Experimental infection of Rhipicephalus sanguineus with Ehrlichia chaffeensis.

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.

Efficacy of a doxycycline treatment regimen initiated during three different phases of experimental ehrlichiosis.

Doxycycline 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 of Ehrlichia canis after 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 experimental E. canis infections from dogs treated during three phases of the disease. Ten dogs were inoculated with blood from E. canis carriers and treated with doxycycline during acute, subclinical, or chronic phases of CME. Daily rectal temperatures and semiweekly blood samples were monitored from each dog, and Rhipicephalus sanguineus ticks were acquisition fed on each dog for xenodiagnosis. Blood collected from dogs treated during acute or subclinical CME became PCR negative for E. canis as clinical parameters improved, but blood samples collected from dogs treated during chronic CME remained intermittently PCR positive. R. sanguineus ticks fed on dogs after doxycycline treatments became PCR positive for E. 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 of E. canis.

The natural history of Anaplasma marginale.

The intracellular pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), described by Sir Arnold Theiler in 1910, is endemic worldwide in tropical and subtropical areas. Infection of cattle with A. marginale causes bovine anaplasmosis, a mild to severe hemolytic disease that results in considerable economic loss to both dairy and beef industries. Transmission of A. marginale to cattle occurs biologically by ticks and mechanically by biting flies and by blood-contaminated fomites. Both male ticks and cattle hosts become persistently infected with A. marginale and serve as reservoirs of infection. While erythrocytes are the major site of infection in cattle, A. marginale undergoes a complex developmental cycle in ticks that begins by infection of gut cells, and transmission to susceptible hosts occurs from salivary glands during feeding. Major surface proteins (MSPs) play a crucial role in the interaction of A. marginale with host cells, and include adhesion proteins and MSPs from multigene families that undergo antigenic change and selection in cattle, thus contributing to maintenance of persistent infections. Many geographic strains of A. marginale have been identified worldwide, which vary in genotype, antigenic composition, morphology and infectivity for ticks. Isolates of A. marginale may be maintained by independent transmission events and a mechanism of infection/exclusion in cattle and ticks. The increasing numbers of A. marginale genotypes identified in some geographic regions most likely resulted from intensive cattle movement. However, concurrent A. marginale strain infections in cattle was reported, but these strains were more distantly related. Phylogenetic studies of selected geographic isolates of A. marginale, using msp4 and msp1alpha, provided information about the biogeography and evolution of A. marginale, and msp1alpha genotypes appear to have evolved under positive selection pressure. Live and killed vaccines have been used for control of anaplasmosis and both types of vaccines have advantages and disadvantages. Vaccines have effectively prevented clinical anaplasmosis in cattle but have failed to block A. marginale infection. Vaccines are needed that can prevent clinical disease and, simultaneously, prevent infection in cattle and ticks, thus eliminating these hosts as reservoirs of infection. Advances in genomics, proteomics, immunology and biochemical and molecular technologies during the last decade have been applied to research on A. marginale and related organisms, and the recent development of a cell culture system for A. marginale has provided a format for studying the pathogen/tick interface. Recent advancements and new research methodologies should provide additional opportunities for development of new strategies for control and prevention of bovine anaplasmosis.

Alternate pathway of infection with Hepatozoon americanum and the epidemiologic importance of predation.

BACKGROUND: The 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. OBJECTIVE: Compare possible sources of infection by field investigations of 2 multiple-dog outbreaks of ACH. ANIMALS: Twenty-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. METHODS: Four 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). RESULTS: Dogs 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 IMPORTANCE: Predation 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.

Antibiotic clearance of Ehrlichia canis from dogs infected by intravenous inoculation of carrier blood.

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.

Experimental infection of dairy calves with Ehrlichia chaffeensis.

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.

Tick acquisition of Ehrlichia canis from dogs treated with doxycycline hyclate.

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.

Molecular characterization of Hepatozoon sp. from Brazilian dogs and its phylogenetic relationship with other Hepatozoon spp.

To characterize phylogenetically the species which causes canine hepatozoonosis at two rural areas of Rio de Janeiro State, Brazil, we used universal or Hepatozoon spp. primer sets for the 18S SSU rRNA coding region. DNA extracts were obtained from blood samples of thirteen dogs naturally infected, from four experimentally infected, and from five puppies infected by vertical transmission from a dam, that was experimentally infected. DNA of sporozoites of Hepatozoon americanum was used as positive control. The amplification of DNA extracts from blood of dogs infected with sporozoites of Hepatozoon spp. was observed in the presence of primers to 18S SSU rRNA gene of Hepatozoon spp., whereas DNA of H. americanum sporozoites was amplified in the presence of either universal or Hepatozoon spp.-specific primer sets; the amplified products were approximately 600bp in size. Cloned PCR products obtained from DNA extracts of blood from two dogs experimentally infected with Hepatozoon sp. were sequenced. The consensus sequence, derived from six sequence data sets, were blasted against sequences of 18S SSU rRNA of Hepatozoon spp. available at GenBank and aligned to homologous sequences to perform the phylogenetic analysis. This analysis clearly showed that our sequence clustered, independently of H. americanum sequences, within a group comprising other Hepatozoon canis sequences. Our results confirmed the hypothesis that the agent causing hepatozoonosis in the areas studied in Brazil is H. canis, supporting previous reports that were based on morphological and morphometric analyses.

Characterization of stages of Hepatozoon americanum and of parasitized canine host cells.

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.

Diagnosis of Hepatozoon spp. in Amblyomma ovale and its experimental transmission in domestic dogs in Brazil.

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.

Sequence variation of the ribosomal DNA second internal transcribed spacer region in two spatially-distinct populations of Amblyomma americanum (L.) (Acari: Ixodidae).

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.

Transstadial and intrastadial experimental transmission of Ehrlichia canis by male Rhipicephalus sanguineus.

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.

Occurrence of the Gulf Coast tick (Acari: Ixodidae) on wild and domestic mammals in north-central Oklahoma.

Parasitic life stages of Amblyomma maculatum Koch were collected from domestic cattle and several species of wild mammals during a 3.5-yr study (May 1998-October 2001) in north-central Oklahoma. Adult ticks were the predominant life stage collected from cattle, white-tailed deer, coyotes, and raccoons, whereas only immature ticks were collected from cotton rats and white-footed mice. The prevalence of adult A. maculatum on white-tailed deer (n = 15) examined in June, July, and August 1998 was 80, 100, 100%, respectively. The prevalence of adult A. maculatum on cattle (n = 84) ranged from 52% in February 1999 to 100% in May 1999. The prevalence of adult A. maculatum on coyotes (n = 16) was 100% in April 1998 and 43% on coyotes (n = 7) examined in January 2001. The prevalence of adult A. maculatum on raccoons (n = 23) examined during May, June, and July 1999 was 13%. No A. maculatum of any life stage were recovered from opossums (n = 7). Nine hundred forty-five rodents were trapped over 294 trap-nights; prevalence of A. maculatum larvae and nymphs on cotton rats (n = 395) was 34 and 15%, respectively, whereas on white-footed mice (n = 517), prevalence was 1.5 and 1.4%, respectively. No A. maculatum were recovered from pack rats (n = 33). There were significant differences (P = 0.0001) in larval infestation prevalence between cotton rats and white-footed mice in the spring, summer, and fall and for nymphs in the spring and summer. Results of A. maculatum parasitism and seasonal occurrence on hosts in this study are compared with previous research conducted in Oklahoma and with collection records of A. maculatum in the Entomology Museum at Oklahoma State University.

Development of a p28-based PCR assay for Ehrlichia chaffeensis.

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.

American canine hepatozoonosis.

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.

Persistence of Hepatozoon americanum (Apicomplexa: Adeleorina) in a naturally infected dog.

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.

American canine hepatozoonosis.

American canine hepatozoonosis is an emerging, tick-transmitted infection of domestic dogs caused by a recently recognized species of apicomplexan parasite, Hepatozoon americanum. The known definitive host of the protozoan is the Gulf Coast tick, Amblyomma maculatum. Presently recognized intermediate hosts include the domestic dog and the coyote, Canis latrans. Laboratory-reared larval or nymphal A. maculatum can be infected readily by feeding to repletion on a parasitemic intermediate host; sporogony requires 35-40 days. Transmission of infection to the dog has been produced experimentally by oral administration of mature oocysts or oocyst-containing ticks. Canine disease follows experimental exposure in 4-6 weeks and is characterized by systemic illness, extreme neutrophilic leukocytosis, muscle and bone pain, and proliferation of periosteal bone. Histopathological findings include multifocal skeletal and cardiac myositis associated with escape of mature merozoites from within the host-cell environment. There is also rapid onset of periosteal activation and osteogenesis and, less frequently, glomerulopathy and amyloidosis. Sequential stages of development of H. americanum in both the dog and the tick have been elucidated. Gamonts potentially infectious to ticks have been observed in peripheral blood leukocytes of the dog in as few as 28 days after exposure to oocysts. Young coyotes experimentally exposed to a canine strain of H. americanum acquired disease indistinguishable from that of similarly exposed young dogs.

Transmission of Hepatozoon americanum (Apicomplexa: Adeleorina) by ixodids (Acari: Ixodidae).

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.

Detection of Ehrlichia canis in canine carrier blood and in individual experimentally infected ticks with a p30-based PCR assay.

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.