Anaplasmataceae: Dichotomous Autophagic Interplay for Infection.

Autophagy is a vital conserved degradative process that maintains cellular homeostasis by recycling or eliminating dysfunctional cellular organelles and proteins. More recently, autophagy has become a well-recognized host defense mechanism against intracellular pathogens through a process known as xenophagy. On the host-microbe battlefield many intracellular bacterial pathogens have developed the ability to subvert xenophagy to establish infection. Obligately intracellular bacterial pathogens of the Anaplasmataceae family, including Ehrlichia chaffeensis, Anaplasma phaogocytophilium and Orientia tsutsugamushi have developed a dichotomous strategy to exploit the host autophagic pathway to obtain nutrients while escaping lysosomal destruction for intracellular survival within the host cell. In this review, the recent findings regarding how these master manipulators engage and inhibit autophagy for infection are explored. Future investigation to understand mechanisms used by Anaplasmataceae to exploit autophagy may advance novel antimicrobial therapies and provide new insights into how intracellular microbes exploit autophagy to survive.

Natural IgM antibodies in the immune defence against neoehrlichiosis.

BACKGROUND: Neoehrlichiosis is an infectious disease caused by the tick-borne bacterium "Candidatus Neoehrlichia mikurensis". Splenectomy and rituximab therapies are risk factors for severe neoehrlichiosis. Our aim was to examine if neoehrlichiosis patients had low levels of natural IgM antibodies and/or were hypogammaglobulinemic, and if such deficiencies were associated with asplenia and vascular complications. METHODS: Neoehrlichiosis patients (n = 9) and control subjects (n = 10) were investigated for serum levels of IgG, IgA, and IgM, and for levels of natural IgM antibodies to pneumococcal polysaccharides (6B, 14), and to the malondialdehyde acetaldehyde epitope of oxidized LDL. The multivariate method Projection to Latent Structures was used to analyze the data. RESULTS: The levels of natural IgM antibodies of various specificities were decreased or not measurable in half of the studied patients with neoehrlichiosis. Only one patient and one control subject were hypogammaglobulinemic. An inverse relationship was noted between the levels of natural IgM antibodies and the development of deep vein thrombosis. Unexpectedly, no association was seen between having or not having a spleen and the levels of natural IgM antibody levels in the circulation. CONCLUSIONS: Neither hypogammaglobulinemia nor lack of natural IgM antibodies alone predisposes for severe neoehrlichiosis. The importance of the spleen in the immune defence against Ca. N. mikurensis probably lies in its capacity to generate or maintain specific antibodies.

Persistent Infections and Immunity in Ruminants to Arthropod-Borne Bacteria in the Family Anaplasmataceae.

Tick-transmitted gram-negative bacteria in the family Anaplasmataceae in the order Rickettsiales cause persistent infection and morbidity and mortality in ruminants. Whereas Anaplasma marginale infection is restricted to ruminants, Anaplasma phagocytophilum is promiscuous and, in addition to causing disease in sheep and cattle, notably causes disease in humans, horses, and dogs. Although the two pathogens invade and replicate in distinct blood cells (erythrocytes and neutrophils, respectively), they have evolved similar mechanisms of antigenic variation in immunodominant major surface protein 2 (MSP2) and MSP2(P44) that result in immune evasion and persistent infection. Furthermore, these bacteria have evolved distinct strategies to cause immune dysfunction, characterized as an antigen-specific CD4 T-cell exhaustion for A. marginale and a generalized immune suppression for A. phagocytophilum, that also facilitate persistence. This indicates highly adapted strategies of Anaplasma spp. to both suppress protective immune responses and evade those that do develop. However, conserved subdominant antigens are potential targets for immunization.

Downregulation of CXCL12 signaling and altered hematopoietic stem and progenitor cell trafficking in a murine model of acute Anaplasma phagocytophilum infection.

Infection with a variety of bacterial pathogens results in hematopoietic stem and progenitor cell (HSPC) mobilization. The mechanism and kinetics of HSPC mobilization during infection are largely unknown. Previously, we found altered HSPC activity in bone marrow, spleen and blood during infection with Anaplasma phagocytophilum, the agent of granulocytic anaplasmosis. We hypothesized that altered CXCL12/CXCR4 signaling, a central pathway for HSPC homing to, and retention within, the bone marrow, plays a role in infection-induced alterations in HSPC number and trafficking. Mice were infected with A. phagocytophilum. Lineage-cKit+ HSPCs were enumerated and proliferation determined. CXCL12 and CXCR4 mRNA were quantified along with CXCL12 protein, and CXCR4 surface, intracellular and total protein expression in HSPCs was determined. Increased bone marrow proliferation of HSPCs began at 2 d post-infection followed by HSPC mobilization and splenic homing. Proliferation of resident HSPCs contributed to increased splenic HSPC numbers. Bone marrow CXCL12 mRNA and protein levels were decreased at 4-8 d post-infection concurrent with HSPC mobilization. CXCR4 protein parameters were decreased in bone marrow HSPCs throughout 2-6 d post-infection. Reduction of CXCL12/CXCR4 signaling simultaneously occurs with HSPC mobilization from bone marrow. Findings suggest that deranged CXCL12/CXCR4 signaling plays a causal role in HSPC mobilization during acute A. phagocytophilum infection.

Two monoclonal antibodies with defined epitopes of P44 major surface proteins neutralize Anaplasma phagocytophilum by distinct mechanisms.

Anaplasma phagocytophilum is an obligatory intracellular bacterium that causes human granulocytic anaplasmosis. The polymorphic 44-kDa major outer membrane proteins of A. phagocytophilum are dominant antigens recognized by patients and infected animals. However, the ability of anti-P44 antibody to neutralize the infection has been unclear due to a mixture of P44 proteins with diverse hypervariable region amino acid sequences expressed by a given bacterial population and lack of epitope-defined antibodies. Monoclonal antibodies (MAbs) 5C11 and 3E65 are directed to different domains of P44 proteins, the N-terminal conserved region and P44-18 central hypervariable region, respectively. Passive immunization with either MAb 5C11 or 3E65 partially protects mice from infection with A. phagocytophilum. In the present study, we demonstrated that the two monoclonal antibodies recognize bacterial surface-exposed epitopes of naturally folded P44 proteins and mapped these epitopes to specific peptide sequences. The two MAbs almost completely blocked the infection of the A. phagocytophilum population that predominantly expressed P44-18 in HL-60 cells by distinct mechanisms: MAb 5C11 blocked the binding, but MAb 3E65 did not block binding or internalization. Instead, MAb 3E65 inhibited internalized A. phagocytophilum to develop into microcolonies called morulae. Some plasma from experimentally infected horses and mice reacted with these two epitopes. Taken together, these data indicate the presence of at least two distinct bacterial surface-exposed neutralization epitopes in P44 proteins. The results indicate that antibodies directed to certain epitopes of P44 proteins have a critical role in inhibiting A. phagocytophilum infection of host cells.

[Prevalence of antibodies against Anaplasma phagocytophilum, Babesia microti i Borrelia burgdorferi in adults in North-Eastern Poland]. / Wystepowanie przeciwcial przeciw Anaplasma phagocytophilum, Babesia microti i Borrelia burgdorferi u doroslych z pólnocno-wschodnich rejonów Polski.

THE AIM: of the study was to evaluate the seroprevalance of antibodies against Anaplasma phagocytophilum and Babesia Microti in healthy north-eastern Poland, adult population. MATERIAL AND METHODS: The study was conducted in a group of 142 healthy adults (mean age 19-22), bitten by ticks within last 2 years. The control group consisted of 50 adults from central Poland (nonendemic area). The antibody levels for A. phagocytophilum (IgG/Ap-Ab) and B. microti (IgM/Bm-Ab) were evaluated in two series of samples from the same persons (interval 5-6 months) by immunoenzymatic tests (Borrelia Biomedica, Austria), immunofluorescence test (Human Granulotic Ehrlichiosis IFA IgG and Babesia microti IFA IgG from MRL Diagnostics). RESULTS: Positive results for A. phagocytophilum were defined as titres > or =1:256 and for B. microti > or =1:64 and B. burgdorferi > or = 11 BBU/ml. Positive results for IgG B. burgdorferi during the first collection were revealed in 16% (n=24/142) of individuals from endemic area and in 4% (n=2/50) of the control group, which was statistically relevant (p<0,05). IgG A. phagocytophilum antibodies were present in 3,5% (n=5/142) of individuals from the endemic area, but for IgG B. microti antibodies (IgG/Bm-Ab) no positive results were found. No IgG antibodies against A. phagocytophilum and B.microti, were found in individuals from non-endemic area. During the second collection, in individuals from the endemic area, the antibodies against B. burgdorferi were found in 9,8% (n=14/142), IgG A. phagocytophilum antibodies (IgG/Ap-Ab) in 4,9% (n=7/142) and against B. microti (IgG/ Bm-Ab) in 1,4% (n=2/142). The antibodies against B. Burgdorferi were found in 2% (n=1/150) of the control group during the second collection, and no IgG against A. phagocytophilum and B. microti were found. CONCLUSION: [corrected] Evaluating the seroprevalance of the studied antibodies in both collections, a conclusion was drawn that there was no significant increase of antibodies levels directly after the highest exposition to tick bites. None of individuals showed 4-fold antibody level increase between the first and second collection. The seroconversion for IgG/Bm-Ab antibodies was present in 1,4% (n=2/142) of individuals, in those 2 cases a 2-fold antibodies level increase was observed. As far as IgG/Ap-Ab antibodies are concerned the seroconversion was observed in 2,1% (n-3/142), but only one case shown a 3-fold antibodies level increase. No seroconversion of B. burgdorferi antibodies were found in the second collection.

Haemobartonellosis in squirrel monkeys (Saimiri sciureus): antagonism between Haemobartonella sp. and experimental Plasmodium falciparum malaria.

A hemotropic parasite of the genus Haemo bartonella (rickettsial parasite of the Family Anaplasmataceae) is responsible for latent asymptomatic infection in colony-born Saimiri monkeys. Indeed, many of these animals develop a patent Haemobartonella infection following splenectomy. Such patent parasitism is characterized by an intense Haemobartonella parasitemia which peaks between days 12 and 14 after removal of the spleen and then decreases to become undetectable between days 25 and 30. During the resolving phase of parasitemia, a moderate anemia associated with monocytosis and erythrophagocytosis is observed. In certain Saimiri monkeys, Haemobartonella parasitemia remains latent following removal of the spleen. This indicates that the spleen plays a role but is not necessary to maintain latent Haemobartonella parasitism. It also suggests the existence of heterogeneity in the host immune reactivity to the parasite. Latent or patent haemobartonellosis might raise a problem when Saimiri monkeys are used as experimental hosts of Plasmodium falciparum asexual blood stages, as already noticed with "rodent malaria." Thus we investigated the relationship between Haemobartonella and P. falci parum in splenectomized monkeys. When animals harboring latent Haemobartonella sp. were infected with P. falciparum, the former remained latent and exerted no influence on the course of the P. falciparum parasitemia. In constrast, when P. falciparum was initiated in animals which were in the process of developing patent haemobarto nellosis, the course of the former was protracted and either the animal resisted longer, or it self-cleared the P. falciparum infection. Conversely, patent haemobartonellosis was delayed when splenectomy was performed at different times after initiation of P. falciparum infection in intact monkeys. Our results do not allow us to draw conclusions as to the mechanism(s) of the antagonism between the two parasites, but they emphasize the need to monitor the presence of Haemobartonella when splenectomized Saimiri monkeys are used as experimentals hosts for P. falciparum parasitism.

Freqüencia de anticorpos anti-Anaplasma marginale em rebanhos leiteiros da Bahia / Frequency of antibodies against Anaplasma marginale in dairy herds of Bahia State, Brazil

Determinou-se a freqüência de bovinos soro-reagentes para Anaplasma marginale em rebanhos leiteiros das microrregiöes de Jequié, Itabuna e Vitória da Conquista, Estado da Bahia, pelas técnicas de imunoadsorçäo enzimática (ELISA), imunofluorescência indireta (IFI) e teste de conglutinaçäo rápida (TCR). Das 324 amostras de soro bovino analisadas, 96,9 por cento, 97,2 por cento e 91,0 por cento foram positivas para anticorpos contra A. marginale, respectivamente pelo ELISA, IFI e TCR. Todas as regiöes caracterizaram-se por estabilidade enzoótica para a anaplasmose. O desempenho dos três testes sorológicos foi bastante similar na detecçäo de anticorpos contra A. marginale

Agglutination of mouse erythrocytes by Eperythrozoon coccoides.

Erythrocytes from blood of mice infected with Eperythrozoon coccoides for 3 or 4 days agglutinated spontaneously. Washed E. coccoides particles agglutinated washed erythrocytes of uninfected mice. E. coccoides-mediated agglutination of normal mouse erythrocytes would be an excellent system for studies of bacterial adhesion.

Immunoconglutinin and antibody against fibrinogen products in hemolytic anemia and nephritis resulting from infection with a Haemobartonella-like agent.

An agent morphologically similar to Haemobartonella muris was isolated from the blood of rats infected with a strain of Trypanosoma lewisi kept at this Department. It caused acute hemolytic anemia, splenomegaly, glomerulonephritis, and death within 5 to 8 days in mature Sprague-Dawley rats. The disease was less severe in weanling rats which usually recovered within 3 to 4 wk. The anemia was accompanied by phagocytosis of erythrocytes by monocytes of the spleen and bone marrow, by high titers of cold-active hemagglutinin, high titers of antibody to the third component of fixed complement (immunoconglutinin), and antibody to fibrinogen/fibrin related products. Filtrates of blood from anemic rats passing a 0.20-micron filter did not produce disease or signs of infections, but filtrate from a 0.45-micron filter was infective. Attempts to grow the agent on rat embryo fibroblast cultures and in embryonated chicken eggs were successful. Tests for bacteria, mycoplasma, and spirochetes gave negative results. Blood of infected rats did not produce signs of infections when inoculated into laboratory mice, and normal rats housed in cages with acutely infected rats did not develop signs of infection or disease. Morphological similarity did not allow differentiation of the agent from H. muris. However, its virulence for mature rats differs markedly from that usually seen in H. muris infection.

Cold-agglutinin hemolytic anemia and Haemobartonella canis infection in a dog.

Cold-agglutinin hemolytic anemia in a dog was manifested by weakness, progressive hemolytic anemia, hematuria, and hemoglobinuria. Corticosteroid therapy failed to alleviate the anemia. The condition became complicated by Haemobartonella canis infection berminally. Prominent postmortem findings included disseminated thrombosis and infarction, glomerulonephritis, and thickened alveolar membranes.

[Latex-Chagestest-reactions of immunsera against Bartonella bacilliformis, Haemobartonella muris and Eperythrozoon coccoides (author's transl)]. / Reaktionen der Immunseren von Bartonella bacilliformis-, Haemobartonella muris- und Eperythrozoon coccoides-Infektionen im Latex-Chagastest.

Sera from 10 patients positive for Carrion's disease were analysed by means of the latex Chagas test. Sera from 40 SPF-rats experimentally infected with Haemobartonella muris and 5 SPF-mice experimentally infected with Eperythrozoon coccoides were similarly tested. Both the human and mice sera were negative whereas positive reaction was observed with rat sera. The nature of this positive reaction has not been clarified.