Danny Morick

Molecular detection of Ehrlichia canis, Anaplasma bovis, Anaplasma platys, Candidatus Midichloria mitochondrii and Babesia canis vogeli in ticks from Israel

: Ticks are vectors of important pathogens of human and animals. Therefore, their microbial carriage capacity is constantly being investigated. The aim of this study was to characterize the diversity of domestic animal pathogens in ticks collected from vegetation and the ground, from different parts of Israel. Non-engorged questing adult ticks were collected from 13 localities. A total of 1196 ticks in 131 pools-83 pools of Rhipicephalus turanicus and 48 of Rhipicephalus sanguineus (with two to ten ticks per pool)-were included in this study. In addition, 13 single free-roaming Hyalomma spp. ticks were collected. Screening by molecular techniques revealed the presence of Ehrlichia canis, Anaplasma platys, Anaplasma bovis and Babesia canis vogeli DNA in R. turanicus ticks. E. canis, A. bovis, B. canis vogeli and Candidatus Midichloria mitochondrii DNA sequences were detected in R. sanguineus ticks. Candidatus Midichloria mitochondrii DNA was also detected in Hyalomma spp. ticks. Neither Hepatozoon spp. nor Bartonella spp. DNA was detected in any of the ticks examined. This study describes the first detection of E. canis in the tick R. turanicus, which may serve as a vector of this canine pathogen; E. canis was the most common pathogen detected in the collected questing ticks. It also describes the first detection of A. bovis and Candidatus Midichloria mitochondrii in Israel. To the best of the authors knowledge, this is the first report describing the detection of DNA of the latter two pathogens in R. sanguineus, and of A. bovis in R. turanicus.

Detection of Bartonella spp. in wild rodents in Israel using HRM real-time PCR

The prevalence of Bartonella spp. in wild rodents was studied in 19 geographical locations in Israel. One hundred and twelve rodents belonging to five species (Mus musculus, Rattus rattus, Microtus socialis, Acomys cahirinus and Apodemus sylvaticus) were included in the survey. In addition, 156 ectoparasites were collected from the rodents. Spleen sample from each rodent and the ectoparasites were examined for the presence of Bartonella DNA using high resolution melt (HRM) real-time PCR. The method was designed for the simultaneous detection and differentiation of eight Bartonella spp. according to the nucleotide variation in each of two gene fragments (rpoB and gltA) and the 16S-23S intergenic spacer (ITS) locus, using the same PCR protocol which allowed the simultaneous amplification of the three different loci. Bartonella DNA was detected in spleen samples of 19 out of 79 (24%) black rats (R. rattus) and in 1 of 4 (25%) Cairo spiny mice (A. cahirinus). In addition, 15 of 34 (44%) flea pools harbored Bartonella DNA. Only rat flea (Xenopsyla cheopis) pools collected from black rats (R. rattus) were positive for Bartonella DNA. The Bartonella sp. detected in spleen samples from black rats (R. rattus) was closely related to both B. tribocorum and B. elizabethae. The species detected in the Cairo spiny mouse (A. cahirinus) spleen sample was closely related to the zoonotic pathogen, B. elizabethae. These results indicate that Bartonella species are highly prevalent in suburban rodent populations and their ectoparasites in Israel. Further investigation of the prevalence and zoonotic potential of the Bartonella species detected in the black rats and the Cairo spiny mouse is warranted.

Molecular detection of Rickettsia massiliae, Rickettsia sibirica mongolitimonae and Rickettsia conorii israelensis in ticks from Israel

Rickettsioses are recognized as important emerging vector-borne infections of humans worldwide. Previous reports documented the presence of two spotted fever group rickettsiae in Israel, Rickettsia conorii israelensis and Rickettsia felis. The aim of this study was to characterize the diversity of rickettsiae in ticks collected from vegetation and the ground, from different parts of Israel. Non-engorged questing adult ticks were collected from 13 localities. A total of 131 tick pools, 83 of Rhipicephalus turanicus and 48 of Rhipicephalus sanguineus (each with 2-10 ticks per pool), were included in this study. In addition, 13 Hyalomma sp. ticks were collected. The ticks were molecularly screened for rickettsiae, targeting the citrate synthase (gltA) and the outer membrane protein A (ompA) gene loci. Rickettsia massiliae ompA DNA (100% sequence identity; 180 bp) was detected in 32 Rh. turanicus and 12 Rh. sanguineus tick pools. R. conorii israelensis was detected in three Rh. sanguineus pools. Rickettsia sibirica mongolitimonae ompA DNA (100% sequence identity; 182 bp) was found in one Hyalomma tick. This study reports the first detection of R. massiliae and R. sibirica mongolitimonae in ticks from Israel. This is the first report describing the presence of these human pathogens in the Middle East.

Investigation of Bartonella acquisition and transmission in Xenopsylla ramesis fleas (Siphonaptera: Pulicidae)

Bartonella are emerging and re‐emerging pathogens affecting humans and a wide variety of animals including rodents. Horizontal transmission of Bartonella species by different hematophagous vectors is well acknowledged but vertical transmission (from mother to offspring) is questionable and was never explored in fleas. The aim of this study was to investigate whether the rodent flea, Xenopsylla ramesis, can acquire native Bartonella from wild rodents and transmit it transovarially. For this aim, Bartonella‐free laboratory‐reared X. ramesis fleas were placed on six naturally Bartonella‐infected rodents and six species‐matched Bartonella‐negative rodents (three Meriones crassus jirds, two Gerbillus nanus gerbils and one Gerbillus dasyurus gerbil) for 7 days, 12–14 h per day. The fleas that were placed on the Bartonella‐positive rodents acquired four different Bartonella genotypes. Eggs and larvae laid and developed, respectively, by fleas from both rodent groups were collected daily for 7 days and molecularly screened for Bartonella. All eggs and larvae from both groups were found to be negative for Bartonella DNA. Interestingly, two of five gut voids regurgitated by Bartonella‐positive fleas contained Bartonella DNA. The naturally infected rodents remained persistently infected with Bartonella for at least 89 days suggesting their capability to serve as competent reservoirs for Bartonella species. The findings in this study indicate that X. ramesis fleas can acquire several Bartonella strains from wild rodents but cannot transmit Bartonella transovarially.

Bartonella Genotypes in Fleas (Insecta: Siphonaptera) Collected from Rodents in the Negev Desert, Israel

ABSTRACT Fleas collected from rodents in the Negev Desert in southern Israel were molecularly screened for Bartonella species. A total of 1,148 fleas, collected from 122 rodents belonging to six species, were pooled in 245 pools based on flea species, sex, and rodent host species. Two Bartonella gene fragments, corresponding to RNA polymerase B (rpoB) and citrate synthase (gltA), were targeted, and 94 and 74 flea pools were found positive by PCR, respectively. The Bartonella 16S-23S internal transcribed spacer (ITS) region was also targeted, and 66 flea pools were found to be positive by PCR. Sixteen different Bartonella gltA genotypes were detected in 94 positive flea pools collected from 5 different rodent species, indicating that fleas collected from each rodent species can harbor several Bartonella genotypes. Based on gltA analysis, identified Bartonella genotypes were highly similar or identical to strains previously detected in rodent species from different parts of the world. A gltA fragment 100% similar to Bartonella henselae was detected in one flea pool. Another 2 flea pools contained gltA fragments that were closely related to B. henselae (98% similarity). The high sequence similarities to the zoonotic pathogen B. henselae warrant further investigation.

Combined MLST and AFLP typing of Bartonella henselae isolated from cats reveals new sequence types and suggests clonal evolution

Bartonella species are Gram-negative, fastidious bacteria. Bartonella henselae is found in cats and transmitted to humans via cat scratches or bites causing cat-scratch disease, characterized by clinical symptoms with varying severity. The prevalence of bartonellosis among humans in Germany appears to be high, and severe clinical cases have been described. However, epidemiological data of B. henselae in cats are rare. In this study we determined the detection rates of Bartonella ssp. in cats by culture and real-time PCR. Furthermore, B. henselae isolates were genetically characterized by highly discriminatory amplified fragment length polymorphism (AFLP) and multilocus sequence typing (MLST). Bartonella spp. were isolated by culture from 11 (2.2%) of 507 blood samples. Out of 169 blood samples additionally analyzed by PCR, 28 (16.6%) were found positive for Bartonella spp., illustrating the advantage of PCR in Bartonella spp. detection. PCR-REA identified B. henselae in 27 cats and Bartonella clarridgeiae in one cat. B. henselae isolates from different geographical regions in Germany were genetically characterized by AFLP and MLST. Both methods confirmed genetic diversity of B. henselae on the strain level. MLST identified 11 new sequence types, all of them assigned to three clonal complexes as determined by eBURST. AFLP typing revealed genetic relation among the B. henselae isolates from the same geographical region. Combining AFLP typing and MLST/eBURST analyses revealed that B. henselae of the same AFLP subcluster belonged to the same clonal complex. Altogether these results indicate that B. henselae may evolve clonally.

The effect of ecological and temporal factors on the composition of Bartonella infection in rodents and their fleas

The composition of Bartonella infection was explored in wild Gerbillus andersoni rodents and their Synosternus cleopatrae fleas. Rodent blood samples and fleas were collected in two periods (two different seasons; 4 months apart) from juveniles and adult hosts, and their bartonellae lineages were identified by a 454-pyrosequencing analysis targeting a specific Bartonella citrate synthase gene (gltA) fragment. The rate of Bartonella spp. co-infection was estimated and the assemblage and distribution of bartonellae lineages across the samples with respect to ecological and phylogenetic distance similarities were analyzed. Moreover, environmental factors that could explain potential differences between samples were investigated. Out of the 91 bartonellae-positive samples, 89% were found to be co-infected with more than two phylogenetically distant Bartonella genotypes and additional closely related (but distinguishable) variants. These bartonellae lineages were distributed in a non-random manner, and a negative interaction between lineages was discovered. Interestingly, the overall composition of those infections greatly varied among samples. This variability was partially explained by factors, such as type of sample (blood versus fleas), flea sex and period of collection. This investigation sheds light on the patterns of Bartonella infection and the organization of Bartonella lineages in fleas and rodents in nature.

Prevalence and Diversity of Bartonella Species in Commensal Rodents and Ectoparasites from Nigeria, West Africa

Background Bartonellae are fastidious bacteria causing persistent bacteremia in humans and a wide variety of animals. In recent years there is an increasing interest in mammalian bartonelloses in general and in rodent bartonelloses in particular. To date, no studies investigating the presence of Bartonella spp. in rodents and ectoparasites from Nigeria were carried out. Methodology/Principal Findings The aim of the current study was to investigate the presence of Bartonella spp. in commensal rodents and their ectoparasites in Nigeria. We report, for the first time, the molecular detection of Bartonella in 26% (46/177) of commensal rodents (Rattus rattus, R. norvegicus and Cricetomys gambianus) and 28% (9/32) of ectoparasite pools (Xenopsylla cheopis, Haemolaelaps spp., Ctenophthalmus spp., Hemimerus talpoides, and Rhipicephalus sanguineus) from Nigeria. Sequence analysis of the citrate synthase gene (gltA) revealed diversity of Bartonella spp. and genotypes in Nigerian rodents and their ectoparasites. Bartonella spp. identical or closely related to Bartonella elizabethae, Bartonella tribocorum and Bartonella grahamii were detected. Conclusions/Significance High prevalence of infection with Bartonella spp. was detected in commensal rodents and ectoparasites from Nigeria. The Bartonella spp. identified were previously associated with human diseases highlighting their importance to public health. Further studies need to be conducted to determine whether the identified Bartonella species could be responsible for human cases of febrile illness in Nigeria.

Prevalence of phocine distemper virus specific antibodies: bracing for the next seal epizootic in north-western Europe

In 1988 and 2002, two major phocine distemper virus (PDV) outbreaks occurred in harbour seals (Phoca vitulina) in north-western European coastal waters, causing the death of tens of thousands seals. Here we investigated whether PDV is still circulating among seals of the Dutch coastal waters and whether seals have protective serum-antibodies against PDV. Therefore seal serum samples, collected from 2002 to 2012, were tested for the presence of PDV-neutralizing antibodies. Antibodies were detected in most seals in 2002 and 2003 while after 2003 antibodies were detected only in seals less than two month-old and adult seals that probably had survived the 2002 PDV-epizootic. We estimated the current proportion of seals with antibodies against PDV at 11%. These findings suggest that at present the vast majority of seals are not immune to PDV infection. PDV re-introduction in this area may cause a major epizootic with infection of >80% and mass-mortality of >50% of the population.

Transmission Dynamics of Bartonella sp. Strain OE 1-1 in Sundevall's Jirds (Meriones crassus)

ABSTRACT A high prevalence of Bartonella infection is found in many natural systems; however, the transmission dynamics leading to observations of these infections is not fully understood. The capability of Xenopsylla ramesis fleas to serve as competent vectors of Bartonella sp. OE 1-1 (a strain closely related to the zoonotic Bartonella elizabethae) to Meriones crassus jirds was investigated. Naïve X. ramesis fleas were placed for 72 h on naïve jirds or jirds that were either experimentally or naturally infected with Bartonella sp. strain OE 1-1, after which they were placed on naïve jirds. Postfeeding, 69 to 100% of the fleas collected from each Bartonella-positive jird contained Bartonella DNA, and all naïve jirds became positive for Bartonella sp. OE 1-1 after infestation with the infected fleas. In addition, maternal transmission of Bartonella sp. OE 1-1 in jirds was tested by mating 5 Bartonella-positive and 5 naïve female jirds with 10 naïve male jirds in the absence of fleas. Fifteen offspring were delivered by each group. Cultures of blood drawn from all offspring on days 35 and 47 postdelivery were found to be negative for Bartonella. A single spleen sample from the offspring of a Bartonella-positive mother was found molecularly positive for Bartonella sp. OE 1-1. This study demonstrates that X. ramesis fleas are competent vectors of Bartonella sp. OE 1-1 to M. crassus jirds and indicates that maternal transmission is probably not the major transmission route from female jirds to their offspring. We suggest that the dynamics of Bartonella sp. OE 1-1 in the M. crassus jird population in nature is mostly dependent on its vectors.