Analysis of risk factors of Leishmania infection in dogs in Gansu Province, China

Background Gansu is a province with the most serious prevalence of canine Leishmaniasis in China, ranked the rst in the number of new cases occurring each year. In order to understand the risk factors of visceral Leishmaniasis in dogs in endemic areas of Gansu province, we conducted an investigation on the risk factors of canine Leishmaniasis in 2 endemic counties from 2018 to 2019, and analyzed the related factors of local CVL infection, so as to provide a basis for exploring new prevention and control methods suitable for Leishmaniasis in China. Methods Leishmaniasis of canine origin was detected in Diebu county and Wenxian County, Gansu province. The risk factors of Leishmania infection in dogs were analyzed by USING IBM SPSS23.0 Pearsonχ2 test and multiple Logistic regression. Results a total of 537 domestic dogs were investigated in the endemic area. The positive rate of Leishmania protozoa nested PCR was 41.15% (221/537). Univariate analysis showed that there were statistically signicant differences in age, height, weight, breed, kennel presence, hair length, custody style, physical condition, exercise status, nail symptoms and other variables with the positive rate of nested PCR (P < 0.05).The results of multivariate analysis showed that there was a signicant correlation between dog ownership [OR=3.051; 95%CI =(0.965 ~ 9.645), dog ownership [OR=1.974; 95%CI =(1.199 ~ 3.252), dog breed [OR=1.819; 95%CI =(0.999 ~ 3.311)] and hair length [0.053 ~ 0.203]. Conclusions there are a large number of asymptomatic infected dogs in Diebu county and Wenxian County of Gansu Province. The infection rate of local short-haired and pure-bred dogs is high. In order to reduce the risk of canine infection, the publicity and education on prevention of canine Leishmaniasis should be strengthened, so as to reduce the risk of human and animal co-contracting visceral Leishmaniasis.


Background
Visceral Leishmaniasis is caused by Leishmania donovani or L. infantum, protozoan parasites that are transmitted to human and animal hosts by the bite of phlebotomine sand ies [1]. Dogs are the main urban reservoirs of L. infantum and represent the major source of contagion for the vectors by virtue of the high prevalence of infection and intense cutaneous parasitism [2,3].
Canine visceral Leishmaniasis (CVL) is present in approximately 50 countries, mainly in South America and the Mediterranean region [4,5]. Epidemiological studies in endemic areas have demonstrated that the prevalence of infection as determined by molecular techniques (50-80%) is higher than the seroprevalence (10-30%) [6,7]. A cohort study conducted by Oliva et al. (2006) showed that most of the study animals were polymerase chain reaction (PCR)-positive months before seroconversion [8]. The CVL infection rate depends on several factors including the length of the transmission season, the vector density, the susceptibility of the dog population, dog behavior, the degree of exposure to vectors, and dog owners' attitudes toward prevention [9,10]. Preventing the expansion and urbanization of zoonotic visceral Leishmaniasis (ZVL) requires that the risk factors associated with human and canine infection be identi ed. Some cross-sectional serological surveys have suggested that canine susceptibility to infection is associated with dog size, fur length, age, and housing conditions [11,14]. During a previously reported cross-sectional study carried out in an urban area of Brazil (Belo Horizonte, southeastern Brazil), They evaluated the prevalence of and risk factors associated with L. infantum infection in dogs, as identi ed by means of a molecular test (polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP) [15], and demonstrated that dogs with PCR positive for L. infantum showed approximately twice the risk of seroconversion as those that were PCR negative [16]; Some foreign studies have evaluated the risk factors associated with L. infantum infection in dogs, however few have used a risk factors in China.
Gansu is a province with the most serious prevalence of canine Kala Azar in China, ranked the rst in the number of new cases occurring each year. Dogs with visceral leishmaniasis are the primary source of infection; patients are mostly infants and young children; the transmission medium is peri-wild Phlebotomus chinensis [17,18]. Following the Wenchuan earthquake in 2008, natural factors and the ecological environment have changed, and the large number of stray dogs have become the source of infection of Kala Azar; as a result, the epidemic situation has recovered and showed a tendency to spread to nonprevalent regions. A previous survey shows that a lot of dogs with latent infection exist in Kala Azar epidemic areas in Gansu Province [19,20]. In order to understand the risk factors of visceral Leishmaniasis in dogs in endemic areas of Gansu province, we conducted an investigation on the risk factors of canine Leishmaniasis in 2 endemic counties from 2018 to 2019, and analyzed the related factors of local CVL infection, so as to provide a basis for exploring new prevention and control methods suitable for Leishmaniasis in China.

Site investigation
Xinglong Village, Zhongzhai Town, Wenxian County, Longnan City, and Luoda Village, Luoda Town, Diebu County, Gannan Prefecture is the visceral leishmaniasis (VL) major epidemic area (Fig. 1), 2 county is located in the mountains, vegetation, rich media suitable for sand y survival quantity many, there is an important channel of tra c travel, business, trade and destination, season more than outsiders to the spread of CVL, ow is frequent, lead to health and infection risk is very big. In recent years, which have reported VL cases, so we were selected as investigation sites. In April of each year, a questionnaire was used to record the gender, age, height, weight, breed, kennel status, custody mode, health status, appearance characteristics and other information of domestic dogs in the survey village, and all dogs were sampled

Site investigation
Xinglong Village, Zhongzhai Town, Wenxian County, Longnan City, and Luoda Village, Luoda Town, Diebu County, Gannan Prefecture is the visceral leishmaniasis (VL) major epidemic area (Fig. 1), 2 county is located in the mountains, vegetation, rich media suitable for sand y survival quantity many, there is an important channel of tra c travel, business, trade and destination, season more than outsiders to the spread of CVL, ow is frequent, lead to health and infection risk is very big. In recent years, which have reported VL cases, so we were selected as investigation sites. In April of each year, a questionnaire was used to record the gender, age, height, weight, breed, kennel status, custody mode, health status, appearance characteristics and other information of domestic dogs in the survey village, and all dogs were sampled by vein. At the same time, venous blood of healthy dogs from Songshan Village, Songshan Town, Tianzhu County, Wuwei city, non-endemic area of Leishmaniasis, was collected for the quality control. All the dogs sampled in the survey had a master dog, and all the owners agreed that the data and samples would be used in the study.

Detection method
Nested PCR was used to detect blood samples with high sensitivity and speci city. DNA was extracted using DNeasy Blood &Tissue Kit from Qiagen, Germany, according to the instructions [21]. The samples were subjected to polymerase chain reaction (PCR) targeting the transcriptional interphase (ITA-1) in vivo of the Leishmanian ribosome, with the target fragment of the expected product being 280-330 bp.The venous blood DNA of the dogs with Leishmania parasite detected by bone marrow puncture smear staining microscopy was used as a positive control. After puri cation, PCR positive products were sequenced by Codon Code Aligner Software V 3.0.1 (Codon Code Corporation, Dedham, Massachusetts, USA), and the quality of each sequence was evaluated and analyzed. Dog Leishmania infection was identi ed by Blast nucleotide standard software (Blast). Quality control The research team and the village doctors at the site of the investigation carried out the eld investigation together, and the eld observation, measurement, inquiry and record formed the questionnaire. The standardization, logic and integrity of the investigation data were checked and improved on the same day to ensure the quality of the on-site investigation. After the experimental detection, the basic information of dogs, PCR detection results and geographic coordinates were matched one by one using Excel.

Statistical Analysis
EpiData3.1 was used to establish a database, and double entry method was used to input data information and compare and verify error correction. IBM Spss 23.0 was used for statistical analysis. The classi ed data were described by the number of cases and rate (n(%)), and chi-square test was used, with the test level = 0.05. A multivariable adjusted model was tted with the variables that were statistically signi cant at p < 0.05 in univariate analyses. A step-by-step backward selection procedure was used to select the variables and to produce the nal multivariate regression model. Only adjusted variables showing a signi cant association (p < 0.05) with the occurrence of infection by L. infantum remained in the nal model. The strength of association was determined by a hazard ratio and a 95% CI.

Sample PCR detection
The study examined 574 dogs. The positive rate of venous blood PCR was 41.15% (221/537) in 537 dogs in Wenxian county and Diebu County, among which the positive rate was 32.30% (126/390) in Diebu County. The positive rate of dogs in Wenxian county was 64.63% (95/147).All the 37 dogs in Tianzhu County were negative.
According to the frequency distribution and results of univariate analysis in Table 1, variables with P < 0.05 were statistically signi cant. Among them, there were signi cant differences between the 12 variables of dogs' sex, age, height, weight, breed, kennel presence, hair length, style of con nement, physical condition, exercise status, mucosal signs and toenail symptoms and those of dogs with positive Leishmentan infection.

Analysis Of Relevant In uencing Factors
Statistically signi cant variables in Table 1 were included in the multivariate Logistic regression analysis, and the results showed that the four variables were the risk factors for the transmission of Leishmaniasis infection in dogs. (Table 2)

Discussion
The survey took place in the rural areas of Wen xian county and Diebu County, Gansu Province. The main purpose of the villagers' dogs was to guard the homes and nursing homes, and most of the dogs were tied up in the corners of the homes.
There are a lot of dogs and very few livestock kept in the villages in study area, so dogs are an easy source of blood meal for sand ies. The survey raises the dog the parasite infection is tied to the dog three times higher risk may be due to putting the dog is tied to the dog range is bigger, raises the dog often follow dog owners into the wild elds and mountain stream, often free access to the village inside and outside activities, and Gansu Leishmaniasis media is sand y mainly wild habitat the sand y, so put the dog activity increases the chances of the sand y, resulting in an increased risk of infection [22,23]. Dogs without kennels were 1.9 times more at risk of infection than dogs with kennels, again due to the greater risk of infection in the open air.
The risk of CVL infection in purebred dogs in this study was 1.8 times higher than that in mongrels (95%CI0.999-3.311).This is consistent with the nding by Solano-Gallego et al that hybrids are a protective factor, that they are less likely to be infected, that hybrids have developed a degree of resistance, and that pure-bred dogs are at risk [24]. Our study also found that shorthaired dogs are more susceptible to infection than long-haired dogs, and there is a signi cant difference, which is consistent with the results that short-haired dogs have a signi cantly higher probability of infection than long-haired dogs [25]. In fact, sand y bite dogs were more likely to choose the face with less hair, around the nostril, muzzle edge, and perianal area. Longhaired dogs in the survey sites were never trimmed, and many long-haired dogs had their face covered with fur, so short-haired dogs were more likely to be bitten and infected by sand ies [26].
In the univariate analysis, the age, height and weight of the dogs were statistically signi cant. There was a signi cant difference in Leishmania infection rates between puppies and adults, and we found a higher risk of Leishmania infection in puppies younger than 2 years of age [27]. And the greater the height and weight of the dog, the greater the exposure, and the higher the risk of Leishmania infection. According to foreign studies, Leishmania infection is more easily acquired in dogs under 3 years of age [28]. Puppies are more susceptible to infection due to congenital immaturity, a lack of internal immune barriers, malnutrition and the sand y's a nity for this group [29]. We found that although the age, height, weight and other factors in the univariate study were statistically signi cant, these factors were not the decisive factors affecting infection in the regression model because of the correlation strength.
Cvl-related symptoms do not actually occur immediately after Leishmania infection in dogs, which is called asymptomatic infection. In this study, a large percentage of infected dogs showed no obvious signs and symptoms, but asymptomatic dogs may exhibit a high parasite load and play an important role in CVL transmission [30,31]. Over time, the physical condition, activity, mucosal signs, nail growth and other indicators of asymptomatic infected dogs changed [32]. Our study found that almost all dog owners did not actually know about CVL and were unaware that mucosal signs, toenail growth, etc., were symptoms of CVL development, thus neglecting the care and selection of dogs and allowing the disease to develop. The persistence of this phenomenon may result in more healthy dogs being infected [33]. Therefore, we believe that promoting owners to master basic KNOWLEDGE of CVL and taking active preventive measures and strengthening the management of sick dogs will help to reduce the spread of CVL.

Conclusions
Research results show that the way of captive, kennels, varieties and dog hair length is Gansu research spot dogs infected with the parasite risk factors, the conclusions will help to improve people's understanding of CVL epidemiology, for the dog to a crowd in endemic areas targeted Suggestions, optimize and develop scienti c dog management measures so as to reduce the CVL transmission has important signi cance.