Canine leishmaniasis caused by Leishmania tropica in southeastern Iran: a case series study

Abstract

Dogs are the primary reservoirs of Leishmania infantum (L. infantum), but Leishmania tropica (L. tropica) infection is also possible in dogs and can transmitted to humans. The southeast of Iran experiences a high prevalence of canine leishmaniasis (CanL), and veterinarians frequently encounter symptomatic cases. Therefore, from December 2017 to November 2022, the present case series was designed to assess the prevalence of CanL in owned dogs with cutaneous manifestations resembling CanL. A total of 500 owned dogs with dermal lesions from two endemic cities, Zabol and Kerman, were enrolled. Impression smears from skin lesions and popliteal lymph nodes were prepared from all cases, whereas blood samples were gathered from amastigote-positive dogs for serological and molecular surveys. Commercial ELISA was done on sera samples, and two-step nested PCR was used on extracted DNA to amplify variable fragments of the Leishmania species’ kinetoplast DNA (kDNA). Leishman bodies were microscopically detected in 7.2% (36/500) of dermal smears. Of the 360 owned dogs from Zabol, 2 have been diagnosed with L. tropica, and 10 have been diagnosed with L. infantum. Out of 140 owned dogs from Kerman, 8 were identified with L. tropica infection, and 16 were infected with L. infantum. Molecular results revealed the presence of 750 full dual-band bases related to the L. tropica species in 10/36 (27.7%) cases, which showed a considerable increase in canine cutaneous leishmaniosis compared with previous studies in southeastern Iran. The noticeable prevalence of L. tropica in owned dogs indicated that the dog’s role in cutaneous leishmaniosis should be re-evaluated as a possible animal reservoir in endemic areas.

Introduction

Six critical tropical diseases have the potential to cause human death, including Visceral Leishmaniasis (VL)^1,2. More than 72 developing countries are affected by leishmaniasis³. It is estimated that Cutaneous Leishmaniasis (CL) is affected yearly by 1-1.5 million people worldwide^4,5. In addition, it is estimated that around 1 billion people are at risk of infection by the disease^6,7. It has been reported that 90% of the global cases of VL are found in Iran, Saudi Arabia, Afghanistan, Algeria, Brazil, Peru, and Syria, among which the incidence of the disease in Iran is among the highest^8–10. Cutaneous leishmaniasis and VL are both prevalent in Iran^11,12. In Iran, CL and VL were reported from many provinces, with emphasis on Kerman^13,14, Razavi Khorasan¹⁵, North Khorasan^16–18, Sistan-va-Baluchestan^19,20, Golestan²¹, Isfahan^22–24, Semnan²⁵, Yazd^26–28, Fars²⁹, Bushehr^30–33, Hormozgan^34–36, Khuzestan^37,38, Ardebil^39,40, Ilam^41–46 and other parts^47–56. Canine leishmaniasis is an important disease reported many times in Zabol and Kerman cities^13,57–70 (Table 1).

Table 1.

Prevalence of CL and VL in animal and human population in Kerman and Sistan & Baluchestan Provinces, southeastern Iran.

Province	Sample	CL/VL	Agent	Infection rate	Methodology	Author & year
Kerman	Human	CL	L. major	4.7%	Direct smear microscopy / nested-PCR	(Khosravi et al., 2013)
Kerman	Human	CL	L. tropica L. major	(95.6%), (4.4%).	Skin scraping smears / nested-PCR	(Sharifi et al., 2012)
Kerman	Rodents / Stray dogs	CL	L. major	33% rodents 0% dogs	Smear microscopy / histopathological / PCR	(Ghaffari et al., 2014)
Kerman	Humans/ owned dogs	VL	L. infantum	3.22% Children 23% dogs	Direct agglutination test	(Mahmoudvand et al., 2011)
Kerman	Stray cat	VL	L. infantum	%6.7, 16.7%	ELISA, PCR	(Akhtardanesh et al., 2017)
Kerman	Stray dog	VL	L. infantum	11.25%	Direct agglutination test / Pathology	(Bamorovat et al., 2014)
Kerman	Humans	CL	L. tropica	5.3%	Smears / cultures / PCR	(Sharifi et al., 2011)
Kerman	Sand flies, Phlebtomus sergenti	CL	L. tropica	(3.6%)	Nested-PCR	(Mozafary et al., 2016)
Kerman	Canine	VL	L. infantum	11% stray dogs 3% sheepdogs	Direct agglutination test	(Afshar et al., 2018)
Kerman	Human	VL	L. infantum	2.6% titers ≥ 1:800 0.6% titers ≥ 1:3200		(Abbaszadeh-Afshar et al., 2015)
Kerman	Human	CL	L. tropica	4.9%	Smear scrapings	(Aflatoonian & Sharifi, 2007)
Kerman	Human	CL	L. major	0.34%	Smear scrapings	(Askari et al., 2018)
Kerman	Human	CL	L. tropica L. major	(88.5%) (11.5%)	Smear scrapings / PCR	(Ramezany et al., 2018)
Kerman	Dogs	CL	L. infantum	15.4%	ELISA	(Mahshid et al., 2014)
Sistan & Baluchestan	Dogs	VL	L. infantum	5.33%	IFA	(Akhtardanesh et al., 2021)

Canine leishmaniasis is a chronic pansystemic disease manifested by many clinical signs and clinical findings^71,72. One of dogs’ most prevalent clinical findings are skin lesions and local or generalized lymphadenomegaly⁷³. Several species of Leishmania can cause various forms of the disease, and leishmaniasis will probably become an emerging zoonosis and a public health problem in the coming years^74,75. It is important to note that while dogs are the primary sources for transmitting L. infantum, clinical diseases associated with L. tropica are also possible in dogs and can be transmitted to humans^76–78. Diagnosing CL in dogs is usually possible by observing the impression smears of the skin lesions, which are stained with Giemsa stain. Molecular methods could confirm the infection and specify the species^79,80. Although PCR is the most effective method of detecting CVL, it is still applied only for research purposes and individual diagnosis because it lacks standardization⁸¹. Several target genes have been identified for detecting Leishmania using the PCR procedure⁸². The most specific tests are used for species identification, while the most sensitive tests are used for generic diagnosis.

To the best of our knowledge, a few cases of L. tropica-infected dogs were reported from Morocco and Iran^83–86. In the current case series, several dogs with skin lesions resembling VL were recently noted in teaching veterinary hospitals in southeastern Iran, and they are being investigated for possible links to leishmaniasis.

Methods and materials

Study area

The study was conducted between December 2017 and November 2022 in the Zabol and Kerman provinces of Iran, situated in the country’s southeast. Sistan and Baluchestan are situated between pivot points of 30 and 18 min in the northern region and 31 and 20 min in the eastern region. Sistan and Baluchestan, from the north and east, are neighbors of Afghanistan. Deserts bound its west and northwest. Sistan and Baluchestan are between 475 and 500 m above sea level. Zabol city is the capital of Sistan and Baluchestan. It has been reported for 20 years that maximum temperatures are between 22 and 49 °C and minimum temperatures are between − 8 °C and 15 °C. Approximately 59 milliliters of rain are reported to fall in Sistan and Baluchestan annually, and the humidity is also 40%.

The latitude of Kerman is 30.283938, and the longitude is 57.083363, with elevations ranging from 1700 to 2700 m above mean sea level and an average annual rainfall of about 120 mm. As a province of 180,726 km² with a population of nearly 3 million, Kerman is Iran’s largest province. The city of Kerman embraces about 80% of the provincial urban population, making it the most developed and the largest city in the province, with a current population of about 550,000 (Fig. 1).

Fig. 1.

Map of Iran. This figure illustrates the sampling area in the present study. The map was drawn by using ArcGIS software version 10.3 (https://enterprise.arcgis.com/en/portal/).

Sampling

A total of 360 owned dogs by Zabol and 140 by Kerman were enrolled in the study. They had skin manifestations resembling leishmaniasis, including focal alopecia, nose and pad hyperkeratosis, dermal lesions on the muzzle, eyelids, pinnae, and onychogryphosis. They were all referred for dermatologic problems to the teaching veterinary hospitals. Demographic data, including age, breed, sex, and location of habitation, were documented. Physical examination data were recorded in a questionnaire.

Microscopic examination

Impression smears from all 500 cases with dermal lesions were prepared. Afterward, 70% ethanol was used to fix the sample for 30 to 60 s. The smear dried, and Giemsa solution was poured onto it for 30–50 min. Finally, the slides were immersed in water for a short time, then quickly removed from the water and air-dried. Microscopic examination was done with eyepieces 10, objective lenses 10, 40, and 100, and immersion oil without coverslips.

Fine needle aspiration of the popliteal lymph node was also prepared for microscopic examination in all 500 cases under short chemical restraint by IV injection of 5 mg/kg ketamine (Alfasan, Nederland) and 0.5 mg/kg midazolam (Caspian Pharmaceutical Company, Iran).

Skin biopsy

In the next step, a full-thickness punch biopsy of the skin lesion was taken under short general anesthesia induced by IV injection of 5 mg/kg ketamine and 0.5 mg/kg midazolam and submitted for PCR (ribosomal DNA testing).

Serological examination

From suspected dogs (36/500; 7.2%), in which amastigotes were seen in their impression smear, 3 ml blood samples were taken from the cephalic veins. Serum samples were separated by centrifugation at 3000 rpm for 3–5 min and stored at -20 °C for serological examination. The ELISA was performed using an indirect kit (ID Screen Canine Leishmaniasis, ID-Vet Company, France) as instructed in the manufacturer’s manual, and the samples were read at 450 nm using an ELISA reader (ELX800 BioTek, USA). Validation of the ELISA test was determined by the difference between the mean optical density of the positive control (ODPC) and the mean optical density of the negative control (ODNC) being > 3 (ODPC/ODNC > 3).

Molecular techniques

DNA extraction

DNA extraction was performed using the Gene All Biotechnology commercial kit (South Korea) on cutaneous ulcer fragments. Following the manufacturer’s instructions, 15µL of proteinase K was initially added to 1.5 ml microtubes. Then, full-thickness skin samples with diameters of 3 mm to 4 mm and 200µL of BL buffer were added to each microtube. The microtubes were vortexed and kept in a Bain-marie at 56 °C for half an hour. Subsequently, the centrifugation steps were carried out according to the kit’s instructions, and the DNA was transferred to a freezer at -18 °C.

Nested PCR assay

Using extracted DNA, Leishmania species’ kinetoplast DNA (kDNA) was amplified by two-step nested PCR. As described in a recent study, the primers were designed specifically for this analysis⁸⁷. In the first step, the external primers CSB2XF (5′-CGAGTAGCAGAAACTCCCGTTCA-3′) and CSB1XR (5′-ATTTTTCGCGATTTT CGCAGAACG-3′) and the second-step, the internal primers 13Z (5′-ACTGGGGGTTGGTGTAAAATAG-3′) and LiR (5′-TCGCAGAACGCCCCT-3′) were utilized. In the first round of each, 25 µl reaction mixture, 5 µl template DNA, 12.5 µl Taq DNA Polymerase Master Mix Red (Ampliqon, Denmark), and 30 picoM of each primer of CSB2XF and CSB1XR were applied together. After 5 min at 95 °C, 35 cycles of 30 s at 94 °C, 1 min at 55 °C, and 1 min at 72 °C were carried out. This was followed by a 5-minute extension at 72 °C. 1 µl of 1:9 diluted PCR product of the first round was used as a template for the second PCR round. LiR and 13Z primers were used in this step, with the same conditions and reaction mixture as in the first. Amplified DNA was electrophoretically separated using an agarose gel electrophoresis, ethidium-bromide pre-staining, and ultra-violet analysis. The presence of 750 bp bands for L. tropica and 560 bp for L. infantum was recorded in positive samples.

Results

Amastigotes were seen in the impression smear of 36 dogs, and FNA of the popliteal lymph node was positive in 28 cases. Molecular investigation of skin biopsy samples showed that among the target population in Zabol, 0.55% (2/360) have been diagnosed with L. tropica, and 2.77% (10/360) have been diagnosed with L. infantum. In Kerman, 5.71% (8/140) were identified with L. tropica, and 11.42% (16/140) were infected with L. infantum (Table 2).

Table 2.

Signalment of 36 leishmania-infected dogs with skin lesions in this study.

Parameter		L. infantum number (%)	L. tropical number (%)
Age	> 3 years	10 (27.7%)	4 (19.9%)
	3–6 years	10 (27.7%)	4 (56.2%)
	6 years <	6 (16.66%)	2 (8.4%)
Sex	Male	16 (10.4%)	6 (49.8%)
	Female	10 (5%)	4 (34.8%)
Location	Kerman	16 (44.44%)	8 (22.22%)
	Zabol	10 (27.77%)	2 (5.55%)
Clinical findings	Focal alopecia	5 (19.23%)	3(30%)
	Hyperkeratosis/foot pad	5(19.23%)	-
	Hyperkeratosis/plenum nasal	10 (38.46%)	1(10%)
	Erosive lesions/ muzzle	6 (23.07%)	5(50%)
	Erosive lesions/ planum nasal	10 (38.46%)	3(30%)
	Erosive lesions/ eyelids	3 (11.53)	1(10%)
	Erosive lesions/ pinnae,	2 (7.69%)	1(10%)
	Onychogryphosis	3 (11.53%)	-
	Pododermatitis	-	1(10%)
	Lymphadenomegaly	22(84.61%)	1(10%)
	Cachexia	5(19.23%)	1(10%)
	Nasal bleeding	3(11.53)	1(10%)
	Hepatosplenomegaly	5(19.23%)	1(10%)

Zinc-responsive alopecia, discoid lupus erythematosus, localized demodicosis, dermatophytosis, and bullous pemphigoid, were the most common other skin disorders that were differentiated in suspected cases in the present study.

One case of L. tropica-infected dogs from Zabol had hyperkeratosis lesions, pustular dermatitis on the muzzles, and pododermatitis, and the second case had mucocutaneous ulceration on the muzzle. Leishmania tropica-infected cases from Kerman had different clinical signs, such as erosive to ulcerative lesions on the planum nasal, lower lip, and supra-ocular regions. Clinical manifestations of all infected dogs with L. tropica were presented in serial images (Fig. 2) (Table 3). Among L. infantum-infected dogs of Zabol, lymphadenomegaly and erosive lesions on the planum nasal and muzzle were the common clinical findings.

Fig. 2.

Case series of the current study. The present study cases that were infected with L. tropica. Location: K = Kerman Z = zabol.

Table 3.

Clinical and cytological findings in 10 Leishmania Tropica-infected dogs.

Dog ID	Signalment (Breed, gendera, ageb)	Clinical findings	ELISA	Popliteal lymph node impressions
1 K	Mix, F, 9yr	Focal erosive lesions on planum nasal and lower lip, focal alopecia	N	P
2k	Mix, M, 2yr	Erosive lesion on supra-ocular regions	N	P
3 K	Pomeranian, F, 3yr	Severe ulcers on nasal planum, generalized lympadenomegaly, cachexia	P	P
4 K	Husky, M, 9yr	Erosive lesions on the planum nasal	N	N
5 K	German shepherd, M, 6yr	Hyperkeratosis in muzzle	N	N
6 K	Alaskan malamute, M, 3yr	severe crusting and ulceration of the face, focal alopecia	N	N
7 K	Labrador, F, 3yr	Mucocutaneous ulceration on the muzzle	N	N
8 K	Mix, M, 2yr	Severe mucocutaneous ulceration on the muzzle, epistaxis, hepatosplenomegaly	P	P
9z	Mix, F, 2yr	Mucocutaneous ulceration on the muzzle, pododermatitis	N	N
10z	Mix, F, 1yr	Mucocutaneous ulceration on the muzzle, focal alopecia	N	N

^aM=Male F = Female.

^byr=Year.

location: K = Kerman Z = zabol.

N = Negative.

P = positive.

Among 16 L. infantum-infected dogs of Kerman, 11 cases showed no other clinical signs except mild skin lesions and generalized lymphadenomegaly. However, 5 cases were in poor health condition and were euthanatized after obtaining owner consent.

All L. infantum-infected cases and two L. tropica-infected cases from Kerman were seropositive in the ELISA test. In these cases, amastigote was seen in a popliteal lymph node in cytology. Based on other systemic signs like cachexia and generalized lymphadenopathy, visceral involvement with L. tropica was suspected, which may cause a false positive reaction in the ELISA test. Bone marrow aspiration was done, which was positive in two cases. However, euthanasia was not accepted by owners (Fig. 3).

Fig. 3.

Bone marrow aspiration. Bone marrow aspiration was positive in case 3.

Molecular results showed the presence of 750 full dual-band bases related to the L. tropica species in 10 infected cases and a band of 560 bp, corresponding to L. infantum in 26 dogs (Figs. 4, 5 and 6).

Fig. 4.

Leishmania spp positive samples of Zabol. Sample 1 is a reference strain, samples 3 and 4 are two Leishmania tropica positive samples of Zabol, and samples 6 to 16 are ten Leishmania infantum positive ones in Zabol.

Fig. 5.

Leishmania tropica-positive samples of Kerman. Sample 2 is a reference strain, and samples 3 to 10 are Eight Leishmania tropica-positive samples of Kerman.

Fig. 6.

Leishmania infantum-positive samples of Kerman. Sixteen Leishmania infantum-positive samples of Kerman.

Excellent clinical improvement and initial response to allopurinol were seen in three cases of L. tropica-infected dogs (cases 1, 4, 7) that participated in a treatment protocol with 10 mg/kg allopurinol, given twice daily after two months. Owners did not report recurrence.

Discussion

In tropical and subtropical regions, leishmaniasis is one of the most important diseases⁸⁸. It has diverse prevalence worldwide, and L. tropica is one of the leading causes of the disease⁸⁹. The CL in dogs is emerging in some parts of the world^90,91. In certain parts of the world, dogs infected with CL are important reservoirs for transmitting the disease from dogs to humans^92,93. Clinical Symptoms are local lesions on the skin that can heal spontaneously or grow into chronic mucocutaneous lesions that eventually cause deformities in affected areas. The epidemiological patterns of CL in Iran have changed over the past two decades due to population migration between urban and rural areas and refugee flow from Afghanistan and Iraq to Iran^94,95.

The results of our study show that among 360 owned dogs from Zabol, two were infected with L. tropica. The mucocutaneous lesion was seen around the lips, nasal planum, and toenails. Infected cases from Kerman had clinical signs such as alopecia, upper and lower lip, erythema, and erosive dermatitis on supra-ocular regions. Nasal planum and hard pads, which are not hair-covered, are an extremely higher risk of bites and injuries than other body parts. Responsible pet ownership has a crucial impact on VL control. Still, unfortunately, the absence of mandatory legal considerations for isolation, treatment, or euthanasia for infected dogs in Iran presented a significant obstacle to the follow-up of many cases. Some owners were not responsive to follow-up efforts, abandoned treatment midway, or faced other challenges that hindered effective monitoring.

According to a study conducted in Morocco, seven dogs were diagnosed with cutaneous leishmaniasis caused by L. tropica⁸⁶. Four dogs in Essaouira and four in Azilal had cutaneous lesions out of 313 dogs examined. Solitary or clustered small ulcers were present around the muzzle. Samples were collected under general anesthesia for examination and culture using an Arquette scalpel. Lymphadenopathy was diagnosed by aspirating lymph nodes. Isoenzyme electrophoresis identified 3 strains from Azilal as L. tropica MON-102 and 4 from Essaouira as L. tropica MON-113. These seven dogs showed no signs of lymphadenopathy. In one dog from Azilal, an ulcer on the muzzle was caused by L. infantum MON-1. The animal also had lymphadenopathy, and L. infantum was isolated.

In the current study, 2 L. tropica-infected cases were positive in the ELISA test, which shows the cross-reaction between the two species; however, molecular tests are needed to differentiate between viscerotropic L. tropica and L. infantum. Furthermore, in another study of 471 dogs in the Kerman province, two were infected with a strain of L. tropic⁸⁵. Clinical examination revealed myiasis, alopecia, and hyperkeratosis of the footpads in one of the dogs. In addition to weight loss, cachexia, splenomegaly, and lymphadenomegaly, these manifestations were particularly prominent in the prescapular and popliteal lymph nodes. The phylogenetic analysis revealed that the positive sample in this research was closely related to the Iranian L. tropica kDNA with accession number AB678350. The present study confirms the emergence of canine CL caused by L. tropica, previously reported in western India⁹⁶.

The study presented herein delves into the prevalence of L. infantum and L. tropica infections in owned dogs exhibiting cutaneous manifestations in southeast Iran. Dogs, particularly in endemic regions, serve as the primary reservoirs for L. infantum, and this study reveals the possibility of L. tropica infections in owned dogs, emphasizing the potential transmission risk to humans. Skin impressions and blood samples were obtained from dogs with dermal lesions for serological and molecular examinations, respectively. The study’s molecular analysis, specifically Two-step nested PCR targeting variable fragments of Leishmania species’ kinetoplast DNA, revealed the presence of L. tropica and L. infantum in the sampled dogs. The results elucidate a significant prevalence of L. tropica in owned dogs, particularly in Kerman province. Moreover, 10 dogs in Zabol and 16 dogs in Kerman were infected with L. infantum, and these cities were considered endemic for canine leishmaniosis in previous studies^13,58.

These findings underscore the importance of re-evaluating dogs as potential animal reservoirs in endemic areas. Given the notable prevalence of L. tropica in owned dogs, the implications extend to public health, highlighting the need for comprehensive surveillance and control measures to mitigate the transmission risk to animals and humans in these regions^97,98. The potential transmission of these infections from dogs to humans underscores the interconnectedness of animal and human health, necessitating comprehensive surveillance and control measures⁹⁹. Canine and human leishmaniasis can be controlled by controlling sand flies and avoiding exposure using repellent antiparasitic collars in endemic areas and vaccinations^100–103. The study’s findings contribute to the scientific understanding of zoonotic diseases and advocate for a holistic approach that recognizes the pivotal role of animals in mitigating the impact of infectious diseases on both veterinary and human populations worldwide.

Conclusion

This case series showed that CL must be noticed as an emerging disease in owned dogs in Iran. Detection of new reservoirs of L. tropica in endemic CL regions is necessary to control and prevent the disease. As dogs may carry L. tropica asymptomatically, further investigation must be performed by aphidological surveys to assess and understand the real prevalence of this species in endemic areas, especially in free-ranging dogs. Complete surveillance of L. tropica-infected dogs for the progression of skin lesions or visceral invasion of this species must be evaluated in experimental studies.

Author contributions

Conceptualization by Baharak AkhtardaneshMethodology by Baharak Akhtardanesh, Soheil Sadr, Javad Khedri, Mehdi Bamorovat, Ehsan Salarkia, Iraj SharifiFormal analysis and investigation by All AuthorsWriting - original draft preparation by Soheil SadrWriting - review and editing by Baharak Akhtardanesh, Soheil Sadr, Javad Khedri, Mehdi Bamorovat, Ehsan Salarkia, Iraj Sharifi.

Data availability

The data presented in this study are contained within the article. Additional data can be provided on request from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed, and we confirm the study was carried out in compliance with the ARRIVE guidelines. Moreover, all experimental protocols were approved by the ethical committee of the Kerman University of Medical Sciences (ID: 910056).