Exploring the diversity and evolutionary strategies of prophages in Hyphomicrobiales, comparing animal-associated with non-animal-associated bacteria.

The Hyphomicrobiales bacterial order (previously Rhizobiales) exhibits a wide range of lifestyle characteristics, including free-living, plant-association, nitrogen-fixing, and association with animals (Bartonella and Brucella). This study explores the diversity and evolutionary strategies of bacteriophages within the Hyphomicrobiales order, comparing animal-associated (AAB) with non-animal-associated bacteria (NAAB). We curated 560 high-quality complete genomes of 58 genera from this order and used the PHASTER server for prophage annotation and classification. For 19 genera with representative genomes, we curated 96 genomes and used the Defense-Finder server to summarize the type of anti-phage systems (APS) found in this order. We analyzed the genetic repertoire and length distributions of prophages, estimating evolutionary rates and comparing intact, questionable, and incomplete prophages in both groups. Analyses of best-fit parameters and bootstrap sensitivity were used to understand the evolutionary processes driving prophage gene content. A total of 1860 prophages distributed in Hyphomicrobiales were found, 695 in AAB and 1165 in the NAAB genera. The results revealed a similar number of prophages per genome in AAB and NAAB and a similar length distribution, suggesting shared mechanisms of genetic acquisition of prophage genes. Changes in the frequency of specific gene classes were observed between incomplete and intact prophages, indicating preferential loss or enrichment in both groups. The analysis of best-fit parameters and bootstrap sensitivity tests indicated a higher selection coefficient, induction rate, and turnover in NAAB genomes. We found 68 types of APS in Hyphomicrobiales; restriction modification (RM) and abortive infection (Abi) were the most frequent APS found for all Hyphomicrobiales, and within the AAB group. This classification of APS showed that NAAB genomes have a greater diversity of defense systems compared to AAB, which could be related to the higher rates of prophage induction and turnover in the latter group. Our study provides insights into the distributions of both prophages and APS in Hyphomicrobiales genomes, demonstrating that NAAB carry more defense systems against phages, while AAB show increased prophage stability and an increased number of incomplete prophages. These results suggest a greater role for domesticated prophages within animal-associated bacteria in Hyphomicrobiales.

Molecular detection of Bartonella quintana, Acinetobacter baumannii and Acinetobacter haemolyticus in Pediculus humanus lice in Nigeria, West Africa.

The human lice Pediculus humanus is distributed worldwide but, it thrives and flourishes under conflict situations where people are forced to live in crowded unhygienic conditions. Molecular methods were used to identify and screen human lice for the DNA of pathogens of public health importance in an area that has been under insurgency related to religious and political conflicts with tens of thousands of internally displaced people (IDP). DNA of Bartonella quintana, Acinetobacter baumannii and Acinetobacter haemolyticus was detected in 18.3%, 40.0% and 1.7%, respectively, of human lice collected from children in Maiduguri, Nigeria. More body lice than head lice were positive for pathogen's DNA (64.3% vs. 44.4%; χ2 = 1.3, p = 0.33), but the difference was not significant. Two lice samples were found to harbour mixed DNA of B. quintana and A. baumannii. Phylogenetic analysis of the cytochrome b (cytb) gene sequences of the positive lice specimens placed them into clades A and E. This is the first report on the molecular identification of human lice and the detection of the DNA of pathogens of public health importance in lice in Nigeria, West Africa. The findings of this study will assist policy makers and medical practitioners in formulating a holistic healthcare delivery to IDPs.

Phylogenetic and network analysis of Pediculus humanus in Nigeria reveal the presences of clade E body lice and novel haplotypes.

An investigation was conducted for the first time to determine the prevalence and genetic diversity of human lice, for the first time in Nigeria, using conventional PCR and sequencing methods. Three mitochondrial genes, cytochrome oxidase subunit 1 (cox1), cytochrome b (cytb), and 12S rRNA of Nigerian human lice, were amplified, sequenced, and analyzed. Overall, high prevalence (72.5%; 103/142) of lice infestation was recorded among the examined volunteers. Head lice infestation was more common 63 (61.2%) than body lice infestation 34 (33.0%). Co-infestation with both head and body lice was recorded in six humans (5.8%). The Nigerian human lice specimens were placed mostly into clade A with few in clade E, including body lice for the first time. Six, three, and eight haplotypes of Nigerian human lice were obtained for the cytb, cox1, and 12S rRNA genes, respectively. Additionally, one (E51), three (A31, A32, and E5), and six (A20, A21, A23, A24, A30, and E1) novel haplotypes were recorded for cox1, cytb, and 12S rRNA, respectively, from the Nigerian specimens which were corroborated by the ML phylogenetic trees and MJ network analyses. Genetic diversity indices indicate minimal variation in the parameters analyzed among the clades of the three genes. However, a statistically significant Snn test, negative Tajima's D test for clade A (cox1 and 12S rRNA genes), and negative Fu and Li's D test in clade A for cox1 gene indicate a geographical structure and the signature of population expansion of the Nigerian human lice. The findings from this study provide additional data on the human lice structure in Africa.

Bartonella infections are prevalent in rodents despite efficient immune responses.

BACKGROUND: Pathogens face strong selection from host immune responses, yet many host populations support pervasive pathogen populations. We investigated this puzzle in a model system of Bartonella and rodents from Israel's northwestern Negev Desert. We chose to study this system because, in this region, 75-100% of rodents are infected with Bartonella at any given time, despite an efficient immunological response. In this region, Bartonella species circulate in three rodent species, and we tested the hypothesis that at least one of these hosts exhibits a waning immune response to Bartonella, which allows reinfections. METHODS: We inoculated captive animals of all three rodent species with the same Bartonella strain, and we quantified the bacterial dynamics and Bartonella-specific immunoglobulin G antibody kinetics over a period of 139 days after the primary inoculation, and then for 60 days following reinoculation with the same strain. RESULTS: Contrary to our hypothesis, we found a strong, long-lasting immunoglobulin G antibody response, with protective immunological memory in all three rodent species. That response prevented reinfection upon exposure of the rodents to the same Bartonella strain. CONCLUSIONS: This study constitutes an initial step toward understanding how the interplay between traits of Bartonella and their hosts influences the epidemiological dynamics of these pathogens in nature.

Mycoplasma wenyonii and Candidatus Mycoplasma Haemobos in Pastoralists Cattle in Nigeria.

PURPOSE: The extensive migration practiced by pastoralists cattle exposes them to a variety of pathogens and vectors which may sometimes lead to severe disease outcomes. Moreover, the synergistic effect of multiple parasitism on the productivity of livestock has been well recognized. This is particularly true where the livestock production system predisposes the animals to constant and heavy infestation with arthropod vectors. METHODS: The presences, prevalence and risk factors for hemotropic Mycoplasma (hemoplasma) infection in cattle in Nigeria was investigated using a PCR and sequencing approach. DNA, extracted from 566 cattle blood samples, collected from 10 states from the three agro-ecological zones (AEZs) of Nigeria, from April 2021 to March 2022, were screened for the presences of hemotropic Mycoplasma spp. DNA. RESULTS: The DNA of hemoplasmas was detected in 48 out of the 566 (8.5%) samples, 12 (25%) of them were identified as Mycoplasma wenyonii and 19 (38.6%) as 'Candidatus Mycoplasma haemobos'. Coinfection with both species was detected in 17 (35.4%) of the samples. High prevalence and risk of hemoplasmas infection was associated with sex of the cattle (bulls were more affected; p = 0.005) and the packed cell volume (p = 0.009), but not with the age (p = 0.08), breed (p = 0.22), body condition (p = 0.052), source of the samples (p = 0.45) or the AEZs (0.59). This is the first nationwide survey of hemotropic mycoplasmas in cattle in Nigeria using this molecular approach. CONCLUSION: Further studies to determine the veterinary and public health significance of these pathogens, which were previously associated with varying degrees of clinical signs and production losses, are recommended in Nigerian cattle.

Molecular detection of Theileria annulata, Theileria mutans and Theileria velifera but no evidence of Theileria parva infected or vaccinated cattle in Nigeria despite extensive transboundary migrations.

The extensive livestock management system predominant in Nigeria necessitates active disease surveillance for the early detection and prompt control of transboundary animal diseases. Theileriae are obligate intracellular protozoa which infect both wild and domestic bovidae throughout much of the world causing East Coast Fever (Theileria parva), Tropical or Mediterranean theileriosis (Theileria annulata) or benign theileriosis (Theileria mutans; Theileria velifera). This study aimed to detect and characterize Theileria spp. infecting cattle in Nigeria using conventional PCR and sequencing approach. Five hundred and twenty-two DNA samples obtained from different cattle blood samples were subjected to PCR targeting the 18S rRNA gene of piroplasmida and specifically, the p104 kDa and Tp1 genes for the evidence of infection or vaccination respectively, with T. parva. A total of 269 out of 522 (51.5%) of the cattle tested PCR- positive for DNA of piroplasmida. Nucleotide sequence and phylogenetic analyses showed that the cattle were infected with T. annulata, T. mutans and T. velifera. Piroplasmida DNA was associated with sex (ꭓ2 = 7.2; p = 0.007), breed (ꭓ2 = 115; p = 0.000002) of animals and the state where the samples were collected (ꭓ2 = 78.8; p = 0.000002). None of the samples tested positive for T. parva DNA or showed evidence of vaccination (Tp1 gene). This is the first report on the molecular detection and characterization of T. annulata in the blood of cattle from Nigeria. Continuous surveillance of Nigerian cattle for East Coast Fever (ECF) is encouraged considering the recent report of the disease in cattle in the neighboring country, Cameroon, where unregulated transboundary cattle movement into Nigeria has been observed.

The "southeastern Europe" lineage of the brown dog tick Rhipicephalus sanguineus (sensu lato) identified as Rhipicephalus rutilus Koch, 1844: Comparison with holotype and generation of mitogenome reference from Israel.

The brown dog tick Rhipicephalus sanguineus (sensu lato) in the southeastern Mediterranean region and the Middle East is difficult to identify due to the presence of multiple mitochondrial DNA haplogroup lineages. The purpose of this study was to clarify the identity of the "southeastern Europe" lineage of this tick species complex. Our research shows that female ticks of the "southeastern Europe" lineage correspond to the morphology of R. rutilus Koch, 1844 as found in type-material at the Museum für Naturkunde Berlin in Germany. We characterised the complete mitogenomes of R. rutilus, R. turanicus Pomerantsev, 1940 and Rhipicephalus sanguineus (Latreille, 1806) in order to improve our understanding of the phylogenetic relationships among species within the R. sanguineus (sensu lato) complex. The material associated with the morphology of R. rutilus was previously labelled as the "southeastern Europe" lineage and found in Israel and Egypt, including Lower Egypt and the Nile Delta, where the original type-material was collected. Based on the morphology, genetic identity, and geographical distribution of the species, we conclude that the name R. rutilus is correctly linked to the "southeastern Europe" lineage of R. sanguineus (sensu lato).

Genomic Characterization of Three Novel Bartonella Strains in a Rodent and Two Bat Species from Mexico.

Rodents and bats are the most diverse mammal group that host Bartonella species. In the Americas, they were described as harboring Bartonella species; however, they were mostly characterized to the genotypic level. We describe here Bartonella isolates obtained from blood samples of one rodent (Peromyscus yucatanicus from San José Pibtuch, Yucatan) and two bat species (Desmodus rotundus from Progreso, and Pteronotus parnellii from Chamela-Cuitzmala) from Mexico. We sequenced and described the genomic features of three Bartonella strains and performed phylogenomic and pangenome analyses to decipher their phylogenetic relationships. The mouse-associated genome was closely related to Bartonella vinsonii. The two bat-associated genomes clustered into a single distinct clade in between lineages 3 and 4, suggesting to be an ancestor of the rodent-associated Bartonella clade (lineage 4). These three genomes showed <95% OrthoANI values compared to any other Bartonella genome, and therefore should be considered as novel species. In addition, our analyses suggest that the B. vinsonii complex should be revised, and all B. vinsonii subspecies need to be renamed and considered as full species. The phylogenomic clustering of the bat-associated Bartonella strains and their virulence factor profile (lack of the Vbh/TraG conjugation system remains of the T4SS) suggest that it should be considered as a new lineage clade (L5) within the Bartonella genus.

Absence of DNA and anti-leishmanial antibodies in dogs (Canis familiaris) in Plateau state, Nigeria.

Dogs are important sentinels for the surveillance of some zoonotic diseases including human leishmaniasis. To obtain information on the role of dogs in the epidemiology of leishmaniasis in Nigeria, 98 sera and 204 DNA samples obtained from dogs were screened for anti-leishmania antibodies and DNA of Leishmania spp. using the enzyme linked immunosorbent assay (ELISA) and PCR, respectively. Initially, three out of the 98 sera samples had ELISA borderline optical density (OD) values and were retested. Two out of the three samples turned out to be negative while one sample gave yet a borderline OD value on a retest. A real time polymerase chain reaction (RT-PCR) targeting the 120-bp fragment of the minicircle kDNA of Leishmania spp. run on DNA extracted from EDTA preserved blood of the borderline positive serum failed to amplify the 120-bp sequence of Leishmania spp. In the second phase of the study, 204 DNA from dog blood samples were subjected to conventional PCR targeting the 300-350 bp of the internal transcribe spacer region 1 (ITS1) of Leishmania spp. None of the samples could be amplified (n = 204, 0%). Our study suggests that L. infantum is not prevalence in Jos, Plateau State, Nigeria and this should be confirmed using a larger sample of local dogs tested using PCR methods in lymphoid tissue samples.

Genomic structural plasticity of rodent-associated Bartonella in nature.

Rodent-associated Bartonella species have shown a remarkable genetic diversity and pathogenic potential. To further explore the extent of the natural intraspecific genomic variation and its potential role as an evolutionary driver, we focused on a single genetically diverse Bartonella species, Bartonella krasnovii, which circulates among gerbils and their associated fleas. Twenty genomes from 16 different B. krasnovii genotypes were fully characterized through a genome sequencing assay (using short and long read sequencing), pulse field gel electrophoresis (PFGE), and PCR validation. Genomic analyses were performed in comparison to the B. krasnovii strain OE 1-1. While, single nucleotide polymorphism represented only a 0.3% of the genome variation, structural diversity was identified in these genomes, with an average of 51 ± 24 structural variation (SV) events per genome. Interestingly, a large proportion of the SVs (>40%) was associated with prophages. Further analyses revealed that most of the SVs, and prophage insertions were found at the chromosome replication termination site (ter), suggesting this site as a plastic zone of the B. krasnovii chromosome. Accordingly, six genomes were found to be unbalanced, and essential genes near the ter showed a shift between the leading and lagging strands, revealing the SV effect on these genomes. In summary, our findings demonstrate the extensive genomic diversity harbored by wild B. krasnovii strains and suggests that its diversification is initially promoted by structural changes, probably driven by phages. These events may constantly feed the system with novel genotypes that ultimately lead to inter- and intraspecies competition and adaptation.

Nucleotide sequence types (ntSTs) of Anaplasma marginale in cattle in Nigeria based on the major surface protein 5 (msp5) gene.

Bovine anaplasmosis caused by Anaplasma marginale is an important endemic disease that exerts negative impact on livestock production with huge socioeconomic consequences in most developing countries. Genetic studies have reported the existence of diverse ntSTs of A. marginale with varying pathogenicity in different countries. Continuous studies to obtain accurate information on disease etiologies is desirable for the formulation of cost-effective control measures. To this end, 582 blood samples from cattle were collected from 10 out of the 36 States of Nigeria from April 2021 to March 2022 and analyzed based on the major surface protein 5 (msp5) gene to determine the ntSTs of A. marginale in Nigeria. In all, 38 out of the 582 samples (6.5%) from cattle in the different Agro-ecological Zones (AEZs) of Nigeria were positive. The Nigerian A. marginale nucleotide sequences were 96.7 to 100% identical to sequences from other countries and were placed in distinct clusters with other A. marginale sequences deposited in GenBank. Network analysis revealed three ntSTs (#2, #4 & #8) of A. marginale from Nigeria with a nucleotide sequence type diversity (Hd) of 0.8, nucleotide diversity (Pi) of 0.015 and average number of nucleotide differences (k) of 7.09. Two different amino acid substitution sites were found in Nigerian and worldwide sequences at positions 148 and 160. This is the first nationwide report on the ntST diversity and genetic characterization of A. marginale in cattle in Nigeria based on the msp5 gene. Bovine anaplasmosis is widespread in Nigeria and deserves further attention.

A road map for in vivo evolution experiments with blood-borne parasitic microbes.

Laboratory experiments in which blood-borne parasitic microbes evolve in their animal hosts offer an opportunity to study parasite evolution and adaptation in real time and under natural settings. The main challenge of these experiments is to establish a protocol that is both practical over multiple passages and accurately reflects natural transmission scenarios and mechanisms. We provide a guide to the steps that should be considered when designing such a protocol, and we demonstrate its use via a case study. We highlight the importance of choosing suitable ancestral genotypes, treatments, number of replicates per treatment, types of negative controls, dependent variables, covariates, and the timing of checkpoints for the experimental design. We also recommend specific preliminary experiments to determine effective methods for parasite quantification, transmission, and preservation. Although these methodological considerations are technical, they also often have conceptual implications. To this end, we encourage other researchers to design and conduct in vivo evolution experiments with blood-borne parasitic microbes, despite the challenges that the work entails.

Treatment of a cat with presumed Bartonella henselae-associated immune-mediated hemolytic anemia, fever, and lymphadenitis.

A 2.5-year-old castrated male cat presented with fever and marked generalized lymphadenopathy of 4-months duration, despite treatment with amoxicillin-clavulanate/marbofloxacin. Abnormalities were not detected on complete blood count, serum chemistry, and FIV/FeLV test apart from a borderline, non-regenerative anemia. Peripheral lymph node fine needle aspirations revealed a marked increase in the percentage of intermediate- and lymphoblastic-lymphocytes in addition to reactive macrophages. Three weeks after presentation, the cat developed a severe, regenerative, immune-mediated hemolytic anemia (IMHA) which responded to immunosuppressive therapy. Fever and lymphadenopathy persisted. Peripheral lymph nodes tested positive for Bartonella henselae DNA in real-time PCR assay and sequencing. Treatment with pradofloxacin and doxycycline resulted in resolution of clinical signs, and negative PCR tests. Despite its reported low pathogenicity, B. henselae infection should also be considered in cats with protracted unexplained fever, lymphadenitis, and IMHA. Furthermore, a combination of pradofloxacin and doxycycline might be considered in cats with bartonellosis given its apparent clinical efficacy.

Molecular detection and genetic characterization of Anaplasma marginale and Anaplasma platys in cattle in Nigeria.

Bovine anaplasmosis poses serious challenge to profitable livestock production in the tropics. Accurate information on the prevalence, distribution and genetic characteristics of Anaplasma spp. infections of cattle is invaluable for the design of cost-effective control measures. Blood samples from 275 cattle in Nigeria were screened for the DNA of Anaplasma spp. using species-specific primers and nucleotide sequence analysis. The DNA of Anaplasmataceae was detected based on 16S rRNA gene in 135 out of the 275 (49.1%) individuals examined, with 31 (23.0%) and 21(15.6%) being positive for Anaplasma marginale based on msp4 and msp2 genes, respectively. DNA of Anaplasma platys was detected in 62 (45.9%) based on groEL gene and in 27 (20.0%) using the A. platys species-specific primers. Presence of Anaplasma spp. DNA was significantly associated (p = 0.011) with the breed of the animals. Anaplasma nucleotide sequences of one group of the infected samples showed high identities of 99.0 to 100% (16S rRNA gene) and 99.6% (groEL gene) with reference sequences of A. platys, while those of another group matched to A. marginale references (msp2 with 98.9% and msp4 with 99.1%). Furthermore, phylogenetic analysis clustered the nucleotide sequences in this study with A. platys and A. marginale sequences in GenBank, confirming these relationships. For the first time, this study revealed the presence of mixed haplotypes in both A. platys and A. marginale in cattle in Nigeria. More studies are needed to elucidate the epidemiology and veterinary and public health significance of Anaplasma spp. infections in cattle in Nigeria.

Tick-borne pathogens in neotropical animals in Trinidad, West Indies.

BACKGROUND: Ticks are important vectors of many pathogens that have contributed to the morbidity and mortality of humans and domestic animals worldwide. Wildlife species have also been implicated as reservoir hosts of a variety of tick-borne pathogens. The objective of this study was to determine which tick-transmitted pathogens were present in the animals harvested from the forest in Trinidad for human consumption. METHODS: Thin blood smears from 43 neotropical animals were examined microscopically for tick-borne pathogens. Additionally, DNA extraction and PCR amplification of the 16S rRNA gene were used for amplification of Anaplasma and Ehrlichia while the gltA gene was used for Bartonella, and Rickettsia spp. and the 18S rRNA gene for Babesia, Hepatozoon and Theileria species. RESULTS: Pathogen DNA was amplified from four samples (a deer, collared peccary and two agoutis). Sequencing of the amplified products from the deer and collared peccary revealed 99.8% homology to Anaplasma bovis and 98.8% homology to Ehrlichia canis, respectively. Sequences from two agoutis revealed 90.4% homology to Theileria spp. DNA of Hepatozoon spp., Bartonella spp. Babesia spp. and Rickettsia spp. was not detected in any of the screened samples. An incidental finding in this study was the presence of bacteria in the blood of animals. CONCLUSIONS: The results indicate that the DNA of tick-transmitted pathogens is present at a frequency of about 10% in the study population and suggests that neotropical mammals may serve as a source for the potential transmission of tick-borne pathogens to domestic animals and humans. In addition, physicians and hunters should be aware of the symptoms associated with zoonotic tick-borne pathogens so that these infections can be recognised, diagnosed and treated promptly. Bacteria present in carcasses can pose a food safety hazard and hunters should be trained in proper harvesting and handling of carcasses.

Molecular detection and characterization of pathogenic Leptospira species in bats (Chiroptera) roosting in human habitats in Nigeria, West Africa.

Leptospirosis is a neglected zoonosis with a nearly global distribution. In order to determine the role of bats in the epidemiology of leptospirosis in Nigeria, a total of 231 bats belonging to three families, Pteropodidae (n = 117), Molossidae (n = 107) and Nycteridae (n = 17), roosting in human habitats were screened by PCR and sequencing for the detection of pathogenic Leptospira species. DNA extracted from the kidneys of bats were subjected to conventional PCR targeting the rrs1, rrs2, flaB and secY genes for the detection of pathogenic Leptospira spp. Overall, 27 out of the 231 (11.7%) of the samples screened were positive for Leptospira spp. High prevalence (>80%) of Leptospira spp. DNA was detected in Chaerophon and Nycteris bat species captures in an abandoned well located within a human habitation. Sequences generated in this study were highly identical to Leptospira borgpetersenii and Leptospira interrogans and clustered with sequences of pathogenic species in GenBank. The detection of pathogenic Leptospira spp. was significantly associated (p < .001) with the bat species, feeding habit, roosting site and study location. To the best of our knowledge, this is the first molecular detection and characterization of pathogenic Leptospira spp. in bats from Nigeria. Results show that bats in Nigeria are infected with diverse Leptospira genotypes phylogenetically related to known pathogenic, including zoonotic taxa. Together, these findings reinforce bats' roles as potential reservoirs of Leptospira spp. and should be considered as a starting point for future comparative studies to improve our understanding of the epidemiology of this bacterial pathogen in Nigeria.

Hedgehogs and Squirrels as Hosts of Zoonotic Bartonella Species.

Free-living animals frequently play a key role in the circulation of various zoonotic vector-borne pathogens. Bacteria of the genus Bartonella are transmitted by blood-feeding arthropods and infect a large range of mammals. Although only several species have been identified as causative agents of human disease, it has been proposed that any Bartonella species found in animals may be capable of infecting humans. Within a wide-ranging survey in various geographical regions of the Czech Republic, cadavers of accidentally killed synurbic mammalian species, namely Eurasian red squirrel (Sciurus vulgaris), European hedgehog (Erinaceus europaeus) and Northern white-breasted hedgehog (Erinaceus roumanicus), were sampled and tested for Bartonella presence using multiple PCR reaction approach targeting several DNA loci. We demonstrate that cadavers constitute an available and highly useful source of biological material for pathogen screening. High infection rates of Bartonella spp., ranging from 24% to 76%, were confirmed for all three tested mammalian species, and spleen, ear, lung and liver tissues were demonstrated as the most suitable for Bartonella DNA detection. The wide spectrum of Bartonella spp. that were identified includes three species with previously validated zoonotic potential, B. grahamii, B. melophagi and B. washoensis, accompanied by 'Candidatus B. rudakovii' and two putative novel species, Bartonella sp. ERIN and Bartonella sp. SCIER.

Adaptation of gltA and ssrA assays for diversity profiling by Illumina sequencing to identify Bartonella henselae, B. clarridgeiae and B. koehlerae.

Introduction. Bartonellosis is an emerging zoonotic disease caused by bacteria of the genus Bartonella. Mixed Bartonella infections are a well-documented phenomenon in mammals and their ectoparasites. The accurate identification of Bartonella species in single and mixed infections is valuable, as different Bartonella species have varying impacts on infected hosts.Gap Statement. Current diagnostic methods are inadequate at identifying the Bartonella species present in mixed infections.Aim. The aim of this study was to adopt a Next Generation Sequencing (NGS) approach using Illumina sequencing technology to identify Bartonella species and demonstrate that this approach can resolve mixed Bartonella infections.Methodology. We used Illumina PCR amplicon NGS to target the ssrA and gltA genes of Bartonella in fleas collected from cats, dogs and a hedgehog in Israel. We included artificially mixed Bartonella samples to demonstrate the ability for NGS to resolve mixed infections and we compared NGS to traditional Sanger sequencing.Results. In total, we identified 74 Ctenocephalides felis, two Ctenocephalides canis, two Pulex irritans and three Archaeopsylla e. erinacei fleas. Real-time PCR of a subset of 48 fleas revealed that twelve were positive for Bartonella, all of which were cat fleas. Sanger sequencing of the ssrA and gltA genes confirmed the presence of Bartonella henselae, Bartonella clarridgeiae and Bartonella koehlerae. Illumina NGS of ssrA and gltA amplicons further confirmed the Bartonella species identity in all 12 flea samples and unambiguously resolved the artificially mixed Bartonella samples.Conclusion. The adaptation and multiplexing of existing PCR assays for diversity profiling via NGS is a feasible approach that is superior to traditional Sanger sequencing for Bartonella speciation and resolving mixed Bartonella infections. The adaptation of other PCR primers for Illumina NGS will be useful in future studies where mixed bacterial infections may be present.

Adaptive Resistance Mutations at Suprainhibitory Concentrations Independent of SOS Mutagenesis.

Emergence of resistant bacteria during antimicrobial treatment is one of the most critical and universal health threats. It is known that several stress-induced mutagenesis and heteroresistance mechanisms can enhance microbial adaptation to antibiotics. Here, we demonstrate that the pathogen Bartonella can undergo stress-induced mutagenesis despite the fact it lacks error-prone polymerases, the rpoS gene and functional UV-induced mutagenesis. We demonstrate that Bartonella acquire de novo single mutations during rifampicin exposure at suprainhibitory concentrations at a much higher rate than expected from spontaneous fluctuations. This is while exhibiting a minimal heteroresistance capacity. The emerged resistant mutants acquired a single rpoB mutation, whereas no other mutations were found in their whole genome. Interestingly, the emergence of resistance in Bartonella occurred only during gradual exposure to the antibiotic, indicating that Bartonella sense and react to the changing environment. Using a mathematical model, we demonstrated that, to reproduce the experimental results, mutation rates should be transiently increased over 1,000-folds, and a larger population size or greater heteroresistance capacity is required. RNA expression analysis suggests that the increased mutation rate is due to downregulation of key DNA repair genes (mutS, mutY, and recA), associated with DNA breaks caused by massive prophage inductions. These results provide new evidence of the hazard of antibiotic overuse in medicine and agriculture.

Ehrlichia canis morulae in peripheral blood lymphocytes of two naturally-infected puppies in Israel.

Ehrlichia canis is the major causative agent of canine monocytic ehrlichiosis (CME). Its morulae might be detected during the acute disease phase, usually within peripheral blood monocytes, but were uncommonly described within peripheral blood lymphocytes. This report describes two unrelated puppies, naturally infected with E. canis. In both, examination of stained peripheral blood smears revealed one to several cytoplasmic inclusions, characteristic of typical E. canis morulae, exclusively within lymphocytes. Ehrlichia canis infection was confirmed in both cases by blood sample real-time PCR. Both dogs were young and had comorbidities. One dog, based on whole blood PCR, was co-infected with Anaplasma platys and Babesia vogeli. The other had no other concurrent tick-borne infection based on PCR, but had bacterial cholangiohepatitis. These comorbidities, and the dogs' young age possibly contributed to the uncommon presence of E. canis morulae within peripheral blood lymphocytes rather than their typical presence in monocytes.