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Full Research Article

First Serological and Molecular Phylogenetic Confirmation of Feline Leishmaniasis in Iraq

A
Amal Hassan Abed1
0000-0002-7189-9733
A
Abbas H.K. Sray2
0000-0003-0076-0600
H
Haider H. Alsaedi2
0009-0002-7824-1480
I
Israa M. Essa3
0000-0002-7379-7503
H
Hasanain A.J. Gharban4,*
0000-0002-6438-1450
1Department of Clinical Laboratory Sciences, College of Pharmacy, University of Al-Qadisiyah, Al-Qadisiyah, Iraq.
2Department of Parasitology, College of Veterinary Medicine, University of Wasit, Wasit, 52001, Iraq.
3Department of Public Health, College of Veterinary Medicine, University of Basrah, Basra, 61001, Iraq.
4Department of Internal and Preventive Veterinary Medicine, College of Veterinary Medicine, University of Wasit, Wasit, 52001, Iraq.

ABSTRACT

Background: Leishmaniasis, caused by different etiological Leishmania species such as L. infantum, represents an important zoonotic disease of various potential hosts as cat whose roles are largely unclear. This present study aims to serological investigation of leishmaniasis and molecular phylogeny of L. infantum.

Materials: Blood samples were collected from totally 91 adult pet cats in Wasit province (Iraq) and examined serologically by the qualitative ELISA, molecularly through PCR and then, the positive DNAs were sequenced and analysed phylogenetically.

Result: Findings of serological ELISA revealed that 15.38% of study cats were positive to leishmaniasis at a Cut Off value of ≥0.230. Respectively, the OD values of seropositively and seronegatively samples were 0.369857±0.025774 and 0.172896±0.003906, respectively. Targeting the ITS1 and 5.8S rRNA gene, molecular PCR reported that 9.89% of study cats were infected with L. infantum. Significant prevalence of leishmaniasis was significantly higher by ELISA than PCR with a level of sensitivity and specificity being 100% and 93.9% respectively. Phylogenetically, the study L. infantum isolates were documented in the NCBI-GenBank database (PQ327040.1, PQ327041.1, PQ327042.1, PQ327043.1, PQ327044.1, PQ327045.1, PQ327046.1, PQ327047.1 and PQ327048.1) and comparative analysis with the NCBI-BLAST L. infantum isolates detected the marked significant identity with the Iranian (ID: KF705514.1) and Brazilian (ID: MH231229.1) isolates obtained from dogs.

INTRODUCTION

Leishmaniasis is a protozoan parasitic disease caused by the genus Leishmania that belongs to Trypanosomatidae family in Kinetoplastea class of Euglenozoa phylum (Silveira et al., 2025). Globally, more than thirty Leishmania species are endemic in nearly 100 countries in Asia, Africa and America (Lorenzetti et al., 2026). Predominantly, Leishmania is acquired throughout infected phlebotomine sandflies that transmit parasitic forms to mammalian hosts; but rarely by alternative routes as congenital, blood transfusion, organ transplantation and laboratory accidents (Shaw, 2025). However, the lifecycle encompasses two main forms are flagellated promastigote that thrives midgut of invertebrate vector and aflagellated amastigote that proliferates within the phagolysosomes of vertebrate (mammalian) macrophages (Arone-Farfán et al., 2024; Liévin-Le and Loiseau, 2016).
       
In tropical and subtropical areas where the incidence is elevated continuously, Leishmania infections present a significant public health challenge (Pal et al., 2022). This directly influenced by accurate detection of Leishmania species and active prevention / treatment (Abbasi, 2025; Cosma et al., 2024). Direct microscopic visualization of amastigotes in smears of bone marrow, lymph nodes, liver and spleen considered as gold standard of confirmation but sensitivity is limited and possible risk of hemorrhage (Pena et al., 2020). Culture can be applied to increasing diagnostic yield; however, it’s time-consumed and prone to bacterial contamination (de Oliveira Filho et al., 2024). Lastly, molecular PCR-based assays have offered a high sensitivity and specificity in determining Leishmania species in various specimens with enabling quantification of parasite burden (Gow et al., 2022; Castelli et al., 2021). Subsequently, phylogenetic analysis demonstrates an efficacy in diagnosis and epidemiological tracking of infections, which essentially help in understanding transmission dynamics and predicting clinical outcomes (Naz et al., 2024; Glidden et al., 2023).
       
In Iraq, several studies mentioned that leishmaniasis is endemic in human; while in animals, online searching detected that rare recent studies have performed on dogs (Fadhil and Ali, 2025; Alseady and Al-Dabbagh, 2024; Alobaidii and Almashhadany, 2023), with lack of such reports in cats. Therefore, the present study was carried out to serological and molecular investigation of leishmaniasis (L. infantum infection) and phylogenetic analysis of study isolates.

MATERIALS AND METHODS

Samples
 
A total of 91 adult pet cats of different ages and sexes were admitted to private veterinary clinics in Wasit province (Iraq) during November (2023) to July (2024). Five ml of venous blood was drained from each cat, divided equally into without and with-anticoagulant tubes and transported to the Laboratory of Clinical Pathology (College of Veterinary Medicine, University of Wasit). The obtained sera and whole blood were kept frozen to be used later for serological and molecular testing (Archana et al., 2026; Al-Eodawee et al., 2024; Al-Abedi et al., 2018; Suzan et al., 2012).
 
Serological examination
 
Qualitative Feline Leishmaniasis ELISA Kit (Cat.No.SL0039Fe, SunLong Biotech, China) was served to screening anti-Leishmania antibodies. Briefly, the serum samples were prepared, processed and optical density (OD) was read. Post evaluation of test effectiveness, calculation of critical value (Cutoff) was done based on the of Negative Control to identify positive samples at ≥ Cutoff value.
 
Molecular examination
 
Initially, the whole blood samples were thawed at room temperature using the water bath and PrestoTM Mini gDNA Bacteria Kit (Geneaid, Taiwan) was utilized to extract the DNAs. Post estimation purity and concentration of DNAs, Master Mix tubes were prepared at a final volume of 25ml using the Accu Power® PCR Pre Mix Kit (Bioneer, Korea) and one set of designated primers [HaF: 5ʹ-CAT CAA GGC CGG ACG GAT AG-3ʹ and HaR: 5ʹ-CCA AGT CAT CCA TCG CGA CA-3ʹ] based on the internal transcribed spacer 1 (ITS1) and 5.8S rRNA gene of the NCBI-GenBank L. infantum isolate (ID: PQ454617.1). Then, the Master Mix tubes were transferred to Thermal Cycler system and subjected to the modified conditions (Table 1). Electrophoresis of PCR products was done in Agarose-gel (1.5%) for 90 minutes at 100 Volts and 80 Am and positive amplified DNAs were observed under UV transilluinator at 291 bp.

Table 1: Conditions for amplification of DNAs.


 
Phylogenetic analysis
 
The positive DNAs in addition to the study primers were sequenced by the Sanger method and study isolates were named and documented in NCBI-GenBank database. Alignment of sequence data of study isolates was constructed through the Clustral W alignment and phylogenetic analysis with the NCBI-BLAST L. infantum isolates was done by the multiple sequence alignment (MSA) in the NCBI website and MEGA-11 Software as well as using the homology sequence identity and phylogenetic tree analysis in MEGA-11 Software.
 
Statistical analysis
 
Among study groups, data were tested statistically by the t-test and One-Way ANOVA in the GraphPad Prism Software to indicate significant differences between study values at p<0.05; whereas, confidence interval 95% (CI95%) was also calculated among different study groups (Al-Hamed et al., 2026; Gharban, 2022; Ajaj et al., 2021).

RESULTS AND DISCUSSION

Serological detection of leishmaniasis
 
After calculation of Cut Off value at ≥ 0.230, the findings of qualitative ELISA detected that 15.38% (14/91) of study cats were serologically positive to leishmaniasis whereas 84.62% were seronegatives (Fig 1).

Fig 1: Serological prevalence of leishmaniasis among 91 pet cats.


       
According to their titers, values seropositives (0.369857±0.025774) were significantly (p<0.0001; 95%CI: 0.9799 to 15.23) higher than recorded for seronegatives (0.172896 ± 0.003906), (Fig 2).

Fig 2: ODs’ levels among the seropositively and seronegatively study of cats.


       
In some endemic areas, serological surveys have been carried out in cats using indirect hemagglutination antibody test (IHAT) in Egypt, Western blot (WB) in France and indirect immunofluorescence antibody test (IFAT) in Italy (Mancianti, 2004). In Egypt, serological testing using an ELISA technique was performed on 104 cats living in Jerusalem area and the seropositive findings were observed in 6.7% (Nasereddin et al., 2008). In Spain, Ayllon et al., (2008) observed that the prevalence rate of leishmaniasis in blood samples of 233 cats tested by IFAT was 4.29% (10/233), which involving 1.29% (3/233) under the cutoff 1:100 and 3% (7/233). In another recent Spanish document, Alcover et al., (2021) revealed leishmaniasis in blood samples of 2.2% (4/179) stray cats tested serologically by ELISA and 14.5% (26/179) by WB. Among different Brazilian reports, Bezerra et al., (2019) detected that the serological prevalence rates of leishmaniasis in diseased cats with feline immunodeficiency virus and feline leukemia virus were 15.38% and 28.57%, respectively. On other hand, Costa-Val et al. (2020) found 29.80% positive cats among 64 blood samples tested serologically by ELISA; while, Leonel et al., (2020) recorded that the seropositive rates of leishmaniasis in blood samples of 94 cats were 31.91% and 29.79% through ELISA and IFAT respectively. In Northern Italy, 12.5% of 152 cats were shown a positive reactivity using IFAT with a titer of ≥1:80 (Urbani et al., 2020). In Iran, a cross-sectional descriptive study involved the testing of blood samples of 60 stray cats by ELISA and the findings recorded that the prevalence of L. infantum was 6.7% (Akhtardanesh et al., 2017). In Turkey, Aksulu et al., (2021) indicated that an overall of 47 out 301 (15.6%) cats were having the anti-Leishmania antibodies using the IFAT. Based on these data, considerable variation among the findings of various studies might be attributed to differences in the parasite species (strains), host immune response, specific test platform used, targeted antigen and geographical endemicity of disease.
 
Molecular identification of L. infantum
 
Targeting the ITS1 and 5.8S rRNA gene, the findings shown that 9.89% (9/91) of study cats were infected with L. infantum while 90.11% (82/91) were negatives (Fig 3).

Fig 3: Molecular prevalence of leishmaniasis (L. infantum) among 91 pet cats.


       
In a previous review article, Mancianti (2004) mentioned that the typing of clinical cases described in several European countries as France, Italy, Portugal and Spain identified L. infantum in all reported cases. Comparatively, L. infantum infection was detected molecularly in 0.43% (Ayllon et al., 2008) and 5.6% (Alcover et al., 2021) of Spanish cats, 16.7% of Iranian stray cats (Akhtardanesh et al., 2017), 10% of Brazilian cats (Costa-Val et al., 2020) and 2.3% of Turkish cats (Aksulu et al., 2021). Among the various molecular targets, the ITS1 and 5.8S rRNA gene has emerged as particularly valuable due to its high variability and conserved flanking regions, which facilitate species-specific amplification and differentiation (Gunderina and Katokhin, 2020). Also, the inherent genetic diversity within the ITS1 region coupled with the stability of the 5.8S rRNA gene enables the development of highly sensitive and specific PCR-based assays capable of identifying Leishmania at the genus and species level, even in samples with low parasite loads (Usmael et al., 2022). This makes them superior to other targets like the 18S rRNA gene that, despite its abundance, requires more intricate amplification methods and often presents challenges in achieving species-level discrimination (Alsulaimany, 2025). Moreover, the tandem repeat organization of rDNA units, which can be present in tens to hundreds of copies per Leishmania cell, significantly enhances the sensitivity of ITS1-based assays, allowing for robust detection even in clinical samples with limited parasite DNA (Salloum et al., 2016).
 
Comparative accuracy of diagnostic assays
 
This study found significantly that the number serologically positive samples (15.38%) was higher (p<0.0474; 95%CI: 22.24 to 47.51) than reported by molecular PCR assay (9.89%), which accordingly indicated that the levels of sensitivity and specificity for ELISA were 100% and 93.9% respectively (Fig 4, Table 2).

Fig 4: Positive serological and molecular results of feline leishmaniasis.



Table 2: Sensitivity and specificity of serology assay (ELISA) according to molecular (PCR) technique.


       
In contrast to our data, Akhtardanesh et al., (2017) obtained that the prevalence rates of positive Leishmania infection by ELISA and PCR were 6.7% and 16.7%, respectively. However, this high sensitivity and specificity might be attributed to the high efficacy of ELISA’s kit applied in current study or to the role of testing similar sample sources in a study. Sherry et al., (2011) determined that serology and PCR results are positively associated and moderate agreement was found between Leishmania ELISA and PCR since the total rate of L. infantum infection derived from seroreactivity and / or positive PCR was 15.4%. Alcover et al., (2021) found that the overall rate of leishmaniasis among 179 stray cats was 15.6% and only two cats tested positive by all the diagnostic methods (IFAT, ELISA, WB and PCR). However, several studies have been reported inconsistent results when using different types of tissue samples for PCR-based diagnosis compared with the parasitological and serological data indicating that sampling from different tissues together with serological analysis is required when screening for leishmaniasis (Rihs et al., 2025; Gow et al., 2022; Castelli et al., 2021; Costa-Val et al., 2020).
 
Phylogenetic analysis of study L. infantum isolates
 
Sequencing data of study positive L. infantum isolates were named as Feline isolates and documented in the NCBI-GenBank database following specified accession numbers (PQ327040.1, PQ327041.1, PQ327042.1, PQ327043.1, PQ327044.1, PQ327045.1, PQ327046.1, PQ327047.1 and PQ327048.1). Comparative analysis of study L. infantum isolates with the NCBI-BLAST L. infantum isolates detected an existence of nucleotide alignment similarities (*) ranged 89-100% and substitution mutations ranged 0.000-0.0583%. However, the marked significant identity was observed between the study isolates named Felin1, Feline 2, Feline6, Feline7 and Feline9 with the NCBI-GenBank L. infantus Iranian (ID: KF705514.1) isolate that identified in ticks of naturally infected dogs; while, the study isolates named Feline3, Feline4, Feline5 and Feline8 were identical with the NCBI-GenBank L. infantus Brazilian (ID: MH231229.1) isolate obtained from the conjunctive of naturally infected dogs (Table 3, Fig 5-9).

Table 3: Homology Sequence identity for study cattle L. infantum isolates and NCBI-BLAST L. infantum isolates.



Fig 5: MSA showing similarity (*) and differences between the local study and NCBI-GenBank L. infantum isolates using the MEGA-11 software.



Fig 6: MSA showing differences between the local study and NCBI-GenBank L. infantum isolates using the NCBI MSA viewer.



Fig 7: MSA showing frequency-based differences between the local study and NCBI-GenBank L. infantum isolates using the NCBI MSA viewer.



Fig 8: MSA showing nucleic acid similarity and differences between the local study and NCBI-GenBank L. infantum isolates using the NCBI MSA viewer.



Fig 9: Phylogenetic tree analysis of the local study and NCBI-GenBank L. infantum isolates using the MEGA-11 software.


       
In the only available study in Iraqi animals, phylogenetic analysis of L. infantum isolates identified in dogs revealed a significant identity to Brazilian, French and American. This demonstrates that many strains of L. infantum might be circulated in Iraq particularly Brazilian ones suggesting that cats could attract their infection from dogs (Fadhil and Ali, 2025). Also, low levels of biosecurity in Iraq allows to introduction of various animals and breeders which may act as a reservoir to infection or infected asymptomatically. Nonetheless, Costa-Val et al. (2020) refer to the genetic complexity of Leishmania spp. populations which circulate in a different geographical area but the same species.

CONCLUSION

For our knowledge, this might represents the first study in Iraq highlighting feline leishmaniasis serologically and molecular phylogenetically. Based on our data, pet cats may carry leishmaniasis (L. infantum) as either a reservoir or an actively asymptomatic suggesting the importance of furthermore epidemiological studies. Additionally, combination of serological ELISA and molecular PCR techniques with phylogenetic analysis by targeting the ITS1 and 5.8S rRNA gene revealed a high efficacy in investigation of feline leishmaniasis and the great role of these diagnostic techniques in detection of such infections. Phylogenetic analysis of study isolates determined a close-relationship with two global Iranian and Brazilian strains of dog source indicating the role of dogs in transmission of infection to cats and other animals or even humans.

ACKNOWLEDGEMENT

Authors thank all veterinarians who facilitate the collection of samples and the owners who allowed to obtaining the venous blood.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures were approved by the Scientific Committee in the College of Veterinary Medicine at University of Wasit (UW.CVM.2023.237) dated 14/11/2023.

CONFLICT OF INTEREST

All authors declare that they have no conflict of interest.

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    First Serological and Molecular Phylogenetic Confirmation of Feline Leishmaniasis in Iraq

    A
    Amal Hassan Abed1
    0000-0002-7189-9733
    A
    Abbas H.K. Sray2
    0000-0003-0076-0600
    H
    Haider H. Alsaedi2
    0009-0002-7824-1480
    I
    Israa M. Essa3
    0000-0002-7379-7503
    H
    Hasanain A.J. Gharban4,*
    0000-0002-6438-1450
    1Department of Clinical Laboratory Sciences, College of Pharmacy, University of Al-Qadisiyah, Al-Qadisiyah, Iraq.
    2Department of Parasitology, College of Veterinary Medicine, University of Wasit, Wasit, 52001, Iraq.
    3Department of Public Health, College of Veterinary Medicine, University of Basrah, Basra, 61001, Iraq.
    4Department of Internal and Preventive Veterinary Medicine, College of Veterinary Medicine, University of Wasit, Wasit, 52001, Iraq.

    ABSTRACT

    Background: Leishmaniasis, caused by different etiological Leishmania species such as L. infantum, represents an important zoonotic disease of various potential hosts as cat whose roles are largely unclear. This present study aims to serological investigation of leishmaniasis and molecular phylogeny of L. infantum.

    Materials: Blood samples were collected from totally 91 adult pet cats in Wasit province (Iraq) and examined serologically by the qualitative ELISA, molecularly through PCR and then, the positive DNAs were sequenced and analysed phylogenetically.

    Result: Findings of serological ELISA revealed that 15.38% of study cats were positive to leishmaniasis at a Cut Off value of ≥0.230. Respectively, the OD values of seropositively and seronegatively samples were 0.369857±0.025774 and 0.172896±0.003906, respectively. Targeting the ITS1 and 5.8S rRNA gene, molecular PCR reported that 9.89% of study cats were infected with L. infantum. Significant prevalence of leishmaniasis was significantly higher by ELISA than PCR with a level of sensitivity and specificity being 100% and 93.9% respectively. Phylogenetically, the study L. infantum isolates were documented in the NCBI-GenBank database (PQ327040.1, PQ327041.1, PQ327042.1, PQ327043.1, PQ327044.1, PQ327045.1, PQ327046.1, PQ327047.1 and PQ327048.1) and comparative analysis with the NCBI-BLAST L. infantum isolates detected the marked significant identity with the Iranian (ID: KF705514.1) and Brazilian (ID: MH231229.1) isolates obtained from dogs.

    INTRODUCTION

    Leishmaniasis is a protozoan parasitic disease caused by the genus Leishmania that belongs to Trypanosomatidae family in Kinetoplastea class of Euglenozoa phylum (Silveira et al., 2025). Globally, more than thirty Leishmania species are endemic in nearly 100 countries in Asia, Africa and America (Lorenzetti et al., 2026). Predominantly, Leishmania is acquired throughout infected phlebotomine sandflies that transmit parasitic forms to mammalian hosts; but rarely by alternative routes as congenital, blood transfusion, organ transplantation and laboratory accidents (Shaw, 2025). However, the lifecycle encompasses two main forms are flagellated promastigote that thrives midgut of invertebrate vector and aflagellated amastigote that proliferates within the phagolysosomes of vertebrate (mammalian) macrophages (Arone-Farfán et al., 2024; Liévin-Le and Loiseau, 2016).
           
    In tropical and subtropical areas where the incidence is elevated continuously, Leishmania infections present a significant public health challenge (Pal et al., 2022). This directly influenced by accurate detection of Leishmania species and active prevention / treatment (Abbasi, 2025; Cosma et al., 2024). Direct microscopic visualization of amastigotes in smears of bone marrow, lymph nodes, liver and spleen considered as gold standard of confirmation but sensitivity is limited and possible risk of hemorrhage (Pena et al., 2020). Culture can be applied to increasing diagnostic yield; however, it’s time-consumed and prone to bacterial contamination (de Oliveira Filho et al., 2024). Lastly, molecular PCR-based assays have offered a high sensitivity and specificity in determining Leishmania species in various specimens with enabling quantification of parasite burden (Gow et al., 2022; Castelli et al., 2021). Subsequently, phylogenetic analysis demonstrates an efficacy in diagnosis and epidemiological tracking of infections, which essentially help in understanding transmission dynamics and predicting clinical outcomes (Naz et al., 2024; Glidden et al., 2023).
           
    In Iraq, several studies mentioned that leishmaniasis is endemic in human; while in animals, online searching detected that rare recent studies have performed on dogs (Fadhil and Ali, 2025; Alseady and Al-Dabbagh, 2024; Alobaidii and Almashhadany, 2023), with lack of such reports in cats. Therefore, the present study was carried out to serological and molecular investigation of leishmaniasis (L. infantum infection) and phylogenetic analysis of study isolates.

    MATERIALS AND METHODS

    Samples
     
    A total of 91 adult pet cats of different ages and sexes were admitted to private veterinary clinics in Wasit province (Iraq) during November (2023) to July (2024). Five ml of venous blood was drained from each cat, divided equally into without and with-anticoagulant tubes and transported to the Laboratory of Clinical Pathology (College of Veterinary Medicine, University of Wasit). The obtained sera and whole blood were kept frozen to be used later for serological and molecular testing (Archana et al., 2026; Al-Eodawee et al., 2024; Al-Abedi et al., 2018; Suzan et al., 2012).
     
    Serological examination
     
    Qualitative Feline Leishmaniasis ELISA Kit (Cat.No.SL0039Fe, SunLong Biotech, China) was served to screening anti-Leishmania antibodies. Briefly, the serum samples were prepared, processed and optical density (OD) was read. Post evaluation of test effectiveness, calculation of critical value (Cutoff) was done based on the of Negative Control to identify positive samples at ≥ Cutoff value.
     
    Molecular examination
     
    Initially, the whole blood samples were thawed at room temperature using the water bath and PrestoTM Mini gDNA Bacteria Kit (Geneaid, Taiwan) was utilized to extract the DNAs. Post estimation purity and concentration of DNAs, Master Mix tubes were prepared at a final volume of 25ml using the Accu Power® PCR Pre Mix Kit (Bioneer, Korea) and one set of designated primers [HaF: 5ʹ-CAT CAA GGC CGG ACG GAT AG-3ʹ and HaR: 5ʹ-CCA AGT CAT CCA TCG CGA CA-3ʹ] based on the internal transcribed spacer 1 (ITS1) and 5.8S rRNA gene of the NCBI-GenBank L. infantum isolate (ID: PQ454617.1). Then, the Master Mix tubes were transferred to Thermal Cycler system and subjected to the modified conditions (Table 1). Electrophoresis of PCR products was done in Agarose-gel (1.5%) for 90 minutes at 100 Volts and 80 Am and positive amplified DNAs were observed under UV transilluinator at 291 bp.

    Table 1: Conditions for amplification of DNAs.


     
    Phylogenetic analysis
     
    The positive DNAs in addition to the study primers were sequenced by the Sanger method and study isolates were named and documented in NCBI-GenBank database. Alignment of sequence data of study isolates was constructed through the Clustral W alignment and phylogenetic analysis with the NCBI-BLAST L. infantum isolates was done by the multiple sequence alignment (MSA) in the NCBI website and MEGA-11 Software as well as using the homology sequence identity and phylogenetic tree analysis in MEGA-11 Software.
     
    Statistical analysis
     
    Among study groups, data were tested statistically by the t-test and One-Way ANOVA in the GraphPad Prism Software to indicate significant differences between study values at p<0.05; whereas, confidence interval 95% (CI95%) was also calculated among different study groups (Al-Hamed et al., 2026; Gharban, 2022; Ajaj et al., 2021).

    RESULTS AND DISCUSSION

    Serological detection of leishmaniasis
     
    After calculation of Cut Off value at ≥ 0.230, the findings of qualitative ELISA detected that 15.38% (14/91) of study cats were serologically positive to leishmaniasis whereas 84.62% were seronegatives (Fig 1).

    Fig 1: Serological prevalence of leishmaniasis among 91 pet cats.


           
    According to their titers, values seropositives (0.369857±0.025774) were significantly (p<0.0001; 95%CI: 0.9799 to 15.23) higher than recorded for seronegatives (0.172896 ± 0.003906), (Fig 2).

    Fig 2: ODs’ levels among the seropositively and seronegatively study of cats.


           
    In some endemic areas, serological surveys have been carried out in cats using indirect hemagglutination antibody test (IHAT) in Egypt, Western blot (WB) in France and indirect immunofluorescence antibody test (IFAT) in Italy (Mancianti, 2004). In Egypt, serological testing using an ELISA technique was performed on 104 cats living in Jerusalem area and the seropositive findings were observed in 6.7% (Nasereddin et al., 2008). In Spain, Ayllon et al., (2008) observed that the prevalence rate of leishmaniasis in blood samples of 233 cats tested by IFAT was 4.29% (10/233), which involving 1.29% (3/233) under the cutoff 1:100 and 3% (7/233). In another recent Spanish document, Alcover et al., (2021) revealed leishmaniasis in blood samples of 2.2% (4/179) stray cats tested serologically by ELISA and 14.5% (26/179) by WB. Among different Brazilian reports, Bezerra et al., (2019) detected that the serological prevalence rates of leishmaniasis in diseased cats with feline immunodeficiency virus and feline leukemia virus were 15.38% and 28.57%, respectively. On other hand, Costa-Val et al. (2020) found 29.80% positive cats among 64 blood samples tested serologically by ELISA; while, Leonel et al., (2020) recorded that the seropositive rates of leishmaniasis in blood samples of 94 cats were 31.91% and 29.79% through ELISA and IFAT respectively. In Northern Italy, 12.5% of 152 cats were shown a positive reactivity using IFAT with a titer of ≥1:80 (Urbani et al., 2020). In Iran, a cross-sectional descriptive study involved the testing of blood samples of 60 stray cats by ELISA and the findings recorded that the prevalence of L. infantum was 6.7% (Akhtardanesh et al., 2017). In Turkey, Aksulu et al., (2021) indicated that an overall of 47 out 301 (15.6%) cats were having the anti-Leishmania antibodies using the IFAT. Based on these data, considerable variation among the findings of various studies might be attributed to differences in the parasite species (strains), host immune response, specific test platform used, targeted antigen and geographical endemicity of disease.
     
    Molecular identification of L. infantum
     
    Targeting the ITS1 and 5.8S rRNA gene, the findings shown that 9.89% (9/91) of study cats were infected with L. infantum while 90.11% (82/91) were negatives (Fig 3).

    Fig 3: Molecular prevalence of leishmaniasis (L. infantum) among 91 pet cats.


           
    In a previous review article, Mancianti (2004) mentioned that the typing of clinical cases described in several European countries as France, Italy, Portugal and Spain identified L. infantum in all reported cases. Comparatively, L. infantum infection was detected molecularly in 0.43% (Ayllon et al., 2008) and 5.6% (Alcover et al., 2021) of Spanish cats, 16.7% of Iranian stray cats (Akhtardanesh et al., 2017), 10% of Brazilian cats (Costa-Val et al., 2020) and 2.3% of Turkish cats (Aksulu et al., 2021). Among the various molecular targets, the ITS1 and 5.8S rRNA gene has emerged as particularly valuable due to its high variability and conserved flanking regions, which facilitate species-specific amplification and differentiation (Gunderina and Katokhin, 2020). Also, the inherent genetic diversity within the ITS1 region coupled with the stability of the 5.8S rRNA gene enables the development of highly sensitive and specific PCR-based assays capable of identifying Leishmania at the genus and species level, even in samples with low parasite loads (Usmael et al., 2022). This makes them superior to other targets like the 18S rRNA gene that, despite its abundance, requires more intricate amplification methods and often presents challenges in achieving species-level discrimination (Alsulaimany, 2025). Moreover, the tandem repeat organization of rDNA units, which can be present in tens to hundreds of copies per Leishmania cell, significantly enhances the sensitivity of ITS1-based assays, allowing for robust detection even in clinical samples with limited parasite DNA (Salloum et al., 2016).
     
    Comparative accuracy of diagnostic assays
     
    This study found significantly that the number serologically positive samples (15.38%) was higher (p<0.0474; 95%CI: 22.24 to 47.51) than reported by molecular PCR assay (9.89%), which accordingly indicated that the levels of sensitivity and specificity for ELISA were 100% and 93.9% respectively (Fig 4, Table 2).

    Fig 4: Positive serological and molecular results of feline leishmaniasis.



    Table 2: Sensitivity and specificity of serology assay (ELISA) according to molecular (PCR) technique.


           
    In contrast to our data, Akhtardanesh et al., (2017) obtained that the prevalence rates of positive Leishmania infection by ELISA and PCR were 6.7% and 16.7%, respectively. However, this high sensitivity and specificity might be attributed to the high efficacy of ELISA’s kit applied in current study or to the role of testing similar sample sources in a study. Sherry et al., (2011) determined that serology and PCR results are positively associated and moderate agreement was found between Leishmania ELISA and PCR since the total rate of L. infantum infection derived from seroreactivity and / or positive PCR was 15.4%. Alcover et al., (2021) found that the overall rate of leishmaniasis among 179 stray cats was 15.6% and only two cats tested positive by all the diagnostic methods (IFAT, ELISA, WB and PCR). However, several studies have been reported inconsistent results when using different types of tissue samples for PCR-based diagnosis compared with the parasitological and serological data indicating that sampling from different tissues together with serological analysis is required when screening for leishmaniasis (Rihs et al., 2025; Gow et al., 2022; Castelli et al., 2021; Costa-Val et al., 2020).
     
    Phylogenetic analysis of study L. infantum isolates
     
    Sequencing data of study positive L. infantum isolates were named as Feline isolates and documented in the NCBI-GenBank database following specified accession numbers (PQ327040.1, PQ327041.1, PQ327042.1, PQ327043.1, PQ327044.1, PQ327045.1, PQ327046.1, PQ327047.1 and PQ327048.1). Comparative analysis of study L. infantum isolates with the NCBI-BLAST L. infantum isolates detected an existence of nucleotide alignment similarities (*) ranged 89-100% and substitution mutations ranged 0.000-0.0583%. However, the marked significant identity was observed between the study isolates named Felin1, Feline 2, Feline6, Feline7 and Feline9 with the NCBI-GenBank L. infantus Iranian (ID: KF705514.1) isolate that identified in ticks of naturally infected dogs; while, the study isolates named Feline3, Feline4, Feline5 and Feline8 were identical with the NCBI-GenBank L. infantus Brazilian (ID: MH231229.1) isolate obtained from the conjunctive of naturally infected dogs (Table 3, Fig 5-9).

    Table 3: Homology Sequence identity for study cattle L. infantum isolates and NCBI-BLAST L. infantum isolates.



    Fig 5: MSA showing similarity (*) and differences between the local study and NCBI-GenBank L. infantum isolates using the MEGA-11 software.



    Fig 6: MSA showing differences between the local study and NCBI-GenBank L. infantum isolates using the NCBI MSA viewer.



    Fig 7: MSA showing frequency-based differences between the local study and NCBI-GenBank L. infantum isolates using the NCBI MSA viewer.



    Fig 8: MSA showing nucleic acid similarity and differences between the local study and NCBI-GenBank L. infantum isolates using the NCBI MSA viewer.



    Fig 9: Phylogenetic tree analysis of the local study and NCBI-GenBank L. infantum isolates using the MEGA-11 software.


           
    In the only available study in Iraqi animals, phylogenetic analysis of L. infantum isolates identified in dogs revealed a significant identity to Brazilian, French and American. This demonstrates that many strains of L. infantum might be circulated in Iraq particularly Brazilian ones suggesting that cats could attract their infection from dogs (Fadhil and Ali, 2025). Also, low levels of biosecurity in Iraq allows to introduction of various animals and breeders which may act as a reservoir to infection or infected asymptomatically. Nonetheless, Costa-Val et al. (2020) refer to the genetic complexity of Leishmania spp. populations which circulate in a different geographical area but the same species.

    CONCLUSION

    For our knowledge, this might represents the first study in Iraq highlighting feline leishmaniasis serologically and molecular phylogenetically. Based on our data, pet cats may carry leishmaniasis (L. infantum) as either a reservoir or an actively asymptomatic suggesting the importance of furthermore epidemiological studies. Additionally, combination of serological ELISA and molecular PCR techniques with phylogenetic analysis by targeting the ITS1 and 5.8S rRNA gene revealed a high efficacy in investigation of feline leishmaniasis and the great role of these diagnostic techniques in detection of such infections. Phylogenetic analysis of study isolates determined a close-relationship with two global Iranian and Brazilian strains of dog source indicating the role of dogs in transmission of infection to cats and other animals or even humans.

    ACKNOWLEDGEMENT

    Authors thank all veterinarians who facilitate the collection of samples and the owners who allowed to obtaining the venous blood.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    All animal procedures were approved by the Scientific Committee in the College of Veterinary Medicine at University of Wasit (UW.CVM.2023.237) dated 14/11/2023.

    CONFLICT OF INTEREST

    All authors declare that they have no conflict of interest.

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