Canine leishmaniasis chemotherapy: dog's clinical condition and risk of Leishmania transmission

The aim of this study was to investigate whether treatment against canine leishmaniasis reduced the presence of Leishmania in the healthy skin of dogs, affecting the capacity of parasite transmission. A total of 37 dogs from an endemic region of leishmaniasis were studied. Thirteen symptomatic animals revealed parasites in the bone marrow and eight had also in the skin. Five of the 22 dogs that had been treated with meglumine antimoniate alone, meglumine antimoniate or trifluralin followed by allopurinol or just with allopurinol had the parasite in bone marrow but none showed Leishmania in the skin. One dog that was treated only with aminosidine was polisymptomatic and had parasites in bone marrow and skin. The different treatments used in this study did not completely eliminate the parasite allowing relapses to occur when the treatment is discontinued, but the use of meglumine antimoniate or allopurinol, alone or combined may improve dogs clinical condition and reduce or eliminate the parasite from the skin decreasing the probability of Leishmania transmission.     

Monitoring the reverse to normal of clinico-pathological findings and the disease free interval time using four different treatment protocols for canine leishmaniosis in an endemic area

Twenty-four dogs naturally infected by Leishmania spp. were treated with four different protocols using meglumine antimoniate (aNm) and allopurinol in combination or in monotherapy. Aiming to compare the efficacy of the different treatments the reverse to normal of clinico-pathological findings and the disease free interval time (DFIT) were evaluated. Treated dogs were monitored for 1 year and, in absence of relapses, the DFIT was postponed to the last available follow-up. Seven dogs treated with aNm alone showed relapses during the year of observation. In the group of dogs treated with the combination of aNm (50 mg/kg/SC 12 hourly up to clinico-pathological recovery) and allopurinol (15 mg/kg/PO 12 hourly administered for 6months) no relapses were registered in the year of monitoring and the DFIT reached up to 65 months. Our results showed that this combination represents the best choice to treat canine leishmaniosis compared to other protocols.     

The effects of prednisone on haemostasis in leishmaniotic dogs treated with meglumine antimoniate and allopurinol

Thirty dogs naturally infected with Leishmania infantum were studied in order to determine the effects of treatment on haemostatic function. The animals were divided randomly into two treatment groups: Group 1 received meglumine antimoniate and allopurinol; Group 2 dogs were given the same treatment plus prednisone. Ten healthy animals were used as untreated controls. Clinical examination and determination of platelet aggregation, coagulation factors and biochemical parameters were undertaken before treatment and after 15, 30 and 60 days. A significant improvement in platelet aggregation was detected after 60 days in Group 1, but only after 15 days in Group 2. In both treated groups, platelet aggregation was lower than in the control group at the end of the study. The results suggest that prednisone may be a useful tool in the treatment of haemostatic disorders during canine leishmaniosis. The potential benefits and risks due to the use of corticosteroids in the treatment of leishmaniosis are discussed.     

Papular dermatitis due to Leishmania spp. infection in dogs with parasite-specific cellular immune responses

Papular dermatitis due to Leishmania spp. infection was diagnosed in three boxers and two Rottweilers with Leishmania-specific cellular immunity. Diagnosis was based on histological and immunohistochemical examination of papules in four dogs and on cytological examination in one dog. Serum protein electrophoresis was within reference ranges and low antibody levels to Leishmania infantum were detected. Delayed-type hypersensitivity (DTH) reaction to leishmanin was evaluated before treatment in three dogs with positive results. After meglumine antimoniate therapy for 3 to 4 weeks and allopurinol treatment for 6 to 10 months, all dogs were clinically normal, had positive DTH reactions to leishmanin and reduced antibody titres. In conclusion, we suggest that this previously unreported cutaneous presentation of canine leishmaniosis appears to be associated with specific immunocompetence and, consequently, with a favourable prognosis.     

Leishmania infantum-specific IgG, IgG1 and IgG2 antibody responses in healthy and ill dogs from endemic areas. Evolution in the course of infection and after treatment

The expression of IgG, IgG1 and IgG2 specific antibodies for Leishmania infantum was studied in five groups of dogs in Catalonia (Spain): I, 99 asymptomatic dogs (infected and uninfected) from a highly endemic area for leishmaniosis; II, 139 untreated dogs with clinically patent leishmaniosis; III, 11 naturally infected asymptomatic dogs monitored for up to 5 years since they were found seropositive to Leishmania antigen and without treatment; IV, 25 naturally infected dogs with clinically patent leishmaniosis and treated with either meglumine antimoniate and allopurinol or allopurinol alone and V, six experimentally infected dogs, treated with meglumine antimoniate and controlled for 5 years. The levels (ELISA units) of IgG, IgG1 and IgG2 in asymptomatic dogs (group I) were very variable (24+/-33, 32+/-31 and 26+/-31, respectively), and, as expected, lower than in ill dogs (group II) (168+/-34, 84+/-71 and 172+/-31, respectively). In both groups, the correlation between IgG and IgG2 levels (r=0.95, P<0.001 in group I and r=0.63, P<0.001 in group II) was higher than between IgG and IgG1 levels (r=0.01, P>0.05 in group I and r=0.31, P<0.001 in group II). In group III, IgG and IgG2 expression increased during infection, while IgG1 expression remained the same. In dogs of group IV, IgG levels after 1 year of treatment decreased more in responsive (mean values, 163+/-42 before treatment (b.t.) and 100+/-36 after treatment (a.t.)) than in unresponsive dogs (158+/-29 b.t. and 124+/-51 a.t.), especially for IgG1 (94+/-89 b.t. and 20+/-21 a.t. in responsive dogs and 35+/-25 b.t. and 22+/-13 a.t. in unresponsive dogs) rather than for IgG2 (156+/-16 b.t. and 114+/-45 a.t. in responsive and 151+/-11 b.t. and 125+/-36 a.t. in unresponsive dogs). Similar results were observed in the evolution of experimentally infected animals after consecutive and specific treatments. Overall results show the great variation in Leishmania-specific IgG1 expression in asymptomatic and symptomatic dogs, their lack of correlation with that of IgG2 and chemotherapy is more effective in dogs with initially high expression of IgG1.     

Use of allopurinol for maintenance of remission in dogs with leishmaniasis

Current treatments for infected dogs with leishmaniasis do not always provide long-term control of the disease and clinical relapses are common. In this study, the usefulness of long-term allopurinol administration in the maintenance of clinical remission in canine leishmaniasis was evaluated. Fifteen dogs with natural leishmania infection were subjected to an initial treatment based on the simultaneous administration of meglumine antimoniate (100 mg/kg/day) and allopurinol (30 mg/kg/day). Once clinical remission was achieved, a maintenance treatment with allopurinol (20 mg/kg/day) administered for one week a month was instituted. Results were compared with those of a retrospective control group comprising 15 infected dogs which only followed the induction treatment. Relapses occurred in 86 per cent of control dogs within 14 months of discontinuing treatment. In contrast, those dogs on intermittent oral allopurinol administration were successfully maintained in clinical remission for a follow-up period of 10 to 44 months. In this latter group, specific antibody titres decreased or were unchanged, no side effects directly attributable to allopurinol were seen and treatment was well accepted by the owners. It is concluded that long-term intermittent administration of allopurinol is an effective way of maintaining clinical remission in dogs with leishmaniasis.     

Real-time PCR assay in Leishmania-infected dogs treated with meglumine antimoniate and allopurinol

A real-time PCR assay was exploited for monitoring the Leishmania DNA load in different tissues from 18 naturally-infected dogs before and after treatment with a combination of meglumine antimoniate (100mg/kg/day, subcutaneously) and allopurinol (10mg/kg/day, orally) for 30 days. After the combined therapy, allopurinol was continued at the same dose until the end of the observation period. Whole blood samples, lymph node aspirates, and skin biopsies were collected at the time of diagnosis, 1 month after starting therapy, and every 3 months for 2 years. In six dogs parasite load assessments continued every 6 months for a further 3 years. At each assessment, the dogs were examined for signs of disease and a clinical score was recorded. At diagnosis, the highest Leishmania DNA load was detected in lymph node aspirates. From 1-6 months post-therapy a general improvement in clinical conditions was recorded in all dogs, which correlated with a decrease in the parasite DNA load in all tested tissues, even though it was less pronounced in lymph node aspirates. In the period from 9-24 months post-therapy, a re-increase in parasite load was observed in the tissues of some dogs, concomitant with a disease relapse. The results show that the combined therapy with meglumine antimoniate and allopurinol promoted a clinical improvement which was accompanied by a reduction in the parasitic load in the blood, skin and lymph nodes but, even after long period of allopurinol administration alone, Leishmania may persist in dog tissues.     

Myocarditis and generalised vasculitis associated with leishmaniosis in a dog

A three-year-old, female bulldog was presented with bilateral uveitis, apathy, listlessness, generalised lymphadenopathy and perivulvar haematoma. The initial laboratory studies showed non-regenerative anaemia, polyclonal gammopathy and a high urine protein:creatinine ratio. Serology for leishmaniosis was positive and treatment with allopurinol and meglumine antimoniate was started. Despite treatment, the dog's clinical condition deteriorated. Signs included cutaneous ecchymosis, respiratory distress and finally cardiorespiratory arrest. Histopathological studies of postmortem tissue samples revealed a generalised vasculitis of several internal organs and severe myocarditis. Leishmania species organisms were identified in affected tissues using immunoperoxidase labelling and PCR techniques.     

Serum cobalamin concentrations in dogs with leishmaniosis before and during treatment

Hypocobalaminemia in dogs is most commonly associated with gastrointestinal disorders leading to impaired absorption and utilization of cobalamin. The objectives of this study were to compare serum cobalamin concentrations between dogs with leishmaniosis and clinically healthy dogs, and to assess possible alterations of serum cobalamin concentrations in dogs with leishmaniosis at different timepoints during treatment. Fifty-five dogs with leishmaniosis and 129 clinically healthy dogs were prospectively enrolled. Diagnosis of leishmaniosis was based on clinical presentation, positive serology and microscopic detection of Leishmania amastigotes in lymph node aspiration smears. Twenty of the dogs with leishmaniosis were treated with a combination of meglumine antimonate and allopurinol for 28 days and serum cobalamin concentrations were measured in blood samples that were collected before initiation of treatment (timepoint 0) and on days 14 and 28. In order to estimate alterations of serum cobalamin concentrations during treatment, cobalamin concentrations were measured in blood samples from 20 out of 55 dogs with leishmaniosis at all timepoints. Serum cobalamin concentrations were significantly lower in dogs with leishmaniosis before treatment (median: 362 ng/L; IQR: 277−477 ng/L) compared to clinically healthy dogs (median: 470 ng/L; IQR: 367−632 ng/L; P = 0.0035). Serum cobalamin concentrations increased significantly in dogs with leishmaniosis on day 14 of treatment compared to timepoint 0 (P = 0.02).In the present study, serum cobalamin concentrations were significantly lower in dogs with leishmaniosis compared to clinically healthy dogs. In addition, there was an increase in serum cobalamin concentrations during treatment. The clinical significance of hypocobalaminemia in dogs with leishmaniosis remains to be determined.     

Serum concentrations of acute-phase proteins in dogs with leishmaniosis during short-term treatment

OBJECTIVE: To evaluate changes in serum concentrations of acute-phase proteins in dogs with leishmaniosis during short-term therapy in accordance with 2 treatment protocols and determine whether concentrations of acute-phase proteins could be used to monitor the initial response of dogs to treatment. ANIMALS: 12 dogs naturally infected with Leishmania infantum. PROCEDURE: Dogs were allocated into 2 groups. Dogs of group 1 were treated by use of meglumine antimonate (100 mg/kg, SC, q 24 h) administered concurrently with allopurinol (15 mg/kg, PO, q 12 h) for 20 days and then with allopurinol alone at the same dosage for the subsequent 30 days. Dogs of group 2 were treated by administration of allopurinol alone (15 mg/kg, PO, q 12 h) for 60 days). Blood samples were obtained before and during treatment for measurement of serum concentrations of acute-phase proteins and determination of CBC counts, serum biochemical analyses, and electropherograms. RESULTS: All dogs evaluated in the study had increased concentrations of C-reactive protein, haptoglobin, and ceruloplasmin at the time of diagnosis of leishmaniosis. Mean concentration of serum amyloid A before treatment was also increased, but some of the dogs had concentrations of serum amyloid A that were within the reference range. Concentrations of C-reactive protein and ceruloplasmin decreased significantly in all dogs at the end of the study period. CONCLUSIONS AND CLINICAL RELEVANCE: Measurement of concentrations of selected acute-phase proteins, such as C-reactive protein or ceruloplasmin, could be used to evaluate the initial response of dogs with leishmaniosis to treatment.     

Comparison of different tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis

In this study, different types of tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis were compared. Skin, whole blood and lymph node samples were collected from 95 naturally infected dogs living in South Italy, where the disease is endemic. Twenty-nine of these 95 dogs, treated with meglumine administered concurrently with allopurinol for 30 days, and then with allopurinol alone, were monitored during a period of 2 years. The DNA extracted from the clinical specimens was amplified by PCR using as target DNA a 116-bp fragment in the constant region of the kinetoplast DNA minicircle. PCR analysis was more sensitive than indirect immunofluorescence antibody test in detecting Leishmania infection in symptomatic dogs: 99% of lymph node samples resulted positive, whereas 94% of blood samples and 95% of skin samples gave a positive result. PCR analysis of samples from dogs followed up 2 years showed that: (1) all subjects resulted positive in at least one of the three types of samples; (2) all time the dogs had a relapse, PCR resulted positive in all three types of samples; (3) when dogs were apparently healthy, PCR analysis was positive on skin and lymph node samples, but not always on blood samples. Since lymph node sampling is invasive and sometimes difficult in healthy asymptomatic dogs, our results suggest that, independently from the presence or not of cutaneous lesions, skin biopsy represents a good substratum for PCR-based diagnosis and follow-up of canine visceral leishmaniosis.     

Subclinical leishmaniasis associated with infertility and chronic prostatitis in a dog

A stud dog was presented for acquired infertility. Haematospermia and teratozoospermia were found on two ejaculates 2 weeks apart. A presumptive diagnosis of prostatitis was made follo-wing ultrasound examination. An ultrasound-guided needle core biopsy was performed under general anaesthesia, revealing a mild chronic macrophagic and plasma cell prostatitis with intracytoplasmic amastigotes consistent with Leishmania spp. infection. Presence of Leishmania infantum, Leishmania donovani or Leishmania chagasi was confirmed by polymerase chain reaction in seminal plasma. Serology and serum protein electrophoresis confirmed the diagnosis of a subclinical active systemic leishmaniasis. A meglumine antimoniate and allopurinol treatment was given which clearly improved within 3 months both general condition and the quality of sperm. To the authors' knowledge, this is the first reported case of a prostatitis secondary to a Leishmania spp. infection. Subclinical systemic leishmaniasis should be considered in the differential diagnosis of infertility in dogs suffering from semen alterations.     

Characterization of circulating lymphocyte subpopulations in canine leishmaniasis throughout treatment with antimonials and allopurinol

Canine leishmaniasis (CL) is a systemic parasitic disease with a wide variability of response to specific therapy: the majority of patients apparently improve with treatment, some of them respond but later relapse, and few of them do not respond at all. It has been demonstrated that the immune response plays a key role in the development and outcome of Leishmania infection in the dog and in the response to the treatment, although this response is not well understood. Some authors have suggested that ill dogs show a reduction in the percentage of circulating CD4+ lymphocytes and in the CD4+/CD8+ ratio, both of which normalize after treatment and clinical recovery. The present paper discusses the variation of the different lymphocyte subpopulations (CD3, CD4, CD8, CD21) of peripheral blood mononuclear cells (PBMC) in 28 dogs diagnosed with CL and submitted to conventional treatment with meglumine antimoniate (Glucantime) for 1 month and with allopurinol (Zyloric) for 1 year, in order to evaluate the usefulness of these parameters as indicators of the immunological condition of the ill animals and of the prognosis of their evolution during the treatment. It is concluded that circulating lymphocyte subpopulations are similar in dogs with leishmaniasis and in healthy dogs and that there is no correlation between the clinical status or response to therapy and the values of the counts of the different lymphocyte subpopulations. Therefore, the percentage of different lymphocyte subpopulations cannot be used as a parameter to predict the evolution of an individual patient in a clinical context.     

Clinical and serological follow-up in dogs with visceral leishmaniosis treated with allopurinol and sodium stibogluconate

Seven dogs with parasitologically proven clinical visceral leishmaniosis (Leishmania infantum infection) were treated with a combination of allopurinol and sodium stibogluconate. The dogs received first orally 15 mg/kg of allopurinol every 12 h until the clinical signs improved, in the following 1 month period allopurinol at same dose and subcutaneously 30 mg/kg of sodium stibogluconate combination were given daily and at the end of the combined treatment, allopurinol was continued alone at the same dose till the end of 8 months. During the treatment period, dogs were supported by additional proteins, vitamins, and minerals. A long acting insecticide (collar or drop) was also used in order to prevent further parasite transmission. Follow-up was maintained by clinical, clinicopathological evaluation, and parasitological examination of lymph node, serology using the indirect immunofluorescent antibody test (IFAT). Before treatment commenced, the most important clinical signs were exfoliative dermatitis, ulcerations, peripheral lymhadenopathy, pale mucous membranes, weight loss, and ocular lesions. Clinicopathological findings included commonly anaemia, hyperproteinaemia, hyperglobulinaemia and hypoalbuminaemia. Before the treatment, amastigotes were seen in six of the seven dogs by examination of lymph node aspiration, and IFAT-titers were positive in all dogs. At the end of 8 months treatment, remission of clinical signs, restoration to normal of clinicopathological abnormalities were noticed. Lymph node aspiration was performed on three out of the seven dogs at the end of the treatment because of the very small sizes of the lymph nodes, and no amastigotes were observed. Although the mean IFAT-titer of the dogs were significantly (P < 0.001) lower compared with pretreatment, IFAT-titers of dogs were still positive. No relapses occurred during treatment period and a 6-24-month duration after the end of therapy. Based on the above results, long-term use of allopurinol combined with sodium stibogluconate together with support treatment concluded to have enough therapeutic efficacies in the treatment of dogs with visceral leishmaniosis. Observations of the cases for possible relapses were still going on and insecticide application was carefully carrying on in order preventing a possible re-infection.     

Study of efficacy of miltefosine and allopurinol in dogs with leishmaniosis

Visceral leishmaniosis is a life-threatening disease of medical, social and economic importance in endemic areas. It is an opportunistic infection in immunocompromised patients, including human immunodeficiency virus-positive subjects. Dogs are the main reservoir of Leishmania infantum. The aim of this study was to evaluate the efficacy of miltefosine and allopurinol for the control of human leishmaniosis using the dog as a model. The study included 28 sick dogs treated with miltefosine (2 mg/kg/day PO) administered concurrently with allopurinol (10 mg/kg/day, PO) for 30 days, and then with allopurinol alone, at the same dosage, for 1 year. Eight dogs (four of which relapsed) received a second cycle of miltefosine within 6 months of the first cycle. Efficacy was measured by real-time polymerase chain reaction assay on whole blood samples and lymph node aspirates, collected at baseline and every 3 months for 12 months. Of the total number of animals (28), two showed renal insufficiency and died after the start of therapy with miltefosine. Two other dogs presented some side effects to treatment, such as nausea, vomiting and reduction in white and red blood cell counts, and these animals were excluded from the follow-up. The results showed that the first cycle of therapy with miltefosine and allopurinol induced a drastic and progressive reduction of L. infantum load in lymph node aspirates but the second cycle did not eliminate the parasite.     

Canine visceral leishmaniasis: comparison of in vitro leishmanicidal activity of marbofloxacin, meglumine antimoniate and sodium stibogluconate

The control of canine leishmaniasis largely depends on the success of treatment. Drugs currently available to treat this disease are toxic and partially effective. The curative effect of marbofloxacin, a third-generation fluoroquinolone developed for veterinarian individual treatment, was evaluated in vitro in the presence of Leishmania infantum promastigotes and dog-monocyte-derived macrophages; meglumine antimoniate and sodium stibogluconate were used as comparative treatments. We observed that the killing of Leishmania promastigotes and intracellular amastigotes by marbofloxacin was dose-dependent. We demonstrated that successful treatment of canine infected macrophages for 48 h was possible with 500 microg/ml of marbofloxacin. Leishmanicidal activity acted through a TNF-alpha and nitric oxide pathway and correlated with the generation of nitric oxide (NO(2)) production by monocytes derived macrophages from infected (23+/-5 microM) or healthy (21+/-6 microM) dogs, in comparison with NO(2) concentration in infected/non-treated macrophages (< 3 microM, P<0.01). This significant induced parasiticidal effect correlated with extensive elimination of amastigotes by macrophages derived from infected (11+/-5) and healthy dogs (6+/-2), when compared to infected/non-treated macrophages (530+/-105 and 472+/-86 amastigotes, respectively, P< 0.01). Marbofloxacin was shown to be non-toxic at 500 microg/ml in vitro and no cell apoptosis was observed. The molecule was able to induce a parasitic process after significant elimination of amastigotes in leishmania-infected dog macrophages. We propose that marbofloxacin, compared to standard chemotherapeutic agents (meglumine antimoniate and sodium stibogluconate), could be an effective and pragmatic oral route alternative to treat canine leishmaniasis.     

Polyarthritis associated with visceral leishmaniasis in a juvenile dog

Canine visceral leishmaniasis (CL) is a zoonosis and a chronic systemic disease characterized by a wide spectrum of clinical signs, including, in rare occasions, polyarthritis. This report describes a case of CL in an 8-month old male boxer dog with a history of lameness, fever and lymphadenopathy. A definitive diagnosis of CL was based on the observation of the Leishmania amastigotes seen concomitantly, and for the first time, in the lymph nodes aspiration smears (in macrophages), synovial fluid (in macrophages and neutrophils) and blood (in neutrophils). Despite this extensive dissemination of the parasite, the animal was successfully treated with a multi-step combination of meglumine antimoniate, aminosidine and allopurinol.     

Effects of allopurinol treatment on the progression of chronic nephritis in Canine leishmaniosis (Leishmania infantum)

Forty dogs with canine leishmaniosis (CL) participated in this study, which was designed to investigate the effect of allopurinol on the progression of the renal lesions associated with this disease. The animals were allocated into 5 groups. Group A dogs (n = 12) had neither proteinuria nor renal insufficiency, group B dogs (n= 10) had asymptomatic proteinuria, and group C dogs (n = 8) were proteinuric and azotemic. Two more groups, CA and CB, comprising 5 dogs each, served as controls for groups A and B, respectively. Group A, B, and C dogs received allopurinol PO (10 mg/kg q12h) for 6 months, whereas group CA and CB dogs were placebo-treated. Serum biochemistry profile, urinalysis, urine protein/creatinine ratio, and glomerular filtration rate (GFR) measurements were carried out at the beginning of the study, the 3rd month, and the 6th month, whereas renal biopsies were carried out only at the beginning and the end of the trial. Membranoproliferative glomerulonephritis was the most common cause of chronic renal failure. Mesangioproliferative and tubulointerstitial nephritis were detected even in group A and CA dogs. Allopurinol not only lowered proteinuria in group B dogs but also prevented the deterioration of GFR and improved the tubulointerstitial, but not the glomerular, lesions in both group A and group B dogs. Further, it resolved the azotemia in 5 of the 8 dogs admitted with 2nd stage chronic renal failure (group C). Consequently, treatment with allopurinol is advisable in CL cases with asymptomatic proteinuria or 1st-2nd stage chronic renal failure.     

Evaluation of renal and hepatic functions in dogs naturally infected by visceral leishmaniasis submitted to treatment with meglumine antimoniate

The present study aimed to evaluate the renal and hepatic responses in eight dogs with visceral leishmaniasis submitted to treatment with meglumine antimoniate and to verify the occurrence of possible side effects. Urinalysis, hepatic and renal function tests were carried out in all animals at up to seven moments. After the end of a six-month observation period, all dogs were euthanized. Before the beginning of the experiment urinary and biochemical alterations were observed in four dogs due to the changes caused by the parasite itself. These alterations included the presence of renal cells, cylindruria, proteinuria, azotemia, hyperproteinemia and hypoalbuminemia. One dog died on the third day after treatment because an aggravation of the clinical picture, probably due to the medication. During the course of the study, an increase in hepatic enzymes was verified in two animals. Sixty days after the beginning of the treatment four dogs showed remission of clinical signs. The other three were asymptomatic with persistent biochemical alterations. From these, two presented recurrence of clinical signs about 150 days after the beginning of the treatment while in the other, hyperproteinemia persisted. Meglumine antimoniate was not efficient to treat dogs with severe renal dysfunction and the side effects observed were pain at the site of injection and the probable transient hepatotoxicity, evidenced by biochemical examinations, but without the presence of clinical signs.