Multiple Ehrlichia chaffeensis genes critical for persistent infection in a vertebrate host are identified as nonessential for its growth in the tick vector; Amblyomma americanum.

Ehrlichia chaffeensis is a tick-transmitted monocytic ehrlichiosis agent primarily causing the disease in people and dogs. We recently described the development and characterization of 55 random mutations in E. chaffeensis, which aided in defining the critical nature of many bacterial genes for its growth in a physiologically relevant canine infection model. In the current study, we tested 45 of the mutants for their infectivity ability to the pathogen's tick vector; Amblyomma americanum. Four mutations resulted in the pathogen's replication deficiency in the tick, similar to the vertebrate host. Mutations causing growth defects in both vertebrate and tick hosts included in genes coding for a predicted alpha/beta hydrolase, a putative dicarboxylate amino acid:cation symporter, a T4SS protein, and predicted membrane-bound proteins. Three mutations caused the bacterial defective growth only in the tick vector, which represented putative membrane proteins. Ten mutations causing no growth defect in the canine host similarly grew well in the tick vector. Mutations in 28 genes/genomic locations causing E. chaffeensis growth attenuation in the canine host were recognized as non-essential for its growth in the tick vector. The tick non-essential genes included genes coding for many metabolic pathway- and outer membrane-associated proteins. This study documents novel vector- and host-specific differences in E. chaffeensis for its functional gene requirements.

Evaluating EcxR for Its Possible Role in Ehrlichia chaffeensis Gene Regulation.

Ehrlichia chaffeensis, a tick-transmitted intraphagosomal bacterium, is the causative agent of human monocytic ehrlichiosis. The pathogen also infects several other vertebrate hosts. E. chaffeensis has a biphasic developmental cycle during its growth in vertebrate monocytes/macrophages and invertebrate tick cells. Host- and vector-specific differences in the gene expression from many genes of E. chaffeensis are well documented. It is unclear how the organism regulates gene expression during its developmental cycle and for its adaptation to vertebrate and tick host cell environments. We previously mapped promoters of several E. chaffeensis genes which are recognized by its only two sigma factors: σ32 and σ70. In the current study, we investigated in assessing five predicted E. chaffeensis transcription regulators; EcxR, CtrA, MerR, HU and Tr1 for their possible roles in regulating the pathogen gene expression. Promoter segments of three genes each transcribed with the RNA polymerase containing σ70 (HU, P28-Omp14 and P28-Omp19) and σ32 (ClpB, DnaK and GroES/L) were evaluated by employing multiple independent molecular methods. We report that EcxR binds to all six promoters tested. Promoter-specific binding of EcxR to several gene promoters results in varying levels of gene expression enhancement. This is the first detailed molecular characterization of transcription regulators where we identified EcxR as a gene regulator having multiple promoter-specific interactions.

Targeted mutagenesis in Anaplasma marginale to define virulence and vaccine development against bovine anaplasmosis.

Tick-borne Anaplasma species are obligate, intracellular, bacterial pathogens that cause important diseases globally in people, agricultural animals, and dogs. Targeted mutagenesis methods are yet to be developed to define genes essential for these pathogens. In addition, vaccines conferring protection against diseases caused by Anaplasma species are not available. Here, we describe a targeted mutagenesis method for deletion of the phage head-to-tail connector protein (phtcp) gene in Anaplasma marginale. The mutant did not cause disease and exhibited attenuated growth in its natural host (cattle). We then assessed its ability to confer protection against wild-type A. marginale infection challenge. Additionally, we compared vaccine protection with the mutant to that of whole cell A. marginale inactivated antigens as a vaccine (WCAV) candidate. Upon infection challenge, non-vaccinated control cattle developed severe disease, with an average 57% drop in packed cell volume (PCV) between days 26-31 post infection, an 11% peak in erythrocytic infection, and apparent anisocytosis. Conversely, following challenge, all animals receiving the live mutant did not develop clinical signs or anemia, or erythrocyte infection. In contrast, the WCAV vaccinees developed similar disease as the non-vaccinees following A. marginale infection, though the peak erythrocyte infection reduced to 6% and the PCV dropped 43%. This is the first study describing targeted mutagenesis and its application in determining in vivo virulence and vaccine development for an Anaplasma species pathogen. This study will pave the way for similar research in related Anaplasma pathogens impacting multiple hosts.

Proteome analysis of Ehrlichia chaffeensis containing phagosome membranes revealed the presence of numerous bacterial and host proteins.

Tick-transmitted Ehrlichia chaffeensis, the causative agent for human monocytic ehrlichiosis, resides and multiplies within a host cell phagosome. Infection progression of E. chaffeensis includes internalization into a host cell by host cell membrane fusion events following engulfment leading to the formation of E. chaffeensis containing vacuole (ECV). Revealing the molecular composition of ECV is important in understanding the host cellular processes, evasion of host defense pathways and in defining host-pathogen interactions. ECVs purified from infected host cells were analyzed to define both host and bacterial proteomes associated with the phagosome membranes. About 160 bacterial proteins and 2,683 host proteins were identified in the ECV membranes. The host proteins included predominantly known phagosome proteins involved in phagocytic trafficking, fusion of vesicles, protein transport, Ras signaling pathway and pathogenic infection. Many highly expressed proteins were similar to the previously documented proteins of phagosome vacuole membranes containing other obligate pathogenic bacteria. The finding of many bacterial membrane proteins is novel; they included multiple outer membrane proteins, such as the p28-Omps, the 120 kDa protein, preprotein translocases, lipoproteins, metal binding proteins, and chaperonins, although the presence of ankyrin repeat proteins, several Type I and IV secretion system proteins is anticipated. This study demonstrates that ECV membrane is extensively modified by the pathogen. This study represents the first and the most comprehensive description of ECV membrane proteome. The identity of many host and Ehrlichia proteins in the ECV membrane will be a valuable to define pathogenic mechanisms critical for the replication of the pathogen within macrophages.

Functional Characterization of Multiple Ehrlichia chaffeensis Sodium (Cation)/Proton Antiporter Genes Involved in the Bacterial pH Homeostasis.

Ehrlichia chaffeensis causes human monocytic ehrlichiosis. Little is known about how this and other related tick-borne rickettsia pathogens maintain pH homeostasis in acidified phagosomes and the extracellular milieu. The membrane-bound sodium (cation)/proton antiporters are found in a wide range of organisms aiding pH homeostasis. We recently reported a mutation in an antiporter gene of E. chaffeensis (ECH_0379) which causes bacterial in vivo attenuation. The E. chaffeensis genome contains 10 protein coding sequences encoding for predicted antiporters. We report here that nine of these genes are transcribed during the bacterial growth in macrophages and tick cells. All E. chaffeensis antiporter genes functionally complemented antiporter deficient Escherichia coli. Antiporter activity for all predicted E. chaffeensis genes was observed at pH 5.5, while gene products of ECH_0179 and ECH_0379 were also active at pH 8.0, and ECH_0179 protein was complemented at pH 7.0. The antiporter activity was independently verified for the ECH_0379 protein by proteoliposome diffusion analysis. This is the first description of antiporters in E. chaffeensis and demonstrates that the pathogen contains multiple antiporters with varying biological functions, which are likely important for the pH homeostasis of the pathogen's replicating and infectious forms.

Experimental Infection of North American Sheep with Ehrlichia ruminantium.

Ehrlichia ruminantium, a tick-borne rickettsial, causes heartwater in ruminants resulting from vascular damage. Severity of heartwater varies greatly in ruminant species and breeds, age of animals and for diverse geographic E. ruminantium strains. E. ruminantium and a tick vector, Amblyomma variegatum, originating from Africa, are well established in certain Caribbean islands two centuries ago. Besides the possibility of introduction of heartwater through African exotic animal importation, presence of the pathogen, and the tick vector in the Caribbean pose a high risk to ruminants in the USA and other western hemisphere countries. Scientific evidence supporting the heartwater threat to nonendemic regions, however, is lacking. We describe the first infection study in sheep reared in the USA with seven E. ruminantium strains. All infected sheep exhibited clinical signs characteristic of subacute to subclinical disease, which included labored breathing, depression, coughing, and nasal discharges. Gross and microscopic lesions consistent with heartwater disease including edema and hemorrhage were observed in several organs. Pathogen-specific IgG antibody response was detected in animals infected with all seven strains, while molecular analysis confirmed the pathogen presence only when infected with in vitro cultures. This is the first infection study demonstrating severe heartwater in sheep reared in North America.

Mutations in Ehrlichia chaffeensis Genes ECH_0660 and ECH_0665 Cause Transcriptional Changes in Response to Zinc or Iron Limitation.

Ehrlichia chaffeensis causes human monocytic ehrlichiosis by replicating within phagosomes of monocytes/macrophages. A function disruption mutation within the pathogen's ECH_0660 gene, which encodes a phage head-to-tail connector protein, resulted in the rapid clearance of the pathogen in vivo, while aiding in induction of sufficient immunity in a host to protect against wild-type infection challenge. In this study, we describe the characterization of a cluster of seven genes spanning from ECH_0659 to ECH_0665, which contained four genes encoding bacterial phage proteins, including the ECH_0660 gene. Assessment of the promoter region upstream of the first gene of the seven genes (ECH_0659) in Escherichia coli demonstrated transcriptional enhancement under zinc and iron starvation conditions. Furthermore, transcription of the seven genes was significantly higher under zinc and iron starvation conditions for E. chaffeensis carrying a mutation in the ECH_0660 gene compared to the wild-type pathogen. In contrast, for the ECH_0665 gene mutant with the function disruption, transcription from the genes was mostly similar to that of the wild type or was moderately downregulated. Recently, we reported that this mutation caused a minimal impact on the pathogen's in vivo growth, as it persisted similarly to the wild type. The current study is the first to describe how zinc and iron contribute to E. chaffeensis biology. Specifically, we demonstrated that the functional disruption in the gene encoding the phage head-to-tail connector protein in E. chaffeensis results in the enhanced transcription of seven genes, including those encoding phage proteins, under zinc and iron limitation. IMPORTANCE Ehrlichia chaffeensis, a tick-transmitted bacterium, causes human monocytic ehrlichiosis by replicating within phagosomes of monocytes/macrophages. A function disruption mutation within the pathogen's gene encoding a phage head-to-tail connector protein resulted in the rapid clearance of the pathogen in vivo, while aiding in induction of sufficient immunity in a host to protect against wild-type infection challenge. In the current study, we investigated if the functional disruption in the phage head-to-tail connector protein gene caused transcriptional changes resulting from metal ion limitations. This is the first study describing how zinc and iron may contribute to E. chaffeensis replication.

Molecular detection and characterization of Anaplasma platys and Ehrlichia canis in dogs from the Caribbean.

Anaplasma platys is a tick-transmitted rickettsial pathogen, which is known to be the etiologic agent for cyclic thrombocytopenia in its primary canine host. Infections with this pathogen are also reported in cats, cattle and people. Similarly, Ehrlichia canis is another tick-borne rickettsial pathogen responsible for canine monocytic ehrlichiosis and is also reported to cause infections in people. We describe infections in dogs with these two pathogens on the Caribbean island of Grenada, West Indies by detection using molecular methods. We utilized a 16S rRNA gene-based PCR assay to detect both Ehrlichia and Anaplasma species by screening 155 canine blood samples from asymptomatic dogs. We found 18.7 % of the dogs to be positive for A. platys and 16.8 % for E. canis. Samples that tested positive for A. platys were further assessed by sequence analysis targeting 16S rRNA, 23S rRNA, citrate synthase (gltA) and heat shock protein (groEL) genes. Phylogenetic analysis revealed high correlation of A. platys 16S rRNA and gltA gene sequences with the geographic origins, while 23S rRNA and groEL gene sequences clustered independent of the geographic origins. This study represents an important step in defining the widespread distribution of active rickettsial infections in Caribbean dogs with no apparent clinical signs, thus posing a high risk for canine health and to a lesser extent to humans, as most dogs in the Caribbean are free-roaming.

Protein and DNA Biosynthesis Demonstrated in Host Cell-Free Phagosomes Containing Anaplasma phagocytophilum or Ehrlichia chaffeensis in Axenic Media.

Rickettsiae belong to the Anaplasmataceae family, which includes mostly tick-transmitted pathogens causing human, canine, and ruminant diseases. Biochemical characterization of the pathogens remains a major challenge because of their obligate parasitism. We investigated the use of an axenic medium for growth of two important pathogens-Anaplasma phagocytophilum and Ehrlichia chaffeensis-in host cell-free phagosomes. We recently reported that the axenic medium promotes protein and DNA biosynthesis in host cell-free replicating form of E. chaffeensis, although the bacterial replication is limited. We now tested the hypothesis that growth on axenic medium can be improved if host cell-free rickettsia-containing phagosomes are used. Purification of phagosomes from A. phagocytophilum- and E. chaffeensis-infected host cells was accomplished by density gradient centrifugation combined with magnet-assisted cell sorting. Protein and DNA synthesis was observed for both organisms in cell-free phagosomes with glucose-6-phosphate and/or ATP. The levels of protein and DNA synthesis were the highest for a medium pH of 7. The data demonstrate bacterial DNA and protein synthesis for the first time in host cell-free phagosomes for two rickettsial pathogens. The host cell support-free axenic growth of obligate pathogenic rickettsiae will be critical in advancing research goals in many important tick-borne diseases impacting human and animal health.

Bovine anaplasmosis herd prevalence and management practices as risk-factors associated with herd disease status.

Bovine anaplasmosis is a hemolytic disease of cattle caused by Anaplasma marginale which can cause anemia, adult mortality, abortion, and performance reduction. The objectives of this study were to estimate herd-level infection prevalence of bovine anaplasmosis in Kansas cow-calf herds and assess management practices associated with herd infection status. Licensed Kansas veterinarians were randomly selected and provided clientele to generate randomly selected participant herds. Blood samples were collected from 10 mature cows during processing of 925 herds between October 1, 2016 and March 1, 2017. A management survey was completed by 780 herd-owners. Sample status was determined by competitive enzyme-linked immunosorbent assay (cELISA); operations indicating vaccination for anaplasmosis were tested with A.marginale-specific polymerase chain reaction (PCR). Survey data underwent logistic regression analysis for calculation of odds ratios and confidence intervals. The herd-level prevalence was 52.5 % of cow-calf herds. Prevalence ranged from 19.1 % of herds in Western Kansas to 87.3 % of herds in Eastern Kansas. Vaccinated herds were more likely (OR = 2.38; CI = 1.16-4.85; p =  0.02) to be positive compared to non-vaccinated herds, and herds that utilized insecticide ear-tags were more likely to be positive (OR = 1.9; CI = 1.42-2.55; p < 0.01) compared to herds which do not. Operations that prescribe-burned 21-50 % and >50 % of their pastures were more likely to be test positive, OR = 5.74 (CI = 3 .14-10.51; p < 0.01) and OR = 4.78 (CI = 2.33-10.17; p < 0.01), respectively, than operations that prescribe-burned <20 % of their pastures. In summary, anaplasmosis is present across Kansas beef herds at varied prevalence levels and selected management practices were found to be associated with herd infection status.

Multiple Ehrlichia chaffeensis Genes Critical for Its Persistent Infection in a Vertebrate Host Are Identified by Random Mutagenesis Coupled with In Vivo Infection Assessment.

Ehrlichia chaffeensis, a tick-transmitted obligate intracellular rickettsial agent, causes human monocytic ehrlichiosis. In recent reports, we described substantial advances in developing random and targeted gene disruption methods to investigate the functions of E. chaffeensis genes. We reported earlier that the Himar1 transposon-based random mutagenesis is a valuable tool in defining E. chaffeensis genes critical for its persistent growth in vivo in reservoir and incidental hosts. The method also aided in extending studies focused on vaccine development and immunity. Here, we describe the generation and mapping of 55 new mutations. To define the critical nature of the bacterial genes, infection experiments were carried out in the canine host with pools of mutant organisms. Infection evaluation in the physiologically relevant host by molecular assays and by xenodiagnoses allowed the identification of many proteins critical for the pathogen's persistent in vivo growth. Genes encoding proteins involved in biotin biosynthesis, protein synthesis and fatty acid biosynthesis, DNA repair, electron transfer, and a component of a multidrug resistance (MDR) efflux pump were concluded to be essential for the pathogen's in vivo growth. Three known immunodominant membrane proteins, i.e., two 28-kDa outer membrane proteins (P28/OMP) and a 120-kDa surface protein, were also recognized as necessary for the pathogen's obligate intracellular life cycle. The discovery of many E. chaffeensis proteins crucial for its continuous in vivo growth will serve as a major resource for investigations aimed at defining pathogenesis and developing novel therapeutics for this and related pathogens of the rickettsial family Anaplasmataceae.

Assessing the current and future potential geographic distribution of the American dog tick, Dermacentor variabilis (Say) (Acari: Ixodidae) in North America.

The American dog tick, Dermacentor variabilis, is a veterinary- and medically- significant tick species that is known to transmit several diseases to animal and human hosts. The spatial distribution of this species in North America is not well understood, however; and knowledge of likely changes to its future geographic distribution owing to ongoing climate change is needed for proper public health planning and messaging. Two recent studies have evaluated these topics for D. variabilis; however, less-rigorous modeling approaches in those studies may have led to erroneous predictions. We evaluated the present and future distribution of this species using a correlative maximum entropy approach, using publicly available occurrence information. Future potential distributions were predicted under two representative concentration pathway (RCP) scenarios; RCP 4.5 for low-emissions and RCP 8.5 for high-emissions. Our results indicated a broader current distribution of this species in all directions relative to its currently known extent, and dramatic potential for westward and northward expansion of suitable areas under both climate change scenarios. Implications for disease ecology and public health are discussed.

AMP-activated protein kinase (AMPK) regulates autophagy, inflammation and immunity and contributes to osteoclast differentiation and functionabs.

Osteoclasts are multinucleated giant cells, responsible for bone resorption. Osteoclast differentiation and function requires a series of cytokines to remove the old bone, which coordinates with the induction of bone remodelling by osteoblast-mediated bone formation. Studies have demonstrated that AMP-activated protein kinase (AMPK) play a negative regulatory role in osteoclast differentiation and function. Research involving AMPK, a nutrient and energy sensor, has primarily focused on osteoclast differentiation and function; thus, its role in autophagy, inflammation and immunity remains poorly understood. Autophagy is a conservative homoeostatic mechanism of eukaryotic cells, and response to osteoclast differentiation and function; however, how it interacts with inflammation remains unclear. Additionally, based on the regulatory function of different AMPK subunits for osteoclast differentiation and function, its activation is regulated by upstream factors to perform bone metabolism. This review summarises the critical role of AMPK-mediated autophagy, inflammation and immunity by upstream and downstream signalling during receptor activator of nuclear factor kappa-B ligand-induced osteoclast differentiation and function. This pathway may provide therapeutic targets for bone-related diseases, as well as function as a biomarker for bone homoeostasis.

Bovine anaplasmosis herd prevalence and management practices as risk-factors associated with herd disease status.

Bovine anaplasmosis is a hemolytic disease of cattle caused by Anaplasma marginale which can cause anemia, adult mortality, abortion, and performance reduction. The objectives of this study were to estimate herd-level infection prevalence of bovine anaplasmosis in Kansas cow-calf herds and assess management practices associated with herd infection status. Licensed Kansas veterinarians were randomly selected and provided clientele to generate randomly selected participant herds. Blood samples were collected from 10 mature cows during processing of 925 herds between October 1, 2016 and March 1, 2017. A management survey was completed by 780 herd-owners. Sample status was determined by competitive enzyme-linked immunosorbent assay (cELISA); operations indicating vaccination for anaplasmosis were tested with A.marginale-specific polymerase chain reaction (PCR). Survey data underwent logistic regression analysis for calculation of odds ratios and confidence intervals. The herd-level prevalence was 52.5 % of cow-calf herds. Prevalence ranged from 19.1 % of herds in Western Kansas to 87.3 % of herds in Eastern Kansas. Vaccinated herds were more likely (OR=2.38; CI=1.16-4.85; p= 0.02) to be positive compared to non-vaccinated herds, and herds that utilized insecticide ear-tags were more likely to be positive (OR = 1.9; CI = 1.42-2.55; p < 0.01) compared to herds which do not. Operations that prescribe-burned 21-50 % and >50 % of their pastures were more likely to be test positive, OR=5.74 (CI=3 .14-10.51; p < 0.01) and OR=4.78 (CI=2.33-10.17; p < 0.01), respectively, than operations that prescribe-burned <20 % of their pastures. In summary, anaplasmosis is present across Kansas beef herds at varied prevalence levels and selected management practices were found to be associated with herd infection status.

Sequence Determinants Spanning -10 Motif and Spacer Region Implicated in Unique Ehrlichia chaffeensis Sigma 32-Dependent Promoter Activity of dnaK Gene.

Ehrlichia chaffeensis is an obligate intracellular tick-borne bacterium that causes human monocytic ehrlichiosis. Studying Ehrlichia gene regulation is challenge, as this and related rickettsiales lack natural plasmids and mutagenesis experiments are of a limited scope. E. chaffeensis contains only two sigma factors, σ32 and σ70. We previously developed Escherichia coli surrogate system to study transcriptional regulation from RNA polymerase (RNAP) containing Ehrlichia σ32 or σ70. We reported that RNAP binding motifs of E. chaffeensis genes recognized by σ32 or σ70 share extensive homology and that transcription may be initiated by either one of the sigma factors, although transcriptional efficiencies differ. In the current study, we investigated mapping the E. chaffeensis dnaK gene promoter using the pathogen σ32 expressed in E. coli lacking its native σ32. The E. coli surrogate system and our previously described in vitro transcription system aided in defining the unique -10 motif and spacer sequence of the dnaK promoter. We also mapped σ32 amino acids/domains engaged in its promoter regulation in E. chaffeensis. The data reported in this study demonstrate that the -10 and -35 motifs and spacer sequence located between the two motifs of dnaK promoter are critical for the RNAP function. Further, we mapped the importance of all six nucleotide positions of the -10 motif and identified critical determinants within it. In addition, we reported that the lack of C-rich sequence upstream to the -10 motif is unique in driving the pathogen-specific transcription by its σ32 from dnaK gene promoter. This is the first study in defining an E. chaffeensis σ32-dependent promoter and it offers insights about how this and other related rickettsial pathogens regulate stress response genes.

Proteome Analysis Revealed Changes in Protein Expression Patterns Caused by Mutations in Ehrlichia chaffeensis.

The tick-borne rickettsial pathogen, Ehrlichia chaffeensis, causes monocytic ehrlichiosis in people and other vertebrate hosts. Mutational analysis in E. chaffeensis genome aids in better understanding of its infection and persistence in host cells and in the development of attenuated vaccines. Our recent RNA deep sequencing study revealed that three genomic mutations caused global changes in the gene expression patterns, which in turn affect the ability of pathogen's survival in a host and the host's ability to induce protection against the pathogen. In this follow-up study, we document the impact of mutations on the pathogen's global protein expression and the influence of protein abundance on a mutant's attenuation and protection of vertebrate host against infection. iTRAQ labeling and mass spectrometry analysis of E. chaffeensis wildtype and mutants identified 564 proteins covering about 63% of the genome. Mutation in ECH_0379 gene encoding for an antiporter protein, causing attenuated growth in vertebrate hosts, led to overexpression of p28 outer membrane proteins, molecular chaperons, and metabolic enzymes, while a mutation downstream to the ECH_0490 gene that caused minimal impact on the pathogen's in vivo growth resulted in major changes in the expression of outer membrane proteins, transcriptional regulators and T4SS proteins. ECH_0660 gene mutation, causing the pathogen's rapid clearance and offering protection against wild type infection challenge in a vertebrate host, had a minimal impact on proteome similar to our prior observations from transcriptome analysis. While the global proteome data revealed fewer translated proteins compared to the transcripts identified from RNA deep sequencing analysis, there is a great deal of correlation noted between the global proteome and transcriptome analysis. Further, global proteome analysis, including the assessment of 2D resolved total and immunoproteomes revealed greater variations in the highly immunogenic p28-Omp proteins.

Rickettsia rickettsii Whole-Cell Antigens Offer Protection against Rocky Mountain Spotted Fever in the Canine Host.

Rocky Mountain spotted fever (RMSF) is a potentially fatal tick-borne disease in people and dogs. RMSF is reported in the United States and several countries in North, Central, and South America. The causative agent of this disease, Rickettsia rickettsii, is transmitted by several species of ticks, including Dermacentor andersoni, Rhipicephalus sanguineus, and Amblyomma americanum RMSF clinical signs generally include fever, headache, nausea, vomiting, muscle pain, lack of appetite, and rash. If untreated, it can quickly progress into a life-threatening illness in people and dogs, with high fatality rates ranging from 30 to 80%. While RMSF has been known for over a century, recent epidemiological data suggest that the numbers of documented cases and the fatality rates remain high in people, particularly during the last two decades in parts of North America. Currently, there are no vaccines available to prevent RMSF in either dogs or people. In this study, we investigated the efficacies of two experimental vaccines, a subunit vaccine containing two recombinant outer membrane proteins as recombinant antigens (RCA) and a whole-cell inactivated antigen vaccine (WCA), in conferring protection against virulent R. rickettsii infection challenge in a newly established canine model for RMSF. Dogs vaccinated with WCA were protected from RMSF, whereas those receiving RCA developed disease similar to that of nonvaccinated R. rickettsii-infected dogs. WCA also reduced the pathogen loads to nearly undetected levels in the blood, lungs, liver, spleen, and brain and induced bacterial antigen-specific immune responses. This study provides the first evidence of the protective ability of WCA against RMSF in dogs.

Antigen-Specific CD4+CD8+ Double-Positive T Cells Are Increased in the Blood and Spleen During Ehrlichia chaffeensis Infection in the Canine Host.

Ehrlichia chaffeensis is an obligate intracellular bacterium belonging to the order, Rickettsiales and is a frequent cause of severe and fatal tick-borne infection in people in North America. The reservoir host for E. chaffeensis is the white-tailed deer, while humans and dogs are regarded as common incidental hosts. In dogs, we and others have shown that E. chaffeensis establishes a chronic infection that persists for several weeks to months, while promoting the development of Th1 and Th17 cellular responses and pathogen-specific humoral immunity. We demonstrate here that vaccination with a live, attenuated clone of E. chaffeensis bearing a targeted mutation in the Ech_0230 gene neither promotes the development of long-lived cellular or humoral immunity, nor confers protection against secondary wild-type E. chaffeensis challenge. In dogs, a population of mature CD4+CD8+ double-positive (DP) T cells exists in the periphery that shares similarities with the DP T cell populations that have been described in humans and swine. Little is known about the function of these cells, particularly in the context of infectious diseases. Here, we demonstrate that canine DP T cells expand significantly in response to E. chaffeensis infection. Using in vitro antigen recall assays, we further demonstrate that canine DP T cells undergo clonal expansion, produce IFNγ and IL-17, and upregulate expression of granzyme B and granulysin. Together, our results demonstrate that DP T cells accumulate in the host during E. chaffeensis infection, and suggest that alternative lymphocyte populations may participate in the immune response to tick-borne infections in the incidental host.

Genetic characterization of extraintestinal Escherichia coli isolates from chicken, cow and swine.

Phenotypic determination of antimicrobial resistance in bacteria is very important for diagnosis and treatment, but sometimes this procedure needs further genetic evaluation. Whole-genome sequencing plays a critical role in deciphering and advancing our understanding of bacterial evolution, transmission, and surveillance of antimicrobial resistance. In this study, whole-genome sequencing was performed on nineteen clinically extraintestinal Escherichia coli isolates from chicken, cows and swine and showing different antimicrobial susceptibility. A total of 44 different genes conferring resistance to 11 classes of antimicrobials were detected in 15 of 19 E. coli isolates (78.9%), and 22 types of plasmids were detected in 15/19 (78.9%) isolates. In addition, whole-genome sequencing of these 19 isolates identified 111 potential virulence factors, and 53 of these VFDB-annotated genes were carried by all these 19 isolates. Twelve different virulence genes were identified while the most frequent ones were gad (glutamate decarboxylase), iss (increased serum survival) and lpfA (long polar fimbriae). All isolates harbored at least one of the virulence genes. The findings from comparative genomic analyses of the 19 diverse E. coli isolates in this study provided insights into molecular basis of the rising multi-drug resistance in E. coli.

Protein and DNA synthesis demonstrated in cell-free Ehrlichia chaffeensis organisms in axenic medium.

Ehrlichia chaffeensis, a tick-transmitted rickettsial bacterium, is the causative agent of human monocytic ehrlichiosis. Biochemical characterization of this and other related Rickettsiales remains a major challenge, as they require a host cell for their replication. We investigated the use of an axenic medium for E. chaffeensis growth, assessed by protein and DNA synthesis, in the absence of a host cell. E. chaffeensis organisms harvested from in vitro cultures grown in a vertebrate cell line were fractionated into infectious dense-core cells (DC) and the non-infectious replicating form, known as reticulate cells (RC) by renografin density gradient centrifugation and incubated in the axenic medium containing amino acids, nucleotides, and different energy sources. Bacterial protein and DNA synthesis were observed in RCs in response to glucose-6-phosphate, although adenosine triphosphate, alpha-ketoglutarate or sodium acetate supported protein synthesis. The biosynthetic activity could not be detected in DCs in the axenic medium. While the data demonstrate de novo protein and DNA synthesis under axenic conditions for E. chaffeensis RCs, additional modifications are required in order to establish conditions that support bacterial replication, and transition to DCs.