Nadia Haddad

Endocarditis in Cattle Caused by Bartonella bovis

This study aimed to determine the role of Bartonella as an endocarditis agent in cattle. Bartonella bovis was identified by PCR, gene sequences analysis, and specific internal transcribed spacer amplicon product size in 2 bovine endocarditis cases with high antibody titers, which demonstrates that B. bovis is a pathogen for cattle.

Opening the black box of Anaplasma phagocytophilum diversity: current situation and future perspectives.

Anaplasma phagocytophilum is a zoonotic obligate intracellular bacterium known to be transmitted by ticks belonging to the Ixodes persulcatus complex. This bacterium can infect several mammalian species, and is known to cause diseases with variable symptoms in many domestic animals. Specifically, it is the causative agent of tick-borne fever (TBF), a disease of important economic impact in European domestic ruminants, and human granulocytic anaplasmosis (HGA), an emerging zoonotic disease in Asia, USA and Europe. A. phagocytophilum epidemiological cycles are complex and involve different ecotypes, vectors, and mammalian host species. Moreover, the epidemiology of A. phagocytophilum infection differs greatly between Europe and the USA. These different epidemiological contexts are associated with considerable variations in bacterial strains. Until recently, few A. phagocytophilum molecular typing tools were available, generating difficulties in completely elucidating the epidemiological cycles of this bacterium. Over the last few years, many A. phagocytophilum typing techniques have been developed, permitting in-depth epidemiological exploration. Here, we review the current knowledge and future perspectives regarding A. phagocytophilum epidemiology and phylogeny, and then focus on the molecular typing tools available for studying A. phagocytophilum genetic diversity.

Molecular Epidemiology of Feline and Human Bartonella henselae Isolates

Multiple locus variable number tandem repeat analysis was performed on 178 Bartonella henselae isolates from 9 countries; 99 profiles were distributed into 2 groups. Human isolates/strains were placed into the second group. Genotype I and II isolates shared no common profile. All genotype I isolates clustered within group B. The evolutive implications are discussed.

A new multiple-locus variable-number tandem repeat analysis reveals different clusters for Anaplasma phagocytophilum circulating in domestic and wild ruminants

BackgroundAnaplasma phagocytophilum is a tick-borne intragranulocytic alpha-proteobacterium. It is the causative agent of tick-borne fever in ruminants, and of human granulocytic anaplasmosis in humans, two diseases which are becoming increasingly recognized in Europe and the USA. However, while several molecular typing tools have been developed over the last years, few of them are appropriate for in-depth exploration of the epidemiological cycle of this bacterium. Therefore we have developed a Multiple-Locus Variable number tandem repeat (VNTR) Analysis typing technique for A. phagocytophilum.MethodsFive VNTRs were selected based on the HZ human-derived strain genome, and were tested on the Webster human-derived strain and on 123 DNA samples: 67 from cattle, 7 from sheep, 15 from roe deer, 4 from red deer, 1 from a reindeer, 2 from horses, 1 from a dog, and 26 from ticks.ResultsFrom these samples, we obtained 84 different profiles, with a diversity index of 0.96 (0.99 for vertebrate samples, i.e. without tick samples). Our technique confirmed that A. phagocytophilum from roe deer or domestic ruminants belong to two different clusters, while A. phagocytophilum from red deer and domestic ruminants locate within the same cluster, questioning the respective roles of roe vs red deer as reservoir hosts for domestic ruminant strains in Europe. As expected, greater diversity was obtained between rather than within cattle herds.ConclusionsOur technique has great potential to provide detailed information on A. phagocytophilum isolates, improving both epidemiological and phylogenic investigations, thereby helping in the development of relevant prevention and control measures.

Prevalence of select vector-borne pathogens in stray and client-owned dogs from Algiers

Data on the prevalence of vector-borne diseases agents infecting canines in Algeria is currently lacking. The purpose of this study is to assess by serological and molecular methods the prevalence of select arthropod borne-bacterial infections in client-owned and stray dogs. Antibodies to Anaplasma phagocytophilum were the most prevalent at 47.7%, followed by Borrelia burgdorferi s.l. at 37.6%, Ehrlichia canis at 30.0%, Bartonella henselae at 32.4% and Bartonella vinsonii subsp. berkhoffii at 27%. Seroprevalence was statistically significantly higher in stray dogs than those owned by clients. Seropositivity was not associated with health status, except for E. canis. Molecular evaluation indicates that 17.8% of the 213 analyzed dogs were positive for Ehrlichia and Anaplasma with a prevalence of 4.2% for E. canis, 14.1% for Anaplama platys and 0% for A. phagocytophilum. Seven (7.1%) of the tested dogs were positive for Bartonella spp. with two characterized as Bartonella rochalimae, four as B. henselae and one as B.v. subsp. berkhoffii.

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.

Differential effects of Bartonella henselae on human and feline macro- and micro-vascular endothelial cells.

Bartonella henselae, a zoonotic agent, induces tumors of endothelial cells (ECs), namely bacillary angiomatosis and peliosis in immunosuppressed humans but not in cats. In vitro studies on ECs represent to date the only way to explore the interactions between Bartonella henselae and vascular endothelium. However, no comparative study of the interactions between Bartonella henselae and human (incidental host) ECs vs feline (reservoir host) ECs has been carried out because of the absence of any available feline endothelial cell lines. To this purpose, we have developed nine feline EC lines which allowed comparing the effects of Bartonella strains on human and feline micro-vascular ECs representative of the infection development sites such as skin, versus macro-vascular ECs, such as umbilical vein. Our model revealed intrinsic differences between human (Human Skin Microvascular ECs –HSkMEC and Human Umbilical Vein ECs – iHUVEC) and feline ECs susceptibility to Bartonella henselae infection. While no effect was observed on the feline ECs upon Bartonella henselae infection, the human ones displayed accelerated angiogenesis and wound healing. Noticeable differences were demonstrated between human micro- and macro-vasculature derived ECs both in terms of pseudo-tube formation and healing. Interestingly, Bartonella henselae effects on human ECs were also elicited by soluble factors. Neither Bartonella henselae-infected Human Skin Microvascular ECs clinically involved in bacillary angiomatosis, nor feline ECs increased cAMP production, as opposed to HUVEC. Bartonella henselae could stimulate the activation of Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) in homologous cellular systems and trigger VEGF production by HSkMECs only, but not iHUVEC or any feline ECs tested. These results may explain the decreased pathogenic potential of Bartonella henselae infection for cats as compared to humans and strongly suggest that an autocrine secretion of VEGF by human skin endothelial cells might induce their growth and ultimately lead to bacillary angiomatosis formation.

A new typing technique for the Rickettsiales Ehrlichia ruminantium: Multiple-locus variable number tandem repeat analysis

Ehrlichia ruminantium (ER) is a member of the order Rickettsiales transmitted by Amblyomma ticks. This obligatory intracellular bacterium is the causative agent of a fatal disease in ruminants, named heartwater. It represents a constraint on breeding development in sub-Saharan Africa and in the Caribbean. The genetic diversity of the strains of ER, which could be a limiting factor to obtain effective vaccines, needs to be better characterized. For this purpose, we developed a molecular typing technique based on the polymorphism of variable number tandem repeat (VNTR) sequences, MLVA (multiple locus VNTR analysis). Eight (out of 21) VNTR candidates were validated using 17 samples representing a panel of ER strains from different geographical origins from West, South Africa, and Caribbean areas and in ER infected ticks and goat tissues. This result demonstrated the ability of these VNTRs to type a wide range of strains. The stability of the selected VNTR markers was very good, at the time scale needed for epidemiological purposes: in particular, no difference in the VNTR profiles was observed between virulent and attenuated strains (for Gardel and Senegal strains) and between strains (Gardel and Blonde strains) isolated in the same area 19years apart. We validated the strong discriminatory power of MLVA for ER and found a high level of polymorphism between the available strains, with 10 different profiles out of 13 ER strains. The MLVA scheme described in this study is a rapid and efficient molecular typing tool for ER, which allows rapid and direct typing of this intracellular pathogen without preliminary culture and gives reliable results that can be used for further epidemiological studies.

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

ABSTRACT 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.

Isolation of Bartonella henselae and Two New Bartonella Subspecies, Bartonellakoehlerae Subspecies boulouisii subsp. nov. and Bartonella koehlerae Subspecies bothieri subsp. nov. from Free-Ranging Californian Mountain Lions and Bobcats

Domestic cats are the natural reservoir of Bartonella henselae, B. clarridgeiae and B. koehlerae. To determine the role of wild felids in the epidemiology of Bartonella infections, blood was collected from 14 free-ranging California mountain lions (Puma concolor) and 19 bobcats (Lynx rufus). Bartonella spp. were isolated from four (29%) mountain lions and seven (37%) bobcats. These isolates were characterized using growth characteristics, biochemical reactions, molecular techniques, including PCR-RFLP of selected genes or interspacer region, pulsed-field gel electrophoresis (PFGE), partial sequencing of several genes, and DNA-DNA hybridization. Two isolates were identical to B. henselae genotype II. All other isolates were distinguished from B. henselae and B. koehlerae by PCR-RFLP of the gltA gene using endonucleases HhaI, TaqI and AciI, with the latter two discriminating between the mountain lion and the bobcat isolates. These two novel isolates displayed specific PFGE profiles distinct from B. henselae, B. koehlerae and B. clarridgeiae. Sequences of amplified gene fragments from the three mountain lion and six bobcat isolates were closely related to, but distinct from, B. henselae and B. koehlerae. Finally, DNA-DNA hybridization studies demonstrated that the mountain lion and bobcat strains are most closely related to B. koehlerae. We propose naming the mountain lion isolates B. koehlerae subsp. boulouisii subsp. nov. (type strain: L-42-94), and the bobcat isolates B. koehlerae subsp. bothieri subsp. nov. (type strain: L-17-96), and to emend B. koehlerae as B. koehlerae subsp. koehlerae. The mode of transmission and the zoonotic potential of these new Bartonella subspecies remain to be determined.