Bartonella in Norway rats (Rattus norvegicus) from the urban slum environment in Brazil.

Bartonella are rodent-borne bacteria that cause varied human etiologies. Studies on synanthropic rodents are rare, causing gaps in epidemiological knowledge. We tested bloodclot samples from 79 rats from an urban slum in Salvador, Brazil through PCR targeting gltA gene. Nine samples (11.4%) were positive: six had 100% identity with Bartonella sp. isolate JF429580 and 99.5% with B. queenslandensis strain AUST/NH8; three were 100% identical to isolate JF429532 and 99.7% to B. tribocorum. This is the second report on urban rat Bartonella indicating bacterial circulation at detectable rates. Its presence in rats from vulnerable human settlements demands public health attention.

Geo-temporal patterns to design cost-effective interventions for zoonotic diseases -the case of brucellosis in the country of Georgia.

Introduction: Control of zoonosis can benefit from geo-referenced procedures. Focusing on brucellosis, here the ability of two methods to distinguish disease dissemination patterns and promote cost-effective interventions was compared. Method: Geographical data on bovine, ovine and human brucellosis reported in the country of Georgia between 2014 and 2019 were investigated with (i) the Hot Spot (HS) analysis and (ii) a bio-geographical (BG) alternative. Results: More than one fourth of all sites reported cases affecting two or more species. While ruminant cases displayed different patterns over time, most human cases described similar geo-temporal features, which were associated with the route used by migrant shepherds. Other human cases showed heterogeneous patterns. The BG approach identified small areas with a case density twice as high as the HS method. The BG method also identified, in 2018, a 2.6-2.99 higher case density in zoonotic (human and non-human) sites than in non-zoonotic sites (which only reported cases affecting a single species) -a finding that, if corroborated, could support cost-effective policy-making. Discussion: Three dissemination hypotheses were supported by the data: (i) human cases induced by sheep-related contacts; (ii) human cases probably mediated by contaminated milk or meat; and (iii) cattle and sheep that infected one another. This proof-of-concept provided a preliminary validation for a method that may support cost-effective interventions oriented to control zoonoses. To expand these findings, additional studies on zoonosis-related decision-making are recommended.

Plague and Trace Metals in Natural Systems.

All pathogenic organisms are exposed to abiotic influences such as the microclimates and chemical constituents of their environments. Even those pathogens that exist primarily within their hosts or vectors can be influenced directly or indirectly. Yersinia pestis, the flea-borne bacterium causing plague, is influenced by climate and its survival in soil suggests a potentially strong influence of soil chemistry. We summarize a series of controlled studies conducted over four decades in Russia by Dr. Evgeny Rotshild and his colleagues that investigated correlations between trace metals in soils, plants, and insects, and the detection of plague in free-ranging small mammals. Trace metal concentrations in plots where plague was detected were up to 20-fold higher or lower compared to associated control plots, and these differences were >2-fold in 22 of 38 comparisons. The results were statistically supported in eight studies involving seven host species in three families and two orders of small mammals. Plague tended to be positively associated with manganese and cobalt, and the plague association was negative for copper, zinc, and molybdenum. In additional studies, these investigators detected similar connections between pasturellosis and concentrations of some chemical elements. A One Health narrative should recognize that the chemistry of soil and water may facilitate or impede epidemics in humans and epizootics in non-human animals.

Molecular Characterization of a Novel Relapsing Fever Borrelia Species from the Desert Cottontail (Sylvilagus audubonii) in New Mexico, USA.

The Borrelia genus comprises vector-borne, spirochete bacteria infecting vertebrates worldwide. We characterized a novel relapsing fever Borrelia species from a desert cottontail (Syvilagus audubonii) from New Mexico, US, using an established multilocus sequence analysis approach. Phylogenetic analysis of the flagellin gene (flaB) and four other protein-coding loci (clpX, pepX, recG, rplB) grouped the novel Borrelia species with hard tick relapsing fever borreliae Borrelia lonestari, Borrelia theileri, and Borrelia miyamotoi. The identity of the vectors and other vertebrate hosts, geographic distribution, and zoonotic potential of this novel Borrelia species deserve further investigation.

Presence of Leptospira spp. and absence of Bartonella spp. in urban rodents of Buenos Aires province, Argentina.

Big cities of Argentina are characterized by a strong social and economic fragmentation. This context enables the presence of urban rodents in close contact to the human population, mostly in the peripheral areas of the cities. Urban rodents can harbor a large variety of zoonotic pathogens. The aim of this study was to molecularly characterize Leptospira spp. and Bartonella spp. in urban rodents from the area of Gran La Plata, Buenos Aires province, Argentina. The species of urban rodents captured and tested were Rattus norvegicus, Rattus rattus, and Mus musculus. Leptospira interrogans and L. borgpetersenii were detected in R. norvegicus and M. musculus respectively. Bartonella spp. DNA was not detected in any of the kidney samples tested. No significant differences were observed between the prevalence of bacteria and rodent and environmental variables such as host sex, presence of stream and season by Generalized Linear Model analysis. These results confirm the role of urban rodents as infection sources of Leptospira spp., suggesting the need to implement public health measures to prevent the transmission of Leptospira spp. and other zoonotic pathogens from rodents to humans. Bartonella was not detected in this set of samples.

Evidence of Extensive Circulation of Yersinia enterocolitica in Rodents and Shrews in Natural Habitats from Retrospective and Perspective Studies in South Caucasus.

Yersinia enterocolitica culture-positive rodents and shrews were reported in different territories across Georgia during 14 of 17 years of investigations conducted for the period of 1981-1997. In total, Y. enterocolitica was isolated from 2052 rodents (15 species) and 33 shrews. Most isolates were obtained from Microtus arvalis, Rattus norvegicus, Mus musculus, and Apodemus spp. During the prospective study (2017-2019), isolates of Yersinia-like bacteria were cultured from 53 rodents collected in four parts of Georgia. All the Yersinia-like isolates were confirmed as Y. enterocolitica based on the API 20E and the BD Phenix50 tests. Whole-genome (WG) sequencing of five rodents and one shrew strain of Y. enterocolitica revealed that they possessed a set of virulence genes characteristic of the potentially pathogenic strains of biogroup 1A. All isolates lacked distinguished virulence determinants for YstA, Ail, TccC, VirF, and virulence plasmid pYV but carried the genes for YstB, YmoA, HemPR-HmuVSTU, YaxAB, PhlA, PldA, ArsCBR, and a flagellar apparatus. One strain contained a gene highly homologous to heat-labile enterotoxin, a chain of E. coli, a function not previously described for Y. enterocolitica. The WG single-nucleotide polymorphism-based typing placed the isolates in four distinct phylogenetic clusters.

Comparison of sand fly trapping approaches for vector surveillance of Leishmania and Bartonella species in ecologically distinct, endemic regions of Peru.

BACKGROUND: In Peru, the information regarding sand fly vectors of leishmaniasis and bartonellosis in the Amazon region is limited. In this study, we carried out sand fly collections in Peruvian lowland and highland jungle areas using different trap type configurations and screened them for Leishmania and Bartonella DNA. METHODOLOGY/PRINCIPAL FINDINGS: Phlebotomine sand flies were collected in Peruvian Amazon jungle and inter Andean regions using CDC light trap, UV and color LED traps, Mosquito Magnet trap, BG Sentinel trap, and a Shannon trap placed outside the houses. Leishmania spp. screening was performed by kDNA PCR and confirmed by a nested cytochrome B gene (cytB) PCR. Bartonella spp. screening was performed by ITS PCR and confirmed by citrate synthase gene (gltA). The PCR amplicons were sequenced to identify Leishmania and Bartonella species. UV and Blue LED traps collected the highest average number of sand flies per hour in low jungle; UV, Mosquito Magnet and Shannon traps in high jungle; and Mosquito Magnet in inter Andean region. Leishmania guyanensis in Lutzomyia carrerai carrerai and L. naiffi in Lu. hirsuta hirsuta were identified based on cytB sequencing. Bartonella spp. related to Bartonella bacilliformis in Lu. whitmani, Lu. nevesi, Lu. hirsuta hirsuta and Lu. sherlocki, and a Bartonella sp. related to Candidatus B. rondoniensis in Lu. nevesi and Lu. maranonensis were identified based on gltA gene sequencing. CONCLUSIONS/SIGNIFICANCE: UV, Blue LED, Mosquito Magnet and Shannon traps were more efficient than the BG-Sentinel, Green, and Red LED traps. This is the first report of L. naiffi and of two genotypes of Bartonella spp. related to B. bacilliformis and Candidatus B. rondoniensis infecting sand fly species from the Amazon region in Peru.

Exposure of Domestic Cats to Three Zoonotic Bartonella Species in the United States.

Cat-associated Bartonella species, which include B. henselae, B. koehlerae, and B. clarridgeiae, can cause mild to severe illness in humans. In the present study, we evaluated 1362 serum samples obtained from domestic cats across the U.S. for seroreactivity against three species and two strain types of Bartonella associated with cats (B. henselae type 1, B. henselae type 2, B. koehlerae, and B. clarridgeiae) using an indirect immunofluorescent assay (IFA). Overall, the seroprevalence at the cutoff titer level of ≥1:64 was 23.1%. Seroreactivity was 11.1% and 3.7% at the titer level cutoff of ≥1:128 and at the cutoff of ≥1:256, respectively. The highest observation of seroreactivity occurred in the East South-Central, South Atlantic, West North-Central, and West South-Central regions. The lowest seroreactivity was detected in the East North-Central, Middle Atlantic, Mountain, New England, and Pacific regions. We observed reactivity against all four Bartonella spp. antigens in samples from eight out of the nine U.S. geographic regions.

Bats are key hosts in the radiation of mammal-associated Bartonella bacteria.

Bats are notorious reservoirs of several zoonotic diseases and may be uniquely tolerant of infection among mammals. Broad sampling has revealed the importance of bats in the diversification and spread of viruses and eukaryotes to other animal hosts. Vector-borne bacteria of the genus Bartonella are prevalent and diverse in mammals globally and recent surveys have revealed numerous Bartonella lineages in bats. We assembled a sequence database of Bartonella strains, consisting of nine genetic loci from 209 previously characterized Bartonella lineages and 121 new cultured isolates from bats, and used these data to perform a comprehensive phylogenetic analysis of the Bartonella genus. This analysis included estimation of divergence dates using a molecular clock and ancestral reconstruction of host associations and geography. We estimate that Bartonella began infecting mammals 62 million years ago near the Cretaceous-Paleogene boundary. Additionally, the radiation of particular Bartonella clades correlate strongly to the timing of diversification and biogeography of mammalian hosts. Bats were inferred to be the ancestral hosts of all mammal-associated Bartonella and appear to be responsible for the early geographic expansion of the genus. We conclude that bats have had a deep influence on the evolutionary radiation of Bartonella bacteria and their spread to other mammalian orders. These results support a 'bat seeding' hypothesis that could explain similar evolutionary patterns in other mammalian parasite taxa. Application of such phylogenetic tools as we have used to other taxa may reveal the general importance of bats in the ancient diversification of mammalian parasites.

Flea Presence and Abundance Are not Predictors of Bartonella tribocorum Carriage in Norway Rats (Rattus norvegicus) from an Underserved Neighborhood of Vancouver, Canada.

Urban Norway rats (Rattus norvegicus) carry pathogenic Bartonella spp. that are transmitted among rats and from rats to people through arthropod vectors, particularly fleas. There is marked temporospatial variation in Bartonella spp. carriage among Norway rats in Vancouver, Canada, and we investigated whether this variation is associated with flea presence or abundance. Bartonella triborocum was isolated from 96/370 (35%) rats and 211 (57%) rats had fleas with an average of one flea per rat. All fleas were identified as Nosopsyllus fasciatus. There was no significant relationship between B. tribocorum carriage and flea presence or abundance, suggesting that, in contrast to other rat-associated zoonoses transmitted by fleas (e.g., Yersinia pestis) flea indices may not be informative for understanding the ecology of Bartonella spp. in rats, particularly for N. fasciatus.

Trypanosoma (Herpetosoma) diversity in rodents and lagomorphs of New Mexico with a focus on epizootological aspects of infection in Southern Plains woodrats (Neotoma micropus).

Protozoan parasites of the genus Trypanosoma infect a broad diversity of vertebrates and several species cause significant illness in humans. However, understanding of the phylogenetic diversity, host associations, and infection dynamics of Trypanosoma species in naturally infected animals is incomplete. This study investigated the presence of Trypanosoma spp. in wild rodents and lagomorphs in northern New Mexico, United States, as well as phylogenetic relationships among these parasites. A total of 458 samples from 13 rodent and one lagomorph species collected between November 2002 and July 2004 were tested by nested PCR targeting the 18S ribosomal RNA gene (18S rRNA). Trypanosoma DNA was detected in 25.1% of all samples, with the highest rates of 50% in Sylvilagus audubonii, 33.1% in Neotoma micropus, and 32% in Peromyscus leucopus. Phylogenetic analysis of Trypanosoma sequences revealed five haplotypes within the subgenus Herpetosoma (T. lewisi clade). Focused analysis on the large number of samples from N. micropus showed that Trypanosoma infection varied by age class and that the same Trypanosoma haplotype could be detected in recaptured individuals over multiple months. This is the first report of Trypanosoma infections in Dipodomys ordii and Otospermophilus variegatus, and the first detection of a haplotype phylogenetically related to T. nabiasi in North America in S. audubonii. This study lends important new insight into the diversity of Trypanosoma species, their geographic ranges and host associations, and the dynamics of infection in natural populations.

Molecular detection of Bartonella henselae, Bartonella clarridgeiae and Rickettsia felis in cat and dog fleas in Tenerife, Canary Islands, Spain.

The cat flea Ctenocephalides felis is the main vector of Bartonella henselae and Bartonella clarridgeiae, the causative agents of cat-scratch disease (CSD) and the spotted-fever agent Rickettsia felis. In spite of their worldwide distribution, there are no data on the occurrence of CSD-causing Bartonella species or the prevalence of Rickettsia species in the Canary Islands, Spain. Therefore, the aim of our study was to screen cat and dog fleas for both pathogens. A total of 128 C. felis from cats and dogs were screened for Bartonella and Rickettsia by PCR. Bartonella henselae (2.3%) and B. clarridgeiae (3.9%) were found in fleas infesting cats, whereas R. felis was identified in both cat (36.6%) and dog (40.7%) fleas. Further, co-infections were observed. This work constitutes the first finding of CSD-causing Bartonella species and the first study on the prevalence of R. felis in fleas from domestic animals in the Canary Islands. These results indicate public health importance, as associated infections could be misdiagnosed in the Archipelago despite their clinical relevance. Establishing human and animal routine diagnosis procedures for these pathogens along with improving vector control in shelters is necessary in order to prevent the spread of the infections among animals.

Pentaplex real-time PCR for differential detection of Yersinia pestis and Y. pseudotuberculosis and application for testing fleas collected during plague epizootics.

Upon acquiring two unique plasmids (pMT1 and pPCP1) and genome rearrangement during the evolution from Yersinia pseudotuberculosis, the plague causative agent Y. pestis is closely related to Y. pseudotuberculosis genetically but became highly virulent. We developed a pentaplex real-time PCR assay that not only detects both Yersinia species but also differentiates Y. pestis strains regarding their plasmid profiles. The five targets used were Y. pestis-specific ypo2088, caf1, and pst located on the chromosome, plasmids pMT1 and pPCP1, respectively; Y. pseudotuberculosis-specific chromosomal gene opgG; and 18S ribosomal RNA gene as an internal control for flea DNA. All targets showed 100% specificity and high sensitivity with limits of detection ranging from 1 fg to 100 fg, with Y. pestis-specific pst as the most sensitive target. Using the assay, Y. pestis strains were differentiated 100% by their known plasmid profiles. Testing Y. pestis and Y. pseudotuberculosis-spiked flea DNA showed there is no interference from flea DNA on the amplification of targeted genes. Finally, we applied the assay for testing 102 fleas collected from prairie dog burrows where prairie dog die-off was reported months before flea collection. All flea DNA was amplified by 18S rRNA; no Y. pseudotuberculosis was detected; one flea was positive for all Y. pestis-specific targets, confirming local Y. pestis transmission. Our results indicated the assay is sensitive and specific for the detection and differentiation of Y. pestis and Y. pseudotuberculosis. The assay can be used in field investigations for the rapid identification of the plague causative agent.

Identification of a Novel Yersinia enterocolitica Strain from Bats in Association with a Bat Die-Off That Occurred in Georgia (Caucasus).

Yersinia entercolitica is a bacterial species within the genus Yersinia, mostly known as a human enteric pathogen, but also recognized as a zoonotic agent widespread in domestic pigs. Findings of this bacterium in wild animals are very limited. The current report presents results of the identification of cultures of Y. entercolitica from dead bats after a massive bat die-off in a cave in western Georgia. The growth of bacterial colonies morphologically suspected as Yersinia was observed from three intestine tissues of 11 bats belonging to the Miniopterus schreibersii species. These three isolates were identified as Y. enterocolitica based on the API29 assay. No growth of Brucella or Francisella bacteria was observed from tissues of dead bats. Full genomes (a size between 4.6-4.7 Mbp) of the Yersinia strains isolated from bats were analyzed. The phylogenetic sequence analyses of the genomes demonstrated that all strains were nearly identical and formed a distinct cluster with the closest similarity to the environmental isolate O:36/1A. The bat isolates represent low-pathogenicity Biotype 1A strains lacking the genes for the Ail, Yst-a, Ysa, and virulence plasmid pYV, while containing the genes for Inv, YstB, and MyfA. Further characterization of the novel strains cultured from bats can provide a clue for the determination of the pathogenic properties of those strains.

Bartonella species in medically important mosquitoes, Central Europe.

Here, we provide the first mass molecular screening of medically important mosquitoes for Bartonella species using multiple genetic markers. We examined a total of 72,115 mosquito specimens, morphologically attributed to Aedes vexans (61,050 individuals), Culex pipiens (10,484 individuals) and species of the Anopheles maculipennis complex (581 individuals) for Bartonella spp. The initial screening yielded 63 Bartonella-positive A. vexans mosquitoes (mean prevalence 0.1%), 34 Bartonella-positive C. pipiens mosquitoes (mean prevalence 0.3%) and 158 Bartonella-positive A. maculipennis group mosquitoes (mean prevalence 27.2%). Several different Bartonella ITS sequences were recovered. This study highlights the need for molecular screening of mosquitoes, the most important vectors of arthropod-borne pathogens, for potential bacterial agents.

Longitudinal Study of Bacterial Infectious Agents in a Community of Small Mammals in New Mexico.

Background and Objectives: Vector-borne bacterial diseases represent a substantial public health burden and rodents have been recognized as important reservoir hosts for many zoonotic pathogens. This study investigates bacterial pathogens in a small mammal community of the southwestern United States of America. Methods: A total of 473 samples from 13 wild rodent and 1 lagomorph species were tested for pathogens of public health significance: Bartonella, Brucella, Yersinia, Borrelia, Rickettsia spp., and Anaplasma phagocytophilum. Results: Three animals were positive for Yersinia pestis, and one Sylvilagus audubonii had a novel Borrelia sp. of the relapsing fever group. No Brucella, Rickettsia, or A. phagocytophilum infections were detected. Bartonella prevalence ranged between 0% and 87.5% by animal species, with 74.3% in the predominant Neotoma micropus and 78% in the second most abundant N. albigula. The mean duration of Bartonella bacteremia in mark-recaptured N. micropus and N. albigula was 4.4 months, ranging from <1 to 18 months, and differed among Bartonella genogroups. Phylogenetic analysis of the Bartonella citrate synthase gene (gltA) revealed 9 genogroups and 13 subgroups. Seven genogroups clustered with known or previously reported Bartonella species and strains while two were distant enough to represent new Bartonella species. We report, for the first time, the detection of Bartonella alsatica in North America in Sylvilagus audubonii and expand the known host range of Bartonella washoensis to include Otospermophilus variegatus. Interpretation and Conclusion: This work broadens our knowledge of the hosts and geographic range of bacterial pathogens that could guide future surveillance efforts and improves our understanding of the dynamics of Bartonella infection in wild small mammals.

Identification of Bartonella rochalimae in Guinea Pigs (Cavia porcellus) and Fleas Collected from Rural Peruvian Households.

In the present study, we tested 391 fleas collected from guinea pigs (Cavia porcellus) (241 Pulex species, 110 Ctenocephalides felis, and 40 Tiamastus cavicola) and 194 fleas collected from human bedding and clothing (142 Pulex species, 43 C. felis, five T. cavicola, and four Ctenocephalides canis) for the presence of Bartonella DNA. We also tested 83 blood spots collected on Flinders Technology Associates (FTA) cards from guinea pigs inhabiting 338 Peruvian households. Bartonella DNA was detected in 81 (20.7%) of 391 guinea pig fleas, in five (2.6%) of 194 human fleas, and in 16 (19.3%) of 83 guinea pig blood spots. Among identified Bartonella species, B. rochalimae was the most prevalent in fleas (89.5%) and the only species found in the blood spots from guinea pigs. Other Bartonella species detected in fleas included B. henselae (3.5%), B. clarridgeiae (2.3%), and an undescribed Bartonella species (4.7%). Our results demonstrated a high prevalence of zoonotic B. rochalimae in households in rural areas where the research was conducted and suggested a potential role of guinea pigs as a reservoir of this bacterium.

Janibacter species with evidence of genomic polymorphism isolated from resected heart valve in a patient with aortic stenosis.

The authors report isolation and identification of two strains of bacteria belonging to the genus Janibacter from a human patient with aortic stenosis from a rural area of the country of Georgia. The microorganisms were isolated from aortic heart valve. Two isolates with slightly distinct colony morphologies were harvested after sub-culturing from an original agar plate. Preliminary identification of the isolates is based on amplification and sequencing of a fragment of 16SrRNA. Whole genome sequencing was performed using the Illumina MiSeq instrument. Both isolates were identified as undistinguished strains of the genus Janibacter. Characterization of whole genome sequences of each culture has revealed a 15% difference in gene profile between the cultures and confirmed that both strains belong to the genus Janibacter with the closest match to J. terrae. Genomic comparison of cultures of Janibacter obtained from human cases and from environmental sources presents a promising direction for evaluating a role of these bacteria as human pathogens.

Marmots and Yersinia pestis Strains in Two Plague Endemic Areas of Tien Shan Mountains.

The main purpose of this study was to clarify the role of gray marmots (Marmota baibacina) in the long-term maintenance of highly virulent strains of Yersinia pestis in two plague endemic foci of the Tien Shan Mountains in Kyrgyzstan. We present data from regular observations of populations of M. baibacina and small rodents cohabiting with marmots in the mountainous grasslands of the Sari-Dzhas (east of Issyk-Kul Lake) and the Upper-Naryn (south of Issyk-Kul Lake) natural foci. During 2012-2017, an abundance of marmots and their ectoparasites (fleas and ticks) was significantly higher in Upper-Naryn comparing to Sari-Dzhas, although there were no differences in a number and diversity of small rodents cohabiting with marmots. The plague bacterium was detected either in marmots or in their ectoparasites collected during 4 of 6 years of observation in Sari-Dzhas and during 2 of 4 years of observation in Upper-Naryn. Plague was found in three sectors situated closely to each other in Sari-Dzhas and in 1 of 8 repeatedly surveyed sectors in Upper-Naryn. During 6 years, we isolated 9 strains of Y. pestis from marmots, two from their fleas Oropsylla silantiewi, one from an unidentified tick, and one from the gray hamster (Cricetulus migratorius). All plague strains isolated from the rodents and their ectoparasites in this study were similar to Antiqua biovar specific for marmots. The results indicate that plague can circulate continuously in the Tien Shan Mountains in populations of gray marmots and their ectoparasites with a facultative involvement of other rodent species after significant changes in rodent communities that happened in Kyrgyzstan during the previous two decades. The simultaneous field survey of two natural foci of plague, Sari-Dzhas, and Upper-Naryn, would be important for further analysis of circulation of Y. pestis strains belonging to Antiqua biovar in the Tien Shan Mountains.