Isolation and molecular identification of cutaneous leishmaniasis in humans and dogs in middle Euphrates, Iraq

1. Identification of tropica and L. major from human and dog skin lesions using kDNA by nested PCR.
2. There were non-significant differences p˃0.05 according to topica and L. major in humans, while there were significant differences p˂0.05 according to L. tropica and L. major in dogs.
3. 10 humans and 5 dogs ( tropica and L. major) local isolates skin lesion products were subjected to phylogenetic tree analyses.
4. Sequens analyses determined large number of major in humans and dogs, which give an indicator that dog is the principal zoonotic transmission disease to humans.

Introduction

Intracellular flagellate protozoa of the Leishmania genus causes Leishmaniasis, the vector-borne illness (1). All Leishmania species are spread to people and other animals by bites from infected insects, sandflies (Phlebotominae) (2,3). There are three basic types of Leishmaniasis: cutaneous (CL), mucocutaneous (ML), and visceral (VL) (4). Worldwide, CL is the most prevalent kind of Leishmaniasis. More than 350 million humans are at risk of contracting CL, which has been detected in 98 countries (5,6). Worldwide travel has increased the frequency of leishmaniasis cases in non-native nations. Anthropogenic Leishmaniasis and zoonotic Leishmaniasis are the two types of cutaneous Leishmaniasis, and they are both brought on by L. tropica and L. major subsequently (4,7). Unusual CL forms like ocular Leishmaniasis and uncommon forms with many nodular, ulcerative lesions that had distribution to the face, trunk, and limbs, as well as ulcers on the lower lip and around the penis, are occasionally caused by L. major (8,9). The infection caused by L. tropica is sometimes referred to as the Delhi boil, Baghdad boil, or oriental sore; it is recognized by the appearance of a skin lesion varying in size from a little pimple to a huge sore, usually found on the face, hands and legs (10). The amastigote and promastigote are the two types of the parasite Leishmania that exist; amastigote is situated in vertebral (intracellular) hosts, whereas promastigote is present in vector (extracellular) in addition to in cultural media (11). The prevalence of cutaneous Leishmaniasis in Iraq has been the subject of several investigations Al Samarai and AlObaidi (12) found that the infection rate was 57%. Salah Al-Din had 468 instances in other places (13).

Leishmaniasis management programs in areas with endemic Leishmaniasis require epidemiological investigations, accurate parasite species identification, and research into the genetic diversity of the parasites. Despite the disease's endemicity, few studies in Iraq focus on phylogenetic analysis and molecular characterization of L. tropica and L. major.

Material and methods

Ethical approval

It was obtained from the guidance of research, Publication and Ethics of College of Medicine, University of Babylon, Iraq (No. BMS/0231/016), which complies with the relevant Iraqi legislations.

Samples collection

One hundred and forty skin lesion samples were collected from patients of both sexes and different ages from four hospitals (Al-Diwaniyah teaching, Al-Hashimih general, Al-Sad,r and Al-Kafil) from single and multiple lesions. Sixty dog skin lesion samples were randomly collected from Middle Euphrates provinces (Babylon, Al-Diwaniyah, Al-Najaf AL Ashraf, and Karbala) of both sexes and different ages during the period from 1 September 2021 to 31 March 2022 for both humans and dogs.

Normal saline was injected into anticipated skin infection lesions (5-10 mm in diameter), drawn out again and the fluid was then maintained in the plain tube (14), transported to the Laboratory of Medical Laboratory Techniques, and stored in the refrigerator for DNA extraction.

DNA extraction

The DNA mini kit from Geneaid in the USA was used to extract genomic DNA from frozen wound skin lesion samples. Proteinase K was used for cell lysis during the extraction per the manufacturer's instructions. The extracted gDNA was then verified using a Nanodrop spectrophotometer before being kept in a refrigerator at -20C until it was utilized in PCR amplification.

PCR (polymerase chain reaction)

The nested PCR test was accomplished utilizing a specific primer for the genus leishmania's kinetoplast DNA (kDNA), which comprises the 750 bp PCR product for L. tropica and the 560 bp PCR product for L. major were amplified utilizing the external primers CSB2XF and CSB1XR and the internal primers 13Z and LiR (Table 1).

Table 1: Contain the name, type, and sequence of the primers utilized in the current study

Sequence analyses

Sequencing DNA sequencing analyses were achieved utilizing Molecular Evolutionary Genetics Analyses version 6.0 (Mega 6.0), and multiple sequence alignment analyses of the partial kDNA gene depend Clustal W alignment analysis evolutionary distances Composite Likelihood manner by phylogenetic tree UPGMA approach.

Statistical analyses

Chi-square (X2) was used in statistical studies to determine whether variables were calculated utilizing the Maximum independence. Values of less than or equal to 0.05 were considered statistically significant by the IBM Statistical Package for Social Sciences (SPSS) statistical soft war, version 31.0 (16).

Results

The total infection rate of cutaneous leishmaniasis in humans and dogs according to the species and lesions shown in (Figures 1 and 2). Nested PCR showed that the overall prevalence of cutaneous Leishmaniasis in humans was 35%, while 88.33% in dogs (Table 2).

Figure 1: In Humans; A- Scaly crusted lesion in the face, B- Single scaly lesion in hand, C- Ulcerated scaly lesion on foot.

Figure 2: In Dogs, A- Scaly multiple lesions in the nose, B- Single scaly lesion in the neck, and C- multiple lesions in legs.

Table 2: Total infection rate of cutaneous Leishmaniasis in humans and dogs using nested PCR

Findings showed that the total infection rate of Leishmania major in humans was 55.1% compared to Leishmania tropica which was 44.89% without significant differences P>0.05. In dogs, Leishmania major was 71.49%, while Leishmania tropica was 28.35%, with significant differences P<0.05 (Table 3).

Table 3: Total infection rate of cutaneous Leishmaniasis in humans and dogs according to the species

NS: No significant differences at P<0.05. S: Significant differences at P<0.05.

To identify the species of cutaneous Leishmaniasis, the amplified gene products from skin lesion samples from humans and dogs were subjected to nested PCR utilizing kDNA gene primers. Agarose gel runs validated the results and showed separate bands of L. major and L. tropica at 560 and 750 bp, respectively (Figure 3 and 4).

Figure 3: Image from an agarose gel electrophoresis showing the detection of kDNA by nested PCR in isolates from human skin lesion samples that are positive for Cutaneous Leishmania. Where M is a marker (2000-100 bp), lanes 1-7 include positive L. major PCR products (560 bp), and lanes 8-10 contain positive L. tropica PCR products (750 bp).

Figure 4: Image from an agarose gel electrophoresis showing the detection of kDNA by nested PCR in isolates from dog skin lesion samples that are positive for Cutaneous Leishmania. Where M is a marker (2000-100 bp), lanes 1-4 include positive L. major PCR products (560 bp), and lane (5) contain positive L. tropica PCR products (750 bp).

Genotype of Leishmania species in humans and dogs

Fifteen samples were positive from each (humans and dogs) after n-PCR amplification of the kDNA gene of Leishmania species was successfully sequenced. The genomic homologous sequencing identity ranged from 99.74-99.88% between isolates of human L. major (OM275415.1 to OM275421.1), and NCBI-Genebank linked L. major (OP046364.1) (Table 4). L. tropica human isolates (OM275422.1 to OM275424.1) were determined to be closely linked to NCBI- BLAST L. tropica (OK359041.1) noticed genomic homologous sequencing identity ranged from 99.74-99.87% (Table 5). Genomic homologous sequencing identity ranged from 99.17-99.67% between isolates of dogs L. major (ON745766.1 to ON745768.1), and NCBI-Gene-bank linked L. major (OP046364.1). L. tropica dog isolates (ON745770.1) were shown to be nearly linked to NCBI -BLAST L. tropica (MH511156.1), which showed genomic homologous sequencing identity 99.34% (Table 6).

Table 4: The NCBI-BLAST Homology Sequence identity (%) between local Leishmania major Human isolates and NCBI-BLAST isolates

Table 5: the NCBI-BLAST Homology Sequence identity (%) between local Leishmania tropica Human isolates and NCBI-BLAST isolates

Table 6: the NCBI-BLAST Homology Sequence identity (%) between local Leishmania major and Leishmania tropica dog isolates and NCBI-BLAST isolates

Phylogenetic analyses

Phylogenetic tree analyses depend on the kDNA gene partial sequence in Leishmania species local isolates and related Leishmania species isolates from the NCBI Gene bank. Local L. major human isolates OM275415.1 to OM275421.1 were shown an identity to NCBI -BLAST L. major isolate IQ-KUT Iraq isolates (OP046364.1) (Figure 5 and 6) and local L. tropica human isolates OM275422.1 to OM275424.1 were shown an identity to NCBI -BLAST L. tropica isolate IQ3 Iraq (OK359041.1) (Figure 7 and 8). Local L. major dog isolates ON745766.1 to ON745768.1 were shown an identity to NCBI -BLAST L. major isolate IQ-KUT kinetoplast, Iraq isolates (OP046364.1) and local L. tropica dog isolates ON745770.1 were shown an identity to NCBI -BLAST L. tropica isolates IQ-7 isolate Iraq (MH511156.1) (Figure 9 and 10).

Figure 5: Leishmania major human isolates from the local area and NCBI-Gen-bank L. major isolates underwent multiple sequence alignment analysis using the Clustal W alignment tool in the MEGA 6.0 version, which revealed substitution mutations on the mitochondrial kinetoplast gene and nucleotide alignment similarity as (*).

Figure 6: Leishmania major human isolates were analyzed for genetic relationships using a phylogenetic tree based on the partial sequence of the mitochondrial kinetoplast gene. A Phylogenetic tree was created using the Un-weighted Pairs Groups Manners with Arithmetic Means (URGMA Tree) in the Mega 6.0 Version. The local L. major isolates IQ-Human-1 - IQ-Human-7 isolates were shown to be nearly related to the NCBI-BLAST L. major isolate IQ-KUT Iraq isolate (OP046364.1) at total genomic changes of 0.008 to 0.0020 percent.

Figure 7: Leishmania tropica human isolates from the local area and NCBI-Genbank L. tropica isolates underwent multiple sequence alignment analysis utilizing the ClustalW alignment tool in MEGA 6.0 version, which revealed substitution mutations on the mitochondrial kinetoplast gene and nucleotide alignment similarity as (*).

Figure 8: Leishmania tropica human isolates were analyzed for genetic relationships using a phylogenetic tree based on the partial sequence of the mitochondrial kinetoplast gene. A Phylogenetic tree was created using the Un-weighted Pairs Groups Manners with Arithmetic Means (URGMA Tree) in the Mega 6.0 Version. The local L. tropica isolates IQ-Human-1 - IQ-Human- 3 isolates were shown to be nearly related to the NCBI-BLAST L. tropica isolate IQ3 Iraq isolate (OK359041.1) at total genomic changes of 0.03 to 0.01 percent.

Figure 9: Leishmania major and Leishmania tropica IQ- dog isolates from the local area and NCBI-Genbank L.major and L. tropica genotypes isolates underwent multiple sequence alignment analyses utilizing the Clustal W alignment tool in MEGA 6.0 version, which revealed substitution mutations on the mitochondrial kinetoplast gene and nucleotide alignment similarity as (*).

Figure 10: Leishmania major and Leishmania tropica IQ- dog isolates were analyzed for genetic relationships utilizing a phylogenetic tree based on the mitochondrial kinetoplast gene partial sequence. A Phylogenetic tree was created using the Un-weighted Pairs Groups Manners with Arithmetic Means (URGMA Tree) in the Mega 6.0 Version. The local L. major isolates IQ-dog -1-IQ dog-4 and L. tropica isolates were shown to be nearly relation to the NCBI-BLAST L. major isolate IQ-KUT kinetoplast, Iraq isolates (OP046364.1) and the L. tropica isolate IQ-dog isolate was shown to be nearly relation to NCBI-BLAST L. tropica isolate IQ-7 Iraq isolate (MH511156.1) at total genomic changes of 1 to 0.0020 percent.

Discussion

The total infection rate of cutaneous Leishmaniasis in humans examined by nested PCR was 35%, which was in agreement with Alsaad and Kawan (17,18), which detected 38.30% with CL in patients who had direct contact with dogs. On the other hand, lower than that detected 67.58% (18); 54.7% (19); 81.25% in Thi-Qar (20), and higher than other studies such as Amro et al. (21) in Libya recorded 25.96%; 15.1% in Al-Diwaniyah, 10.8% in Diyala and Salah-Edin ,13.1% in Thi-Qar ,11.2% in Baghdad (22); 25.9% in Sudi Arabia (23). The spread of the vector population is still linked to the geographic expansion and worldwide escalation of Leishmaniasis (24).

Due to sand flies low in pursuit of prey, environmental and sociological conditions, and people's sleeping patterns all impact the spread of the disease. The province's inhabitants frequently experience power outages, which causes them to sleep outside their homes or on their roofs. During summer, high temperatures and high humidity are considered favorable conditions that significantly increase the reproduction of insects and their contact with human skin. The prevalence of Leishmaniasis in rural areas is higher than in urban as a result of both the rural areas' underdeveloped social customs, culture, and lifestyles, which are probably to blame for the absence of hygienic education and the existence of animals living nearby or inside of people's homes. In addition to facilitating the distribution of infection, rodents, bulk dogs, tankers, and insects in rural parts of the province significantly contribute to the disease's reservoir hosts. In addition, vast plantations and ranches in villages are ideal habitats for insects containing sand flies, which play a role in disease transmission (25). Cutaneous leishmaniasis was determined in rural regions more than in urban regions. This finding is compatible with other studies, such as Al-Difaie (14) and Al-Samarai et al. (26) in Al-Qadisiya governorate, Al-Nahhas and Kaldas (27) in Syria, Mehdi et al. (28) in Iran, Fahriye et al. (29) in Turkey, Khudhur (30) in Iraq. However, different research was detected Al-Hucheimi (31) and Al-Atabi (32).

Findings showed that prevalence in dogs was 88.33%, it was similar to those recorded 80.9% in Nineveh province, Iraq (33), and higher than Alsaad and Kawan (17) recorded 27.71% in dogs 1.6% (23). The canine family in this study is considered a key parasite reservoir and contributes significantly to the spread of the disease. The bulk and distribution of dogs play a big part in the infection's propagation. This occurs due to insufficient of health and municipal services and a decline in public health across the provinces (25). Dogs were exposed to sandflies more than once, and since dogs are usually damp, they provide an ideal habitat for the insect vector to grow and reproduce. As a result, illness developed in the dogs. This may be due to repeated infections, an overabundance of sandflies, and a lack of treatment and follow-up in the area, which led to frequent infection (34). The most frequent association between patients and the reservoir host (dogs) was direct contact, whereas indirect contact was reported in 28.211% of cases and related to stray or neighborhood dogs located close to residences. 33.486% of those with CL reported having no contact with dogs. Due to their outbreaks and the prevalence of the sand fly, patients who owned or lived near domestic dogs had a high infection risk. Some owned one dog, two, three, or even fifteen dogs (17).

Prevalence of cutaneous Leishmania in humans and dogs according to the species; among 140 skin lesion patient samples, numerically, the highest rate recorded with L. major was 55.1% compare to the lowest with L. tropica 44.89%. These results were compatible with other researches that conducted 54% L. major and 46% L. tropica in Iran (18); 49.5% L. major and 28.6% L. tropica (35); 54.7% L. major, 45.3% L. tropica (19); 98.8% L. major, 1.2% L. tropica in Iran (36) and not agree with other studies detected in Iraq (37), Flaih et al. (20) and in Iran Hajjaran et al. (38) who reached that L. tropica had highest prevalence than L. major. The nested PCR approach amplified two different sized fragments from a particular area of the kinetoplast minicircle flanked by two primer sets (CSB and LiR). According to the nested PCR results, L. major is predominant species over L. tropica in Iraqi Hosts.

Current findings were close to the study reached that according to the available epidemiological data, all forms of CL in Jordan are primarily zoonotic diseases, with L. major accounting for 75% of cases (39,40). L. major is a zoonotic infection retained by the reservoir hosts Meriones hibiscus, Psammomys obesus, and sand fly vector P. papatasi. Cases of Leishmania tropica are uncommon. The sand fly P. sergenti is thought to be the reservoir host for L. tropica. However, data suggests that canines or hyraxes found in all L. tropica foci in Jordan may also be reservoir hosts (41). For dogs, current results were in contrast with Alanazi et al. (23), which identified that all dogs isolated were L. tropica. Earlier investigations conducted in Saudi Arabia using enzymatic biochemical techniques have documented that the natural infection by Leishmania is L. major in dogs (42,43).

The results of our study were in agreement with those in Saudi Arabia and other Middle Eastern nations, showed L. major and L. tropica have been identified as agents of cutaneous Leishmaniasis according to molecular evidence of their circulation in human and dog populations from the study locations (44,45). Also, L. major is more common nationwide and may be found in Saudi Arabia's wide desert regions (46,47). The findings revealed that L. major had a higher prevalence than L. tropica. This was in agreement with Al-Ghabban et al. (48) in Diyala province, Iraq, in Iran Yadav and Shrestha (49) and Namazi et al. (50) which detected that L. major was the principal cause of CL. On the contrary, a study in Ramadi, Iraq, recorded that L. tropica had a higher prevalence than L. major (51). This could be due to the abundance of animals, notably rodents and stray dogs, which act as reservoirs and natural hosts for L. major. In addition, several vectors, such as sand flies, are probably major determinants of the high prevalence of human infection.

Phylogenetic analyses and sequencing: The current study showed a divergence of genotypes of cutaneous Leishmaniasis, and this may be due to different methods of transmission, choice of a specific gene for genotyping, various environmental conditions and diagnostic techniques, presence of sand fly and reservoir hosts, although noted limited diversity among the investigated local isolated comparable with other international studies and this could be due to a significant obstacle to understanding the transmission cycles is the lack of information on the interactions between the disease, the reservoirs, and the vectors especially given that the patterns of distribution can easily change over time in particular geographic areas (52). In order to prevent epidemiology and the distribution of CL, data submitted here help close knowledge gaps. Extrachromosomal DNA, such as kinetoplast DNA, is one of the most popular techniques used to identify infections of Leishmania species (53). The kDNA gene has high sensitivity, and the kinetoplast contains almost 10,000 circular and convoluted kDNA minicircles per cell (54).

Previous research detected sequence analyses of kDNA in Leishmania evidence that five positive human samples were all L. major with the identity of nucleotide ranging 99.3-100% with NCBI L. major from Iraq (MN313423) and two positive human samples found identity 99.7-100% with NCBI L. tropica from Iraq (MF166799) in human in Saudi Arabia. Also, five positive dog lesion samples were the identity of nucleotide 99.33-99.80% with NCBI L. tropica from Iraq (MF166800, MN334661), UK (AF308689) (23). Azmi et al. (55) Determined that L. tropica is a highly heterogeneous species confirmed by little bootstrap values accomplished on the phylogenetic tree. Data showed an incidence of L. tropica in dogs and humans, which has been previously confirmed about the disposal of Phlebotomus sergenti, a suitable vector for L. tropica (56). All local L. tropica isolates noticed approximately more similar nitrogen bases sequence and genetically nearly relation to NCBI-BLAST L. tropica registered (K4680852.1) in Iraq (57), (AB678350.1) from patients registered in Iraq (20); (KY612611) registered in Iraq (48); all local L. major isolates noticed approximately more similarly nitrogen bases sequence and genetically be nearly relation to NCBI-BLAST L. major (MH428844.1) registered in Iraq (57), (KP773404.1) registered in Iraq (48).

Conclusion

Nested PCR based on the kDNA gene detected non-significant differences P>0.05 according to L. major and L. tropica in humans, while there were significant differences P<0.05in dogs. Phylogenetic tree analyses determined that 7 of local isolates skin lesion human samples (OM275415.1 to OM275421.1) belong to L. major isolates IQ Kut isolates, Iraq (OP046364.1) and three isolates (OM275422.1 to OM275424.1) belongs to L. tropica isolates IQ3, Iraq (OK359041.1). Four of the local isolates skin lesion dog samples (ON745766.1 to ON745768.1) belong to L. major isolates IQKut, Iraq (OP046364.1), and one isolates (ON745770.1) belong to L. tropica isolates IQ-7 Iraq (MH511156.1). Determining the large number of L. major in humans and dogs indicates that dogs are key parasite reservoirs and significant zoonotic contributes to the transmission of disease to humans in the Middle Euphrates, Iraq.

Acknowledgments

I want to show my sincere appreciation to the Technical Institute of Babylon, Al-Furat Al-Awsat University, Iraq, for supporting our research. I would like to thank all employees in Al-Diwaniyah Teaching, Al-Hashimih General, Al-Sadr, and Al-Kafil hospitals for cooperating with me in collecting patients’ skin lesion samples.

Conflict of interest

The authors declare that there are no conflicts of interest regarding the publication and no funding of this manuscript.