Prevalence of Rickettsia and Bartonella species in Spanish cats and their fleas

The aim of this study was to determine the prevalence of Bartonella henselae, Rickettsia felis, and Rickettsia typhi in fleas and companion cats (serum and claws) and to assess their presence as a function of host, host habitat, and level of parasitism. Eighty‐nine serum and claw samples and 90 flea pools were collected. Cat sera were assayed by IFA for Bartonella henselae and Rickettssia species IgG antibodies. Conventional PCRs were performed on DNA extracted from nails and fleas collected from cats. A large portion (55.8%) of the feline population sampled was exposed to at least one of the three tested vector‐borne pathogens. Seroreactivity to B. henselae was found in 50% of the feline studied population, and to R. felis in 16.3%. R. typhi antibodies were not found in any cat. No Bartonella sp. DNA was amplified from the claws. Flea samples from 41 cats (46%) showed molecular evidence for at least one pathogen; our study demonstrated a prevalence rate of 43.3 % of Rickettsia sp and 4.4% of Bartonella sp. in the studied flea population. None of the risk factors studied (cat's features, host habitat, and level of parasitation) was associated with either the serology or the PCR results for Bartonella sp. and Rickettsia sp.. Flea‐associated infectious agents are common in cats and fleas and support the recommendation that stringent flea control should be maintained on cats.     

Molecular detection of zoonotic bartonellae (B. henselae,B. elizabethae and B. rochalimae) in fleas collected from dogs in Israel

Fleas represent an acknowledged burden on dogs worldwide. The characterization of flea species infesting kennel dogs from two localities in Israel (Rehovot and Jerusalem) and their molecular screening for Bartonella species (Rhizobiales: Bartonellaceae) was investigated. A total of 355 fleas were collected from 107 dogs. The fleas were morphologically classified and molecularly screened targeting the Bartonella 16S–23S internal transcribed spacer (ITS). Of the 107 dogs examined, 80 (74.8%) were infested with Ctenocephalides canis (Siphonaptera: Pulicidae), 68 (63.6%) with Ctenocephalides felis, 15 (14.0%) with Pulex irritans (Siphonaptera: Pulicidae) and one (0.9%) with Xenopsylla cheopis (Siphonaptera: Pulicidae). Fleas were grouped into 166 pools (one to nine fleas per pool) according to species and host. Thirteen of the 166 flea pools (7.8%) were found to be positive for Bartonella DNA. Detected ITS sequences were 99–100% similar to those of four Bartonella species: Bartonella henselae (six pools); Bartonella elizabethae (five pools); Bartonella rochalimae (one pool), and Bartonella bovis (one pool). The present study indicates the occurrence of a variety of flea species in dogs in Israel; these flea species are, in turn, carriers of several zoonotic Bartonella species. Physicians, veterinarians and public health workers should be aware of the presence of these pathogens in dog fleas in Israel and preventive measures should be implemented.     

Coexistence of Bartonella henselae and B. clarridgeiae in populations of cats and their fleas in Guatemala

Cats and their fleas collected in Guatemala were investigated for the presence of Bartonella infections. Bartonella bacteria were cultured from 8.2% (13/159) of cats, and all cultures were identified as B. henselae. Molecular analysis allowed detection of Bartonella DNA in 33.8% (48/142) of cats and in 22.4% (34/152) of cat fleas using gltA, nuoG, and 16S–23S internal transcribed spacer targets. Two Bartonella species, B. henselae and B. clarridgeiae, were identified in cats and cat fleas by molecular analysis, with B. henselae being more common than B. clarridgeiae in the cats (68.1%; 32/47 vs 31.9%; 15/47). The nuoG was found to be less sensitive for detecting B. clarridgeiae compared with other molecular targets and could detect only two of the 15 B. clarridgeiae‐infected cats. No significant differences were observed for prevalence between male and female cats and between different age groups. No evident association was observed between the presence of Bartonella species in cats and in their fleas.     

Real‐time and multiplex real‐time polymerase chain reactions for the detection of Bartonella henselae within cat flea,Ctenocephalides felis,samples

Bartonella henselae (Rhizobiales: Bartonellacae), the agent of cat‐scratch disease, is an emerging bacterial pathogen which can be transmitted via infective faecal material of Ctenocephalides felis Bouché (Siphonaptera: Pulicidae). Worldwide, B. henselae has been identified in 1–53% of felines and 2.9–17.4% of fleas. Although culture is the routine method for detection, the procedure is time‐consuming and is rarely used for isolation directly from flea vectors. The current study reports the development of a quantitative real‐time polymerase chain reaction (qPCR) to detect and quantify B. henselae organisms from vector samples. The qPCR is specific and detects as few as 2.5 genome copies. To enable direct quantification of Bartonella organisms in different vector samples, we developed a qPCR to detect C. felis DNA that also acts as an extraction control. Combining both PCRs into a multiplex format validates B. henselae results when sampling flea populations, although there is a reduction in sensitivity. This reduction might be counteracted by a different combination of probe fluorophores.     

Detection of Rickettsia spp. and Bartonella spp. in Ctenocephalides felis fleas from free‐ranging crab‐eating foxes (Cerdocyon thous)

Fleas are insects with a worldwide distribution that have been implicated in the transmission of several pathogens. The present study aimed to investigate the presence of Rickettsia spp. (Rickettsiales: Rickettsiaceae) and Bartonella spp. (Rhizobiales: Bartonellaceae) in fleas from free‐ranging crab‐eating foxes Cerdocyon thous (Linnaeus, 1766) (Carnivora: Canidae) from Rio Grande do Sul, southern Brazil. Fleas were collected manually from animals and used for the molecular detection of Rickettsia spp. and Bartonella spp. Twenty‐nine C. thous were sampled in six municipalities. Four foxes were parasitized by 10 fleas, all of which were identified as Ctenocephalides felis (Bouché, 1935) (Siphonaptera: Pulicidae). DNA from Rickettsia felis Bouyer et al., 2001 and Rickettsia asembonensis Maina et al., 2016 were found in three and eight fleas, respectively. In four fleas, DNA of Bartonella sp. was identified. Phylogenetic analysis grouped Bartonella sp. together with other genotypes previously reported in C. felis worldwide. The scenario described in the present study highlights a Neotropical canid parasitized by the invasive cosmopolitan cat flea, which in turn, is carrying potentially invasive vector‐borne microorganisms. These findings suggest that C. felis is adapted to wild hosts in wilderness areas in southern Brazil, hypothetically exposing the Neotropical fauna to unknown ecological and health disturbances.     

Comparison of the abilities of proteins from Bartonella bacilliformis and Bartonella henselae to deform red cell membranes and to bind to red cell ghost proteins

Infections in humans by Bartonella bacilliformis, but not Bartonella henselae, are characterized by invasion of red cells. Supernatants of culture medium from B. bacilliformis and B. henselae each contain a protein which causes invagination of membranes of human red cells and formation of intracellular vacuoles. These two proteins are very similar in molecular mass, heat stability and mechanism of action. B. henselae does not bind to human red cells, but human red cell ghost membrane proteins were recognized by both bacteria, five by B. bacilliformis and the same five, and one additional protein by B. henselae. Two of these proteins had molecular masses consistent with actin and spectrin. Actin binds to five electroblotted outer membrane proteins from B. henselae and four of these proteins are retained on an actin-Sepharose column.     

Flea species infesting dogs in Spain: updated spatial and seasonal distribution patterns

This entomological survey examines the spatial and seasonal distribution patterns of flea species infesting dogs in Spain. Bioclimatic zones covering broad climate and vegetation ranges were surveyed according to size. In a cross‐sectional spatial survey carried out from late May 2013 to mid‐July 2015, 1084 dogs from 42 different locations were examined. A total of 3032 fleas were collected and identified as belonging to the following species: Ctenocephalides felis (Siphonaptera: Pulicidae) (81.7%, 2476 fleas); Ctenocephalides canis (11.4%, 347 fleas); Pulex irritans (Siphonaptera: Pulicidae) (6.9%, 208 fleas), and Echidnophaga gallinacea (Siphonaptera: Pulicidae) (0.03%, one flea). Variables observed to have effects on flea abundance were animal weight, sex, length of hair and habitat. In the seasonal survey conducted from June 2014 to June 2015, 1014 fleas were collected from 239 dogs at 30 veterinary practices across Spain. Peaks in C. felis abundance were observed in early summer and late autumn, whereas high numbers of P. irritans and C. canis were recorded in autumn. Numbers of fleas detected in winter were low overall. Based on these findings, the present study updates the spatial and seasonal distributions of flea species in Spain and assesses the impacts of host and habitat variables on flea infestation.     

Mixed infections, cryptic diversity, and vector-borne pathogens: evidence from Polygenis fleas and Bartonella species

Coinfections within hosts present opportunities for horizontal gene transfer between strains and competitive interactions between genotypes and thus can be a critical element of the lifestyles of pathogens. Bartonella spp. are Alphaproteobacteria that parasitize mammalian erythrocytes and endothelial cells. Their vectors are thought to be various biting arthropods, such as fleas, ticks, mites, and lice, and they are commonly cited as agents of various emerging diseases. Coinfections by different Bartonella strains and species can be common in mammals, but little is known about specificity and coinfections in arthropod vectors. We surveyed the rate of mixed infections of Bartonella in flea vectors (Polygenis gwyni) parasitizing cotton rats (Sigmodon hispidus) in which previous surveys indicated high rates of infection. We found that nearly all fleas (20 of 21) harbored one or more strains of Bartonella, with rates of coinfection approaching 90%. A strain previously identified as common in cotton rats was also common in their fleas. However, another common strain in cotton rats was absent from P. gwyni, while a rare cotton rat strain was quite common in P. gwyni. Surprisingly, some samples were also coinfected with a strain phylogenetically related to Bartonella clarridgeiae, which is typically associated with felids and ruminants. Finally, a locus (pap31) that is characteristically borne on phage in Bartonella was successfully sequenced from most samples. However, sequence diversity in pap31 was novel in the P. gwyni samples, relative to other Bartonella previously typed with pap31, emphasizing the likelihood of large reservoirs of cryptic diversity in natural populations of the pathogen.     

Detection and identification of Bartonella sp. in fleas from carnivorous mammals in Andalusia,Spain

A total of 559 fleas representing four species (Pulex irritans, Ctenocephalides felis, Ctenocephalides canis and Spilopsyllus cuniculi) collected on carnivores (five Iberian lynx Lynx pardinus, six European wildcat Felis silvestris, 10 common genet Genetta genetta, three Eurasian badger Meles meles, 22 red fox Vulpes vulpes, 87 dogs and 23 cats) in Andalusia, southern Spain, were distributed in 156 pools of monospecific flea from each carnivore, and tested for Bartonella infection in an assay based on polymerase chain reaction (PCR) amplification of the 16 S–23 S rRNA intergenic spacer region. Twenty‐one samples (13.5%) were positive and the sequence data showed the presence of four different Bartonella species. Bartonella henselae was detected in nine pools of Ctenocephalides felis from cats and dogs and in three pools of Ctenocephalides canis from cats; Bartonella clarridgeiae in Ctenocephalides felis from a cat, and Bartonella alsatica in Spilopsyllus cuniculi from a wildcat. DNA of Bartonella sp., closely related to Bartonella rochalimae, was found in seven pools of Pulex irritans from foxes. This is the first detection of B. alsatica and Bartonella sp. in the Iberian Peninsula. All of these Bartonella species have been implicated as agents of human diseases. The present survey confirms that carnivores are major reservoirs for Bartonella spp.     

Bartonella henselae in eastern Poland: the relationship between tick infection rates and the serological response of individuals occupationally exposed to tick bites

To explore the potential role of Ixodes ricinus as the presumed vector of Bartonella henselae in eastern Poland, ticks collected in various geographic locations were examined for the presence of B. henselae, and the results were matched against the prevalence of anti‐B. henselae antibodies in individuals occupationally exposed to tick bites. The presence of Bartonella DNA was investigated by PCR in a total of 1,603 unfed Ixodes ricinus ticks. The presence of IgG antibodies against B. henselae was investigated in serum samples from 332 people occupationally exposed to tick bites (94 farmers and 238 forestry workers). The total prevalence of B. henselae in ticks was 1.7%; the infection rates in males (3.1%) and females (2.7%) were nearly ten times greater than in nymphs (0.3%). The prevalence of seropositive results in the risk group (30.4%), farmers (27.7%) and forestry workers (31.5%), was significantly greater compared to the control group (8.9%). The results showed a weak positive correlation between the degree of infection of ticks and humans living in the same geographic region. The lack of a direct relationship indicates that exposure to tick bites is only one of the factors contributing to the significant preponderance of a seropositive response to B. henselae in the forestry workers and farmers over the control group. Other factors must be considered, such as contact with cats, which are popular domestic animals in Polish villages, and exposure to cat fleas.     

巴尔通体在滇西南蝙蝠中高度流行并具有丰富的遗传变异特征

蝙蝠是很多病原微生物的自然宿主, 全球多项研究表明蝙蝠是巴尔通体(Bartonella species)的主要宿主。为了解滇西南地区蝙蝠中巴尔通体的流行特征, 我们于2015-2017年间在云南省4个地区应用网捕法捕获蝙蝠3种305只。经种类鉴定后采集肝脾组织, 提取核酸, 通过TaqMan实时荧光定量PCR方法检测巴尔通体的tmRNA基因ssrA, 并进行测序鉴定和系统发育分析。结果发现172只蝙蝠检出该基因, 总感染率为56.4%; 其中临沧、西双版纳、保山和瑞丽4个采样点的蝙蝠感染率分别为50.0% (22/44)、61.7% (29/47)、62.1% (18/29)和55.7% (103/185)。中菊头蝠(Rhinolophus affinis)、小菊头蝠(R. blythi)和棕果蝠(Rousettus leschenaultii)的感染率分别为50.0% (22/44)、62.1% (18/29)和56.9% (132/232), 差异没有统计学意义(χ² = 1.135, P = 0.567), 表明巴尔通体在云南当地的蝙蝠种群中高度流行。定量PCR扩增产物2次扩增后测序获得37个巴尔通体ssrA序列, 属于10个系统发育分支, 其中1个为伊丽莎白巴尔通体(B. elizabethae)、特利波契巴尔通体(B. tribocorum)和克拉斯诺夫巴尔通体(B. krasnovii)的近缘种。其余序列与已知巴尔通体距离较远, 与亚洲、欧洲和美洲等其他地域来源于蝙蝠的巴尔通体近缘。遗传多样性分析显示, ssrA基因的核苷酸多样性指数(π)为0.11381 ± 0.00928, 基因型多样性指数(Hd)为0.985 ± 0.010, 形成29个基因型(单倍型), 说明云南蝙蝠巴尔通体具有丰富的遗传多样性。通过对本研究标本与全球相关序列的系统发育网络重建, 分析全球蝙蝠巴尔通体的地理和宿主分布特征, 可以看出巴尔通体与蝙蝠之间存在显著的宿主特异性关联。因此可初步确定蝙蝠-巴尔通体具有协同进化特征, 同时受到地理隔离的影响。     

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, 12-14h 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.     

Molecular detection of Bartonella in fleas (Hexapoda,Siphonaptera) collected from wild rodents (Cricetidae,Sigmodontinae) from Argentina

Bartonella are facultative intracellular Gram‐negative bacteria, transmitted mainly by hematophagous arthropods, and the rodents act as a natural reservoir. Different species of Bartonella associated with rodents have been implicated as causing human disease. Studies from Argentina are scarce and no Bartonella from fleas have been reported previously. The present study investigated the presence of Bartonella spp. in fleas associated with sigmodontine rodents in four localities of the Santa Cruz Province, Argentina. In total, 51 fleas (four species) were analysed of which 41.2% were found to be positive for the gltA gene fragment via a nested polymerase chain reaction. All positive fleas were of the species Neotyphloceras crackensis from three different localities. Eight of the 21 amplified samples were sequenced, and the presence of three different genotypes was detected with an identity of 95.5–98.8% amongst themselves. Bartonella genotypes from American rodents and rodent fleas were recovered in a monophyletic group. Similarly, most of the Peruvian and all Argentinean variants constitute a natural group sister of the American remainder. The importance of the Bartonella spp. with respect to public health is unknown, although future studies could provide evidence of the possible involvement of N. crackensis in the Bartonella transmission cycles.     

Observations on the susceptibility of Ctenocephalides felis (Siphonaptera: Pulicidae) to malathion and permethrin in Tanzania

Laboratory-reared Ctenocephalides felis (Bouche) adults were tested with 0.5% malathion and 0.5% permethrin, using the standard WHO methods. After 24 h exposure to malathion (3.6 mg/cm2), 92% of the fleas died. The LT50 for malathion was approximately 8 h. Permethrin (0.45 mg/cm2) produced 100% mortality of exposed insects after 24 h while with a higher dose (0.9 mg/cm2) all fleas died after 8 h exposure. LT50 for the two doses of permethrin were 7.7 and 1.05 h, respectively. The failure of the diagnostic dose of malathion to kill 100% of the population was attributed to resistance. Permethrin is a suitable pesticide for controlling fleas of domestic animals in Tanzania.     

Phylogenetic relationships and genetic diversity among members of theFestuca-Lolium complex (Poaceae) based on ITS sequence data

There have been few DNA sequencebased studies of phylogenetic relationships within theFestuca-Lolium complex. Here we infer the phylogeny of 31Festuca-Lolium taxa with a dataset of 116 ITS sequences. The results are consistent with previous studies that resolved two majorFestuca clades: one clade of fine fescues and another clade that containsLolium and associatedFestuca species. This study is unique in suggesting a third, basalFestuca clade, but support for the basal position of this group is low. Extensive sampling permitted investigation of the effects of lineage sorting and reticulate events on the evolution of the complex. Roughly half of the taxa show evidence of lineage sorting or reticulation, and the monophyly ofLolium has likely been obscured by reticulate events. Overall, polyploid species harbor higher levels of ITS sequence diversity than diploids; ITS sequence variants may provide clues to the identity of allopolyploid parents.     

Prevalence and genetic diversity of coronaviruses in bats from China

Coronaviruses can infect a variety of animals including poultry, livestock, and humans and are currently classified into three groups. The interspecies transmissions of coronaviruses between different hosts form a complex ecosystem of which little is known. The outbreak of severe acute respiratory syndrome (SARS) and the recent identification of new coronaviruses have highlighted the necessity for further investigation of coronavirus ecology, in particular the role of bats and other wild animals. In this study, we sampled bat populations in 15 provinces of China and reveal that approximately 6.5% of the bats, from diverse species distributed throughout the region, harbor coronaviruses. Full genomes of four coronavirues from bats were sequenced and analyzed. Phylogenetic analyses of the spike, envelope, membrane, and nucleoprotein structural proteins and the two conserved replicase domains, putative RNA-dependent RNA polymerase and RNA helicase, revealed that bat coronaviruses cluster in three different groups: group 1, another group that includes all SARS and SARS-like coronaviruses (putative group 4), and an independent bat coronavirus group (putative group 5). Further genetic analyses showed that different species of bats maintain coronaviruses from different groups and that a single bat species from different geographic locations supports similar coronaviruses. Thus, the findings of this study suggest that bats may play an integral role in the ecology and evolution of coronaviruses.     

Fleas from domestic dogs and rodents in Rwanda carry Rickettsia asembonensis and Bartonella tribocorum

Fleas (Siphonaptera) are ubiquitous blood‐sucking parasites that transmit a range of vector‐borne pathogens. The present study examined rodents (n = 29) and domestic dogs (n = 7) living in the vicinity of the Volcanoes National Park, Rwanda, for fleas, identified flea species from these hosts, and detected Bartonella (Rhizobiales: Bartonellaceae) and Rickettsia (Rickettsiales: Rickettsiaceae) DNA. The most frequently encountered flea on rodents was Xenopsylla brasiliensis (Siphonaptera: Pulicidae). In addition, Ctenophthalmus (Ethioctenophthalmus) calceatus cabirus (Siphonaptera: Hystrichopsyllidae) and Ctenocephalides felis strongylus (Siphonaptera: Pulicidae) were determined using morphology and sequencing of the cytochrome c oxidase subunit I and cytochrome c oxidase subunit II genes (cox1 and cox2, respectively). Bartonella tribocorum DNA was detected in X. brasiliensis and Rickettsia asembonensis DNA (a Rickettsia felis‐like organism) was detected in C. felis strongylus. The present work complements studies that clarify the distributions of flea‐borne pathogens and potential role of fleas in disease transmission in sub‐Saharan Africa. In the context of high‐density housing in central sub‐Saharan Africa, the detection of B. tribocorum and R. asembonensis highlights the need for surveillance in both rural and urban areas to identify likely reservoirs.     

Chemical and genetic diversity of Ligularia latihastata and Ligularia villosa in Yunnan Province of China

The chemical constituents of the root extracts and the nucleotide sequences of the atpB-rbcL intergenic region of Ligularia latihastata and L. villosa, collected in northwestern Yunnan Province, were studied. In the twelve collected samples of L. latihastata, two major benzofurans, 5,6-dimethoxy-2-(1-methylethenyl)-1-benzofuran (1) and euparin (2) were detected as major components. The minor compound (2R*,3S*)-5-acetyl-2,3-dihydro-6-hydroxy-2-(1-methylethenyl)-1-benzofuran-3-yl (2Z)-2-[(acetoxy)methyl]but-2-enoate (4) was found to be susceptible to artifact formation upon extraction with EtOH. The intra-specific diversity in chemical composition of the samples was small, but the diversity in the atpB-rbcL sequence was fairly large. Compounds 1 and 2 were also found in the three collected samples of L. villosa, indicating that the two species are chemically close to each other, in agreement with morphological taxonomy.     

First isolation of Bartonella henselae type I from a cat-scratch disease patient in Japan and its molecular analysis

We isolated Bartonella henselae from an inguinal lymph node of a 36-year-old male patient with cat-scratch disease. The patient had many areas of erythema on his body, swelling of the left inguinal lymph nodes with pain and slight fever. The diagnosis was made on the basis of polymerase chain reaction for B. henselae DNA from the lymph node biopsies and blood sample, and isolation of the organism, histology of the lymph node and serology with an indirect immunofluorescent antibody test. We also analyzed the genome profiles for five strains of 90 isolates from the lymph node by pulsed-field gel electrophoresis after Not I endonuclease digestion. We found two different genomic profiles. These results suggest that the patient had been either co-infected or re-infected with two genetically different strains of B. henselae.