Fibroblast growth factor-23 and Alpha-Klotho concentrations in dogs with canine Leishmaniasis.

This study aimed to assess the concentrations of Fibroblast Growth Factor-23 (FGF-23) and α-Klotho in healthy dogs and dogs at different stages of Canine Leishmaniasis (CanL), and investigate the changes of these parameters in relation to renal function and calcium­phosphorus metabolism. A total of 74 dogs (22 healthy and 52 with CanL) of varying ages, sexes, and medium-sized breeds were included. Dogs with CanL were categorized into different stages (Stage I-IV) based on Leishvet recommendations. In addition to routine hematological parameters, plasma FGF-23, serum α-Klotho, urea, creatinine, phosphorus, calcium, parathormone, vitamin D concentrations, and urine protein/creatinine ratio were measured. Data from healthy dogs were compared to dogs with CanL overall and by stage. Dogs with CanL exhibited higher concentrations of FGF-23 (p < 0.05), α-Klotho, and parathormone (p < 0.001), as well as lower concentrations of vitamin D and calcium (p < 0.001). FGF-23 concentration was particularly elevated in Stage IV compared to other stages. However, no significant differences in α-Klotho levels were observed among the stages. FGF-23 levels showed a weak positive correlation with urea and creatinine concentrations and a moderate positive correlation with urine protein/creatinine ratio. This study demonstrated increased levels of FGF-23 and α-Klotho in dogs with CanL for the first time. The increase in FGF-23 levels was more prominent in advanced stages of the disease and correlated with higher urea and creatinine concentrations. These findings may serve as a basis for future diagnostic and therapeutic investigations, contributing to the understanding of the pathophysiology of kidney disease in CanL.

In vitro evaluation of antileishmanial activity of Boswellia serrata essential oil nanoliposome.

BACKGROUND: Leishmaniasis poses a significant health risk. OBJECTIVES: This study aimed to evaluate the effects of Boswellia serrata (B. serrata) essential oil nanoliposomes on Leishmania tropica (L. tropica) in vitro. METHODS: A mixture of B. serrata essential oil, phosphatidylcholine and Tween 80 were used to prepare B. serrata essential oil nanoliposomes, followed by drying, hydration and size characterisation. The promastigotes of L. tropica were cultured in Roswell Park Memorial Institute medium (RPMI-1640) containing streptomycin, penicillin and fetal bovine serum. Different concentrations of B. serrata essential and nanoliposomes were tested for their antileishmanial properties by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide tests (MTT). RESULTS: Results of Dynamic Light Scattering (DLS) for B. serrata nanoliposomes indicate that they are successful at producing nanoliposomes with dimensions of 74.8 nm. At 1 µg/mL dose, B. serrata essence caused 17 ± 1.73% mortality, while B. serrata nanoliposomes induced 26 ± 1.15% mortality. B. serrata essence achieved a mortality of 55 ± 2.88% at 10 µg/mL, whereas B. serrata nanoliposomes demonstrated a mortality of 63.66±0.88% at 10 µg/mL. Furthermore, there was a significant difference between similar concentrations of B. serrata and B. serrata nanoliposomes. The LC50 of B. serrata essential oil is 7.26 µg/mL in the 95% confidence interval (12.13-5.25). The LC90 value of B. serrata essential oil is 129.37 µg/mL in the 95% confidence interval (50.07-852.58). The LC50 value of B. serrata nanoliposome is 4.20 µg/mL in the 95% confidence interval (6.13-3.10). LC90 value for B. serrata nanoliposome is calculated as 91.89 µg/mL in the 95% confidence interval (37.09-583.29). CONCLUSIONS: In vitro experiments have shown that B. serrata oil and the nanoliposome suppress the proliferation of L. tropica promastigotes, which suggests it may be a promising option for treating leishmaniasis.

Vitamin d and leishmaniasis: Neither seasonal nor risk factor in canine host but potential adjuvant treatment through cbd103 expression.

Vitamin D (VitD) deficiency has been shown to be a risk factor for a plethora of disorders. We have shown that dogs with clinical leishmaniasis presented lower VitD serum levels than non-infected dogs, and even lower than those with asymptomatic infection. However, if VitD deficiency is a risk factor to develop clinical leishmaniasis remains to be answered. It is also unknown if VitD participates in Leishmania control. First, we retrospectively analysed VitD concentration in serum samples from 36 healthy dogs collected in different periods of the year concluding that there isn't a seasonal variation of this vitamin in dogs. We also included 9 dogs with clinical leishmaniasis and 10 non-infected healthy dogs, in which we measured VitD levels at the beginning of the study, when all dogs were negative for serology and qPCR, and 1 year later. Whereas non-infected dogs showed no change in VitD levels along the study, those developing clinical leishmaniasis showed a significant VitD reduction at the end of the study (35%). When we compared VitD concentration between the two groups at the beginning of the study, no differences were detected (43.6 (38-59) ng/mL, P = 0.962). Furthermore, an in vitro model using a canine macrophage cell line proved that adding active VitD leads to a significant reduction in L. infantum load (31.4%). Analyzing expression of genes related to VitD pathway on primary canine monocytes, we showed that CBD103 expression was significantly enhanced after 1,25(OH)2D addition. Our results show that VitD concentration is neither seasonal nor a risk factor for developing canine leishmaniasis, but it diminishes with the onset of clinical disease suggesting a role in parasitic control. Our in vitro results corroborate this hypothesis and point out that VitD regulates infection through CBD103 expression. These results open the possibility for studies testing VitD as an adjuvant in leishmaniasis therapy.

Influence of domperidone supplementation on short-term changes in C-reactive protein and paraoxonase-1 in dogs with leishmaniasis undergoing meglumine antimoniate and allopurinol therapy.

BACKGROUND: C-reactive protein (CRP) and paraoxonase 1 (PON1) might increase and decrease in canine leishmaniasis (CanL), , and both can rapidly normalize after therapy. Recently, supplementation of domperidone with conventional therapy , increasing the activity of cells involved in acute phase responses in vitro. This combined therapy has been recommended to treat mild forms of CanL; however, no studies have investigated the effects of domperidone supplementation on early CRP or PON1 changes in dogs with CanL. OBJECTIVES: The aim of this study was to evaluate whether domperidone, added to conventional treatments, modifies CRP concentration and PON1 activity kinetics in CanL dogs responsive to conventional therapy. METHODS: Serum CRP concentrations and PON1 activities were measured in dogs with mild CanL before (t-0) and 3 (t-1), 7 (t-2), 14 (t-3), and 21 (t-4) days after treatment with N-methylglucamine antimoniate and allopurinol alone (n = 18) or combined with domperidone (n = 18). RESULTS: C-reactive protein concentrations increased at t-1 in the domperidone group, especially when the CRP concentration at t-0 was normal. However, the concentrations normalized at t-4 in 18/18 dogs compared with 14/18 dogs not receiving domperidone. The median PON1 activity decreased at t-1 in the domperidone group, and this decrease was more significant in dogs with normal PON1 activity at t-0. CONCLUSIONS: Based on these results, transient increases in CRP concentrations or decreases in PON1 activities after domperidone administration should not be erroneously interpreted as signs of a worsening disease process.

Controlling phlebotomine sand flies to prevent canine Leishmania infantum infection: A case of knowing your enemy.

Leishmaniosis caused by Leishmania infantum is a widespread zoonotic disease that can be transmitted to animals and humans by their vectors, blood-sucking phlebotomine sand flies. To prevent canine leishmaniosis across the whole Mediterranean region, vector control is essential. Because of phlebotomine breeding sites are diverse, environmental larval controls have limited practical value. Control methods of adults are being evaluated, such as selective baits based on sugar feeding of males and females or Attractive Toxic Sugar Baits (ATSB), and the indoor use of Long-Lasting-Insecticidal Nets (LLINs) treated with permethrin to prevent sand fly bites complementing the Indoor Residual Spraying (IRS) approach suggested by WHO. Although several strategies exist, the best control measure to prevent canine Leishmania infantum is to treat dogs using biocidal topical formulations based on legal insecticides (PTs18) or repellents (PTs19) (as collars, spot-ons and/or sprays) during the period when the vectors are active. This means we need to really know the biology and life cycle of the sand fly vector. According to available data, by mapping ambient temperatures we can already predict high risk areas where vector densities will be higher. In ongoing research, new candidates are emerging to fight against sand flies including natural plant extracts with low impacts on the environment and host animal. Other options in the future could be systemic insecticides to help reduce sand fly populations in high density areas. In parallel, health authorities and professionals involved in animal and public health (veterinarians, physicians, entomologists and epidemiologists) must work together in a One Health approach to minimize Leishmania infection. Veterinarians play a crucial role in liaising between key stake holders and dog owners to ensure the latter act responsibly in using repellents as a preventive measure against sand fly bites.

Artemisia annua contra la leishmaniosis canina: cuatro casos clínicos / Artemisia annua contra a leishmaniose canina: quatro casos clínicos / Artemisia annua against canine leishmaniasis: four clinical cases

La leishmaniosis canina es una enfermedad ampliamente extendida. En España afecta principalmente a la cuenca mediterránea, pero debido a su constante expansión, las zonas endémicas son cada vez mayores. La necesidad de controlarla hace que constantemente surjan nuevos tratamientos y protocolos terapéuticos. En ese sentido, la existencia de remedios naturales con probada efectividad terapéutica y carente de efectos secundarios abre una puerta valiosa en el control y tratamiento de la enfermedad. Artemisia annua contiene artemisinina, una lactona sesquiterpénica que actúa induciendo estrés oxidativo en el parásito, inhibiendo su crecimiento e induciendo su apoptosis. Se presentan cuatro casos clínicos de leishmaniosis canina tratados con preparados de A. annua en los que se han obtenido mejorías significativas (AU)

A leishmaniose e uma doenga com ampla distribuiçáo geográfica: em Espanha, afecta principalmente a bacia do Mediterráneo mas, devido a sua constante expansáo, as suas áreas endémicas sao cada vez maiores. A necessidadede controlar esta doença leva a que surjam constantemente novos tratamentos e protocolos terapéuticos. Nesse sentido, a existencia de fármacos naturais, com eficacia comprovada e desprovidos de efeitos secundários, abre uma porta inestimável para o controle e tratamento da doença. A planta medicinal Artemisia annua contém uma lactona sesquiterpénica, a artemisinina, que actua através de indugáo de stress oxidativo no parasita, inibindo o seu crescimento e induzindo a respectiva apoptose. Neste artigo sao apresentados quatro casos de leishmaniose canina tratados com preparaçóes de A. annua em que foram obtidas melhorias significativas (AU)

Canine leishmaniasis is a widespread disease: in Spain, it mainly affects the Mediterranean basin; but due to its constant expansion, the endemic areas are increasing. Due to the need of control, new treatments and therapeutic protocols constantly appear. In this context, the existence of natural remedies with preven therapeutic effectiveness and lacking side effects opens a valuable gateway in the control and treatment of the disease. Artemisia annua contains artemisinin, a sesquiterpene lactone which induces oxidative stress, inhibits the growth and induces the apoptosis in the parasite. Four clinical cases of canine leishmaniasis treated with A. annua preparations are presented. Significant improvements have been obtained (AU)

TLR-2 and TLR-4 transcriptions in unstimulated blood from dogs with leishmaniosis due to Leishmania infantum at the time of diagnosis and during follow-up treatment.

Innate immunity, in particular, the role of toll-like receptors (TLRs), has not been extensively studied in canine L. infantum infection. The main aim of this study was to determine the transcription of TLR2 and TLR4 in the blood of dogs with natural clinical leishmaniosis at the time of diagnosis and during treatment follow-up and subsequently correlate these findings with clinical, serological and parasitological data. Forty-six Leishmania-seropositive sick dogs with a high antibody level at the time of diagnosis were studied and compared with 34 healthy seronegative dogs. Twenty-two of these sick dogs were treated with meglumine antimoniate and allopurinol and followed-up at 30, 180 and 365days following the start of treatment. Clinical status was defined by a thorough physical examination, complete blood count, biochemistry profile, electrophoresis of serum proteins, and urinary protein/creatinine ratio (UPC). EDTA blood was stored in RNAlater® solution before RNA extraction and cDNA production were performed. TLR2, TLR4 and three reference genes (HPRT-1, CG14980 and SDHA) were studied in each blood sample by real time PCR. The relative quantification of TLR2 was higher (mean 3.5) in sick dogs when compared with seronegative healthy dogs (mean 1.3; P=0.0001) while the relative quantification of TLR4 was similar in both groups. In addition, the relative quantification of TLR2 significantly decreased during follow-up at all time points compared with day 0 whereas no changes were observed with TLR4 transcription. A significant positive correlation was noted between TLR2 and UPC, total protein, beta and gamma globulins, specific L. infantum antibodies and blood parasite load while a negative correlation was observed with albumin, albumin/globulin ratio, hematocrit and hemoglobin. TLR4 transcript did not correlate with any parameter. These findings indicate an up-regulation of TLR2 transcription in unstimulated blood in naturally infected sick dogs as compared to healthy dogs suggesting active innate immune and proinflammatory responses. In addition, TLR2 transcription is reduced with clinical improvement during treatment. In contrast, TLR4 transcription appears to be similar among groups at the time of diagnosis with no changes during treatment follow-up suggesting a less important role for this TLR in clinical canine leishmaniosis.

An immune-modulating diet increases the regulatory T cells and reduces T helper 1 inflammatory response in Leishmaniosis affected dogs treated with standard therapy.

BACKGROUND: Clinical appearance and evolution of Canine Leishmaniosis (CL) are the consequence of complex interactions between the parasite and the genetic and immunological backgrounds. We investigated the effect of an immune-modulating diet in CL. Dogs were treated with anti- Leishmania pharmacological therapy combined with standard diet (SD Group) or with the immune-modulating diet (IMMD Group). CD3+ CD4+ Foxp3+ Regulatory T cells (Treg) and CD3+ CD4+ IFN-γ + T helper 1 (Th1) were analyzed by flow cytometry. RESULTS: All sick dogs showed low platelet number at diagnosis (T0). A platelet increase was observed after six months (T6) SD Group, with still remaining in the normal range at twelve months (T12). IMMD Group showed an increase in platelet number becoming similar to healthy dogs at T6 and T12. An increase of CD4/CD8 ratio was revealed in SD Group after three months (T3), while at T6 and at T12 the values resembled to T0. The increase in CD4/CD8 ratio at T3 was maintained at T6 and T12 in IMMD Group. A reduction in the percentage of Treg of all sick dogs was observed at T0. A recovery of Treg percentage was observed only at T3 in SD Group, while this effect disappeared at T6 and T12. In contrast, Treg percentage became similar to healthy animals in IMDD Group at T3, T6 and T12. Sick dogs showed an increase of Th1 cells at T0 as compared with healthy dogs. We observed the occurrence of a decrease of Th1 cells from T3 to T12 in SD Group, although a trend of increase was observed at T6 and T12. At variance, IMMD Group dogs showed a progressive decrease of Th1 cells, whose levels became similar to healthy controls at T6 and T12. CONCLUSION: The immune-modulating diet appears to regulate the immune response in CL during the standard pharmacological treatment. The presence of nutraceuticals in the diet correlates with the decrease of Th1 cells and with the increase of Treg in sick dogs. Therefore, the administration of the specific dietary supplement improved the clinical response to the standard treatment in a model of CL.

Serum phosphorus concentrations in dogs with leishmaniosis at different stages of chronic kidney disease.

Serum phosphorus concentrations were measured in 155 dogs with leishmaniosis at different stages of chronic kidney disease (CKD), and in 54 healthy dogs. CKD was classified into six stages, as follows: stage 0 (dogs with no evidence of CKD), serum creatinine (SCr) less than 125 micromol/l and urine protein:creatinine ratio (UPC) less than 0.2; stage 1A, SCr less than 125 micromol/l and UPC 0.2 to 0.5; stage 1B, SCr less than 125 micromol/l and UPC over 0.5; stage 2, SCr 125 micromol/l to 180 micromol/l; stage 3, SCr 181 micromol/l to 440 micromol/l; stage 4, SCr over 440 micromol/l. The dogs' serum phosphorus concentrations correlated significantly with the severity of CKD (P<0.001), and hyperphosphataemia (>1.8 mmol/l) affected 12 per cent, 11.8 per cent, 50 per cent, 76.9 per cent and 100 per cent of the dogs at stages 1A, 1B, 2, 3 and 4, respectively.

The efficacy of enrofloxacin, alone or combined with metronidazole, in the therapy of canine leishmaniasis.

We evaluated the efficacy of enrofloxacin, alone or combined with metronidazole, against Leishmania infantum. The in vitro activity of this fluoroquinolone was assessed using two different methods: a direct test aimed at assessing the drug activity on the parasite, and an indirect test aimed at evaluating the drug effect on macrophage killing, lymphomonocyte activation and nitric oxide production. An in vivo test was also performed on 36 dogs with leishmaniasis, subdivided into three groups, one treated with enrofloxacin, another with enrofloxacin plus metronidazole, and a control group with meglumine antimoniate. The direct test did not show any action of enrofloxacin on the parasite, while the indirect testing showed an enhancement of macrophage killing and an increase in nitric oxide production. These findings show that enrofloxacin does not exert a direct anti-leishmanial activity in vitro. However, on the basis of the positive immunostimulation results shown in vitro and the clinical improvement, particularly of the cutaneous lesions, obtained in several dogs in the in vivo trial, the use of enrofloxacin in association with a specific anti-leishmanial drug can be proposed in the therapeutic protocol of canine leishmaniasis.