Enterobacterales high-risk clones and plasmids spreading blaESBL/AmpC and blaOXA-48 genes within and between hospitalized dogs and their environment.

BACKGROUND: Compared with healthcare settings, the role of veterinary hospitals in the spread of extended-spectrum cephalosporin- and carbapenem-resistant (ESC-R/CP-R) bacteria has been overlooked. OBJECTIVES: To investigate using genome-based approaches the dynamics of ESC-R and CP-R Enterobacterales among 125 dogs admitted to the same veterinary hospital over a 4 month period. METHODS: Dogs (n = 125) were sampled within 48 h of admission and at discharge. ESC-R/CP-R were phenotypically characterized and whole-genome sequenced using short- and long-read technologies. Phylogenetic analyses were performed using appropriate pipelines. RESULTS: ESC-R/CP-R prevalence in dogs was 4.8% (6/125) upon admission and reached 24.8% (31/125) at discharge, reflecting multiple acquisitions of ESBL/AmpC and OXA-48-positive Enterobacterales during hospitalization. Indistinguishable or closely related isolates were found within dogs, shared between dogs, and shared between dogs and their environment, suggesting numerous clonal and plasmid spreads. Even though carbapenems are not licensed for use in companion animals, a wide distribution of the blaOXA-48/IncL plasmid was evidenced across different bacterial species and dogs. CONCLUSIONS: This study highlights nosocomial acquisitions of ESBL/AmpC and carbapenemase-producing Enterobacterales by companion animals and the risk of further transmission within the community in a One Health perspective. Reinforced infection prevention and control measures and screening procedures are urgently needed in small animal veterinary settings where advanced therapeutics and intensive care is provided.

Bovine Organospecific Microvascular Endothelial Cell Lines as New and Relevant In Vitro Models to Study Viral Infections.

Microvascular endothelial cells constitute potential targets for exogenous microorganisms, in particular for vector-borne pathogens. Their phenotypic and functional variations according to the organs they are coming from provide an explanation of the organ selectivity expressed in vivo by pathogens. In order to make available relevant tools for in vitro studies of infection mechanisms, our aim was to immortalize bovine organospecific endothelial cells but also to assess their permissivity to viral infection. Using transfection with SV40 large T antigen, six bovine microvascular endothelial cell lines from various organs and one macrovascular cell line from an umbilical cord were established. They display their own panel of endothelial progenitor/mature markers, as assessed by flow cytometry and RT-qPCR, as well as the typical angiogenesis capacity. Using both Bluetongue and foot-and-mouth disease viruses, we demonstrate that some cell lines are preferentially infected. In addition, they can be transfected and are able to express viral proteins such as BTV8-NS3. Such microvascular endothelial cell lines bring innovative tools for in vitro studies of infection by viruses or bacteria, allowing for the study of host-pathogen interaction mechanisms with the actual in vivo target cells. They are also suitable for applications linked to microvascularization, such as anti-angiogenic and anti-tumor research, growing fields in veterinary medicine.

Potentially Zoonotic Bartonella in Bats from France and Spain.

We detected Bartonella in 11 of 109 insectivorous bats from France and 1 of 26 bats from Spain. These genetic variants are closely related to bat-associated Bartonella described in Finland and the United Kingdom and to B. mayotimonensis, the agent of a human endocarditis case in the United States.

Bartonella Infection among Cats Adopted from a San Francisco Shelter, Revisited.

Bartonella infection among cats from shelters can pose a health risk to adopters. Bartonella henselae is the most common species, with B. clarridgeiae and B. koehlerae being less common. The lower rates of infection by the latter species may reflect their rarity or an inefficiency of culture techniques. To assess the incidence of infection, blood cultures, serology, and PCR testing were performed on 193 kittens (6 to 17 weeks old) and 158 young adult cats (5 to 12 months old) from a modern regional shelter. Classical B. henselae culture medium was compared to a medium supplemented with insect cell growth factors. Bartonella colonies were isolated from 115 (32.8%) animals, including 50 (25.9%) kittens and 65 (41.1%) young adults. Therefore, young adults were twice as likely to be culture positive as kittens. Enhanced culture methods did not improve either the isolation rate or species profile. B. henselae was isolated from 40 kittens and 55 young adults, while B. clarridgeiae was cultured from 10 animals in each group. B. koehlerae was detected in one young adult by PCR only. B. henselae genotype II was more commonly isolated from young adults, and genotype I was more frequently isolated from kittens. Kittens were 4.7 times more likely to have a very high bacterial load than young adults. A significantly higher incidence of bacteremia in the fall and winter than in the spring and summer was observed. Bartonella antibodies were detected in 10% (19/193) of kittens and 46.2% (73/158) of young adults, with culture-positive kittens being 9.4 times more likely to be seronegative than young adults.

The efficacy of a selamectin (Stronghold ®) spot on treatment in the prevention of Bartonella henselae transmission by Ctenocephalides felis in cats, using a new high-challenge model.

Bartonella henselae is the causative agent of cat scratch disease in humans, which is recognized as an emerging zoonotic disease. Ctenocephalides felis is the main vector, and transmission of B. henselae infection between cats and humans occurs mainly through infected flea feces. Control of feline infestation with this arthropod vector therefore provides an important strategy for the prevention of infection of both humans and cats. In the present study, a new challenge model is used to evaluate the efficacy of selamectin (Stronghold(®) spot on) in the prevention of B. henselae transmission by C. felis. In this new challenge model, domestic cats were infected by direct application of B. henselae-positive fleas. The fleas used for infestation were infected by feeding on blood that contained in vitro-cultured B. henselae. The direct application of the fleas to the animals and the use of different B. henselae strains ensured a high and consistent challenge. Two groups of six cats were randomly allocated on pre-treatment flea counts to either control (untreated cats) or the selamectin-treated group with one pipette per cat according to the label instruction. Stronghold (selamectin 6 % spot on solution) was administered on days 0 and 32. On days 3, 10, 19, 25, and 31, each cat was infested by direct application of 20 fleas that fed on blood inoculated with B. henselae. Polymerase chain reaction (PCR) on pooled fleas confirmed that the fleas were infected. Blood samples were collected from each cat on days -3 (prior to flea infestation and treatment), 9, 17, 24, 30, 37, and 44 and assayed for B. henselae antibodies using an indirect immunofluorescence (IFA), for the presence of bacteria by bacterial culture and for B. henselae DNA presence by PCR. Cats were also assessed on a daily basis for general health. There were no abnormal health observations during the study and none of the animals required concomitant treatment. None of the cats displayed any clinical signs of bartonellosis during the study. In the untreated group, all cats became bacteremic within 17 to 44 days. None of the selamectin-treated cats became positive during the study. It was concluded that Stronghold(®) spot on administered to cats was efficacious in the prevention of the transmission of B. henselae by fleas to cats in a high-challenge model.

[Zoonoses (synthetic tables): animal reservoirs of pathogens and way of transmission to man]. / Les zoonoses (tableaux synthétiques: animaux réservoirs de pathogènes et modes de transmission).

The knowledge of animals which act as reservoirs for human pathogenic agents is essential for the understanding of the epidemiology of zoonotic diseases and for the application of relevant methods of control. In the present article, synthetic tables present the main zoonoses due to viruses, bacteria, protozoa, helminths, arthropods and fungi. A list of the main pathogenic agents, the animal reservoirs and the way of transmission to man is detailed in each table.

Comparative genomics of first available bovine Anaplasma phagocytophilum genome obtained with targeted sequence capture.

BACKGROUND: Anaplasma phagocytophilum is a zoonotic and obligate intracellular bacterium transmitted by ticks. In domestic ruminants, it is the causative agent of tick-borne fever, which causes significant economic losses in Europe. As A. phagocytophilum is difficult to isolate and cultivate, only nine genome sequences have been published to date, none of which originate from a bovine strain.Our goals were to; 1/ develop a sequencing methodology which efficiently circumvents the difficulties associated with A. phagocytophilum isolation and culture; 2/ describe the first genome of a bovine strain; and 3/ compare it with available genomes, in order to both explore key genomic features at the species level, and to identify candidate genes that could be specific to bovine strains. RESULTS: DNA was extracted from a bovine blood sample infected by A. phagocytophilum. Following a whole genome capture approach, A. phagocytophilum DNA was enriched 197-fold in the sample and then sequenced using Illumina technology. In total, 58.9% of obtained reads corresponded to the A. phagocytophilum genome, covering 85.3% of the HZ genome. Then by performing comparisons with nine previously-sequenced A. phagocytophilum genomes, we determined the core genome of these ten strains. Following analysis, 1281 coding DNA sequences, including 1001 complete sequences, were detected in the A. phagocytophilum bovine genome, of which four appeared to be unique to the bovine isolate. These four coding DNA sequences coded for "hypothetical proteins of unknown function" and require further analysis. We also identified nine proteins common to both European domestic ruminants tested. CONCLUSION: Using a whole genome capture approach, we have sequenced the first A. phagocytophilum genome isolated from a cow. To the best of our knowledge, this is the first time that this method has been used to selectively enrich pathogenic bacterial DNA from samples also containing host DNA. The four proteins unique to the A. phagocytophilum bovine genome could be involved in host tropism, therefore their functions need to be explored.

Multilocus sequence analysis of Anaplasma phagocytophilum reveals three distinct lineages with different host ranges in clinically ill French cattle.

Molecular epidemiology represents a powerful approach to elucidate the complex epidemiological cycles of multi-host pathogens, such as Anaplasma phagocytophilum. A. phagocytophilum is a tick-borne bacterium that affects a wide range of wild and domesticated animals. Here, we characterized its genetic diversity in populations of French cattle; we then compared the observed genotypes with those found in horses, dogs, and roe deer to determine whether genotypes of A. phagocytophilum are shared among different hosts. We sampled 120 domesticated animals (104 cattle, 13 horses, and 3 dogs) and 40 wild animals (roe deer) and used multilocus sequence analysis on nine loci (ankA, msp4, groESL, typA, pled, gyrA, recG, polA, and an intergenic region) to characterize the genotypes of A. phagocytophilum present. Phylogenic analysis revealed three genetic clusters of bacterial variants in domesticated animals. The two principal clusters included 98% of the bacterial genotypes found in cattle, which were only distantly related to those in roe deer. One cluster comprised only cattle genotypes, while the second contained genotypes from cattle, horses, and dogs. The third contained all roe deer genotypes and three cattle genotypes. Geographical factors could not explain this clustering pattern. These results suggest that roe deer do not contribute to the spread of A. phagocytophilum in cattle in France. Further studies should explore if these different clusters are associated with differing disease severity in domesticated hosts. Additionally, it remains to be seen if the three clusters of A. phagocytophilum genotypes in cattle correspond to distinct epidemiological cycles, potentially involving different reservoir hosts.

[Not Available]. / Risques sanitaires liés aux nouveaux animaux de compagnie d'origine sauvage.

Exotic and pocket pets are becoming more and more popular in developed countries. These animals can carry zoonotic pathogens and young children and elderly people as well as immunocompromised persons are at highest risk to contract severe or even lethal infections. Despite the small number of such incidents or outbreaks compared to the large number of exotic and pocket pets owned by households, risk of infection still exist. Basic hygienic rules and a regular veterinary and medical check-up should be apply regularly to reduce such a risk.

Tick-borne diseases in Europe: Current prevention, control tools and the promise of aptamers.

In Europe, tick-borne diseases (TBDs) cause significant morbidity and mortality, affecting both human and animal health. Ticks can transmit a wide variety of pathogens (bacteria, viruses, and parasites) and feed on many vertebrate hosts. The incidence and public health burden of TBDs are tending to intensify in Europe due to various factors, mainly anthropogenic and often combined. Early detection of tick-borne pathogens (TBPs), preventive measures and treatment are of great importance to control TBDs and their expansion. However, there are various limitations in terms of the sensitivity and/or specificity of detection and prevention methods, and even in terms of feasibility. Aptamers are single-stranded DNA or RNA that could address these issues as they are able to bind with high affinity and specificity to a wide range of targets (e.g., proteins, small compounds, and cells) due to their unique three-dimensional structure. To date, aptamers have been selected against TBPs such as tick-borne encephalitis virus, Francisella tularensis, and Rickettsia typhi. These studies have demonstrated the benefits of aptamer-based assays for pathogen detection and medical diagnosis. In this review, we address the applications of aptamers to TBDs and discuss their potential for improving prevention measures (use of chemical acaricides, vaccination), diagnosis and therapeutic strategies to control TBDs.

Aptamer selection against cell extracts containing the zoonotic obligate intracellular bacterium, Anaplasma phagocytophilum.

A. phagocytophilum is a zoonotic and tick-borne bacterium, threatening human and animal health. Many questions persist concerning the variability of strains and the mechanisms governing the interactions with its different hosts. These gaps can be explained by the difficulty to cultivate and study A. phagocytophilum because of its strict intracellular location and the lack of specific tools, in particular monoclonal antibodies, currently unavailable. The objective of our study was to develop DNA aptamers against A. phagocytophilum, or molecules expressed during the infection, as new study and/or capture tools. Selecting aptamers was a major challenge due to the strict intracellular location of the bacterium. To meet this challenge, we set up a customized selection protocol against an enriched suspension of A. phagocytophilum NY18 strain, cultivated in HL-60 cells. The implementation of SELEX allowed the selection of three aptamers, characterized by a high affinity for HL-60 cells infected with A. phagocytophilum NY18 strain. Interestingly, the targets of these three aptamers are most likely proteins expressed at different times of infection. The selected aptamers could contribute to increase our understanding of the interactions between A. phagocytophilum and its hosts, as well as permit the development of new diagnostic, therapeutic or drug delivery appliances.

Anaplasma phagocytophilum in urban and peri-urban passerine birds in Ile-de-France.

Wild animals in general, birds in particular, play a key role in transporting ticks and propagating tick-borne pathogens. Several studies have confirmed the infection of birds with Anaplasma phagocytophilum, with overall prevalence varying widely from country to country and/or study to study. This zoonotic bacterium, transmitted mainly by ticks of the genus Ixodes, is responsible for granulocytic anaplasmosis in humans (HGA) and domestic animals (cats, dogs, horses). The disease is also called tick-borne fever (TBF) in ruminants. Extremely rare in the USA, TBF is very common in Europe, where it causes economic losses in livestock. Conversely, HGA is well established in the USA whereas only a few less severe cases have been observed in Europe. Current typing techniques support the existence of multiple variants with differences in virulence/pathogenicity and tropism for certain tick and host species. However, epidemiological cycles remain difficult to characterize in Europe. Several studies describe a cycle apparently involving only birds in Europe, but no such study has been conducted in mainland France. Our objectives were to search for A. phagocytophilum in passerine birds in the Ile-de-France region and to explore their diversity using groEL and ankA gene typing and multilocus sequence typing (MLST). Various tissues (spleen, liver, and skin) were collected from cadavers of 680 passerines between March and December 2021. The presence of A. phagocytophilum was detected by qPCR Taqman targeting the msp2 gene. Three blackbirds (Turdus merula) were found positive, representing detection rates of 0.4 % in all birds tested and 3.3 % in blackbirds. The higher frequency of detection in blackbirds could be at least partially explained by their lifestyle, as they feed on the ground. Analysis of the results of groEL and ankA typing and MLST from positive blackbirds support the hypothesis that the avian A. phagocytophilum strains in Ile-de-France are distinct from those found in mammals, and that they form their own cluster in Europe.

Extended-spectrum ß-lactamase CTX-M-15-producing Klebsiella pneumoniae of sequence type ST274 in companion animals.

Screening of extended-spectrum ß-lactamase (ESBL)-producing Gram-negative bacteria in companion animals living in the Paris area in France identified a high rate of CTX-M-15-producing Klebsiella pneumoniae. Those isolates were recovered during the 2010-2011 period from both infections and asymptomatic colonizations. Sequence typing revealed that most of these isolates belonged to sequence type ST274. Interestingly, the bla(CTX-M-15) gene was located on a specific and novel plasmid scaffold. These findings highlight that companion animals may be reservoirs for CTX-M-15-producing K. pneumoniae evolving separately from the human reservoir of CTX-M-15 producers.

Assessment of persistence of Bartonella henselae in Ctenocephalides felis.

Bartonella henselae (Rhizobiales: Bartonellaceae) is a Gram-negative fastidious bacterium of veterinary and zoonotic importance. The cat flea Ctenocephalides felis (Siphonaptera: Pulicidae) is the main recognized vector of B. henselae, and transmission among cats and humans occurs mainly through infected flea feces. The present study documents the use of a quantitative molecular approach to follow the daily kinetics of B. henselae within the cat flea and its excreted feces after exposure to infected blood for 48 h in an artificial membrane system. B. henselae DNA was detected in both fleas and feces for the entire life span of the fleas (i.e., 12 days) starting from 24 h after initiation of the blood meal.

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.

Identification of a novel nanoRNase in Bartonella.

In Escherichia coli, only one essential oligoribonuclease (Orn) can degrade oligoribonucleotides of five residues and shorter in length (nanoRNA). In Bacillus subtilis, NrnA and NrnB, which do not show any sequence similarity to Orn, have been identified as functional analogues of Orn. Sequence comparisons did not identify orn, nrnA or nrnB homologues in the genomes of the Chlamydia/Cyanobacteria and Alphaproteobacteria family members. Screening a genomic library from Bartonella birtlesii, a member of the Alphaproteobacteria, for genes that can complement a conditional orn mutant in E. coli, we identified BA0969 (NrnC) as a functional analogue of Orn. NrnC is highly conserved (more than 80 % identity) in the Bartonella genomes sequenced to date. Biochemical characterization showed that this protein exhibits oligo RNA degradation activity (nanoRNase activity). Like Orn from E. coli, NrnC is inhibited by micromolar amounts of 3'-phosphoadenosine 5'-phosphate in vitro. NrnC homologues are widely present in genomes of Alphaproteobacteria. Knock down of nrnC decreases the growth ability of Bartonella henselae, demonstrating the importance of nanoRNase activity in this bacterium.

The Trw type IV secretion system of Bartonella mediates host-specific adhesion to erythrocytes.

Bacterial pathogens typically infect only a limited range of hosts; however, the genetic mechanisms governing host-specificity are poorly understood. The alpha-proteobacterial genus Bartonella comprises 21 species that cause host-specific intraerythrocytic bacteremia as hallmark of infection in their respective mammalian reservoirs, including the human-specific pathogens Bartonella quintana and Bartonella bacilliformis that cause trench fever and Oroya fever, respectively. Here, we have identified bacterial factors that mediate host-specific erythrocyte colonization in the mammalian reservoirs. Using mouse-specific Bartonella birtlesii, human-specific Bartonella quintana, cat-specific Bartonella henselae and rat-specific Bartonella tribocorum, we established in vitro adhesion and invasion assays with isolated erythrocytes that fully reproduce the host-specificity of erythrocyte infection as observed in vivo. By signature-tagged mutagenesis of B. birtlesii and mutant selection in a mouse infection model we identified mutants impaired in establishing intraerythrocytic bacteremia. Among 45 abacteremic mutants, five failed to adhere to and invade mouse erythrocytes in vitro. The corresponding genes encode components of the type IV secretion system (T4SS) Trw, demonstrating that this virulence factor laterally acquired by the Bartonella lineage is directly involved in adherence to erythrocytes. Strikingly, ectopic expression of Trw of rat-specific B. tribocorum in cat-specific B. henselae or human-specific B. quintana expanded their host range for erythrocyte infection to rat, demonstrating that Trw mediates host-specific erythrocyte infection. A molecular evolutionary analysis of the trw locus further indicated that the variable, surface-located TrwL and TrwJ might represent the T4SS components that determine host-specificity of erythrocyte parasitism. In conclusion, we show that the laterally acquired Trw T4SS diversified in the Bartonella lineage to facilitate host-restricted adhesion to erythrocytes in a wide range of mammals.

Optimized Lambda Exonuclease Digestion or Purification Using Streptavidin-Coated Beads: Which One Is Best for Successful DNA Aptamer Selection?

The high failure rate of the in vitro aptamer selection process by SELEX (Systematic Evolution of Ligands by EXponential enrichment) limits the production of these innovative oligonucleotides and, consequently, limits their potential applications. The generation of single-stranded DNA (ssDNA) is a critical step of SELEX, directly affecting the enrichment and the selection of potential binding sequences. The main goal of this study was to confirm the best method for generating ssDNA by comparing the purification of ssDNA, using streptavidin-coated beads, and lambda exonuclease digestion, and by improving ssDNA recovery through protocol improvements. In addition, three techniques for quantifying the ssDNA generated (Qubit vs. NanodropTM vs. gel quantification) were compared, and these demonstrated the accuracy of the gel-based quantification method. Lambda exonuclease digestion was found to be more efficient for ssDNA recovery than purification using streptavidin-coated beads, both quantitatively and qualitatively. In conclusion, this work provides a detailed and rigorous protocol for generating ssDNA, improving the chances of a successful aptamer selection process.