Introduction: Ehrlichia chaffeensis is an emerging zoonotic tick‐borne rickettsial pathogen that has been detected in a wide range of vertebrate hosts, including ruminants, canids and primates. Although white‐tailed deer ( Odocoileus virginianus ) are considered the primary reservoir of E. chaffeensis , this pathogen was also reported in other naturally infected cervids, including Korean spotted or sika deer ( Cervus nippon ) and Brazilian marsh deer ( Blastocerus dichotomus ). Case presentation: A captive adult bull elk ( Cervus elaphus ) from east‐central Missouri was submitted for post‐mortem analysis. The elk was in poor body condition with easily palpable ribs and vertebral spinal processes. Excoriations were noted on the occipital region of the head and on the left scapula, which had moderate amounts of maggots within the lesions. Large numbers of ticks were scattered over the body. Novel and established PCR assays were used to detect E. chaffeensis in blood and spleen samples from this elk, but the pathogen was not detected in Dermacentor albipictus ticks collected at necropsy. Portions of several gene sequences were analysed from the infecting agent. Conclusion To the best of our knowledge this is the first report of E. chaffeensis infection in an elk. It was not determined whether the pathogen contributed to cause of death. Notably, the pathogen was not detected in D. albipictus ticks collected from the elk.

Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis, is a tick-borne rickettsial pathogen that is infective to a wide range of mammals, including dogs and people. Amblyomma americanum, the lone star tick, is considered the primary vector of E. chaffeensis, but this pathogen has been detected in other tick species, including the brown dog tick, Rhipicephalus sanguineus. We hypothesized that the Arkansas strain of E. chaffeensis is infective to R. sanguineus, and used a novel PCR assay to test for acquisition of this pathogen by R. sanguineus and A. americanum ticks that were simultaneously fed on experimentally infected dogs. Although E. chaffeensis was not frequently detected in peripheral blood of these dogs, the pathogen was detected in both tick species and in canine lung, kidney, lymph node, bone marrow and frontal lobe samples. One dog (AFL) was maintained for several years, and ticks again acquired E. chaffeensis from this dog 566 days after intradermal inoculation with E. chaffeensis, but the pathogen was not detected in ticks fed on the same dog at 764 or 1086 days after the intradermal inoculation.

This chapter first gives a brief history of human monocytotropic ehrlichiosis (HME), which is a zoonotic disease. From this brief history of HME, it is clear that rapid progress in understanding this newly emerged tick-borne zoonosis was facilitated both by its successful cultivation and by experimental studies of ticks linked to field observations. Epidemiological studies were furthered through the use of molecular techniques, and knowledge of HME expanded rapidly in the early to mid-1990s. Next, the chapter talks about biology of the etiologic agent. In the eastern United States, Ehrlichia chaffeensis is maintained in an epizootic cycle involving the lone star tick (Amblyomma americanum) as the principal vector and the white-tailed deer (Odocoileus virginianus) as a major vertebrate reservoir. This critical vector-host association places these two species at the core of E. chaffeensis epizootiology and strongly influences its geographic distribution and seasonality of transmission. HME caused by E. chaffeensis is endemic to the United States, with residents in the southeastern and south-central states at highest risk. The clinical recognition of HME can be difficult, since the spectrum of manifestations is broad and nonspecific, ranging from asymptomatic infection to mild self-limited symptoms to rapidly fatal disease. HME typically presents as an uncomplicated, acute, nonspecific febrile illness that resolves rapidly if treated with doxycycline, characteristic of many rickettsial infections. The chapter ends with a discussion on laboratory diagnosis, treatment and prevention of HME.

ABSTRACTDoxycycline is the treatment of choice for canine monocytic ehrlichiosis (CME), a well-characterized disease and valuable model for tick-borne zoonoses. Conflicting reports of clearance ofEhrlichia canisafter treatment with doxycycline suggested that the disease phase during which treatment is initiated influences outcomes of these treatments. The purpose of this study was to evaluate the efficacy of a 28-day doxycycline regimen for clearance of experimentalE. canisinfections from dogs treated during three phases of the disease. Ten dogs were inoculated with blood fromE. caniscarriers and treated with doxycycline during acute, subclinical, or chronic phases of CME. Daily rectal temperatures and semiweekly blood samples were monitored from each dog, andRhipicephalus sanguineusticks were acquisition fed on each dog for xenodiagnosis. Blood collected from dogs treated during acute or subclinical CME became PCR negative forE. canisas clinical parameters improved, but blood samples collected from dogs treated during chronic CME remained intermittently PCR positive.R. sanguineusticks fed on dogs after doxycycline treatments became PCR positive forE. canis, regardless of when treatment was initiated. However, fewer ticks became PCR positive after feeding on two persistently infected dogs treated with doxycycline followed by rifampin, suggesting that antibiotic therapy can reduce tick acquisition ofE. canis.

BackgroundThe range of American canine hepatozoonosis (ACH) is expanding from the southern USA northward. Transmission of Hepatozoon americanum occurs by ingestion of infected Gulf Coast ticks, Amblyomma maculatum. The source of the protozoan for the tick remains undetermined; infected dogs are unusual hosts for the tick.ObjectiveCompare possible sources of infection by field investigations of 2 multiple-dog outbreaks of ACH.AnimalsTwenty-eight privately owned dogs (Canis familiaris), 1 coyote (Canis latrans), 31 wild-trapped cotton rats (Sigmodon hispidus), 24 wild-trapped field mice (Peromyscus leucopus), and 9 wild-caught rabbits (Sylvilagus spp.) from sites in eastern Oklahoma were monitored for hepatozoonosis. Six laboratory-raised cotton rats (S. hispidus), 6 Sprague-Dawley rats (Rattus norvegicus), 6 C57BL/6J-Lystbg-J/J mice (Mus musculus), 6 outbred white mice (M. musculus), 6 New Zealand white rabbits (Oryctolagus cuniculus), and 2 dogs were acquired through commercial vendors for experimental transmission trials of H. americanum.MethodsFour of 15 dogs in a rural neighborhood and 5/12 hunting Beagles were confirmed to be infected by blood smear examination, muscle biopsy, and polymerase chain reaction assay of the 18S rRNA gene of Hepatozoon species. Histories and tick host preferences led to field collections of common prey of canids and experimental transmission trials of H. americanum to selected prey (M. musculus, S. hispidus, R. norvegicus, and O. cuniculus).ResultsDogs with ready access to prey (4/15 dogs) or that were fed prey retrieved from hunts (5/12 hunting Beagles) became infected, providing evidence that predation is an important epidemiologic component of ACH infection. Experimental transmission studies identified a quiescent, infectious stage (cystozoite) of the parasite that provides an alternate mode of transmission to canids through predation, demonstrating that cotton rats, mice, and rabbits but not brown rats may act as paratenic hosts of H. americanum.Conclusions and Clinical ImportancePredation of prey harboring infected A. maculatum or containing cystozoites of H. americanum in their tissues provide 2 modes of transmission of ACH to dogs, putting unconfined dogs at increased risk of infection in endemic areas.

Ehrlichia canis is the etiologic agent of canine monocytic ehrlichiosis (CME) and is a useful model for zoonotic tick‐borne pathogens, many of which infect dogs. The purpose of this study was to evaluate rifampin and doxycycline regimens for clearance of E. canis infections in addition to alleviation of CME. Beagles were infected with E. canis by intravenous inoculation with carrier blood and treated with either rifampin or doxycycline after the acute phase of CME. Improved hematological values demonstrated that both treatments effectively relieved signs of the disease. Peripheral blood from all dogs became PCR negative after antibiotic treatment, suggesting that these infections were eliminated and that rifampin is an effective alternative chemotherapeutic agent for treatment of CME.

ABSTRACT Doxycycline generally alleviates clinical monocytic ehrlichiosis, but its efficacy in the control of monocytotropic ehrlichial pathogens requires further investigation. In this study, Ehrlichia canis was detected in dogs treated with doxycycline for 14 days and in ticks fed on these dogs, suggesting that treated dogs can remain reservoirs for E. canis .

Human monocytic ehrlichiosis (HME) is a zoonotic emerging tick-borne disease with clinical signs that range from mild symptoms to multiple organ failure and death. Ehrlichia chaffeensis, the aetiologic agent of HME, is reported to infect a divergent range of mammals. Although cattle are common hosts of the primary vector of this pathogen, the susceptibility of this host to E. chaffeensis has not been reported to date. This study was undertaken to determine if cattle could provide a useful infection model of E. chaffeensis. Dairy calves were injected with DH82 cells infected with the Arkansas, St Vincent or 91HE17 strain of E. chaffeensis, and monitored for signs of clinical ehrlichiosis and for infection of peripheral blood and ticks by PCR assay. Splenectomized and spleen-intact calves were injected with cryopreserved stabilates of E. chaffeensis-infected DH82 cells for the first experiment. Mild clinical signs were occasionally observed among these calves, and only two blood samples were PCR-positive, while several ticks fed on each calf tested PCR-positive. The second experiment involved injection of normal calves with active cultures of the same E chaffeensis strains. Interestingly, three of six calves inoculated with active cultures became recumbent and died or had to be euthanized. All of the surviving calves in this experiment tested PCR-positive on multiple dates, but fewer ticks fed on these calves were PCR-positive. These results suggest that a bovine disease model could facilitate the understanding of factors that affect the severity of HME.

Sequence analysis of the ribosomal DNA second internal transcribed spacer (ITS 2) region in 2 spatially distinct populations of Amblyomma americanum (L.) revealed intraspecific variation. Nucleotide sequences from multiple DNA extractions and several polymerase chain reaction amplifications of eggs from mixed-parentage samples from both populations of ticks revealed that 12 of 1,145 (1.0%) sites varied. Three of the 12 sites of variation were distinct between the 2 A. americanum populations, which corresponded to a rate of 0.26%. Phylogenetic analysis based on ITS 2 sequences provided strong support (i.e., bootstrap value of 80%) that wild A. americanum clustered into a distinguishable group separate from those derived from colony ticks.

The acquisition and transmission of rickettsial pathogens by different tick developmental stages has important epidemiological implications. The purpose of this study was to determine if male Rhipicephalus sanguineus can experimentally acquire and transmit Ehrlichia canis in the absence of female ticks. Two trials were performed where nymphal and male R. sanguineus were simultaneously acquisition fed on the same infected donor hosts, and transstadially or intrastadially exposed male ticks were fed on separate pathogen-free dogs as a test for transmission. A single-step p30-based PCR assay was used to test canine and tick hosts for E. canis infections before and after tick feeding. E. canis was detected after either intrastadial or transstadial passage in male ticks, the organism remained detectable in both tick groups after transmission feeding, and both tick groups transmitted the rickettsia to susceptible dogs. Infection of dogs via tick feeding resulted in milder clinical signs and lower antibody titers than intravenous inoculation of carrier blood, but further investigation is needed to understand the mechanisms responsible for this observation. These results demonstrate that male R. sanguineus can take multiple feedings, and that they can both acquire and transmit E. canis in the absence of female ticks. This tick development stage could be important in transmission of E. canis, and perhaps related pathogens, between vertebrate hosts under natural and experimental conditions.

American canine hepatozoonosis is caused by Hepatozoon americanum, a protozoan parasite, the definitive host of which is the tick, Amblyomma maculatum. Infection of the dog follows ingestion of ticks that harbor sporulated H. americanum oocysts. Following penetration of the intestinal mucosa, sporozoites are disseminated systemically and give rise to extensive asexual multiplication in cells located predominantly in striated muscle. The parasitized canine cells in “onion skin” cysts and in granulomas situated within skeletal muscle, as well as those in peripheral blood leukocytes (PBL), were identified as macrophages by use of fine structure morphology and/or immunohistochemical reactivity with macrophage markers. Additionally, two basic morphologic forms of the parasite were observed in macrophages of granulomas and PBLs. The forms were presumptively identified as merozoites and gamonts. The presence of a “tail” in some gamonts in PBLs indicated differentiation toward microgametes. Recognition of merozoites in PBLs supports the contention that hematogenously redistributed merozoites initiate repeated asexual cycles and could explain persistence of infection for long periods in the vertebrate host. Failure to clearly demonstrate a host cell membrane defining a parasitophorous vacuole may indicate that the parasite actively penetrates the host cell membrane rather than being engulfed by the host cell, as is characteristic of some protozoans.

Transmission of Hepatozoon spp. to dogs was investigated using four species of ixodid ticks: Rhipicephalus sanguineus, Amblyomma aureolatum, Amblyomma ovale and Amblyomma cajennense. We collected completely or partially engorged adult ticks of these species from dogs that were naturally infested and positive for Hepatozoon spp. We selected some of these ixodids and inoculated them orally in four negative dogs. The other ticks were dissected and examined for oocysts. Of all dogs inoculated orally with R. sanguineus, A. aureolatum, A. cajennense and A. ovale, only the animal that received the macerate of A. ovale was positive; evidence (gametocytes in peripheral blood) of infection was found 63 days after inoculation. Among all dissected ticks, we found only two oocysts; these were similar to those of Hepatozoon canis, and both were recovered from a single A. ovale specimen. We inoculated sporozoites recovered from the oocysts intraperitoneally into a Hepatozoon spp. negative dog, and circulating gametocytes were detected 84 days later. Our study demonstrated that A. ovale can be a vector of Hepatozoon spp. in Brazil.

Detection of Ehrlichia chaffeensis is necessary to study interactions between the parasite and its vertebrate and invertebrate hosts. The purpose of this study was to develop a sensitive, specific PCR assay for E. chaffeensis based on the outer membrane protein gene, p28. Candidate primer sets were identified and ranked based on annealing scores, similarities to three major p28 sequence clusters, dissimilarity to E. canis p30, an ortholog of p28, and the proximities of flanking primer sequences for nested PCR. The relative sensitivities of five optimized single-step and two nested PCR assays were determined, and the most sensitive assay was found to be a single-step PCR that was as much as 1000-fold more sensitive than a standard 16S rDNA-based nested PCR assay. This p28-based PCR assay amplified the target amplicon from isolates representative of all three major clusters of known p28 sequences, and this assay did not amplify template prepared from either of the two species most closely related to E. chaffeensis, E. canis and E. muris. These results indicate that this sensitive, specific and isolate-universal single-step PCR assay will be a useful tool in characterizing the transmission of this important zoonotic pathogen.

Hepatozoonosis is caused by apicomplexan haemoparasites of the genus Hepatozoon, which are closely related to Plasmodium spp. and piroplasms. Recent research revealed that two tick-borne Hepatozoon spp. infect dogs and cause distinct syndromes. Comparisons of these related species illustrates that whereas Hepatozoon canis appears to be well adapted to its canine host, Hepatozoon americanum, an emerging pathogen producing severe and frequently fatal myositis, is highly virulent and might have recently crossed the species barrier from a wild host.

American canine hepatozoonosis (ACH) is a tick-borne disease that is spreading in the southeastern and south-central United States. Characterized by marked leukocytosis and periosteal bone proliferation, ACH is very debilitating and often fatal. Dogs acquire infection by ingesting nymphal or adult Gulf Coast ticks (Amblyomma maculatum) that, in a previous life stage, ingested the parasite in a blood meal taken from some vertebrate intermediate host. ACH is caused by the apicomplexan Hepatozoon americanum and has been differentiated from Old World canine hepatozoonosis caused by H. canis. Unlike H. canis, which is transmitted by the ubiquitous brown dog tick (Rhipicephalus sanguineus), H. americanum is essentially an accidental parasite of dogs, for which Gulf Coast ticks are not favored hosts. The geographic portrait of the disease parallels the known distribution of the Gulf Coast tick, which has expanded in recent years. Thus, the endemic cycle of H. americanum involves A. maculatum as definitive host and some vertebrate intermediate host(s) yet to be identified. Although coyotes (Canis latrans) are known to be infected, it is not known how important this host is in maintaining the endemic cycle. This review covers the biology of the parasite and of the tick that transmits it and contrasts ACH with classical canine hepatozoonosis. Clinical aspects of the disease are discussed, including diagnosis and treatment, and puzzling epidemiologic issues are examined. Brief consideration is given to the potential for ACH to be used as a model for study of angiogenesis and of hypertrophic osteoarthropathy.

To determine the persistence of Hepatozoon americanum in a naturally infected dog, skeletal muscle biopsies were performed at approximately 6-mo intervals over a period of 5.5 yr, and the samples were examined for presence of lesions of American canine hepatozoonosis (ACH). Nymphal Amblyomma maculatum (Gulf Coast tick) were allowed to feed to repletion on the dog periodically over the 5.5-yr period, and adult ticks were dissected and examined for presence of H. americanum oocysts. With 3 exceptions, the biopsied muscle contained lesions characteristic of ACH; no evidence of infection was found at 36, 54, and 67 mo after the original diagnosis. In every instance, nymphal Gulf Coast ticks became infected, indicating that dogs naturally infected with H. americanum can remain infectious for Gulf Coast ticks for at least 5.5 yr. Skeletal muscle biopsy is a reasonably reliable method of determining whether dogs are infected with the parasite. Xenodiagnosis using nymphal Gulf Coast ticks is an even more sensitive method, but the procedure is practicable only experimentally. Design of prevention and control measures for ACH must take into account knowledge that the parasite can survive in dogs, and presumably other vertebrate host(s), for long periods. Preventing ingestion of Gulf Coast ticks is an effective control measure.

Laboratory-reared larval Gulf Coast ticks (GCTs) (Amblyomma maculatum) were exposed experimentally and found to acquire Hepatozoon americanum infection while feeding on parasitemic dogs. These ticks supported gamogonic and sporogonic development of the apicomplexan, and oocysts from newly molted nymphs were infectious for a dog. Other nymphs from this cohort that were allowed to feed on a blood-parasite naive sheep molted normally; the resulting adult ticks contained oocysts that were infectious for another dog. Merogonic development of H. americanum in the dogs and the resulting lesions/disease appeared similar, irrespective of whether infectious oocysts were derived from nymphal or adult ticks that acquired infection as larvae. In the system previously known, nymphal ticks acquire infection and adults harbor infective oocysts, which vertebrate hosts ingest. Given that larval A. maculatum can acquire infection and nymphs can harbor viable oocysts as demonstrated by this study, the potential variety of vertebrate hosts that can alternate with GCTs in maintaining an endemic cycle is considerably expanded.

ABSTRACT Detection of vector-borne pathogens is necessary for investigation of their association with vertebrate and invertebrate hosts. The ability to detect Ehrlichia spp. within individual experimentally infected ticks would be valuable for studies to evaluate the relative competence of different vector species and transmission scenarios. The purpose of this study was to develop a sensitive PCR assay based on oligonucleotide sequences from the unique Ehrlichia canis gene, p30 , to facilitate studies that require monitoring this pathogen in canine and tick hosts during experimental transmission. Homologous sequences for Ehrlichia chaffeensis p28 were compared to sequences of primers derived from a sequence conserved among E. canis isolates. Criteria for primer selection included annealing scores, identity of the primers to homologous E. chaffeensis sequences, and the availability of similarly optimal primers that were nested within the target template sequence. The p30- based assay was at least 100-fold more sensitive than a previously reported nested 16S ribosomal DNA (rDNA)-based assay and did not amplify the 200-bp target amplicon from E. chaffeensis, the human granulocytic ehrlichiosis agent, or Ehrlichia muris DNA. The assay was used to detect E. canis in canine carrier blood and in experimentally infected Rhipicephalus sanguineus ticks. Optimized procedures for preparing tissues from these hosts for PCR assay are described. Our results indicated that this p30 -based PCR assay will be useful for experimental investigations, that it has potential as a routine test, and that this approach to PCR assay design may be applicable to other pathogens that occur at low levels in affected hosts.

American canine hepatozoonosis (ACH) caused by Hepatozoon americanum Vincent-Johnson, Macintire, Lindsay, Lenz, Baneth, and Shkap is an emerging, often fatal, tick-borne protozoal disease of dogs in the United States of America. Dogs acquire infection by ingesting ticks that contain oocysts. To understand the invertebrate (definitive) host range of H. americanum, experiments were carried out using four ixodids, Rhipicephalus sanguineus (Latreille), Dermacentor variabilis Say, Amblyomma americanum (L.), and Amblyomma maculatum Koch. Laboratory-reared nymphal ticks were fed on dogs that were either naturally or experimentally infected with H. americanum; when these ticks molted to the adult stage they were either fed to susceptible dogs or were dissected and examined for the presence of oocysts. Mature H. americanum oocysts were found in >90% of A. maculatum (both males and females), whereas oocysts were not found in any of the other three species. These results confirm that A. maculatum is an excellent host and vector for H. americanum and also suggest that this apicomplexan may have a narrow invertebrate host range, at least among ixodid ticks that are likely candidate vectors in the United States.

Histologic examination of eyes and brains of 27 dogs experimentally infected with either Ehrlichia canis, E. ewingii, E. chaffeensis, or human granulocytic ehrlichia (HGE) was conducted in the course of several experiments, the primary objectives of which were to investigate the susceptibility of the domestic dog to infection with various ehrlichial species and to assess the ability of ixodid tick species to acquire and transmit those infections. Uveitis and meningitis occurred in each of the dogs infected with E. canis but was not observed in dogs infected with the other Ehrlichia species. The inflammatory infiltrate was predominantly lymphocytic, monocytic, and plasmacytic; granulocytes were notably few. Ocular inflammation was most common and most intense in the ciliary body, becoming less intense in the choroid, iris, and retina, respectively. Meningitis was often accompanied by mild neuroparenchymal vascular cuffing and gliosis. The meningeal inflammatory cell infiltrate included a prominent monocyte population. Ocular and meningeal lesions were present in all E. canis-infected dogs from 22 through 200 days postexposure. Neither ocular nor brain lesions were observed with any of the other ehrlichial infections.