Declines in large wildlife increase landscape-level prevalence of rodent-borne disease in Africa.

Populations of large wildlife are declining on local and global scales. The impacts of this pulse of size-selective defaunation include cascading changes to smaller animals, particularly rodents, and alteration of many ecosystem processes and services, potentially involving changes to prevalence and transmission of zoonotic disease. Understanding linkages between biodiversity loss and zoonotic disease is important for both public health and nature conservation programs, and has been a source of much recent scientific debate. In the case of rodent-borne zoonoses, there is strong conceptual support, but limited empirical evidence, for the hypothesis that defaunation, the loss of large wildlife, increases zoonotic disease risk by directly or indirectly releasing controls on rodent density. We tested this hypothesis by experimentally excluding large wildlife from a savanna ecosystem in East Africa, and examining changes in prevalence and abundance of Bartonella spp. infection in rodents and their flea vectors. We found no effect of wildlife removal on per capita prevalence of Bartonella infection in either rodents or fleas. However, because rodent and, consequently, flea abundance doubled following experimental defaunation, the density of infected hosts and infected fleas was roughly twofold higher in sites where large wildlife was absent. Thus, defaunation represents an elevated risk in Bartonella transmission to humans (bartonellosis). Our results (i) provide experimental evidence of large wildlife defaunation increasing landscape-level disease prevalence, (ii) highlight the importance of susceptible host regulation pathways and host/vector density responses in biodiversity-disease relationships, and (iii) suggest that rodent-borne disease responses to large wildlife loss may represent an important context where this relationship is largely negative.

Bartonella spp. in rats and zoonoses, Los Angeles, California, USA.

Bartonella spp. were detected in rats (Rattus norvegicus) trapped in downtown Los Angeles, California, USA. Of 200 rats tested, putative human pathogens, B. rochalimae and B. tribocorum were found in 37 (18.5%) and 115 (57.5%) rats, respectively. These bacteria among rodents in a densely populated urban area are a public health concern.

Bartonella species in dogs and their ectoparasites from Khon Kaen Province, Thailand.

In order to access the prevalence of Bartonella species in dogs, whole blood and any associated ectoparasites were collected from 164 dogs with owners in 25 villages throughout Khon Kaen Province. DNA was extracted from dog blood, 92 ticks (Rhipicephalus sanguineus) and 137 fleas (Ctenocephalides spp) and screened by PCR using intergenic spacer region and citrate synthase gene primers. B. clarridgeiae DNA was detected in blood of 3 dogs, 4 C. felis and 1 C. canis; B. rochalimae DNA was found in 1 tick; and B. vinsonii subsp vinsonii DNA was found in 2 C. felis. The findings indicate that dogs residing in northeast Thailand are exposed to diverse Bartonella species that are also potential human pathogens.

A Review of Bartonella Infections in California-Implications for Public and Veterinary Health.

Bartonella are vector-transmitted, intracellular bacteria that infect a wide variety of blood-feeding arthropods and their vertebrate hosts. In California, more than 13 species of Bartonella have been described from companion animals, livestock, and wildlife, of which four have been associated with human disease. Infections in humans cause a range of symptoms from relatively mild to severe, especially in immunocompromised individuals. Exposure to infected domestic animals and wildlife, and their ectoparasites, may increase the risk of cross-species transmission. The objective of this review was to compile and summarize published materials on human and animal Bartonella infections in California. Medical and veterinary case reports of bartonellosis were highlighted in an effort to increase the awareness of this poorly understood and potentially under-recognized disease among healthcare professionals and veterinarians.

Molecular detection and identification of Bartonella species in Xenopsylla cheopis fleas (Siphonaptera: Pulicidae) collected from Rattus norvegicus rats in Los Angeles, California.

Of 200 individual Xenopsylla cheopis fleas removed from Rattus norvegicus rats trapped in downtown Los Angeles, CA, 190 (95%) were positive for the presence of Bartonella DNA. Ninety-one amplicons were sequenced: Bartonella rochalimae-like DNA was detected in 66 examined fleas, and Bartonella tribocorum-like DNA was identified in 25 fleas. The data obtained from this study demonstrate an extremely high prevalence of Bartonella DNA in rat-associated fleas.

Molecular detection of Bartonella species in ticks from Peru.

A total of 103 ticks, collected from canines, horses, donkeys, and snakes from Peru, were screened for the presence of Bartonella DNA by polymerase chain reaction analysis. Bartonella DNA was detected in two ticks using Bartonella 16S-23S intergenic spacer region primers and in an additional two ticks using Bartonella NADH dehydrogenase gamma subunit gene (nuoG) primers. Bartonella rochalimae Eremeeva et al., B. quintana Schmincke, and B. elizabethae Daly et al. DNA was detected in a Rhipicephalus sanguineus Latreille (Acari: Ixodidae) female tick removed from a dog and B. quintana DNA was present in a Dermacentor nitens Neumann (Acari: Ixodidae) pool of five larvae, one nymph, and one adult male tick collected from donkeys. This is the first study to report the detection of B. rochalimae, B. quintana, and B. elizabethae DNA in ticks from Peru. Further investigations must be performed to decipher the role ticks may play in the transmission of Bartonella species.

Potential for tick-borne bartonelloses.

As worldwide vectors of human infectious diseases, ticks are considered to be second only to mosquitoes. Each tick species has preferred environmental conditions and biotopes that determine its geographic distribution, the pathogens it vectors, and the areas that pose risk for tick-borne diseases. Researchers have identified an increasing number of bacterial pathogens that are transmitted by ticks, including Anaplasma, Borrelia, Ehrlichia, and Rickettsia spp. Recent reports involving humans and canines suggest that ticks should be considered as potential vectors of Bartonella spp. To strengthen this suggestion, numerous molecular surveys to detect Bartonella DNA in ticks have been conducted. However, there is little evidence that Bartonella spp. can replicate within ticks and no definitive evidence of transmission by a tick to a vertebrate host.

Insecticide Resistance Status of Aedes aegypti (Diptera: Culicidae) in California by Biochemical Assays.

Insecticide resistance in Aedes aegypti mosquitoes poses a major threat to public health worldwide. There are two primary biological mechanisms that can lead to insecticide resistance, target site and metabolic resistance, both of which confer resistance to specific classes of insecticides. Due to the limited number of chemical compounds available for mosquito control, it is important to determine current enzymatic profiles among mosquito populations. This study assessed resistance profiles for three metabolic pathways, α-esterases, ß-esterases, and mixed-function oxidases (MFOs), as well as insensitivity of the acetylcholinesterase (iAChE) enzyme in the presence of propoxur, among Ae. aegypti from the Central Valley and southern California. All field-collected Ae. aegypti demonstrated elevated MFOs and iAChE activity, indicating potential development of pyrethroid and organophosphate resistance, respectively. Although regional variations were found among α-esterase and ß-esterase activity, levels were generally elevated, further suggesting additional mechanisms for developing organophosphate resistance. Furthermore, mosquito samples from southern California exhibited a higher expression level to all three metabolic enzymes and iAChE activity in comparison to mosquitoes from the central region. These results could help guide future mosquito control efforts, directing the effective use of insecticides while limiting the spread of resistance.

Infection and replication of Bartonella species within a tick cell line.

Bartonella species are fastidious, gram negative bacteria, some of which are transmitted by arthropod vectors, including fleas, sandflies, and lice. There is very little information regarding the interaction and/or transmission capabilities of Bartonella species by ticks. In the present study, we demonstrate successful infection of the Amblyomma americanum cell line, AAE12, by seven Bartonella isolates and three Candidatus Bartonella species by electron or light microscopy. With the exception of Bartonella bovis, infection with all other examined Bartonella species induced cytopathic effects characterized by heavy cellular vacuolization and eventually cell lysis. Furthermore, using quantitative real time PCR (qPCR), we demonstrated significant amplification of two B. henselae genotype I isolates in the A. americanum cell line over a 5 days period. Ultimately, tick-cell derived Bartonella antigens may prove useful for the development of more sensitive diagnostic reagents and may assist in the development of an effective vaccine to prevent the further spread of disease caused by these organisms.

Identification of Molecular Determinants of Resistance to Pyrethroid Insecticides in Aedes aegypti (Diptera: Culicidae) Populations in California, USA.

The first breeding populations of Aedes aegypti (Linnaeus) were identified in California in 2013, and have since been detected in 13 counties. Recent studies suggest two introductions likely occurred, with genetically distinct populations in the central and southern regions of the state. Given the threat of dengue, chikungunya, and Zika virus transmission, it is imperative to understand if these populations harbor genes that could confer resistance to pyrethrin-based insecticides, known as pyrethroids, the most commonly used class of adulticides in the state. In 2017, the California Department of Public Health initiated a pesticide resistance screening program for Ae. aegypti to assess the presence of specific mutations on the sodium channel gene (V1016I and F1534C) associated with knockdown resistance to pyrethroids. Mosquitoes collected between 2015 and 2017 from 11 counties were screened for mutations using real-time polymerase chain reaction assays. Results revealed distinctly different resistance profiles between the central and southern regions. The central population displayed nearly fixed resistant mutations at both loci, whereas the southern population was more variable. The relative proportion of resistant alleles observed in sampled mosquitoes collected in southern California increased each year from 2015 through 2017, indicating potential increases in resistance across this region. The presence of these mutations indicates that these mosquitoes may be predisposed to surviving pyrethroid treatments. Additional biological and biochemical assays will help better elucidate the mechanisms underlying insecticide resistance in California Ae. aegypti and prompt the use of pesticides that are most effective at controlling these mosquitoes.

Detection of two Bartonella tamiae-like sequences in Amblyomma americanum (Acari: Ixodidae) using 16S-23S intergenic spacer region-specific primers.

Four hundred and sixty-six questing Amblyomma americanum (L.) (Acari: Ixodidae) from Carolina County, VA, and 98 questing A. americanum from Chatham County, NC, were screened by polymerase chain reaction (PCR) for the Bartonella 16S-23S intergenic spacer region. Two amplicons, approximately 270-280 bp, were detected in two ticks from Virginia. Based upon PCR and sequencing, an adult male and adult female tick harbored DNA sequences closely related to Bartonella tamiae (DQ395180). Bartonella DNA was not detected in A. americanum from North Carolina. Potential transmission of Bartonella spp. by A. americanum should be the focus of future experimental studies.

Suspected and Confirmed Vector-Borne Rickettsioses of North America Associated with Human Diseases.

The identification of pathogenic rickettsial agents has expanded over the last two decades. In North America, the majority of human cases are caused by tick-borne rickettsioses but rickettsiae transmitted by lice, fleas, mites and other arthropods are also responsible for clinical disease. Symptoms are generally nonspecific or mimic other infectious diseases; therefore, diagnosis and treatment may be delayed. While infection with most rickettsioses is relatively mild, delayed diagnosis and treatment may lead to increased morbidity and mortality. This review will discuss the ecology, epidemiology and public health importance of suspected and confirmed vector-transmitted Rickettsia species of North America associated with human diseases.

MOLECULAR SURVEILLANCE FOR BARTONELLA, BORRELIA, AND RICKETTSIA SPECIES IN TICKS FROM DESERT BIGHORN SHEEP ( OVIS CANADENSIS) AND MULE DEER ( ODOCOILEUS HEMIONUS) IN SOUTHERN CALIFORNIA, USA.

: Ticks (Acari: Ixodidae) were collected from 44 desert bighorn sheep ( Ovis canadensis) and 10 mule deer ( Odocoileus hemionus) in southern California, US during health inspections in 2015-16. Specimens were identified and screened by PCR analysis to determine the presence and prevalence of Bartonella, Borrelia, and Rickettsia species in ticks associated with these wild ruminants. None of the 60 Dermacentor hunteri and 15 Dermacentor albipictus ticks tested yielded positive PCR results. Additional tick specimens should be collected and tested to determine the prevalence of these confirmed or suspected tickborne pathogens within ruminant populations.

Assessing Cat Flea Microbiomes in Northern and Southern California by 16S rRNA Next-Generation Sequencing.

Flea-borne diseases (FBDs) impact both human and animal health worldwide. Because adult fleas are obligately hematophagous and can harbor potential pathogens, fleas act as ectoparasites of vertebrates, as well as zoonotic disease vectors. Cat fleas (Ctenocephalides felis) are important vectors of two zoonotic bacterial genera listed as priority pathogens by the National Institute of Allergy and Infectious Diseases (NIAID-USA): Bartonella spp. and Rickettsia spp., causative agents of bartonelloses and rickettsioses, respectively. In this study, we introduce the first microbiome analysis of C. felis samples from California, determining the presence and abundance of relevant pathogenic genera by characterizing the cat flea microbiome through 16S rRNA next-generation sequencing (16S-NGS). Samples from both northern (NoCal) and southern (SoCal) California were assessed to expand current knowledge regarding FBDs in the state. We identified Rickettsia and Bartonella, as well as the endosymbiont Wolbachia, as the most abundant genera, followed by less abundant taxa. In comparison to our previous study screening Californian cat fleas for rickettsiae using PCR/digestion/sequencing of the ompB gene, the 16S-NGS approach applied herein showed a 95% level of agreement in detecting Rickettsia spp. There was no overall difference in microbiome diversity between NoCal and SoCal samples. Bacterial taxa identified by 16S-NGS in this study may help to improve epidemiological investigations, pathogen surveillance efforts, and clinical diagnostics of FBDs in California and elsewhere.

Detection of Rickettsia amblyommii in association with a tick bite rash.

In the summer of 2006, an Amblyomma americanum tick was removed from a woman in central North Carolina, who subsequently developed a rash at the site of tick attachment. When examined by polymerase chain reaction (PCR) for Borrelia, Anaplasma, Ehrlichia, Babesia, Rickettsia, and Bartonella DNA, only the Rickettsia primers generated an amplicon, which was identified as "R. amblyommii" by sequencing. To our knowledge, this is the first case in which R. amblyommii was temporally associated with a rash.

Prevalence of Anaplasma phagocytophilum in domestic felines in the United States.

Anaplasma phagocytophilum is among the more common tick-borne disease agents in the United States. It is of veterinary and public health significance as dogs, cats, and human beings are known to be susceptible. A. phagocytophilum is transmitted trans-stadially by either nymphs or adults of either the black-legged tick (Ixodes scapularis) or the western black-legged tick (Ixodes pacificus). Little information is available regarding either the prevalence of this agent in cats or the dynamics of vector transmission. Four hundred and sixty feline blood samples from sites throughout the United States were assayed for antibodies to A. phagocytophilum using an indirect immunofluorescence assay (IFA). Results of the prevalence study showed that 20 samples (4.3%) were positive for A. phagocytophilum antibodies by IFA at a 1:50 dilution, however these results could not be confirmed by PCR analysis. PCR analysis for other cross-reacting Ehrlichia/Anaplasma spp. was also negative. These results demonstrate that natural infection of A. phagocytophilum in cats is uncommon.

Limited Evidence for Rickettsia felis as a Cause of Zoonotic Flea-Borne Rickettsiosis in Southern California.

Over 90% of human flea-borne rickettsioses cases in California are reported from suburban communities of Los Angeles and Orange counties and are presumed to be associated with either Rickettsia typhi or Rickettsia felis infection. Ctenocephalides felis (Bouché) is considered the principal vector for both rickettsiae, and R. felis has largely replaced R. typhi as the presumptive etiologic agent based on the widespread incidence of R. felis in cat flea populations. However, with no evidence to confirm R. felis as the cause of human illness in southern California, coupled with recent findings that showed R. felis to be widespread in cat fleas statewide, we propose that this hypothesis should be reconsidered. Evidence of only limited numbers of R. typhi-infected cat fleas in the environment may indicate a very rare infection and explain why so few cases of flea-borne rickettsioses are reported each year in southern California relative to the population.

Distribution and Diversity of Bartonella washoensis Strains in Ground Squirrels from California and Their Potential Link to Human Cases.

We investigated the prevalence of Bartonella washoensis in California ground squirrels (Otospermophilus beecheyi) and their fleas from parks and campgrounds located in seven counties of California. Ninety-seven of 140 (69.3%) ground squirrels were culture positive and the infection prevalence by location ranged from 25% to 100%. In fleas, 60 of 194 (30.9%) Oropsylla montana were found to harbor Bartonella spp. when screened using citrate synthase (gltA) specific primers, whereas Bartonella DNA was not found in two other flea species, Hoplopsyllus anomalus (n = 86) and Echidnophaga gallinacea (n = 6). The prevalence of B. washoensis in O. montana by location ranged from 0% to 58.8%. A majority of the gltA sequences (92.0%) recovered from ground squirrels and fleas were closely related (similarity 99.4-100%) to one of two previously described strains isolated from human patients, B. washoensis NVH1 (myocarditis case in Nevada) and B. washoensis 08S-0475 (meningitis case in California). The results from this study support the supposition that O. beecheyi and the flea, O. montana, serve as a vertebrate reservoir and a vector, respectively, of zoonotic B. washoensis in California.

Detection of Rickettsia Species in Fleas Collected from Cats in Regions Endemic and Nonendemic for Flea-Borne Rickettsioses in California.

Rickettsia typhi, transmitted by rat fleas, causes most human flea-borne rickettsioses worldwide. Another rickettsia, Rickettsia felis, found in cat fleas, Ctenocephalides felis, has also been implicated as a potential human pathogen. In the continental United States, human cases of flea-borne rickettsioses are reported primarily from the southern regions of Texas and California where the cat flea is considered the principal vector. In California, more than 90% of locally acquired human cases are reported from suburban communities within Los Angeles and Orange counties despite the almost ubiquitous presence of cat fleas and their hosts throughout the state. The objective of this study is to assess the presence and infection rate of Rickettsia species in cat fleas from selected endemic and nonendemic regions of California. Cat fleas were collected from cats in Los Angeles County (endemic region) and Sacramento and Contra Costa counties (nonendemic region). Sequencing of 17 amplicons confirmed the presence of R. felis in both the endemic and non-endemic regions with a calculated maximum likelihood estimation of 131 and 234 per 1000 fleas, respectively. R. typhi was not detected in any flea pools. Two R. felis-like genotypes were also detected in fleas from Los Angeles County; Genotype 1 was detected in 1 flea pool and Genotype 2 was found in 10 flea pools. Genotype 1 was also detected in a single flea pool from Sacramento County. Results from this study show that R. felis is widespread in cat flea populations in both flea-borne rickettsioses endemic and nonendemic regions of California, suggesting that a high prevalence of this bacterium in cat fleas does not predispose to increased risk of human infection. Further studies are needed to elucidate the role of R. felis and the two R. felis-like organisms as etiologic agents of human flea-borne rickettsioses in California.

Use of imidacloprid-permethrin to prevent transmission of Anaplasma phagocytophilum from naturally infected Ixodes scapularis ticks to dogs.

One group of eight beagles was treated with a combination of imidacloprid and permethrin 7 days before exposure to Ixodes scapularis ticks that were naturally infected with Anaplasma phagocytophilum. A second group of eight beagles was not treated and was also exposed to infected ticks. Seven of eight non-treated dogs--but none of the treated dogs--developed specific antibodies to A. phagocytophilum. Results of this study indicate that a combination of imidacloprid and permethrin can prevent transmission of A. phagocytophilum to dogs if administered before exposure to infected ticks.