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Equine granulocytic anaplasmosis
Research in Veterinary Science 95 (2013) 316–320
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Equine granulocytic anaplasmosis Beata Dzie˛giel, Łukasz Adaszek ⇑, Marcin Kalinowski, Stanisław Winiarczyk Department of Epizootiology and Clinic of Infectious Diseases, University of Life Sciences, Głe˛boka St. 30, 20-612 Lublin, Poland
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Article history: Received 19 February 2013 Accepted 22 May 2013
Keywords: Anaplasma phagocytophilum Horses Tick-borne disease
a b s t r a c t Anaplasma phagocytophilum is an emerging pathogen of horses that is transmitted by Ixodid ticks. Recent studies suggest that multiple strains of A. phagocytophilum may be circulating in wild and domestic animal populations, and these strains may have differential host tropisms and pathogenicity. The organism infects and survives within neutrophils. Co-infections with other tick-borne pathogens may occur, especially Borrelia burgdorferi. A. phagocytophilum causes an acute febrile illness in horses with lethargy, inappetence, lameness and hemorrhages. Diagnosis is based on finding morulae within granulocytes in the peripheral blood, and detection of the DNA of A. phagocytophilum using specific polymerase chain reaction assays. Most reports suggesting that anaplasmosis is a self-limiting disease that responds well to a tetracycline therapy. Ó 2013 Elsevier Ltd. All rights reserved.
1. Equine granulocytic anaplasmosis Equine Granulocytic Anaplasmosis (EGA) is a transmissible, multisystem human and animal disease associated with thrombocytopenia (Gribble, 1969; Chen et al., 1994). The disease occurs during spring and autumn – seasons of tick activity (Madigan and Gribble, 1987; Bown et al., 2003) and its etiological factor is Anaplasma phagocytophilum. It is a small, gram-negative bacteria, spheroid or pleomorphic in shape, present inside the granulocytes of the infected organism (Rikihisa, 1991; Bjöersdorff et al., 2002). It is classified as Anaplasma genus belonging to the Rickettsiales order. The order of Rickettsiales was created by merging of two bacteria families: Anaplasmataceae and Rickettsiaceae. Based on the molecular studies A. phagocytophilum includes pathogens which were previously considered as separate species: Ehrlichia phagocytophila, Ehrlichia equi and Human Granulocytic Ehrlichiosis agent – HGE agent (Dumler et al., 1995, 2001). 2. Epidemiology The first case of EGA was described in 1969 in California, USA (Gribble, 1969; Stannard et al., 1969). In Europe, the disease has been identified for many years: In 1984 equine anaplasmosis was diagnosed in Germany (Büscher et al., 1984), in 1985 in Switzerland (Hermann et al., 1985) and in 1990 in Sweden (Bjöersdorff et al., 1990). The disease has also been reported in Great Britain (Korbutiak and Schneiders, 1994), Denmark (Eriksen et al., 1997), Austria (Fröhlich and Edelhofer, 1998), the Czech Republic (Jahn et al., 2010), Holland (Butler et al., 2008), France (Bermann et al., ⇑ Corresponding author. Tel.: +48 4456192; fax: +48 4456202. E-mail addresses: [email protected], [email protected] (Ł. Adaszek). 0034-5288/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2013.05.010
2002) and Italy (Lillini et al., 2006). In Poland, the first case of EGA was described by Adaszek et al. (2009). Aside from America and Europe, the bibliographical data also report the occurrence of equine anaplasmosis in Asia (Chan et al., 2010) and Africa (M’ghirbi et al., 2012). The host of A. phagocytophilum may be invertebrates or vertebrates (Silaghi et al., 2011). The studies on the etiological factor of equine granulocytic anaplasmosis development conducted in Poland prove that the disease is caused by a relatively genetically stable genotype of Rickettsia (Adaszek et al., 2009, 2012). The following ticks: Ixodes spp., Dermacentor spp., Rhipicephalus spp., Hyalomma spp. and Haemaphysalis spp. are vectors of A. phagocytophilum, depending on the continent. The primary vector of Rickettsia in Europe is Ixodes ricinus (Stuen, 2007). In the regions endemic for anaplasmosis the disease is often accompanied by such infections as borreliosis or piroplasmosis (Stan´czak et al., 2004). As the pathogen is transmitted in the tick population transstadially and not transovarially (Bown et al., 2003; Dunning Hotopp et al., 2006), their habitat must contain the bacteria reservoir consisting of domestic and wild animals, including rodents (Walls et al., 1997; Nieto and Foley, 2008; Adaszek et al., 2012), in order to infect arachnids. Disease transmission from ticks to a sensitive mammal may occur only when ticks feed on the surface of the host’s body for 2–36 h (Bakken and Dumler, 2008; Ismail et al., 2010). 3. Pathogenesis Pathogenesis of EGA is not well known. A. phagocytophilum may inhibit processes of the host cells’ apoptosis and has the capacity to modify mechanisms related to energy generation, signal transduc-
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tion in a cell, as well as protective immune reactions (Lin et al., 2011; Ojogun et al., 2011). The microorganism proliferates in the cell lines of human myeloid leukemia (HL-60, KG-1, THP-1), in ovarian cells of Cricetulus griseus and IDE8 tick cell line (Munderloh et al., 1996; Dumler et al., 2001). Rickettsia may infect progenitor cells of bone marrow and endothelium (Klein et al., 1997; Herron et al., 2005) as well as bone marrow-derived mast cells (BMMCs) and human skin cells, which suggests that A. phagocytophilum may attack skin mast cells in the location of a tick bite (Ojogun et al., 2011). The pathogen demonstrates tropism to the cells of hematopoietic and phagocyte systems. Rickettsiae spread all over the host with bloodstream, causing damage of bone marrow and pancytopenia, especially thrombocytopenia (Gribble, 1969; Reubel et al., 1998; Dumler et al., 2001). The mechanisms causing platelet number reduction remain unexplained and may be related to their destruction by the immune system, increased phagocytosis by macrophages and their intensified disintegration in the spleen. Bone marrow hypoplasia may also result in the decrease of thrombocyte production. A. phagocytophilum attacks many organs and tissues leading to inflammatory lesions, mainly in the spleen, lungs, liver, kidneys and heart (Lepidi et al., 2000). Bench studies conducted on BMMCs cell lines and human skin lines proved that the pathogen blocks the release of TNF-a, IL-6, IL-13 and reduces the IgE-dependent activation of mastocytes (Ojogun et al., 2011). The bacterium does not induce much inflammation, which results from the fact that the cell wall of A. phagocytophilum does not contain LPS or peptidoglycan (Rikihisa, 2010).
4. Clinical symptoms The course of equine granulocytic anaplasmosis may be subclinical or acute. The incubation period of the acute form of anaplasmosis is approximately 10 days (Gribble, 1969; Madigan, 1993; Barlough et al., 1995). Initially the symptoms are atypical, the infected animal develops: apathy, weakening and increased body temperature. Next, an aversion to movement appears, stiff gait, painful edema of fetlock joints and subcutaneous tissue of an inflammatory character, and sometimes lameness (Adaszek et al., 2009; Silaghi et al., 2011). The subcutaneous tissue edemas may persist even for 2 weeks after complete regression of the other symptoms (Madigan and Gribble, 1987) or disappear directly following the antibiotic therapy (Pusterla et al., 1998). Intensification of clinical symptoms demonstrates a considerable diversification and, to a great extent, depends on the condition and age of the infected animal. A typical course of EGA is observed in horses of above 3 years of age. The infected animals develop fever (39–40 °C) which may persist for 6–12 days. It is accompanied by general symptoms and limb swelling. The disease in one-yearold and younger horses is of non-serious nature with nonspecific symptoms such as lack of appetite and aversion to movement (Madigan and Gribble, 1987). Other symptoms of acute anaplasmosis are high fever, bleeding from mucosa, weight loss, enlarged spleen and lymph nodes. During the febrile period, the heart rate is increased up to 50–60 beats per minute and the respiratory rate up to 30 breaths per minute. Aside from the above, the following symptoms may be observed: diarrhea, vomiting, inflammation of joints, neurological symptoms such as convulsions and paralysis (Reubel et al., 1998; Adaszek et al., 2009). Hematological tests can reveal anemia, which is a result of bone marrow hypoplasia and may be of a non-regenerative form (Madigan and Gribble, 1987; Madigan et al., 1996). A specific hematological disorder observed in the course of anaplasmosis is thrombocytopenia, which leads to petechiae on the
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mucous membranes, in particular on the inner surface of the lips and gums. Apart from thrombocytopenia, a hematological test may reveal leucocytosis or leucopenia with neutropenia and hematocrit reduction. Biochemical tests of the blood serum of the infected animals are used to determine concentration increases of total bilirubin, urea and creatinine (Reubel et al., 1998; Lepidi et al., 2000; Adaszek et al., 2009; Silaghi et al., 2011). The concentration of plasma fibrinogen may be increased (Berrington et al., 1996; Adaszek et al., 2009) or remain within the limits of physiological norm (Pusterla et al., 1998). Sometimes proteinuria appears (Lepidi et al., 2000). The course of EGA is rarely subclinical. Chang et al. (1998) applied the PCR method to detect the DNA of A. phagocytophilum in poorly vascularized tissues 38 days after the experimental infection of horses. Franzén et al. (2009) indicated that experimental infection with A. phagocytophilum may not induce any clinical symptoms and the pathogen itself, after being introduced into the host, can survive in it for at least 129 days. Sometimes infected animals will collapse as a consequence; however, cases of self-recovery following the primary infection are observed more frequently (Gribble, 1969). The results of the studies conducted in the Netherlands confirm that the disease in horses is characterized by a considerably diversified course. A 4-year-old horse with acute anaplasmosis died despite implementing proper causal treatment (Butler et al., 2008). A rapid, incurable course of infection was also noted in a 19-year-old horse experimentally infected with A. phagocytophilum (Franzén et al., 2007). On the other hand in one study, all horses in the 9–15 years age group survived the disease without treatment (Butler et al., 2008). Histological tests demonstrated vasculitis necroticans in fragments of organs collected from the dead animals (Madigan and Gribble, 1987). It was also found that the infected animals had blood extravasations and petechiae on the mucous and serous membranes, edema of subcutaneous tissue as well as inflammatory lesions of internal organs, particularly the spleen, liver, lungs, kidneys and heart (Lepidi et al., 2000; Franzén et al., 2007), which indicates at the fact that the disease is of generalized nature and may lead to development of multiple organ failure.
5. Diagnosis Diagnosis of equine granulocytic anaplasmosis is based on the analysis of data from the epizootic history, as well as results of clinical and laboratory tests (Amusategui et al., 2006). Differential diagnosis of the disease should primarily consider borreliosis, equine infectious anemia, liver diseases and equine viral arteritis (Madigan, 1993; Lepidi et al., 2000; Silaghi et al., 2011). The microscopic tests of blood smears taken from the infected animals stained using the Diff-Quick, Wright or Giemsa methods can detect the presence of A. phagocytophilum morulae in the cytoplasm of neutrophils and, at times, eosinophils (Sells et al., 1976; Pusterla et al., 1998). The morulae appear in granulocytes in the acute stage of the disease, usually 2–4 days after infection (Gribble, 1969; Barlough et al., 1995; Adaszek and Winiarczyk, 2011). They are inclusion bodies of dark blue to violet color, consisting of many tiny initial bodies of circular, oval or rod shape and size of 0.18– 1.4 lm (Fig. 1). Sometimes they disintegrate into single elementary bodies (Sells et al., 1976). Despite the common belief that in infected horses the presence of morulae may be observed only in 1–6% of neutrophils (Berrington et al., 1996), the level of bacteraemia may be higher sometimes. Łukaszewska et al. (2008) proved the presence of these structures in 3–8% of neutrophils, whereas Uehlinger et al. (2011) in ca. 16%. Administration of dexamethasone to infected horses resulted in an increase of the infected neutrophils from 4% to 10%
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(Łukaszewska et al., 2008). Studies conducted in Poland have not proved the presence of Anaplasma in eosinophils of horses suffering from anaplasmosis (Adaszek and Winiarczyk, 2011). It is becoming increasingly popular to apply molecular biology methods, particularly polymerasis chain reaction (PCR), to the diagnosis of granulocytic anaplasmosis and the assessment of epizootic situations, as well as the detection of subclinical infections with Anaplasma (Madigan et al., 1996; Adaszek et al., 2009, 2013). Determination of a sequence of the obtained amplicons provides valuable information for epidemiological and taxonomic analysis. The PCR reaction and sequencing of the obtained amplicons allows, for example, the distinguishing of A. phagocytophilum from Ehrlichia ewingii. Both pathogens induce the development of morulae in neutrophils, which cannot be distinguished in the microscopic blood smear tests (Łukaszewska et al., 2008). Material from which it is possible to isolate DNA for PCR includes full blood, leukocytes, bone marrow or fragments of the spleen. The PCR reaction is performed using specific primers limiting the fragment of a selected rickettsial pathogen gene. The presence of the amplicon of a specific volume in a given sample is confirmed electrophoretically and the obtained products are subject to sequencing, enabling the determination of which bacterium caused the disease and to what extent it differs from the cases described so far (Adaszek et al., 2009, 2013). This method allows the early diagnosis of the disease. Barlough et al. (1995) with the use of the nested PCR method confirmed the presence of genetic material of A. phagocytophilum in the blood of a horse 9 days after infection, a day after developing fever and 2 days before the first observation of morulae in the blood. This shows that the technique is the most sensitive of all the diagnostic methods used to diagnose EGA. Other laboratory techniques, less significant than PCR, applied in equine anaplasmosis diagnostics are serological tests (van Andel et al., 1998; Amusategui et al., 2006). Most veterinary laboratories use the indirect fluorescent antibody test or ELISA test (Magnarelli et al., 2001), however, these tests are of limited applicability in the diagnosis of the acute stage of the infection, when the antibodies specific for A. phagocytophilum have not yet developed in the plasma of the infected animal. There were also attempts to culture A. phagocytophilum ‘‘in vitro’’ in the cell lines. However, this test is costly and of little diagnostic value due to the extended time needed for the result (Munderloh et al., 1996).
Fig. 1. Presence of Anaplasma inclusion bodies in neutrophils.
6. Treatment and prevention Because A. phagocytophilum is an intracellular pathogen, not many chemotherapeutics are effective in fighting this infection. The most effective group of antibiotics in causal therapy of anaplasmosis is the tetracyclines. In case of EGA, the drug of choice is oxytetracycline, its mechanism consisting in blocking protein synthesis through bonding with subunits 30S of the bacterial ribosomes (Madigan, 1993; Maurin et al., 2003). Pusterla et al. (1998) administered oxytetracycline at a dose of 10 mg/kg body weight to a 12-year-old mare for 5 days. The efficiency of the antibiotic and the selected dose was confirmed by Adaszek et al. (2009), who administered the mentioned chemotherapeutic to mares of 4 and 5 years of age with confirmed A. phagocytophilum infection for 10 days. The results of other studies conducted by this team indicate the effectiveness of oxytetracycline administered intravenously to a 7-year-old English full-blood gelding with symptoms of anaplasmosis for 7 days at a dose of 8 mg/kg body weight along with dexametasone i.m. at a dose of 20 mg/horse for 3 days. The therapy resulted in a gradual return of appetite and, after 3 days, the disappearance of limb swelling and problems with mobility. The neurological symptoms also subsided after another two days, and after 10 days from the beginning of treatment the horses were fully recovered (Adaszek et al., 2009). In Switzerland, horses were successfully treated with oxytetracycline i.v. administered for 7 days at a dose of 7 mg/kg body weight. Usually, a distinct decrease of body temperature, return of appetite and improvement of general condition of the patient were observed after the first day of therapy. The therapy was continued orally for a week (Hermann et al., 1985). A similar treatment regimen has been applied in the USA, where significant health improvement of the infected horses was noted 24 h from commencing the tetracycline therapy (Madigan and Gribble, 1987). These results are corroborated by recent studies conducted in Poland, during which the condition of the infected animals improved after 24 h from administration of the drugs and full recovery was reported after 7 days of oxytetracycline i.v. therapy at a dose of 8 mg/kg body weight (Adaszek and Winiarczyk, 2011). In some cases, recovery has been observed as soon as 12 h from the start of the treatment (Pusterla et al., 1998). In Virginia a sick mare was administered oxytetracycline intravenously at a dose of 6.6 mg/kg body weight, which resulted in the overall health improvement of the animal; however, perivascular swelling appeared in the place of injection (Lewis et al., 2009). Adverse reactions of this chemotherapeutic include hepatotoxic and neurotoxic effects, which are observed after long-term therapy. Moreover, administration of the antibiotic has a suppressive impact on the immune system and may result in gastro-intestinal disorders in the form of diarrheas and discoloration of dental enamel due to its capacity to chelate the formation of metal ions (Klein et al., 1997; Maurin et al., 2003). In the event of intolerance of oxytetracycline in horses, granulocytic anaplasmosis may be treated with other antibiotics. In the USA, EGA was effectively treated with doxycycline (p.o. 10 mg/kg every 12 h) (Lewis et al., 2009). Furthermore, the in vitro studies revealed that A. phagocytophilum is sensitive to new generations of fluorochinolones and riphampicine (Klein et al., 1997). Corticosteroids can be applied in the supportive treatment of autoimmune hemolytic anemia. They stimulate the bone marrow to produce red blood cells and stabilize the membranes of erythrocytes (Peek, 2009). Non-steroidal anti-inflammatory drugs and infusion fluids are applied only in justified cases. Prophylaxis of equine granulocytic anaplasmosis includes tick control and preventing them from feeding on the integuments of animals (Parola and Raoult, 2001). Removal of a tick from the skin
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does not exclude the possibility of infection (Oteo et al., 2001). Currently there is no vaccine against EGA on the market. The studies on development of vaccine against anaplasmosis on the basis of Anaplasma marginale antigens are in progress (Dark et al., 2011; Palmer et al., 2012).
7. Summary In Poland, EGA is a relatively new disease which may cause both therapeutic and diagnostic difficulties in veterinary medicine. The disease should be considered in horses in every case of acute symptoms, accompanied by thrombocytopenia or leucopenia with neutropenia revealed by hematological test performed after horse contact with ticks. Finally, numerous Anaplasma spp. have been identified in ticks and vertebrates throughout the world. Although their pathogenicity for people has yet to be demonstrated, they are good candidates to be involved in human diseases since many of them have been detected in ticks that readily bite people (Stan´czak et al., 2004; Cisak et al., 2005). Therefore, more worldwide studies could lead to the description of emerging human anaplasmosis in the future (Parola et al., 2005). References Adaszek, Ł., Winiarczyk, S., 2011. Identification of Anaplasma spp. Rickettsia isolated from horses from clinical disease cases in Poland. Zoonoses and Public Health 58, 514–518. Adaszek, Ł., Winiarczyk, S., Łukaszewska, J., 2009. A first case of ehrlichiosis in a horse in Poland. Deutsche Tierarztliche Wochenschrift 116, 330–334. Adaszek, Ł., Klimiuk, P., Skrzypczak, M., Górna, M., Zie˛tek, J., Winiarczyk, S., 2012. The identification of Anaplasma spp. isolated from fallow deer (Dama dama) on a free-range farm in eastern Poland. Polish Journal of Veterinary Science 15, 393– 394. Adaszek, Ł., Górna, M., Skrzypczak, M., Buczek, K., Balicki, I., Winiarczyk, S., 2013. Three clinical cases of Anaplasma phagocytophilum infection in cats in Poland. Journal of Feline Medicine and Surgery 15, 333–337. Amusategui, I., Sainz, A., Tesouro, M.A., 2006. Serological evaluation of Anaplasma phagocytophilum infection in livestock in northwestern Spain. Annals of the New York Academy of Science 1078, 487–490. Bakken, J.S., Dumler, J.S., 2008. Human granulocytic anaplasmosis. Infectious Disease Clinics of North America 22, 433–448. Barlough, J.E., Madigan, J.E., DeRock, E., Dumler, J.S., Bakken, J.S., 1995. Protection against Ehrlichia equi is conferred by prior infection with the Human granulocytotropic ehrlichia (HGE agent). Journal of Clinical Microbiology 33, 3333–3334. Bermann, F., Davoust, B., Fournier, P.E., Brisou-Lapointe, A.V., Brouqui, P., 2002. Ehrlichia equi (Anaplasma phagocytophila) infection in an adult horse in France. Veterinary Record 150, 787–788. Berrington, A., Moats, R., Lester, S., 1996. A case of Ehrlichia equi in an adult horse in British Columbia. Canadian Veterinary Journal 37, 174–175. Bjöersdorff, A., Christenson, D., Johnsson, A., Sjöström, A.C., Madigan, J.E., 1990. Ehrlichia equi–infektion diagnostiserat hos häst. Svensk Veterinärtidning 42, 357–360. Bjöersdorff, A., Bagert, B., Massung, R.F., Gusa, A., Eliasson, I., 2002. Isolation and characterization of two European strains of Ehrlichia phagocytophila of equine origin. Clinical and Diagnostic Laboratory Immunology 9, 341–343. Bown, K.J., Begon, M., Bennett, M., Woldehiwet, Z., Ogden, N.H., 2003. Seasonal dynamics of Anaplasma phagocytophila in a rodent-tick (Ixodes trianguliceps) system, United Kingdom. Emerging Infectious Diseases 9, 63–70. Büscher, G., Gandras, R., Apel, G., Friedhoff, K.T., 1984. Der erste Fall von Ehrlichiosis beim Pferd in Deutschland (Kurzmitteilung). Deutsche Tierarztliche Wochenschrift 91, 408–409. Butler, C.M., Nijhof, A.M., Jongejan, F., van der Kolk, J.H., 2008. Anaplasma phagocytophilum infection in horses in the Netherlands. Veterinary Record 162, 216–218. Chan, K.Y., Wang, Ch.H., Wu, Y.L., 2010. Serological survey of equine piroplasmosis, equine granulocytic anaplasmosis, and equine lyme disease in Taiwan. Taiwan Veterinary Journal 36, 261–267. Chang, Y.F., Novosel, V., Dubovi, E., Wong, S.J., Chu, F.K., Chang, C.F., Del Piero, F., Shin, S., Lein, D.H., 1998. Experimental infection of the human granulocytic ehrlichiosis agent in horses. Veterinary Parasitology 78, 137–145. Chen, S.M., Dumler, J.S., Bakken, J.S., Walker, D.H., 1994. Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. Journal of Clinical Microbiology 32, 589–595. Cisak, E., Chmielewska-Badora, J., Zwolin´ski, J., Wójcik-Fatla, A., Polak, J., Dutkiewicz, J., 2005. Risk of tick-borne bacterial diseases among workers of Roztocze National Park (south-eastern Poland). Annals of Agricultural and Environmental Medicine 12, 127–132.
319
Dark, M.J., Al-Khedery, B., Barbet, A.F., 2011. Multistrain genome analysis identifies candidate vaccine antigens of Anaplasma marginale. Vaccine 29, 4923–4932. Dumler, J.S., Asanovich, K.M., Bakken, J.S., Richter, P., Kimsey, R., Madigan, J.E., 1995. Serologic cross-reactions among Ehrlichia equi, Ehrlichia phagocytophila and human granulocytic Ehrlichia. Journal of Clinical Microbiology 33, 1098–1103. Dumler, J.S., Barbet, A.F., Bekker, C.P., Dasch, G.A., Palmer, G.H., Ray, S.C., Rikihisa, Y., Rurangirwa, F.R., 2001. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichia phagocytophila. International Journal of Systematic and Evolutionary Microbiology 51, 2145–2165. Dunning Hotopp, J.C., Lin, M., Madupu, R., Crabtree, J., Angiuoli, S.V., Eisen, J.A., Seshadri, R., Ren, Q., Wu, M., Utterback, T.R., Smith, S., Lewis, M., Khouri, H., Zhang, C., Niu, H., Lin, Q., Ohashi, N., Zhi, N., Nelson, W., Brinkac, L.M., Dodson, R.J., Rosovitz, M.J., Sundaram, J., Daugherty, S.C., Davidsen, T., Durkin, A.S., Gwinn, M., Haft, D.H., Selengut, J.D., Sullivan, S.A., Zafar, N., Zhou, L., Benahmed, F., Forberger, H., Halpin, R., Mulligan, S., Robinson, J., White, O., Rikihisa, Y., Tettelin, H., 2006. Comparative genomics of emerging human ehrlichiosis agents. PLoS Genetics 2, E21. Eriksen, L., Hansen, J.F., Abildtrup, E., Engvall, E.O., 1997. Equine granulocytic ehrlichiosis diagnosed in Denmark. Dansk Veterinærtidsskrift 80, 231–234. Franzén, P., Berg, A.-L., Aspan, A., Gunnarsson, A., Pringle, J.D., 2007. Death of a horse infected experimentally with Anaplasma phagocytophilum. Veterinary Record 160, 122–125. Franzén, P., Aspan, A., Egenvall, A., Gunnarsson, A., Karlstam, E., Pringle, J., 2009. Molecular evidence for persistence of Anaplasma phagocytophilum in the absence of clinical abnormalities in horses after recovery from acute experimental infection. Journal of Veterinary Internal Medicine 23, 636–642. Fröhlich, W., Edelhofer, R., 1998. Erstbeschreibung der equinen granulozytären Ehrlichiose (EGE) bei einem Pferd in Ös terreich. Wiener Tierärztliche Monatsschrift 11, 389–394. Gribble, D.H., 1969. Equine ehrlichiosis. Journal of the American Veterinary Medical Association 155, 462–469. Hermann, M., Baumann, D., Lutz, H., Wild, P., 1985. Erster diagnostizierter Fall von equiner Ehrlichiose in der Schweiz. Pferdeheilkunde 1, 247–250. Herron, M.J., Ericson, M.E., Kurtti, T.J., Munderloh, U.G., 2005. The interactions of Anaplasma phagocytophilum, endothelial cells, and human neutrophils. Annals of the New York Academy of Science 1063, 374–382. Ismail, N., Bloch, K.C., McBride, J.W., 2010. Human ehrlichiosis and anaplasmosis. Journal of Laboratory and Clinical Medicine 30, 261–292. Jahn, P., Zeman, P., Bezdekova, B., Praskova, I., 2010. Equine granulocytic anaplasmosis in the Czech Republic. Veterinary Record 166, 646–649. Klein, M.B., Miller, J.S., Nelson, C.M., Goodman, J.L., 1997. Primary bone marrow progenitors of both granulocytic and monocytic lineages are susceptible to infection with the agent of human granulocytic ehrlichiosis. Journal of Infectious Diseases 176, 1405–1409. Korbutiak, E., Schneiders, D.H., 1994. First confirmed case of equine ehrlichiosis in Great Britain. Equine Veterinary Education 6, 303–304. Lepidi, H., Bunnell, J.E., Martin, M.E., Madigan, J.E., Stuen, S., Dumler, J.S., 2000. Comparative pathology and immunohistology associated with clinical illness after Ehrlichia phagocytophila-group infections. The American Journal of Tropical Medicine and Hygiene 62, 29–37. Lewis, S.R., Zimmerman, K., Dascanio, J.J., Pleasant, R.S., Witonsky, S.G., 2009. Equine granulocytic anaplasmosis: a case report and review. Journal of Equine Veterinary Science 29, 160–166. Lillini, E., Marcì, G., Proietti, G., Scarpulla, M., 2006. New findings on anaplasmosis caused by infection with Anaplasma phagocytophilum. Annals of the New York Academy of Science 1081, 360–370. Lin, M., Kikuchi, T., Brewer, H.M., Norbeck, A.D., Rikihisa, Y., 2011. Global proteomic analysis of two tick-borne emerging zoonotic agents: Anaplasma phagocytophilum and Ehrlichia chaffeensis. Frontiers in Microbiology 2, 24. Łukaszewska, J., Adaszek, Ł., Winiarczyk, S., 2008. Hematological changes in _ granulocytic anaplasmosis in dogs and horses. Zycie Weterynaryjne 83, 827– 831. M’ghirbi, Y., Yaïch, H., Ghorbel, A., Bouattour, A., 2012. Anaplasma phagocytophilum in horses and ticks in Tunisia. Parasites & Vectors 5, 180. Madigan, J.E., 1993. Equine ehrlichiosis. Veterinary Clinics of North America 9, 423– 428. Madigan, J.E., Gribble, D., 1987. Equine ehrlichiosis in northern California: 49 cases (1968–1981). Journal of the American Veterinary Medical Association 190, 445– 448. Madigan, J.E., Barlough, J.E., Dumler, J.S., Schankman, N.S., DeRock, E., 1996. Equine granulocytic ehrlichiosis in Connecticut caused by an agent resembling the human granulocytotropic ehrlichia. Journal of Clinical Microbiology 34, 434– 435. Magnarelli, L.A., Ijdo, J.W., van Andel, A.E., Wu, C., Fikrig, E., 2001. Evaluation of a polyvalent enzyme-linked immunosorbent assay incorporating a recombinant p44 antigen for diagnosis of granulocytic ehrlichiosis in dogs and horses. American Journal of Veterinary Research 62, 29–32. Maurin, M., Bakken, J.S., Dumler, J.S., 2003. Antibiotic susceptibilities of Anaplasma (Ehrlichia) phagocytophilum strains from various geographic areas in the United States. Antimicrobial Agents and Chemotherapy 47, 413–415. Munderloh, U.G., Madigan, J.E., Dumler, J.S., Goodman, J.L., Hayes, S.F., Barlough, J.E., Nelson, C.M., Kurtti, T.J., 1996. Isolation of the equine granulocytic ehrlichiosis
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agent, Ehrlichia equi, in tick cell culture. Journal of Clinical Microbiology 34, 664–670. Nieto, N.C., Foley, J.E., 2008. Evaluation of squirrels (Rodentia: Sciuridae) as ecologically significant hosts for Anaplasma phagocytophilum in California. Journal of Medical Entomology 45, 763–769. Ojogun, N., Barnstein, B., Huang, B., Oskeritzian, C.A., Homeister, J.W., Miller, D., Ryan, J.J., Carlyon, J.A., 2011. Anaplasma phagocytophilum infects mast cells via alpha1,3-fucosylated but not sialylated glycans and inhibits IgE-mediated cytokine production and histamine release. Infection and Immunity 79, 2717– 2726. Oteo, J.A., Blanco, J.R., Ibarra, V., 2001. Can we prevent tickborne transmission disease? Enfermedades Infecciosas y Microbiología Clinica 19, 509–513. Palmer, G.H., Brown, W.C., Noh, S.M., Brayton, K.A., 2012. Genome-wide screening and identification of antigens for rickettsial vaccine development. FEMS Immunology and Medical Microbiology 64, 115–119. Parola, P., Raoult, D., 2001. Tick-borne bacterial diseases emerging in Europe. Clinical Microbiology and Infection 7, 80–83. Parola, P., Davoust, B., Raoult, D., 2005. Tick- and flea-borne rickettsial emerging zoonoses. Veterinary Research 36, 469–492. Peek, S.F., 2009. Icterus. In: Robinson, N.E., Sprayberry, K.A. (Eds.), Current Therapy in Equine Medicine. Saunders Elsevier Inc., St. Louis, MO, USA, pp. 456–460. Pusterla, N., Lutz, H., Braun, U., 1998. Experimental infection of four horses with Ehrlichia phagocytophila. Veterinary Record 143, 303–305. Reubel, G.H., Kimsey, R.B., Barlough, J.E., Madigan, J.E., 1998. Experimental transmission of Ehrlichia equi to horses through naturally infected ticks (Ixodes pacificus) from northern California. Journal of Clinical Microbiology 36, 2131–2134.
Rikihisa, Y., 1991. The tribe Ehrlichieae and ehrlichial diseases. Clinical Microbiology Reviews 4, 286–308. Rikihisa, Y., 2010. Anaplasma phagocytophilum and Ehrlichia chaffeensis: subversive manipulators of host cells. Nature Reviews Microbiology 8, 328–339. Sells, D.M., Hildebrandt, P.K., Lewis, G.E., Nyindo, M.B., Ristic, M., 1976. Ultrastructural observations on Ehrlichia equi organisms in equine granulocytes. Infection and Immunity 13, 273. Silaghi, C., Liebisch, G., Pfister, K., 2011. Genetic variants of Anaplasma phagocytophilum from 14 equine granulocytic anaplasmosis cases. Parasites & Vectors 4, 161. Stan´czak, J., Gabre, R.M., Kruminis-Łozowska, W., Racewicz, M., Kubica-Biernat, B., 2004. Ixodes ricinus as a vector of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Babesia microti in urban and suburban forests. Annals of Agricultural and Environmental Medicine 11, 109–114. Stannard, A.A., Gribble, D.H., Smith, R.S., 1969. Equine ehrlichiosis: a disease with similarities to tick-borne fever and bovine petechial fever. Veterinary Record 84, 149–150. Stuen, S., 2007. Anaplasma phagocytophilum – the most widespread tick-borne infection in animals in Europe. Veterinary Research Communications 31, 79–84. Uehlinger, F.D., Clancey, N.P., Lofstedt, J., 2011. Granulocytic anaplasmosis in a horse from Nova Scotia caused by infection with Anaplasma phagocytophilum. Canadian Veterinary Journal 52, 537–540. van Andel, A.E., Magnarelli, L.A., Heimer, R., Wilson, M.L., 1998. Development and duration of antibody response against Ehrlichia equi in horses. Journal of the American Veterinary Medical Association 212, 1910–1914. Walls, J.J., Greig, B., Neitzel, D.F., Dumler, J.S., 1997. Natural infection of small mammal species in Minnesota with the agent of human granulocytic ehrlichiosis. Journal of Clinical Microbiology 35, 853–855.
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Equine granulocytic anaplasmosis Beata Dzie˛giel, Łukasz Adaszek ⇑, Marcin Kalinowski, Stanisław Winiarczyk Department of Epizootiology and Clinic of Infectious Diseases, University of Life Sciences, Głe˛boka St. 30, 20-612 Lublin, Poland
a r t i c l e
i n f o
Article history: Received 19 February 2013 Accepted 22 May 2013
Keywords: Anaplasma phagocytophilum Horses Tick-borne disease
a b s t r a c t Anaplasma phagocytophilum is an emerging pathogen of horses that is transmitted by Ixodid ticks. Recent studies suggest that multiple strains of A. phagocytophilum may be circulating in wild and domestic animal populations, and these strains may have differential host tropisms and pathogenicity. The organism infects and survives within neutrophils. Co-infections with other tick-borne pathogens may occur, especially Borrelia burgdorferi. A. phagocytophilum causes an acute febrile illness in horses with lethargy, inappetence, lameness and hemorrhages. Diagnosis is based on finding morulae within granulocytes in the peripheral blood, and detection of the DNA of A. phagocytophilum using specific polymerase chain reaction assays. Most reports suggesting that anaplasmosis is a self-limiting disease that responds well to a tetracycline therapy. Ó 2013 Elsevier Ltd. All rights reserved.
1. Equine granulocytic anaplasmosis Equine Granulocytic Anaplasmosis (EGA) is a transmissible, multisystem human and animal disease associated with thrombocytopenia (Gribble, 1969; Chen et al., 1994). The disease occurs during spring and autumn – seasons of tick activity (Madigan and Gribble, 1987; Bown et al., 2003) and its etiological factor is Anaplasma phagocytophilum. It is a small, gram-negative bacteria, spheroid or pleomorphic in shape, present inside the granulocytes of the infected organism (Rikihisa, 1991; Bjöersdorff et al., 2002). It is classified as Anaplasma genus belonging to the Rickettsiales order. The order of Rickettsiales was created by merging of two bacteria families: Anaplasmataceae and Rickettsiaceae. Based on the molecular studies A. phagocytophilum includes pathogens which were previously considered as separate species: Ehrlichia phagocytophila, Ehrlichia equi and Human Granulocytic Ehrlichiosis agent – HGE agent (Dumler et al., 1995, 2001). 2. Epidemiology The first case of EGA was described in 1969 in California, USA (Gribble, 1969; Stannard et al., 1969). In Europe, the disease has been identified for many years: In 1984 equine anaplasmosis was diagnosed in Germany (Büscher et al., 1984), in 1985 in Switzerland (Hermann et al., 1985) and in 1990 in Sweden (Bjöersdorff et al., 1990). The disease has also been reported in Great Britain (Korbutiak and Schneiders, 1994), Denmark (Eriksen et al., 1997), Austria (Fröhlich and Edelhofer, 1998), the Czech Republic (Jahn et al., 2010), Holland (Butler et al., 2008), France (Bermann et al., ⇑ Corresponding author. Tel.: +48 4456192; fax: +48 4456202. E-mail addresses: [email protected], [email protected] (Ł. Adaszek). 0034-5288/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2013.05.010
2002) and Italy (Lillini et al., 2006). In Poland, the first case of EGA was described by Adaszek et al. (2009). Aside from America and Europe, the bibliographical data also report the occurrence of equine anaplasmosis in Asia (Chan et al., 2010) and Africa (M’ghirbi et al., 2012). The host of A. phagocytophilum may be invertebrates or vertebrates (Silaghi et al., 2011). The studies on the etiological factor of equine granulocytic anaplasmosis development conducted in Poland prove that the disease is caused by a relatively genetically stable genotype of Rickettsia (Adaszek et al., 2009, 2012). The following ticks: Ixodes spp., Dermacentor spp., Rhipicephalus spp., Hyalomma spp. and Haemaphysalis spp. are vectors of A. phagocytophilum, depending on the continent. The primary vector of Rickettsia in Europe is Ixodes ricinus (Stuen, 2007). In the regions endemic for anaplasmosis the disease is often accompanied by such infections as borreliosis or piroplasmosis (Stan´czak et al., 2004). As the pathogen is transmitted in the tick population transstadially and not transovarially (Bown et al., 2003; Dunning Hotopp et al., 2006), their habitat must contain the bacteria reservoir consisting of domestic and wild animals, including rodents (Walls et al., 1997; Nieto and Foley, 2008; Adaszek et al., 2012), in order to infect arachnids. Disease transmission from ticks to a sensitive mammal may occur only when ticks feed on the surface of the host’s body for 2–36 h (Bakken and Dumler, 2008; Ismail et al., 2010). 3. Pathogenesis Pathogenesis of EGA is not well known. A. phagocytophilum may inhibit processes of the host cells’ apoptosis and has the capacity to modify mechanisms related to energy generation, signal transduc-
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tion in a cell, as well as protective immune reactions (Lin et al., 2011; Ojogun et al., 2011). The microorganism proliferates in the cell lines of human myeloid leukemia (HL-60, KG-1, THP-1), in ovarian cells of Cricetulus griseus and IDE8 tick cell line (Munderloh et al., 1996; Dumler et al., 2001). Rickettsia may infect progenitor cells of bone marrow and endothelium (Klein et al., 1997; Herron et al., 2005) as well as bone marrow-derived mast cells (BMMCs) and human skin cells, which suggests that A. phagocytophilum may attack skin mast cells in the location of a tick bite (Ojogun et al., 2011). The pathogen demonstrates tropism to the cells of hematopoietic and phagocyte systems. Rickettsiae spread all over the host with bloodstream, causing damage of bone marrow and pancytopenia, especially thrombocytopenia (Gribble, 1969; Reubel et al., 1998; Dumler et al., 2001). The mechanisms causing platelet number reduction remain unexplained and may be related to their destruction by the immune system, increased phagocytosis by macrophages and their intensified disintegration in the spleen. Bone marrow hypoplasia may also result in the decrease of thrombocyte production. A. phagocytophilum attacks many organs and tissues leading to inflammatory lesions, mainly in the spleen, lungs, liver, kidneys and heart (Lepidi et al., 2000). Bench studies conducted on BMMCs cell lines and human skin lines proved that the pathogen blocks the release of TNF-a, IL-6, IL-13 and reduces the IgE-dependent activation of mastocytes (Ojogun et al., 2011). The bacterium does not induce much inflammation, which results from the fact that the cell wall of A. phagocytophilum does not contain LPS or peptidoglycan (Rikihisa, 2010).
4. Clinical symptoms The course of equine granulocytic anaplasmosis may be subclinical or acute. The incubation period of the acute form of anaplasmosis is approximately 10 days (Gribble, 1969; Madigan, 1993; Barlough et al., 1995). Initially the symptoms are atypical, the infected animal develops: apathy, weakening and increased body temperature. Next, an aversion to movement appears, stiff gait, painful edema of fetlock joints and subcutaneous tissue of an inflammatory character, and sometimes lameness (Adaszek et al., 2009; Silaghi et al., 2011). The subcutaneous tissue edemas may persist even for 2 weeks after complete regression of the other symptoms (Madigan and Gribble, 1987) or disappear directly following the antibiotic therapy (Pusterla et al., 1998). Intensification of clinical symptoms demonstrates a considerable diversification and, to a great extent, depends on the condition and age of the infected animal. A typical course of EGA is observed in horses of above 3 years of age. The infected animals develop fever (39–40 °C) which may persist for 6–12 days. It is accompanied by general symptoms and limb swelling. The disease in one-yearold and younger horses is of non-serious nature with nonspecific symptoms such as lack of appetite and aversion to movement (Madigan and Gribble, 1987). Other symptoms of acute anaplasmosis are high fever, bleeding from mucosa, weight loss, enlarged spleen and lymph nodes. During the febrile period, the heart rate is increased up to 50–60 beats per minute and the respiratory rate up to 30 breaths per minute. Aside from the above, the following symptoms may be observed: diarrhea, vomiting, inflammation of joints, neurological symptoms such as convulsions and paralysis (Reubel et al., 1998; Adaszek et al., 2009). Hematological tests can reveal anemia, which is a result of bone marrow hypoplasia and may be of a non-regenerative form (Madigan and Gribble, 1987; Madigan et al., 1996). A specific hematological disorder observed in the course of anaplasmosis is thrombocytopenia, which leads to petechiae on the
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mucous membranes, in particular on the inner surface of the lips and gums. Apart from thrombocytopenia, a hematological test may reveal leucocytosis or leucopenia with neutropenia and hematocrit reduction. Biochemical tests of the blood serum of the infected animals are used to determine concentration increases of total bilirubin, urea and creatinine (Reubel et al., 1998; Lepidi et al., 2000; Adaszek et al., 2009; Silaghi et al., 2011). The concentration of plasma fibrinogen may be increased (Berrington et al., 1996; Adaszek et al., 2009) or remain within the limits of physiological norm (Pusterla et al., 1998). Sometimes proteinuria appears (Lepidi et al., 2000). The course of EGA is rarely subclinical. Chang et al. (1998) applied the PCR method to detect the DNA of A. phagocytophilum in poorly vascularized tissues 38 days after the experimental infection of horses. Franzén et al. (2009) indicated that experimental infection with A. phagocytophilum may not induce any clinical symptoms and the pathogen itself, after being introduced into the host, can survive in it for at least 129 days. Sometimes infected animals will collapse as a consequence; however, cases of self-recovery following the primary infection are observed more frequently (Gribble, 1969). The results of the studies conducted in the Netherlands confirm that the disease in horses is characterized by a considerably diversified course. A 4-year-old horse with acute anaplasmosis died despite implementing proper causal treatment (Butler et al., 2008). A rapid, incurable course of infection was also noted in a 19-year-old horse experimentally infected with A. phagocytophilum (Franzén et al., 2007). On the other hand in one study, all horses in the 9–15 years age group survived the disease without treatment (Butler et al., 2008). Histological tests demonstrated vasculitis necroticans in fragments of organs collected from the dead animals (Madigan and Gribble, 1987). It was also found that the infected animals had blood extravasations and petechiae on the mucous and serous membranes, edema of subcutaneous tissue as well as inflammatory lesions of internal organs, particularly the spleen, liver, lungs, kidneys and heart (Lepidi et al., 2000; Franzén et al., 2007), which indicates at the fact that the disease is of generalized nature and may lead to development of multiple organ failure.
5. Diagnosis Diagnosis of equine granulocytic anaplasmosis is based on the analysis of data from the epizootic history, as well as results of clinical and laboratory tests (Amusategui et al., 2006). Differential diagnosis of the disease should primarily consider borreliosis, equine infectious anemia, liver diseases and equine viral arteritis (Madigan, 1993; Lepidi et al., 2000; Silaghi et al., 2011). The microscopic tests of blood smears taken from the infected animals stained using the Diff-Quick, Wright or Giemsa methods can detect the presence of A. phagocytophilum morulae in the cytoplasm of neutrophils and, at times, eosinophils (Sells et al., 1976; Pusterla et al., 1998). The morulae appear in granulocytes in the acute stage of the disease, usually 2–4 days after infection (Gribble, 1969; Barlough et al., 1995; Adaszek and Winiarczyk, 2011). They are inclusion bodies of dark blue to violet color, consisting of many tiny initial bodies of circular, oval or rod shape and size of 0.18– 1.4 lm (Fig. 1). Sometimes they disintegrate into single elementary bodies (Sells et al., 1976). Despite the common belief that in infected horses the presence of morulae may be observed only in 1–6% of neutrophils (Berrington et al., 1996), the level of bacteraemia may be higher sometimes. Łukaszewska et al. (2008) proved the presence of these structures in 3–8% of neutrophils, whereas Uehlinger et al. (2011) in ca. 16%. Administration of dexamethasone to infected horses resulted in an increase of the infected neutrophils from 4% to 10%
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(Łukaszewska et al., 2008). Studies conducted in Poland have not proved the presence of Anaplasma in eosinophils of horses suffering from anaplasmosis (Adaszek and Winiarczyk, 2011). It is becoming increasingly popular to apply molecular biology methods, particularly polymerasis chain reaction (PCR), to the diagnosis of granulocytic anaplasmosis and the assessment of epizootic situations, as well as the detection of subclinical infections with Anaplasma (Madigan et al., 1996; Adaszek et al., 2009, 2013). Determination of a sequence of the obtained amplicons provides valuable information for epidemiological and taxonomic analysis. The PCR reaction and sequencing of the obtained amplicons allows, for example, the distinguishing of A. phagocytophilum from Ehrlichia ewingii. Both pathogens induce the development of morulae in neutrophils, which cannot be distinguished in the microscopic blood smear tests (Łukaszewska et al., 2008). Material from which it is possible to isolate DNA for PCR includes full blood, leukocytes, bone marrow or fragments of the spleen. The PCR reaction is performed using specific primers limiting the fragment of a selected rickettsial pathogen gene. The presence of the amplicon of a specific volume in a given sample is confirmed electrophoretically and the obtained products are subject to sequencing, enabling the determination of which bacterium caused the disease and to what extent it differs from the cases described so far (Adaszek et al., 2009, 2013). This method allows the early diagnosis of the disease. Barlough et al. (1995) with the use of the nested PCR method confirmed the presence of genetic material of A. phagocytophilum in the blood of a horse 9 days after infection, a day after developing fever and 2 days before the first observation of morulae in the blood. This shows that the technique is the most sensitive of all the diagnostic methods used to diagnose EGA. Other laboratory techniques, less significant than PCR, applied in equine anaplasmosis diagnostics are serological tests (van Andel et al., 1998; Amusategui et al., 2006). Most veterinary laboratories use the indirect fluorescent antibody test or ELISA test (Magnarelli et al., 2001), however, these tests are of limited applicability in the diagnosis of the acute stage of the infection, when the antibodies specific for A. phagocytophilum have not yet developed in the plasma of the infected animal. There were also attempts to culture A. phagocytophilum ‘‘in vitro’’ in the cell lines. However, this test is costly and of little diagnostic value due to the extended time needed for the result (Munderloh et al., 1996).
Fig. 1. Presence of Anaplasma inclusion bodies in neutrophils.
6. Treatment and prevention Because A. phagocytophilum is an intracellular pathogen, not many chemotherapeutics are effective in fighting this infection. The most effective group of antibiotics in causal therapy of anaplasmosis is the tetracyclines. In case of EGA, the drug of choice is oxytetracycline, its mechanism consisting in blocking protein synthesis through bonding with subunits 30S of the bacterial ribosomes (Madigan, 1993; Maurin et al., 2003). Pusterla et al. (1998) administered oxytetracycline at a dose of 10 mg/kg body weight to a 12-year-old mare for 5 days. The efficiency of the antibiotic and the selected dose was confirmed by Adaszek et al. (2009), who administered the mentioned chemotherapeutic to mares of 4 and 5 years of age with confirmed A. phagocytophilum infection for 10 days. The results of other studies conducted by this team indicate the effectiveness of oxytetracycline administered intravenously to a 7-year-old English full-blood gelding with symptoms of anaplasmosis for 7 days at a dose of 8 mg/kg body weight along with dexametasone i.m. at a dose of 20 mg/horse for 3 days. The therapy resulted in a gradual return of appetite and, after 3 days, the disappearance of limb swelling and problems with mobility. The neurological symptoms also subsided after another two days, and after 10 days from the beginning of treatment the horses were fully recovered (Adaszek et al., 2009). In Switzerland, horses were successfully treated with oxytetracycline i.v. administered for 7 days at a dose of 7 mg/kg body weight. Usually, a distinct decrease of body temperature, return of appetite and improvement of general condition of the patient were observed after the first day of therapy. The therapy was continued orally for a week (Hermann et al., 1985). A similar treatment regimen has been applied in the USA, where significant health improvement of the infected horses was noted 24 h from commencing the tetracycline therapy (Madigan and Gribble, 1987). These results are corroborated by recent studies conducted in Poland, during which the condition of the infected animals improved after 24 h from administration of the drugs and full recovery was reported after 7 days of oxytetracycline i.v. therapy at a dose of 8 mg/kg body weight (Adaszek and Winiarczyk, 2011). In some cases, recovery has been observed as soon as 12 h from the start of the treatment (Pusterla et al., 1998). In Virginia a sick mare was administered oxytetracycline intravenously at a dose of 6.6 mg/kg body weight, which resulted in the overall health improvement of the animal; however, perivascular swelling appeared in the place of injection (Lewis et al., 2009). Adverse reactions of this chemotherapeutic include hepatotoxic and neurotoxic effects, which are observed after long-term therapy. Moreover, administration of the antibiotic has a suppressive impact on the immune system and may result in gastro-intestinal disorders in the form of diarrheas and discoloration of dental enamel due to its capacity to chelate the formation of metal ions (Klein et al., 1997; Maurin et al., 2003). In the event of intolerance of oxytetracycline in horses, granulocytic anaplasmosis may be treated with other antibiotics. In the USA, EGA was effectively treated with doxycycline (p.o. 10 mg/kg every 12 h) (Lewis et al., 2009). Furthermore, the in vitro studies revealed that A. phagocytophilum is sensitive to new generations of fluorochinolones and riphampicine (Klein et al., 1997). Corticosteroids can be applied in the supportive treatment of autoimmune hemolytic anemia. They stimulate the bone marrow to produce red blood cells and stabilize the membranes of erythrocytes (Peek, 2009). Non-steroidal anti-inflammatory drugs and infusion fluids are applied only in justified cases. Prophylaxis of equine granulocytic anaplasmosis includes tick control and preventing them from feeding on the integuments of animals (Parola and Raoult, 2001). Removal of a tick from the skin
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does not exclude the possibility of infection (Oteo et al., 2001). Currently there is no vaccine against EGA on the market. The studies on development of vaccine against anaplasmosis on the basis of Anaplasma marginale antigens are in progress (Dark et al., 2011; Palmer et al., 2012).
7. Summary In Poland, EGA is a relatively new disease which may cause both therapeutic and diagnostic difficulties in veterinary medicine. The disease should be considered in horses in every case of acute symptoms, accompanied by thrombocytopenia or leucopenia with neutropenia revealed by hematological test performed after horse contact with ticks. Finally, numerous Anaplasma spp. have been identified in ticks and vertebrates throughout the world. Although their pathogenicity for people has yet to be demonstrated, they are good candidates to be involved in human diseases since many of them have been detected in ticks that readily bite people (Stan´czak et al., 2004; Cisak et al., 2005). Therefore, more worldwide studies could lead to the description of emerging human anaplasmosis in the future (Parola et al., 2005). References Adaszek, Ł., Winiarczyk, S., 2011. Identification of Anaplasma spp. Rickettsia isolated from horses from clinical disease cases in Poland. Zoonoses and Public Health 58, 514–518. Adaszek, Ł., Winiarczyk, S., Łukaszewska, J., 2009. A first case of ehrlichiosis in a horse in Poland. Deutsche Tierarztliche Wochenschrift 116, 330–334. Adaszek, Ł., Klimiuk, P., Skrzypczak, M., Górna, M., Zie˛tek, J., Winiarczyk, S., 2012. The identification of Anaplasma spp. isolated from fallow deer (Dama dama) on a free-range farm in eastern Poland. Polish Journal of Veterinary Science 15, 393– 394. Adaszek, Ł., Górna, M., Skrzypczak, M., Buczek, K., Balicki, I., Winiarczyk, S., 2013. Three clinical cases of Anaplasma phagocytophilum infection in cats in Poland. Journal of Feline Medicine and Surgery 15, 333–337. Amusategui, I., Sainz, A., Tesouro, M.A., 2006. Serological evaluation of Anaplasma phagocytophilum infection in livestock in northwestern Spain. Annals of the New York Academy of Science 1078, 487–490. Bakken, J.S., Dumler, J.S., 2008. Human granulocytic anaplasmosis. Infectious Disease Clinics of North America 22, 433–448. Barlough, J.E., Madigan, J.E., DeRock, E., Dumler, J.S., Bakken, J.S., 1995. Protection against Ehrlichia equi is conferred by prior infection with the Human granulocytotropic ehrlichia (HGE agent). Journal of Clinical Microbiology 33, 3333–3334. Bermann, F., Davoust, B., Fournier, P.E., Brisou-Lapointe, A.V., Brouqui, P., 2002. Ehrlichia equi (Anaplasma phagocytophila) infection in an adult horse in France. Veterinary Record 150, 787–788. Berrington, A., Moats, R., Lester, S., 1996. A case of Ehrlichia equi in an adult horse in British Columbia. Canadian Veterinary Journal 37, 174–175. Bjöersdorff, A., Christenson, D., Johnsson, A., Sjöström, A.C., Madigan, J.E., 1990. Ehrlichia equi–infektion diagnostiserat hos häst. Svensk Veterinärtidning 42, 357–360. Bjöersdorff, A., Bagert, B., Massung, R.F., Gusa, A., Eliasson, I., 2002. Isolation and characterization of two European strains of Ehrlichia phagocytophila of equine origin. Clinical and Diagnostic Laboratory Immunology 9, 341–343. Bown, K.J., Begon, M., Bennett, M., Woldehiwet, Z., Ogden, N.H., 2003. Seasonal dynamics of Anaplasma phagocytophila in a rodent-tick (Ixodes trianguliceps) system, United Kingdom. Emerging Infectious Diseases 9, 63–70. Büscher, G., Gandras, R., Apel, G., Friedhoff, K.T., 1984. Der erste Fall von Ehrlichiosis beim Pferd in Deutschland (Kurzmitteilung). Deutsche Tierarztliche Wochenschrift 91, 408–409. Butler, C.M., Nijhof, A.M., Jongejan, F., van der Kolk, J.H., 2008. Anaplasma phagocytophilum infection in horses in the Netherlands. Veterinary Record 162, 216–218. Chan, K.Y., Wang, Ch.H., Wu, Y.L., 2010. Serological survey of equine piroplasmosis, equine granulocytic anaplasmosis, and equine lyme disease in Taiwan. Taiwan Veterinary Journal 36, 261–267. Chang, Y.F., Novosel, V., Dubovi, E., Wong, S.J., Chu, F.K., Chang, C.F., Del Piero, F., Shin, S., Lein, D.H., 1998. Experimental infection of the human granulocytic ehrlichiosis agent in horses. Veterinary Parasitology 78, 137–145. Chen, S.M., Dumler, J.S., Bakken, J.S., Walker, D.H., 1994. Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. Journal of Clinical Microbiology 32, 589–595. Cisak, E., Chmielewska-Badora, J., Zwolin´ski, J., Wójcik-Fatla, A., Polak, J., Dutkiewicz, J., 2005. Risk of tick-borne bacterial diseases among workers of Roztocze National Park (south-eastern Poland). Annals of Agricultural and Environmental Medicine 12, 127–132.
319
Dark, M.J., Al-Khedery, B., Barbet, A.F., 2011. Multistrain genome analysis identifies candidate vaccine antigens of Anaplasma marginale. Vaccine 29, 4923–4932. Dumler, J.S., Asanovich, K.M., Bakken, J.S., Richter, P., Kimsey, R., Madigan, J.E., 1995. Serologic cross-reactions among Ehrlichia equi, Ehrlichia phagocytophila and human granulocytic Ehrlichia. Journal of Clinical Microbiology 33, 1098–1103. Dumler, J.S., Barbet, A.F., Bekker, C.P., Dasch, G.A., Palmer, G.H., Ray, S.C., Rikihisa, Y., Rurangirwa, F.R., 2001. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichia phagocytophila. International Journal of Systematic and Evolutionary Microbiology 51, 2145–2165. Dunning Hotopp, J.C., Lin, M., Madupu, R., Crabtree, J., Angiuoli, S.V., Eisen, J.A., Seshadri, R., Ren, Q., Wu, M., Utterback, T.R., Smith, S., Lewis, M., Khouri, H., Zhang, C., Niu, H., Lin, Q., Ohashi, N., Zhi, N., Nelson, W., Brinkac, L.M., Dodson, R.J., Rosovitz, M.J., Sundaram, J., Daugherty, S.C., Davidsen, T., Durkin, A.S., Gwinn, M., Haft, D.H., Selengut, J.D., Sullivan, S.A., Zafar, N., Zhou, L., Benahmed, F., Forberger, H., Halpin, R., Mulligan, S., Robinson, J., White, O., Rikihisa, Y., Tettelin, H., 2006. Comparative genomics of emerging human ehrlichiosis agents. PLoS Genetics 2, E21. Eriksen, L., Hansen, J.F., Abildtrup, E., Engvall, E.O., 1997. Equine granulocytic ehrlichiosis diagnosed in Denmark. Dansk Veterinærtidsskrift 80, 231–234. Franzén, P., Berg, A.-L., Aspan, A., Gunnarsson, A., Pringle, J.D., 2007. Death of a horse infected experimentally with Anaplasma phagocytophilum. Veterinary Record 160, 122–125. Franzén, P., Aspan, A., Egenvall, A., Gunnarsson, A., Karlstam, E., Pringle, J., 2009. Molecular evidence for persistence of Anaplasma phagocytophilum in the absence of clinical abnormalities in horses after recovery from acute experimental infection. Journal of Veterinary Internal Medicine 23, 636–642. Fröhlich, W., Edelhofer, R., 1998. Erstbeschreibung der equinen granulozytären Ehrlichiose (EGE) bei einem Pferd in Ös terreich. Wiener Tierärztliche Monatsschrift 11, 389–394. Gribble, D.H., 1969. Equine ehrlichiosis. Journal of the American Veterinary Medical Association 155, 462–469. Hermann, M., Baumann, D., Lutz, H., Wild, P., 1985. Erster diagnostizierter Fall von equiner Ehrlichiose in der Schweiz. Pferdeheilkunde 1, 247–250. Herron, M.J., Ericson, M.E., Kurtti, T.J., Munderloh, U.G., 2005. The interactions of Anaplasma phagocytophilum, endothelial cells, and human neutrophils. Annals of the New York Academy of Science 1063, 374–382. Ismail, N., Bloch, K.C., McBride, J.W., 2010. Human ehrlichiosis and anaplasmosis. Journal of Laboratory and Clinical Medicine 30, 261–292. Jahn, P., Zeman, P., Bezdekova, B., Praskova, I., 2010. Equine granulocytic anaplasmosis in the Czech Republic. Veterinary Record 166, 646–649. Klein, M.B., Miller, J.S., Nelson, C.M., Goodman, J.L., 1997. Primary bone marrow progenitors of both granulocytic and monocytic lineages are susceptible to infection with the agent of human granulocytic ehrlichiosis. Journal of Infectious Diseases 176, 1405–1409. Korbutiak, E., Schneiders, D.H., 1994. First confirmed case of equine ehrlichiosis in Great Britain. Equine Veterinary Education 6, 303–304. Lepidi, H., Bunnell, J.E., Martin, M.E., Madigan, J.E., Stuen, S., Dumler, J.S., 2000. Comparative pathology and immunohistology associated with clinical illness after Ehrlichia phagocytophila-group infections. The American Journal of Tropical Medicine and Hygiene 62, 29–37. Lewis, S.R., Zimmerman, K., Dascanio, J.J., Pleasant, R.S., Witonsky, S.G., 2009. Equine granulocytic anaplasmosis: a case report and review. Journal of Equine Veterinary Science 29, 160–166. Lillini, E., Marcì, G., Proietti, G., Scarpulla, M., 2006. New findings on anaplasmosis caused by infection with Anaplasma phagocytophilum. Annals of the New York Academy of Science 1081, 360–370. Lin, M., Kikuchi, T., Brewer, H.M., Norbeck, A.D., Rikihisa, Y., 2011. Global proteomic analysis of two tick-borne emerging zoonotic agents: Anaplasma phagocytophilum and Ehrlichia chaffeensis. Frontiers in Microbiology 2, 24. Łukaszewska, J., Adaszek, Ł., Winiarczyk, S., 2008. Hematological changes in _ granulocytic anaplasmosis in dogs and horses. Zycie Weterynaryjne 83, 827– 831. M’ghirbi, Y., Yaïch, H., Ghorbel, A., Bouattour, A., 2012. Anaplasma phagocytophilum in horses and ticks in Tunisia. Parasites & Vectors 5, 180. Madigan, J.E., 1993. Equine ehrlichiosis. Veterinary Clinics of North America 9, 423– 428. Madigan, J.E., Gribble, D., 1987. Equine ehrlichiosis in northern California: 49 cases (1968–1981). Journal of the American Veterinary Medical Association 190, 445– 448. Madigan, J.E., Barlough, J.E., Dumler, J.S., Schankman, N.S., DeRock, E., 1996. Equine granulocytic ehrlichiosis in Connecticut caused by an agent resembling the human granulocytotropic ehrlichia. Journal of Clinical Microbiology 34, 434– 435. Magnarelli, L.A., Ijdo, J.W., van Andel, A.E., Wu, C., Fikrig, E., 2001. Evaluation of a polyvalent enzyme-linked immunosorbent assay incorporating a recombinant p44 antigen for diagnosis of granulocytic ehrlichiosis in dogs and horses. American Journal of Veterinary Research 62, 29–32. Maurin, M., Bakken, J.S., Dumler, J.S., 2003. Antibiotic susceptibilities of Anaplasma (Ehrlichia) phagocytophilum strains from various geographic areas in the United States. Antimicrobial Agents and Chemotherapy 47, 413–415. Munderloh, U.G., Madigan, J.E., Dumler, J.S., Goodman, J.L., Hayes, S.F., Barlough, J.E., Nelson, C.M., Kurtti, T.J., 1996. Isolation of the equine granulocytic ehrlichiosis
320
B. Dzie˛giel et al. / Research in Veterinary Science 95 (2013) 316–320
agent, Ehrlichia equi, in tick cell culture. Journal of Clinical Microbiology 34, 664–670. Nieto, N.C., Foley, J.E., 2008. Evaluation of squirrels (Rodentia: Sciuridae) as ecologically significant hosts for Anaplasma phagocytophilum in California. Journal of Medical Entomology 45, 763–769. Ojogun, N., Barnstein, B., Huang, B., Oskeritzian, C.A., Homeister, J.W., Miller, D., Ryan, J.J., Carlyon, J.A., 2011. Anaplasma phagocytophilum infects mast cells via alpha1,3-fucosylated but not sialylated glycans and inhibits IgE-mediated cytokine production and histamine release. Infection and Immunity 79, 2717– 2726. Oteo, J.A., Blanco, J.R., Ibarra, V., 2001. Can we prevent tickborne transmission disease? Enfermedades Infecciosas y Microbiología Clinica 19, 509–513. Palmer, G.H., Brown, W.C., Noh, S.M., Brayton, K.A., 2012. Genome-wide screening and identification of antigens for rickettsial vaccine development. FEMS Immunology and Medical Microbiology 64, 115–119. Parola, P., Raoult, D., 2001. Tick-borne bacterial diseases emerging in Europe. Clinical Microbiology and Infection 7, 80–83. Parola, P., Davoust, B., Raoult, D., 2005. Tick- and flea-borne rickettsial emerging zoonoses. Veterinary Research 36, 469–492. Peek, S.F., 2009. Icterus. In: Robinson, N.E., Sprayberry, K.A. (Eds.), Current Therapy in Equine Medicine. Saunders Elsevier Inc., St. Louis, MO, USA, pp. 456–460. Pusterla, N., Lutz, H., Braun, U., 1998. Experimental infection of four horses with Ehrlichia phagocytophila. Veterinary Record 143, 303–305. Reubel, G.H., Kimsey, R.B., Barlough, J.E., Madigan, J.E., 1998. Experimental transmission of Ehrlichia equi to horses through naturally infected ticks (Ixodes pacificus) from northern California. Journal of Clinical Microbiology 36, 2131–2134.
Rikihisa, Y., 1991. The tribe Ehrlichieae and ehrlichial diseases. Clinical Microbiology Reviews 4, 286–308. Rikihisa, Y., 2010. Anaplasma phagocytophilum and Ehrlichia chaffeensis: subversive manipulators of host cells. Nature Reviews Microbiology 8, 328–339. Sells, D.M., Hildebrandt, P.K., Lewis, G.E., Nyindo, M.B., Ristic, M., 1976. Ultrastructural observations on Ehrlichia equi organisms in equine granulocytes. Infection and Immunity 13, 273. Silaghi, C., Liebisch, G., Pfister, K., 2011. Genetic variants of Anaplasma phagocytophilum from 14 equine granulocytic anaplasmosis cases. Parasites & Vectors 4, 161. Stan´czak, J., Gabre, R.M., Kruminis-Łozowska, W., Racewicz, M., Kubica-Biernat, B., 2004. Ixodes ricinus as a vector of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Babesia microti in urban and suburban forests. Annals of Agricultural and Environmental Medicine 11, 109–114. Stannard, A.A., Gribble, D.H., Smith, R.S., 1969. Equine ehrlichiosis: a disease with similarities to tick-borne fever and bovine petechial fever. Veterinary Record 84, 149–150. Stuen, S., 2007. Anaplasma phagocytophilum – the most widespread tick-borne infection in animals in Europe. Veterinary Research Communications 31, 79–84. Uehlinger, F.D., Clancey, N.P., Lofstedt, J., 2011. Granulocytic anaplasmosis in a horse from Nova Scotia caused by infection with Anaplasma phagocytophilum. Canadian Veterinary Journal 52, 537–540. van Andel, A.E., Magnarelli, L.A., Heimer, R., Wilson, M.L., 1998. Development and duration of antibody response against Ehrlichia equi in horses. Journal of the American Veterinary Medical Association 212, 1910–1914. Walls, J.J., Greig, B., Neitzel, D.F., Dumler, J.S., 1997. Natural infection of small mammal species in Minnesota with the agent of human granulocytic ehrlichiosis. Journal of Clinical Microbiology 35, 853–855.