Prospective serological and molecular cross-sectional study focusing on Bartonella and other blood-borne organisms in cats from Catalonia (Spain)

Background There is limited clinical or epidemiological knowledge regarding Bartonella infection in cats, and no serological studies have compared the presence of antibodies against different Bartonella species. Moreover, there are limited feline Bartonella studies investigating co-infections with other vector-borne pathogens and the associated risk factors. Therefore, the objective of this study was to investigate Bartonella spp. infections and co-infections with other pathogens in cats from Barcelona (Spain) based on serological and/or molecular techniques and to determine associated risk factors. Methods We studied colony and owned cats (n = 135). Sera were tested for Bartonella henselae-, Bartonella quintana-, and Bartonella koehlerae-specific antibodies using endpoint in-house immunofluorescence antibody assays. Bartonella real-time PCR (qPCR) and conventional PCR (cPCR) were performed. In addition, cPCR followed by DNA sequencing was performed for other pathogenic organisms (Anaplasma, Babesia, Cytauxzoon, Ehrlichia, Hepatozoon, hemotropic Mycoplasma, and Theileria spp.). Results From 135 cats studied, 80.7% were seroreactive against at least one Bartonella species. Bartonella quintana, B. koehlerae, and B. henselae seroreactivity was 67.4, 77.0, and 80.7%, respectively. Substantial to almost perfect serological agreement was found between the three Bartonella species. Colony cats were more likely to be Bartonella spp.-seroreactive than owned cats. Moreover, cats aged ≤ 2 years were more likely to be Bartonella spp.-seroreactive. Bartonella spp. DNA was detected in the blood of 11.9% (n = 16) of cats. Cats were infected with B. henselae (n = 12), B. clarridgeiae (n = 3), and B. koehlerae (n = 1). Mycoplasma spp. DNA was amplified from 14% (n = 19) of cat blood specimens. Cats were infected with Mycoplasma haemofelis (n = 8), Candidatus M. haemominutum (n = 6), Candidatus Mycoplasma turicensis (n = 4), and Mycoplasma wenyonii (n = 1). Anaplasma, Babesia, Cytauxzoon, Ehrlichia spp., Hepatozoon, and Theileria spp. DNA was not amplified from any blood sample. Of the 16 Bartonella spp.-infected cats based on PCR results, six (37%) were co-infected with Mycoplasma spp. Conclusions Bartonella spp. and hemoplasma infections are prevalent in cats from the Barcelona area, whereas infection with Anaplasma spp., Babesia, Cytauxzoon, Ehrlichia spp., Hepatozoon, and Theileria infections were not detected. Co-infection with hemotropic Mycoplasma appears to be common in Bartonella-infected cats. To our knowledge, this study is the first to document M. wenyonii is infection in cats. Graphical Abstract

Role of the CtrA cell cycle regulator in the bloodborne bacterial pathogen, Bartonella quintana.

<p>Bartonella quintana is an important re-emerging human pathogen and the causative agent of trench fever. It utilizes a stealth invasion strategy to infect hosts and is transmitted by lice. Throughout infection it is crucial for the bacteria to maintain a tight regulation of cell division, to prevent immune detection and allow for transmission to new hosts. CtrA is an essential master cell cycle regulatory protein found in the alpha-proteobacteria. It regulates many genes, ensuring the appropriate timing of gene expression and DNA replication. In the model organism Caulobacter crescentus, it regulates 26% of cell cycle-regulated genes. CtrA has been reported to bind two specific DNA motifs in gene promoter regions, TTAAN7TTAAC, and TTAACCAT. Genes regulated by CtrA encode proteins with a wide range of activities, including initiation of DNA replication, cell division, DNA methylation, polar morphogenesis, flagellar biosynthesis, and cell wall metabolism. However, the role of the CtrA homologue in Bartonella spp. has not been investigated. In this project we aimed to make an initial characterisation of the master cell cycle regulator CtrA. This was done by identifying gene regulatory regions containing putative CtrA binding sites and testing for direct interactions via a -galactosidase assay. It was found B. quintana CtrA shared 81 % amino acid identity with its C. crescentus homologue. Within the genome of B. quintana str. Toulouse we discovered 21 genes containing putative CtrA binding sites in their regulatory regions. Of these genes we demonstrated interactions between CtrA and the promoter region of ftsE a cell division gene [1], hemS, and hbpC, two heme regulatory genes. We also found no evidence of CtrA regulating its own expression, which was unexpected because CtrA autoregulation has been demonstrated in C. crescentus.</p>

Role of the CtrA cell cycle regulator in the bloodborne bacterial pathogen, Bartonella quintana.

<p>Bartonella quintana is an important re-emerging human pathogen and the causative agent of trench fever. It utilizes a stealth invasion strategy to infect hosts and is transmitted by lice. Throughout infection it is crucial for the bacteria to maintain a tight regulation of cell division, to prevent immune detection and allow for transmission to new hosts. CtrA is an essential master cell cycle regulatory protein found in the alpha-proteobacteria. It regulates many genes, ensuring the appropriate timing of gene expression and DNA replication. In the model organism Caulobacter crescentus, it regulates 26% of cell cycle-regulated genes. CtrA has been reported to bind two specific DNA motifs in gene promoter regions, TTAAN7TTAAC, and TTAACCAT. Genes regulated by CtrA encode proteins with a wide range of activities, including initiation of DNA replication, cell division, DNA methylation, polar morphogenesis, flagellar biosynthesis, and cell wall metabolism. However, the role of the CtrA homologue in Bartonella spp. has not been investigated. In this project we aimed to make an initial characterisation of the master cell cycle regulator CtrA. This was done by identifying gene regulatory regions containing putative CtrA binding sites and testing for direct interactions via a -galactosidase assay. It was found B. quintana CtrA shared 81 % amino acid identity with its C. crescentus homologue. Within the genome of B. quintana str. Toulouse we discovered 21 genes containing putative CtrA binding sites in their regulatory regions. Of these genes we demonstrated interactions between CtrA and the promoter region of ftsE a cell division gene [1], hemS, and hbpC, two heme regulatory genes. We also found no evidence of CtrA regulating its own expression, which was unexpected because CtrA autoregulation has been demonstrated in C. crescentus.</p>

Investigation of Bartonella quintana Host Immune Evasion

<p>Bartonella is a genus of gram-negative alphaproteobacteria that infect mammals, causing both acute and chronic disease. Bartonella are re-emerging infectious pathogens that cause a variety of clinical syndromes in humans worldwide, including cat scratch disease, trench fever, bacillary angiomatosis, and endocarditis. Bartonella spp. are spread by biting arthropods such as the sand fly, cat flea, and body louse, and have been isolated from almost all mammalian species tested. Bartonella are a re-emerging concern as the number of confirmed Bartonella diagnoses are increasing, primarily in immunocompromised groups, homeless populations, refugee camps, and in veterinary workers. The three primary human disease-causing Bartonella spp. are B. henselae, B. quintana, and B. bacilliformis. Bartonella are known to subvert the host immune system and persist within the host, often causing bacteraemia which is difficult to effectively diagnose and treat. B. quintana infects humans; after introduction to the skin the bacteria implement numerous immune evasion mechanisms to enter the bloodstream and invade erythrocytes. The mechanisms by which B. quintana modulates and evades the immune system during early infection are almost entirely unknown. Following exposure to B. quintana, the bacteria encounter host immune cells but survive, evading these cells and disseminating into the lymphatic system and eventually bloodstream. This thesis project aimed to dissect the interactions between B. quintana and the human innate immune system to better understand the early stages of infection. A gentamicin protection assay was developed to investigate the ability of THP-1 macrophages, representing human macrophages present in the skin, to internalise B. quintana. These data revealed THP-1 cells were unable to effectively internalise B. quintana, although the mechanism responsible was not determined. Subsequent experiments investigated the role of the B. quintana Type IV secreted effector protein BepA1 in the inhibition of internalisation. Bacterial effector proteins often pathogenically modulate host cell signalling to benefit the bacteria, i.e., altering the actin cytoskeleton to inhibit phagocytosis or supressing immune responses. It was hypothesised BepA1 could play a role in inhibiting phagocytosis; therefore, the host cell target of BepA1 was investigated with a yeast two-hybrid system assay. The human protein Myozap was uncovered as a potential protein that interacts with BepA1. Myozap is expressed in cardiac and lung tissue as well as epithelial and endothelial cells, where it modulates Rho-dependent actin signalling, potentially affecting the actin cytoskeleton and the transcription factor MRTF-A, which influences immune reaction through modulation of NF-κB. To investigate the functional effects of BepA1 activity in host cells, HeLa cells were transfected with BepA1; cell migration and cytokine secretion were assessed, revealing a decrease in pro-inflammatory cytokines in BepA1-transfected cells in response to TNF-a stimulation. These data suggest BepA1 may be deployed by B. quintana during infection to suppress the host immune response and avoid clearance from the site of infection. This research addressed a major gap in our understanding of B. quintana infections. Improving our understanding of the interactions between Bartonella and the host immune system is an essential first step in the development of improved diagnostic techniques and treatments. </p>

Investigation of Bartonella quintana Host Immune Evasion

<p>Bartonella is a genus of gram-negative alphaproteobacteria that infect mammals, causing both acute and chronic disease. Bartonella are re-emerging infectious pathogens that cause a variety of clinical syndromes in humans worldwide, including cat scratch disease, trench fever, bacillary angiomatosis, and endocarditis. Bartonella spp. are spread by biting arthropods such as the sand fly, cat flea, and body louse, and have been isolated from almost all mammalian species tested. Bartonella are a re-emerging concern as the number of confirmed Bartonella diagnoses are increasing, primarily in immunocompromised groups, homeless populations, refugee camps, and in veterinary workers. The three primary human disease-causing Bartonella spp. are B. henselae, B. quintana, and B. bacilliformis. Bartonella are known to subvert the host immune system and persist within the host, often causing bacteraemia which is difficult to effectively diagnose and treat. B. quintana infects humans; after introduction to the skin the bacteria implement numerous immune evasion mechanisms to enter the bloodstream and invade erythrocytes. The mechanisms by which B. quintana modulates and evades the immune system during early infection are almost entirely unknown. Following exposure to B. quintana, the bacteria encounter host immune cells but survive, evading these cells and disseminating into the lymphatic system and eventually bloodstream. This thesis project aimed to dissect the interactions between B. quintana and the human innate immune system to better understand the early stages of infection. A gentamicin protection assay was developed to investigate the ability of THP-1 macrophages, representing human macrophages present in the skin, to internalise B. quintana. These data revealed THP-1 cells were unable to effectively internalise B. quintana, although the mechanism responsible was not determined. Subsequent experiments investigated the role of the B. quintana Type IV secreted effector protein BepA1 in the inhibition of internalisation. Bacterial effector proteins often pathogenically modulate host cell signalling to benefit the bacteria, i.e., altering the actin cytoskeleton to inhibit phagocytosis or supressing immune responses. It was hypothesised BepA1 could play a role in inhibiting phagocytosis; therefore, the host cell target of BepA1 was investigated with a yeast two-hybrid system assay. The human protein Myozap was uncovered as a potential protein that interacts with BepA1. Myozap is expressed in cardiac and lung tissue as well as epithelial and endothelial cells, where it modulates Rho-dependent actin signalling, potentially affecting the actin cytoskeleton and the transcription factor MRTF-A, which influences immune reaction through modulation of NF-κB. To investigate the functional effects of BepA1 activity in host cells, HeLa cells were transfected with BepA1; cell migration and cytokine secretion were assessed, revealing a decrease in pro-inflammatory cytokines in BepA1-transfected cells in response to TNF-a stimulation. These data suggest BepA1 may be deployed by B. quintana during infection to suppress the host immune response and avoid clearance from the site of infection. This research addressed a major gap in our understanding of B. quintana infections. Improving our understanding of the interactions between Bartonella and the host immune system is an essential first step in the development of improved diagnostic techniques and treatments. </p>

СЕРОЛОГІЧНА ДІАГНОСТИКА КЛІЩОВИХ ІНФЕКЦІЙ У ХВОРИХ НА ЛОКАЛІЗОВАНУ СКЛЕРОДЕРМІЮ

Мета дослідження – встановити частоту виявлення специфічних антитіл IgM і/чи IgG до Borrelia burgdorferi s. l., B. miyamotoi, Bartonella henselae та B. quintana у сироватці крові хворих на локалізовану склеродермію. Пацієнти і методи. Під спостереженням було 78 хворих із локалізованою склеродермією, віком від 18 до 74 років, які протягом 2015-2021 рр. лікувались амбулаторно і стаціонарно в КУТОР «Тернопільський обласний клінічний шкірно-венерологічний диспансер». Чоловіків було 17 (21,8 %), жінок – 61 (78,2 %). Для виявлення специфічних IgM і/чи IgG до B. burgdorferi s. l. (збудників Лайм-бореліозу) у сироватці крові використали двохетапний метод (ІФА та імуноблот) за допомогою тест-систем компанії Euroimmun AG (Німеччина). Отримані результати аналізували відповідно до рекомендацій виробника. Антитіла IgM і IgG до B. miyamotoi (одного зі збудників кліщових поворотних гарячок) визначали в сироватці крові методом імуноблоту в лабораторії «IGeneX Inc.» (Мілпітас, Каліфорнія, США). Специфічні антитіла IgG до Bartonella henselae та Bartonella quintana (збудників бартонельозу) визначали у сироватці крові пацієнтів за допомогою методу мультиплексної непрямої імунофлуоресценції, застосувавши тест-системи «Mosaic for Bartonella henselae / Bartonella quintana (IgG)», компанії Euroimmun AG (Німеччина), із використанням технології «Біочіп», які містили мічені флуоресцеїном антигени вказаних видів бартонел. Результати досліджень та їх обговорення. Позитивні або проміжні результати пошуку специфічних IgM і/чи IgG до комплексу B. burgdorferi s. l. (хоча б одного класу антитіл) за допомогою ІФА отримано в 29 (37,2 %) із 78 пацієнтів з локалізованою склеродермією. Підтвердити отримані дані методом імуноблоту вдалося у 25 (86,2 %) хворих. Антитіла лише класу IgM одночасно до B. burgdorferi s. l. та B. miyamotoi методом імуноблоту діагностовано у 4 (11,1 %), IgG у – у 5 (13,9 %) із 36 пацієнтів із локалізованою склеродермією. За допомогою методу непрямої імунофлуоресценції специфічні антитіла класу G лише до B. henselae виявлено в сироватці крові 4 (15,3 %) із 26 пацієнтів із локалізованою склеродермією. Серологічну діагностику кліщових поворотних гарячок і бартонельозу (наявних або перенесених у минулому) в пацієнтів із локалізованою склеродермією, мешканців Тернопільської області, проведено вперше. Висновки. Застосування двохетапного методу серологічної діагностики ЛБ (ELISA та імуноблот) дозволило виявити антитіла IgM і/чи IgG до B. burgdorferi s. l. у 32,1 % хворих із локалізованою склеродермією. Встановлено причетність B. miyamotoi до клінічних проявів локалізованої склеродермії у 13,9 % пацієнтів шляхом виявлення у них антитіл IgG одночасно до B. miyamotoi та B. burgdorferi s. l. методом імуноблоту. Специфічні антитіла IgG лише до B. henselae діагностовано в сироватці крові 15,3 % пацієнтів із локалізованою склеродермією.

Real time micro-organisms PCR in 104 patients with polymorphic signs and symptoms that may be related to a tick bite

Introduction Ticks are frequently polyinfected and can thus transmit numerous microorganisms. A large number of bacteria, parasites and viruses are transmitted by tick bites and could cause different signs and symptoms in patients. The main goal of this study was to search for these numerous microorganisms in patients presenting with persistent polymorphic syndrome possibly due to a tick bite (SPPT). Patients and methods The following microorganisms were searched for in saliva, urine, venous and capillary blood by using real time PCR: Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Borrelia hermsii, Bartonella spp., Bartonella quintana, Bartonella henselae, Ehrlichia spp., Anaplasma spp., Rickettsia spp., Coxiella burnetii, Brucella spp., Francisella tularensis, Mycoplasma spp., Chlamydia spp., Babesia spp., Theileria spp. Results 104 patients were included. 48% of the patients were poly-infected, and 25% harboured at least three different microorganisms. Borrelia spp. were not the most frequent bacteria observed, observed far behind Mycoplasma spp., Rickettsia spp. and Ehrlichia spp. which were the most frequent microorganisms observed. Piroplasms were found in a significant number of patients. The most sensitive matrix was saliva, followed by urine, capillary blood and venous blood. Conclusion Our prospective study has shown that patients with SPPT, a syndrome close to fibromyalgia, could harbour several tick borne microorganisms.

Characterisation of the Bartonella quintana YopJ Homologue

<p><b>Bartonella is a genus of re-emerging bacterial pathogens that typically cause asymptomatic, intra-erythrocytic bacteraemia in their reservoir hosts and are highly specialised to evade host immunity. One of the many mechanisms by which Bartonella spp. modulate the host immune system is the type-IV-secretion system, a protein complex that delivers effector proteins directly into host cells to modulate their function.</b></p> <p>Some Bartonella species, including B. quintana, the causative agent of trench fever, possess an effector protein that is homologous to the effector YopJ of Yersinia species. Yersinia YopJ inhibits the MAPK and NF-kB pathways, and YopJ homologues in other species have similar effects, though through different targets. Very little is known about the function of the B. quintana YopJ homologue, but it may play a role in immune modulation by the bacteria.</p> <p>My aim was to characterise the function of the B. quintana YopJ homologue.</p> <p>I had evidence that it inhibits the NF-kB pathway, so I investigated which step of signalling activation is the target of this inhibition. I also sought to determine whether it impacts signalling pathways other than NF-kB, and to identify the specific host protein that it targets.</p> <p>I performed these investigations using ELISA, high-throughput fluorescence microscopy, Western blotting, LC/MS proteomic screening, and a yeast two-hybrid screen.</p> <p>I found that the B. quintana YopJ homologue inhibits the NF-kB pathway at or upstream of IKK activity, may also impact JNK signalling, the cell cycle, and the mTOR complex, and interacts with the host protein DCNL1, a component of neddylation machinery. Additionally, I determined that this interaction does not impact the neddylation of the protein Cullin-1.</p>

Characterisation of the Bartonella quintana YopJ Homologue

<p><b>Bartonella is a genus of re-emerging bacterial pathogens that typically cause asymptomatic, intra-erythrocytic bacteraemia in their reservoir hosts and are highly specialised to evade host immunity. One of the many mechanisms by which Bartonella spp. modulate the host immune system is the type-IV-secretion system, a protein complex that delivers effector proteins directly into host cells to modulate their function.</b></p> <p>Some Bartonella species, including B. quintana, the causative agent of trench fever, possess an effector protein that is homologous to the effector YopJ of Yersinia species. Yersinia YopJ inhibits the MAPK and NF-kB pathways, and YopJ homologues in other species have similar effects, though through different targets. Very little is known about the function of the B. quintana YopJ homologue, but it may play a role in immune modulation by the bacteria.</p> <p>My aim was to characterise the function of the B. quintana YopJ homologue.</p> <p>I had evidence that it inhibits the NF-kB pathway, so I investigated which step of signalling activation is the target of this inhibition. I also sought to determine whether it impacts signalling pathways other than NF-kB, and to identify the specific host protein that it targets.</p> <p>I performed these investigations using ELISA, high-throughput fluorescence microscopy, Western blotting, LC/MS proteomic screening, and a yeast two-hybrid screen.</p> <p>I found that the B. quintana YopJ homologue inhibits the NF-kB pathway at or upstream of IKK activity, may also impact JNK signalling, the cell cycle, and the mTOR complex, and interacts with the host protein DCNL1, a component of neddylation machinery. Additionally, I determined that this interaction does not impact the neddylation of the protein Cullin-1.</p>

Bartonella spp. seroprevalence in tick-exposed Swedish patients with persistent symptoms

Background Bartonella spp. are emerging pathogens transmitted by arthropod vectors, possibly including ticks. We have investigated signs of bartonellosis in Swedish patients with presumed tick-bite exposure and symptom duration of at least 6 months. Methods Serological testing for Bartonella henselae and Bartonella quintana was performed in 224 patients. Symptoms, tick exposure, evidence of co-infection and previous treatments were evaluated. Seropositive patients were compared to a matched group (twofold larger and negative serology) from the same study cohort. Results Seroprevalence was 7% for B. henselae and 1% for B. quintana, with one patient testing positive to both agents. Tick bites were reported by 63% of the patients in the seropositive group and 88% in the seronegative group and presumed tick exposure was more common in the seronegative group. Animal contact was equally common in both groups, along with reported symptoms. The most common symptoms were fatigue, muscular symptoms, arthralgia and cognitive symptoms. Exposure to co-infections was evenly distributed in the seropositive and seronegative groups. Conclusions Antibodies to Bartonella were more common in this cohort of patients than in cohorts of healthy Swedish blood donors in previous studies but lower than those in blood donors from southern Europe. Positive Bartonella serology was not linked to any specific symptom, nor to (suspected) tick-bite exposure. Graphical abstract