Efficacy of Different Treatment Regimens of Marbofloxacin In

Available online at www.sciencedirect.com

Veterinary Parasitology 153 (2008) 244–254 www.elsevier.com/locate/vetpar

Efficacy of different treatment regimens of marbofloxacin in canine visceral leishmaniosis: A pilot study Sandrine Rougier a,*, Ioannis Vouldoukis b, Sandrine Fournel a, Sylvie Pe´re`s a, Fre´de´rique Woehrle´ a a Ve´toquinol Laboratories, BP 189, 70204 Lure, France b INSERM U511, Immunobiologie cellulaire et molećulaire des infections parasitaires, Universite´ Paris VI, CHU-Pitie´ Salpe´trie`re, 75013 Paris, France Received 6 September 2007; received in revised form 17 January 2008; accepted 22 January 2008

Abstract This phase II, randomized, open-label field trial was designed to evaluate and compare the safety and efficacy of four treatment durations (10, 20, 28 or 40 days) with marbofloxacin administered orally at the dosage of 2 mg/kg once a day for canine visceral leishmaniosis. Twenty-four dogs naturally infected with visceral leishmaniosis and without biochemical disorder evidences of renal insufficiency, were recruited by two Greek veterinarian clinics. They were also randomly assigned to one of the four treatment duration groups, and have been clinically, haematologically, biochemically and parasitologically followed-up regularly for 9 months. Efficacy was achieved for 5/6 dogs treated for 28 days, 4/6 dogs treated for 10 or 20 days and for 3/6 dogs treated for 40 days. Moreover, efficacy was reached more quickly (58.4 days) in dogs treated for 28 days. Improvement of clinical signs tended to be better and faster in the 28 days treatment group too. After 9 months of follow-up, a total of three cases could be considered as relapsing (two dogs treated for 40 days and one dog treated for 28 days). There was a significant reduction in amastigotes density in macrophages after 3 months in the four groups when compared with the parasite density at inclusion. No adverse effects were noticed during this 9 months study. Results obtained with marbofloxacin at the dosage of 2 mg/kg once a day for 28 days seem encouraging and may offer a safe alternative for treating canine visceral leishmaniosis. # 2008 Elsevier B.V. All rights reserved.

Keywords: Marboﬂoxacin; Visceral leishmaniosis; Dogs; Efﬁcacy; Treatment duration

1. Introduction around the world (WHO, 2002). In the Mediterranean region, including Southern France, Italy, Greece, Spain, Leishmaniosis is an infectious disease caused by a Portugal and Northern Africa, Leishmania infantum is protozoan parasite of the genus Leishmania that is an important species in producing both human and transmitted by the bite of phlebotomine sand ﬂy vectors. animal visceral leishmaniosis. In the United States, In 2001, the World Health Organization (WHO) canine visceral leishmaniosis (CVL) due to L. infantum estimated that there were «2.4 million disability- recently emerged (Rosypal et al., 2003, 2005a,b). The adjusted life years» and 59,000 deaths in 88 countries dog is the domestic reservoir of human visceral leishmaniosis caused by L. infantum. Leishmaniosis is always a serious and life-threatening disease, both for * Corresponding author. Tel.: +33 384625592; fax: +33 384625500. humans and dogs. Domestic dogs and humans affected E-mail address: [email protected] (S. Rougier). with leishmaniosis are often treated with the same or

0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.01.041 S. Rougier et al. / Veterinary Parasitology 153 (2008) 244–254 245 related drugs, such as meglumine antimoniate or a trend in an optimal treatment duration of marbo- sodium stibogluconate. Moreover, due to the serious- floxacin in the treatment of CVL. Efficacy and safety of ness of the disease, the prolonged inconvenient the product administered once a day at the registered administration, drug toxicity and high costs of products, dosage (for other indications) of 2 mg/kg for 10, 20, 28 euthanasia is often the final outcome for dogs. The and 40 days, to dogs naturally infected with visceral parasite’s sensitivity towards the antimonials is further- leishmaniosis, were compared. more tending to decline, even in humans, and relapses are not uncommon. These facts increase the risk of 2. Materials and methods transmission and reduce owner compliance to treatment (Alvar et al., 1994). This emphasises the importance of 2.1. Dogs searching for alternative anti-leishmanial drugs. Marbofloxacin (Marbocyl1,Ve´toquinol, Lure, Twenty-four dogs of both sexes and aged from 2 to 9 France) is a synthetic, third generation fluoroquinolone years were recruited between July and September 2003 developed for veterinary use only. It presents potent to participate in this randomized and comparative pilot activity against several bacterial agents and is fre- study. They presented at two veterinarian clinics in quently used in the treatment of a wide range of gram- Athens (Greece) with clinical signs compatible with positive and gram-negative bacterial infections (Pre- CVL. Dogs were eligible for the study if they met the scott et al., 2000; Meunier et al., 2004). Like other following inclusion criteria: having displayed at least quinolones, marbofloxacin inhibits the bacterial one cutaneo-mucosal sign, lymphadenomegaly, weight enzyme DNA gyrase (or topoisomerase II), but does loss, and parasitologically confirmed CVL from lymph not act on mammalian topoisomerases, due to their node examinations (positive cultures and more than structural differences from the bacterial topoisomerase 50% of infected macrophages). Exclusion criteria were: (Slunt et al., 1996; Prescott et al., 2000). The L. dogs having received antibiotic, corticosteroid and/or donovani type I enzyme resembles bacterial topoi- anti-leishmanial treatment within the 30 days preceding somerase I in its requirement for divalent cations and its inclusion or 2 months in the case of long-acting inability to relax positively supercoiled DNA (Chakra- corticosteroids, presenting with biochemical evidence borty et al., 1993). Trypanosomidae, such as L. of renal and/or liver failure, a concomitant infection (for infantum, present a genomic structure which exhibits example ehrlichiosis, dirofilariosis, demodicosis or major similarities with bacteria (Chakraborty and mange) or at least one nodule requiring surgical Majumder, 1988; Slunt et al., 1996). Some previous exeresis. Informed consent was obtained from the studies demonstrated inhibition of L. donovani amas- owners of all dogs prior to their participation in this tigote forms with various fluoroquinolones (Raether study. et al., 1989; Furet and Peche`re, 1991). Moreover, fluoroquinolones are known to have immunomodula- 2.2. Treatment tory properties, attenuating cytokine response in particular (Dalhoff and Shalit, 2003); and there is a All dogs were equally randomly distributed to one of long-held assumption that the success of anti-leishma- the four treatment duration groups. They all received nial chemotherapies depends on the potential immu- marbofloxacin tablets (Marbocyl1,Ve´toquinol Labora- nological response of the host, which determines the tories, Lure, France) at the dosage of 2 mg/kg, once a outcome infection (Murray et al., 1989; Alberola et al., day, for 10 (group A), 20 (group B), 28 (group C) or 40 2004). As a result of all these considerations associated days (group D). with a frequent presence of secondary bacterial Use of repellent, insecticide spot-on and collars infections in the skin lesions in animals suffering against phlebotomine sandflies were recommended primarily from leishmaniosis (Koutinas et al., 1993), the during the 9 months study in order to prevent a fluoroquinolone, marbofloxacin, is being tested for leishmanial re-infection. treatment of CVL. Previous in vitro studies demonstrated a direct and indirect leishmanicidal activity of 2.3. Follow-up marbofloxacin via the TNF-a and NO synthase path- ways, and correlated with NO2 production (Vouldoukis All animals were visited at regular intervals for 3 et al., 2006). months, on days 0 (D0), 10 (D10), 20 (D20), 28 (D28), The purpose of the present pilot field clinical study 40 (D40), 56 (D56), 70 (D70) and 84 (D84). Moreover, conducted in Athens (Greece) was aimed at determining a last visit was planned nine months (M9 Æ 2 weeks) 246 S. Rougier et al. / Veterinary Parasitology 153 (2008) 244–254 after inclusion in order to assess a possible clinical used as the negative control. After washing in phosphate relapse. If clinical relapse was suspected before M9, this buffer saline (PBS), cells were incubated for 30 min at last visit could be brought forward at any time between 37 8C with FITC-conjugated rabbit anti-mouse IgG M3 (D84) and M9. (Sigma), diluted in Evans blue (1/1000) for counter- staining, and the slides were examined under a 2.4. Clinical evaluations fluorescence microscope (Monjour et al., 1987). Thirdly, a culture analysis at 28 8C of lymph node At each examination, dogs were assessed for these aspiration was performed, in order to detect the clinical signs: presence of Leishmania parasites. Lymph node samples were transferred into 45 ml culture flasks (Becton Systemic signs: weight loss, hyperthermia, listless- Dickinson) containing 10 ml of RPMI 1640 medium ness and lymphadenomegaly. supplemented with 2 mM L-glutamine, 1% sodium Cutaneo-mucosal signs: alopecia/lunettes, squamo- pyruvate, 1% amino acids, penicillin and streptomycin sis/hyperkeratosis, seborrhea, pyodermatitis, ulcers, antibiotics (all Gibco, Paisley, UK) and 20% of foetal ulcerated stomatitis, epistaxis, onychogryphosis and calf serum (FCS) (Sera Lab, Sussex, UK) (Vouldoukis nodules. et al., 1986). Cultures were maintained at 28 8C for 12 Musculo-skeletal signs: limping/arthritis and amyo- days and regularly examined under inversed micro- trophy; scopy and immersion optical microscopy, to assess the Gastro-intestinal signs: diarrhoea, hepatomegaly, potential of amastigote forms to differentiate into splenomegaly, intestinal haemorrhage; and promastigotes (Neogy et al., 1994). Finally, to evaluate Ocular signs: conjunctivitis, keratitis, uveitis. Leishmania sensitivity or resistance to marbofloxacin in vitro at D0 and D84, the 030-D canine cells line (Pinelli Each clinical parameter was classified according to et al., 2000) were cultured in RPMI-1640 with 10% FCS its severity on a numerical scale from 0 to 3, as follows: complete medium. They were then infected with 0, absent; 1, mild; 2, moderate and 3, important, except cultured promastigote parasites from the dogs’ lymph lymphadenomegaly which was scored as 0, absent; 1, node in eight chamber Lab Tek tissue culture slides local and 2, generalised. (Nunc) at a parasite cell ratio of 3:1 at 37 8C and under a In order to assess the need of a complementary 5% CO2 atmosphere. After 48 h of incubation, cells treatment for ocular lesions, ocular signs were only were washed and 100 mg/ml dilution of marbofloxacin followed-up during the study. They did not have been were added in wells containing canine cells previously taken into account for efficacy evaluation. infected by dogs’ parasites (parasites from D0 and D84 samples). After 72 h of exposure to a single drug 2.5. Parasitological examinations concentration, cells were washed, fixed with methanol and stained with Giemsa. The parasite load, expressed In all animals included in this study, popliteal lymph as a percentage according to infected cells and number node aspirate was performed for different parasitolo- of amastigotes, was compared with that of untreated gical laboratory assays, on D0 and at each examination controls on D0 and on D84 (Sereno et al., 2001). time up to D84 except on D20. Firstly, for direct On M9 visit, or at any time during the period D84- parasitological examination, smears were prepared, M9, only dogs which were clinically suspected of being fixed in methanol and Giemsa stained. The presence of in relapse had to be parasitologically examined again, as infected macrophages (%) in 500 macrophage cell previously described. counts and the number of intact parasites per 100 macrophage cells were microscopically determined 2.6. Haemato-biochemical tests (Neogy et al., 1994). Secondly, to confirm the presence of infected macrophages and the number of amasti- All dogs were checked for complete blood count gotes, other smears were fixed for 10 min in acetone and (granulocyte count, lymphocyte count, platelet count, were incubated for 30 min with monoclonal antibodies haemoglobin concentration, mean corpuscular haemo- (MAbs) against infected macrophages (clone H35, mice globin concentration (MCHC), packed cell volume IgG1 isotype) and amastigote forms (clone H19, mice (PCV), and white blood cells count (WBC)), liver IgG1 isotype). The MAbs (clone H0, mice IgG1 function tests (ASAT and total proteins) and renal isotype) produced against Leishmania non-infected function tests (urea and creatinine), on D0, D28, D56 canine macrophages (INSERM unit collection), were and D84. Only dogs suspected of being in relapse on M9 S. Rougier et al. / Veterinary Parasitology 153 (2008) 244–254 247 were sampled again. Haemato-biochemical analyses the frequencies and percentages for each class for the were performed at each veterinarian practice. scores and other qualitative variables. For the main criterion (time to efficacy), the four 2.7. Efficacy criteria treatment groups were compared using a 1-factor (group) analysis of variance, followed, if there was a The therapeutic protocol was classified as effective significant group effect, by a Newman–Keuls test. The if, between D0 and D84: 2-sided 95% confidence interval of the mean of the four groups was calculated. The dog became asymptomatic (possibly except for ocular signs) and its weight increased. 3. Results Parasites could not be obtained from lymph node material cultures. 3.1. Dogs’ characteristics

The treatment was considered as partially effective if A total of 24 dogs were enrolled (6 per group). The the dog showed an improvement in at least half of the majority of them were crossbreeds (14/24–58.3%), clinical parameters initially affected on D0, associated male (64.9%) and of medium size (Table 1). Thirteen with failure of parasite cultures from lymph node. dogs (from 2/6 in group A to 5/6 in group C) had The treatment was classified as ineffective if less previously presented with one to four episodes of than half of the initial clinical signs had improved and/ leishmaniosis, treated in each case with meglumine or at least one clinical sign had deteriorated and/or the antimoniate at the dosage of either 150 mg/kg once a parasite cultures from lymph node succeeded. day or 75 mg/kg twice a day, for one month, except for We also analysed the relapse rate. Clinical relapse of one dog (group C) which was treated with allopurinol leishmaniosis was evaluated at least on M9 for all dogs. only, at the dosage of 10 mg/kg/day for 3 months. It was defined as an initial therapeutic response Fifteen months (Æ11.4 months) separated the end of the (efficacy or partial efficacy) followed by the reappear- last anti-leishmanial treatement and the first adminis- ance of clinical signs, and possibly confirmed by tration of marbofloxacin. Leishmanial relapse occurred parasitological exams. within 2–45 months after the end of the last treatment. The frequency of adverse effects attributable to the treatment was calculated for each group. 3.2. Physical examination on D0 (Fig. 1)

2.8. Statistical analysis All dogs presented at the inclusion with a slight weight loss (<25% of the normal weight), except one A descriptive analysis was given for all the variables dog with a marked weight loss, and lymphadenomegaly concerning the included population and the secondary diagnosed as generalised for the majority of them criteria, including the mean, standard deviation, (83%). Splenomegaly and seborrhea were diagnosed in minimum and maximum for the quantitative variables, 21 (87.5%) dogs. With seborrhea, onychogryphosis

Table 1 Characteristics of dogs included in the study before treatment with marboﬂoxacin (Marbocyl1) Treatment groups ABCD Age (years) Median 5445 Range 3–9 2–7 2.5–6 3–9 Sex (male/female) 4/2 4/2 3/3 4/2 Body weight (kg) Median 27.8 38.7 31.2 33.2 Range 18–52 20–52 19–37 19–42 Previous episodes (yes/no) 2/4 3/3 5/1 3/3 Relapse rank (1st/2nd/3rd/4th) 1/1/0/0 2/1/0/0 4/1/0/0 2/0/0/1 248 S. Rougier et al. / Veterinary Parasitology 153 (2008) 244–254

Fig. 1. Clinical signs observed on D0 according to the four treatment duration groups.

(79.2%), squamosis and hyperkeratosis (50%), alopecia required by the protocol. In vitro, sensitivity of and/or ‘‘lunettes’’ (54.2%) and pyodermatitis (25%) amastigotes to marbofloxacin was quite good in the were the most common skin lesions; only one dog (in four groups with a percentage of parasitic index group D) presented with skin ulcers. Nodules had never inhibition of over 80% in the majority of cases (18/ been diagnosed. Listlessness (79.2%), limping (62.5%), 24: 5 cases each in groups A, B and C, and 3 cases in diarrhoea (37.5%) and amyotrophy (20.8%) were also group D). well-represented in our dog population. Hepatomegaly, Thus, according to all characteristics at inclusion, epistaxis and stomatitis were observed in only a few dogs were statistically considered as equally distributed animals. Eye symptoms were observed in 10 dogs among the four groups. (41.7%) including one case with conjunctivitis, one case with keratitis and 8 cases with both conjunctivitis and 3.4. Response to treatment protocols and time to keratitis. Uveitis was present in five dogs and combined efficacy in each case with keratitis and conjunctivitis. The severity of ocular lesions varied but the most severe Efficacy was obtained for five dogs treated for 28 lesions were seen in group D, statistically considered days, for four dogs treated for 10 or 20 days, and for without any significance compared to the other groups. three dogs treated for 40 days. Moreover, clinical condition of one dog, treated for 20 and 40 days, 3.3. Laboratory findings and parasitological data respectively, regularly improved during the study. Thus, on D0 the treatment was ineffective in only one case in groups B and C and in two cases in groups A and D. A trend to a All included dogs presented biochemical values better time to efficacy was revealed for dogs treated for within the normal range. Six dogs had a high 28 days (58.4 days), closely followed by dogs treated lymphocyte count (>40%), two had a low PCV rate for 20 days (59.5 days, not statistically significant, (<35%), two a low platelet count (<2 Â 1011/L) and p = 0.95). one dog a high WBC (>20 Â 109/L); MCHC, Considering dogs which already had previous haemoglobin and granulocyte count were in the normal episode(s) of leishmaniosis or not, we observed a range for all dogs. statistically significant better response when marbo- In the four compared groups, a mean of about 300 floxacin was used as second or third treatment. Indeed, amastigotes per 100 infected macrophages was found, of the 16 dogs for which the treatment was efficient, and all cultures of promastigotes were positive as twelve (75.0%) had ever received at least one previous S. Rougier et al. / Veterinary Parasitology 153 (2008) 244–254 249 anti-leishmanial treatment ( p = 0.008). Moreover, time M9 for dogs in group A. Three dogs in this group were to efficacy showed a difference between these two clinically cured on M9 and one still presented with populations and was reached in a mean of 57 days for ocular lesions only. A slight improvement was seen dogs already treated for leishmaniosis versus 73.5 days between D84 and M9 for dogs in group D: two of them for dogs for whom marbofloxacin was the first anti- were clinically cured on M9 and two presented with leishmanial treatment ( p = 0.15). ocular lesions only. Marbofloxacin treatment had a moderate effect on 3.5. Clinical parameter development ocular lesions. Indeed, of the 10 dogs which presented with ocular signs on D0, half of them had no ocular A clinical improvement to treatments was visible for signs on D84 (all dogs from groups B and C and one dog veterinarians only after D20 and particularly on skin from groups A and D), four dogs showed an lesions, regardless of the duration of marbofloxacin improvement in their ocular lesions and one (in group treatment. Owners reported an appetite recovery less A) showed a deterioration of these signs. The first than 10 days after the beginning of the treatment. Body ophthalmological improvements were observed from weight increased in the four groups during the whole D20 but improvement progressed slowly during the study, but the difference of mean body weight gain study. Uveitis was the most difficult sign to improve. between the four groups at the end of the follow-up was To summarize, it appeared that animals treated for 20 not statistically significant ( p = 0.996). Listlessness or 28 days with marbofloxacin firstly clinically disappeared on D28 for all dogs from groups C and D improved and presented concomitantly with the highest and on D40 for dogs from group B. Lymphadenome- rate of clinical cure. Thus, according to clinical galy, onychogryphosis and splenomegaly were symp- considerations only, 20 or 28 days treatment duration toms which took the longest time to improve. appears to be more effective than 10 or 40 days Disappearance of clinical signs on D84 was particularly treatment duration. visible in groups B and C (Fig. 2). At the last visit performed on M9, clinical signs continued to disappear, 3.6. Parasitological changes (Fig. 4) particularly in groups B and C (Fig. 3). Of these 12 dogs, four were clinically cured on M9 in each group An important decrease in the number of amastigotes and one presented only with a slight weight loss in each was visible in the four treatment groups, from a mean of group. Apart from a reappearance of pyodermatitis in 300 parasites per 100 macrophage cells on D0 to 30 one case on M9, no variation was seen between D84 and ones on D84. A trend to a better decrease was seen in

Fig. 2. Clinical signs observed on D84 according to the four treatment duration groups. 250 S. Rougier et al. / Veterinary Parasitology 153 (2008) 244–254

Fig. 3. Clinical signs observed on M9 according to the four treatment duration groups. groups B and C, whereas no statistical difference directly correlated with the clinical and parasitological between groups was found on D84 (Fischer exact test, results previously described. All parasites from dogs in p = 0.194). Moreover, first negative cultures of pro- treatment failure or in relapse were less susceptible to mastigotes were found on D28 in the four treatment marbofloxacin, both on D0 and D84, as compared with groups. parasites from dogs which responded. Interestingly, As on D0, susceptibility testing to marbofloxacin on these six dogs presented with a leishmanial episode for D84 revealed six out of 24 dogs with 80% or less of the first time, i.e. they had never received any anti- inhibition of amastigote forms, and concerned the same leishmanial treatment before marbofloxacin. All dogs dogs between the two dates (Table 2). These results are which presented amastigotes with a good susceptibility

Fig. 4. Decrease in the number of amastigotes per 100 infected macrophage cells observed under a microscope and induced by four different treatment durations with marboﬂoxacin at 2 mg/kg/day. S. Rougier et al. / Veterinary Parasitology 153 (2008) 244–254 251

Table 2 D84: clinical signs were still present in these two dogs Percentage of parasitic index inhibition assessed at D0 and D84 after a after 3 months. 72 h treatment with marbofloxacin at the dosage of 100 mg/ml Treatment group No. of dogs Percentage of parasitic 3.8. Safety considerations and treatment index inhibition evaluations D0 D84 A188Æ 387Æ 4 No adverse effect was noted during the whole 284Æ 586Æ 2 follow-up. 583Æ 382Æ 3 Tablets appetence was considered as satisfactory. 13 84 Æ 387Æ 2 Owners appreciated the ease with which treatment was 15 84 Æ 482Æ 4 administered with marbofloxacin tablets. 16 80 Æ 375Æ 3 B383Æ 485Æ 3 4. Discussion 785Æ 484Æ 2 12 88 Æ 390Æ 3 18 81 Æ 482Æ 3 In this preliminary study, we compared four 21 90 Æ 387Æ 4 treatment durations with a third generation fluoroqui- 23 79 Æ 480Æ 3 nolone, marbofloxacin, in canine visceral leishmaniosis C687Æ 489Æ 4 (CVL). This pilot study was aimed at finding a trend in 992Æ 494Æ 2 the optimal treatment duration of the product. To be 10 75 Æ 477Æ 3 included, dogs had to present with various clinical signs 19 87 Æ 390Æ 4 compatible with CVL. The diagnosis also had to be 22 90 Æ 392Æ 3 24 82 Æ 386Æ 3 parasitologically confirmed. The traditional drug used to treat CVL is meglumine D483Æ 285Æ 4 1 887Æ 386Æ 4 antimoniate (Glucantime ), nowadays often combined 11 89 Æ 388Æ 3 with allopurinol (Ginel et al., 1998; Denerolle and 14 77 Æ 274Æ 3 Bourdoiseau, 1999; Noli and Auxilia, 2005). Whereas 17 73 Æ 467Æ 3 relapses are common with meglumine antimoniate, 20 76 Æ 372Æ 2 allopurinol tends to increase the time to relapse significantly (Ginel et al., 1998; Cavaliero et al., 1999; Denerolle and Bourdoiseau, 1999; Noli and to marbofloxacin on D0 remained susceptible on D84. Auxilia, 2005). However, this combined treatment is No resistance development appeared after marboflox- expensive: allopurinol is administered for a long time, acin treatment, regardless of the duration of treatment. daily injections of meglumine antimoniate are needed and adverse effects are not rare. An orally safe treatment 3.7. Relapse rate administered for a short time appears to be the treatment of choice. Some products currently used to treat CVL Between D0 and D84, 3 dogs relapsed (12.5%): one try to correspond to this scheme, such as the in group C and 2 in group D. The dog treated 28 days combination of spiramycin and metronidazole (Pennisi was cured on D28 but was clinically relapsing on D56; et al., 2005) or miltefosine (Miro et al., 2005). Another however, no parasitological relapse was observed for fluoroquinolone, enrofloxacin, has recently been tested this case. One dog from group D relapsed on D84. The as a single treatment in leishmaniosis but without any other dog from group D relapsed on D56 but a clinical significant results (Bianciardi et al., 2004), in contrast to cure was noted on M9 whereas no alternative treatment encouraging results previously found for treatment of had been administered. CVL with fluoroquinolones (Raether et al., 1989; Furet All dogs were finally visited on M9: no comple- and Peche`re, 1991). Twelve dogs naturally infected with mentary visit between M3 and M9 was needed. On M9, L. infantum were then treated with enrofloxacin alone at according to investigators, two other cases were the dosage of 20 mg/kg for 30 days. A good clinical suspected of being in relapse based on clinical signs, improvement, particularly in the disappearance of skin and concerned two cases treated for 10 days (group A). lesions (3/6), was reported, but also a relapse rate of Only one case was parasitologically positive on M9. 50%, 60–90 days post-treatment. However, according to the protocol definition, these two It is interesting to note that results observed with dogs considered as relapsing on M9 were not cured on marbofloxacin look to be more promising. In contrast to 252 S. Rougier et al. / Veterinary Parasitology 153 (2008) 244–254 enrofloxacin (Bianciardi et al., 2004), we previously It is interesting to observe that a longer treatment observed in vitro activity on canine infected macro- period (i.e. 40 days) led to a result which is the opposite phages with marbofloxacin, through a TNF-a and nitric of what we expected: insufficient clinical improvement oxide pathway (Vouldoukis et al., 2006). In this study, when compared with a 20 or 28 days treatment duration skin lesions were the first symptoms to disappear, as and two relapses were observed. No explanation can be seen with enrofloxacin (Bianciardi et al., 2004), but it given at present for this observation but only hypothesis seems that the disappearance of all leishmanial lesions can be made. Indeed, we wonder if administering was longer-lasting with marbofloxacin: only three marbofloxacin for too long could block the dog’s relapses were observed during the 9 month study and immunomodulatory system involved in the control of concerned two dogs treated for 40 days and one dog this evolutive pathology (Murray et al., 1989). This treated for 28 days. Unlike enrofloxacin, and in regard hypothesis was put forward in a preliminar study where to results obtained here, we may think that marbo- we observed disappointing results with a too high floxacin, as a single treatment, could be effective dosage of marbofloxacin (at 10 mg/kg/day) (data not against CVL. This study supports the place of shown). But, in group D, amastigotes from half of the marbofloxacin in the treatment of the disease. dogs had less than 80% of susceptibility to marbo- Others treatments have been tested, such as floxacin at inclusion; this was only found for one dog in immunotherapy (Bourdoiseau et al., 2004) or the use each other groups. This difference between group D and of antimicrobial peptides (Alberola et al., 2004), but the others could also partially explain the disappointing complementary data are needed to determine their results obtained with this treatment duration, but further interest in the therapeutic arsenal of CVL. studies with a higher number of dogs are needed to When the four treatment durations were compared, confirm these hypotheses. in terms of efficacy and time to efficacy, no statistically Conversely, the poor results obtained in group A (i.e. significant differences between the groups were found, treated for 10 days) are probably due to an insufficient probably due to the low number of dogs included in duration of treatment, as we previously observed each group. However, a trend towards better efficacy (personal data), but may be also due to the presence was visible in group C: next to a better efficacy rate, the in this group of 4/6 dogs which had never been treated time to efficacy was shorter in this group (about 58.4 for CVL. days) and an advantage in resolution of clinical signs Only cases suspected by veterinarians of being in was seen, particularly for lymphadenomegaly, ony- relapse were lymph node sampled again on M9 for chogryphosis, limping and splenomegaly. Results parasitological tests. However, it would have been obtained in groups B and C were very close and interesting to confirm the clinical cure or the progres- further studies with more dogs are required to establish sion of the clinical improvement with parasitological the optimal treatment duration based on statistical tests on the other dogs. We can hypothesise some considerations. parasitological relapse on M9, without any reappear- Poorer results were observed in this study with dogs ance of clinical signs, but as a sign heralding a future never treated previously for leishmaniosis: efficacy rate clinical relapse, as has already been seen with the usual was significantly lower, time to efficacy was longer, two anti-leishmanial treatments (Poli et al., 1997; Oliva relapse cases concerned dogs which had not been et al., 1998). Nevertheless, more than one year after the treated previously, and lower susceptibility to marbo- end of this study, none of the included dogs considered floxacin was seen in six dogs in this category. This as cured on M3 and M9 clinically relapsed, confirming difference needs however, to be confirmed in further thereby the usefulness of marbofloxacin in this disease studies and on a greater number of dogs too. and its value regarding monotherapy. Ocular lesions disappeared in groups B and C, but It is now well-known that the host immune response these groups were less affected than the two others (not plays a very important role in the efficacy of drugs used statistically significant for all ocular parameters, to treat leishmaniosis (Murray et al., 1989). The p = 0.45 for conjunctivitis, p = 0.64 for keratitis and development and progression of the disease depend p = 0.25 for uveitis). However, although it was not on interactions between the parasite and the immune tested here, and in the absence of demonstrative efficacy system (Alberola et al., 2004). Resistance and suscept- on eye symptoms, we should recommend a comple- ibility to the disease are associated with the develop- mentary local treatment, appropriate for the ocular ment of a Th-1 or Th-2 type response, respectively. It is lesions observed, in order to prevent irreversible ocular interesting to note that, in this study, efficacy was lesions possibly leading to blindness. reached a few weeks after the end of treatment with S. Rougier et al. / Veterinary Parasitology 153 (2008) 244–254 253 marbofloxacin, regardless of the length of treatment. It Acknowledgement could suggest that the immunomodulatory system of dogs took over from chemotherapeutic treatment. While The authors would like to thank Dr. Thomas Svigas there are no data on the immunomodulatory effect of and Dr. Theodoros Bablenis for their active contribution marbofloxacin, quinolones are known to have some to this study. immunomodulatory properties (Dalhoff and Shalit, 2003). Marbofloxacin in vitro leishmanicidal activity References was demonstrated to act through a TNF-a and nitric oxide pathway and correlated with the generation of Alberola, J., Rodriguez, A., Francino, O., Roura, X., Rivas, L., Andreu, D., 2004. Safety and efficacy of antimicrobial peptides NO2 production by macrophages derived from dog against naturally acquired leishmaniosis. Antimicrob. Agents Che- monocytes (Vouldoukis et al., 2006). Considering the mother. 48, 641–643. promising results shown in vitro and the clinical Alvar, J., Molina, R., San Andres, M., Tesouro, M., Nieto, J., Vitutia, improvement in the 24 dogs included, obtained during M., Gonzalez, F., San Andres, M.D., Boggio, J., Rodriguez, F., the first three months of the study in particular, and as no Sainz, A., Escacenas, C., 1994. Canine leishmaniosis: clinical, parasitological and entomological follow-up after chemotherapy. immunological tests have been performed in this study, Ann. Trop. Med. Parasitol. 88, 371–378. we can only hypothesise that the immunological Bianciardi, P., Fasanella, A., Manzillo, V.F., Trotta, T., Aceti, A., activity of marbofloxacin could be comparable to other Pagano, A., Gradoni, L., Oliva, G., 2004. The efficacy of enro- fluoroquinolones and regulate the protective immune floxacin, alone or combined with metronidazole, in the therapy of response. The molecular mechanisms of action still canine leishmaniosis. Parasitol. Res. 93, 486–492. Bourdoiseau, G., Hugnet, C., Papierok, G.M., Lemesre, J.L., 2004. La remain unknown and further studies are necessary to leishmaniose canine a` Leishmania infantum: essais d’immunothe´r- elucidate them. apie. Bull. Acad. Ve´t. France 157, 63–67. Although further complementary studies are needed, Cavaliero, T., Arnold, P., Mathis, A., Glaus, T., Hofmann-Lehmann, R., which will have to be carried out in more investigation Deplazes, P., 1999. Clinical, serologic, and parasitologic follow-up sites, with a greater number of dogs, and possibly with a after long-term allopurinol therapy of dogs naturally infected with Leishmania infantum. J. Vet. Intern. Med. 13, 330–334. randomization based on previous anti-leishmanial Chakraborty, A.K., Majumder, H.K., 1988. Mode of action of penta- treatments(s) versus no previous anti-leishmanial valent antimonials: specific inhibition of type I DNA topoisome- treatment, the fluoroquinolone marbofloxacin, adminis- rase of Leishmania donovani. Biochem. Biophys. Res. Commun. tered at the dosage of 2 mg/kg once a day for 28 days, 152, 605–611. may prove to be a good pragmatic opportunity for the Chakraborty, A.K., Gupta, A., Majumder, H.K., 1993. A type 1 DNA topoisomerase from the kinetoplast hemoflagellate Leishmania treatment of CVL without renal insufficiency. Indeed, donovani. Indian J. Biochem. Biophys. 30, 257–263. marbofloxacin proved to be safe (no adverse effects Dalhoff, A., Shalit, I., 2003. Immunomodulatory effects of quino- reported), clinically efficacious, decreased the parasitic lones. Lancet Infect. Dis. 3, 359–371. load, was easy to administer (tablet form), convenient Denerolle, P., Bourdoiseau, G., 1999. Combination allopurinol and (once a day) and administered for a short time, which antimony treatment versus antimony alone and allopurinol alone in the treatment of leishmaniosis (96 cases). J. Vet. Intern. Med. encourages owner compliance and involves a reason- 13, 413–415. able cost for the majority of owners. All these data Furet, Y.X., Peche`re, J.C., 1991. Newly documented antimicrobial support the place of marbofloxacin in the treatment of activity of quinolones. Eur. J. Clin. Microbiol. Infect. Dis. 10, 249– the disease. Moreover, as marbofloxacin is known to 254. ensure renal safety (Lefebvre et al., 1998a,b), and Ginel, P.J., Lucena, R., Lopez, R., Molleda, J.M., 1998. Use of allopurinol for maintenance of remission in dogs with leishma- although further studies on dogs with renal insufficiency niosis. J. Small Anim. Pract. 39, 271–274. may be necessary, we may think that marbofloxacin Koutinas, A.F., Scott, D.W., Kantos, V., Lekkas, S., 1993. Skin lesions could be efficient and safe for all stages of CVL. in canine leishmaniosis (Kala–Azar): a clinical and histopatholo- In conclusion, our results suggest that marbofloxacin gical study on 22 spontaneous cases in Greece. Vet. Dermatol. 3, (Marbocyl1,Ve´toquinol, Lure, France) could be used 121–130. Lefebvre, H.P., Dupouy, V., Schneider, M., Laroute, V., Toutain, P.L., as a single treatment, at a dosage regimen of 2 mg/kg 1998a. Pharmacokinetics of marbofloxacin in renal impairment in once a day for 28 days, to treat CVL safely and the dog. Vet. Quart. 20, S104. efficiently. We claim an association only for the eye Lefebvre, H.P., Schneider, M., Dupouy, V., Laroute, V., Costes, G., lesions for which the efficacy of marbofloxacin alone Delesalle, L., Toutain, P.L., 1998b. Effect of experimental renal turned to be moderate. We also recommend the impairment on disposition of marbofloxacin and its metabolites in the dog. J. Vet. Pharmacol. Ther. 21, 453–461. concomitant use of repellent and insecticide spot-on Meunier, D., Acar, J.F., Martel, J.L., Kroemer, S., Valle´, M., 2004. A and collars against phlebotomine sandflies to prevent seven-year survey of susceptibility to marbofloxacin of pathogenic leishmanial spreading and re-infection. strains isolated from pets. Int. J. Antimicrob. Agents 24, 592–598. 254 S. Rougier et al. / Veterinary Parasitology 153 (2008) 244–254

Miro, G., Mateo, M., Cruz, I., Canavate, C., Nieto, J., Montoya, A., Raether, W., Seidenath, H., Hofmann, J., 1989. Potent antibacterial Galy, S., Medaille, C., Alvar, J., 2005. Miltefosine: a new treat- fluoroquinolones with marked activity against Leishmania dono- ment for canine leishmaniosis. In: Proceedings of the 3rd World vani in vivo. Parasitol. Res. 75, 412–413. Congress on Leishmaniosis, Palermo, Sicily, Italy, p. 171. Rosypal, A.C., Troy, G.C., Gogal, R., Zajac, A.M., Lindsay, D.S., Monjour, L., Vouldoukis, I., Debons-Guillemin, M.C., Alfred, C., 2003. Emergence of zoonotic canine leishmaniosis in the United Chopin, C., Ploton, I., Segovia, M., Roseto, A., 1987. Application States: isolation and immunohistochemical detection of Leishma- of an in vivo culture system for rapid demonstration of anti- nia infantum from foxhounds from Virginia. J. Eukaryot. Micro- Leishmania activity of monoclonal antibodies. Trans. Roy. Soc. biol. 50, S691–S693. Trop. Med. Hyg. 81, 210–211. Rosypal, A.C., Zajac, A.M., Troy, G.C., Lindsay, D.S., 2005a. Infec- Murray, H.W., Oca, M.J., Granger, A.M., Schreiber, R.D., 1989. tions in immunocompetent and immune deficient mice with Requirement for T cells and effect of lymphokines in successful promastigotes of a North American isolate of Leishmania infan- chemotherapy for an intracellular infection. Experimental visceral tum. Vet. Parasitol. 130, 19–27. leishmaniosis. J. Clin. Invest. 83, 1253–1257. Rosypal, A.C., Gogal Jr., R.M., Zajac, A.M., Troy, G.C., Lindsay, Neogy, A.B., Vouldoukis, I., Da Costa, J.M., Monjour, L., 1994. D.S., 2005b. Cellular immune responses in dogs experimentally Exploitation of parasite-derived antigen in therapeutic success infected with a North American isolate of Leishmania infantum. against canine visceral leishmaniosis. Veterinary group of Lupino. Vet. Parasitol. 131, 45–51. Vet. Parasitol. 54, 367–373. Sereno, D., Guilvard, E., Maquaire, S., Cavaleyra, M., Holzmuller, Noli, C., Auxilia, S.T., 2005. Treatment of canine old world visceral P., Ouaissi, A., Lemesre, J.L., 2001. Experimental studies on leishmaniosis: a systematic review. Vet. Dermatol. 16, 213–232. the evolution of antimony-resistant phenotype during the Oliva, G., Gradoni, L., Cortese, L., Orsini, S., Ciaramella, P., Scalone, in vitro life cycle of Leishmania infantum: implications for A., De Luna, R., Persechino, A., 1998. Comparative efficacy of the spread of chemoresistance in endemic areas. Acta Trop. meglumine antimoniate and aminosidine sulphate, alone or in 80, 195–205. combination, in canine leishmaniosis. Ann. Trop. Med. Parasitol. Slunt, K.M., Grace, J.M., MacDonald, T.L., Pearson, R.D., 1996. 92, 165–171. Effect of mitonafide analogs on topoisomerase II of Leishmania Pennisi, M.G., De Majo, M., Masucci, M., Britti, D., Vitale, F., Del chagasi. Antimicrob. Agents Chemother. 40, 706–709. Maso, R., 2005. Efficacy of the treatment of dogs with leishma- Vouldoukis, I., Alfred, C., Monjour, L., Mazier, D., Brandicourt, O., niosis with a combination of metronidazole and spiramycin. Vet. Ploton, I., Tselentis, Y., Nzuzi, K.K., Gentilini, M., 1986. Rec. 156, 346–349. Milieux de culture pour la production des formes promastigotes Pinelli, E., Gebhard, D., Mommaas, A.M., Van Hoeij, M., Langer- et amastigotes de Leishmania. Application au diagnostic se´ro- mans, J.A., Ruitenberg, E.J., Rutten, V.P., 2000. Infection of a logique et essais the´rapeutiques. Ann. Parasitol. Hum. Comp. 61, canine macrophage cell line with Leishmania infantum: determi- 147–154. nation of nitric oxide production and anti-leishmanial activity. Vet. Vouldoukis, I., Rougier, S., Dugas, B., Pino, P., Mazier, D., Woehrle´, Parasitol. 92, 181–189. F., 2006. Canine visceral leishmaniosis: comparison of in vitro Poli, A., Sozzi, S., Guidi, G., Bandinelli, P., Mancianti, F., 1997. leishmanicidal activity of marbofloxacin, meglumine antimoniate Comparison of aminosidine (paromomycin) and sodium stiboglu- and sodium stibogluconate. Vet. Parasitol. 135, 137–146. conate for treatment of canine leishmaniosis. Vet. Parasitol. 71, World Health Organization, 2002. Annex 3. Burden of disease in 263–271. DALYs by cause, sex and mortality stratum in WHO regions, Prescott, J.F., Desmond Baggot, J., Walker, R.D., 2000. Fluoroqui- estimates in 2001. In: The World Health Report 2002, p.192–197. nolones. In: Antimicrobial Therapy in Veterinary Medicine, 3rd World Health Organization, Geneva, Switzerland. [Online.] http:// ed. Iowa State University Press/Ames, Iowa, pp. 315–338. www.who.int/whr/2002/en/whr2002annex3.pdf.