Abstract
Anaplasma phagocytophilum is an emergent hemoparasite in regions where ticks are regularly found. In order to investigate the rate of anti-A. phagocytophilum antibody in horses of the Mounted Police of Rio de Janeiro state and therefore the presence of this agent, 41 horses from the Cavalry Squadron (CS) and 50 from the Regiment of Mounted Police (RMP) were selected. For the serologic diagnosis the Indirect Fluorescent Antibody test was performed. In the present work, among 91 equine sera samples anti-A. phagocytophilum antibodies, with titer ≥ 1:80, were detected in 11 animals (12%), being five (5%) from males and six (7%) females, with no statistical difference. The adult animals with ages varying between five and 14 years presented the highest rate of positive reaction, although antibodies were detected in animals of all ages. We did not observe statistic differences in relation to the presence of anti-A. phagocytophilum antibodies among the animals bred in the CS and RMP. The presence of anti-A.phagocytophilim antibodies in horses of the Mounted Police with no clinical signs is indicative that the parasite is present in the enzootic form among the horses of the urban area. The circulation of the parasite among the animals is not dependent of the presence of tick infestations.
Keywords
equine; granulocytic anaplasmosis; hemoparasites; IFA; tick
Resumo
Anaplasma phagocytophilum é uma hemoparasitose emergente em regiões onde existem carrapatos. Com o objetivo de investigar a frequência de anticorpos anti-A. phagocytophilum em cavalos da Policia Montada do Estado do Rio de Janeiro e, consequentemente, os indícios da presença desse agente em áreas urbanas, foram selecionados 41 cavalos do Esquadrão de Cavalaria (EC) e 50 do Regimento de Polícia Montada (RPM). Para o diagnóstico sorológico, foi utilizado o método de Imunofluorescência Indireta. No presente trabalho, dentre 91 amostras de soro de equinos, foram detectados anticorpos anti-A. phagocytophilum com título ≥ 1:80 em 11 animais (12%), sendo que cinco (5%) eram machos e seis (7%) fêmeas, sem diferenças estatísticas. Os animais adultos com faixa etária entre cinco e 14 anos foram os que apresentaram maior frequência de reação positiva; no entanto, foram detectados anticorpos em animais de todas as faixas etárias. Não foram observadas diferenças estatísticas quanto à presença de anticorpos anti-A. phagocytophilum nos animais criados no EC e no RPM. A presença de anticorpos anti-A. phagocytophilum em cavalos da Policia Montada sem sinais clínicos aparentes é indicativo que A. phagocytophilum está presente de forma enzóotica na região urbana e a circulação do parasita entres os animais independe da presença de infestações aparentes de carrapatos nos animais.
anaplasmose granulocítica
carrapatos; equino; hemoparasitos; RIFI
Introduction
Anaplasma phagocytophilum is an obligate intracellular bacterium of neutrophils in wild and domestic mammals, birds and humans(11 Rikihisa, Y. The tribe Ehrlichieae and ehrlichial diseases. Clinical Microbiology Reviews. 1991 jul; 4(3):286-308.,22 Bjöersdorff A, Bergström S, Massung RF, Haemig PD, Olsen B. Ehrlichia-infected ticks on migrating birds. Emerging Infectious Diseases. 2001 sep-oct; 7(5):877-879.). In Brazil, it has been described in horses(33 Mesquita Neto F De, Araujo T, Vieira RM, Gouveia AMG. Ehrlichia equi in horses from the municipality of Betim (Brazil). Arquivo Brasileiro de Medicina Veterinária e Zootecnia. 1984 apr; 36(2):203-207.), cervids(44 Sacchi AB, Duarte JM, André MR, Machado RZ. Prevalence and molecular characterization of Anaplasmataceae agents in free-ranging Brazilian marsh deer (Blastocerus dichotomus). Comparative Immunology, Microbiology and Infectious Diseases. 2012 jul; 35(4):325-334.), dogs(55 Santos HA, Pires MS, Vilela JA, Santos TM, Faccini JL, Baldani CD, Thomé SM, Sanavria A, Massard CL. Detection of Anaplasma phagocytophilum in brazilian dogs by real-time PCR. Journal of veterinary diagnostic investigation. 2011 jul; 23(4):770-774.), migratory birds(66 Machado RZ, André MR, Werther K, De Sousa E, Gavioli FA, Alves Junior JR. Migratory and Carnivorous Birds in Brazil: Reservoirs for Anaplasma and Ehrlichia Species? Vector Borne Zoonotic Diseases. 2012 aug; 12(8):705-708.) and wild carnivores(77 André MR, Dumler JS, Scorpio DG, Teixeira RH, Allegretti SM, Machado RZ. Molecular detection of tick-borne bacterial agents in Brazilian and exotic captive carnivores. Ticks and tick-borne diseases. 2012 sep; 3(4):257-253.).
Even though the disease caused by A. phagocytophilum, granulocytic anaplasmosis (GA), is considered a zoonosis, the transmission dynamics of this bacterium, which involve ticks, reservoirs and hosts, have not been extensively studied in Brazil(44 Sacchi AB, Duarte JM, André MR, Machado RZ. Prevalence and molecular characterization of Anaplasmataceae agents in free-ranging Brazilian marsh deer (Blastocerus dichotomus). Comparative Immunology, Microbiology and Infectious Diseases. 2012 jul; 35(4):325-334., 77 André MR, Dumler JS, Scorpio DG, Teixeira RH, Allegretti SM, Machado RZ. Molecular detection of tick-borne bacterial agents in Brazilian and exotic captive carnivores. Ticks and tick-borne diseases. 2012 sep; 3(4):257-253., 88 Blanco JR, Oteo JA. Human granulocytic ehrlichiosis in Europe. Clinical Microbiology and Infectious Diseases. 2002 dec; 8(12):763-772.), especially in animals that are in close contact with humans, such as horses. GA is characterized by an acute febrile syndrome with clinical and laboratorial characteristics that are common to both humans and horses(99 Pusterla N, Chae JS, Kimsey RB, Berger Pusterla J, Derock E, Dumler JS, Madigan JE. Transmission of Anaplasma phagocytophila (human granulocytic ehrlichiosis agent) in horses using experimentally infected ticks (Ixodes scapularis). Journal of Veterinary Medicine Series B-infectious Diseases and Veterinary Public Health. 2002 dec; 49(10):484-488.). The clinical form of this condition in horses is associated with poor performance, depression, petechiae in oral, vaginal, nasal and conjunctive mucosae, limb edema and ataxia(1010 Madigan JE. Equine ehrlichiosis. Veterinary Clinics of North America: Equine Practic. 1993 aug; 9(2):423-428.,1111 Madigan JE, Pusterla N. Ehrlichial diseases. Veterinary Clinics of North America: Equine Practic. 2000 dec; 16(3):487-499.). GA incidence in horses is poorly known and, additionally, infection by A. phagocytophilum has been frequently diagnosed as a self-limiting disease with no apparent clinical signs(1010 Madigan JE. Equine ehrlichiosis. Veterinary Clinics of North America: Equine Practic. 1993 aug; 9(2):423-428.,1212 Franzen P, Aspan A, Egenvall A, Gunnarsson A, Aberg L, Pringle J. Acute clinical, hematologic, serologic, and polymerase chain reaction findings in horses experimentally infected with a European strain of Anaplasma phagocytophilum. Journal of Veterinary Internal Medicine. 2005 mar-apr; 19(2):232-239.,1313 Parra AC. Investigação diagnóstica de doença concomitante babesiose anaplasmose em rebanho equino por pcr e elisa. [Internet]. São Paulo: Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia de São Paulo; 2009. [citado 2014 Dez 14] Available from: file://localhost/C:/Users/Flavio/Downloads/Andrea_Cristina_Parra_doutorado(1).pdf
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). These data highlight the potential role of horses as a reservoir for this bacterium(1414 Ebani V, Cerri D, Fratini F, Ampola M, Andreani E. Seroprevalence of Anaplasma phagocytophilum in domestic and wild animals from central Italy. New Microbiologica. 2008 ju; 31(3):371-375.). Additionally, the capability of horses to produce anti-A. phagocytophilum antibodies that can be detected by commercial tests for up to six months enables the use of this species as sentinels to prevent outbreaks in humans(1515 Van Andel AE, Magnarelli LA, Heimer R Wilson, M.L. Development and duration of antibody response against Ehrlichia equi in horses. The Journal of the American Medical Association. 1998 jun; 15(212):1910-1914.).
Anaplasma phagocytophilum should be included in the differential diagnosis in cases of suspected equine babesiosis, especially when horses exhibit clinical signs and either the protozoan is not detected in peripheral blood smears or there is no response to chemotherapy(1616 Salvagni CA, Dagnone AS, Gomes TS, Mota JS, Andrade GM, Baldani CD, Machado RZ. Serologic evidence of equine granulocytic anaplasmosis in horses from central West Brazil. Brazilian Journal of Veterinary Parasitology. 2010 jul-sep; 19(3):135-140.).
The main A. phagocytophilum vectors are ticks of the Ixodes genus, which include I. persulcatus, I. ricinus, I. pacificus and I. scapularis(1717 Macleod J. Ticks and disease in domestic stock in Great Britain. Symposium of the Zoological Society of London. 1962; 6:29-50.
18 Holden K, Boothby JT, Anand S, Massung RF. Detection of Borrelia burgdorferi,Ehrlichia chaffeensis and Anaplasma phagocytophilum in ticks (Acari: Ixodidae) from a coastal region of California. Journal of Medical Entomology. 2003 jul; 40(4):534-539.
19 Cao WC, Zhan L, He J, Foley JE, De Vlas SJ, Xiao-Mingwu, Yang H, Richardus JH, Habbema JDF. Natural Anaplasma phagocytophilum infection of ticks and rodents from a forest area of Jilin Province, China. The American journal of tropical medicine and hygiene. 2006 oct; 75(4):664-668.-2020 Yoshimoto K, Matsuyama Y, Matsuda H, Sakamoto L, Matsumoto K, Yokoyama N, Inokuma H. Detection of Anaplasma bovis and Anaplasma phagocytophilum DNA from Haemaphysalis megaspinosa in Hokkaido, Japan. Veterinary parasitology. 2010 feb; 26(168):170-172.). Researchers have identified differences in transmissibility within this genus, which can explain variations not only in GA incidence and severity, but also in the vertebrate hosts that are affected.(2121 Teglas MB, Foley J. Differences in the transmissibility of two Anaplasma phagocytophilum strains by the North American tick vector species, Ixodes pacificus and Ixodes scapularis (Acari: Ixodidae). Experimental and Applied Acarology. 2006 jul; 38(1):47-58.) In Central America some thick species were reported as probable vectors of A. phagocytophilum, like Amblyomma cajennense, Anocentor nittens e Riphicephalus microti(2222 Teglas M, Matern E, Lein S, Foley P, Mahan SM, Foley J. Ticks and tick-borne disease in Guatemalan cattle and horses. Veterinary Parasitology. 2005; 15(23):119-127.).
Considering that Brazil's Southeastern Region has the largest population of horses in the country due to the intensification of activities that put these animals in close contact with humans, the objectives of this study were to assess the rate of anti-A. phagocytophilum antibodies in the horses of the mounted police of Rio de Janeiro state, which are used in activities such as patrolling, classic equestrian and hippotherapy; to determine possible risks of infection by the parasite associated to breedings, and also to draw attention to the importance of the detection of A. phagocytophilum.
Materials and Methods
During January and February of 2010, we collected blood samples of 91 of the 182 horses of the mounted police of Rio de Janeiro state, from which 154 were males and 28 females; 50 samples came from the Mounted Police Regiment (RMP) of Rio de Janeiro state and 41 from the Cavalry Squadron (CS). This investigation was performed in the western region of the metropolitan area of Rio de Janeiro state, southeast of Brazil. Sample size was calculated using the statistical software Epi-info 2000, with expected anti-A. phagocytophilum antibodies rate of 17.03% and acceptable up to 25% with confidence interval of 95%.
The horses were raised in a semi-intensive breeding system; they were directly examed by veterinarians, subjected to sanitary handling, tested for equine infectious anemia (EIA), treated for the control of endo- and ectoparasites, and vaccinated against equine influenza (EI), equine herpesvirus Type-1 (EH-1), equine encephalomyelitis, tetanus, and rabies. Blood smears examination for hematozoa was performed in association to hemogram as a protocol for animals with reduced performance, apathy or fever syndrome. Blood samples were obtained from jugular vein punctures with needles and adapters for vacutainer tubes; samples were later centrifuged for serum extraction. Serum samples were stored in duplicates in plastic 2.0 ml eppendorf tubes and kept at -20 °C. For serological diagnosis, the indirect fluorescent antibody test (IFA) method was used. Specifically, samples were tested with the Anaplasma phagocytophilum IFA Equine Antibody Kit (Fuller Laboratories©, Fullerton, California, USA). Serum samples were diluted 1:80 in phosphate buffered saline solution (PBS) with a pH of 7.2 in the proportion of 10 pL of sample for 790 pL of PBS. 10 pL of the diluted samples were pipetted in each well of slides containing antigenic substrate for A. phagocytophilum (HL60 cells containing A. phagocytophilum morulae), reserving two wells for the addition of positive and negative control samples containing, respectively, reactive equine serum diluted from 1:80 to 1:640 and non-reactive equine serum diluted at 1:80. The slides were incubated at 37 °C for 60 minutes in a humid chamber, washed in PBS, and then dried. Next, we added 10 pL of conjugate (equine anti-IgG isothiocyanate marked with fluorescein to each well (Fuller Laboratories©, Fullerton, California, USA). The slides were incubated for 60 minutes at 37 °C in a moist room. After three washings with PBS and drying, slides were mounted with 50% glycerol diluted in PBS. Results were visualized by examining the slides using a Zeiss immunofluorescence microscope (AXIOSHOOP 40) with a Carl Zeiss digital camera (AXIO CAM MRC - HAL100) for image capture that was coupled to a microcomputer with Zeiss AxionVision LE 4.8.2 software. Positive samples reacted at the dilution of 1:80; this titer was the cut-off and allowed the observation of fluorescence in the morulae (Figure 1).
Indirect Fluorescent antibody test (IFA) in sera of horses of the Rio de Janeiro State Militaiy Police, Brazil. In A, arrows point to fluorescent morulae in positive samples, contrasting with a negative reaction observed in B.
Additionally, we carried out an epidemiological investigation, observing aspects associated to animal identification (breed, age and sex), animal origin, work type, environment, nutritional and sanitary handling, breeding system, transit, participation in events, animal commercialization, reproduction types, history of hemoparasites, presence of ectoparasites and wild animals, and the presence of other species of domestic animals.
From the data collected during the epidemiological investigation we calculated the rate of positive animals associated to the ages. The relative risk for A. phagocytophilum infection associated to gender was calculated using Fisher's Exact Test with Katz approximation and to the origin we used the Chi-Square test with Yates correction and Katz approximation. All calculations were made with 95% confidence interval using the software Graphpad Instat.
Results
In the present study, 91 equine serum samples were tested, and anti-A. phagocytophilum antibodies were detected in 11 (12%) animals being 1 (1%), 0 (0%), 9 (10%) and 1 (1%) in the range of 0 to 2, 3 to 4, 5 to 14 and over 15 years old, respectively. Although adult animals aged between five and fourteen years had the the highest frequency of positive reactions, antibodies against A. phagocytophilum were detected in animals of all ages (Figure 2). There were no statistical differences observed with regard to the presence of anti-A. phagocytophilum antibodies between the animals raised at the CS and RMP (Table 1). Of the positive animals, five (5%) were males and six (7%) were females (Table 1); no statistical difference was observed (p=0.0868).
Rates of seropositive and negative horses for antibodies anti-A. phagocytophilum antibodies in breedings of tlie Momited Police of Rio de Janeiro state, Brazil, according to age ranges 0 to 2 years old, (1%, 1%), 3 to 4 (0.0%, 1%), 5 to 14 (10%, 70%) and over 15 (1%, 16%) compared by Chi-square test (p= 0,8828). respectively.
Relative risk for the presence of anti-Anaplasma phagocytophilum antibodies associated to sex and origin of the horses of the Cavalry Squadron (CS) and Regiment of Mounted Police (RMP) of Rio de Janeiro State, Brazil
The horses of the military police participate in the activities of mounted policing in Rio de Janeiro state, horseback riding throughout Brazil and hippotherapy with special children. Horses in both squadrons presented fifteen cases of babesiosis in the last five years, all diagnosed by blood smear. During this period, two animals that had reduced performance, fever, leucopenia and were suspected for babesiosis and did not respond to specific therapeutic treatment, were tested for the presence of antibodies against A. phagocytophilum using IFA. After positive test response, animals were treated with oxytetracycline hydrochloride (10 mg/kg) and recovered after three days.
Discussion
We observed that 12% of the examined horses of Rio de Janeiro state Mounted Police that were raised at a urban region of the city of Rio de Janeiro tested positive for the presence of anti-A. phagocytophilum antibodies. However, with the exception of one animal that had already been serologically tested and treated, there were no clinical signs reported in the epidemiological investigation that could lead to clinical suspicion of the disease. In Brazil, three reports on the presence of anti-A. phagocytophilum antibodies in horses have been published. In 1984, MESQUITA NETO et al(33 Mesquita Neto F De, Araujo T, Vieira RM, Gouveia AMG. Ehrlichia equi in horses from the municipality of Betim (Brazil). Arquivo Brasileiro de Medicina Veterinária e Zootecnia. 1984 apr; 36(2):203-207.). reported Ehrlichia equi in horses from the municipality of Betim, Minas Gerais state. In 2010, SALVAGNI et al.(1616 Salvagni CA, Dagnone AS, Gomes TS, Mota JS, Andrade GM, Baldani CD, Machado RZ. Serologic evidence of equine granulocytic anaplasmosis in horses from central West Brazil. Brazilian Journal of Veterinary Parasitology. 2010 jul-sep; 19(3):135-140.) reported 65% seropositivity for A. phagocytophilum using an ELISA-based test; however, negative results were reported when PCR was used for pathogen detection. In contrast to the present study, blood samples were collected from equines in military use in the central region of the city of Goiania with clinical symptoms suggestive of infection. Previously, PARRA(1313 Parra AC. Investigação diagnóstica de doença concomitante babesiose anaplasmose em rebanho equino por pcr e elisa. [Internet]. São Paulo: Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia de São Paulo; 2009. [citado 2014 Dez 14] Available from: file://localhost/C:/Users/Flavio/Downloads/Andrea_Cristina_Parra_doutorado(1).pdf
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) reported that 3% of 250 horses in the southeastern Brazilian state of São Paulo were seropositive by ELISA for anti-A. phagocytophilum antibodies, however, all animals were negative for hemoparasites in the PCR test. Thus, EGA is possibly being neglected in Brazil due to the difficulty of conclusive diagnosis and its self-limiting nature. This hypothesis is reinforced if we compare the 12% of seropositive equines observed in this research to the levels reported for horses in some European countries, such as Holland (83.3%) and Denmark (22.3%)(2323 Franzen P, Aspan A, Egenvall A, Gunnarsson A, Karlstam E., Pringle J Molecular evidence for persistence of abre-italicAnaplasma phagocytophilumfecha-italic in the absence of clinical abnormalities in horses after recovery from acute experimental infection. Journal of Veterinary Internal Medicine. 2009 may-jun; 23(3):636-642.). High frequencies of A. phagocytophilumseropositivity in horses was also reported by Frazen et al.(1212 Franzen P, Aspan A, Egenvall A, Gunnarsson A, Aberg L, Pringle J. Acute clinical, hematologic, serologic, and polymerase chain reaction findings in horses experimentally infected with a European strain of Anaplasma phagocytophilum. Journal of Veterinary Internal Medicine. 2005 mar-apr; 19(2):232-239.) and Passamonti et al.(2424 Passamonti F, Veronesi F, Cappelli K, Capomaccio S, Coppola G, Marenzoni ML, Piergili FD, Verini SA, Coletti, M. Anaplasma phagocytophilum in horses and ticks: A preliminary survey of Central Italy. Comparative Immunology, Microbiology and Infectious Disease. 2010 jan; 33(1):73-83.) in Sweden (16.6%) and Italy (17.3%); however, these same authors reported lower seropositive frequencies in animals in France (11.3%) and Spain (6.5%) compared to the horses of the Regiment of the Mounted Police of Rio de Janeiro. In the United States of America (USA), the seroprevalence for A. phagocytophilum in horses in eastern cities ranged from 17.6% to 29% in regions where ticks are endemic; seroprevalence was 3.8% in areas where the presence of ticks was insignificant(2525 Foley JE, Foley P, Brown RN, Lane RS, Dumlers JS, Madigan JE. Ecology of Anaplasma phagocytophilum and Borrelia burgdorferi in the western United States. Journal of Vector Ecology. 2004 jun; 29(1):41-50.). This could be an explanation for the few suspect cases among horses of the Mounted Police of Rio de Janeiro, in which AGE test is applied; these animals are submitted to a rigorous ectoparasite control regimen.
Even with the periodic parasite control on horses of the Mounted Police, one cannot discard the importance of ticks in the epidemiologic chain for the analyzed breedings, as some researchers have verified differences in the tutorial capacity of different species of ticks, identified as possible transmitters, what could explain the ecologic variations and the emerging of granulocytic anaplasmosis in different demographic regions in a way that corroborates with the incidence and gravity of the disease, besides the affected vertebrate hosts(88 Blanco JR, Oteo JA. Human granulocytic ehrlichiosis in Europe. Clinical Microbiology and Infectious Diseases. 2002 dec; 8(12):763-772.,2121 Teglas MB, Foley J. Differences in the transmissibility of two Anaplasma phagocytophilum strains by the North American tick vector species, Ixodes pacificus and Ixodes scapularis (Acari: Ixodidae). Experimental and Applied Acarology. 2006 jul; 38(1):47-58.,2626 Li H, Zhou Y, Wang W, Guo D, Huang S, Jie S. The clinical characteristics and outcomes of patients with human granulocytic anaplasmosis in China. International Journal of Infectious Diseases. 2011 dec; 15(12):859-866.,2727 Scharf W, Schauer S, Freyburger F, Petrovec M, Schaarschmidt-Kiener D, Liebisch G, Runge M, Ganter M, Kehl A, Dumler JS, Garcia-Perez AL, Jensen J, Fingerle V, Meli ML, Ensser A, Stuen S, Von Loewenich FD. Distinct Host Species Correlate with Anaplasma phagocytophilum ankA Gene Clusters. Journal of Clinical Microbiology. 2011 mar; 49(3):790-796.). These researchers reinforce this hypothesis because in Europe the infection was detected in ruminants, in California in equids, in Asia in humans and not in domestic animals, and in East USA in dogs and humans.
The most efficient transmitter of A. phagocytophilum to horses is the adult tick; it is known that nymphs most frequently transmit the pathogen from animals to humans(2525 Foley JE, Foley P, Brown RN, Lane RS, Dumlers JS, Madigan JE. Ecology of Anaplasma phagocytophilum and Borrelia burgdorferi in the western United States. Journal of Vector Ecology. 2004 jun; 29(1):41-50.,2828 Ogden NH, Woldehiwet Z, Hart CA. Granulocytic ehrlichiosis: an emerging or rediscovered tick-borne disease? Journal of Medical Microbiology. 1998 jun; 47(6):475-482.). Due to the predominantly tropical climate in Rio de Janeiro, all of the stages of the tick life cycle are observed throughout the year on various animals, increasing the probability of horses acquiring hemoparasites.
Samples were collected from horses of the Military Police of Rio de Janeiro during the summer, when conditions are propitious for tick infection; however, observations of ticks in the facilities or on the horses were rare. The principal reservoirs for A. phagocytophilum in Europe and USA are wild animals, rodents and deer in addition to the same vectors for domestic animals.(2929 Vorou RM, Pavassiliou VG, Tsiodras S. Emerging zoonoses and vector-borne infections affecting humans in Europe. Epidemiology and Infection. 2007 nov; 135(8):1231-1247.,3030 Baldridg GD, Scoles GA, Burkhardt NY, Schloeder B, Kurtti TJ, Munderloh UG. Transovarial Transmission of Francisella-Like Endosymbionts and Anaplasma phagocytophilum Variants in Dermacentor albipictus (Acari: Ixodidae). Journal of Medical Entomology. 2009 may; 46(3):625-632.) Anaplasma phagocytophilum was detected in dogs in Brazil by PCR(55 Santos HA, Pires MS, Vilela JA, Santos TM, Faccini JL, Baldani CD, Thomé SM, Sanavria A, Massard CL. Detection of Anaplasma phagocytophilum in brazilian dogs by real-time PCR. Journal of veterinary diagnostic investigation. 2011 jul; 23(4):770-774.), signs of the presence of the agent in goats and sheeps in the northeastern region were reported by RAMOS et al. (3131 Ramos RA, Ramos CA, Araújo FR, Melo ES, Tembue AA, Faustino MA, Alves LC, Rosinha GM, Elisei C, Soares CO. Detection of antibodies against Anaplasma sp. in small ruminants from the semi-arid region against Pernambuco State, Brazil. Revista Brasileira de Parasitologia Veterinária. 2008 apr-jun; 17(2):115-117.), besides migrating birds(66 Machado RZ, André MR, Werther K, De Sousa E, Gavioli FA, Alves Junior JR. Migratory and Carnivorous Birds in Brazil: Reservoirs for Anaplasma and Ehrlichia Species? Vector Borne Zoonotic Diseases. 2012 aug; 12(8):705-708.), cervids(44 Sacchi AB, Duarte JM, André MR, Machado RZ. Prevalence and molecular characterization of Anaplasmataceae agents in free-ranging Brazilian marsh deer (Blastocerus dichotomus). Comparative Immunology, Microbiology and Infectious Diseases. 2012 jul; 35(4):325-334.) and wild carnivores(77 André MR, Dumler JS, Scorpio DG, Teixeira RH, Allegretti SM, Machado RZ. Molecular detection of tick-borne bacterial agents in Brazilian and exotic captive carnivores. Ticks and tick-borne diseases. 2012 sep; 3(4):257-253.). In this epidemiologic investigation we noted the presence of wild animals such as opossums, armadillos, and birds in both analyzed breedings. As there was no difference in these observations for both breedings, these variables were not considered in the risk analysis of the infection by Anaplasma phagocytophilum. But one cannot discard the importance of these animals for the infection in equines of the Military Police of Rio de Janeiro. For horses in rural regions, other natural reservoirs may be involved in anaplasmosis transmission, including human infection, which in Brazil is still a rarely studied disease(66 Machado RZ, André MR, Werther K, De Sousa E, Gavioli FA, Alves Junior JR. Migratory and Carnivorous Birds in Brazil: Reservoirs for Anaplasma and Ehrlichia Species? Vector Borne Zoonotic Diseases. 2012 aug; 12(8):705-708.,44 Sacchi AB, Duarte JM, André MR, Machado RZ. Prevalence and molecular characterization of Anaplasmataceae agents in free-ranging Brazilian marsh deer (Blastocerus dichotomus). Comparative Immunology, Microbiology and Infectious Diseases. 2012 jul; 35(4):325-334.).
The results concerning the ages (Figure 1) revealed a higher number of seropositive adult animals and fewer young and elderly animals, but other authors(3232 Hansen MG, Christoffersen M, Thuesen LR, Petersen MR, Bojesen AM. Seroprevalence of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in Danish horses. Acta Veterinaria Scandinavica, 2010 jan; 18(1):52-53.) found significant correlation (p< 0,03) between age of equids over 11 years old and the presence of anti-A. phagocytophilum antibodies. Engvall & Egenvall(3333 Engvall EO, Egenvall A. Granulocytic ehrlichiosis in Swedish dogs and horses. International Journal of Medical Microbiology. 2002 jun; 291(33):100-103.) concluded, differently from the results for the Rio de Janeiro Mounted Police, the prevalence increases with the age what turns it a risk factor due to the higher rate of exposition to the tick during life. This age is also the most commonly used for work and sport; therefore, these animals are more often exposed to potential risk factors including journeys, exposure to animals from other regions, and stress(3333 Engvall EO, Egenvall A. Granulocytic ehrlichiosis in Swedish dogs and horses. International Journal of Medical Microbiology. 2002 jun; 291(33):100-103.,3434 Bullock PM, Ames TR, Robinson RA, Greig B, Mellencamp MA, Dumler JS. Ehrlichia equi infection of horses from Minnesota and Wisconsin: detection of seroconversion and acute disease investigation. Journal of Veterinary Internal Medicine. 2000; 14(3):252-257.).
In this study we did not verify significant difference for males and females in the seropositivity for anti-A. phagocytophilum antibodies, what means sex is not a risk factor for A.phagocytophilum infection. This corroborates with the findings of Bullock et al.(3434 Bullock PM, Ames TR, Robinson RA, Greig B, Mellencamp MA, Dumler JS. Ehrlichia equi infection of horses from Minnesota and Wisconsin: detection of seroconversion and acute disease investigation. Journal of Veterinary Internal Medicine. 2000; 14(3):252-257.) in 733 equids studied in the region of Minnesota and Wisconsin.
There was no significant variation (p=0.0596) in the seropositivity for anti-A. phagocytophilum antibodies between CS and RMP (Table 1), what can be explained by the free transit of animals between both environments as verified in the epidemiological report.
The origin of animals was not considered a risk factor for infection with A. phagocytophilum in horses of the Mounted Police of Rio de Janeiro state; however, the bleach of seropositive animals allows suspecting the existence of possible outbreaks of infection located at the other properties or microregions of the city of Rio de Janeiro.
The occurrence of A. phagocytophilum infection in horses from the urban area of the city of Rio de Janeiro, principally those in close quarters with other domestic animals and humans, may suggest a potential risk for emergent zoonoses in Brazil, similar to what has already been observed in the USA and in Europe(2525 Foley JE, Foley P, Brown RN, Lane RS, Dumlers JS, Madigan JE. Ecology of Anaplasma phagocytophilum and Borrelia burgdorferi in the western United States. Journal of Vector Ecology. 2004 jun; 29(1):41-50.,3535 Strle, F. Human granulocytic ehrlichiosis in Europe. International Journal of Medical Microbiology. 2004; 293:27-35.). However, this parasitosis should be better investigated, including studies that confirm diagnosis through molecular biology. Molecular-based diagnosis represents a challenge because of the genetic diversity of surface proteins displayed by various strains in accordance with the geographic distribution of A. phagocytophilum.Additionally, cross-reactivity with other Anaplasma spp. also poses difficulties for molecular identification(3636 De La Fuente J, Torina A, Caracappa S, Tumino G, Furla R, Almaza NC, Kocan KM. Serologic and molecular characterization of Anaplasma species infection in farm animals and ticks from Sicily. Veterinary Parasitology. 2005 nov; 133(4):357-362.).
In Brazil, equine monocytic erlichiosis caused by Neorickettsia risticii was described in Rio Grande do Sul, and has its cycle dependent of a trematode and snail as intermediate hosts(3737 Dutra F, Schuch LF, Delucchi E, Curcio BR, Coimbra H, Raffi MB, Dellagostin O, Riet-Correa F. Equine monocytic Ehrlichiosis (Potomac horse fever) in horses in Uruguay and southern Brazil. Journal of veterinary diagnostic investigation. 2001 sep; 13(5):433-437.,3838 Coimbra HS, Schuch LFD, Veitenheimer-Mendes IL, Meireles MCA. Neorickettsia (ehrlichia) risticii no sul do brasil: Heleobia spp.(mollusca: Hydrobilidae) e parapleurolophocecous cercariae (trematoda: Digenea) como possíveis vetores. Arquivos do Instituto Biologico (Sao Paulo). 2005; 72(3): 325-329.). The pathological clinical manifestation, with severe enterocolitis, presents differences when compared to equine granulocytic erlichiosis(3939 Oliver OE, Stämpfli H. Acute diarrhea in the adult horse: case example and review. Veterinary Clinics of North America: Equine Practic. 2006 apr; 22(1):73-84.). In horses the cross-reaction in serological tests among natural species of N. risticii and A. phagocytophilum was not yet proved(11 Rikihisa, Y. The tribe Ehrlichieae and ehrlichial diseases. Clinical Microbiology Reviews. 1991 jul; 4(3):286-308.). The control of parasites and care taken in the handling of horses of the Mounted Police of Rio de Janeiro made the identification of the species of ticks involved in the transmission of A. phagocytophilum to these animals impossible. Although the cases of equines with symptoms and seropositivity in Brazil documented by SALVAGNI et al. in 2010(1616 Salvagni CA, Dagnone AS, Gomes TS, Mota JS, Andrade GM, Baldani CD, Machado RZ. Serologic evidence of equine granulocytic anaplasmosis in horses from central West Brazil. Brazilian Journal of Veterinary Parasitology. 2010 jul-sep; 19(3):135-140.) presented with histories of infestation by Amblyomma spp. we have not found scientific studies demonstrating the importance of these arthropod species in transmission. In 2004, two HGA cases of suspected anaplasmosis in Brazil were described and diagnosed through clinical findings and indirect immunofluorescence assays; however, both individuals were only sero-reactive for Ehrlichia chaffeensis(4040 Calic SB, Galvão MAM, Bacella F, Rocha CM, Mafra CL, Leite RC, Walker DH. Human ehrlichiosis in Brazil: first suspect case. The Brazilian Journal of infection disease. 2004 jun; 8(3):259-262.). HGA is a zoonosis that is found in various countries in Europe and the USA that is transmitted by ticks of the genus Ixodes. With the increasing incidence of HGA in the USA, especially in regions of the Midwest and Northeast, some experimental studies have shown that the agent of HGA may induce clinical disease in horses, besides protecting the subsequent infection, which is important when considering the possibility of horses act as reservoir and source of infection for human granulocytic anaplasmosis(4141 Barlough JE, Madigan JE, Derock E, Dumler JS, Bakken J.S. Protection against Ehrlichia equi is conferred by prior infection with the human granulocytotropic Ehrlichia (HGE agent). Journal of Clinical Microbiology. 1995 dec; 33(12):3333). Various mammals, such as horses, cattle, goats, dogs, and cats, have important roles in the propagation of A. phagocytophilum and its transmission to humans.
Conclusion
The presence of anti-A. phagocytophilum antibodies in horses of the Mounted Police of Rio de Janeiro State, regardless of the place they are bred, shows that the parasite is enzootically present in horses of the urban region of Rio de Janeiro and that animal sex does not interfere in the susceptibility to the parasite.
Acknowledgements
We would like to thank the Military Police of Rio de Janeiro state for making their animals available for the present study. Thanks also to the Fundação Carlos Chagas Filho de amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
References
-
1Rikihisa, Y. The tribe Ehrlichieae and ehrlichial diseases. Clinical Microbiology Reviews. 1991 jul; 4(3):286-308.
-
2Bjöersdorff A, Bergström S, Massung RF, Haemig PD, Olsen B. Ehrlichia-infected ticks on migrating birds. Emerging Infectious Diseases. 2001 sep-oct; 7(5):877-879.
-
3Mesquita Neto F De, Araujo T, Vieira RM, Gouveia AMG. Ehrlichia equi in horses from the municipality of Betim (Brazil). Arquivo Brasileiro de Medicina Veterinária e Zootecnia. 1984 apr; 36(2):203-207.
-
4Sacchi AB, Duarte JM, André MR, Machado RZ. Prevalence and molecular characterization of Anaplasmataceae agents in free-ranging Brazilian marsh deer (Blastocerus dichotomus). Comparative Immunology, Microbiology and Infectious Diseases. 2012 jul; 35(4):325-334.
-
5Santos HA, Pires MS, Vilela JA, Santos TM, Faccini JL, Baldani CD, Thomé SM, Sanavria A, Massard CL. Detection of Anaplasma phagocytophilum in brazilian dogs by real-time PCR. Journal of veterinary diagnostic investigation. 2011 jul; 23(4):770-774.
-
6Machado RZ, André MR, Werther K, De Sousa E, Gavioli FA, Alves Junior JR. Migratory and Carnivorous Birds in Brazil: Reservoirs for Anaplasma and Ehrlichia Species? Vector Borne Zoonotic Diseases. 2012 aug; 12(8):705-708.
-
7André MR, Dumler JS, Scorpio DG, Teixeira RH, Allegretti SM, Machado RZ. Molecular detection of tick-borne bacterial agents in Brazilian and exotic captive carnivores. Ticks and tick-borne diseases. 2012 sep; 3(4):257-253.
-
8Blanco JR, Oteo JA. Human granulocytic ehrlichiosis in Europe. Clinical Microbiology and Infectious Diseases. 2002 dec; 8(12):763-772.
-
9Pusterla N, Chae JS, Kimsey RB, Berger Pusterla J, Derock E, Dumler JS, Madigan JE. Transmission of Anaplasma phagocytophila (human granulocytic ehrlichiosis agent) in horses using experimentally infected ticks (Ixodes scapularis). Journal of Veterinary Medicine Series B-infectious Diseases and Veterinary Public Health. 2002 dec; 49(10):484-488.
-
10Madigan JE. Equine ehrlichiosis. Veterinary Clinics of North America: Equine Practic. 1993 aug; 9(2):423-428.
-
11Madigan JE, Pusterla N. Ehrlichial diseases. Veterinary Clinics of North America: Equine Practic. 2000 dec; 16(3):487-499.
-
12Franzen P, Aspan A, Egenvall A, Gunnarsson A, Aberg L, Pringle J. Acute clinical, hematologic, serologic, and polymerase chain reaction findings in horses experimentally infected with a European strain of Anaplasma phagocytophilum. Journal of Veterinary Internal Medicine. 2005 mar-apr; 19(2):232-239.
-
13Parra AC. Investigação diagnóstica de doença concomitante babesiose anaplasmose em rebanho equino por pcr e elisa. [Internet]. São Paulo: Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia de São Paulo; 2009. [citado 2014 Dez 14] Available from: file://localhost/C:/Users/Flavio/Downloads/Andrea_Cristina_Parra_doutorado(1).pdf
» file://localhost/C:/Users/Flavio/Downloads/Andrea_Cristina_Parra_doutorado(1).pdf -
14Ebani V, Cerri D, Fratini F, Ampola M, Andreani E. Seroprevalence of Anaplasma phagocytophilum in domestic and wild animals from central Italy. New Microbiologica. 2008 ju; 31(3):371-375.
-
15Van Andel AE, Magnarelli LA, Heimer R Wilson, M.L. Development and duration of antibody response against Ehrlichia equi in horses. The Journal of the American Medical Association. 1998 jun; 15(212):1910-1914.
-
16Salvagni CA, Dagnone AS, Gomes TS, Mota JS, Andrade GM, Baldani CD, Machado RZ. Serologic evidence of equine granulocytic anaplasmosis in horses from central West Brazil. Brazilian Journal of Veterinary Parasitology. 2010 jul-sep; 19(3):135-140.
-
17Macleod J. Ticks and disease in domestic stock in Great Britain. Symposium of the Zoological Society of London. 1962; 6:29-50.
-
18Holden K, Boothby JT, Anand S, Massung RF. Detection of Borrelia burgdorferi,Ehrlichia chaffeensis and Anaplasma phagocytophilum in ticks (Acari: Ixodidae) from a coastal region of California. Journal of Medical Entomology. 2003 jul; 40(4):534-539.
-
19Cao WC, Zhan L, He J, Foley JE, De Vlas SJ, Xiao-Mingwu, Yang H, Richardus JH, Habbema JDF. Natural Anaplasma phagocytophilum infection of ticks and rodents from a forest area of Jilin Province, China. The American journal of tropical medicine and hygiene. 2006 oct; 75(4):664-668.
-
20Yoshimoto K, Matsuyama Y, Matsuda H, Sakamoto L, Matsumoto K, Yokoyama N, Inokuma H. Detection of Anaplasma bovis and Anaplasma phagocytophilum DNA from Haemaphysalis megaspinosa in Hokkaido, Japan. Veterinary parasitology. 2010 feb; 26(168):170-172.
-
21Teglas MB, Foley J. Differences in the transmissibility of two Anaplasma phagocytophilum strains by the North American tick vector species, Ixodes pacificus and Ixodes scapularis (Acari: Ixodidae). Experimental and Applied Acarology. 2006 jul; 38(1):47-58.
-
22Teglas M, Matern E, Lein S, Foley P, Mahan SM, Foley J. Ticks and tick-borne disease in Guatemalan cattle and horses. Veterinary Parasitology. 2005; 15(23):119-127.
-
23Franzen P, Aspan A, Egenvall A, Gunnarsson A, Karlstam E., Pringle J Molecular evidence for persistence of abre-italicAnaplasma phagocytophilumfecha-italic in the absence of clinical abnormalities in horses after recovery from acute experimental infection. Journal of Veterinary Internal Medicine. 2009 may-jun; 23(3):636-642.
-
24Passamonti F, Veronesi F, Cappelli K, Capomaccio S, Coppola G, Marenzoni ML, Piergili FD, Verini SA, Coletti, M. Anaplasma phagocytophilum in horses and ticks: A preliminary survey of Central Italy. Comparative Immunology, Microbiology and Infectious Disease. 2010 jan; 33(1):73-83.
-
25Foley JE, Foley P, Brown RN, Lane RS, Dumlers JS, Madigan JE. Ecology of Anaplasma phagocytophilum and Borrelia burgdorferi in the western United States. Journal of Vector Ecology. 2004 jun; 29(1):41-50.
-
26Li H, Zhou Y, Wang W, Guo D, Huang S, Jie S. The clinical characteristics and outcomes of patients with human granulocytic anaplasmosis in China. International Journal of Infectious Diseases. 2011 dec; 15(12):859-866.
-
27Scharf W, Schauer S, Freyburger F, Petrovec M, Schaarschmidt-Kiener D, Liebisch G, Runge M, Ganter M, Kehl A, Dumler JS, Garcia-Perez AL, Jensen J, Fingerle V, Meli ML, Ensser A, Stuen S, Von Loewenich FD. Distinct Host Species Correlate with Anaplasma phagocytophilum ankA Gene Clusters. Journal of Clinical Microbiology. 2011 mar; 49(3):790-796.
-
28Ogden NH, Woldehiwet Z, Hart CA. Granulocytic ehrlichiosis: an emerging or rediscovered tick-borne disease? Journal of Medical Microbiology. 1998 jun; 47(6):475-482.
-
29Vorou RM, Pavassiliou VG, Tsiodras S. Emerging zoonoses and vector-borne infections affecting humans in Europe. Epidemiology and Infection. 2007 nov; 135(8):1231-1247.
-
30Baldridg GD, Scoles GA, Burkhardt NY, Schloeder B, Kurtti TJ, Munderloh UG. Transovarial Transmission of Francisella-Like Endosymbionts and Anaplasma phagocytophilum Variants in Dermacentor albipictus (Acari: Ixodidae). Journal of Medical Entomology. 2009 may; 46(3):625-632.
-
31Ramos RA, Ramos CA, Araújo FR, Melo ES, Tembue AA, Faustino MA, Alves LC, Rosinha GM, Elisei C, Soares CO. Detection of antibodies against Anaplasma sp. in small ruminants from the semi-arid region against Pernambuco State, Brazil. Revista Brasileira de Parasitologia Veterinária. 2008 apr-jun; 17(2):115-117.
-
32Hansen MG, Christoffersen M, Thuesen LR, Petersen MR, Bojesen AM. Seroprevalence of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in Danish horses. Acta Veterinaria Scandinavica, 2010 jan; 18(1):52-53.
-
33Engvall EO, Egenvall A. Granulocytic ehrlichiosis in Swedish dogs and horses. International Journal of Medical Microbiology. 2002 jun; 291(33):100-103.
-
34Bullock PM, Ames TR, Robinson RA, Greig B, Mellencamp MA, Dumler JS. Ehrlichia equi infection of horses from Minnesota and Wisconsin: detection of seroconversion and acute disease investigation. Journal of Veterinary Internal Medicine. 2000; 14(3):252-257.
-
35Strle, F. Human granulocytic ehrlichiosis in Europe. International Journal of Medical Microbiology. 2004; 293:27-35.
-
36De La Fuente J, Torina A, Caracappa S, Tumino G, Furla R, Almaza NC, Kocan KM. Serologic and molecular characterization of Anaplasma species infection in farm animals and ticks from Sicily. Veterinary Parasitology. 2005 nov; 133(4):357-362.
-
37Dutra F, Schuch LF, Delucchi E, Curcio BR, Coimbra H, Raffi MB, Dellagostin O, Riet-Correa F. Equine monocytic Ehrlichiosis (Potomac horse fever) in horses in Uruguay and southern Brazil. Journal of veterinary diagnostic investigation. 2001 sep; 13(5):433-437.
-
38Coimbra HS, Schuch LFD, Veitenheimer-Mendes IL, Meireles MCA. Neorickettsia (ehrlichia) risticii no sul do brasil: Heleobia spp.(mollusca: Hydrobilidae) e parapleurolophocecous cercariae (trematoda: Digenea) como possíveis vetores. Arquivos do Instituto Biologico (Sao Paulo). 2005; 72(3): 325-329.
-
39Oliver OE, Stämpfli H. Acute diarrhea in the adult horse: case example and review. Veterinary Clinics of North America: Equine Practic. 2006 apr; 22(1):73-84.
-
40Calic SB, Galvão MAM, Bacella F, Rocha CM, Mafra CL, Leite RC, Walker DH. Human ehrlichiosis in Brazil: first suspect case. The Brazilian Journal of infection disease. 2004 jun; 8(3):259-262.
-
41Barlough JE, Madigan JE, Derock E, Dumler JS, Bakken J.S. Protection against Ehrlichia equi is conferred by prior infection with the human granulocytotropic Ehrlichia (HGE agent). Journal of Clinical Microbiology. 1995 dec; 33(12):3333
Publication Dates
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Publication in this collection
Jul-Sep 2015
History
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Received
29 Aug 2012 -
Accepted
11 Dec 2014