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

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


Introduction
The Family Anaplasmataceae has four genera -Ehrlichia, Anaplasma, Neorickettsia and Wolbachia -which include small obligatory intracellular bacteria with unique host cell specificities (DUMLER et al., 2001;RIKIHISA, 1991;2003).Horses can be naturally infected by the ehrlichial agent Anaplasma phagocytophilum (formerly E. equi) causing the Equine Granulocytic Anaplasmosis (EGA) that is a seasonal disease of horses, first reported in 1969 in California (STANNARD et al., 1969;GRIBBLE, 1969).Since then, it has been described worldwide.This agent was found first in humans in 1994 in the United States (BAKKEN et al., 1994) and 16S rRNA gene sequence of A. phagocytophilum described in horses and dogs seems to be identical to the A. phagocytophilum strain that infects humans (JOHANSSON et al., 1995) and described in many countries including Chile (LÓPEZ et al., 2003), Italy (RUSCIO;CINCO, 2003) and Korea (HEO et al., 2002).The vectors are Ixodes ticks (PANCHOLI et al., 1995).A. phagocytophilum is presented in various tick-rodent cycles and wild reservoirs in nature (FOLEY et al., 2004).The clinical signs of A. phagocytophilum in horses include high fever, depression, partial anorexia, limboedema, petechiae, icterus, ataxia and a reluctance to move (MADIGAN;GRIBBLE, 1987).However, clinical signs are not specific for the disease and it has been posed that different hosts demonstrate several degrees of susceptibility to regional strains of A. phagocytophilum.In South America a few cases of human erlichiosis are described due to E. canis and E. chaffeensis in Venezuela (PEREZ et al., 2006); and serologic evidence of human erlichiosis is found in Chile and Argentina (RIPOLL et al., 1999;LÓPEZ et al., 2003).In Brazil, suspected cases of human were demonstrated with clinical and laboratory findings and with positive serology for human granulocytic anaplasmosis (HGA) by Calic et al. (2004).However, Brazilian spotted fever (BSF) is the most common form of human Rickettsia infection in our country.Ehrlichia chaffeensis was detected for the first time in blood samples from Brazilian marsh deer (Blastocerus dichotomus) captured in the marshes of the Parana River in Southeast Brasil in 1998 (MACHADO et al., 2006), but no human case have been diagnosed to the present time.
Therefore, general diagnosis is based on association of clinical signs in horses, with seasonality and geographic location associated with characteristic gram-negative inclusion bodies/morulae in the cytoplasm of circulating neutrophils in blood smears (MADIGAN, 1993).And the presence of the vector Ixodes ticks is important in the acute phase.The acute phase of the disease can be diagnosed directly by microscopic identification of inclusion bodies in peripheral granulocytes, polymerase chain reaction (PCR) and blood culture (BAKKEN et al., 1994;GOODMAN et al., 1996).PCR methods have been shown to be effective for the diagnosis of acute granulocytic ehrlichial infections (MASSUNG;SLATER, 2003).Antibody detection is also used to support the clinical diagnosis (AGUERO-ROSENFELD et al., 2000).Indirect immunofluorescent antibody assay (IFA) is the most commonly test used in the laboratory to confirm a diagnosis of A. phagocytophilum infection in humans (Human granulocytic ehrlichiosis, HGE).In horses, a diferential diagnostic approach should be made because of such other diseases as encephalitis, liver disease, purpura hemorrhagica, and equine infectious anemia, which can have similar clinical conditions (MADIGAN, 1993).The present study reports the first serologic evidence of granulocytic ehrlichiosis in horses from Brazil.And considering that cases of equine ehrlichiosis can be due to Neorickettsia risticii, a screening PCR for the Genus was performed.Additionally, the serodiagnosis and molecular detection of Theileria equi were carried out once equine piroplasmosis became widespread and a cause of serious concern in Brazil.

Horses
Twenty cases suspected of equine granulocytic ehrlichiosis were selected from horses with clinical signs suggestive of A. phagocytophilum infection.Nineteen mixed-breed horses came from from the Brazilian Army (1º Regimento da Cavalaria de Guarda, Dragões da Independência, Brasilia -DF, Brazil) and one horse from Goiânia County -GO, Brazil.At the time of initial examination the clinical signs were fever, anorexia, lethargy, pale mucous membranes and edema of the limbs.Tick exposure or tick infestation were commonly observed in all horses.

Samples
Whole blood was collected aseptically via jugular venipuncture and an aliquot was allowed to clot, then centrifuged and serum was separated.Another part of the sample was EDTA-whole blood that was used as a source for buffy coat smears stained with Giemsa.The remaining blood was stored at -20 °C until assayed.

A. phagocytophilum serology
An enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies against A. phagocytophilum was performed (Ehrlichia equi IgG antibody Kit -ELISA -Helica Biosystems, Inc.).Briefly, equine serum samples were diluted to 1:100 and allowed to react with E. equi / (A.phagocytophilum) whole antigens coated on specially treated micro-well plates provided with the kit.The reactions were performed according to the manufacter's instructions and the results were based on color intensity (absorbance) developed with an optical density (OD) of 450 nm.The visual determinations were made with aid of the color chart provided by the kit.ODs over 0.32 were interpreted as positive.

Theileria equi serology
Indirect immnufluorescent antibody assays (IFA) were performed to detect IgG antibodies against T. equi as previously described by Machado et al. (1994), with a serum dilution of 1: 80. Also, an ELISA test was performed as reported elsewhere (BALDANI et al., 2004) with an antigen concentration of 10 µg.mL -1 and a serum dilution of 1:100.The immunological activity of each serum was calculated by determining the sample-to-positive serum ratio (S/P), considering positive and negative sera as reference.Horses were considered positives when S/P values at an optical density of 405 nm were equal to or greater than 0.278.

DNA isolation
DNA was extracted from 200 µL of EDTA whole-blood EDTA samples using the QIAamp DNA Blood Mini Kit (QIAGEN ™ ), according to the manufacturer´s instructions.Samples from the thirteen horses tested as serologically positive (OD ≥ 0.32) for A. phagocytophilum antibodies by the ELISA Kit were analysed.These horse blood samples (n = 13) were then collected before (rest, n = 13) and after exercise (stress, n = 13), and were submitted to buffy coat smear preparations stained with Giemsa for microscopic examination, and to PCR amplifications for A. phagocytophilum and Ehrlichia spp.DNA extraction was also performed for the detection of T. equi by nPCR.
The nPCR for the diagnosis of T. equi were performed as previously described by Baldani et al. (2008).

Clinical signs and presence of ticks
The most common clinical signs found in all tested horses were anemia (pale mucous membranes), fever, anorexia, lethargy and limbs edema.Amblyomma cajennense and Amblyomma spp.were the tick species found in these animals.

Buffy coat smears
None of the samples examined showed any evidence of Anaplamataceae infection as inclusion bodies and/or morulae structures.

Anaplasma phagocytophilum serology
Thirteen (65%) of 20 equine serum samples included in this study showed positive reactions (OD ≥ 0.32) by the ELISA Kit (Helica ™ ).These 13 positive samples were then collected again (after exercise), as previously described, and tested by PCR and nPCR in two sets: before exercise (n = 13) and after exercise (n = 13).

Anaplamataceae agents PCR and nPCR
None of the blood samples tested (n = 26) were positive by PCR amplification using primers ECC/ECB for Ehrlichia ssp.Also, the amplifications PCR and nPCR for the A. phagocytophilum 16srRNA gene (Figure 1) and msp2 gene were all negative.All the results are summarized in Table 1.

Theileria equi serology and nPCR
Eighteen ( 90%) equine serum samples tested were positive by the IFA test (cut off titer ≥ 80) and 19 (95%) by the ELISA assay for the same agent.T. equi was detected by nPCR in 9 (45%) horses.

Discussion
In the present study none of the twenty equines with clinical signs demonstrated granulocitic morulae or initial bodies in Giemsa blood smears.Clinical anaplasmosis in horses is probably still underdiagnosed in Brazil, especially those horses taking part in sports and traveling to others regions outside of the country where the disease is endemic.On the other hand, clinical anaplasmosis is similar to those caused by infections with other pathogens such as Babesia caballi, Theileria equi, equine infectious anaemia virus, equine herbevirus, equine arteritis virus and Leptospiraceae.Recently, in Brazil, a spontaneous outbreak of Equine Monocytic Ehrlichiosis (EME) was described, caused by Neorickettsia risticii, affecting horses 1-5 years old in the State of Rio Grande do Sul.The most important clinical sign was profuse acute diarrhea, accompanied by anorexia and depression (COIMBRA et al., 2006).
Acute equine ehrlichiosis is evaluated observing the characteristic inclusion bodies in blood smears as well as clinical signs, but chronic cases are not clinically detected.Also, in acute disease a differential diagnosis pattern should be performed because of the nonspecific clinical signs observed.Diseases such as encephalitis, liver disease, purpura hemorrhagica, equine infectious anemia and equine viral arteritis should be ruled out (MADIGAN;GRIBBLE, 1987).The severity of clinical signs of EGA is a function of the age of the horse and the duration of the illness (MADIGAN, 1993).This  None of the tested animals showed positive reaction in PCR assays to A. phagocytophilum, for either the 16s rRNA gene or msp2 gene.However, positive nPCR in nine animals to T. equi confirmed the presence of the agent by molecular detection.In addition, 90% of all animals tested were positive by the IFA test and 95% by the ELISA assay for the same agent.Previous studies in our laboratory have not confirmed the presence of A. phagocytophilum in blood samples tested from two hundred horses with clinical signs of babesiosis or suspected of anaplasmosis by PCR or nPCR (data not shown).In Brazil, the prevalence of equine piroplasmosis is of serious concern, considered as an endemic disease.To detect anti-T.equi antibodies, Heuchert et al. (1999) tested 752 horse serum samples from São Paulo State, by IFA and a complementary fixation test (CFT), and reported prevalence rates of 29.6 and 17.6%, respectively.Serum samples from horses from different regions of Brazil were tested by CTF and the results demonstrated the prevalence of T. equi infections were 42.48% (KERBER et al., 1999).Also Xuan et al. (2001) examined serum samples from horses in the State of São Paulo and Mato Grosso do Sul, Brazil, and showed that the positive rate for T. equi was 81%.Baldani et al. (2004) demonstrated that 75% (67/90) of the horses from the state of São Paulo presented anti-T.equi antibodies by IFA and ELISA tests.The prevalence of equine piroplasmosis reported in the present study is higher and may be due to differences in serological tests used, horse breeding operations, intensity of tick infestation or even number of serum samples examined.This study is the first report on the serological evidence of co-infection with Equine Granulocytic Ehrlichiosis and equine piroplasmosis agents in horses from the Brazilian Army and Goiânia county.Amblyomma cajennense and Amblyomma spp.were the most common tick species found in our animals in this study.
Our data may be considered as important information contributing to understanding the occurrence of these diseases in centralwest Brazil.