absence of Bartonella spp. in Thaumapsylla sp. fleas collected from these same bats. This represents the first documented finding of Bartonella infection in these species of bat and bat fly, as well as a new geographic record for Thaumapsylla sp. We further relate the absence of both Bartonella spp. and ectoparasites in sympatrically sampled Pteropus rufus, thus suggestive of a potential role for bat flies in Bartonella spp. transmission. These findings shed light on transmission ecology of bat-borne Bartonella spp., recently demonstrated as a potentially zoonotic pathogen.     

Bartonella spp. Bacteremia in Blood Donors from Campinas,Brazil

Bartonella species are blood-borne, re-emerging organisms, capable of causing prolonged infection with diverse disease manifestations, from asymptomatic bacteremia to chronic debilitating disease and death. This pathogen can survive for over a month in stored blood. However, its prevalence among blood donors is unknown, and screening of blood supplies for this pathogen is not routinely performed. We investigated Bartonella spp. prevalence in 500 blood donors from Campinas, Brazil, based on a cross-sectional design. Blood samples were inoculated into an enrichment liquid growth medium and sub-inoculated onto blood agar. Liquid culture samples and Gram-negative isolates were tested using a genus specific ITS PCR with amplicons sequenced for species identification. Bartonella henselae and Bartonella quintana antibodies were assayed by indirect immunofluorescence. B. henselae was isolated from six donors (1.2%). Sixteen donors (3.2%) were Bartonella-PCR positive after culture in liquid or on solid media, with 15 donors infected with B. henselae and one donor infected with Bartonella clarridgeiae. Antibodies against B. henselae or B. quintana were found in 16% and 32% of 500 blood donors, respectively. Serology was not associated with infection, with only three of 16 Bartonella-infected subjects seropositive for B. henselae or B. quintana. Bartonella DNA was present in the bloodstream of approximately one out of 30 donors from a major blood bank in South America. Negative serology does not rule out Bartonella spp. infection in healthy subjects. Using a combination of liquid and solid cultures, PCR, and DNA sequencing, this study documents for the first time that Bartonella spp. bacteremia occurs in asymptomatic blood donors. Our findings support further evaluation of Bartonella spp. transmission which can occur through blood transfusions.     

Persistence of Bartonella spp. stealth pathogens: from subclinical infections to vasoproliferative tumor formation

Bartonella spp. are facultative intracellular bacteria that typically cause a long-lasting intraerythrocytic bacteremia in their mammalian reservoir hosts, thereby favoring transmission by blood-sucking arthropods. In most cases, natural reservoir host infections are subclinical and the relapsing intraerythrocytic bacteremia may last weeks, months, or even years. In this review, we will follow the infection cycle of Bartonella spp. in a reservoir host, which typically starts with an intradermal inoculation of bacteria that are superficially scratched into the skin from arthropod feces and terminates with the pathogen exit by the blood-sucking arthropod. The current knowledge of bacterial countermeasures against mammalian immune response will be presented for each critical step of the pathogenesis. The prevailing models of the still-enigmatic primary niche and the anatomical location where bacteria reside, persist, and are periodically seeded into the bloodstream to cause the typical relapsing Bartonella spp. bacteremia will also be critically discussed. The review will end up with a discussion of the ability of Bartonella spp., namely Bartonella henselae, Bartonella quintana, and Bartonella bacilliformis, to induce tumor-like vascular deformations in humans having compromised immune response such as in patients with AIDS.     

Role of spleen in ANF-induced reduction in plasma volume.

Atrial natriuretic factor (ANF) causes an increase in hematocrit that cannot be accounted for by urinary losses. The mechanism behind this phenomenon was studied in intact and splenectomized rats. Rat ANF 99-126 was infused i.v. for 30 min into conscious rats at rates of 0 (saline control), 0.05, or 0.1 microgram/min. Plasma volume was then determined by dilution of the dye, Evan's Blue. In one group of rats, red cell volume was determined using 51Cr-labelled erythrocytes. ANF infusion was continued uninterrupted throughout the experiments. In the intact rats, ANF (0.10 microgram/min) caused hematocrit to increase from 38.9 +/- 0.5 to 41.2 +/- 0.4% (p < 0.005). Splenectomy so attenuated this response to ANF that it failed to reach significance. Similarly, ANF (0.10 microgram/min) caused plasma volume to fall from 5.1 +/- 0.1 to 4.5 +/- 0.1 mL/100 g body wt. (p < 0.005) in the intact rats, but did not affect plasma volume in the splenectomized rats. As a result, blood volume was significantly reduced by ANF in the intact rats, but remained unchanged in the splenectomized rats. Red cell volume did not change in response to infusion of ANF, nor did ANF affect the rate of clearance of Evan's Blue out of the plasma. It is concluded that the spleen is an important site of movement of protein-poor fluid out of the vasculature, and that this exchange is influenced by ANF.     

Role of the flaR gene in flagellar hook formation in Salmonella spp.

Flagellar filaments were reconstituted by polymerization with exogenously supplied flagellin monomers at the tips of normal hooks on Salmonella cells which were missing the filaments because of mutations in either the flaL or flaU gene or the flagellin genes H1 and H2. Reconstitution did not occur at the tips of polyhooks of the flaR mutant cells. Thus, the absence of flagellar filaments in the flaR mutant cells was probably caused by the inability of the polyhooks to work as polymerization nuclei for flagellin. A Phf+ mutant which produced polyhooks with flagellar filaments was isolated from a flaR polyhook mutant. Genetic analysis of the Phf+ mutant showed that it carried an intracistronic suppressor mutation of the original flaR mutation. This result indicated that the flaR gene regulates hook length and initiates flagellin formation.     

Detection of Rickettsia spp. and Bartonella spp. in Ctenocephalides felis fleas from free‐ranging crab‐eating foxes (Cerdocyon thous)

Fleas are insects with a worldwide distribution that have been implicated in the transmission of several pathogens. The present study aimed to investigate the presence of Rickettsia spp. (Rickettsiales: Rickettsiaceae) and Bartonella spp. (Rhizobiales: Bartonellaceae) in fleas from free‐ranging crab‐eating foxes Cerdocyon thous (Linnaeus, 1766) (Carnivora: Canidae) from Rio Grande do Sul, southern Brazil. Fleas were collected manually from animals and used for the molecular detection of Rickettsia spp. and Bartonella spp. Twenty‐nine C. thous were sampled in six municipalities. Four foxes were parasitized by 10 fleas, all of which were identified as Ctenocephalides felis (Bouché, 1935) (Siphonaptera: Pulicidae). DNA from Rickettsia felis Bouyer et al., 2001 and Rickettsia asembonensis Maina et al., 2016 were found in three and eight fleas, respectively. In four fleas, DNA of Bartonella sp. was identified. Phylogenetic analysis grouped Bartonella sp. together with other genotypes previously reported in C. felis worldwide. The scenario described in the present study highlights a Neotropical canid parasitized by the invasive cosmopolitan cat flea, which in turn, is carrying potentially invasive vector‐borne microorganisms. These findings suggest that C. felis is adapted to wild hosts in wilderness areas in southern Brazil, hypothetically exposing the Neotropical fauna to unknown ecological and health disturbances.     

Diversity of Bartonella and Rickettsia spp. in Bats and Their Blood-Feeding Ectoparasites from South Africa and Swaziland

In addition to several emerging viruses, bats have been reported to host multiple bacteria but their zoonotic threats remain poorly understood, especially in Africa where the diversity of bats is important. Here, we investigated the presence and diversity of Bartonella and Rickettsia spp. in bats and their ectoparasites (Diptera and Siphonaptera) collected across South Africa and Swaziland. We collected 384 blood samples and 14 ectoparasites across 29 different bat species and found positive samples in four insectivorous and two frugivorous bat species, as well as their Nycteribiidae flies. Phylogenetic analyses revealed diverse Bartonella genotypes and one main group of Rickettsia, distinct from those previously reported in bats and their ectoparasites, and for some closely related to human pathogens. Our results suggest a differential pattern of host specificity depending on bat species. Bartonella spp. identified in bat flies and blood were identical supporting that bat flies may serve as vectors. Our results represent the first report of bat-borne Bartonella and Rickettsia spp. in these countries and highlight the potential role of bats as reservoirs of human bacterial pathogens.     

Studies on the maturation of immune responsiveness in the mouse. II. Role of the spleen.

Experiments were designed to investigate the role of the spleen in the development of the murine immune system. By using mice splenectomized within 24 hr of birth, as well as mice with a hereditary, congenital absence of the spleen, the primary immune response to sheep erythrocytes was examined. The immunocompetence of lymph node cells from spleenless or control mice was assessed in vitro, in organ and in cell suspension cultures, and in vivo, by transfer into lethally irradiated syngeneic recipients followed by antigenic stimulation. The immunologic capacities of thymus and bone marrow cells were similarly tested by injection separately or in combination into irradiated syngeneic mice. Lymph node cells from spleenless animals appeared fully competent both in vitro and in transfer experiments. Neither neonatal splenectomy nor congenital absence of the spleen significantly reduced the capacity of bone marrow or thymus cells to participate in the immune response to sheep erythrocytes.     

Kinetics of Encephalitozoon spp. infection of human macrophages

Microsporidia are obligate intracellular, eukaryotic parasites that are known to infect a variety of invertebrate and vertebrate species and have been reported to include a broad range of host specificities for various cell types. Although it is clear that some species of microsporidia have the ability to disseminate, causing multiorgan infections, it is not understood how dissemination occurs. One hypothesis suggests that mononuclear phagocytes engulf the pathogen and migrate to various organs while the parasite persists and proliferates. This implies that microsporidia have developed methods by which to escape intracellular degradation and can, instead, use the host as a source of nourishment and a vehicle for dissemination. In our study, we investigated the infection kinetics of 2 Encephalitozoon spp. known to cause disseminated disease in humans. Using fluorescence and scanning electron microscopy, it was determined that spore adherence to the host was rapid (3-6 hr), as was the uptake and organization of internal parasitophorous vacuoles (24 hr). Furthermore, replication was shown to occur within macrophages at 72 hr, as measured by the bromodeoxyuridine proliferation assay, and the production of mature spores occurred in host cells at 120 hr. Parasitic replication could be reduced by pretreatment of macrophages with interferon-gamma and bacterial lipopolysaccharide.     

Role of spleen in immune response to polyvalent pneumococcal vaccine.

The immune response of lymphocytes to subcutaneously administered pneumococcal vaccine was studied in five patients without spleens and in five healthy subjects. Seven days after immunisation circulating B cells synthesising IgG antipneumococcal capsular polysaccharides (anti-PCP) appeared in both groups. Twenty one days after vaccination this B cell population had disappeared and a B cell subset which secreted IgM and IgG anti-PCP in the presence of pokeweed mitogen was detected in the normal but not in the splenectomised subjects. In the splenectomised group polyclonal IgM synthesis induced by pokeweed mitogen was defective. It was concluded that the early events of the immune response to PCP may be mediated by lymph nodes but that, later, the spleen acquires a central role in producing lymphocyte subsets capable of synthesising specific antibodies and that this might explain the increased sensitivity of splenectomised subjects to pneumococcal infection.