Genetic diversity in Leishmania infantum and Leishmania tropica isolates from human and canine hosts in northern Morocco.

This study investigated nine provinces in northern Morocco and collected 275 skin scraping, 22 bone marrow aspirates, and 89 fine needle aspirations from suspected cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) patients and potentially infected dogs. Molecular analysis using ITS1 RFLP PCR and RT-PCR revealed a higher prevalence of L. infantum (66.18 %; χ2 = 28.804; df = 1; P-value = 8.01e-08) than L. tropica in skin scraping, with L. infantum being the sole causative agent for both VL and canine leishmaniasis. L. infantum was predominantly found in most provinces, while L. tropica was relatively more dominant in Taza Province. Discriminant Analysis of Principal Components (DAPC) revealed distinct clustering between L. tropica and the other three species. However, no small subset of SNPs could clearly differentiate between Infantum_CL, Infantum_VL, and CanL, as they likely share a significant genetic background. The high rate of L. infantum could be attributed to the abundance of sand fly species transmitting VL. In Taza Province, Phlebotomus sergenti, responsible for anthroponotic CL, is the most abundant species. DNA sequencing demonstrated sequence heterogeneity in L. infantum (variants 1 to 9) and L. tropica (variants 1 to 7). Phylogenetic analysis showed a distinct separation between L. tropica and L. infantum strains, with an overlap among L. infantum strains isolated from cutaneous, visceral, and canine cases, and dogs serving as the central population for L. infantum.

Refinement of the rKLi8.3-Based Serodiagnostic ELISA Allows Detection of Canine Leishmaniosis in Dogs with Low Antibody Titers.

The diagnosis of canine leishmaniasis (CanL) still represents a challenge due to the variable clinical manifestations and the large number of asymptomatic dogs. Serological tests are most commonly used to detect infected animals, revealing anti-Leishmania antibodies, mainly of the IgG isotype. Recently, a new diagnostic antigen, rKLi8.3, containing 8.3 kinesin tandem repeats (TR) from a Leishmania infantum strain from Sudan, has been shown to provide excellent specificity and sensitivity for the detection of Leishmania-infected humans and dogs. However, asymptomatic animals with very low antibody titers are often difficult to detect by serodiagnosis. Thus, we wondered whether the addition of an anti-IgG-enhancing step in the protein A/G-based rKLi8.3-ELISA will improve the diagnostic performance without decreasing the specificity. For this, parasitologically confirmed CanL cases with low or high clinical scores, uninfected healthy controls and dogs with other infections were tested by rKLi8.3-ELISA as well as two different immunochromatographic rapid tests, rKLi8.3-lateral flow test (LFT) and Dual Path Platform (DPP®) based on the rK28 antigen. Our results show that the diagnostic accuracies of the rKLi8.3-ELISA and LFT were similar to that of DPP, missing several asymptomatic animals. However, the addition of a secondary, amplifying anti-dog IgG antibody in the protein A/G-based rKLi8.3-ELISA enabled the detection of nearly all asymptomatic dogs without compromising its specificity.

Clinical importance of neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio and systemic immune-inflammation index in dogs with leishmaniasis.

Leishmaniasis is a zoonotic disease caused by Leishmania spp., impacts multiple systems and organs. While hematological and biochemical profiles aren't definitive for diagnosis, recent studies have identified the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and systemic immune-inflammation index (SII) as predictors of morbidity and mortality in critically ill human and dog patients. This study examined 100 dogs diagnosed with leishmaniasis, categorized by the International Renal Interest Society (IRIS) stages 1-4. Additionally, the dogs were divided based on whether they survived less or more than one year (L1Y and G1Y). Control group consisted of 43 dogs. The NLR increased as the disease progressed (IRIS 1-4), presenting statistically significant differences (P<0.05) when compared to the control group (2,37±2,08) IRIS 3 and 4 (4,59±13,39 and 6,99±12,86, respectively), and G1Y and L1Y (3,60±4,02 and 4,87±5,82, respectively). Significant changes in SII were only evident in short-term survivors (L1Y 951,93±1402) and advanced renal disease cases (IRIS 4 stage 1073,68±1901,09). Conversely, PLR remained largely unchanged. In conclusion, these results suggest that the neutrophil-to-lymphocyte ratio (NLR) and systemic immune-inflammation index (SII) may serve as potential markers for assessing disease progression and prognosis in dogs diagnosed with leishmaniasis.

A non-replicative antibiotic resistance-free DNA vaccine delivered by the intranasal route protects against canine leishmaniasis.

Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis (ZVL). The disease is endemic in Central and South America, Central and South East Asia, and the Mediterranean basin. Dogs are the main reservoir, with an estimated prevalence of approximately 2.5 million dogs in Southern Europe. Current treatments cause side effects, disease recurrence, and drug resistance. Therefore, the development of vaccines against canine leishmaniasis is necessary. We have generated a DNA vaccine based on the non-replicative antibiotic resistance marker-free plasmid vector pPAL that contains the encoding gene for the L. infantum activated protein kinase C receptor analog (LACK). Homologous pPAL-LACK prime-boost intranasal administration confers efficacious protection in Beagle dogs with a reduction of clinical signs and a statistically significant reduction of the parasite burden in the bone marrow of more than 90% of dogs after experimental infection with highly infective promastigotes. This DNA vaccine elicits a robust cellular immune response skewed towards the Th1 profile.

Canine Leishmania spp. infection in two distinct foci of visceral and cutaneous leishmaniasis in Tunisia.

Visceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania (L.) infantum and L. major, respectively, are endemic in Tunisia. The aim of the study was to assess canine Leishmania spp. infection prevalence as well as to identify the Leishmania species involved in two well-documented and geographically distinct VL and ZCL foci. One hundred seventy-six dogs were randomly recruited in the VL focus of Sbikha-Zaghouan (n = 100) and the ZCL focus of Echrarda-Nasrallah (n = 76). Physical examination and blood collection were systemically performed. Needle aspiration was done in case of lymph node (LN) enlargement. All sera were tested by ELISA. kDNA RT-PCR was performed on DNA extracts from (i) buffy coats of seropositive dogs and (ii) LN aspirates. Leishmania species identification was done by ITS1 PCR-sequencing. Thirty-three dogs (18.8%) were infected by Leishmania; 30 having anti-Leishmania antibodies and 3 were seronegative dogs with Leishmania DNA in LN aspirates. Prevalence of infection was significantly higher in VL foci than in ZCL foci (27% versus 7.9%, p = 0.002). Leishmania species was identified in 11 dogs and corresponded to L. infantum. Combination of serology and qPCR on LN aspirates seems to be the best option for canine leishmaniasis diagnosis. Infection is more frequent in VL foci and L. infantum is the only identified species.

Rectal Administration of Leishmania Cells Elicits a Specific, Th1-Associated IgG2a Response in Mice: New Perspectives for Mucosal Vaccination against Leishmaniasis, after the Repurposing of a Study on an Anti-Viral Vaccine Candidate.

The mucosal immune system plays a pivotal role in the control of infections, as it represents the first line of defense against most pathogens, from respiratory viruses to intestinal parasites. Mucosal vaccination is thus regarded as a promising strategy to protect animals, including humans, from infections that are acquired by ingestion, inhalation or through the urogenital system. In addition, antigens delivered at the mucosal level can also elicit systemic immune responses. Therefore, mucosal vaccination is potentially effective also against systemic infections acquired through non-mucosal routes, for example, through the bite of hematophagous insects, as in the case of leishmaniasis, a widespread disease that affects humans and dogs. Here, we explored the potential of antigen rectal administration for the generation of anti-Leishmania immunity. Mice were immunized through rectal administration of whole cells of the model parasite Leishmania tarentolae (using a clone engineered to express the spike protein of the SARS-CoV-2 virus generated in a previous study). A specific anti-Leishmania IgG antibody response was detected. In addition, the recorded IgG2a/IgG1 ratio was higher than that of animals injected subcutaneously; therefore, suggesting a shift to a Th1-biased immune response. Considering the importance of a Th1 polarization as a protective response against Leishmania infections, we suggest that further investigation should be focused on the development of novel types of vaccines against these parasites based on rectal immunization.

Occurrence of Leishmaniasis in Iberian Wolves in Northwestern Spain.

Canine leishmaniasis is an important vector-borne protozoan disease in dogs that is responsible for serious deterioration in their health. In the Iberian Peninsula, as in most countries surrounding the Mediterranean Sea, canine leishmaniasis is caused by Leishmania infantum (zymodeme MON-1), a digenetic trypanosomatid that harbors in the parasitophorous vacuoles of host macrophages, causing severe lesions that can lead to death if the animals do not receive adequate treatment. Canine leishmaniasis is highly prevalent in Spain, especially in the Mediterranean coastal regions (Levante, Andalusia and the Balearic Islands), where the population of domestic dogs is very high. However, the presence of this disease has been spreading to other rural and sparsely populated latitudes, and cases of leishmaniasis have been reported for years in wildlife in northwestern Spain. This work describes for the first time the presence of wolves that tested positive for leishmaniasis in the vicinity of the Sierra de la Culebra (Zamora province, northwestern Spain), a protected sanctuary of this canid species, using PCR amplification of L. infantum DNA from different non-invasive samples such as buccal mucosa and those from both ears and hair. In addition to live animals (21), samples from carcasses of mainly roadkill animals (18) were also included and analyzed using the same technique, obtaining a positivity rate of 18 of the 39 wolves sampled (46.1%) regardless of their origin.

Bayesian multivariate longitudinal model for immune responses to Leishmania: A tick-borne co-infection study.

While many Bayesian state-space models for infectious disease processes focus on population infection dynamics (eg, compartmental models), in this work we examine the evolution of infection processes and the complexities of the immune responses within the host using these techniques. We present a joint Bayesian state-space model to better understand how the immune system contributes to the control of Leishmania infantum infections over the disease course. We use longitudinal molecular diagnostic and clinical data of a cohort of dogs to describe population progression rates and present evidence for important drivers of clinical disease. Among these results, we find evidence for the importance of co-infection in disease progression. We also show that as dogs progress through the infection, parasite load is influenced by their age, ectoparasiticide treatment status, and serology. Furthermore, we present evidence that pathogen load information from an earlier point in time influences its future value and that the size of this effect varies depending on the clinical stage of the dog. In addition to characterizing the processes driving disease progression, we predict individual and aggregate patterns of Canine Leishmaniasis progression. Both our findings and the application to individual-level predictions are of direct clinical relevance, presenting possible opportunities for application in veterinary practice and motivating lines of additional investigation to better understand and predict disease progression. Finally, as an important zoonotic human pathogen, these results may support future efforts to prevent and treat human Leishmaniosis.

Urinary cystatin C and N-acetyl-beta-D-glucosaminidase (NAG) as early biomarkers for renal disease in dogs with leishmaniosis.

Canine leishmaniasis (CanL) is a disease caused by Leishmania infantum that can vary from a subclinical infection to a severe disease. Dogs affected with CanL present varying degrees of renal dysfunction. Unfortunately, traditional biomarkers such as urea and creatinine detect renal damage in advanced stages of the disease, so more accurate biomarkers are needed. Hence, we aimed to study how urinary cystatin C (CysC) and N-acetyl-beta-D-glucosaminidase (NAG), behave in dogs with CanL at different stages of the disease. Eighty-six CanL infected dogs were classified according to LeishVet stages: LI (16 dogs), LIIa (12 dogs), LIIb (12 dogs), LIII (16 dogs) and LIV (30 dogs); as a control, 17 healthy dogs were studied. Blood samples were collected for complete haematological and biochemistry analysis including plasma cystatin C. Urine analysis included urine specific gravity (USG), urine protein to creatinine ratio (UPC), CysC and NAG expressed as a ratio with creatinine uCysCc (µg/g) and uNAGc (IU/g). The haematological, biochemical and urinary analysis coincided with the LeishVet guidelines. The statistical study of the uCysCc ratio and the uNAGc, showed significant increase when compared against control starting from group LI (p < 0.05). Interestingly, when the cut-off values were calculated using the ROC curve, uCysCc (258.85 µg/g) and uNAGc (2.25 IU/g) 75 % of the dogs included in LI groups surpassed the threshold. Hence our study indicates that uCysCc and uNAGc, could help to detect early renal damage in CanL affected dogs.

Arginine, symmetric and asymmetric dimethylarginine levels in canine leishmaniasis.

The study aimed to investigate the levels of arginine, symmetric dimethylarginine (SDMA), and asymmetric dimethylarginine (ADMA) in dogs with canine leishmaniasis (CanL) and their relationship with some renal and cardiovascular parameters. A total of 60 dogs were enrolled, including 40 with CanL and 20 healthy controls. The CanL group was divided into four stages based on clinical and laboratory findings. The levels of plasma arginine, SDMA, and ADMA were determined by high performance liquid chromatography (HPLC). The data from the healthy group were compared with those from the CanL group, and according to the stages. In dogs with CanL, systolic and diastolic blood pressure, plasma creatinine, cystatin-C, phosphorus, potassium, and low-density lipoprotein concentrations, the urine protein/creatinine ratio, the amount of nitric oxide, and creatine kinase-MB activity were higher, while the high-density lipoprotein concentration was lower compared to healthy controls. The concentration of arginine was low (p < 0.05) and the levels of ADMA (p < 0.001) and SDMA (p < 0.05) were high in dogs with CanL. There were no statistically significant differences in arginine concentration among the different stages of CanL. However, the concentration of plasma ADMA was higher in all stages of CanL compared to the healthy group, and the concentration of plasma SDMA was higher in Stage IV compared to the healthy group and Stage III. The present study demonstrates for the first time a decrease in arginine concentration and an increase in ADMA concentration in dogs with CanL. The increase in SDMA concentration in dogs with CanL was consistent with previous studies. However, compared to other renal parameters, SDMA exhibited limited performance distinguishing between clinical stages of CanL. These findings could be a source for future diagnostic and therapeutic studies to explain the renal and cardiovascular pathophysiology of CanL. Additional clinical studies that include treatment and patient follow-up with an assessment of the acute phase response are needed to provide a more detailed understanding of the changes observed in dogs with CanL.

Serodiagnosis of leishmaniasis in asymptomatic and symptomatic dogs by use of the recombinant dynamin-1-like protein from Leishmania infantum: A preliminary study.

Visceral leishmaniasis (VL) is a fatal manifestation of an infection caused by intracellular protozoa of the Leishmania genus. In New World countries, VL is classified as a zoonotic disease with domestic dogs acting as its main reservoir. Asymptomatic dogs are as competent to transmit Leishmania to the vectors as symptomatic dogs, however current diagnostic tests are limited and present low sensitivity for this important group. The development of accurate tests is fundamental to the early diagnosis, treatment, and control of canine leishmaniasis. In this study, we investigated the use of a recombinant protein (dynamin-1-like protein, Dyn-1) from L. infantum, as a potential target antigen for leishmaniasis serodiagnosis in both symptomatic and asymptomatic dogs. The antigenic performance of the protein was evaluated by means of ELISA assays using sera from symptomatic (n = 25), asymptomatic (n = 34) and non-infected dogs (n = 36) using ELISA. In addition, sera from dogs experimentally infected with Trypanosoma cruzi (n = 49) and naturally infected with Babesia sp. (n = 8) were tested to evaluate possible cross-reactivity. A crude soluble antigen (CSA) of Leishmania was used as an antigen control and K39 and K26 were used as reference antigens because they are already widely used in commercial tests. rDyn-1-based assay showed the highest sensitivity (97%) compared to the antigens K39 (88%), K26 (86%) and crude extract (95%). The highest specificity among the tests was also obtained with the protein rDyn-1 (94%), compared with the other antigens K39 (81%), K26 (87%), and crude extract (77%). This study showed that the rDyn-1 ELISA assay was able to identify 100% of asymptomatic dogs, establishing its potential as a target for the diagnosis of canine leishmaniasis.

Molecular Detection of Canine Leishmaniasis in Northern Anatolia, Turkiye.

Background: Canin leishmaniasis (CanL), mostly caused by Leishmania infantum, is one of the most important vector-borne diseases in dogs in the Mediterranean region. In this study, we aimed to determine the disease profile in this region by firstly making microscopic and then molecular analyzes in the samples taken from the dogs. Methods: Overall, 112 whole blood samples taken from dogs for clinical applications by a veterinarian in Cankiri between December 2021 and November 2022 were used. After blood collection, both thin and thick drop blood smear preparations were prepared and evaluated for Giemsa staining. L. infantum was investigated by Real time-PCR (RT-PCR) method from all blood samples. Sequence analysis and phylogenetic tree study were performed on positive samples. Results: Both microscopic and RT-PCR analyzes were performed. In both studies, 3 of the 112 samples were positive. Because of the sequence analysis, they were L. infantum. Sequence analysis was performed from the samples found 3 positive. The phylogenetic tree was drawn by making NCBI (National Center for Biotechnology) data entries of the positive samples (Accession numbers: OQ184728, OQ184729, OQ184730). Conclusion: Dogs are important, as they are reservoir of this disease. In this study, 3 (2.7%) positive Leishmaniasis was detected in dogs in Cankiri. Ultimately, this should prompt discussion about new strategies going forward to combat infection caused by Leishmania.

Can domestic dogs be considered a good reservoir of Leishmania (L.) infantum chagasi in an endemic area of nonulcerated cutaneous leishmaniasis in Southern Honduras?

ABSTRACT Dogs are considered to be the main domestic reservoir associated with the transmission of Leishmania (L.) infantum chagasi to humans in endemic areas of visceral leishmaniasis in America. However, little is known about the role of canines as a source of infection in endemic areas of nonulcerated cutaneous leishmaniasis (NUCL). Therefore, the objective of the present study was to investigate the role of dogs as a possible reservoir of the parasite in Southern Honduras. Dogs (n = 107) living with individuals affected by NUCL were clinically examined and biological material was collected for parasitological and immunological diagnosis. Most animals showed a healthy appearance and a few presented slight weight loss (64%), alopecia (7%), onychogryphosis (5%) and skin lesions (1%). The overall seroprevalence of Leishmania infection based on the DDP ® quick test and/or in-house ELISA serological test was 41%. The presence of the parasite's DNA was confirmed in 94% of the dogs; however, the average parasite load in the buffy coat was low at 6.09 parasites/µL, ranging between 0.221 and 50.2. The skin of seropositive dogs examined by histopathology using paraffin sections stained by hematoxylin and immunohistochemistry did not show cutaneous lesions or parasite amastigotes. Based on the absence of parasites in the skin and the low parasite load detected in the buffy coat, it seems that the dog does not represent a good source of infection for the vector in the endemic area of NUCL transmission in Southern Honduras. Other domestic and/or wild animals should be investigated.

Urinary phoretograms performed by capillary electrophoresis in dogs with chronic disease with or without Leishmania infantum infection.

Introduction: The study of early markers to detect kidney malfunction has increased in recent years since serum markers, such as creatinine increase when there is a 75% loss of renal mass. Urinary capillary electrophoresis (UCE) is an available laboratory technique that provides an easily interpretable electrophoretic pattern. This pattern in our study has been divided into five fractions as it is done in serum: fraction 1 migrating in the albumin zone, fraction 2 in the alpha1-globulins zone, fraction 3 in the alpha2-globulins zone, fraction 4 in the beta-globulins zone, and fraction 5 in the gamma globulins zone. UCE can be useful in the early diagnosis of renal disease. Material and methods: In this study, UCE was performed in dogs with azotemia and proteinuria due to chronic kidney disease (CKD) not related to Leishmania infantum (L. infantum) infection (G1, n = 11) and dogs with CKD related to L. infantum infection (G2, n = 17) and compared with reference intervals from healthy dogs (G0, n = 123), with the aim of comparing their phoretograms and assessing changes in the fractions of the phoretograms based on the health status of individuals. Results: Fraction 2 was statistically augmented in dogs with CKD (G1) when compared with the healthy population (G0) and dogs infected by L. infantum (G2). Fraction 3 was statistically increased in dogs with CKD (G1) and dogs infected by L. infantum (G2) compared with G0. Fraction 4 was found to be statistically decreased in dogs with CKD (G1) and dogs infected by L. infantum (G2) compared with G0. Fraction 5 was statistically higher in dogs with L. infantum (G2) compared with G0 and dogs with CKD (G1). No statistical relationship was found between the protein to creatinine ratio and different fractions from the urinary phoretogram in the study population. No statistical relationship was found between serum and urine fractions in the study population. Discussion: The results of the present study suggest that UCE is a promising non-invasive technique that might be used as a part of the diagnostic and follow-up in dogs with kidney disease due to different pathologies.

Prevalence and risk factors of canine leishmaniasis in Morocco: a systematic review and meta-analysis.

Human visceral leishmaniasis has long been associated with canine leishmaniasis (CL). However, to date, there is no clear information on the status of the disease in dogs in Morocco that could be used by policymakers for the prevention of human cases. This study aims to assess the status of CL in Morocco and its risk factors through an exhaustive literature search. The meta-analysis was performed using RevMan 5.4. The main results showed that the overall prevalence of CL in Morocco is 17% (95% CI: 0.12-0.22), caused by two strains of Leishmania parasite: Leishmania tropica and L. infantum. According to the region, the maximum prevalence was reported in the coastal provinces and in the central part of the country; while, the CL risk was higher in rural area (18% [95% CI: 0.14-0.23]) and at altitude above 1000 m (23% [95% CI: - 0.08-0.53]). Regarding the intrinsic factors, the prevalence of the disease increased with the age of the dog, (30% [95% CI - 0.09-0.68) and the risk was very high in clinically asymptomatic dogs (RR = 2.08 [95% CI: 1.15-3.76]). This study is the first in Morocco indicating the CL prevalence, its geographical distribution and detailing its risk factors. These results are needed to improve management strategies for the canine reservoir of leishmaniasis in Morocco and interrupt the local transmission cycle to humans. Supplementary Information: The online version contains supplementary material available at 10.1007/s12639-022-01521-2.

Leishmaniasis.

Leishmaniasis caused by Leishmania infantum is an important zoonotic disease transmitted by sand flies with a high prevalence of infection in dogs and cats in regions whereby transmission occurs. Clinical disease is systemic with variable presenting signs and degrees of severity. It affects the skin, lymph nodes, eyes, bone marrow, kidneys, and other organs. The clinical findings in dogs and cats with L. infantum infection are generally similar. Subclinical infection of canines and felines in endemic areas is frequent. Long-term treatment of the disease with allopurinol, or combination of allopurinol with meglumine antimoniate or miltefosine, is needed, and clinical relapse is probable.

Laboratory validation of an ELISA method to measure circulating immune complexes levels in canine visceral leishmaniasis.

Susceptible dogs suffering from canine leishmaniasis (CanL) develop an ineffective humoral immune response that leads to the formation of circulating immune complexes (CIC). These CIC are aggregates of Leishmania proteins and anti-Leishmania immunoglobulins. Their deposition in different tissues is considered the main cause of mortality. For this reason, CIC have been suggested as an excellent CanL biomarker for measuring the progression of the disease and the effectiveness of specific treatments. The present study aims to perform a laboratory validation of a Leishmania-specific method to isolate and quantify CIC in dog serum samples. CIC isolated from serum samples of infected dogs, grouped according to the LeishVet classification, were quantified following a PEG-ELISA procedure. The validation established a cut-off of 0.274 OD. All the parameters analyzed (including linearity, specificity, precision, and robustness) fulfilled the defined criteria, confirmed by statistical analyses. The results also proved the reproducibility and reliability of the method when samples were tested under the same conditions, and the consistency and usefulness of the method for an optimal staging of infected dogs. In conclusion, the laboratory validated method offers a potent tool to clinicians for a proper CanL management and to measure the progression of the disease.

Diagnosis and prevalence of canine leishmaniasis in the Atlas shepherd dog.

The aim of this study was to determine the prevalence of canine leishmaniasis in the Atlas shepherd dogs from the Tiaret region of Algeria. A total of 161 dogs were included in this study and four diagnostic techniques were used, namely lymph node cytology, PCR, IFAT and ELISA. 110 out of 161 dogs were positive by at least one diagnostic technique, a percentage of 68.32% of the total number, which represent very high prevalence of canine leishmaniasis in this canine breed. 152 dogs underwent the two serological tests namely IFAT and ELISA among which 137 dogs underwent in addition the molecular examination by PCR. IFAT was positive in 9.86% of the dogs, ELISA was positive in 12.5% while 68% of the dogs proved positive by PCR. 47 dogs with lymph node enlargement underwent lymph node cytology, of which 26 dogs showed forms of amastigotes in their smears after microscopic examination, confirming the usefulness of this approach for the diagnosis of canine leishmaniasis. The clinical signs of the disease in this breed are similar to those of other canine breeds The dominant clinical signs in these dogs were onychogryphosis, poor general condition and lymph node hypertrophy. The autopsy was carried out on 7 dogs that died of canine leishmaniasis, all of which showed splenomegaly, while 5 dogs also showed hepatomegaly. Renal damage was found in 1 dog with frank jaundice. The splenic cytology was positive in all 7 dogs with the presence of amastigote forms in the smears after microscopic examination.

Report of the presence of Leishmania infantum in the milk of a naturally infected female dog in Brazil.

Dogs are the most important reservoir of Leishmania infantum, the causal agent of visceral leishmaniasis in Brazil. Although lymphoid tissue is the most important biological tissue where amastigotes can be found, this paper describes the presence of L. infantum DNA in the milk of a lactating naturally infected female dog. This finding suggests the need for further studies to elucidate whether breastfeeding can be a route of infection.

Pre-test probability and likelihood ratios for clinical findings in canine leishmaniasis.

Canine leishmaniasis is a parasitic zoonosis mainly caused by L. infantum; an obligate intracellular protozoan transmitted by haematophagous insects of the genus Phlebotomus, which affects dogs and wild canids. The clinical implications of this disease are highly variable, since infected animals may remain asymptomatic (absence of observable clinical signs) or present a wide spectrum of clinical alterations and degrees of severity, including the death of the animal. Symptoms such as lymphadenomegaly, alopecia, weight loss, keratoconjunctivitis and onychogryphosis are usually the first diagnostic reference available. The objectives of this study are to evaluate the validity (sensitivity, specificity and likelihood ratios) and diagnostic utility (pre-test probability) of the clinical signs commonly associated with canine leishmaniasis based on the prevalence in the area and to explore the combination of symptoms that best predicts the diagnosis of canine leishmaniasis. It is a matched case-control study in the canine population of southern Spain based on the comparison of the findings collected in the clinical history and the results of the LeisSCAN quantitative ELISA. A total of 39 cases and 78 controls were analysed. Approximately 80% of the infected animals showed signs compatible with the disease. The most frequent alterations were cutaneous (64.1%), systemic (51.3%) and oculo-nasal (30.7%). The most useful signs to support this diagnosis were alopecia and epistaxis (LR+ 6.69 and 6.0, respectively) (pre-test leishmaniasis probability is ≥70% for prevalence ≥28% when alopecia or epistaxis is present), followed by lameness (LR+ 5.0). The combinations of signs that showed greater validity were alopecia with hyperkeratosis of the snout and alopecia with onychogryphosis (LR+ > 10). None of the observed signs or their combinations resulted useful to rule out the diagnosis (LR- 0.55 to 1.15). The results found show notable differences in the diagnostic value of the clinical signs, individually and in combination, so we believe that medical decisions should be based on their diagnostic validity (LR+) and the estimation of the pre-test and post-test probability.