Identification of Bartonella strains isolated from wild and domestic ruminants by a single-step PCR analysis of the 16S-23S intergenic spacer region

Of the 20 species or subspecies of Bartonella currently known, 7 cause various diseases in humans with many being zoonotic. However, some Bartonella species appear only to cause asymptomatic bacteraemia in their hosts. In ruminants, three Bartonella species (B. bovis, B. capreoli and B. schoenbuchensis) have recently been described. However, limited or no information has yet been published concerning their mode of transmission and their possible pathogenicity for domestic cattle. The phylogenetic relationship of these species with other bacteria of the Bartonella genus has only been recently investigated. It is therefore necessary to develop appropriate tools that will easily allow identification of these ruminant strains for epidemiological and clinical studies. A single-step PCR assay, based on the amplification of a fragment of the 16S-23S rRNA intergenic spacer (ITS), was evaluated for identification of Bartonella isolated from domestic cattle and from free-ranging or captive cervids. For each Bartonella species tested, the PCR assay led to a product that was unique either for its length or its sequence. All ruminant isolates tested could be easily differentiated among themselves and from the other Bartonella species. Furthermore, sequence analysis of the PCR products revealed a close relationship between all ruminant Bartonella strains. Therefore, ITS PCR testing appears to be a convenient tool for a quick diagnosis of ruminant Bartonella species.     

巴尔通体体外侵染巨噬细胞特性研究

巴尔通体（Bartonella）是一类革兰氏阴性、营养需求苛刻的兼性胞内需氧菌,感染导致人类罹患猫抓病及杆菌样血管瘤。组织病理学研究结果发现淋巴结内存在大量巴尔通体,提示巴尔通体具有能逃避宿主先天免疫吞噬的功能。然而目前关于巴尔通体与巨噬细胞间相互作用的研究尚未深入开展。本研究利用鼠源巴尔通体（Bartonella tribocorum）体外感染J774A、RAW264.7及C57小鼠腹腔巨噬细胞,探索巴尔通体在巨噬细胞内存活特性。免疫荧光试验结果显示巨噬细胞能有效吞噬巴尔通体,细胞形态未发生显著变化。庆大霉素保护试验结果证实被吞噬的巴尔通体在不同巨噬细胞内均能增殖及存活至48 h,且能在LPS诱导活化的巨噬细胞中存活。     

Bacillary hemoglobinuria in a free-ranging elk calf.

A dead elk (Cervus elaphus roosevelti) calf was diagnosed with bacillary hemoglobinuria, a toxemia caused by the bacterium Clostridium haemolyticum. The mortality occurred in southwest Washington, USA (46 degrees 13'N, 123 degrees 22'W), in an area in which several previous mortalities, suspected but not conclusively diagnosed to be either bacillary hemoglobinuria, enterotoxemia, or leptospirosis, occurred. This is the first reported incidence of mortality attributable to bacillary hemoglobinuria in free-ranging elk. Similar deaths of young elk in the area suggest that mortality from this disease may be common locally.     

Identification of novel Bartonella spp. in bats and evidence of Asian gray shrew as a new potential reservoir of Bartonella

Many studies indicated that small mammals are important reservoirs for Bartonella species. Using molecular methods, several studies have documented that bats could harbor Bartonella. This study was conducted to investigate the relationship of Bartonella spp. identified in bats and small mammals living in the same ecological environment. During May 2009 and March 2010, a total of 102 blood specimens were collected. By whole blood culture and molecular identification, a total of 6 bats, 1 rodent and 9 shrews were shown to be infected by Bartonella species. After sequencing and phylogenetic analyses of the sequences of gltA, ftsZ, rpoB and ribC genes, these specific isolates from bats were not similar to the known Bartonella species (the similarity values were less than 91.2%, 90.5%, 88.8%, and 82.2%, respectively); these isolates formed an independent clade away from other known Bartonella type strains. The Bartonella spp. isolated from small mammals, which were closely related to Bartonella tribocorum, Bartonella elizabethae, Bartonella grahamii, Bartonella rattimassiliensis and Bartonella queenslandensis, were similar to the findings in previous studies worldwide. Therefore, the results implied that the species of Bartonella strains isolated from small mammals were different from those identified in bats. Our results strongly suggested that the bat isolate could be a new Bartonella species. This study is also the first one to isolate Bartonella organisms from Asian gray shrews, Crocidura attenuata tanakae.     

Flea-borne pathogens in the cat flea Ctenocephalides felis and their association with mtDNA diversity of the flea host

Flea-borne pathogens were screened from 100 individual cat fleas using a PCR approach, of which 38 % were infected with at least one bacterium. Overall, 28 % of the flea samples were positive for Bartonella as inferred from ITS DNA region. Of these, 25 % (7/28) were identified as Bartonella clarridgeiae, 42.9 % (12/28) as Bartonella henselae consisted of two different strains, and 32.1 % (9/28) as Bartonella koehlerae, which was detected for the first time in Malaysia. Sequencing of gltA amplicons detected Rickettsia DNA in 14 % of cat flea samples, all of them identified as Rickettsia asembonensis (100 %). None of the flea samples were positive for Mycoplasma DNA in 16S rRNA gene detection. Four fleas were co-infected with Bartonella and Rickettsia DNAs. Statistical analyses reveal no significant association between bacterial infection and mtDNA diversity of the cat flea. Nevertheless, in all types of pathogen infections, infected populations demonstrated lower nucleotide and haplotype diversities compared to uninfected populations. Moreover, lower haplotype numbers were observed in infected populations.     

Differential detection of Bartonella species and strains in cat scratch disease diagnostics by polymerase chain reaction amplification of 16S ribosomal RNA gene.

Cat scratch disease (CSD) has been difficult to diagnose in animals because of the protracted clinical course of infection and the quiescent phases when the microbial culprit lies dormant. The causative agent in CSD appears to be multiple species and strains of Bartonella. Using polymerase chain reaction (PCR) techniques for amplification of highly variable regions of the 16S ribosomal RNA (rRNA) gene sequence, a very sensitive species- and strain-specific assay for CSD-causing Bartonella species was developed. PCR primers were designed to specifically amplify the 16S rRNA gene of Bartonella species but not of other microbial pathogens. This initial PCR was multiplexed with a universal primer set, based on conserved sequence regions in the 16S rRNA gene, that provides a 162-bp fragment in all species tested. Subsequently, 3 distinct nested PCR primer sets enabled the individual amplification and specific detection of Bartonella henselae type 1, B. henselae type II, and B. clarridgeae. Thus, this 2-step PCR procedure enabled the sensitive detection and identification of these species and the B. henselae genotype by exploiting minor sequences differences. Verification of these results were demonstrated with both sequencing and ligase chain reaction techniques. The diagnostic usefulness of this CSD test has been demonstrated by the analysis of specimens from control and infected cats. The diagnosis was confirmed and the specific B. henselae strain was correctly identified in peripheral blood specimens obtained from control and strain-specific CSD-infected cats. Such an accurate and sensitive diagnostic tool for the detection and identification of CSD causative agents should be a useful for the medical, veterinary, and scientific communities.     

麋鹿重组朊蛋白单克隆抗体的制备及初步鉴定

为研制鹿慢性消耗性疾病(chronic wasting disease,CWD)单克隆抗体,本研究以原核表达后经纯化的麋鹿重组成熟朊蛋白(PrP^c)为免疫原免疫PrP^c基因敲除小鼠(PrP^c-null mice)。两次加强免疫后,利用淋巴细胞杂交瘤技术,取脾细胞与SP2/0骨髓瘤细胞进行细胞融合,间接ELISA方法筛选阳性杂交瘤细胞,采用3次有限稀释法实现杂交瘤细胞的亚克隆,筛选出5株能稳定分泌针对麋鹿重组成熟朊蛋白特异性单克隆杭体(McAbs)的杂交瘤细胞株5A5、3B2、6D12、5E3、1F5,其腹水ELISA效价均达到1∶10000以上。经鉴定,5株单抗均为IgG抗体,5A5、6D12、5E3株为IgG1亚类,3B2、1F5株为IgG2a亚类。Western blotting鉴定结果表明,获得的McAbs均能特异性识别麋鹿重组成熟朊蛋白和健康麋鹿脑组织匀浆中的PrP^c。本研究制备了CWD腹水McAbs,同时也为CWD的研究及其诊断方法的建立奠定了基础。     

Canine bartonellosis: serological and molecular prevalence in Brazil and evidence of co-infection with Bartonella henselae and Bartonella vinsonii subsp. berkhoffii

The purpose of this study was to determine the serological and molecular prevalence of Bartonella spp. infection in a sick dog population from Brazil. At the S?o Paulo State University Veterinary Teaching Hospital in Botucatu, 198 consecutive dogs with clinicopathological abnormalities consistent with tick-borne infections were sampled. Antibodies to Bartonella henselae and Bartonella vinsonii subsp. berkhoffii were detected in 2.0% (4/197) and 1.5% (3/197) of the dogs, respectively. Using 16S-23S rRNA intergenic transcribed spacer (ITS) primers, Bartonella DNA was amplified from only 1/198 blood samples. Bartonella seroreactive and/or PCR positive blood samples (n=8) were inoculated into a liquid pre-enrichment growth medium (BAPGM) and subsequently sub-inoculated onto BAPGM/blood-agar plates. PCR targeting the ITS region, pap31 and rpoB genes amplified B. henselae from the blood and/or isolates of the PCR positive dog (ITS: DQ346666; pap31 gene: DQ351240; rpoB: EF196806). B. henselae and B. vinsonii subsp. berkhoffii (pap31: DQ906160; rpoB: EF196805) co-infection was found in one of the B. vinsonii subsp. berkhoffii seroreactive dogs. We conclude that dogs in this study population were infrequently exposed to or infected with a Bartonella species. The B. henselae and B. vinsonii subsp. berkhoffii strains identified in this study are genetically similar to strains isolated from septicemic cats, dogs, coyotes and human beings from other parts of the world. To our knowledge, these isolates provide the first Brazilian DNA sequences from these Bartonella species and the first evidence of Bartonella co-infection in dogs.     

Abomasal parasite syndrome in North American elk (Cervus elaphus canadensis)

CASE HISTORY An 18-month clinical course of chronic ill-thrift, weight loss and emaciation, and eventual death occurred in a group of 520 translocated elk of mixed age and sex. Trans-location was carried out without regard to animal welfare or health risks associated with the translocation. Mortality was approximately 84% (436/520) despite supportive nutritional and medical treatment.

PATHOLOGICAL FINDINGS: General clinical and post-mortem examinations indicated only Se and Cu imbalances and nutritional inadequacy. Additional purposeful post-mortem examination and histological evaluation of tissue sections from four of the affected adult elk demonstrated elevated abomasal pH and proliferative abomasal lesions as the most significant findings, consistent with Type-II ostertagiosis; intra-lesional nematodes were seen in the abomasum of two animals.

DIAGNOSIS: Fading elk syndrome, or abomasal parasite syndrome in elk.

CLINICAL RELEVANCE: Abomasal parasite syndrome initiated by Type-II ostertagiosis should be considered as a differential diagnosis in cases of ill-thrift and wasting in elk or elk-red deer hybrids. Changes to the architecture and secretory function of the abomasal wall lead to apparently irreversible digestive pathophysiology and nutritional disease.     

Bartonella species in wild rodents and fleas from them in Japan

The purpose of this study was to assess the role of fleas for transmission of Bartonella species among wild rodents in Japan. Flea samples were collected from wild rodents and examined genetically for Bartonella infection. Bartonella DNA was detected from 16 of 40 (40.0%) flea samples. Sequence analysis demonstrated that 3 of 16 (18.8%) of the Bartonella-positive animals were infested with fleas from which the closely related Bartonella DNA sequence was detected, indicating that the fleas acquired Bartonella from the infested rodents. The DNA was detected in hemolymph, the midgut and the ovary (only in female), indicating that Bartonella might be colonized through the midgut and distributed into the body.     

Prevalence of Bartonella species DNA and antibodies in cats (Felis catus) submitted to a spay/neuter program in Rio de Janeiro, Brazil

The prevalence of Bartonella species DNA and antibodies for Bartonella henselae were studied in 40 clinically healthy cats (Felis catus, Linnaeus 1758) submitted to a spay/neuter program in Rio de Janeiro, Brazil. Additionally, the prevalence of Bartonella species DNA was investigated in the fleas found parasitizing the subject cats. For this purpose, blood samples were obtained from all cats, and DNA extraction was performed on the blood, and blood clotted samples, as well as on pools of fleas obtained from them. Antibodies for B henselae were detected on serum samples. Bartonella species DNA was detected in 17 cats, whereas serum reactivity for B henselae was found in 19. A total of 20 cats were flea-infested and nine of these 20 had Bartonella species DNA in their blood. In four of the 20 flea-infested cats, Bartonella species DNA was detected in the fleas obtained from those cats, but only one of these four cats had Bartonella species DNA in its blood.     

A comparative study of the interaction of Bartonella henselae strains with human endothelial cells

Bartonella henselae can cause a wide range of clinical outcomes and may lead to severe disease, especially in patients with acquired immunodeficiency syndrome. It is well-known that B. henselae-induced cell proliferation is mediated by anti-apoptotic activity; however, the detailed mechanism is still unclear. In this study, the cellular responses of endothelial cells after infection with four B. henselae strains were compared and protein candidates that may be involved in the interaction between cells and bacteria were determined. The Houston-1 strain elicited the fastest response in terms of stimulating endothelial cell proliferation, and the JK-40 strain had the strongest ability to induce cell proliferation. By Western blot analysis, it was demonstrated that B. henselae-induced cell proliferation involved the mitochondria intrinsic apoptotic pathway. In addition, the adhesion abilities of the U-4 and JK-40 strains were much greater than those of the Houston-1 and JK-47 strains; however, the ability of Houston-1 to invade host cells was high. By two-dimensional gel electrophoresis analysis, it was found that succinyl-CoA synthetase subunit beta, phage-related protein, and ATP synthase subunit alpha might be involved in the invasion process. The expression of superoxide dismutase [Cu-Zn] precursor increased with infection time for all four strains but was significantly higher in the Houston-1 strain, which may increase the competitive advantage of Houston-1 in terms of survival in host cells and render it successful in invading host cells and stimulating cell proliferation. Our data suggest that the interaction of B. henselae and endothelial cells differed between strains, and the results indicated possible candidate proteins that may play a role in the pathogenesis of B. henselae infection.     

Increased risk of chronic wasting disease in Rocky Mountain elk associated with decreased magnesium and increased manganese in brain tissue

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of Rocky Mountain elk in North America. Recent studies suggest that tissue and blood mineral levels may be valuable in assessing TSE infection in sheep and cattle. The objectives of this study were to examine baseline levels of copper, manganese, magnesium, zinc, selenium, and molybdenum in the brains of Rocky Mountain elk with differing prion genotypes and to assess the association of mineral levels with CWD infection. Elk with leucine at prion position 132 had significantly lower magnesium levels than elk with 2 copies of methionine. Chronic wasting disease-positive elk had significantly lower magnesium than control elk. The incorporation of manganese levels in addition to magnesium significantly refined explanatory ability, even though manganese alone was not significantly associated with CWD. This study demonstrated that mineral analysis may provide an additional disease correlate for assessing CWD risk, particularly in conjunction with genotype.     

Molecular cloning and genetic polymorphism of Babesia capreoli gene Bcp37/41, an ortholog of Babesia divergens merozoite surface antigen Bd37

Babesia divergens and Babesia capreoli are closely related species with distinct host ranges, a zoonotic feature being described only for B. divergens. The two species are 99.8% similar in the 18S rDNA gene sequence and indistinguishable by morphological or serological means, leading to confusion as to their species status. The phylogenetic relatedness between the two species, and the frequent involvement of surface components in serological cross-reactions led us to postulate that an ortholog of Bd37, the merozoite surface antigen described for B. divergens, could also exist in B. capreoli. We were able to amplify a single partial PCR product from B. capreoli genomic DNA using primers specific for the B. divergens merozoite surface protein coding gene Bd37, and sequencing confirmed the presence of a Bd37 ortholog in B. capreoli, named Bcp37/41. The full sequences of the Bcp37/41 genes and their intron-exon structures were obtained for two cloned lines of B. capreoli. They suggest functional homologies between Bd37 and Bcp37/41 such as their surface localization, their role in immune escape mechanism and in the initial non-specific attachment to the erythrocyte. Restriction fragment length polymorphism (RFLP) analysis of the amplicons and partial sequencing revealed an extreme polymorphism within B. capreoli, greater than the one observed for its ortholog Bd37. Such a marker could thus be useful in epidemiological as well as phylogenetic studies.     

Effects of a Rest–Rotation Grazing System on Wintering Elk Distributions at Wall Creek,Montana

The large-scale influence of livestock grazing in the western United States generates a need to integrate landscape management to incorporate both wildlife and livestock. The purpose of this project was to evaluate the effects of four different grazing cells (spring grazing, summer growing-season grazing, fall grazing, and resting) on wintering elk resource selection within the Wall Creek range in southwest Montana. We collected biweekly observations of elk (Cervus elaphus) numbers and distributions across the winter range from 1988 to 2007. Using a matched-case control logistic regression model to estimate selection coefficients, we evaluated the effects of annual green-up conditions, winter conditions, landscape features, and grazing treatment on elk group resource selection within the grazing system. We found that within the grazing system, elk groups preferentially selected for rested pastures over pastures that were grazed during the previous spring (1 May–1 June), summer (1 June–15 July), and fall (15 September–30 September). The strength of selection against the pasture grazed during the summer growing season was strongest, and pastures grazed during the spring and fall were selected for over the pasture grazed during the summer. The number of elk utilizing the grazing system increased in the 19 yr following implementation of the grazing system; however, total elk herd size also increased during this time. We found no evidence that the proportion of the elk herd utilizing the grazing system changed following implementation of the rest–rotation grazing system. Wintering elk group preference for rested pastures suggests rested pastures play an important role in rotation grazing systems by conserving forage for wintering elk. Additionally, rested pastures provide important cover for a host of other wildlife species. We recommend wildlife managers maintain rested pastures within rotation grazing systems existing on ungulate winter range.     

Pathogens at the livestock-wildlife interface in Western Alberta: does transmission route matter?

In southwestern Alberta, interactions between beef cattle and free-ranging elk (Cervus elaphus) may provide opportunities for pathogen transmission. To assess the importance of the transmission route on the potential for interspecies transmission, we conducted a cross-sectional study on four endemic livestock pathogens with three different transmission routes: Bovine Viral Diarrhea Virus and Bovine Herpesvirus 1 (predominantly direct transmission), Mycobacterium avium subsp. paratuberculosis (MAP) (indirect fecal-oral transmission), Neospora caninum (indirect transmission with definitive host). We assessed the occurrence of these pathogens in 28 cow-calf operations exposed or non-exposed to elk, and in 10 elk herds exposed or not to cattle. We characterized the effect of species commingling as a risk factor of pathogen exposure and documented the perceived risk of pathogen transmission at this wildlife-livestock interface in the rural community. Herpesviruses found in elk were elk-specific gamma-herpesviruses unrelated to cattle viruses. Pestivirus exposure in elk could not be ascertained to be of livestock origin. Evidence of MAP circulation was found in both elk and cattle, but there was no statistical effect of the species commingling. Finally, N. caninum was more frequently detected in elk exposed to cattle and this association was still significant after adjustment for herd and sampling year clustering, and individual elk age and sex. Only indirectly transmitted pathogens co-occurred in cattle and elk, indicating the potential importance of the transmission route in assessing the risk of pathogen transmission in multi-species grazing systems.     

Transdisciplinary habitat models for elk and cattle as a proxy for bovine tuberculosis transmission risk

Zoonotic diseases such as bovine tuberculosis (TB) that infect wildlife and livestock are particularly difficult to eradicate where wild animals make extensive use of agricultural landscapes. Transmission of TB between cattle (Bos taurus) and wild elk (Cervus elaphus) in southwestern Manitoba, Canada remains poorly understood but there is a risk when comingling occurs on summer pasture. Elk use of cattle summer pastures was assessed using ecological data (187 VHF and 25 GPS collared elk monitored over four years representing 8% of the elk population). Local knowledge was documented by conducting interviews and participatory mapping exercises with 86 cattle producers (98% of those within the study area). Of the 294 cattle pastures mapped by farmers, 13% were used by radio-collared elk, 38% were reported by farmers as being used by elk, and 42% were identified as used by elk when both when all datasets were combined. Cattle pastures that had been used by elk and those that had no elk were compared using binary logistic regression based on each of the three datasets (i.e. farmer observations, radio-collared elk on pasture, and combined dataset). For all three datasets, distance to protected area and proportion of forest cover on the cattle pasture were identified as the most and second most important predictor variables, respectively. There was strong agreement among the relative probabilities of elk occurrence on each pasture derived from the resource selection function (RSF) models developed using farmer interviews and elk collaring data. The farmer interview and collar datasets were then combined to generate a final integrated RSF map summarizing the probability of elk–cattle comingling and were contrasted over each of four cattle grazing seasons (spring, early summer, late summer, and autumn). These predictive maps indicate that use of cattle pastures by elk is extensive, particularly in spring and early summer. Farmer observations indicate that elk and cattle share water sources and livestock mineral supplements on pasture. Local knowledge and conventional ecological data complement and validate one another and help us better understand the temporospatial aspects of shared space use among wildlife and livestock and more generally the risks of disease transmission in agricultural landscapes.     

Evaluation of the anaplasmosis rapid card agglutination test for detecting experimentally infected elk

Anaplasmosis was experimentally transmitted from cattle to elk to cattle. Six non-splenectomized adult elk (Cervus canadensis canadensis) that were inoculated with freshly collected heparinized blood from cattle chronically infected with Anaplasma marginale became asymptomatic carriers. Although the exposed elk did not develop clinical or hematologic evidence of infection they become seropositive by the serum and plasma rapid card agglutination (RCA) tests. Blood from the experimentally infected elk produced disease in splenectomized bovine calves and the carrier state persisted for at least one year. Infection did not occur when two elk were inoculated with 0.5 ml of frozen blood from known bovine carriers. The blood had been frozen for four weeks in liquid nitrogen with six percent dimethyl-sulfoxide.     

Bartonella bovis and Candidatus Bartonella davousti in cattle from Senegal

In Senegal, domestic ruminants play a vital role in the economy and agriculture and as a food source for people. Bartonellosis in animals is a neglected disease in the tropical regions, and little information is available about the occurrence of this disease in African ruminants. Human bartonellosis due to Bartonella quintana has been previously reported in Senegal. In this study, 199 domestic ruminants, including 104 cattle, 43 sheep, and 52 goats were sampled in villages from the Senegalese regions of Sine Saloum and Casamance. We isolated 29 Bartonella strains, all exclusively from cattle. Molecular and genetic characterization of isolated strains identified 27 strains as Bartonella bovis and two strains as potentially new species. The strains described here represent the first Bartonella strains isolated from domestic ruminants in Senegal and the first putative new Bartonella sp. isolated from cattle in Africa.     

Candidatus Bartonella antechini: a novel Bartonella species detected in fleas and ticks from the yellow-footed antechinus (Antechinus flavipes), an Australian marsupial

Bartonella are fastidious, Gram-negative, aerobic bacilli belonging to the Alphaproteobacteria group. In the last ten years, the discovery of new Bartonella species from a variety of mammalian hosts, arthropod vectors and geographical areas has increased. More than 20 species of Bartonella have been identified, of which approximately thirteen are associated with disease in humans and animals. Recently, four novel species of Bartonella were isolated from mammalian hosts in Australia: Bartonella australis from eastern grey kangaroos (Macropus giganteus) and Bartonella rattaustraliani, Bartonella queenslandensis and Bartonella coopersplainsensis from rodents. Bartonella-like organisms have also been detected from Ixodes tasmani ticks collected from koalas (Phascolarctos cinereus). However, very little is known about Bartonella spp. in other marsupials in Australia. We report the identification of a novel Bartonella species detected from fleas (Acanthopsylla jordani) and ticks (Ixodes antechini) collected from a small carnivorous marsupial, Antechinus flavipes (Mardos or Yellow-footed antechinus) in the southwest of Western Australia. New nested-PCRs targeting the gltA gene and the ribosomal ITS region were developed as part of the present study. DNA sequencing of the 16S rRNA, gltA, ftsZ and rpoB genes and the ribosomal ITS region revealed that this detection is a distinct Bartonella species and is related to B. australis isolated from kangaroos. This is the first report of two different possible arthropod vectors in Australia (ticks and fleas) being infected with the same species of Bartonella. We propose the name Candidatus Bartonella antechini n. sp. for the recently characterized organism.