Latest trends in L. infantum infection in dogs in Spain (Part II): current clinical management and control according to a national survey in practitioners

Background Canine leishmaniosis (CanL) is a parasitic zoonotic disease, endemic in the Mediterranean basin including Spain. While knowledge about CanL, its management, treatment, prevention and control mounts, it remains unclear whether all clinical veterinarians follow the same international recommendations, such as those of the LeishVet group. This study was thus designed to assess recent trends in the clinical management of CanL in veterinary clinics across Spain through a questionnaire-based survey. Results were compared with those of our prior national multicenter questionnaire administered by our research team in 2005. Methods A questionnaire consisting of 28 questions about CanL was developed using Google Forms and distributed by email to 1428 veterinary clinics in Spain. Questions were designed to obtain data on common clinical signs, techniques and complementary exams used to diagnose the disease, and on its monitoring, treatment and control measures. Data were collected in a database for statistical analysis. Results Completed questionnaires were returned by 295 clinics. Compared to the situation in 2005, replies indicate that clinical signs of CanL have not changed significantly, cutaneous lesions being still the most prevalent sign observed by practitioners. Quantitative serological techniques are considered an adequate approach to diagnosis, provided their results are supported by the findings of a thorough physical exam, as well as complementary tests (complete blood count (CBC), biochemical profile, plasma protein electrophoretogram and complete urinalysis). Treatment protocols and check-ups follow international recommendations. Finally, a multimodal approach is being endorsed to adequately control CanL including preventive measures such as: annual serological check-ups and the combination of repellents and vaccines. Besides, owners are being better informed about CanL by veterinarians, which translates to the improved control of this


zoonosis.
Conclusions The clinical management of CanL has recently undergone significant changes owing to improvements in clinical knowledge of the disease, more unified international criteria, improved diagnostic techniques and their adequate interpretation, as well as a greater awareness of the disease transmitted to owners.

Bullet Points
The incidence of the endemic zoonotic disease CanL has not decreased in recent years despite new control measures available (e.g. vaccines). While protocols of treatment and diagnosis are more standardized, there is still a need to unify recommendations regarding the global management of CanL in veterinary clinics.
To improve clinical management and control strategies against CanL, reports of previous knowledge of CanL by veterinarians are essential.
The "One Health" approach to leishmaniosis stresses the need for multidisciplinary collaboration in the fight against this important zoonosis.

Background Canine leishmaniosis (CanL) is an important parasitic zoonotic disease caused by
Leishmania infantum, endemic in the Mediterranean basin including Spain. The disease is transmitted by female blood-feeding phlebotomine sand flies, and dogs (Canis familiaris) are both its natural host and the major reservoir of infection for humans and other animals [1,2]. The presence of L. infantum in felids and other animals has also been confirmed, although so far there are few data on these species despite being considered a potential secondary reservoir for the infection of humans and other animals [3,4,5,6,7]. The closest example is the largest outbreak of human leishmaniasis known in Europe occurring in 2009 in the southwest of Madrid [8] in which hares (Lepus granatensis) and rabbits (Oryctolagus cuniculus) were identified in xenodiagnostic and molecular diagnostic studies as reservoirs and held responsible for the transmission of human leishmaniosis [9,10].
The epidemiological role of wildlife species has not yet been established [4,11,12,13] yet several authors propose that these animals can act as sentinels as they indicate the risk of transmission to other animals (domestic and wild) or even to humans, highlighting the importance of the concept of "One Health" for the control of leishmaniosis [14,15,16].
In Spain, the seroprevalence of CanL differs from one area to another, depending on environmental factors such as temperature, humidity, geographical location, density and dispersion of the vector (see Part I of this study; submitted) [1,17]. In cats, seroprevalence rates provided in numerous studies have not been negligible, yet significantly lower than those observed in the canine population [18].
The most important characteristic of CanL is its extraordinary clinical polymorphism. This determines that a thorough assessment including medical history and physical examination are mandatory to confirm a causal relationship between Leishmania infection and the clinical signs presented by the animal. The diagnostic techniques used are based on the detection of the parasite (cytology, culture, molecular techniques, etc.) and anti-L.
infantum antibodies (serological techniques). However, complementary diagnostic tests such as blood tests (blood count and biochemical profile), urine tests (e.g. urinalysis, UPC), ultrasound, etc. are required to identify the clinicopathological abnormalities associated with the disease and thus assess the general disease status of the animal and monitor its clinical progression after treatment [2,19,20].
With regard to the treatment of CanL, progress has been limited. Although treatment and clinical follow-up protocols have markedly advanced, there is currently no treatment capable of parasitological cure or of avoiding relapse. Prevention is the best way to fight the disease, greatly helping to stop the spread of infection to other animals and humans.
However, at present, no preventive measure offers 100% guarantee. Thus, recommendations are adequate control of the vector, early diagnosis, and the treatment of sick dogs according to their clinical stage depending on their clinical signs [2,21,22].
The development and appearance of new vaccines for CanL is a new strategy for the control of this important zoonosis [21,23,24].
Knowledge about CanL, its management, treatment, prevention and control is on the increase. However, it is unknown whether all clinical veterinarians follow the same international recommendations such as those of the LeishVet group [2,21]. Consequently, the aim of the present study was determine how CanL is clinically managed via a multicentre questionnaire completed by veterinarians throughout Spain. Results were then compared with those of a similar national multicentre questionnaire developed by our research team in 2005 [25].
This study is Part II of a larger investigation addressing the current situation of CanL is Spain. In Part I, we map seroprevalences of infection in dogs across the country based on reported and our own more recent data, and also provide sand fly species distributions and address factors affecting their distribution and density.

Questionnaire
The questionnaire, consisting of 28 questions about CanL clinical management, was developed through Google Forms. The items included were the same as in a previous national multicenter questionnaire developed by our research team in 2005 [25]. In this questionnaire, information is obtained about: characteristics of the veterinary clinics, incidence and prevalence of L. infantum infection, the clinical signs observed, the diagnostic techniques and the complementary analyses used for the diagnosis of CanL and its monitoring, treatment, disease progression, control measures, vaccination and information provided to the owner.
Replies were anonymous and it was assured by collecting email addresses that questionnaires were not completed by more than one veterinarian at the same veterinary clinic. Email addresses were obtained through the Association of Spanish Veterinarian Specialists in Small Animals (AVEPA), the Veterinary Colleges of the different provinces and in a web search.

Statistical analysis
All data were collected in a database (Microsoft Excel 2010) for statistical analysis. In addition to descriptive statistics of the survey responses, we analysed the data in an effort to understand possible associations between the incidence and progression of L.
infantum infection and geographical area. Differences were also explored in the responses regarding diagnostic techniques, management of leishmaniosis (treatment and follow-up) and prophylactic measures provided by veterinarians who indicated they used LeishVet guidelines versus those who did not. For this purpose, we used the Chi-square test (SPSS 21.0). Significance was set at p ≤ 0.05.

General characteristics
The questionnaire sent by email to 1428 veterinary clinics in Spain was completed by 295  Few data about feline leishmaniosis (FelL) were reported. 24 of 295 (8.1%) participants indicated that they had diagnosed at least one cat infected by L. infantum. No significant differences in FelL were observed with respect geographical area (p=0.592).

Clinical signs
The clinical signs of CanL often observed by veterinarians were loss of weight, adenopathy, exfoliative dermatitis and renal disease ( Figure 2). Some veterinarians also described sporadic clinical signs: diarrhoea (n=25), neurological signs (n=5) and lameness (n=18).
Only eight veterinarians described clinical signs in cats testing seropositive for L.
infantum. The most common of these were: nodular cutaneous lesions, ulcerative lesions, and chronic nasal discharge. In two seropositive cats, a concomitant retrovirus infection was identified.

Diagnostic methods
The items of the questionnaire were designed to differentiate between i) an etiological and immunological diagnosis (serology), and ii) complementary diagnostic tests (laboratory findings) ( Figure 3).
For an etiological diagnosis, 35.4-59.5% of veterinarians have never used cytological tests (lymph node, bone marrow, cutaneous imprinting). Further, 58.4-66.4% also reported they did not carry out PCR techniques on bone marrow or lymph node aspirates, while a blood sample was used by around 25% of veterinarians sometimes or even systematically for a PCR diagnosis. Also, some vets used other biological samples such as hair, ear swabs for a PCR diagnosis.
Of the serological techniques used, quantitative methods such as IFAT and ELISA were used always or frequently by 66.7% and 61.8%, respectively. Replies, however, indicated that qualitative methods such as a rapid test were used by 70.5% as the first diagnostic tool.
CBC and biochemical profiles were used frequently or systematically by around 94% of veterinarians, as well as serum electrophoretogram (89%). This was followed by urinalysis and UPC used frequently or always by around 58.9-61.2% although 8.4-11.7% had never used this parameter to assess CanL infection status. Additional proof such as an abdominal ultrasound was frequently used by 21% of veterinarians. Besides, 63.9% of vets diagnosed CanL while conducting tests for other CVBDs (Figure 4).

Treatment and follow-up of CanL
More than 90% of veterinarians described they used meglumine antimoniate as treatment for CanL. However, 31.9% and 23.6% stated they had never used the doses recommended by LeishVet of 50 mg/kg/BID/28d and 100 mg/kg/SID/28d. Miltefosine was frequently used by around 50% of veterinarians, and usually at the dose recommended by the manufacturer. Allopurinol was used systemically practically by 100% of participants, the most common dose being 10 mg/kg. Immunomodulators such as domperidone were often or sometimes added by 30.6% and 34.3% of veterinarians, respectively; and systematically used by 20.9%. Regarding the use of Impromune® (Bioibérica, Spain), 50.6% reported they had never used it compared to 4.2% and 13.6% who did so systematically or often, respectively. Finally, 95% of the survey respondents indicated they had never used autovaccine for the treatment of CanL ( Figure 5).

Follow-up
Replies to questions about the frequency of follow-up of dogs with CanL were: visits every 3 or 6 months respectively in 26.8% and 32.3%, and yearly in 5.2%. Replies by 35.7% were that they only scheduled visits if there was clinical recurrence. Figure 6 shows the survival rates of dogs with CanL. Few (0-25%) dogs showed a survival of less than 3 months, 6 months or 1 year. While 45.4%, 65.8% and 76.8% of the veterinarians surveyed considered that more than 50% of dogs have a survival rate of more than 1, 2 and 5 years, respectively.

Survival and euthanasia
As reasons for euthanasia, the main ones given were recommended by the vet or requested by the owner (Table 1).
When we asked about the detection of antibodies against L. infantum before primary vaccination, 85.2% stated they undertook rapid tests while 11.7% conducted a quantitative test (IFAT or ELISA). Before re-vaccination, 58.7% admitted they only used rapid tests, and 7.6% quantitative tests.
Veterinarians considered that vaccination was safe, as associated clinical signs were not usually observed. The most common clinical signs reported were pain and erythema at the point of inoculation.

Public health considerations
One of the last questions was whether the veterinarians explained to owners the zoonotic impacts of CanL. Replies were 73.4% always, 12. Before asking about CanL, a question was included about other CVBD, and many of the veterinarians indicated they mainly detected CanL and ehrlichiosis. Nevertheless, other CVBD (dirofilariosis, piroplasmosis, ehrlichiosis, etc.) were also reported. According to previous epidemiological studies, the presence of some vector borne diseases (VBD) was associated with geographical distribution. Indeed, in this survey, dirofilariosis (S Spain, E Spain and Canary Islands) and piroplasmosis (N Spain) were associated with this variable as suggested by others [28,29,30,31]. According to these new results, CanL seems to be the most widely VBD distributed in Spain. However, veterinarians working in areas showing a high or medium endemicity considered that the incidence of CanL remains stable, while those working in low CanL endemic areas indicated that cases of CanL have increased. This perception is similar to that described in other European countries that were until recently free of CanL, such as United Kingdom or Germany [32,33,34,35]. The reason for this is unknown, but could be due to (i) the increased transfer of infected dogs from endemic areas, such as rehomed or hunting dogs; (ii) more dogs travelling overseas to Leishmania-endemic areas (e.g. taken there by their owners on holiday); (iii) other nonvector routes of transmission such as vertical, venereal, blood transfusion or dog-to-dog [36,37,38,39] which may be contributing to the spread of autochthonous cases. The reason could also be a combination of these factors.
The clinical manifestations of CanL vary from subclinical to severe disease. In the present study, weight loss, lymphadenomegaly and exfoliative dermatitis were frequently observed in agreement with the findings of other studies and surveys [25,26,40,41,42,43]. While renal disease, vasculitis, ocular and join disease have been less frequently observed, these are indicators of a worse prognosis because they are clinical signs related to immunocomplex deposition [44,45,46,47,48]. Besides, other clinical signs have been sporadically observed such as neurological signs [49,50,51,52,53] or digestive disorders [54,55,56]. Although these signs could due to CanL, other diseases must be considered in the differential diagnosis.
Regarding the diagnosis questions, the present survey differentiated between an etiological diagnosis (microscopic observation and/or PCR), serology and complementary tests. It should be noted that more than 35% of the practitioners surveyed mentioned they had never performed an etiological diagnosis, and if this was done, the most common was PCR on blood (13.2-36.8%) or cytology of lymph node aspirates (13.7-47.9%), while cytology of bone marrow aspirates (5.3%) or PCR (3.9%) were not frequently used for diagnostic purposes. Similar results have been obtained in questionnaire surveys by other authors [26,42]. However, according to the LeishVet group, blood is one of the least sensitive biological samples, and the use of bone marrow or lymph node aspirates is strongly recommended [2,57]. Bourdeau suggested veterinarians could reject the more invasive techniques due to the extra costs and the fact they are time consuming as in some cases it will be necessary to sedate the animal [26]. Other authors suggest a lack of referral laboratories close to veterinary practices surveyed as the reason for the reduced use of molecular techniques [58]. However, we found no difference in the approach used according to the location of the veterinary practice.
Quantitative serological diagnosis is essential in the diagnosis and monitoring of CanL, and although many veterinarians used the IFAT technique (66.7%) and quantitative ELISA (51.8%) frequently or systematically, there were also many clinics that used rapid tests (70.5%) as the first diagnostic approach, in agreement with the results of similar surveys [25,26,59]. While a rapid test may help confirm clinically suspected cases as they show high specificity in sick dogs, the indirect fluorescent antibody test and the enzyme-linked immunosorbent assay are the most suitable serological tools according the LeishVet group and OIE [2,60], as confirmed by several authors [61,62,63]. In other surveys conducted in Spain and other CanL endemic countries (eg, Italy), IFAT was considered the gold standard by veterinary practices surveyed [41,42,43].
According to the replies, almost all veterinarians used CBC and biochemical profiles to monitor infected dogs, and around 80% also used electrophoretograms. These rates are significantly higher than those of our previous survey [25], and indicate the improved clinical management of CanL as suggested by the LeishVet group [2,21]. Regarding the use of urinalysis, ranges of 25.5-33.9% to 21.5-37.4% used urine strip and urinalysis, respectively. Also, 27.2-34% frequently or systematically, respectively, determined the UPC (urine protein/creatinine ratio). This is important, as according to LeishVet and IRIS guidelines, UPC is a highly suitable biomarker for a diagnosis of CanL because immunocomplex deposits in the renal glomerulus may induce immune-mediated glomerulonephritis. In sick dogs, this serious kidney lesion causes proteinuria with or without azotemia and chronic renal disease or even nephrotic syndrome of worse prognosis [64,65]. disease stage II or III [2,21]. The results of the present survey suggest veterinarians frequently use the combination of meglumine antimoniate and allopurinol, followed by miltefosine, in accordance with a previous questionnaire survey [43]. The long-term use of allopurinol, in combination with n-methylglucamine antimoniate or miltefosine, has proved to be effective for maintaining clinical and parasitological improvement and delay relapses in treated dogs. Miltefosine is better tolerated with regards to liver and kidney function than methylglucamine antimoniate, but disease relapses occur much earlier than in dogs treated with antimonials. Therefore, miltefosine is recommended for the treatment of canine leishmaniosis patients with renal or liver disease, and/or when subcutaneous or parenteral therapy may not be administered or is not indicated [66,67,68,69].
When veterinarians were asked about regimens of n-methylglucamine antimoniate, they indicated the 100 mg/kg/SID/28d dose was more used than 50 mg/kg/BID/28d, although differences were not significant. This could be related to the fact that some owners prefer to inject their dogs once a day rather than twice.
Most veterinarians stated they used allopurinol in combination with leishmanicide drugs, as allopurinol has been shown to prevent recurrence [70]. Likewise, it has been shown that dogs treated with allopurinol are not able to transmit the parasite to sand flies [71].
However, some dogs develop xanthinuria due to the inhibition of the enzyme xanthine oxidase, so it is strongly recommended to include frequent urinalysis and abdominal ultrasound for monitoring dogs under long term therapy with allopurinol [72], and assess renal mineralisation and urolithiasis [73]. The veterinarians surveyed responded they used urinalysis (58.9-61.2%) and ultrasound (21%) as preventative of adverse urinary effects.
Avoiding sand flies bite is one of the best ways to stop the spread of L. infantum infection [22,74]. Results obtained in this survey indicate that veterinarians recommend the use of repellents against the vector (96.7%), mainly collars and spot-ons as in other studies [41,58,75]. Other measures to avoid sand flies bites were not often recommended such as avoiding dogs going outside during hours of sand fly activity (from dusk to dawn) and using mosquito nets. These preventive measures have increased in comparison to the 2005 survey [25]. 66% of veterinarians recommended keeping dogs indoors during the risk period and 32% recommended the use of mosquito nets.
Lastly, another preventative measure recommended was vaccination. In the 2005 survey, 100% of veterinarians asked about vaccination answered that if there were a vaccine available, they would use it. In this survey, 87.6% replied they recommended vaccination as a preventative measure. Further, it is remarkable that the most common vaccine used by veterinarians surveyed is LetiFend ® and not Canileish ® . This lower use than initially expected could be because its percentage effectiveness is around 70% [76]. In addition, Canileish ® needs to be given for primovaccination as three consecutive doses three weeks apart to develop an adequate immune response while LetiFend ® is a single dose even for primary vaccination. This vaccine is also a DIVA (Differentiating Infected from Vaccinated Animals) vaccine, allowing to discriminate vaccinated from infected dogs. For all these reasons, LetiFend ® is at the first-line vaccine option over Canileish ® , as reflected in the results of other surveys [77,78,79].  [40]. Further, to date, no dogs receiving LetiFend ® have shown any associated local or systemic adverse events [79,81]. However, in our survey the appearance of clinical signs could be correlated with the vaccine used (CaniLeish ® or LetiFend ® ).
Vaccine manufacturers recommend performing a serological test before vaccination. In this survey almost all veterinarians mentioned they undertook a pre-primovaccination test. However, a high percentage of veterinarians stated they never performed any test before revaccination so it cannot be known if a dog has been infected during the intervaccination period. This test is strictly necessary since vaccines do not protect 100% [76]. On the other hand, the results of the present survey indicate that more veterinarians carry out rapid tests before vaccination. Although rapid tests have a good sensitivity and specificity, quantitative techniques are considered more sensitive. Thus, a quantitative test is always recommended before vaccination to ensure the vaccination of healthy animals, as sometimes dogs with low antibody titres are not detected using qualitative techniques [76,82]  highlighting the importance of control and prevention measures [21,22]. Veterinarians also need to make owners aware that culling CanL positive dogs is not an adequate disease control measure, as confirmed in studies conducted in Brazil where culling seropositive dogs failed to reduce the incidence of canine or human leishmaniosis [83,84,85].
Another aspect to consider is that the dog is not the only reservoir capable of transmitting L. infantum. Several studies have shown that Leishmania-competent vectors feed on cats naturally infected with L. infantum and through xenodiagnosis have confirmed that infected cats can transmit the infection to P. perniciosus and L. longipalpis [86,87,88].
However, in endemic areas of CanL, subclinical feline infections are common, although clinical cases are so far exceptional [18,89,90]. Indeed, in the present questionnaire, 8% cases of feline leishmaniosis were reported although these data are insufficient to reveal the role of cats as a possible reservoir as the veterinarians did not specify the diagnosis technique or clinical signs.

Availability of data and materials
Not applicable

Competing interests
The authors declare they have no competing interests

Funding
This study was funded in part by Bayer Animal Health.

Authors' contributions
AM participated in the design of the questionnaire, performed the statistical analysis, and drafted the manuscript. RG participated in the design of the questionnaire, collected questionnaires and drafted the manuscript. RC, JPB, VM and JS participated in collecting questionnaires and database completion. AP participated in database completion and helped to draft the manuscript. GM conceived and coordinated the study, participated in its design and drafted the manuscript. All authors read and approved the final manuscript.      Active principles and frequency of use Survival rate of dogs with CanL treated with the different agents