Equine Granulocytic Ehrlichiosis Research Articles

Temporal and spatial variation inAnaplasma phagocytophiluminfection in Swedish moose (Alces alces)

The occurrence of Anaplasma phagocytophilum was investigated in spleen and serum samples from Swedish moose (Alces alces) in southern Sweden (island and mainland). Samples were analysed for presence of A. phagocytophilum DNA by real-time PCR (n = 263), and for Anaplasma antibodies with ELISA serology (n = 234). All serum samples had antibodies against A. phagocytophilum. The mean DNA-based prevalence was 26·3%, and significant (P < 0·01) temporal, and spatial variation was found. Island moose had significantly (P < 0·001) higher prevalence of A. phagocytophilum DNA than moose from the mainland areas. Two samples were sequenced to determine genetic variation in the 16S rRNA and groESL genes. Genetic sequence similarity with the human granulocytic anaplasmosis agent, equine granulocytic ehrlichiosis agent, and different wildlife-associated A. phagocytophilum variants were observed in the 16S rRNA and groESL genes. Our study shows that moose are exposed to A. phagocytophilum in Sweden, and represent a potential wildlife reservoir of the pathogen.

Serologic evidence of equine granulocytic anaplasmosis in horses from central West Brazil

Ehrlichiosis is a zoonotic disease caused by gram-negative and intracellular obligatory bacterial organisms. Equine Granulocytic Anaplasmosis - EGA (formerly Equine Granulocytic Ehrlichiosis, EGE) is a seasonal disease, normally self-limited in horses. There are few reports in Brazil about this ehrlichial agent, as well as its natural vectors. Nowadays, veterinarians are considering the suspicion of EGA in horses with suggestive symptoms of ehrlichiosis and which do not respond to piroplasmosis treatment. The aim of the present study was to identify horses exposed to the agent A. phagocytophilum by serological and molecular techniques. Twenty equine blood and serum samples from the central West region of Brazil were evaluated by microscopic examination of buffy coat smear, enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody test (IFAT) and nested polymerase chain reaction (nPCR). Additionally, the serodiagnosis of Theileria equi by IFA and ELISA were carried out, as well as molecular diagnosis by nPCR. Thirteen (65%) serum samples were positive for A. phagocytophilum by ELISA, but none of them were positive by buffy-coat smear examination or nPCR. Antibodies IgG anti-T. equi were detected in 18 (90%) and 17 (85%) horses by IFA and ELISA, respectively and the agent was detected in 9 (45%) animals by nPCR. Our data may be considered as important information to understanding the occurrence of EGA and equine piroplasmosis in central West Brazil.

Detection of Anaplasma phagocytophilum in wild boar in Slovenia

Human granulocytic anaplasmosis (HGA) comprises a group of emerging tick-borne infectious diseases and it is caused by the intracellular bacteria, Anaplasma phagocytophilum. Infections with Anaplasma spp. have been described in humans and animals. Many studies have been performed to elucidate the natural cycle of A. phagocytophilum, but a competent reservoir host has not been clearly established in Europe [1]. Roe and red deer represent a very important host for the adult tick I. ricinus, a vector of A. phagocytophilum, and could therefore also serve as a natural reservoir for A. phagocytophilum in enzootic transmission of this bacterium [1]. Two genetic lineages of groESL operon were described among isolates of A. phagocytophilum from deer in Slovenia. However, both genetic lineages differed from a single variant, found in all Slovenian HGA patients [2]. PCR screening of different wild animals from Austria and the Czech Republic showed that wild boar (Sus scrofa) were infected with A. phagocytophilum [3]. Nucleotide sequences of groESL operon of A. phagocytophilum obtained from these infected animals were identical to the sequences from Slovenian patients [3]. Interestingly, a German horse with equine granulocytic ehrlichiosis was also infected with an identical variant of A. phagocytophilum to that detected in Slovenian patients [4]. In the years 2002 and 2007, 248 wild boars (Sus scrofa) from different locations in Slovenia were shot by professional hunters. Blood and spleen samples collected by hunters were analysed for infection with Anaplasma species in our laboratory. DNA was extracted from 135 spleen (year 2002) and 113 blood (year 2007) samples as described previously [1]. For the initial screening of all samples a segment of the 16S rDNA of A. phagocytophilum was used [1]. All positive samples were additionally amplified with a nested PCR targeting groESL operon of variants of A. phagocytophilum [1]. The gene for 16S rDNA is very conservative, therefore only the amplicons of the groESL operon were further analysed by sequencing. To obtain the genetic variation of A. phagocytophilum, all amplicons of groESL operon were further sequenced on both strands and analysed by using TREECON software. A phylogenetic tree was constructed with the neighbour-joining method. Support for the tree nodes was calculated with 1000 bootstrap replicates. Of 135 spleen samples (2002) and 113 blood (2007) samples, six (4.4%) and four (3.5%) tested positive for the presence of Anaplasmae spp. DNA, respectively. The groESL gene was subsequently amplified from all positive samples. The homology search and the alignment of groESL sequences showed 100% identity with A. phagocytophilum from a human patient (acc. no. AF033101) and from the Ixodes ricinus tick (acc. no. EU246961) from Slovenia (Fig. 1). Genetic sequences from wild boar were also 100% identical among each other. Our study confirms that wild boar is naturally infected with A. phagocytophilum. The Anaplasmae spp. DNA was detected in 10 of 248 (4.0%) samples, collected from different locations in Slovenia in two different years. Anaplasma phagocytophilum was previously detected in wild boar samples. Interestingly, the wild boar were hunted in a different geographical area (Czech Republic) but the results were in accordance with our findings [3]. Scandinavian groESL sequences from dogs and horses differ from Slovenian in six nucleotides, but are identical among each other. These differences might suggest geographical variability of A. phagocytophilum and contribute Corresponding author and reprint requests: Tatjana Avsic Županc, Institute of Microbiology and Immunology, Faculty of Medicine, Zaloska 4, SI–1000 Ljubljana, Slovenia E-mail: tatjana.avsic@mf.uni-lj.si

Death of a horse infected experimentally with Anaplasma phagocytophilum

A 19-year-old horse that was one of a group of six horses infected experimentally with Anaplasma phagocytophilum for a study of the pathogenesis of equine granulocytic ehrlichiosis died suddenly two...

Clinical signs and clinical pathology of horses with equine granulocytic ehrlichiosis

Clinical signs and clinical pathology of horses with equine granulocytic ehrlichiosis

Molecular Diagnosis of Granulocytic Anaplasmosis and Infectious Cyclic Thrombocytopenia by PCR‐RFLP

Anaplasma phagocytophilum (A. phagocytophilum, formerly Ehrlichia phagocytophila) is a tick-borne pathogen responsible for tick-borne fever in ruminants, equine granulocytic ehrlichiosis (EGE) in horses, canine granulocytic ehrlichiosis (CGE) in dogs, and for human granulocytic ehrlichiosis (HGE). Human cases have been registered in many countries with a broad range of symptoms and pathogenicity. This article focused on Sardinia as the prevalence in humans was almost seven times higher than in the rest of Italy. To evaluate the risk, blood samples were collected from dogs and horses on the island. Genomic DNA was extracted from the buffy coat and amplified by heminested polymerase chain reaction (PCR) using the groEL gene primers. The first PCR reaction amplified a 624-bp fragment for both A. phagocytophilum and A. platys while the second PCR reaction amplified 573-bp and 515-bp fragments for the above two pathogens, respectively. Six A. phagocytophilum samples were PCR positive (3 dogs and 3 horses) while another dog was A. platys PCR positive. A phylogenetic analysis was conducted with A. phagocytophilum sequences in GenBank from the United States, Slovenia, Switzerland, Germany, UK, Austria, and Czech Republic. Surprisingly, the related phylogenetic tree showed that the Sardinian isolates were closer to the American isolates, which were showing highest mortality rates than from the other two European lineages.

New Findings on Anaplasmosis Caused by Infection with Anaplasma phagocytophilum

Ixodes ricinus (I. ricinus) is one of the vectors of Anaplasma phagocytophilum (A. phagocytophilum) in Europe, in which rates of infection range from 1.9% to 34%. In 1998, human granulocytic ehrlichiosis-like (HGE-like) Ehrlichia DNA was detected in Italy, by PCR technique in one I. ricinus nymph out of 55 ticks that were examined. In 1996, 6.3% of 310 human sera in high-risk subjects from Italy were found positive for antibodies to Ehrlichia phagocytophila (E. phagocytophila). In the same year, the authors reported the first case of equine granulocytic ehrlichiosis. In 1997, only 2 out of 563 equine blood samples examined were found positive for antibodies to E. phagocytophila in the Latium region. In 1998, serological positivity was not observed in 14 symptomatic race horses. In 2002, a symptomatic horse living in Rome was found positive for Ehrlichia equi (E. equi) antibodies, as confirmed by PCR. E. equi was also demonstrated in horses by detection of specific antibodies from two asymptomatic ponies. We tested 128 sera from sheep in different flocks, and antibodies to E. phagocytophila were detected in 17 sera (13.3%) of these sheep. From 2000 to 2004, 147 dog sera were tested for antibodies against A. phagocytophilum, and 7 of these sera were positive (4.8%). These data confirm the presence of the infection in human, domestic animals, and pets in Italy. Studies are under way to correlate the distributions of the disease and tick vector, I. ricinus.

Equine ehrlichiosis in Italy.

The authors review equine granulocytic ehrlichiosis (EGE) in Italy from 1996 to 2002. In 1996, the first case of EGE has been observed in a horse affected with specific symptomatology (fever, lethargy, anorexia, limb edema, thrombocytopenia, and petechiae). In 1997, a seroepidemiological survey was performed in the province of Rome on 563 animals using IFAT. The authors describe the last case, which occurred on 2002 in a 15-year-old male, bay, half-breed, tick-infested horse. Clinical features included fever, lethargy, limb edema, icterus, leukocytopenia, and thrombocytopenia. Laboratory tests were positive for ELISA and IFAT and several morulae were seen in the cytoplasm of neutrophils in buffy coat smears. The authors think that in Italy the prevalence of EGE is underestimated because of the nonpathognomonic clinical symptoms, the diffusion of specific infected vector, and the nonroutine nature of specific laboratory tests.

Ehrlichial Diseases

Equine granulocytic and monocytic ehrlichiosis caused by Ehrlichia equi and E. risticii, respectively, are seasonal diseases in horses that occur throughout the United States E. equi is transmitted by lxodes ticks and causes high fever, depression, anorexia, limb edema, petechiation, icterus, ataxia, and stiffness in gait. E. risticii, also known as the agent of Potomac horse fever, causes a febrile illness with a colitis of variable severity. Its occurrence is associated with aquatic habitats. The natural route of transmission is oral, through the ingestion of E. risticii infected trematode stages either free in water or in an intermediate host, such as aquatic animals.

Ehrlichia equi infection of horses from Minnesota and Wisconsin: detection of seroconversion and acute disease investigation.

Equine granulocytic ehrlichiosis (EGE) is caused by infection with Ehrlichia equi. EGE has been reported primarily in northern California, where E equi is transmitted by the tick Ixodes pacificus. Reports of EGE and the emergence of human granulocytic ehrlichia in Minnesota prompted a seroprevalence study of E equi in horses of Minnesota and Wisconsin. Tick (Ixodes scapularis) endemic areas of Minnesota and Wisconsin were compared to nonendemic regions of Minnesota. Indirect fluorescent antibody was used to detect the presence of serum antibodies to E equi. Serum samples from healthy horses, 375 samples from I scapularis endemic counties, and 366 samples from nonendemic counties were screened at a 1:40 dilution. Results demonstrated a seroprevalence of 17.6% in endemic areas versus 3.8% in nonendemic areas. Ehrlichial DNA from 2 samples was successfully amplified by polymerase chain reaction and 919 base pairs were sequenced. The DNA sequence of 1 Minnesota/Wisconsin strain differed from the GenBank strain (M73223) of E equi at positions 84 and 886 and from the MRK strain of E equi at position 84, and was identical to the human granulocytic ehrlichiosis (HGE) agent. The 2nd Minnesota/Wisconsin strain was identical to the 1st with the exception of a substitution of "A" at position 453 that is not present in E phagocytophila, E equi, or HGE agent strain sequences. Based on the results of this study, we concluded that E equi is present and causes infection in horses in Minnesota and Wisconsin. The occurrence of infection is higher in tick endemic regions.

Equine granulocytic ehrlichiosis: a case report with DNA analysis and species comparison.

Equine granulocytic ehrlichiosis: a case report with DNA analysis and species comparison.

A serological and clinical follow-up in horses with confirmed equine granulocytic ehrlichiosis.

For diagnosis of equine granulocytic ehrlichiosis (EGE) serological testing of antibodies to Ehrlichia equi is frequently used. An elevated antibody level is often misinterpreted as confirmative of active infection and results in treatment with antibiotics. If only seropositivity is considered as the diagnostic criterium, many horses showing convalescence titres will be treated. This study was undertaken to obtain information about the kinetics of antibodies during the course of infection and, for this purpose, 45 horses with clinical signs of EGE and confirmed ehrlichiaemia were monitored serologically and clinically over time. For a correct handling of cases with suspected EGE, the following results should be helpful: (i) 44% of the horses in the acute ehrlichiaemic stage were found to be serologically positive to E. equi; (ii) all horses showed a rapid increase in antibody titre, reaching maximum value within a month after the ehrlichiaemic stage; (iii) when 8 months had passed, titres had decreased, but 18 of 24 examined horses were still serologically positive; (iv) after 12-15 months most of the horses (n = 10) were serologically negative; and (v) the period required for complete clinical recovery varied from one day up to 6 months after antibiotic treatment.

Association of Ixodes pacificus (Acari: ixodidae) with the spatial and temporal distribution of equine granulocytic ehrlichiosis in California.

This study was conducted to determine if the biology of certain ticks associated with horses regulates the spatial and temporal distribution of equine granulocytic ehrlichiosis (EGE) in California north of Monterey County. We compared the spatial and temporal distribution of EGE cases with the seasons of activity and life histories of ticks that infest horses. Spatially, cases collected from equine veterinarians clustered around each other in a manner different from the way in which control cities of practice were distributed, with foci limited to the Sierra Nevada and coastal foothills. Cases also clustered seasonally: most were diagnosed between November and April. The spatial and temporal pattern of EGE cases closely parallels the well-characterized life history and distribution of Ixodes pacificus Cooley & Kohls, but not other ticks commonly associated with horses. Building on previous studies, there is compelling evidence that this tick has the vectorial capacity to transmit Ehrlichia equi to horses. Based on the life history and distribution of I. pacificus in relation to EGE cases, we reason that this tick is the only biologically plausible vector of E. equi in California, and provide evidence for a tightly linked association between I. pacificus and the epidemiology of EGE.

Serologic testing of horses for granulocytic ehrlichiosis, using indirect fluorescent antibody staining and immunoblot analysis

Abstract Objective To diagnose granulocytic ehrlichiosis in horses, compare results of indirect fluorescent antibody (IFA) staining procedures with those of immunoblot analysis, and compare serologic test findings with polymerase chain reaction (PCR) results. Animals 69 horses with high rectal temperatures (≥ 39 C) and lethargy, anorexia, or limb edema. Procedure 43 convalescent serum samples obtained from 38 horses 2 to 18 weeks after onset of illness were analyzed by use of immunoblot procedures and IFA staining methods, using the NCH-1 or BDS ehrlichial strains. Blood samples from 69 acutely ill horses were tested by PCR to detect ehrlichial DNA. Results Antibodies to Erlichial equi were detected in serum samples obtained during all seasons; seropositivity rates ranged from 50 to 93%. In IFA assays using the BDS or NCH-1 strain, seropositivity rates were 70 and 79%, respectively, whereas in immunoblot analyses using the NCH-1 strain, a seropositivity value of 79% was recorded. By immunoblot analysis, all serum samples of all seropositive horses were reactive to a protein having molecular mass of about 44 kd. Blood samples from 29 of 69 (42%) acutely ill horses contained ehrlichial DNA. Conclusion and Clinical Relevance Results of the various serologic testing procedures were in close agreement with each other. All serologic testing methods are suitable for laboratory diagnosis of equine granulocytic ehrlichiosis. (Am J Vet Res 1999; 60:631–635)

Development and duration of antibody response against Ehrlichia equi in horses

Objective To characterize antibody response in horses with clinical signs of Ehrlichia equi infection. Design Prospective study. Animals 13 horses with confirmed acute E equi infection. Procedure Sequential serum sampling was performed in Connecticut and New York during 1995 and 1996 to identify horses with naturally acquired equine granulocytic ehrlichiosis (EGE). Horses with clinical signs of EGE (ie, fever without respiratory involvement) were confirmed as having E equi infection by polymerase chain reaction detection of ehrlichial DNA and by a minimum fourfold increase in total antibody titer by indirect fluorescent antibody staining methods. Infection was corroborated by use of DNA sequencing. Results 11 of 13 horses did not have detectable antibody in serum samples obtained at onset of disease. Seroconversion was evident in samples obtained 19 to 81 days thereafter. Median time to peak antibody response was 46 days after onset and median titer was 1:320. For 11 of 13 horses, antibody titers were ≤ 1:40 by 215 days after onset. Clinical Implications E equi was found in horses in the northeastern United States and caused EGE. Concentration of antibodies to E equi increased within 19 to 81 days of disease onset and were low during early weeks of infection. Therefore, antibody detection may be of limited value for early serologic diagnosis. We suggest that disease may develop from a reinfection, and retrospective serologic studies to determine exposure to E equi may reflect a disproportionate number of negative reactions. (J Am Vet Med Assoc 1998;212:1910–1914)

Ehrlichial DNA amplified from Ixodes ricinus (Acari: Ixodidae) in France.

Granulocytic ehrlichia 16S rDNA was amplified for the 1st time from an Ixodes ricinus (Linne) tick collected in Europe. Sequence analysis of polymerase chain reaction products from the 16S rRNA gene demonstrated the organism from which it originated to be closely related to the agent of human granulocytic ehrlichiosis, an emerging disease that was recently described in the United States; Ehrlichia phagocytophila, the agent of tick-borne fever of ruminants in Europe; and Ehrlichia equi. the agent of the worldwide equine granulocytic ehrlichiosis. These granulocytic ehrlichiae have been associated with Ixodes spp. ticks that may act as vectors. It remains to be determined if each of these granulocytic ehrlichiae, that may constitute variants of the same species, is responsible for a specific disease in animals and in humans.

Isolation of the equine granulocytic ehrlichiosis agent, Ehrlichia equi, in tick cell culture.

The equine granulocytic ehrlichiosis agent, Ehrlichia equi, is closely related or identical to the human granulocytic ehrlichiosis (HGE) agent. Both are suspected of being transmitted by ticks. We have successfully isolated E. equi in a cell line, IDE8, derived from a putative vector, the tick Ixodes scapularis. Peripheral blood leukocytes from an experimentally infected horse were inoculated onto IDE8 monolayers. Cultures were incubated in a candle jar at 34 degrees C in tick cell culture medium with NaHCO3 and an organic buffer [3-(N-morpholino)-propanesulfonic acid] (MOPS). Within 2 weeks, infected cells were detected in Giemsa-stained culture samples, and the organisms subsequently spread to uninfected cells in the cultures. E. equi was passaged serially by transferring a portion of an infected culture to new cell layers every 2 to 3 weeks. The identity of the organisms was confirmed by PCR using oligonucleotide primers specific for E. equi and the HGE agent and by immunocytology. Homologous equine antibodies and human anti-HGE convalescent serum recognized E. equi grown in tick cell culture. Electron microscopy revealed electron-lucent and -dense ehrlichia-like forms developing within host cell endosomes. E. equi passaged twice in tick cell culture retained infectivity and pathogenicity for the equine host, as demonstrated by intravenous inoculation of a suspension of infected tick cells and subsequent reisolation from peripheral blood, in fulfillment of Koch's postulates. The horse developed severe clinical signs, i.e., fever, inappetence, thrombocytopenia, icterus, and limb edema, typical of granulocytic equine ehrlichiosis, within 1 week.

Equine granulocytic ehrlichiosis in Connecticut caused by an agent resembling the human granulocytotropic ehrlichia

The first recognized cases of equine granulocytic ehrlichiosis in New England are described. The DNA sequence of the 16S rRNA gene of the causative ehrlichia was found to be identical to that of the human granulocytotropic ehrlichia, the agent of human granulocytic ehrlichiosis.

Ixodes pacificus (Acari: Ixodidae) as a vector of Ehrlichia equi (Rickettsiales: Ehrlichieae).

Ehrlichia equi, a rickettsia described from horses in California 30 yr ago, causes equine granulocytic ehrlichiosis throughout the Americas and possibly Europe. Here, we report experimental transmission of E. equi from infected to susceptible horses through bites of western blacklegged ticks, Ixodes pacificus (Cooley & Kohls). In preliminary field studies, only I. pacificus consistently infested horses and vegetation at 3 locations with contemporary cases of equine ehrlichosis, and in particular, I. pacificus was the only species found attached to all of the infected horses. Exposure to bites of ticks in the genus Ixodes poses previously unrecognized and serious health risks to humans and animals.