Dogs may carry Leishmania tropica and Leishmania major in their blood circulation: a molecular and hematological study

Abstract

Background

Dogs may be infected with species of Leishmania parasites that are disseminated through blood circulation and invade the internal organs. In this study, we aim to detect the parasite in the blood of dogs using the PCR technique. The present work was performed from February 2022 to May 2023 in Fars Province, southern Iran, where the disease is endemic.

Results

In total, 7(5.1%) out of 135 blood samples, six were identified as Leishmania tropica and one as Leishmania major. We found no trace of Leishmania infantum, which is always known for visceral infection. In addition, no sign of cutaneous lesions or a significant disease was seen in the animals infected with both species. Of 48 dogs with anemia, two were Leishmania positive. The mean value of hematological parameters in the infected dogs was within the normal range except for a significant reduction in the platelet measures (p < 0.05).

Conclusions

Our data revealed that both Leishmania species, tropica and major, may manifest as viscerotropic leishmaniasis. More investigations are needed to understand the conditions under which these species choose the type of infection. Moreover, our data emphasize the role of asymptomatic dogs in carrying these parasites, a crucial factor in spreading the disease.

Background

Leishmaniasis is a contagious disease that infects both people and domestic and wild dogs worldwide. The disease has been epidemiologically expanded, and new methods of diagnosis have corroborated that the Leishmania species are progressively spreading to different hosts [1, 2]. Compared to conventional diagnostic methods, the molecular techniques are very accurate, sensitive, and reliable ways to find infections at the early stage, especially in people and reservoirs that are hiding infections or without any clinical signs [3, 4]. The usefulness of the PCR-based methods has been assessed for a range of samples, such as skin, bone marrow and blood. Among different genes, the internal transcribed spacer (ITS) region has different sizes and nucleotide sequences for each species. Therefore, it is a suitable molecular marker for tracking and identifying Leishmania species in dog hosts [5].

Based on the clinical manifestations, domestic dogs may show cutaneous symptoms, mainly associated with Leishmania tropica and Leishmania major, while Leishmania infantum is the main cause of visceral leishmaniasis (VL). Dogs are usually thought to be the principal domestic vectors of L. infantum, which causes zoonotic VL in humans [6].

Leishmaniasis, due to the three species mentioned, has been frequently reported and is endemic in the Middle East region of Asia [7]. A recent systematic review in Iran showed that canine visceral leishmaniasis (CVL) is endemic in at least half of the country's provinces, with an estimated overall prevalence of 16% [8]. According to the available molecular studies, L. infantum is the main species involved in visceral infection in canid hosts [8, 9]. In other research, molecular data implicated that dogs with visceral or blood infection may carry the other species, particularly L. tropica [10,11,12]. In addition, the detection of Leishmania in the blood could be explained by the fact that they should be suspected as a reservoir for cutaneous disease. Such findings help to understand the transmission of this disease (as the reservoir host) and, therefore, to prevent and control the spread of leishmaniasis [13].

The present cross-sectional study aimed to investigate and identify the Leishmania parasite in dogs' blood circulation using the molecular approach. The sequence data of the two highly variable ITS sections (ITS-1 and ITS-2) were used to characterize the Leishmania species. In addition, we attempted to determine any possible associations between the parasite contamination and the hematological alterations.

Materials and methods

Sample collection and DNA extraction

This study was conducted on domestic dogs referred to the Clinic of Veterinary Medicine, Shiraz University, Fars Province, Iran. This area is located in an arid and semi-arid region of southern Iran and covers a 52,069-square-mile area (8.09% of the entire country) at 27°03′ and 31°42′N latitudes and 50°30′ and 55°36′E longitudes (Fig. 1).

Fig. 1

Map of sampling area in Fars province, southern Iran

A total of 135 blood samples were taken through the jugular vein and stored in EDTA tubes. Sampling was done without anesthesia and with the owner's consent. A complete blood count was performed by a Veterinary Hematology Analyzer (Nihon Kohden, MEK-6450 Celltac Alpha, Tokyo, Japan). Standard blood smears were also prepared, and the remaining blood samples were used for DNA extraction. Genomic DNA was extracted from 200 μl of whole blood using the commercial Blood Genomic DNA Purification Kit (Parstous®, Iran). The quantity and purity of the extracted DNA were checked using a NanoDrop spectrophotometer (Thermo Scientific, NanoDrop, USA). The collected DNA was stored at − 20 ◦C until use.

Polymerase Chain Reaction (PCR)

In this study, the infection with Leishmania was determined by PCR reactions targeting the ribosomal DNA regions. Primers LSGITS1-F1: 5’-CATTTTCCGATGATTACAC-3’, LSGITS1R1: 5’-CGTTATGTGAGCCGTTATC-3’, and LGITSF2: 5’-GCATGCCATATTCTCAGTGT-3’, LGITSR2: 5’-GGCCAACGCGAAGTTGAATTC-3’ were used for the identification of ITS1 and ITS2 regions [14]. The amplification reaction was carried out in a total volume of 20 μl containing 10 μl PCR premix (Ampliqon, Denmark), 1 μl (10 pmol) of each primer, 5 μl H2O, and 3 μl (~ 30 ng) of DNA as a template. In this study, a touchdown PCR program was used to delete irrelevant products or smears. The amplification condition was as follows: An initial denaturation at 95°C for 5 min, then one cycle of denaturation (1 min at 94°C), annealing (1 min at 58°C), and extension (45 s at 72°C), continued with three PCR cycles again with annealing temperatures at 57°C, 56°C, and 55°C (for 1 min), followed by 35 cycles as above except a final annealing temperature at 54°C (for 1 min). After the last cycle, an extension step was applied for a further 5 min at 72°C. The PCR products were subjected to electrophoresis on a 3% agarose gel stained with Safe DNA Gel Stain (Sinaclone®, Iran) and visualized under ultraviolet light.

Products were directly sequenced (Pishgam Biotech Co., Tehran, Iran), and the results were compared with other available sequences in the NCBI using the BLAST search. Our sequences were aligned with homologous sequences existing in the GenBank database using the Clustal W program performed by MEGA software (version 11). The phylogenetic tree was constructed based on the maximum likelihood method, and analyses were carried out using the Kimura 2-parameter distance estimate [15].

Statistical analysis

Animals included in this study were assigned to Leishmania positive and negative groups, according to the PCR results. The mean values of the haematological parameters were compared between the groups. In order to test whether the leishmania infection may relate to anemia in dogs, the data were also divided into two groups according to the hematocrit (Hct) being under or above the normal percentage of 37. Based on the normal or abnormal spread of each data set, a parametric or non-parametric statistical approach was applied using an independent sample t-test and Mann–Whitney analysis, respectively.

Results

Among the 135 dogs sampled, seven (5.1%) were positive for Leishmania DNA. Of 48 anemic dogs, only two cases showed the parasite in their blood circulation. The Hct Value (%) in those two dogs were 31.2% and 26.7%. The infected animals did not show any signs of cutaneous invasion.

The results of measured hematological parameters in Leishmania positive and negative dogs are represented in Tables 1 and 2. Analyses on RBC-related parameters did not accompany with significant differences. In the Leishmania positive group, means of RBC, PCV and hemoglobin were all in the range of normal values (Table 1). In this study, the platelet count was the only hematological factor significantly lower in the infected dogs (p < 0.05). However, compared to the non-infected group, we did not conclude any remarkable difference in white blood cell counts of the infected dogs (Table 2).

Table 1 Mean ± SEM of the hematological parameters related to the RBC (Red blood cell) value

Table 2 Mean ± SEM of Platelet count, Mean Platelet Volume (MPV), and the total WBCs with its differential measurements

Molecular analysis

The PCR amplification based on the ITS region revealed seven Leishmania-positive samples. Fragments about 260 and 450 bp were successfully amplified and sequenced using primers specific to ITS-1 and ITS-2 regions, respectively. The sequence analysis of the ITS-1 region showed that six samples were identified as L. tropica and one was L. major. The ITS-1 sequences obtained for L. tropica (252 bp) and L. major (266 bp) were recorded in the GenBank under the accession numbers OR237833 and OR237832, respectively.. No intraspecific variation was observed among Iranian obtained sequences. The infection with both species was also corroborated by the sequencing of ITS-2 amplicons, which were recorded in the GenBank as OR229790 (L. tropica) and PP094785 (L. major). The expected size for the ITS-2 bands of L. tropica (415 bp) and L. major (434 bp) was observed on the agarose gel. However, L. infantum yields 418 bp-sized fragments, close to L. tropica. Here, sequencing helped us to distinguish the species.

The phylogenetic trees inferred from data on ITS-1 and ITS-2 are demonstrated in Fig. 2 (A and B, respectively). Both the cladograms show that the ITS region has separated different species well and placed them in a different clade. According to the pairwise distance analysis of the ITS-1 region, the present L. tropica strains had a distance value of 0.00 to 0.016 from the other reported Iranian isolates. This value reached the 0.025 with other GenBank records (Table 3). Similar results were obtained considering the ITS-2 region, except for much higher distance values of 0.078 and 0.041 with records from human specimens in Ethiopia and Phlebotomus sergenti from Israel, respectively. The analysis of the ITS region showed that the only L. major specimen identified in this study resembled other reports from geographical and host resources (Table 4).

Fig. 2

Phylogenetic tree of the Leishmania species found in blood of dogs based on analyzes on the ITS1 (A) and ITS2 (B) regions using the Maximum likelihood method. The numbers associated with nodes represent the percentage of 2000 bootstrap reps and the horizontal distance is proportional to hypothesized evolutionary change (scale bar)

Table 3 Pairwise distance between sequences of the L. tropica obtained in the present study and some of the similar species recorded before in the GenBank. Other Leishmania species are included for more comparisons

Table 4 Pairwise distance between sequences of the L. major obtained in the present study and some of the similar species recorded before in the GenBank. Other Leishmania species are included for more comparisons

Discussion

In the present study, the infection rate of the blood samples was 5.1%. In terms of species involved, L. tropica and L. major were confirmed using the molecular test. In a previous study in southern Iran, 23% of blood samples of domestic dogs were PCR-positive and the only identified species was L. infantum [16]. A few years later, the infection with L. infantum and L. tropica was detected in three (5%) buffy coat samples of dogs living in a similar region [17]; however, the sera infection rate of those animals was 46.7%. In the North of Iran, three species of L. infantum (18%), L. tropica (4%), and Crithidia spp. (3%) were also detected in the blood of dogs [12]. However, Bamorovat et al. [10] detected only two L. tropica-positive specimens from visceral tissue samples taken from stray dogs [10]. These variations in the frequency of the diseased animals and the species detected may originate from differences in sample size, geographical location and climate, method of detection and gene regions used, season of sampling and activity of vectors [18].

A comparative investigation of the mentioned studies reveals that L. tropica and, according to our study, L. major are specified to establish visceral infection. Dogs have been regarded as the significant reservoirs of VL for human infection [9, 19]. In older literature, the etiology of CVL in domestic and wild canines was mainly attributed to L. infantum in the old and new worlds [20]. This species was not traced in the current study. Therefore, the presence of L. tropica and L. major in blood circulation should be highlighted because both species are often detected in skin scrapes. This is a crucial result, as those infected dogs could be a suspected reservoir for cutaneous leishmaniasis and form a basis for transmission of the parasite to canine and human hosts. In our study, an animal with blood infection with L. major was detected. However, the animal had no sign of skin lesions. According to the literature at least in Iran, this was a new finding that L. major is detected in the blood of infected dogs. Previously, L. tropica was reported to have the potential to be an etiologic agent of visceral Leishmaniasis in Morocco [21, 22]. This species has been shown before for its capability of organ invasion known as the Kala-azar in humans [23].

The Leishmania-positive animals in the present study represented alterations in thrombocyte count. Also, mild anemia was seen in two animals, but no evident cutaneous lesion or significant visceral disease was seen in them. In line with our observations, meta-analysis of studies in Iran indicates that 81% of the infected dogs were asymptomatic [8]. Other surveys from other parts of the world also show that exposure of dogs to Leishmania is mostly not coincident with clinical disease [24]. Thus, in asymptomatic dogs, the focus should be on their possible role in parasite transmission. In addition, the low infection rate in the present study is maybe because the animals were households and probably had much less exposure to the vector [25].

In the present study, the values of most hematological parameters were within the normal range. This finding is consistent with the lack of significant clinical signs in the infected dogs. Of all parameters measured, only the platelet value was significantly lower in PCR-positive dogs. In several clinical and experimental studies of Leishmania-infected dogs with or without epistaxis [26, 27], hemostatic abnormalities and thrombocytopenia was not severe enough to cause spontaneous bleeding. Still, one of the observed hemostatic abnormalities was described as thrombocytopathy [27, 28]. A number of previous studies reported a low frequency of thrombocytopenia in the infected dogs, especially in asymptomatic cases [29,30,31]. In contrast, thrombocytopenia and anemia were more common in dogs with acute leishmaniasis and the clinical symptoms were due to bone marrow and renal and other organ dysfunctions [32]. Since anti-platelet antibodies have been found in dogs with leishmaniasis, an immune-mediated peripheral elimination of circulating platelets is the most plausible cause of thrombocytopenia [33,34,35]. In this study, a significant decrease was seen in platelet numbers in three out of seven infected dogs. They were clinically healthy, but no information was available regarding the co-infection disease. Therefore, additional studies are suggested to investigate the extracted DNA material for the presence of other protozoans using the appropriate molecular approach.

Conclusion

To estimate the prevalence of Leishmania parasite infection, our study used the PCR method based on the ITS region, investigating visceral infection in blood samples of dogs in an endemic area in Fars Province, southern Iran. The results of this study did not show any trace of L. infantum. However, the other two detected species, L. tropica and L. major were found in the blood specimens with no history of notable disease. Therefore, clinically healthy dogs can transfer the parasite to other hosts and owners in areas with densities of sandflies.