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Seroconversion for Anaplasma phagocytophilum in a Mare with Concomitant Piroplasmosis
Journal of Equine Veterinary Science 31 (2011) 185-187
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Short Communication
Seroconversion for Anaplasma phagocytophilum in a Mare with Concomitant Piroplasmosis Elisabetta Giudice DVM, PhD a, Claudia Giannetto DVM, PhD b , Alessandra Torina BS c, Pietro P. Niutta DVM a a b c
Dipartimento di Sanità Pubblica Veterinaria, Università degli Studi di Messina, Polo Universitario dell’Annunziata, Messina, Italy Dipartimento di Scienze Sperimentali e Biotecnologie Applicate, Università degli Studi di Messina, Polo Universitario dell’Annunziata, Messina, Italy Centro di Referenza Nazionale per Babesia, Anaplasma, Rickettsia, Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri”, Palermo, Italy
a b s t r a c t Keywords: EGA Theileria equi TBD Horse Limbs edema Tetracyclines
In this article, we describe the clinical course of a 3-year-old purebred mare at the third month of gestation found to be positive for Theileria equi (indirect immunofluorescence antibody test and polymerase chain reaction) at the time of the first evaluation. At the clinical examination, the mare showed profound depression, pale mucous membranes, enlarged lymph nodes, febrile temperature, hind limbs edema, and swelling of the right carpus. Anemia and thrombocytopenia were also observed. Because of worsening of clinical signs, treatment with oxytetracycline (6 mg/kg) was started. An improvement of clinical conditions was observed within 12 hours, with a full recovery in 2 weeks. Two months after the first hospitalization, new serological tests showed seroconversion to Anaplasma phagocytophilum. In conclusion, the severity of clinical signs was attributed to the concomitant infections, usually subclinical in Sicily. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Equine granulocytic anaplasmosis (EGA) is a rickettsial disease of horses that was first reported in the late 1960s in California. The causative agent is Anaplasma phagocytophilum, member of the A phagocytophilum complex, and has since long been recognized as a worldwide tick-borne agent for several species of wild and domestic mammals, including rodents, carnivores, equids, and ruminants. They are obligate intracellular bacteria that reside within vacuoles in the cytoplasm of granulocytotropic cell lines. In horses, EGA is recognized as an acute disease with an incubation period of <14 days, characterized by high fever, lethargy, inappetence, staggering or ataxia, distal limb edema, petechiation, icterus, and hematological alteration
Corresponding author at: Claudia Giannetto, DVM, PhD, Dipartimento di Scienze Sperimentali e Biotecnologie Applicate, Facoltà di Medicina Veterinaria, Università degli Studi di Messina, Polo Universitario dell’Annunziata, Messina 98168, Italy. E-mail address: [email protected] (C. Giannetto). 0737-0806/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2011.02.004
such as thrombocytopenia, neutropenia, lymphopenia, and mild anemia [1-3]. A recent report documented sudden death in a horse associated with experimental infection with the bacterium [4]. Horses aged <4 years tend to develop a milder form of the disease, whereas in those aged <1 year, the clinical signs may be difficult to recognize [5]. Horses experimentally infected by A phagocytophilum develop clinical manifestations that are either typical of natural virulent disease when infected with low-passage bacteria, or with significantly diminished clinical signs and laboratory features when infected with high-passage bacteria [6]. In naturally infected horses, immunity persists for a minimum of 2 years and does not seem to depend on latent infection or carrier status [5]. Equine piroplasmosis is a tick-borne intraerythrocytic protozoal disease caused by Theileria equi and Babesia caballi, or even by a mixed infection. Piroplasmosis is endemic to most tropical and subtropical areas and seems to be spreading to more temperate zones. Clinical signs of the disease are not pathognomonic and may vary considerably between horses. Affected animals may exhibit apathy, lethargy, pyrexia, anemia, icterus, hemoglobinuria,
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enlarged lymph nodes, weight loss, and edema of distal limbs, leading to, in some cases, death. Subclinical infections may occur commonly in horses living in endemic areas. The presence of both parasites may also persist in clinically recovered horses for a minimum of several years, thus acting as reservoirs for subsequent infecting ticks [7]. 2. Clinical Case A 3-year-old purebred English mare at the third month of gestation was referred to the Faculty of Veterinary Medicine of Messina University (Italy), with a history of lack of appetite, slight weight loss, depression, and reluctance to move since a week. The mare lived in a stable declared free of equine infectious anemia. Because of a progressive worsening of clinical manifestation during the last 2 days, the referred veterinarian decided to administer oxytetracycline (Pan Terramicina, Pfizer, Italy) intravenously at a dosage of 6 mg/kg without performing laboratory tests, although its use in pregnant mares may be discouraged [8]. Six hours later, the horse was admitted to our clinic. At the clinical inspection, the horse was found to be profoundly depressed, the mucous membranes were extremely pale, lymph nodes were enlarged, and heart and respiratory rates were slowly increased. The horse at this time had a rectal temperature of 39.5 C. Hind limbs edema and swelling of the right carpus were both observed. Carpus was also painful and hot. The horse showed stiff movement, almost like she did not want to move, and manifested right forelimb lameness. The mare had not displayed similar clinical signs before the acute onset of this episode. Differential diagnosis based on clinical findings was tick-borne diseases (TBDs), viral arteritis, equine adenitis, and septic arthritis. Blood samples were collected from the animal by jugular vein puncture into vacutainer tubes (Terumo Corporation, Japan) containing ethylenediamine tetraacetic acid and without anticoagulant. Using the ethylenediamine tetraacetic acid sample, emocromocytometric test was assessed by means of an automated hematology analyzer (HecoVet C, SEAC, Florence, Italy). Blood samples with no additives were centrifuged at 3,000 rpm for 10 minutes and sera separated in aliquots. Using one aliquot, biochemical parameters were assessed by means of an ultraviolet spectrophotometer (SEAC, Slim, Florence, Italy); the other aliquots were stored at 20 C pending further analysis. Fine-needle aspiration biopsy of the submandibular lymph nodes and carpal arthrocentesis were performed. Bacterial culture from lymphonodular and synovial specimens was done. Abnormal laboratory findings included low hematocrit (19.7%), low red blood cell count (4.46 M/mL), low hemoglobin level (6.8 g/dL), and thrombocytopenia (35.00 K/mL). Biochemical parameters were within the physiological range and no bacterial colonies growing on culture medium were observed. Samples included in the study (blood, serum, lymph nodes, and synovial fluid) were supplied to the Centro Nazionale di Referenza per Anaplasma, Babesia, Rickettsia e Theileria at the Istituto Zooprofilattico Sperimentale della Sicilia. Indirect immunofluorescence antibody test (Fuller
Laboratories, Fullerton, CA) and polymerase chain reaction (Amersham, Piscataway, NJ) for T equi, B caballi, and A phagocytophilum were performed, as described previously [9]. At the same time, the sample was tested for equine viral arteritis by means of seroneutralization on RK13 confluent cell monolayer. An improvement of clinical conditions was observed within 12 hours after the first tetracycline injection. After 24 hours, the horse showed normal appetite and sensory function, and rectal temperature was 37.5 C. Reduction of hind limbs edema and carpal swelling were also observed. Tetracycline treatment was continued once a day for 7 days. Positive serological tests were found for T equi and B caballi, no antibodies were found for A phagocytophilum. T equi deoxyribonucleic acid was found in blood, lymph nodes, and synovial fluid. Imidocarb dipropionate, the drug of choice for piroplasmosis, was not administered because clinical improvement had occurred and its parasympathetic effect could compromise the gestation state of the horse. Clinical signs had completely resolved 20 days after the first treatment and did not relapse. At this time, all hematological parameters were within the physiological range. Two months after the first hospitalization, a new serological test was conducted. T equi antibodies were confirmed, B caballi antibodies were not found, and antibodies for A phagocytophilum were observed. The owner confirmed that the horse had no further clinical signs in the next 6 months. The mare delivered a living and viable full-term foal without any discoloration of teeth. 3. Discussion The mare developed the infection in June, which is comparable with the seasonal peak of the vector seen in Sicily. This horse was native to this region and had not travelled outside the island. Native animals in Sicily are often infected by T equi and B caballi, but do not exhibit any sign of disease. The everlasting presence of the parasites induces an efficient immune response in the habitual host. Subclinical anaplasmosis may be common in endemic areas, based on studies of horse farms where a single case of clinical A phagocytophilum may occur yearly and still nearly 50% of horses on that farm had evidence of antibody to A phagocytophilum [10]. In most horses, EGA recovery occurs spontaneously and thus does not attract the attention of clinicians [11]. Viral arteritis, equine adenitis, and septic arthritis were excluded by laboratory findings. The controversy on the diagnosis was attributed to the absence of pathognomonic symptoms of TBD symptoms and the presence of multiple infections. Hypertrophy and inflammation of the lymph nodes are more pronounced in the case of T equi, and have not been observed in EGA. Edema owing to a proliferative and necrotizing vasculitis of small arteries and veins in the skeletal muscle and tendons of legs has been commonly described in horses suffering from EGA [12] and less frequently observed during piroplasmosis. Limb edema is generally the most delayed clinical signs in horses experimentally infected with A phagocytophilum [13]. It occurs in 83% of naturally infected
E. Giudice et al. / Journal of Equine Veterinary Science 31 (2011) 185-187
horses [11] and its frequency is lower if prompt treatment is established [3]. In our case, limb edema appeared a week after the onset of clinical signs but regressed suddenly after tetracycline administration. Thrombocytopenia and varying degrees of anemia are typically found both during EGA [3,11] and piroplasmosis [14]. Although at the first evaluation, positive serological tests were found for T equi and B caballi, and no antibodies were found for A phagocytophilum, the improvement of clinical signs after oxytetracycline administration suggested EGA infection. In fact, A phagocytophilum is susceptible to tetracyclines [15-18], whereas B caballi is not susceptible and T equi is susceptible only during the pre-erythrocytic phase [19]. The high level of suspicion for EGA was confirmed by the seroconversion observed 2 months after the infection. In the early phase of infection, antibodies production against A phagocytophilum is not detectable, and 50% of infected horses are negative on serological test [20]. Cell-mediated and humoral immune responses are characterized by the production of immunoglobulin G, which became serologically detectable 19 days after the infection date, reached their peak approximately 8 weeks after the infection, and tend to become serologically negative, unless reinfections intervene [5,21]. Seropositivity for B caballi was probably because of a past dated infection. This hypothesis was confirmed by the seroreversion observed 2 months after the clinical onset. The absence of intragranulocytic inclusions was not in disagreement with EGA suspicion. Morulae or elementary bodies are present for only 3 to 7 days and the degree of bacteremia is variable [1]. A phagocytophilum deoxyribonucleic acid was intermittently detected in experimentally infected horses [1,13] and this could explain the negative polymerase chain reaction results, even though the degree of bacteremia seems to be related to the clinical signs [22]. In addition, tetracycline treatment, initiated several hours before our sampling, could have altered microscopic and molecular assays. In conclusion, the severity of clinical signs was attributed to the concomitant presence of both pathogens. Piroplasmosis may have worsened A phagocytophilum infection, which in horses aged <4 years has a generally less severe course. Conversely, A phagocytophilum may have exacerbated the clinical signs of piroplasmosis, usually asymptomatic in this area, which again became subclinical after the EGA treatment. The concomitant infection of piroplasms and A phagocytophilum probably is a common event in Sicily. The clinical signs are usually attributed to an acute piroplasmosis, treated with imidocarb dipropionate. Because of this drug efficacy for both pathologies, EGA could be underestimated. This aspect would remind practitioners of the need to consider the possibility of more than one infectious agent when evaluating similar cases and their treatment.
187
References [1] Franzén P, Aspan A, Egenvall A, Gunnarsson A, Karlstam E, Pringle J. Molecular evidence for persistence of Anaplasma phagocytophilum in the absence of clinical abnormalities in horses after recovery from acute experimental infection. J Vet Intern Med 2009;23:636-42. [2] Hilton H, Madigan JE, Aleman M. Rhabdomyolysis associated with Anaplasma phagocytophilum infection in a horse. J Vet Intern Med 2008;22:1061-4. [3] Jahn P, Zeman P, Bezdekova B, Praskova I. Equine granulocytic anaplasmosis in the Czech Republic. Vet Rec 2010;166:646-9. [4] Franzén P, Berg AL, Aspan A, Gunnarsson A, Pringle J. Death of a horse infected experimentally with Anaplasma phagocytophilum. Vet Rec 2007;160:122-5. [5] Sellon CD, Long MT. Equine infectious diseases. St Louis, MO: Saunders; 2007. p. 354-7. [6] Pusterla N, Madigan JE, Asanovich KM, Chae JS, Derock E, Leutenegger CM, et al. Experimental inoculation with human granulocytic Ehrlichia agent derived from high- and low-passage cell culture in horses. J Clin Microbiol 2000;38:1276-8. [7] Pitel PH, Pronost S, Scrive T, Léon A, Richard E, Fortier G. Molecular detection of Theileria equi and Babesia caballi in the bone marrow of asymptomatic horses. Vet Parasitol 2010;170:182-4. [8] Dowling PM. Antimicrobial therapy. In: Berton JJ, Horspool LJI, editors. Equine clinical pharmacology. London: WB Saunders; 2004. p. 38-40. [9] Torina A, Vicente J, Alongi A, Scimena S, Turlà R, Nicosia S, et al. Observed prevalence of tick-borne pathogens in domestic animals in Sicily, Italy during 2003-2005. Zoonoses Public Health 2007;54:8-15. [10] Madigan JE, Hietala S, Chalmers S, DeRock E. Seroepidemiologic survey of antibodies to Ehrlichia equi in horses of northern California. J Am Vet Med Assoc 1964;196:1962-4. [11] Butler CM, Nijhof AM, Jongejan F, van der Kolk JH. Anaplasma phagocytophilum infection in horses in the Netherlands. Vet Rec 2008;162:216-7. [12] Lepidi H, Bunnell JE, Martin ME, Madiga JE, Stuen S, Dumler S. Comparative pathology and immunohistology associated with clinical illness after Ehrlichia phagocytophilum group infections. Am J Trop Med Hyg 2000;62:29-37. [13] Franzèn P, Aspan A, Egenvall A, Gunnarsson A, Aberg L, Pringle J. Acute clinical, haematological, serologic, and polymerase chain reaction findings in horses experimentally infected with a European strain of Anaplasma phagocytophilum. J Vet Intern Med 2005;19:232-9. [14] Diana A, Guglielmini C, Candini D, Pietra M, Cipone M. Cardiac arrhythmias associated with piroplasmosis in the horse: a case report. Vet J 2007;174:193-5. [15] Klein MB, Hu S, Chao CC, Goodman JL. Antibiotic susceptibility of the newly cultivated agent of human granulocytic ehrlichiosis: promising activity of quinolones and rifamycins. Antimicrob Agents Chemother 1997;41:76-9. [16] Horowitz HW, Hsieh TC, Aguero-Rosenfeld ME, Kalantarpour F, Chowdhury I, Wormser GP, et al. Antimicrobial susceptibility of Ehrlichia phagocytophila. Antimicrob Agents Chemother 2001;45:786-8. [17] Maurin M, Abergel C, Raoult D. Antibiotic susceptibilities of Anaplasma (Ehrlichia) phagocytophilum strains from various geographical areas in the United States. Antimicrob Agents Chemother 2003;47:413-5. [18] Branger S, Rolain JM, Raoult D. Evaluation of antibiotic susceptibility of Ehrlichia canis, Ehrlichia chaffeensis, and Anaplasma phagocytophilum by Real-Time PCR. Antimicrob Agents Chemother 2004;48:4822-8. [19] Zweygarth E, Ahmed JS, Rehbein G. The effect of halofuginone, Wellcome 993 C, oxytetracycline, and diminazene diaceturate on Babesia equi-infected lymphoblastoid cell cultures. J Parasitol 1984;70:542-4. [20] Artursson K, Gunnarsson A, Wikström UB, Engvall EO. A serological and clinical follow-up in horses with confirmed equine granulocytic ehrlichiosis. Equine Vet J 1999;31:473-7. [21] Van Andel AE, Magnarelli LA, Hiemer R, Wilson ML. Development and duration of antibody response against Ehrlichia equi in horses. J Am Vet Med Assoc 1998;212:1910-4. [22] Stuen S. Anaplasma phagocytophilumdthe most widespread tick borne infection in animals in Europe. Vet Res Commun 2007; 31(Suppl 1):79-84.
Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Short Communication
Seroconversion for Anaplasma phagocytophilum in a Mare with Concomitant Piroplasmosis Elisabetta Giudice DVM, PhD a, Claudia Giannetto DVM, PhD b , Alessandra Torina BS c, Pietro P. Niutta DVM a a b c
Dipartimento di Sanità Pubblica Veterinaria, Università degli Studi di Messina, Polo Universitario dell’Annunziata, Messina, Italy Dipartimento di Scienze Sperimentali e Biotecnologie Applicate, Università degli Studi di Messina, Polo Universitario dell’Annunziata, Messina, Italy Centro di Referenza Nazionale per Babesia, Anaplasma, Rickettsia, Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri”, Palermo, Italy
a b s t r a c t Keywords: EGA Theileria equi TBD Horse Limbs edema Tetracyclines
In this article, we describe the clinical course of a 3-year-old purebred mare at the third month of gestation found to be positive for Theileria equi (indirect immunofluorescence antibody test and polymerase chain reaction) at the time of the first evaluation. At the clinical examination, the mare showed profound depression, pale mucous membranes, enlarged lymph nodes, febrile temperature, hind limbs edema, and swelling of the right carpus. Anemia and thrombocytopenia were also observed. Because of worsening of clinical signs, treatment with oxytetracycline (6 mg/kg) was started. An improvement of clinical conditions was observed within 12 hours, with a full recovery in 2 weeks. Two months after the first hospitalization, new serological tests showed seroconversion to Anaplasma phagocytophilum. In conclusion, the severity of clinical signs was attributed to the concomitant infections, usually subclinical in Sicily. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Equine granulocytic anaplasmosis (EGA) is a rickettsial disease of horses that was first reported in the late 1960s in California. The causative agent is Anaplasma phagocytophilum, member of the A phagocytophilum complex, and has since long been recognized as a worldwide tick-borne agent for several species of wild and domestic mammals, including rodents, carnivores, equids, and ruminants. They are obligate intracellular bacteria that reside within vacuoles in the cytoplasm of granulocytotropic cell lines. In horses, EGA is recognized as an acute disease with an incubation period of <14 days, characterized by high fever, lethargy, inappetence, staggering or ataxia, distal limb edema, petechiation, icterus, and hematological alteration
Corresponding author at: Claudia Giannetto, DVM, PhD, Dipartimento di Scienze Sperimentali e Biotecnologie Applicate, Facoltà di Medicina Veterinaria, Università degli Studi di Messina, Polo Universitario dell’Annunziata, Messina 98168, Italy. E-mail address: [email protected] (C. Giannetto). 0737-0806/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2011.02.004
such as thrombocytopenia, neutropenia, lymphopenia, and mild anemia [1-3]. A recent report documented sudden death in a horse associated with experimental infection with the bacterium [4]. Horses aged <4 years tend to develop a milder form of the disease, whereas in those aged <1 year, the clinical signs may be difficult to recognize [5]. Horses experimentally infected by A phagocytophilum develop clinical manifestations that are either typical of natural virulent disease when infected with low-passage bacteria, or with significantly diminished clinical signs and laboratory features when infected with high-passage bacteria [6]. In naturally infected horses, immunity persists for a minimum of 2 years and does not seem to depend on latent infection or carrier status [5]. Equine piroplasmosis is a tick-borne intraerythrocytic protozoal disease caused by Theileria equi and Babesia caballi, or even by a mixed infection. Piroplasmosis is endemic to most tropical and subtropical areas and seems to be spreading to more temperate zones. Clinical signs of the disease are not pathognomonic and may vary considerably between horses. Affected animals may exhibit apathy, lethargy, pyrexia, anemia, icterus, hemoglobinuria,
186
E. Giudice et al. / Journal of Equine Veterinary Science 31 (2011) 185-187
enlarged lymph nodes, weight loss, and edema of distal limbs, leading to, in some cases, death. Subclinical infections may occur commonly in horses living in endemic areas. The presence of both parasites may also persist in clinically recovered horses for a minimum of several years, thus acting as reservoirs for subsequent infecting ticks [7]. 2. Clinical Case A 3-year-old purebred English mare at the third month of gestation was referred to the Faculty of Veterinary Medicine of Messina University (Italy), with a history of lack of appetite, slight weight loss, depression, and reluctance to move since a week. The mare lived in a stable declared free of equine infectious anemia. Because of a progressive worsening of clinical manifestation during the last 2 days, the referred veterinarian decided to administer oxytetracycline (Pan Terramicina, Pfizer, Italy) intravenously at a dosage of 6 mg/kg without performing laboratory tests, although its use in pregnant mares may be discouraged [8]. Six hours later, the horse was admitted to our clinic. At the clinical inspection, the horse was found to be profoundly depressed, the mucous membranes were extremely pale, lymph nodes were enlarged, and heart and respiratory rates were slowly increased. The horse at this time had a rectal temperature of 39.5 C. Hind limbs edema and swelling of the right carpus were both observed. Carpus was also painful and hot. The horse showed stiff movement, almost like she did not want to move, and manifested right forelimb lameness. The mare had not displayed similar clinical signs before the acute onset of this episode. Differential diagnosis based on clinical findings was tick-borne diseases (TBDs), viral arteritis, equine adenitis, and septic arthritis. Blood samples were collected from the animal by jugular vein puncture into vacutainer tubes (Terumo Corporation, Japan) containing ethylenediamine tetraacetic acid and without anticoagulant. Using the ethylenediamine tetraacetic acid sample, emocromocytometric test was assessed by means of an automated hematology analyzer (HecoVet C, SEAC, Florence, Italy). Blood samples with no additives were centrifuged at 3,000 rpm for 10 minutes and sera separated in aliquots. Using one aliquot, biochemical parameters were assessed by means of an ultraviolet spectrophotometer (SEAC, Slim, Florence, Italy); the other aliquots were stored at 20 C pending further analysis. Fine-needle aspiration biopsy of the submandibular lymph nodes and carpal arthrocentesis were performed. Bacterial culture from lymphonodular and synovial specimens was done. Abnormal laboratory findings included low hematocrit (19.7%), low red blood cell count (4.46 M/mL), low hemoglobin level (6.8 g/dL), and thrombocytopenia (35.00 K/mL). Biochemical parameters were within the physiological range and no bacterial colonies growing on culture medium were observed. Samples included in the study (blood, serum, lymph nodes, and synovial fluid) were supplied to the Centro Nazionale di Referenza per Anaplasma, Babesia, Rickettsia e Theileria at the Istituto Zooprofilattico Sperimentale della Sicilia. Indirect immunofluorescence antibody test (Fuller
Laboratories, Fullerton, CA) and polymerase chain reaction (Amersham, Piscataway, NJ) for T equi, B caballi, and A phagocytophilum were performed, as described previously [9]. At the same time, the sample was tested for equine viral arteritis by means of seroneutralization on RK13 confluent cell monolayer. An improvement of clinical conditions was observed within 12 hours after the first tetracycline injection. After 24 hours, the horse showed normal appetite and sensory function, and rectal temperature was 37.5 C. Reduction of hind limbs edema and carpal swelling were also observed. Tetracycline treatment was continued once a day for 7 days. Positive serological tests were found for T equi and B caballi, no antibodies were found for A phagocytophilum. T equi deoxyribonucleic acid was found in blood, lymph nodes, and synovial fluid. Imidocarb dipropionate, the drug of choice for piroplasmosis, was not administered because clinical improvement had occurred and its parasympathetic effect could compromise the gestation state of the horse. Clinical signs had completely resolved 20 days after the first treatment and did not relapse. At this time, all hematological parameters were within the physiological range. Two months after the first hospitalization, a new serological test was conducted. T equi antibodies were confirmed, B caballi antibodies were not found, and antibodies for A phagocytophilum were observed. The owner confirmed that the horse had no further clinical signs in the next 6 months. The mare delivered a living and viable full-term foal without any discoloration of teeth. 3. Discussion The mare developed the infection in June, which is comparable with the seasonal peak of the vector seen in Sicily. This horse was native to this region and had not travelled outside the island. Native animals in Sicily are often infected by T equi and B caballi, but do not exhibit any sign of disease. The everlasting presence of the parasites induces an efficient immune response in the habitual host. Subclinical anaplasmosis may be common in endemic areas, based on studies of horse farms where a single case of clinical A phagocytophilum may occur yearly and still nearly 50% of horses on that farm had evidence of antibody to A phagocytophilum [10]. In most horses, EGA recovery occurs spontaneously and thus does not attract the attention of clinicians [11]. Viral arteritis, equine adenitis, and septic arthritis were excluded by laboratory findings. The controversy on the diagnosis was attributed to the absence of pathognomonic symptoms of TBD symptoms and the presence of multiple infections. Hypertrophy and inflammation of the lymph nodes are more pronounced in the case of T equi, and have not been observed in EGA. Edema owing to a proliferative and necrotizing vasculitis of small arteries and veins in the skeletal muscle and tendons of legs has been commonly described in horses suffering from EGA [12] and less frequently observed during piroplasmosis. Limb edema is generally the most delayed clinical signs in horses experimentally infected with A phagocytophilum [13]. It occurs in 83% of naturally infected
E. Giudice et al. / Journal of Equine Veterinary Science 31 (2011) 185-187
horses [11] and its frequency is lower if prompt treatment is established [3]. In our case, limb edema appeared a week after the onset of clinical signs but regressed suddenly after tetracycline administration. Thrombocytopenia and varying degrees of anemia are typically found both during EGA [3,11] and piroplasmosis [14]. Although at the first evaluation, positive serological tests were found for T equi and B caballi, and no antibodies were found for A phagocytophilum, the improvement of clinical signs after oxytetracycline administration suggested EGA infection. In fact, A phagocytophilum is susceptible to tetracyclines [15-18], whereas B caballi is not susceptible and T equi is susceptible only during the pre-erythrocytic phase [19]. The high level of suspicion for EGA was confirmed by the seroconversion observed 2 months after the infection. In the early phase of infection, antibodies production against A phagocytophilum is not detectable, and 50% of infected horses are negative on serological test [20]. Cell-mediated and humoral immune responses are characterized by the production of immunoglobulin G, which became serologically detectable 19 days after the infection date, reached their peak approximately 8 weeks after the infection, and tend to become serologically negative, unless reinfections intervene [5,21]. Seropositivity for B caballi was probably because of a past dated infection. This hypothesis was confirmed by the seroreversion observed 2 months after the clinical onset. The absence of intragranulocytic inclusions was not in disagreement with EGA suspicion. Morulae or elementary bodies are present for only 3 to 7 days and the degree of bacteremia is variable [1]. A phagocytophilum deoxyribonucleic acid was intermittently detected in experimentally infected horses [1,13] and this could explain the negative polymerase chain reaction results, even though the degree of bacteremia seems to be related to the clinical signs [22]. In addition, tetracycline treatment, initiated several hours before our sampling, could have altered microscopic and molecular assays. In conclusion, the severity of clinical signs was attributed to the concomitant presence of both pathogens. Piroplasmosis may have worsened A phagocytophilum infection, which in horses aged <4 years has a generally less severe course. Conversely, A phagocytophilum may have exacerbated the clinical signs of piroplasmosis, usually asymptomatic in this area, which again became subclinical after the EGA treatment. The concomitant infection of piroplasms and A phagocytophilum probably is a common event in Sicily. The clinical signs are usually attributed to an acute piroplasmosis, treated with imidocarb dipropionate. Because of this drug efficacy for both pathologies, EGA could be underestimated. This aspect would remind practitioners of the need to consider the possibility of more than one infectious agent when evaluating similar cases and their treatment.
187
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