Detection of Leishmania infantum in a naturally infected horse in an endemic area for Leishmaniasis in Northeast Brazil

Introduction:

Introduction: Leishmaniasis is an anthropozoonosis caused by a protozoan of the genus Leishmania and transmitted by vectors of the species Lutzomyia longipalpis.The disease can present in two forms: the cutaneous form (Cutaneous Leishmaniasis) and the visceral form (Visceral Leishmaniasis).Its main hosts are domestic canids, but it can parasitize other diverse species such as rodents, possums, foxes, non-human primates, edentulous primates, domestic cats, and even equids, although this is infrequent.Study objective: Due to the scarcity of reports on Leishmaniasis in horses.Added to this, taking into account the susceptibility of horses to infection by many species of Leishmania and the fact that the vast majority of cases are asymptomatic, the objective of this study was to report a case of equine leishmaniasis in the state of Pernambuco.Results and discussion: A male horse, of the Manga Larga Machador breed, four years old, residing in the municipality of Bezerros-PE, Northeast of Brazil, was treated.On physical examination, the presence of ulcerated and non-ulcerated nodular lesions was observed on the face close to the masseter muscle, in the ventrodorsal region, and below the humeroradiounar joint.Presence of papules in the neck region.In the blood count, normocytic normochromic anemia, and leukocytosis Brazilian Journal of Animal and Environmental Research, Curitiba, v.7, n.1, p. [704][705][706][707][708][709][710][711][712][713][714][715][716]2024 due to neutrophilia and eosinophilia were observed.In the parasitological diagnosis through immunohistochemistry, the presence of Leishmania sp.amastigotes was observed.As a therapeutic protocol, injectable marbofloxacin was used for this patient at a dosage of 2mg/kg once a day, intramuscularly for 28 days.Conclusion: Horses infected by L. infantum may present dermatological lesions.Therefore, early diagnosis and treatment are recommended, as it is suggested that the use of marbofloxacin provides good results in the regression of skin lesions in these cases.However, more studies are needed to investigate the epidemiology and pathophysiology of this disease in horses.

INTRODUCTION
Leishmaniasis are anthropozoonotic disease of veterinary medical interest caused by protozoa of the genus Leishmania and transmitted by sandflies of the genus Lutzomyia (Silva et al., 2017).The clinical presentation of the infection caused by these parasites is diverse, with two main forms known as Visceral Leishmaniasis (VL) and Cutaneous Leishmaniasis (CL), which include the diffuse cutaneous, mucus cutaneous and cutaneous forms (Gontijo and Carvalho, 2003).
For a long time, the domestic dog was considered the main reservoir in urban areas (Maia and Campino, 2011).However, it is important to highlight that many other species of animals (for example, rodents, opossums, foxes, non-human primates, edentulous animals, domestic cats, and horses) may be involved, especially in rural areas or in regions highly marked by anthropogenic actions ( Caldart et al., 2017;Miró and Vélez, 2018).
The detection of protozoa of the genus Leishmania in horses is an unusual finding, characterized mainly by the presence of Leishmania braziliensis (Benassi et al., 2018).The proximity of equids to humans, the low socioeconomic level of the population, and the insertion of these animals in endemic areas increase the risk of infection throughout the zoonotic cycle of CL, playing an important role in the epidemiology of the disease, acting as potential reservoirs or food sources for sandflies (Feitosa et al., 2012;Limeira et al., 2019).
Lymphadenopathy can be observed, but more rarely (Escobar et al., 2019).However, studies using immunological and/or molecular techniques show that the majority of horses are asymptomatic, due to the complexity of modulating the immune response in these animals.(Mukhtar et al., 2000;Kenubih et al., 2015;Rolão et al., 2005;Soares et al., 2013).
The impact of L. infantum infection in horses and epidemiological conversion remains uncertain.Added to this, the susceptibility of horses to infection by many species of Leishmania spp.and the fact that the vast majority of cases are asymptomatic, the objective of this study was to report the detection of Leishmania infantum in a naturally infected horse in an endemic area for Leishmaniasis in Northeast Brazil.

CASE REPORT
A male horse, of the Manga Larga Machador breed, four years old, residing in the municipality of Bezerros (-8 ° 14´00´´ S 37 ° 47´49´´ W.GR), state of Pernambuco, Northeast, was treated in Brazil (Figure 1).On physical examination, the presence of ulcerated and non-ulcerated nodular lesions was observed on the face close to the masseter muscle (Figure 2A), in the ventral region (Figure 2B), and below the humeroradiounar joint (Figure 2C).Presence of papules in the scapular region (Figure 2D), in addition to an enlarged submandibular lymph node (Figure 2E).All lesions evolved over 4 months.Initially, Habronemosis was ruled out and the differential diagnosis was carried out for Leishmaniasis, as the region had cases in dogs and humans.
For diagnosis, two biopsies of the ulcerated skin on the face were performed with the aid of a punch (4mm).One of the biological samples was stored in 10% buffered formalin and the other in Eppendorf and frozen at -20°.An exfoliative cytology of the skin was performed, which made it possible to visualize amastigote forms of Leishmania sp.In addition to blood collection for subsequent biochemical, hematological, and serological analysis.
In the hematological evaluation, a complete blood count was then performed using an automatic cell counter (Labtest SDH-3 vet).Normocytic normochromic anemia and leukocytosis due to neutrophilia and eosinophilia were detected (Table 1).The biochemical analysis was performed using the Bioclin 1000 automated analyzer, where it was possible to evaluate urea, creatinine, alanine aminotransferase (ALT), and alkaline phosphatase (AF), in addition to total protein, albumin, and globulin.As a result, their respective levels were unchanged, except for the high urea levels.For immunohistochemical analysis (IHC), a skin fragment was collected and placed in an individual vial containing a 10% formaldehyde solution buffered with 0.01M sodium phosphate and pH 7.3 for 48 hours for fixation and subsequently stored in solution of 70% ethyl alcohol.The Endogenous peroxidase was blocked by adding 4% hydrogen peroxide (30 volumes) to the PBS bath for 30 minutes at room temperature.To block nonspecific responses, 6% skimmed milk was added and the slides were incubated in a humid chamber for 30 minutes at room temperature.
For amastigote forms, serum from a naturally infected dog was used as primary antibody at a dilution of 1:100 in sufficient quantity to cover the fragments.
The slides were then incubated in a humid chamber for 18 hours at 4°C and then the secondary antibody was added and incubated in a humid chamber for 30 minutes at room temperature.Next, work was carried out with streptoavidin-peroxidase and, as a revealed solution, hydrogen peroxide was used for five minutes at room temperature.Then, the slides were washed in running water, dehydrated in increasing amounts of alcohol (70% absolute), clarified in xylene and mounted with Entelam TM balm.
For the positive control, a slide with a histological section of organs from naturally infected dogs with high parasitism was used.The immunohistochemistry technique was performed to detect amastigote forms of L. infantum, according to Tafuri et al., (2004) this is an alternative method for detecting Leishmania in tissues.During the parasitological diagnosis, a typical positive immunohistochemical reaction was observed in the patient's biopsy, indicating stained amastigotes (Figure 3).
For molecular diagnosis, DNA extraction was performed from a fragment of a skin biopsy sample obtained with the Qiagen QIamp ® DNA Blood Mini Kit (Qiagen, CA, USA) according to the manufacturer's instructions.The sample was amplified for kDNA using primers MC1 and MC2, specific for the Leishmania donovani complex according to Cortes et al., (2004).Amplicons were purified using ExoSAP-IT PCR Product Cleanup Reagent (Applied Biosystems from Thermo Fisher Scientific -BR).

RESULTS AND DISCUSSION
In this work, infection by L. infantum in a horse in an endemic area for canine visceral leishmaniasis is reported.It is known that infections in horses caused by the agent Leishmania infantum have been described on some continents, mainly in South America and Europe (Rolão, 2005;Müller et al., 2009;Müller, 2005).
Regarding clinical manifestations, equine leishmaniasis generally presents papular or nodular lesions in the vectors' usual feeding sites, lesions compatible with the patient in this report (Mhadhbi and Sassi, 2020).In some cases, the host's immune system develops several mechanisms modulating its immune response, making the host asymptomatic, thus avoiding clinical suspicion of leishmaniasis (Koehler et al., 2002;Soares et al., 2013;Müller et al., 2009 ;Feitosa et al., 2012).
As for the eating habits of vectors, when choosing the host that will be their food source, it is more related to availability than to the sandflies' preference for a specific species, according to studies by Afonso et al., (2005).
Brazilian Journal of Animal and Environmental Research, Curitiba, v.7, n.1, p. 704-716, 2024 However, as horses on rural properties have greater contact with canines, and knowing that these are the main domestic reservoirs of the infectious agent, they become more susceptible to infection (Escobar et al., 2019).
Horses with leishmaniasis present changes such as anemia, neutropenia accompanied by uremia, due to anemia in the patient leading to acute kidney injury, neutropenia occurs through the ability of the infectious agent to modify the oxidative metabolism of its host, inducing programmed cell death of immune system cells (Escobar et al., 2019).
For the diagnosis of leishmaniasis, the immunohistochemistry technique has been used, which consists of a technique based on the detection of the parasite, through staining in tissue fragments (Rosa, 2020).Morales et al., (2010) prove symbolic lesions of Tegumentary Leishmaniasis in an Equus asinus.This diagnostic method provided accurate results in diagnosing the disease (Kenner et al., 1999).Be assertive in identifying the infection in the patient in this report.
Although parasitological techniques are capable of identifying parasites, they have low sensitivity when compared to molecular biology techniques (Reithinger and Dujardin, 2007).
Diagnosis through Polymerase Chain Reaction (PCR), which allows the identification of the subgenus or even the species, thus enabling better treatment targeting (Faber et al., 2003).
The treatment of leishmaniasis in horses occurs in two stages, one of which is the surgical approach in which there is excision of the nodules associated with the use of medications such as fluconazole and amphotericin, in addition to the use of pentavalent antimonials (Reuss, 2013).
The therapy recommended in this report was through the administration of marbofloxacin, since this medication has very promising leishmanicidal properties in combating L. infantum, as in non-cytotoxic concentrations it exerts an immunomodulatory effect on infected phagocytic cells (Amante et al., 2020).
Regarding its prophylaxis and control in horses, it is carried out through early diagnosis, use of repellents and insecticides against insect vectors, preventing blood meal and subsequent infection, in addition to a therapeutic approach in sick animals (Mhadhbi and Sassi, 2020).

CONCLUSION
Horses infected by L. infantum may present dermatological lesions.Therefore, early diagnosis and treatment are recommended, as it is suggested that the use of marbofloxacin provides good results in the regression of skin lesions in these cases.However, more studies are needed to investigate the epidemiology and pathophysiology of this disease in horses.

V
.C.M= Average Corpuscular Volume C.H.C.M= Average Corpuscular Hemoglobin Concentration Brazilian Journal of Animal and Environmental Research, Curitiba, v.7, n.1, p. 704-716, 2024 paraffin block slides were initially dewaxed in two different xylene solutions for 15 minutes each and hydrated in alcohol (3 minutes each in 70% Absolute II alcohol).
As a therapeutic protocol, injectable marbofloxacin was used to treat this patient at a dosage of 2mg/kg once a day, intramuscularly for 28 days, seeking clinical improvement (Figure2f-D7, D14, D28).

Figure 2 :
Figure 2: A) Ulcerated and non-ulcerated nodular lesions on the face close to the masseter muscle, 5cm in diameter; B) ulcer below the region of the humeroradiounar joint (20 mm); C) Ulcer in the ventral region (40 mm) and metacarpal region (10 mm); D) Presence of papules in the neck region; E) Enlarged submandibular lymph node F) Clinical evolution of a horse with Leishmaniasis: D7-seven days after the start of treatment; D14-fourteen days after the start of treatment; D28-twenty-eight days of treatment.

Table 1 :
Hematological parameters in a horse with Leishmaniasis.