clinical evolution of naturally infected and treated (meglumine antimoniate plus allopurinol) dogs. Canine leishmaniasis is a widespread disease in all Med-.
Am. J. Trop. Med. Hyg., 61(4), 1999, pp. 652–653 Copyright q 1999 by The American Society of Tropical Medicine and Hygiene
SHORT REPORT: IMPROVED DIAGNOSIS AND FOLLOW-UP OF CANINE LEISHMANIASIS USING AMASTIGOTE-BASED INDIRECT IMMUNOFLUORESCENCE ´ NDEZ-PE ´ REZ, S. ME ´ NDEZ, C. DE LA FUENTE, M. T. GO ´ MEZ-MUN ˜ OZ, F. J. FERNA M. CUQUERELLA, AND J. M. ALUNDA Departamento de Patologı´a Animal I, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain
Abstract. A comparison of an indirect immunofluorescence test using promastigotes (IFATp) or cultured amastigotes (IFATa) in the diagnosis and follow-up of canine leishmaniasis caused by Leishmania infantum was carried out. Results obtained with both diagnostic methods were in good agreement although the IFATa titration was more sensitive than the currently used IFATp without losing specificity. The higher sensitivity of the amastigote-based IFAT resulted in an earlier diagnosis in the absence of clinical signs. Both methods showed comparable results for monitoring the clinical evolution of naturally infected and treated (meglumine antimoniate plus allopurinol) dogs. Canine leishmaniasis is a widespread disease in all Mediterranean countries, with an average prevalence of 5–8% in the total dog population and exceeding 30% in some areas.1,2 In human populations, the infection is strongly linked to individuals positive for human immunodeficiency virus.3 Since killing of infected animals is not compulsory, most affected dogs are treated, and success is extremely dependent on early diagnosis. Some diagnostic methods, such as an ELISA4,5 or Western blotting are more sensitive,6,7 but an indirect immunofluorescence test (IFAT) has been found to be suitable for follow-up of recovery after chemotherapy,8 and is by far the test most accepted by veterinary practitioners. Leishmania amastigotes have been identified in canine tissues by immunoperoxidase staining9 and amastigotes of some human Leishmania species have been used in IFAT,10,11 but to our knowledge, there is no information on L. infantum. The results presented here compare an IFAT using promastigotes (IFATp) or amastigotes (IFATa) in the diagnosis of canine leishmaniasis and in the monitoring of dogs treated with a combination of allopurinol and antimonials. Sera from asymptomatic dogs were obtained from the Armed Forces Central Veterinary Depot (Madrid) (no. 1–18) and the Leishmaniasis Diagnosis External Service (Veterinary School, Universidad Complutense de Madrid [UCM], Madrid) (no. 31, 32, 34 and 35). Sera from naturally infected dogs (no. 19–30) were obtained from the Clinical Services of Infectious and Parasitic Diseases (Veterinary School, UCM, Madrid) and a private practitioner (no. 33) (Clı´nica Veterinaria Lobo, Aranda de Duero, Burgos, Spain). After clinical and serologic (IFATp) diagnosis, dogs no. 19–27 were treated with allopurinol plus antimoniate meglumine at different dosages and schedules, according to clinicians’ criteria. Animal No. 20 did not tolerate allopurinol and received only the antimonial drug. Serum samples from these treated animals were obtained at different times after treatment, depending on availability. The IFAT titration with promastigotes (IFATp) was carried out using a local L. infantum strain (MCAN/ES/88/ISS441, DOBA) cultured in Grace’s medium using the procedure described previously;7 the cut-off titer was . 1/100. The IFAT titration with amastigotes (IFATa) was performed with macrophage-like line J774.G8 cells infected with stationary phase promastigotes of the same strain.12 Briefly, cultures were maintained at 338C for 96 hr, washed three times in ice-cold phosphate-buffered saline, and 10 ml of a 1.5 3 105
cells/ml suspension was allowed to settle on immunofluorescence slides for 30 min. Comparison of IFATp and IFATa results in the diagnosis of canine leishmaniasis is shown in Table 1. There was a good agreement, despite some individual variations, between the two IFAT methods and the clinical status of the animals. The IFATa values were comparable with those obtained using promastigotes (IFATp), although a slightly higher (1 serum dilution) was observed in 9 dogs, and a clearly higher response was observed in 4 dogs. All clinically healthy (asymptomatic) dogs (22 animals) had IFATp values below the cut-off titer (. 1/100), whereas two dogs (no. 34 and 35) were positive (1/200). The possibility that these results in asymptomatic animals represented false positive findings was ruled out using the more sensitive Western blot technique in which sera from both animals reacted with some L. infantum antigens. In addition, animal no. 35 was sampled one month later, by which time it had seroconverted (IFATp titer 5 1/200). All animals with clinical manifestations had titers above the cut-off value (. 1/100) in the IFATa, whereas only one of the affected dogs (no. 33) had a titer of 1/50 in the IFATp. After two repetitions of IFATp titration with similar results, a bone marrow aspirate was performed and Leishmania-infected macrophages were found. Variations in sensitivity and specificity of the IFAT in diagnosing some human leishmaniases, depending on the life cycle stage (amastigotes, promastigotes), have been reported.10,11 Our results showed that the IFATa is equally specific and more sensitive than the currently used IFATp in detecting canine leishmaniasis by L. infantum. Nine of the infected dogs (no. 19–27) received chemotherapy (allopurinol plus meglumine antimoniate, no. 19, 21–27 or meglumine antimoniate, no. 20), and some serum samples were available after the initiation of treatment. The IFATp titration has been found to be useful in monitoring the clinical evolution of infected dogs7 and infected dogs that were treated.8 Our results showed that clinical improvement was accompanied by a tendency towards lower IFAT values with both parasite stages (Table 2). Of the 8 treated dogs, 5 showed a four-fold or more decrease in IFATp and IFATa titers (no. 19, 20, 21, 24, and 25) and one showed a twofold decrease (no. 27). These results confirm the value of the IFAT in following-up the treatment of infected dogs,8 and are in contrast with those obtained previously in similarly treated animals (glucantime plus allopurinol).13
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CANINE LEISHMANIASIS DIAGNOSIS AND FOLLOW-UP
TABLE 1 Comparative indirect immunofluorescence test (IFAT) titration with promastigotes (IFATp) and amastigotes (IFATa) of Leishmania infantum Clinical status
IFATp titer
IFATa titer
Asymptomatic
Negative
Negative
Asymptomatic Asymptomatic Asymptomatic Asymptomatic Symptomatic Symptomatic Symptomatic Symptomatic Symptomatic Symptomatic Symptomatic Symptomatic Symptomatic Symptomatic Symptomatic Symptomatic Symptomatic
1/50 Negative 1/50 1/50 1/400 1/400 1/6,400 1/200 1/1,600 1/800 1/6,400 1/6,400 1/800 1/800 1/6,400 1/6,400 1/50
1/50 1/50 Negative 1/200 1/3,200 1/800 1/12,800 1/1,600 1/800 1/800 1/12,800 1/3,200 1/1,600 1/3,200 1/6,400 1/3,200 1/200
Dog number
1, 3, 6, 8, 10–14, 17, 18 4, 7, 9, 32 5, 15, 31 2, 16 34, 35 19 20 21 22 23 24 25 26 27 28 29 30 33
We conclude that IFATa is overall a more sensitive method than the IFATp for diagnosing canine leishmaniasis without loss of specificity. This increased sensitivity allows earlier diagnosis than the IFATp in the absence of clinical signs, and could be used advantageously to start chemotherapeutic treatment. The value of the IFAT in monitoring the clinical evolution of infected and treated dogs is, at least, comparable to that of the IFATp. TABLE 2 Comparative indirect immunofluorescent test (IFAT) titration (IFATp and IFATa) of Leishmania infantum–infected dogs during combined (allopurinol plus meglumine antimoniate) treatment Dog number
Days after treatment
IFATp titer
IFATa titer
19 19 19 20 20 20 21 21 21 21 22 22 22 23 23 24 24 25 25 26 26 26 26 27 27
0 84 135 0 105 285 0 41 69 139 0 46 142 0 193 0 129 0 69 0 76 237 302 0 85
1/400 1/400 1/50 1/400 1/400 1/100 1/6,400 1/6,400 1/800 1/100 1/200 1/400 1/400 1/1,600 1/400 1/800 1/50 1/6,400 1/1,600 1/6,400 1/400 1/6,400 1/800 1/800 1/400
1/3,200 1/1,600 1/50 1/800 1/400 1/200 1/12,800 1/3,200 1/1,600 1/50 1/1,600 1/3,200 1/1,600 1/800 1/800 1/800 1/50 1/12,800 1/800 1/3,200 1/200 1/12,800 1/6,400 1/1,600 1/800
Acknowledgments: We thank Dr. G. Miro´, C. Rupe´rez, C. Fraile, and P. Sagredo (Clinical Services of Infectious and Parasitic Diseases, Veterinary School, UCM), and Major J. L. Iglesias, and Captains J. A. Ferna´ndez-Martı´n and J. L. Arceiz (Armed Forces Central Veterinary Depot, Madrid, Spain) for providing some of the canine sera. We also thank C. Mark for reviewing the manuscript. Financial support: This work was partially supported by grant 08.2/ 0006/1997 (Comunidad de Madrid). F. J. Ferna´ndez-Pe´rez was the recipient of an Oficina de Transferencia Resultados de Investigacio´n–UCM fellowship. Authors’ addresses: F. J. Ferna´ndez-Pe´rez, S. Me´ndez, C. de la Fuente, M. T. Go´mez-Mun˜oz, M. Cuquerella, and J. M. Alunda, Dpto. Patologı´a Animal I, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain. REFERENCES
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