First Report of the Use of Meglumine Antimoniate for Treatment of Canine Leishmaniasis in a Pregnant Dog

Introduction

The leishmaniases are a group of infectious diseases caused by species of the genus Leishmania, such as L. infantum, that affect people and domestic and wild animals worldwide. The infection is transmitted mainly indirectly by sand flies of the genus Phlebotomus in the Old World and Lutzomyia in the New World. Regions endemic for this disease include the Mediterranean countries and tropical and subtropical areas (i.e., the Middle East, SouthWest Asia, and Latin America) where dogs represent the main reservoirs.¹ Canine leishmaniasis is also sometimes found in nonendemic countries due to pet travel or dog importation.² In the last few years, new foci of infections were identified in countries not endemic for canine leishmaniasis, including North America where the infection appears to be widespread in foxhounds. Spread of the disease in North America appears to be limited to dog-to-dog mechanisms of transmission.³ Alternative methods of transmission include direct transmission via coitus⁴ and blood transfusion from infected donors.⁵ Vertical transmission from mother to puppies has also been demonstrated in an experimentally infected beagle and in BALB/c mice.^6,7

Cases of neonatal infection or disease are infrequently reported in veterinary medicine. Transplacental transmission may be difficult to demonstrate in a natural setting due to possible alternative explanations (e.g., infection is transmitted during passage through the birth canal or shortly after birth).^8–11 Conversely, neonatal cases of transplacental transmission are well-documented in humans even though they occur only sporadically.^12,13

In humans, amphotericin B is considered the most effective and least toxic drug for treatment of leishmaniasis during pregnancy.^12,13 Treatment of bitches with leishmaniasis is problematic because amphotericin B is not commonly used to treat canine leishmaniasis and there are no data about the safety of antimonial drugs, an alternate group of drugs used to treat leishmaniasis, during pregnancy. To the authors’ knowledge, this is the first report that describes the diagnosis, management, and outcome of canine leishmaniasis in a pregnant bitch treated with meglumine antimoniate.

Case Report

A 4 yr old, 11 kg, intact female mixed-breed dog, fully vaccinated against CDV, CPV, leptospirosis, and ICH with prophylaxis against endo- (ivermectin plus pirantel^a) and ectoparasites (imidacloprid plus permetrina^b) was referred to the Internal Medicine Service of the School of Veterinary Medicine of Milan with a 15 day history of sneezing and right epistaxis. The dog had been found when she was a few months old in South Italy (Sardinia Island), an endemic region for canine leishmaniasis. The dog had returned to South Italy every summer on holiday. The referring veterinarian performed a retroflex rhinoscopy revealing nasal cavities with moderate mucoid exudate, mild hyperemia, depigmentation, and hemorrhagic tendency of the mucous membrane.

On referral examination, 1 wk after performing the rhinoscopy by the referring veterinarian, the bitch was in estrus and in good body condition (body condition score=5/9). She showed a generalized moderate and painless lymphadenomegaly, an exfoliative, dry, nonpruritic dermatitis with scaling, alopecia of the elbows and hocks, and vulvar enlargement compatible with estrus. The lymphadenomegaly, skin involvement, and epistaxis were all compatible with canine leishmaniasis.¹ Other possible differential diagnoses were an infectious disease (e.g., ehrlichiosis), a neoplastic disordes (e.g., lymphoma), an autoimmune disorder (e.g., pemphigus foliaceus), a keratinization defect (e.g., canine seborrhea), or a coagulative disorder (e.g., anticoagulant poisoning). Routine hematology, serum biochemistry and complete urinalysis were performed. Abnormal CBC results included a normochromic, microcytic, nonregenerative anemia (RBC=4.0 × 10⁶/μL (reference range, 5.7–8.8 × 10⁶/μL); Hb=8 g/L (reference range, 12.9 g/L–18.4 g/L); PCV=22.6% (reference range, 37%–57%); mean corpuscular volume (MCV) = 55.5 fL (reference range, 58.8–71.2 fL); mean corpuscular hemoglobin (MCH) = 19.6 pg (reference range, 20.5–24.2 pg); mean corpuscular hemoglobin concentration (MCHC) = 35.3% (reference range, 31.0%–36%); and thrombocytopenia (99 × 10³/μL (reference range, 143.3–400 × 10³/μL). Biochemical analysis detected hyperproteinemia with hypoalbuminemia, hypergammaglobulinemia, and a decreased albumin-globulin ratio (A/G) ratio (0.18; reference range, 0.8–1.7). Specifically, total protein was 12.1 g/dL (reference range, 6–8 g/dL), albumin was 1.86 g/dL (reference range, 2.7–4 g/dL), alpha-1 globulins were 0.16 g/dL (reference range, 0.25–0.6 g/dL), alpha-2 globulins were 0.92 g/dL (reference range, 0.7–1.5 g/dL), beta globulins were 1.21 g/dL (reference range, 1.2–1.9 g/dL), gamma globulins were 7.95 g/dL (reference range, 0.5–1.1 g/dL). Urinalysis revealed a urine specific gravity of 1.025 and proteinuria with a urine protein-to-creatinine (UPC) ratio of 0.8 (reference range, <0.4). The serum indirect immunofluorescence antibody test (IFAT) for L. infantum-specific antibodies yielded a positive titer of 1:640 (reference range, ≤1:80).¹⁴ The diagnosis of leishmaniasis was confirmed by positive real-time polymerase chain reaction (PCR) analysis for L. infantum (the primary cause of canine leishmaniasis) on aspirates of superficial cervical lymph nodes. No parasites were identified during cytologic evaluation of the lymph nodes.

Treatment was immediately initiated with the subcutaneous administration of 100 mg/kg q 24 hr of meglumine antimoniate^c. Two days after therapy was initiated, the dog was accidentally bred to a 10 yr old male mixed-breed dog. The male dog appeared healthy and the IFAT and real-time PCR analysis on blood collected from the dog postcoitus were negative for canine leishmaniasis. The test was performed 2 wks postcoitus to establish if the male had already been infected with L. infantum rather than determining if the male dog had been infected during coitus. In this case, testing the male dog for L. infantum was intended to exclude the possibility of paternal transmission of infection. The owners of the bitch declined preventing an eventual pregnancy with either drugs (i.e., progestins) or surgery (i.e., ovariohysterectomy) as recommended by the veterinarians (the authors of this study). Instead, the owners elected to continue with the antimonial treatment of the bitch. Thirty days after mating (i.e., 32 days after initiating treatment for leishmaniasis), an abdominal ultrasonography identified vital fetuses. The bitch showed a resolution of epistaxis but persistence of mild lymphadenopathy and localized exfoliative dermatitis. Blood evaluation revealed a general improvement in the biochemical and hematologic abnormalities; however, some abnormalities persisted including a microcytic nonregenerative anemia (RBC=4.8 × 10⁶/μL; Hb=9.4 g/L; PCV=27.2%; MCV=56.9 fL; MCH=19.7 pg; MCHC=34.6%), thrombocytopenia (113 × 10³/μL), hyperproteinemia (total protein=11.9 g/dL), hypoalbuminemia (2.2 g/dL), hypergammaglobulinemia (5.22 g/dL), and a reduced A/G ratio (0.26). Antimonial treatment was prescribed for another thirty days.

Five puppies were naturally delivered on day 58 of gestation. The placentas were not collected because they were consumed by the bitch. Two puppies died due to crushing by the mother at the time of parturition and one puppy had signs of suffocation 2 days after parturition. The puppies that died had normal body weights and conformation and necropsies did not identify any macroscopic or histologic lesions consistent with leishmaniasis. Leishmania amastigotes were not detected in sections of internal fetal tissues (i.e., spleen, liver, kidney, pancreas, tongue, stomach, intestine, bone marrow, and skin). Real-time PCR of sternal bone marrow and spleen of the puppies were all negative for L. infantum. Antimonial treatment of the bitch was discontinued when the puppies were delivered (i.e., after 60 days of antimonial therapy) because clinical abnormalities had improved and the owner declined further treatment. Blood tests performed 60 days from the start of the antimonial therapy, immediately prior to the delivery of the puppies, revealed continued improvement in hematologic and biochemical abnormalities. Most notably, the RBC was 5.0 × 10⁶/μL, Hb was 10.5 g/L, PCV was 29.7%, MCV was 59.0 fL, MCH was 20.9 pg, MCHC was 35.4%, the platelet count was 126 × 10³/μL, total protein was 10.6 g/dL, albumin was 2.3 g/dL; gamma globulins were 7.16 g/dL, and the A/G ratio was 0.24. The 2 surviving puppies showed no signs of leishmaniasis during lactation. At 2, 4, and 12 mo of age, the surviving puppies were clinically healthy and negative for L. infantum infection (tested by IFAT and PCR on blood samples).

After the puppies were weaned at 40 days of age, a hematologic survey of the bitch showed resolution of the anemia and thrombocytopenia, but persistence of the hyperproteinemia (total protein was 9.7 g/dL) with hypergammaglobulinemia (gamma globulin was 3.41 g/dL) and a low A/G ratio (0.5). Antimonial therapy was resumed at the same dose described above. After an additional 30 days of antimonial therapy (total antimonial therapy was 90 days), there was a normal proteinemia but the hypergammaglobulinemia (gamma globulin=1.74 g/dL) and the low A/G ratio (0.68) persisted. On urinalysis, urine specific gravity was 1.028 and the UPC ratio was normal (0.2).

Hemorrhagic vulvar discharge persisted for 20 days, which was considered beyond the physiologic period of lochia expulsion (2 wk of sanguineous vulvar discharge after whelping is considered normal). Syncytial vacuolated cells were detected in vaginal cytologic samples 2 mo after parturition indicating subinvolution of the placental sites.¹⁵ Antimonial treatment was again discontinued and the bitch underwent an ovariohysterectectomy to prevent further pregnancy and to treat the pathologic uterine condition.

The uterus had an abnormal appearance and a diameter of 2 cm with five swollen areas. These round bilateral enlargements measured 2.5 cm × 3 cm and represented the residual uterine placental sites characterized by a visible narrow brown zone at the serosal surface. Gross examination of the ovaries detected structures typical of the end of the anestrus phase with the corpora lutea regressed at the cortical surface. Detection of L. infantum in the uterine tissue was possible using real-time PCR (1,203 parasites/g); however, a simultaneous PCR test of peripheral blood from the bitch was negative. Parasites were not identified on histologic examination of the uterus.

The dog was examined 1 mo after discontinuing the antimonial therapy at the time of the ovariohysterectomy. The bitch was clinically normal with no CBC alterations; however, the mild hypergammaglobulinemia (1.68 g/dL) and low A/G ratio (0.63) persisted. On urinalysis, the UPC ratio and urine specific gravity were normal.

Examinations performed 4 and 6 mo after completing the second round of antimonial therapy, all hematologic and biochemical alterations had resolved and the IFAT titer for L. infantum was 1:160. Total antimonial therapy in this dog was 90 days: 60 days for the first round of antimonial therapy followed by 40 days of lactation with no antimonial therapy then a second round of antimonial therapy for 30 days until the ovariohysterectomy was performed.

Discussion

Canine leishmaniasis during pregnancy is rarely reported, even in places where the infection in dogs is endemic, such as Mediterranean countries.^8,9 Although no evidence of parasites was found in 36 puppies naturally born from 18 bitches with leishmaniasis due to L. chagasi, vertical transmission has been experimentally demonstrated and supported by a number of case reports.⁶,^8–11,16 Maternal transmission of a North American isolate of L. infantum from an experimentally infected bitch bred to a male was demonstrated with semen that tested negative for L. infantum. In this report,⁶ the puppies were born via cesarean section and immediately euthanatized and analyzed. DNA of L. infantum was found in several organs from all of the three puppies analyzed. Thus, the possibility of acquiring the infection via male semen, during parturition, or intimate contact with the mother or colostrum was excluded.⁶

The first report of a natural case of congenital leishmaniasis in puppies from a bitch with leishmaniasis was in 1995.⁸ Since then, there have been reports of leishmaniasis in newborn puppies from seven bitches with natural Leishmania infection in Sicily. From a sample of 31 puppies examined between 3 and 30 days of age, PCR for L. infantum was positive in eight puppies. This result provided a confirmation of experimentally demonstrated vertical transmission especially since the puppies were very young at the time of examination and the environmental and climatic conditions were not favorable for activity of the phlebotomine vector.⁹ More recently there has been a report of disseminated L. infantum natural infection in two sibling foxhounds due to possible vertical transmission in North America.¹¹ In all these cases the species of Leishmania was L. infantum. It has been hypothesized that only some species of the genus Leishmania, such as L. infantum, have the ability to cross the placenta, but more studies are required to validate this theory.⁶ In the majority of these cases the infected bitch was either not treated for canine leishmaniasis during pregnancy and died before, during, or soon after delivery or presented with a worsening of the infection. In the present report, antimonial treatment of a bitch during pregnancy led to clinical improvement of clinical signs and may have prevented transmission of the infection to the offspring.

In humans, examination of the placentas of infected mothers to investigate the presence of these protozoa is important to predict infection in the newborn.^12,13 Also, in some veterinary reports Leishmania amastigotes have been found in the canine placenta.^10,17 In the present case the bitch consumed all the placentas so the authors could not examine them. Important data for confirming infection with Leishmania in the present study were the positive results of real time PCR on uterine tissue when the bitch tested negative for PCR on blood. This result confirmed that, even if undetected in the histologic section of the examined portion of the uterus, the parasite was localized in the uterus and the antimonial treatment may have prevented the parasite from crossing the placenta and reaching the fetus.

It has been shown experimentally that pregnancy impairs the resistance of mice to Leishmania major infection.^18,19 In this case report there was not a worsening of the infection during the pregnancy, probably due to the antimonial treatment. Spread of infection and immunosuppression are two potential complications of a bitch with leishmaniasis becoming pregnant, but in the present case, the owners did not want to prevent or interrupt the pregnancy. Instead, the bitch was treated medically in an attempt to cure the bitch and prevent spread of infection to the puppies. In a recent case report, a pregnant bitch with leishmaniasis gave birth to puppies positive for L. infantum. The bitch died of renal failure secondary to visceral leishmaniasis 2 wk following weaning of the pups.¹¹

Another important aspect to consider when treating pregnant patients for leishmaniasis is the toxicity of the antimonial compounds to both mother and fetus. The most commonly used drugs for the treatment of canine leishmaniasis are antimonial drugs. The veterinary-labeled product with meglumine antimoniate^c is commercially available in some Mediterranean and South American countries. Meglumine antimoniate selectively inhibits the leishmanial enzymes required for glycolytic and fatty acid oxidation. After subcutaneous or intramuscular injections in dogs the systemic bioavailability is about 92%; highest tissue concentrations are found in the liver, spleen, and skin. Within 9 hr of dosing, 80% of the antimony is excreted in the urine. Primary adverse effects noted in dogs are injection site reactions, lethargy, and gastrointestinal effects (i.e., inappetence, vomiting). Transient increases in liver enzymes have been reported.²⁰

Although antimonial compounds have been used for more than 50 yr to treat human visceral leishmaniasis, little information is currently available regarding safety during pregnancy. In 2001, the embryotoxicity/fetotoxicity of meglumine antimoniate was experimentally evaluated in the rat. No maternal toxicity and no reduction of fetal weight were noted in the treatment group, but repeated administration of meglumine antimoniate enhanced embryolethality, with a higher incidence of atlas bone anomaly in newborn rats.²¹ For these reasons, antimonial compounds are not considered safe for the treatment of human visceral leishmaniasis during pregnancy. Many authors recommend amphotericin B formulations as an alternative treatment of pregnant patients with leishmaniasis because of its efficacy and safety for both mothers and fetus.^13,22 In veterinary medicine, there are no veterinary-labeled products with amphotericin B. This drug must be administered intravenously and nephrotoxicity is a major concern, particularly with the deoxycholate form (the patient's renal function should be aggressively monitored during therapy). Newer lipid-based amphotericin B products are less nephrotoxic and penetrate tissues better, but more expensive.²⁰ There are no data available regarding the safety of amphotericin B in canine pregnancy.

In the last decade there have been attempts to find alternatives to antimonials in the treatment of canine leishmaniasis (e.g., pentamidine, buparvaquone, enrofloxacin, metronidazole, spiramicyn, and ketoconazole), but nothing has proven as effective as meglumine antimoniate.²³ Recently, miltefosine^d has been registered as a veterinary-labeled product with leishmanicidal effect for the therapy of canine leishmaniasis, but this compound is a proven teratogen.²⁴

Meglumine antimoniate is one of the most widely used drugs in the treatment of canine leishmaniasis and, in spite of the high prevalence of this disease in dogs in the Mediterranean, there are no data pertaining to the safety and efficacy of this compound during pregnancy.²⁵ There has only been one report on treatment of a bitch with leishmaniasis with antimonials before and during pregnancy. All six puppies were asymptomatic at birth and all but one puppy tested negative for L. infantum by PCR on blood and/or lymph nodes at 26 days of age. No data were provided on the clinical condition of the pregnant bitch during or after the antimonial therapy.⁹ In the present case report, no apparent toxicity was apparent in either the bitch or puppies.

In the present case, in which a female dog with leishmaniasis mated and became pregnant during treatment of canine leishmaniasis, no vertical transmission was observed even though DNA of L. infantum was found in the uterus after ovariohysterectomy of the bitch. The puppies were followed clinically, serologically, and by PCR until one year of age and were consistently negative for canine leishmaniasis.

Conclusion

It is possible for Leishmania amastigotes to pass from a positive bitch to the offspring during gestation.⁶ Thus, when interruption of pregnancy is not an option for the owner or breeder, antimonial treatment could be considered to reduce the risk of vertical transmission and worsening of the infection in the bitch. Further reports concerning the potential teratological effects of antimonial therapy are needed.