Seasonal Prevalence and Risk Factors of canine Leishmania Infection in Beijing, China

Seasonal Prevalence and Risk Factors of canine Leishmania Infection in Beijing, China | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Seasonal Prevalence and Risk Factors of canine Leishmania Infection in Beijing, China Yujuan Chen, Han Tang, Yonglan Yu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4482352/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Leishmaniasis, a severe zoonotic disease where dogs act as the main reservoir for Leishmania infantum ( L. infantum ), poses a significant public health threat. Despite recent cases in Beijing, China, there is a lack of comprehensive studies on canine leishmaniasis (CanL) in this region. This study aimed to determine the prevalence of CanL in Beijing and identify associated risk factors. Methods Enzyme-linked immunosorbent assay (ELISA) was used to detect anti-L. infantum antibodies, and quantitative real-time PCR (qPCR) was used to identify Leishmania DNA. The study included 737 dogs: 575 were tested with both ELISA using serum samples and qPCR using whole blood samples, while the remaining 162 were tested only with qPCR. Variables related to the dogs were collected and evaluated through univariate analysis (the Chi-square test) and multivariate logistical regression analysis. For dogs that tested positive on ELISA or qPCR and exhibited suspicious clinical symptoms, we collected lymph node punctures and skin scrape material to reconfirm Leishmania infection by qPCR. Results ELISA and qPCR prevalence rates were 4.87% (28/575) and 3.26% (24/737), respectively, with all qPCR-positive samples confirmed as L. infantum by PCR amplification of the internal transcribed spacer (ITS) gene. QPCR and ELISA demonstrated strong consistency ( P = 0.00) in detecting Leishmania among the 575 dogs subjected to both tests. Multivariate analysis revealed a higher prevalence of CanL during springtime in Beijing (ELISA: OR = 9.31, 95% CI = 2.75–31.47; qPCR: OR = 7.22, 95% CI = 2.49–20.94), and among younger dogs (0-2Y) (ELISA: OR = 5.49, 95% CI = 1.70–17.80; qPCR: OR = 4.51, 95% CI = 1.25–16.24). Univariate analysis identified suburban areas ( P = 0.02) and medium to large-sized dogs ( P = 0.00) as secondary risk factors. Among the dogs that tested positive on ELISA or qPCR, 16 were subsequently confirmed to have CanL through qPCR test of lymph node aspirates and skin scrapings. The majority of these cases were found in suburban areas (68.75%), with 43.75% detected during springtime. Additionally, 50% of the cases involved medium-sized dogs. Conclusion Seroprevalence and molecular prevalence of CanL in Beijing was 4.87% and 3.26% respectively, with a higher prevalence observed in spring and among younger dogs (0-2Y), emphasizing the importance of comprehensive studies to address public health threats. canine leishmaniasis epidemiology prevalence risk factors Figures Figure 1 Figure 2 Figure 3 Background Leishmaniasis, a vector-borne zoonotic disease, is instigated by protozoa of the genus Leishmania , primarily transmitted through the bite of sandflies. Dogs are the principal domestic reservoir for Leishmania infantum ( L. infantum ), making canine leishmaniasis (CanL) a focal point of public health concern. Notably, leishmaniasis has achieved endemic status in over 98 countries [ 1 ] , endangering approximately 350 million people, with an estimated 700,000 to 1 million new cases reported annually [ 2 ] . Regions reporting endemic prevalence of leishmaniasis include the Americas, East Africa, North Africa, West Asia, and Southeast Asia [ 2 ] . L. infantum in humans is accountable for various forms of leishmaniasis, such as visceral, cutaneous, or mucosal [ 3 ] . CanL is often asymptomatic, although it can sometimes manifest in generalized cutaneous and ovarian forms that are spontaneously fatal [ 4 ] . This asymptomatic nature combined with limited laboratory diagnostic capabilities exacerbates the difficulty of detecting infected animals [ 5 ] . Diagnostic methodologies for leishmaniasis often encompass serological and molecular techniques. Serological assays, including enzyme-linked immunosorbent assay (ELISA) and direct agglutination test (DAT), provide a non-invasive means of detecting specific antibodies, but fail to differentiate between past and ongoing infections [ 6 ] . Conversely, PCR-based assays have proven to be highly reliable [ 7 ] , leading many veterinarians to opt for a dual approach: combining molecular analyses with serological diagnosis to verify infections and identify the Leishmania species involved. Existing reports from China, specifically endemic areas such as Shanxi [ 8 ] , Sichuan [ 9 ] , and Gansu Provinces [ 10 ] , provide limited seroprevalence and molecular data, having included fewer than 300 samples. Moreover, while the number of CanL cases in Beijing has seen a steady rise over the past two years, comprehensive data detailing the prevalence of CanL in Beijing remains scarce. Studies have indicated that the prevalence of CanL varies between suburban and urban areas [ 11 , 12 ] , with seasonal trends diverging across different regions [ 12 ] , yet no data elucidating the spatial distribution and seasonal variations of CanL in Beijing has been reported. It is also uncertain whether individual factors such as age, sex, breed, and body size influence the incidence of CanL. In light of these gaps, we sought to gain a clearer understanding of the prevalence of leishmaniasis in domestic dogs in Beijing. For this purpose, we collected serum samples from 575 dogs for the detection of anti- L. infantum Ig G antibodies by ELISA, and anticoagulated whole blood samples from 737 dogs for the detection of Leishmania DNA via quantitative real-time PCR (qPCR). Subsequently, we analyzed the risk factors associated with CanL. Additionally, we scrutinized the epidemiological data of 16 dogs diagnosed with CanL to identify potential risk factors. This study aims to generate data that can be instrumental in devising strategies for leishmaniasis prevention and control. Methods Study area Beijing is located in northern China, positioned between 115°25' and 117°30' E and 39°26' and 41°03' N. The city encompasses 16 districts and is predominantly characterized by plains and hills, with mountainous regions to the northwest. Beijing experiences a temperate continental monsoon climate, featuring hot, rainy summers and cold, dry winters. Ethical Approval and Sample Collection The study, having been approved by the Laboratory Animal Welfare and Animal Experimental Ethics Committee, China Agricultural University (permit number: AW21012020-2), included a total of 737 dogs from March 2021 to February 2022. Serum and anticoagulated whole blood samples of these dogs were collected from China Agricultural University Veterinary Teaching Hospital and Weiwei International Diagnostic Center in Beijing. From the 737 dogs, both serum and anticoagulated whole blood samples were collected from 575 individuals, besides, only serum samples were collected from 162 dogs. Serum samples from 575 dogs were centrifuged at 1000×g for 10 min, and 200 µL of the serum samples was stored at -80°C for ELISA testing; EDTA-anticoagulated whole blood samples from 737 dogs were collected for qPCR testing. Epidemiological Data Collection Epidemiological data for all 737 dogs, including factors such as region, season, breed, body size, sex, and age, was obtained from the owners through face-to-face questionnaires or telephone interviews. ELISA Testing IgG antibodies against L. infantum in dogs were detected using the ID Screen® leishmaniasis Indirect Test (VET-Innovate ID Diagnostics, France). DNA Extraction DNA was extracted from blood, lymph node aspirates, and skin scrapings of dogs using the TianGen® DNA Kit for Blood, Cell and Tissues (Genomics Bioscience and Technology Co., Ltd., Beijing, China) according to the manufacturer’s protocol. The extracted DNA was stored at -80°C for molecular testing. QPCR Testing The kinetoplast DNA (kDNA) of Leishmania was amplified using the extracted genomic DNA as a template [ 13 ] . The reaction mixture consisted of PowerUp™ SYBR™ Green Master Mix, forward and reverse primers, extracted DNA, and nuclease-free water. A pre-specified Ct value of ≤ 35 was considered positive. Leishmania Species Identification The Leishmania ITS gene was amplified from the extracted DNA of qPCR-positive blood samples using the ITS primers [ 14 ] . The PCR products were sequenced and compared with the molecular profiles of Leishmania reference strains, namely L. braziliensis (FJ753376), L. guyanensis (FJ753387), L. panamensis (FJ948438), L. mexicana (FJ948432), L. amazonensis (FJ753371), L. donovani (FJ753386), L. infantum (GU045592), L. major (FJ753391), L. tropica (FJ948447) and L. aethiopica (GQ920673). Statistical Analysis Statistical analysis of infection risk factors was conducted using SPSS version 23 software. The Kappa test was used to analyze the consistency of qPCR and ELISA on detecting Leishmania . The Chi-square (χ2) test was applied to assess risk factors associated with Leishmania infection diagnosed by ELISA or qPCR. Variables exhibiting a statistically significant association ( P < 0.05) were then included in a multivariable logistic regression model to determine risk factors, odds ratios (ORs) and confidence intervals (CIs) for each significant variable. The Spearman's rank correlation coefficient was used to assess the correlation between two variables. The Hosmer-Lemeshow goodness-of-fit test was utilized to evaluate the fit of the multivariable logistic regression model. During the construction of the multivariable model concerning variables associated with the ELISA positive rate of Leishmania , we excluded body size due to its correlation with age according to the Spearman's rank correlation coefficient. Epidemiological Analysis of Diagnosed CanL Cases Based on risk factors identified in preceding statistical analysis, dogs diagnosed with CanL were analyzed further. Symptoms such as poor general condition, weight loss, swollen lymph nodes, onychogryphosis, and cutaneous lesions were used to select dogs for additional testing. Lymph node punctures were performed on swollen lymph nodes, including the pre-scapular, popliteal, and mandibular lymph nodes. Skin scrapings were obtained from cutaneous lesions, and both sample types were assessed using qPCR to confirm Leishmania infection. Results Overall Prevalence of canine leishmaniasis Out of 575 serum samples tested for anti- Leishmania infantum antibodies, 28 (4.87%) were reactive in the ELISA test. Additionally, out of 737 whole blood samples, 24 (3.26%) were Leishmania DNA positive (Fig. 1 ). All 24 qPCR-positive samples matched ≥ 99.28% similarity with the L. infantum species after sequencing analysis (Fig. 2 ). QPCR and ELISA showed remarkable consistency ( P = 0.00) in detecting Leishmania among the 575 dogs subjected to both tests. Out of the total, 36 dogs tested positive either by qPCR or ELISA. Among them, 16 were positive by both ELISA and qPCR, 8 were positive by qPCR but negative by ELISA, and 12 were positive by ELISA but negative by qPCR. (Table 1 ). Table 1 Prevalence of canine leishmaniasis in Beijing tested by qPCR and ELISA. QPCR ELISA No. of tested Kappa-value P -value Positive Positive 16 0.60 0.00** Positive Nagative 8 Negative Positive 12 Negative Nagative 539 Negative Not tested 162 **: The result is significant at P < 0.01; ELISA: Enzyme-linked immunosorbent assay; qPCR: quantitative real-time PCR. Prevalence of canine leishmaniasis in Different Sites Suburban areas showed a higher prevalence of CanL (7.38% by ELISA, 4.56% by qPCR) compared to urban areas (3.02% by ELISA, 2.33% by qPCR) (Table 2 ). Dogs in Mentougou District had the highest serology (50%) and qPCR (37.0%) positive rates (Fig. 3 ). Table 2 Univariate analysis of risk factors associated with canine leishmaniasis. Risk factors ELISA qPCR No. of tested dogs (n = 575) No. of positive dogs (%) χ2 P -value No. of tested dogs (n = 737) No. of positive dogs (%) χ2 P -value Living region 5.75 0.02* 2.839 0.09 Urban 331 10(3.02%) 430 10(2.33%) Suburban 244 18(7.38%) 307 14(4.56%) Season 31.83 0.00** 39.801 0.00** Spring 69 11(15.94%) 69 11(15.94%) Summer 248 6(2.42%) 248 1(0.40%) Autum 201 4(2.00%) 246 6(2.44%) Winter 57 7(12.28%) 174 6(3.45%) Breed 1.06 0.30 0.075 0.79 Purebred 499 22 (4.41%) 643 20 (3.11%) Mixed 76 6 (7.89%) 94 4 (4.26%) Body size 13.56 0.00** 3.815 0.15 Small 336 7 (2.08%) 439 10 (2.28%) Medium 169 15 (8.88%) 213 11 (5.16%) Large 70 6 (8.57%) 85 3 (3.53%) Sex 0.08 0.77 0.256 0.61 Male 334 17 (5.09%) 436 13 (2.98%) Female 241 11 (4.56%) 301 11 (3.65%) Age 27.63 0.00** 18.899 0.00** 0–2 Y 33 6 (18.18%) 42 4 (18.18%) 2–4 Y 46 5 (10.87%) 58 6 (10.87%) 4–6 Y 63 7 (11.11%) 82 4 (4.88%) > 6 Y 433 10 (2.31%) 555 10 (1.80%) χ2: Chi-square. *: The result is significant at P < 0.05; **: The result is significant at P < 0.01; ELISA, qPCR: the same as Table 1 . Risk Factors associated with canine leishmaniasis Based on univariate analysis, residence in suburban areas was linked to a higher risk of CanL seroprevalence ( P = 0.02), but not significantly correlated with molecular positive rates ( P = 0.09) (Table 2 ). Additionally, a heightened prevalence of CanL was observed during spring (15.94%) compared to other seasons (0.40%-12.28%) ( P < 0.00). Among individual risk factors, regardless of the testing method used, age was a significant risk factor for Leishmania infection. Dogs aged 0–2 years showed a significantly higher positive rate (18.18%) compared to other age groups (1.80–11.11%) ( P < 0.01). Medium-sized and large-sized dogs had higher ELISA-positive rates (8.57–8.88%) compared to small-sized dogs (2.08%) ( P = 0.00), but no significant size-related difference was found with qPCR testing ( P = 0.15). Breed and sex did not significantly impact Leishmania -positive rates in either test ( P = 0.30 to 0.79). Application of a multivariate logistical regression model identified season and age as definitive predictors of both ELISA positivity and qPCR positivity of L. infantuma in dogs (Table 3 ). According to the multivariate model, the prevalence of CanL significantly increased in spring (ELISA: OR = 9.31, 95% CI = 2.75–31.47; qPCR: OR = 7.22, 95% CI = 2.49–20.94). Moreover, the risk of infection increased in young age, peaking in dogs aged 0–2 years old (ELISA: OR = 5.49, 95% CI = 1.70–17.80; qPCR: OR = 4.51, 95% CI = 1.25–16.24). Whereas no significant variation was observed among different living regions in the final model. Table 3 Multivariable logistic analysis for risk factors associated with canine leishmaniasis. Risk factors ELISA qPCR OR 95% CI P -value OR 95% CI P -value Living region Urban 1 Suburban 1.78 0.74–4.28 0.20 Season Spring 9.31 2.75–31.47 0.00** 7.22 2.49–20.94 0.00** Summer 1.33 0.36–4.86 0.67 0.17 0.02–1.45 0.11 Autum 1 1 Winter 5.05 1.30-19.66 0.02* 1.26 0.39–4.11 0.70 Age 0–2 Y 5.49 1.70–17.80 0.00** 4.51 1.25–16.24 0.02* 2–4 Y 4.20 1.27–13.87 0.02* 5.77 1.91–17.46 0.00** 4–6 Y 5.53 1.93–15.85 0.00** 2.84 0.83–9.70 0.10 > 6 Y 1 1 OR: Odds ratio; CI: Confidence interval; *: The result is significant at P < 0.05; **: The result is significant at P < 0.01; ELISA, qPCR: the same as Table 1 . Epidemiological Trends in Diagnosed CanL Cases Lymph node aspirates and skin scrapings were collected from 16 dogs that tested positive in either ELISA or qPCR using blood samples. All samples tested positive for Leishmania infection via qPCR. Among these confirmed CanL cases, more cases were confirmed in spring (43.75%) compared to other seasons (12.5–25%). Most of them (68.75%) resided in suburban areas. Medium-sized dogs (50%) had more infections than small-sized (25%) or large-sized (25%) dogs. Age-wise, 12.50% were 0–2 years old, 31.25% were 2–4 years, 31.25% were 4–6 years, and 25.00% were over 6 years old (Table 4 ). Table 4 Epidemiological trend of canine leishmaniasis positive cases. Epidemiological factors Category The number of cases Percent Season Spring (Mar.-May.) 7 43.75% Summer (Jun.-Aug.) 2 12.50% Autumn (Sept.-Nov.) 4 25.00% Winter (Dec.-Feb.) 3 18.75% Living region Urban 5 31.25% Suburban 11 68.75% Body size Small 4 25.00% Medium 8 50.00% Large 4 25.00% Age 0–2 Y 2 12.50% 2–4 Y 5 31.25% 4–6 Y 5 31.25% > 6 Y 4 25.00% Discussion This study is the first to investigate the prevalence of canine Leishmania infection in Beijing using both ELISA and qPCR methods. Epidemiological studies performed in different regions of China report seroprevalences of Leishmania ranging from 5.97–40% [ 8 – 10 ] , molecular positive rate ranging from 28.6–86% [ 9 , 10 ] . In this study, the serology and molecular positive rates in Beijing were 4.87% and 3.26%. While the prevalence rates were lower than some other parts of China, the findings suggest that the luxuriant vegetation in Beijing's hilly areas, which makes up 62% of the city, could favor the reproduction of sandflies and the transmission of Leishmania [ 15 ] . Moreover, since canine Leishmania was first detected in Beijing in 2018, the number of CanL cases has showed an increasing tendency along with population mobility. Thus, it emphasizes the necessity for continued monitoring and prevention strategies, especially in suburban regions like the Mentougou District, known for higher rates of CanL. Our study demonstrated a strong consistency ( P = 0.00) between ELISA and qPCR in detecting Leishmania . In other words, the distribution of positive and negative results was similar for both methods across all samples, despite 20 cases where the results did not align. Additionally, we found the serology positive rate to be slightly higher than the molecular positive rate in blood samples, similar to results reported in previous literature [ 16 , 17 ] . This can be attributed to the longer persistence of antibodies in the blood as compared to the rapid degradation of Leishmania DNA [ 6 ] . Our results underscore the effectiveness of serological assays, particularly ELISA, due to their high sensitivity and ease of use with serum or plasma samples. This study detected L. infantum antigen-specific IgG level in dog’s serum samples by ELISA. Researches have shown that Leishmania infection caused a strong IgG response, and using IgG as an immune marker could achieve a sensitivity of 93.4% and a specificity of 93.7%, but IgM response was not significantly higher in leishmaniasis patients compared to Leishmania -negative individuals, so detection of antigen-specific IgG is recommended for diagnosis of leishmaniasis [ 18 ] . However, ELISA does not always provide accurate results. For instance, in our study, we found that there were 8 samples that tested positive for Leishmania using PCR but tested negative using ELISA. The discrepancy may be due to the fact that PCR can detect Leishmania DNA in the blood within a few weeks after infection [ 19 ] . On the other hand, ELISA requires a seroconversion time of 4–22 months before specific IgG antibodies can be detected in the serum samples [ 20 ] . It is possible that these 8 dogs, which tested positive using PCR but negative using ELISA, had not yet seroconverted, making it difficult for the ELISA method to detect specific antibodies. This hypothesis is supported by Quinnell RJ et al [ 21 ] , who demonstrated that the sensitivity of ELISA is relatively low during the early stages of Leishmania infection. Additionally, a study by Martínez V et al [ 22 ] detected medium to high parasitemia using qPCR in nine dogs with clinical signs but inconclusive or low ELISA results. Therefore, qPCR proves to be an effective diagnostic tool for cases when the dog has not yet seroconverted or symptomatic cases of inconclusive ELISA results. Among the 16 diagnosed cases of CanL in this study, one case tested positive for Leishmania by qPCR using whole blood but tested negative by ELISA using serum samples. This particular case exhibited symptoms such as corneal degeneration, polydipsia, frequent urination, and epistaxis, which had only recently developed. It is possible that this case has not seroconverted yet, resulting in the inability of ELISA to detect the specific antibodies. On the other hand, among the 16 cases, there was one case that tested negative for Leishmania by qPCR using whole blood but tested positive by ELISA using serum samples. This case had previously been treated for focal alopecia and itching symptoms in the dog's ears and paws 2–3 years ago. It is likely that the blood parasitemia disappeared after the treatment, but the specific antibody still remains detectable by ELISA. Besides, lymph node aspirates and skin scrapings from the 16 Leishmania infected animals were also tested by qPCR, and the ITS gene of Leishmania was positive in all the samples. There is evidence that PCR method was highly sensitive when detecting lymph node aspirates and skin scrapings [ 7 , 23 ] . Therefore, combining serology with molecular methods is recommended to enhance diagnostic efficiency. In terms of risk factors, the multivariate logistical regression model indicated that season and age group are significant. The disease had a seasonal trend that varied in different region [ 12 ] . In this study, the disease showed higher incidence in spring, which coincides with the peak breeding period of sandflies in Beijing and the timing of seroconversion following Leishmania infection. Untreated Leishmania infection can lead to lifelong persistence of specific antibodies [ 6 ] . However, if treated, the levels of blood parasitemia decrease significantly after a month [ 24 ] , and gradually becoming undetectable; specific antibodies may persist for more than a year [ 25 ] . In addition to detecting previous infections using ELISA, there is also a seasonal increase in serological positivity attributed to the seasonal increase in new infections. The peak breeding period of sandflies in Beijing primarily occurs in May-June, which is followed by a median seroconversion time of 10.5 months (4–22 months) in naturally infected dogs [ 26 ] . This timeframe aligns with the high seropositivity rate observed in spring (March-May). The high qPCR positivity rate of Leishmania in spring may be attributed to the presence of Leishmania -carrying sandflies during this season. Additionally, the increase in outdoor activities of humans and dogs during spring makes them more likely to come into contact with sandflies. Moreover, younger dogs (0-2Y) showed a higher prevalence rate, likely reflecting their less mature immune systems [ 27 ] . Despite these findings, univariate analysis revealed associations between location and body size with serology positive rates. Although these factors did not maintain significance in the final multivariate model, they should still be regarded as secondary risk factors. The higher prevalence in suburban areas was consistent with the research of Braz et al. [ 11 ] , and this variable reinforced the dynamics of endemicity of visceral leishmaniasis in suburban areas. Medium-sized and large-sized dogs were more affected, possibly due to their greater exposure to sandflies during outdoor activities [ 28 ] . Our analysis of the diagnosed CanL cases reiterates the influence of season, location, and body size as risk factors. Particularly, more cases were confirmed during the spring season and in suburban areas. Medium-sized dogs were most affected, suggesting the need for heightened attention to this group when diagnosing CanL. However, this study has several limitations that should be acknowledged. For example, while it covered both urban and suburban areas of Beijing, the samples are collected exclusively from one animal hospital and one animal disease diagnostic institution in Beijing. Future research could consider expanding the sample selection to include dogs from various settings, such as animal shelters, rescue organizations, and community collaborations. This would provide a more comprehensive understanding of the Leishmania prevalence in the canine population of the entire Beijing region. In conclusion, our findings indicate a definite presence of CanL in Beijing, particularly in the suburban regions. Therefore, a reinforcement of surveillance measures and preventive strategies against canine Leishmania infection is warranted. Conclusions This study represents the first comprehensive serological and molecular examination of canine leishmaniasis (CanL) and associated risk factors in Beijing, China. The findings revealed a seroprevalence of 4.87% and a molecular prevalence of 3.26% for L. infantum among the tested dogs. The study identified a higher prevalence of CanL in spring and among younger dogs (0–2 years), with suburban areas and medium to large-sized dogs also showing higher infection rates. These results highlight the need for targeted control measures and further research to mitigate the public health threat posed by CanL in Beijing. Declarations Acknowledgements We would like to thank participating veterinarians from China Agricultural University Veterinary Teaching Hospital, and Weiwei International Diagnostic Center for their assistance in sample collection in Beijing. The positive control DNA sample of L eishmania was kindly provided by China Agricultural University Veterinary Teaching Hospital. Funding This project was supported by the China Agricultural University Animal Hospital Foundation for the Talents (Project No. 2222005). Availability of data and materials The data that support the findings of this study are available from the corresponding author upon reasonable request. Author contributions Analysis and interpretation of data, drafting the article and revising article for intellectual content: Yujuan Chen. Conception and design, acquisition and analysis of data: Han Tang. Conception and design, analysis and interpretation of data, and revising article for intellectual content: Yonglan Yu. Ethics approval and consent to participate The study has been approved by the Laboratory Animal Welfare and Animal Experimental Ethics Committee, China Agricultural University (permit number: AW21012020-2). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details All the authors are from the College of Veterinary Medicine, China Agricultural University, Beijing, China. References Abdullah AYM, Dewan A, Shogib MRI, Rahman MM, Hossain MF. 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Parasitology. 2001; 122: 253-61. http://10.1017/s0031182001007363 Martínez V, Quilez J, Sanchez A, Roura X, Francino O, Altet L. Canine leishmaniasis: the key points for qPCR result interpretation . Parasit Vectors. 2011; 4: 57. http://10.1186/1756-3305-4-57 Manna L, Vitale F, Reale S, Caracappa S, Pavone LM, Morte RD, et al. Comparison of different tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis . Vet Parasitol. 2004; 125: 251-62. http://10.1016/j.vetpar.2004.07.019 Solano-Gallego L, Di Filippo L, Ordeix L, Planellas M, Roura X, Altet L, et al. Early reduction of Leishmania infantum -specific antibodies and blood parasitemia during treatment in dogs with moderate or severe disease . Parasit Vectors. 2016; 9: 235. http://10.1186/s13071-016-1519-0 Solano-Gallego L, Riera C, Roura X, Iniesta L, Gallego M, Valladares JE, et al. Leishmania infantum -specific IgG, IgG1 and IgG2 antibody responses in healthy and ill dogs from endemic areas. Evolution in the course of infection and after treatment . Vet Parasitol. 2001; 96: 265-76. http://10.1016/s0304-4017(00)00446-5 Oliva G, Scalone A, Foglia Manzillo V, Gramiccia M, Pagano A, Di Muccio T, et al. Incidence and time course of Leishmania infantum infections examined by parasitological, serologic, and nested-PCR techniques in a cohort of naive dogs exposed to three consecutive transmission seasons . J Clin Microbiol. 2006; 44: 1318-22. http://10.1128/jcm.44.4.1318-1322.2006 de Almeida Ado B, Sousa VR, da Cruz FA, Dahroug MA, Figueiredo FB, Madeira Mde F. Canine visceral leishmaniasis: seroprevalence and risk factors in Cuiabá, Mato Grosso, Brazil . Rev Bras Parasitol Vet. 2012; 21: 359-65. http://10.1590/s1984-29612012005000005 Leal GGA, Carneiro M, Pinheiro ADC, Marques LA, Ker HG, Reis AB, et al. Risk profile for Leishmania infection in dogs coming from an area of visceral leishmaniasis reemergence . Prev Vet Med. 2018; 150: 1-7. http://10.1016/j.prevetmed.2017.11.022 Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.jpg Graphical abstract Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4482352","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":310738037,"identity":"a29e64bb-c31a-449f-9ca1-b2d18ea6b7ff","order_by":0,"name":"Yujuan Chen","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yujuan","middleName":"","lastName":"Chen","suffix":""},{"id":310738038,"identity":"2f6e9eec-2fb1-48d5-b064-5be13a6fe4cf","order_by":1,"name":"Han Tang","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"","lastName":"Tang","suffix":""},{"id":310738039,"identity":"4f516d4f-1bfd-47a3-b51d-1454cad2ea2a","order_by":2,"name":"Yonglan Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYJACiQQbBh4DBgbGBxB+AjFa0sBamA2I18KQxsAAVM4mQZQWeffDB288SLCRMWc/fKziR81hBn72HAOGnztwazE8k5ZskZCQxmPZk5Z2s+fYYQbJnjcGjL1n8GhpyDGTSPxxmMfgQI7ZbQa2wwwGN3IMmBnb8Gjpf2MmkZDwn8fg/BuzYoZ/hxnsCWmRl8gBaTnAAzTcDKgSaIsEAS0GEs9AfkkGanmWLNnbl84jceZZwcFefLb0Jx+8+SPBzt7gfPLBDz++WcvxtydvfPATny0H0AR4QAS6IKotDfhkR8EoGAWjYBSAAACT3lB0/mcgKQAAAABJRU5ErkJggg==","orcid":"","institution":"China Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Yonglan","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2024-05-27 05:08:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4482352/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4482352/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58224865,"identity":"e5ecf23b-760b-4e29-86d8-d8c057cde99b","added_by":"auto","created_at":"2024-06-12 17:41:58","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2898610,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAmplification curve of quantitative real-time PCR positive blood samples.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4482352/v1/d59b2273919d22e4889cb954.jpg"},{"id":58224863,"identity":"df823c5f-491d-48a5-b2f8-b889dda04db0","added_by":"auto","created_at":"2024-06-12 17:41:58","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1520410,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe specific fragment of the ITS gene of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLeishmania\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM: DNA marker; Lane 1, 25: Negative control; Lane 2, 26: Positive control; Lane 3~24, 27~29: target gene fragments.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4482352/v1/afc1740e2b6dfed79f6be260.jpg"},{"id":58224864,"identity":"aa655a9e-e33b-47a7-a4cc-fb95337c8e4a","added_by":"auto","created_at":"2024-06-12 17:41:58","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":338388,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrevalence of canine leishmaniasis in Beijing.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSP: seroprevalence detected by ELISA; MP: molecular prevalence detected by qPCR.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4482352/v1/07f87425569ddc6f7311038b.jpg"},{"id":62993981,"identity":"868e77d9-f2ed-4e9a-a8fe-c90f6196c890","added_by":"auto","created_at":"2024-08-22 00:22:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5687885,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4482352/v1/c8796746-2f48-4743-81aa-3993ca0ed0c8.pdf"},{"id":58225652,"identity":"bdbb813d-6d24-41b2-9350-76ce0ad2faa5","added_by":"auto","created_at":"2024-06-12 17:49:58","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2623196,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"GraphicalAbstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4482352/v1/414d50d4ed2946d24ba53961.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Seasonal Prevalence and Risk Factors of canine Leishmania Infection in Beijing, China","fulltext":[{"header":"Background","content":"\u003cp\u003eLeishmaniasis, a vector-borne zoonotic disease, is instigated by protozoa of the genus \u003cem\u003eLeishmania\u003c/em\u003e, primarily transmitted through the bite of sandflies. Dogs are the principal domestic reservoir for \u003cem\u003eLeishmania infantum\u003c/em\u003e (\u003cem\u003eL. infantum\u003c/em\u003e), making canine leishmaniasis (CanL) a focal point of public health concern. Notably, leishmaniasis has achieved endemic status in over 98 countries \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e, endangering approximately 350\u0026nbsp;million people, with an estimated 700,000 to 1\u0026nbsp;million new cases reported annually \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Regions reporting endemic prevalence of leishmaniasis include the Americas, East Africa, North Africa, West Asia, and Southeast Asia \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eL. infantum\u003c/em\u003e in humans is accountable for various forms of leishmaniasis, such as visceral, cutaneous, or mucosal \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. CanL is often asymptomatic, although it can sometimes manifest in generalized cutaneous and ovarian forms that are spontaneously fatal \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. This asymptomatic nature combined with limited laboratory diagnostic capabilities exacerbates the difficulty of detecting infected animals \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDiagnostic methodologies for leishmaniasis often encompass serological and molecular techniques. Serological assays, including enzyme-linked immunosorbent assay (ELISA) and direct agglutination test (DAT), provide a non-invasive means of detecting specific antibodies, but fail to differentiate between past and ongoing infections \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Conversely, PCR-based assays have proven to be highly reliable \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e, leading many veterinarians to opt for a dual approach: combining molecular analyses with serological diagnosis to verify infections and identify the \u003cem\u003eLeishmania\u003c/em\u003e species involved.\u003c/p\u003e \u003cp\u003eExisting reports from China, specifically endemic areas such as Shanxi \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, Sichuan \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, and Gansu Provinces \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, provide limited seroprevalence and molecular data, having included fewer than 300 samples. Moreover, while the number of CanL cases in Beijing has seen a steady rise over the past two years, comprehensive data detailing the prevalence of CanL in Beijing remains scarce. Studies have indicated that the prevalence of CanL varies between suburban and urban areas \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e, with seasonal trends diverging across different regions \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e, yet no data elucidating the spatial distribution and seasonal variations of CanL in Beijing has been reported. It is also uncertain whether individual factors such as age, sex, breed, and body size influence the incidence of CanL.\u003c/p\u003e \u003cp\u003eIn light of these gaps, we sought to gain a clearer understanding of the prevalence of leishmaniasis in domestic dogs in Beijing. For this purpose, we collected serum samples from 575 dogs for the detection of anti-\u003cem\u003eL. infantum\u003c/em\u003e Ig G antibodies by ELISA, and anticoagulated whole blood samples from 737 dogs for the detection of \u003cem\u003eLeishmania\u003c/em\u003e DNA via quantitative real-time PCR (qPCR). Subsequently, we analyzed the risk factors associated with CanL. Additionally, we scrutinized the epidemiological data of 16 dogs diagnosed with CanL to identify potential risk factors. This study aims to generate data that can be instrumental in devising strategies for leishmaniasis prevention and control.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy area\u003c/h2\u003e \u003cp\u003eBeijing is located in northern China, positioned between 115\u0026deg;25' and 117\u0026deg;30' E and 39\u0026deg;26' and 41\u0026deg;03' N. The city encompasses 16 districts and is predominantly characterized by plains and hills, with mountainous regions to the northwest. Beijing experiences a temperate continental monsoon climate, featuring hot, rainy summers and cold, dry winters.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthical Approval and Sample Collection\u003c/strong\u003e \u003c/p\u003e\u003cp\u003e The study, having been approved by the Laboratory Animal Welfare and Animal Experimental Ethics Committee, China Agricultural University (permit number: AW21012020-2), included a total of 737 dogs from March 2021 to February 2022. Serum and anticoagulated whole blood samples of these dogs were collected from China Agricultural University Veterinary Teaching Hospital and Weiwei International Diagnostic Center in Beijing. From the 737 dogs, both serum and anticoagulated whole blood samples were collected from 575 individuals, besides, only serum samples were collected from 162 dogs. Serum samples from 575 dogs were centrifuged at 1000\u0026times;g for 10 min, and 200 \u0026micro;L of the serum samples was stored at -80\u0026deg;C for ELISA testing; EDTA-anticoagulated whole blood samples from 737 dogs were collected for qPCR testing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eEpidemiological Data Collection\u003c/h2\u003e \u003cp\u003eEpidemiological data for all 737 dogs, including factors such as region, season, breed, body size, sex, and age, was obtained from the owners through face-to-face questionnaires or telephone interviews.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eELISA Testing\u003c/h2\u003e \u003cp\u003eIgG antibodies against \u003cem\u003eL. infantum\u003c/em\u003e in dogs were detected using the ID Screen\u0026reg; leishmaniasis Indirect Test (VET-Innovate ID Diagnostics, France).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDNA Extraction\u003c/h2\u003e \u003cp\u003eDNA was extracted from blood, lymph node aspirates, and skin scrapings of dogs using the TianGen\u0026reg; DNA Kit for Blood, Cell and Tissues (Genomics Bioscience and Technology Co., Ltd., Beijing, China) according to the manufacturer\u0026rsquo;s protocol. The extracted DNA was stored at -80\u0026deg;C for molecular testing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eQPCR Testing\u003c/h2\u003e \u003cp\u003eThe kinetoplast DNA (kDNA) of \u003cem\u003eLeishmania\u003c/em\u003e was amplified using the extracted genomic DNA as a template \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. The reaction mixture consisted of PowerUp\u0026trade; SYBR\u0026trade; Green Master Mix, forward and reverse primers, extracted DNA, and nuclease-free water. A pre-specified Ct value of \u0026le;\u0026thinsp;35 was considered positive.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLeishmania Species Identification\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eLeishmania\u003c/em\u003e ITS gene was amplified from the extracted DNA of qPCR-positive blood samples using the ITS primers \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. The PCR products were sequenced and compared with the molecular profiles of \u003cem\u003eLeishmania\u003c/em\u003e reference strains, namely \u003cem\u003eL. braziliensis\u003c/em\u003e (FJ753376), \u003cem\u003eL. guyanensis\u003c/em\u003e (FJ753387), \u003cem\u003eL. panamensis\u003c/em\u003e (FJ948438), \u003cem\u003eL. mexicana\u003c/em\u003e (FJ948432), \u003cem\u003eL. amazonensis\u003c/em\u003e (FJ753371), \u003cem\u003eL. donovani\u003c/em\u003e (FJ753386), \u003cem\u003eL. infantum\u003c/em\u003e (GU045592), \u003cem\u003eL. major\u003c/em\u003e (FJ753391), \u003cem\u003eL. tropica\u003c/em\u003e (FJ948447) and \u003cem\u003eL. aethiopica\u003c/em\u003e (GQ920673).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis of infection risk factors was conducted using SPSS version 23 software. The Kappa test was used to analyze the consistency of qPCR and ELISA on detecting \u003cem\u003eLeishmania\u003c/em\u003e. The Chi-square (χ2) test was applied to assess risk factors associated with \u003cem\u003eLeishmania\u003c/em\u003e infection diagnosed by ELISA or qPCR. Variables exhibiting a statistically significant association (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were then included in a multivariable logistic regression model to determine risk factors, odds ratios (ORs) and confidence intervals (CIs) for each significant variable. The Spearman's rank correlation coefficient was used to assess the correlation between two variables. The Hosmer-Lemeshow goodness-of-fit test was utilized to evaluate the fit of the multivariable logistic regression model. During the construction of the multivariable model concerning variables associated with the ELISA positive rate of \u003cem\u003eLeishmania\u003c/em\u003e, we excluded body size due to its correlation with age according to the Spearman's rank correlation coefficient.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEpidemiological Analysis of Diagnosed CanL Cases\u003c/h2\u003e \u003cp\u003eBased on risk factors identified in preceding statistical analysis, dogs diagnosed with CanL were analyzed further. Symptoms such as poor general condition, weight loss, swollen lymph nodes, onychogryphosis, and cutaneous lesions were used to select dogs for additional testing. Lymph node punctures were performed on swollen lymph nodes, including the pre-scapular, popliteal, and mandibular lymph nodes. Skin scrapings were obtained from cutaneous lesions, and both sample types were assessed using qPCR to confirm \u003cem\u003eLeishmania\u003c/em\u003e infection.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOverall Prevalence of canine leishmaniasis\u003c/h2\u003e \u003cp\u003eOut of 575 serum samples tested for anti-\u003cem\u003eLeishmania infantum\u003c/em\u003e antibodies, 28 (4.87%) were reactive in the ELISA test. Additionally, out of 737 whole blood samples, 24 (3.26%) were \u003cem\u003eLeishmania\u003c/em\u003e DNA positive (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All 24 qPCR-positive samples matched\u0026thinsp;\u0026ge;\u0026thinsp;99.28% similarity with the \u003cem\u003eL. infantum\u003c/em\u003e species after sequencing analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). QPCR and ELISA showed remarkable consistency (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00) in detecting Leishmania among the 575 dogs subjected to both tests. Out of the total, 36 dogs tested positive either by qPCR or ELISA. Among them, 16 were positive by both ELISA and qPCR, 8 were positive by qPCR but negative by ELISA, and 12 were positive by ELISA but negative by qPCR. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrevalence of canine leishmaniasis in Beijing tested by qPCR and ELISA.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQPCR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eELISA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo. of tested\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKappa-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.00**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNagative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNagative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e539\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNot tested\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e**: The result is significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ELISA: Enzyme-linked immunosorbent assay; qPCR: quantitative real-time PCR.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePrevalence of canine leishmaniasis in Different Sites\u003c/h2\u003e \u003cp\u003eSuburban areas showed a higher prevalence of CanL (7.38% by ELISA, 4.56% by qPCR) compared to urban areas (3.02% by ELISA, 2.33% by qPCR) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Dogs in Mentougou District had the highest serology (50%) and qPCR (37.0%) positive rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eUnivariate analysis of risk factors associated with canine leishmaniasis.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRisk factors\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eELISA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e \u003cp\u003eqPCR\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo. of tested dogs (n\u0026thinsp;=\u0026thinsp;575)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo. of positive dogs (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eχ2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo. of tested dogs (n\u0026thinsp;=\u0026thinsp;737)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNo. of positive dogs (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eχ2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLiving region\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.02*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.839\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrban\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e331\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10(3.02%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e430\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e10(2.33%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSuburban\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e244\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18(7.38%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e307\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e14(4.56%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSeason\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.00**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e39.801\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.00**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpring\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11(15.94%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e11(15.94%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSummer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e248\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6(2.42%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e248\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1(0.40%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAutum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e201\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4(2.00%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e246\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6(2.44%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWinter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7(12.28%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6(3.45%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBreed\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.075\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePurebred\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e499\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e22 (4.41%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e643\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e20 (3.11%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMixed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6 (7.89%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4 (4.26%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBody size\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.00**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.815\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmall\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e336\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7 (2.08%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e439\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e10 (2.28%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15 (8.88%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e213\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e11 (5.16%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLarge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6 (8.57%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3 (3.53%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSex\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e334\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17 (5.09%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e436\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e13 (2.98%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e241\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11 (4.56%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e301\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e11 (3.65%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.00**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e18.899\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.00**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u0026ndash;2 Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6 (18.18%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4 (18.18%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u0026ndash;4 Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5 (10.87%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6 (10.87%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u0026ndash;6 Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7 (11.11%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4 (4.88%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;6 Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e433\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10 (2.31%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e10 (1.80%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eχ2: Chi-square. *: The result is significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **: The result is significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ELISA, qPCR: the same as Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRisk Factors associated with canine leishmaniasis\u003c/h2\u003e \u003cp\u003eBased on univariate analysis, residence in suburban areas was linked to a higher risk of CanL seroprevalence (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02), but not significantly correlated with molecular positive rates (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.09) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Additionally, a heightened prevalence of CanL was observed during spring (15.94%) compared to other seasons (0.40%-12.28%) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.00). Among individual risk factors, regardless of the testing method used, age was a significant risk factor for \u003cem\u003eLeishmania\u003c/em\u003e infection. Dogs aged 0\u0026ndash;2 years showed a significantly higher positive rate (18.18%) compared to other age groups (1.80\u0026ndash;11.11%) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Medium-sized and large-sized dogs had higher ELISA-positive rates (8.57\u0026ndash;8.88%) compared to small-sized dogs (2.08%) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00), but no significant size-related difference was found with qPCR testing (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.15). Breed and sex did not significantly impact \u003cem\u003eLeishmania\u003c/em\u003e-positive rates in either test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.30 to 0.79).\u003c/p\u003e \u003cp\u003eApplication of a multivariate logistical regression model identified season and age as definitive predictors of both ELISA positivity and qPCR positivity of \u003cem\u003eL. infantuma\u003c/em\u003e in dogs (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). According to the multivariate model, the prevalence of CanL significantly increased in spring (ELISA: OR\u0026thinsp;=\u0026thinsp;9.31, 95% CI\u0026thinsp;=\u0026thinsp;2.75\u0026ndash;31.47; qPCR: OR\u0026thinsp;=\u0026thinsp;7.22, 95% CI\u0026thinsp;=\u0026thinsp;2.49\u0026ndash;20.94). Moreover, the risk of infection increased in young age, peaking in dogs aged 0\u0026ndash;2 years old (ELISA: OR\u0026thinsp;=\u0026thinsp;5.49, 95% CI\u0026thinsp;=\u0026thinsp;1.70\u0026ndash;17.80; qPCR: OR\u0026thinsp;=\u0026thinsp;4.51, 95% CI\u0026thinsp;=\u0026thinsp;1.25\u0026ndash;16.24). Whereas no significant variation was observed among different living regions in the final model.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMultivariable logistic analysis for risk factors associated with canine leishmaniasis.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRisk factors\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eELISA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eqPCR\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLiving region\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrban\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSuburban\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.74\u0026ndash;4.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSeason\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpring\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.75\u0026ndash;31.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.00**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.49\u0026ndash;20.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.00**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSummer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.36\u0026ndash;4.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.02\u0026ndash;1.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAutum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWinter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.30-19.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.02*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.39\u0026ndash;4.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u0026ndash;2 Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.70\u0026ndash;17.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.00**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.25\u0026ndash;16.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.02*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u0026ndash;4 Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.27\u0026ndash;13.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.02*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.91\u0026ndash;17.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.00**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u0026ndash;6 Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.93\u0026ndash;15.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.00**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.83\u0026ndash;9.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;6 Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eOR: Odds ratio; CI: Confidence interval; *: The result is significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **: The result is significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ELISA, qPCR: the same as Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEpidemiological Trends in Diagnosed CanL Cases\u003c/h2\u003e \u003cp\u003eLymph node aspirates and skin scrapings were collected from 16 dogs that tested positive in either ELISA or qPCR using blood samples. All samples tested positive for \u003cem\u003eLeishmania\u003c/em\u003e infection via qPCR. Among these confirmed CanL cases, more cases were confirmed in spring (43.75%) compared to other seasons (12.5\u0026ndash;25%). Most of them (68.75%) resided in suburban areas. Medium-sized dogs (50%) had more infections than small-sized (25%) or large-sized (25%) dogs. Age-wise, 12.50% were 0\u0026ndash;2 years old, 31.25% were 2\u0026ndash;4 years, 31.25% were 4\u0026ndash;6 years, and 25.00% were over 6 years old (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEpidemiological trend of canine leishmaniasis positive cases.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEpidemiological factors\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCategory\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThe number of cases\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePercent\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSeason\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpring (Mar.-May.)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e43.75%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSummer (Jun.-Aug.)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAutumn (Sept.-Nov.)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWinter (Dec.-Feb.)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18.75%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiving region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUrban\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31.25%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSuburban\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e68.75%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBody size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSmall\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLarge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u0026ndash;2 Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u0026ndash;4 Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31.25%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u0026ndash;6 Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31.25%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;6 Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study is the first to investigate the prevalence of canine \u003cem\u003eLeishmania\u003c/em\u003e infection in Beijing using both ELISA and qPCR methods. Epidemiological studies performed in different regions of China report seroprevalences of Leishmania ranging from 5.97\u0026ndash;40% \u003csup\u003e[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, molecular positive rate ranging from 28.6\u0026ndash;86% \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. In this study, the serology and molecular positive rates in Beijing were 4.87% and 3.26%. While the prevalence rates were lower than some other parts of China, the findings suggest that the luxuriant vegetation in Beijing's hilly areas, which makes up 62% of the city, could favor the reproduction of sandflies and the transmission of \u003cem\u003eLeishmania\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Moreover, since canine \u003cem\u003eLeishmania\u003c/em\u003e was first detected in Beijing in 2018, the number of CanL cases has showed an increasing tendency along with population mobility. Thus, it emphasizes the necessity for continued monitoring and prevention strategies, especially in suburban regions like the Mentougou District, known for higher rates of CanL.\u003c/p\u003e \u003cp\u003eOur study demonstrated a strong consistency (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00) between ELISA and qPCR in detecting \u003cem\u003eLeishmania\u003c/em\u003e. In other words, the distribution of positive and negative results was similar for both methods across all samples, despite 20 cases where the results did not align. Additionally, we found the serology positive rate to be slightly higher than the molecular positive rate in blood samples, similar to results reported in previous literature \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. This can be attributed to the longer persistence of antibodies in the blood as compared to the rapid degradation of \u003cem\u003eLeishmania\u003c/em\u003e DNA \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Our results underscore the effectiveness of serological assays, particularly ELISA, due to their high sensitivity and ease of use with serum or plasma samples. This study detected \u003cem\u003eL. infantum\u003c/em\u003e antigen-specific IgG level in dog\u0026rsquo;s serum samples by ELISA. Researches have shown that \u003cem\u003eLeishmania\u003c/em\u003e infection caused a strong IgG response, and using IgG as an immune marker could achieve a sensitivity of 93.4% and a specificity of 93.7%, but IgM response was not significantly higher in leishmaniasis patients compared to \u003cem\u003eLeishmania\u003c/em\u003e-negative individuals, so detection of antigen-specific IgG is recommended for diagnosis of leishmaniasis \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHowever, ELISA does not always provide accurate results. For instance, in our study, we found that there were 8 samples that tested positive for \u003cem\u003eLeishmania\u003c/em\u003e using PCR but tested negative using ELISA. The discrepancy may be due to the fact that PCR can detect \u003cem\u003eLeishmania\u003c/em\u003e DNA in the blood within a few weeks after infection \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. On the other hand, ELISA requires a seroconversion time of 4\u0026ndash;22 months before specific IgG antibodies can be detected in the serum samples \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. It is possible that these 8 dogs, which tested positive using PCR but negative using ELISA, had not yet seroconverted, making it difficult for the ELISA method to detect specific antibodies. This hypothesis is supported by Quinnell RJ et al \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, who demonstrated that the sensitivity of ELISA is relatively low during the early stages of \u003cem\u003eLeishmania\u003c/em\u003e infection. Additionally, a study by Mart\u0026iacute;nez V et al \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e detected medium to high parasitemia using qPCR in nine dogs with clinical signs but inconclusive or low ELISA results. Therefore, qPCR proves to be an effective diagnostic tool for cases when the dog has not yet seroconverted or symptomatic cases of inconclusive ELISA results.\u003c/p\u003e \u003cp\u003eAmong the 16 diagnosed cases of CanL in this study, one case tested positive for \u003cem\u003eLeishmania\u003c/em\u003e by qPCR using whole blood but tested negative by ELISA using serum samples. This particular case exhibited symptoms such as corneal degeneration, polydipsia, frequent urination, and epistaxis, which had only recently developed. It is possible that this case has not seroconverted yet, resulting in the inability of ELISA to detect the specific antibodies. On the other hand, among the 16 cases, there was one case that tested negative for \u003cem\u003eLeishmania\u003c/em\u003e by qPCR using whole blood but tested positive by ELISA using serum samples. This case had previously been treated for focal alopecia and itching symptoms in the dog's ears and paws 2\u0026ndash;3 years ago. It is likely that the blood parasitemia disappeared after the treatment, but the specific antibody still remains detectable by ELISA. Besides, lymph node aspirates and skin scrapings from the 16\u003cem\u003eLeishmania\u003c/em\u003e infected animals were also tested by qPCR, and the ITS gene of \u003cem\u003eLeishmania\u003c/em\u003e was positive in all the samples. There is evidence that PCR method was highly sensitive when detecting lymph node aspirates and skin scrapings \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Therefore, combining serology with molecular methods is recommended to enhance diagnostic efficiency.\u003c/p\u003e \u003cp\u003eIn terms of risk factors, the multivariate logistical regression model indicated that season and age group are significant.\u003c/p\u003e \u003cp\u003eThe disease had a seasonal trend that varied in different region \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. In this study, the disease showed higher incidence in spring, which coincides with the peak breeding period of sandflies in Beijing and the timing of seroconversion following \u003cem\u003eLeishmania\u003c/em\u003e infection. Untreated \u003cem\u003eLeishmania\u003c/em\u003e infection can lead to lifelong persistence of specific antibodies \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. However, if treated, the levels of blood parasitemia decrease significantly after a month \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, and gradually becoming undetectable; specific antibodies may persist for more than a year \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. In addition to detecting previous infections using ELISA, there is also a seasonal increase in serological positivity attributed to the seasonal increase in new infections. The peak breeding period of sandflies in Beijing primarily occurs in May-June, which is followed by a median seroconversion time of 10.5 months (4\u0026ndash;22 months) in naturally infected dogs \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. This timeframe aligns with the high seropositivity rate observed in spring (March-May). The high qPCR positivity rate of \u003cem\u003eLeishmania\u003c/em\u003e in spring may be attributed to the presence of \u003cem\u003eLeishmania\u003c/em\u003e-carrying sandflies during this season. Additionally, the increase in outdoor activities of humans and dogs during spring makes them more likely to come into contact with sandflies.\u003c/p\u003e \u003cp\u003eMoreover, younger dogs (0-2Y) showed a higher prevalence rate, likely reflecting their less mature immune systems \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite these findings, univariate analysis revealed associations between location and body size with serology positive rates. Although these factors did not maintain significance in the final multivariate model, they should still be regarded as secondary risk factors.\u003c/p\u003e \u003cp\u003eThe higher prevalence in suburban areas was consistent with the research of Braz et al. \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e, and this variable reinforced the dynamics of endemicity of visceral leishmaniasis in suburban areas. Medium-sized and large-sized dogs were more affected, possibly due to their greater exposure to sandflies during outdoor activities \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur analysis of the diagnosed CanL cases reiterates the influence of season, location, and body size as risk factors. Particularly, more cases were confirmed during the spring season and in suburban areas. Medium-sized dogs were most affected, suggesting the need for heightened attention to this group when diagnosing CanL.\u003c/p\u003e \u003cp\u003eHowever, this study has several limitations that should be acknowledged. For example, while it covered both urban and suburban areas of Beijing, the samples are collected exclusively from one animal hospital and one animal disease diagnostic institution in Beijing. Future research could consider expanding the sample selection to include dogs from various settings, such as animal shelters, rescue organizations, and community collaborations. This would provide a more comprehensive understanding of the \u003cem\u003eLeishmania\u003c/em\u003e prevalence in the canine population of the entire Beijing region.\u003c/p\u003e \u003cp\u003eIn conclusion, our findings indicate a definite presence of CanL in Beijing, particularly in the suburban regions. Therefore, a reinforcement of surveillance measures and preventive strategies against canine \u003cem\u003eLeishmania\u003c/em\u003e infection is warranted.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study represents the first comprehensive serological and molecular examination of canine leishmaniasis (CanL) and associated risk factors in Beijing, China. The findings revealed a seroprevalence of 4.87% and a molecular prevalence of 3.26% for L. infantum among the tested dogs. The study identified a higher prevalence of CanL in spring and among younger dogs (0\u0026ndash;2 years), with suburban areas and medium to large-sized dogs also showing higher infection rates. These results highlight the need for targeted control measures and further research to mitigate the public health threat posed by CanL in Beijing.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank participating veterinarians from China Agricultural University Veterinary Teaching Hospital, and Weiwei International Diagnostic Center for their assistance in sample collection in Beijing. The positive control DNA sample of \u003cem\u003eL\u003c/em\u003e\u003cem\u003eeishmania\u0026nbsp;\u003c/em\u003ewas kindly provided by China Agricultural University Veterinary Teaching Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was supported by the China Agricultural University Animal Hospital Foundation for the Talents (Project No. 2222005).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalysis and interpretation of data, drafting the article and revising article for intellectual content: Yujuan Chen. Conception and design, acquisition and analysis of data: Han Tang. Conception and design, analysis and interpretation of data, and revising article for intellectual content: Yonglan Yu.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study has been approved by the Laboratory Animal Welfare and Animal Experimental Ethics Committee, China Agricultural University (permit number: AW21012020-2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors are from the College of Veterinary Medicine, China Agricultural University, Beijing, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdullah AYM, Dewan A, Shogib MRI, Rahman MM, Hossain MF. 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Risk profile for \u003cem\u003eLeishmania\u003c/em\u003e infection in dogs coming from an area of visceral leishmaniasis reemergence\u003cem\u003e.\u003c/em\u003e Prev Vet Med. 2018; 150: 1-7. http://10.1016/j.prevetmed.2017.11.022\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"canine leishmaniasis, epidemiology, prevalence, risk factors","lastPublishedDoi":"10.21203/rs.3.rs-4482352/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4482352/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLeishmaniasis, a severe zoonotic disease where dogs act as the main reservoir for \u003cem\u003eLeishmania infantum\u003c/em\u003e (\u003cem\u003eL. infantum\u003c/em\u003e), poses a significant public health threat. Despite recent cases in Beijing, China, there is a lack of comprehensive studies on canine leishmaniasis (CanL) in this region. This study aimed to determine the prevalence of CanL in Beijing and identify associated risk factors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEnzyme-linked immunosorbent assay (ELISA) was used to detect anti-L. infantum antibodies, and quantitative real-time PCR (qPCR) was used to identify Leishmania DNA. The study included 737 dogs: 575 were tested with both ELISA using serum samples and qPCR using whole blood samples, while the remaining 162 were tested only with qPCR. Variables related to the dogs were collected and evaluated through univariate analysis (the Chi-square test) and multivariate logistical regression analysis. For dogs that tested positive on ELISA or qPCR and exhibited suspicious clinical symptoms, we collected lymph node punctures and skin scrape material to reconfirm \u003cem\u003eLeishmania\u003c/em\u003e infection by qPCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eELISA and qPCR prevalence rates were 4.87% (28/575) and 3.26% (24/737), respectively, with all qPCR-positive samples confirmed as \u003cem\u003eL. infantum\u003c/em\u003e by PCR amplification of the internal transcribed spacer (ITS) gene. QPCR and ELISA demonstrated strong consistency (\u003cem\u003eP\u003c/em\u003e = 0.00) in detecting \u003cem\u003eLeishmania\u003c/em\u003e among the 575 dogs subjected to both tests. Multivariate analysis revealed a higher prevalence of CanL during springtime in Beijing (ELISA: OR = 9.31, 95% CI = 2.75–31.47; qPCR: OR = 7.22, 95% CI = 2.49–20.94), and among younger dogs (0-2Y) (ELISA: OR = 5.49, 95% CI = 1.70–17.80; qPCR: OR = 4.51, 95% CI = 1.25–16.24). Univariate analysis identified suburban areas (\u003cem\u003eP\u003c/em\u003e = 0.02) and medium to large-sized dogs (\u003cem\u003eP\u003c/em\u003e = 0.00) as secondary risk factors. Among the dogs that tested positive on ELISA or qPCR, 16 were subsequently confirmed to have CanL through qPCR test of lymph node aspirates and skin scrapings. The majority of these cases were found in suburban areas (68.75%), with 43.75% detected during springtime. Additionally, 50% of the cases involved medium-sized dogs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeroprevalence and molecular prevalence of CanL in Beijing was 4.87% and 3.26% respectively, with a higher prevalence observed in spring and among younger dogs (0-2Y), emphasizing the importance of comprehensive studies to address public health threats.\u003c/p\u003e","manuscriptTitle":"Seasonal Prevalence and Risk Factors of canine Leishmania Infection in Beijing, China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-12 17:41:53","doi":"10.21203/rs.3.rs-4482352/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8b34e774-4841-4f2b-b126-ff98d4af59f7","owner":[],"postedDate":"June 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-22T00:14:09+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-12 17:41:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4482352","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4482352","identity":"rs-4482352","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}