Vitamin D deficiency and inflammation in dogs with visceral leishmaniasis

Deficiência de vitamina D e inflamação em cães com leishmaniose visceral

ABSTRACT:

Low vitamin D (VitD) levels have been reported in dogs with visceral leishmaniasis (VL), suggesting a possible correlation. However, the relationship between hypovitaminosis D and inflammatory markers in these animals remains unclear. This study investigated whether VitD levels correlate with inflammation in dogs with VL. Twenty-four dogs were evaluated: eight healthy controls (control group, CG) and 16 VL-positive dogs (diseased group, DG). VitD, creatinine, albumin, C-reactive protein (CRP), and hemoglobin levels were measured and compared between groups using the Mann-Whitney test. The frequency of VitD deficiency was compared using Fisher’s exact test, and correlations between VitD and inflammatory markers (CRP, albumin, and hemoglobin) were assessed using Spearman’s correlation. The significance level was set at 0.05. All inflammatory markers differed significantly between groups. The DG showed a higher frequency of VitD deficiency (57.14%) compared to the CG (0%). However, no significant correlations were reported between VitD levels and the inflammatory markers evaluated. In conclusion, VitD deficiency is more frequent in dogs with VL; although, no association with inflammatory markers was established.

Key words:
vitamin D; visceral Leishmaniosis; dogs

RESUMO:

Baixos níveis de vitamina D (VitD) foram observados em cães infectados com leishmaniose visceral (LV), sugerindo uma correlação com a gravidade da doença. No entanto, ainda não foi estabelecido se existe uma relação entre a hipovitaminose D e os marcadores inflamatórios nesses animais. O objetivo deste estudo é investigar a possível correlação entre a inflamação em cães com LV e as alterações nos níveis de VitD. Para isso, foram utilizados 24 cães: oito saudáveis, alocados no grupo controle (GC), e 16 positivos para LV, alocados no grupo doente (GD). Os níveis de VitD, creatinina, albumina, proteína C reativa (PCR) e hemoglobina foram dosados e comparados entre os grupos por meio do teste de Mann-Whitney. A frequência de animais com deficiência de VitD foi analisada entre os grupos utilizando o teste exato de Fisher. As correlações entre os níveis de VitD e os marcadores inflamatórios (PCR, albumina e hemoglobina) foram avaliadas pelo teste de correlação de Spearman. O nível de significância adotado foi de 0,05. Todos os marcadores inflamatórios apresentaram diferenças estatísticas em suas concentrações entre os grupos estudados. O GD mostrou uma maior frequência de deficiência de VitD (57,14%) em comparação com o GC (0%). No entanto, não foi observada correlação significativa entre os níveis de vitamina D e os marcadores inflamatórios avaliados. Conclui-se que a deficiência de vitamina D é mais frequente em cães positivos para LV, mas não foi possível estabelecer uma correlação entre os níveis de VitD e os marcadores inflamatórios comumente utilizados para cães.

Palavras-chave:
vitamina D; leishmaniose visceral; cães

INTRODUCTION

Visceral leishmaniasis (VL) is a serious zoonotic disease caused by the protozoan Leishmania infantum. It occurs in the Americas, Asia, Africa, and Europe and is transmitted by sand fly vectors of the genera Phlebotomus and Lutzomyia. Although, several mammalian species can be affected, dogs are considered the primary urban reservoir of the disease (MORALES-YUSTE et al., 2022). Canine VL exhibits significant clinical variation depending on the host’s immune response. Dogs that mount a humoral response mediated by T helper type 2 (Th2) lymphocytes tend to develop more severe clinical signs and have a poorer prognosis compared to those with a cellular response dominated by T helper type 1 (Th1) lymphocytes (SOLANO-GALLEGO et al., 2011).

In dogs with a predominant Th2 response, antibody production against the protozoan is excessive yet ineffective, as Leishmania sp. resides intracellularly in its amastigote form. Conversely, in dogs with a Th1-dominated response, macrophage activation by interferon-gamma (IFN-γ) is more effective at controlling the parasite, leading to milder clinical signs and, in some cases, preventing its dissemination (SOLANO-GALLEGO et al., 2011; MORALES-YUSTE et al., 2022). Therefore, mechanisms that modulate immune response patterns may contribute to the disease’s pathogenesis in dogs and represent potential therapeutic targets.

In recent decades, several studies have highlighted the role of vitamin D in regulating immune function. Low levels of this hormone have been associated with increased susceptibility and severity of various infections (ZAFALON et al., 2020; NANYANG et al., 2021). Many immune cells express vitamin D receptors, with T helper cells showing a particularly strong response, including a shift in differentiation from Th1 to Th2 profiles; thereby, altering the immune response (BOONSTRA et al., 2001; ARANOW, 2011).

Although, more severe clinical presentations of leishmaniasis have been linked to vitamin D deficiency in dogs, the relationship between hypovitaminosis D and inflammatory markers has not been fully established (RODRIGUEZ-CORTES et al., 2017). Therefore, this study investigated the correlation between serum vitamin D (calcidiol) levels and inflammatory markers in dogs with VL, as well as to determine the frequency of hypovitaminosis D in affected animals.

MATERIALS AND METHODS

Dogs of various breeds and sexes were included in this study without any specific predilection. Sampling was conducted by convenience at the Veterinary Hospital of the Universidade Federal de Mato Grosso do Sul (UFMS) and the Veterinary Clinic of the Centro universitário da Grande Dourados (UNIGRAN). The inclusion criteria were: (1) diagnosis of visceral leishmaniasis (VL) confirmed by parasitological examination of lymph node or bone marrow aspirates, or by two positive serological tests (immunochromatographic test/ELISA or ELISA/IFA); and (2) absence of concurrent diseases. Exclusion criteria included: (1) age under eight months; (2) pregnancy or lactation; (3) use of vitamin and/or mineral supplements that could interfere with bone metabolism; (4) ongoing treatment for VL or any other disease; and (5) presence of azotemia, as defined by the IRIS (2023) guidelines. Clinically and laboratory-healthy animals with negative results for VL (based on TR DPP^® or ELISA) were included in the control group (CG).

Blood and urine samples were collected from both diseased and healthy animals. Blood was drawn from the jugular or cephalic vein into tubes with and without EDTA (ethylenediaminetetraacetic acid). Urine was obtained via urethral catheterization or cystocentesis. Blood samples were used to perform complete blood counts and measurements of serum creatinine, albumin, phosphorus, vitamin D (calcidiol), and C-reactive protein (CRP). Urinalysis and urinary protein-to-creatinine ratio (UP/C) were performed on the urine samples. Routine tests were processed on the same day of collection. For vitamin D and CRP analyses, serum was stored at -30 ºC and analyzed within seven days.

Complete blood counts were performed using an automated hematology analyzer (HumanCount80TS^®) following the method described by STOCKHAM & SCOTT (2011). Biochemical analyses were conducted on an automated system (InviStar150^®) using Invitro^® kits. CRP was measured with Labmax Plenno^® equipment and Labtest^® kits. Vitamin D levels were determined via chemiluminescence (Cobas e411^®) using Roche^® kits at a support laboratory. Urinalysis followed the procedures outlined by CHEW et al. (2011), and urinary protein was measured with a semi-automated analyzer (Bioplus-200^®) using Gold Analise^® kits.

Subsequently, levels of vitamin D, CRP, phosphorus, serum creatinine, hemoglobin, albumin, UP/C, and urine-specific gravity were compared between groups using the Mann-Whitney test. The frequency of animals with reduced vitamin D levels was compared between groups using Fisher’s exact test. The inflammatory markers albumin, C-reactive protein, and hemoglobin were individually correlated with vitamin D levels using Spearman’s correlation test. A significance level of 0.05 was adopted for all statistical analyses. Statistical analyses were performed using BioEstat version 5.3^®.

RESULTS

A total of 24 dogs were included in the study, with eight assigned to the control group (CG) and 16 to the diseased group (DG). CG included four females and four males, while DG consisted of nine males and seven females. The age of CG ranged from one to four years, with a median of 3.5 years, while DG ranged from one to eight years, with a median of four years. Regarding breed, DG included 13 mixed-breed dogs, one Dachshund, one Boxer, and one Pitbull. CG consisted of six mixed-breed dogs, one French Bulldog, and one Australian Cattle Dog.

Among the animals in the DG, 93.7% (15/16) exhibited clinical signs, with the most prevalent being integumentary changes-including pinna lesions, scaling, onychogryphosis, and pododermatitis-observed in all symptomatic dogs (15/15, 100%), followed by lymph-adenomegaly (12/15, 80%) and weight loss or cachexia (4/15, 26.6%). Hypoalbuminemia (12/14, 85.7%) and anemia (12/15, 80%) were the most frequent laboratory abnormalities observed. Table 1 presents the median values of vitamin D, UP/C, phosphorus, albumin, CRP, hemoglobin, creatinine, and urine-specific gravity for both groups, along with the statistical comparisons.

Table 1
Median, range, and statistical comparison of serum vitamin D, creatinine, phosphorus, CRP, albumin, hemoglobin, UP/C ratio, and urine specific gravity in healthy dogs and non-azotemic dog’s positive for visceral leishmaniasis.

	-------------------Control Group-----------------			----------------Diseased Group---------------
	N	Med	Range	N	Med	Range	P-value
Vitamin D ng/mL	6	37.90	23.9	14	20.90	73.20	0.069
UP/C	7	0.14	0.28	12	0.59	4.3	0.002
UD	7	1.042	0.048	12	1.034	0.034	0.236
Creatinine mg/dL	8	1.05	0.72	16	0.6	1.0	0.004
Phosphorus mg/dL	8	4.45	3.8	16	4.0	4.1	0.806
Albumin g/dL	8	3.35	1.8	15	2.20	2.3	0.002
CRP mg/L	8	2.0	21.8	15	5.9	31.4	0.025
Hemoglobin g/dL	7	17.40	8.4	15	9.70	15.0	0.008

• UP/C - urinary protein creatinine ratio; UD - Urinary density; CRP - C-reactive protein; Med - median; Range - range; n - number of animals.

No statistical difference in vitamin D levels was observed between the groups (P = 0.069). However, 57.14% (8/14) of the dogs in the DG had vitamin D concentrations below the reference range (24.9 ng/dL), differing significantly from the CG, which showed no cases of hypovitaminosis D (P = 0.0419).

CRP levels also differed significantly between groups (P = 0.025; Table 1); although, only 46.66% (7/15) of affected animals had elevated CRP values indicative of inflammation, despite presenting clinical signs, anemia, and proteinuria. Serum albumin and hemoglobin levels showed significant differences between groups as well (Table 1). However, Spearman’s correlation between vitamin D and hemoglobin (rs = 0.194, P = 0.42), CRP (rs = -0.329, P = 0.156), and albumin (rs = 0.181, P = 0.444) was not statistically significant (Table 2).

Dogs in the DG had significantly higher urinary protein-to-creatinine ratios (UP/C) than those in the CG (Table 1), with 63.63% (7/11) of DG animals showing transient proteinuria (>0.5), as defined by IRIS (2023). No significant difference in urine-specific gravity was observed between groups; however, 36.36% of DG animals had values below 1.030.

Table 2
Spearman’s correlation coefficients between vitamin D levels and C-reactive protein (CRP), albumin, and hemoglobin in dogs positive and negative for visceral leishmaniasis.

Correlation	rs	P-value
Vitamin D and CRP	-0.329	0.156
Vitamin D and albumin	0.181	0.444
Vitamin D and hemoglobin	0.194	0.42

DISCUSSION

RODRIGUEZ-CORTÉS et al. (2017) and MARTORI et al. (2021) also reported reduced vitamin D levels in dogs with VL; however, neither study assessed specific inflammatory markers. The vitamin D levels observed in both studies were comparable to those found in the present study, with mean or median values near 20 ng/dL in diseased animals and approximately 40 ng/dL in healthy controls. Nonetheless, comparisons of calcidiol concentrations across studies should be made cautiously due to differences in measurement methodologies (WINDRICHOVA et al., 2021).

RODRIGUEZ-CORTÉS et al. (2017) suggested that low vitamin D levels may contribute to the progression and increased susceptibility to VL, as lower levels were associated with more severe clinical signs. In contrast, MARTORI et al. (2021) proposed that the reduction in vitamin D was a consequence of the disease. Their findings showed that before VL diagnosis, vitamin D levels did not differ significantly from those of healthy animals, but decreased by approximately 30% following infection.

In the present study, vitamin D was measured after VL diagnosis, with approximately 60% of dogs showing low levels, a result consistent with MARTORI et al. (2021), who observed hypovitaminosis D in one-third of infected animals. However, since no significant correlation was established between vitamin D levels and inflammatory markers, it is not possible to confirm whether the deficiency is a cause or consequence of the disease.

The lack of correlation between vitamin D and inflammatory markers such as albumin and hemoglobin may be attributed to the multiple factors influencing the reduction of these analytes in dogs with VL. Albumin, for instance, can be lost through urine-as indicated by the high frequency of proteinuria-or may be reduced due to impaired hepatic synthesis or chronic blood loss (CHEW et al., 2011; SOLANO-GALLEGO et al., 2011; CACHEIRO-LLAGUNO et al., 2021). Similarly, hemoglobin levels can be affected by chronic bleeding and exhibit considerable individual variability, including breed-related differences, as reported by other authors (STOCKHAM & SCOTT, 2011; MIGLIO et al., 2020). CRP is also highly variable among individuals and is considered a nonspecific marker; its elevation may be secondary to VL but can also be influenced by other inflammatory processes (MALIN; WITKOWSKA-PILASZEWICZ, 2022). These factors likely contributed to the absence of a statistically significant correlation between vitamin D and the inflammatory markers evaluated in this study.

However, the higher frequency of hypovitaminosis D observed in DG, in association with elevated CRP, hypoalbuminemia, and anemia, supports the hypothesis that inflammation may contribute to the reduction of this hormone (BOONSTRA et al., 2001; HELMING et al., 2005; EHRCHEN et al., 2007; FITZPATRICK et al., 2018; ALLISON et al., 2020).

One possible explanation for decreased vitamin D levels in dogs with VL is that clinical signs emerge after the development of a type III hypersensitivity reaction. This reaction involves the formation of immune complexes that accumulate in tissues and trigger an inflammatory response (SOLANO-GALLEGO et al., 2011; TIZARD, 2019; CACHEIRO-LLAGUNO et al., 2021). The resulting inflammation induces the production of fibroblast growth factor 23 (FGF-23) (DAVID et al., 2016; CZAYA & FAUL, 2019), which inhibits the synthesis and promotes the degradation of calcitriol, consequently reducing calcidiol levels as well (SHIMADA et al., 2004; GALVÃO et al., 2013; FITZPATRICK et al., 2018; JENKINSON, 2019). As vitamin D levels decline, the Th1 cellular immune response is upregulated, alongside increased antibody production by plasma cells (BOONSTRA et al., 2001; CHEN et al., 2007). This exaggerated cellular and humoral activation amplifies inflammation, creating a feedback loop that perpetuates both hypovitaminosis D and inflammation. This is supported by the present findings, as all animals with hypovitaminosis D exhibited clinical signs; 87.4% were anemic, 50% had elevated CRP levels, and 62% had hypoalbuminemia-all indicators of inflammatory activity.

CRP levels were higher in DG compared to the CG, with 46.66% (7/15) of the sick animals presenting elevated CRP concentrations (>6.0 mg/dL). These findings are consistent with those of BRAZ et al. (2018), who reported high CRP levels in 38% of dogs’ positive for VL. However, their study used a qualitative method, unlike the present study, which employed a quantitative assay. Similar results were reported by SILVESTRINI et al. (2013), who also observed higher CRP levels in dogs with VL compared to healthy animals, although, their values were notably higher (median: 27.5 mg/L). These differences are likely due to the varying methodologies used for CRP quantification.

CRP is synthesized in the liver under stimulation by interleukin-6 (IL-6). As an acute-phase protein, it plays a key role in activating the classical complement pathway, primarily during acute inflammation (AGUIAR et al., 2013), which explains its elevation in animals with VL.

Serum albumin levels were significantly lower in diseased animals, consistent with findings from other studies (FERREIRA et al., 2021). Albumin is considered a negative acute-phase protein, with serum levels typically reduced during inflammation. This reduction is partly due to its role as an amino acid source for the hepatic production of acute-phase proteins and interleukins during the inflammatory response (TIZAND, 2019).

Hemoglobin concentrations were also lower in VL-affected animals compared to controls. Anemia in dogs with leishmaniasis can result from anemia of inflammatory disease, mediated by pro-inflammatory cytokines (FRAENKEL, 2017). However, other mechanisms may also contribute, including hemorrhages from coagulation disorders or vascular damage, immune-mediated hemolytic anemia, and bone marrow suppression (SOLANO-GALLEGO et al., 2011).

Proteinuria was observed in 58.33% (7/12) of dogs in DG, corroborating the findings of CORTADELLAS et al. (2009), FRAZILIO et al. (2018), and OLIVEIRA et al. (2020). Glomerulonephritis caused by the accumulation of immune complexes in the glomeruli leads to inflammation and glomerular damage, allowing proteins such as albumin to pass into the urine, thereby increasing the UP/C ratio (SOLANO-GALLEGO et al., 2011; CHEW et al., 2011; CACHEIRO-LLAGUNO et al., 2021).

No significant difference in urine-specific gravity was observed between groups, likely due to the inclusion criteria, which excluded animals with severe renal dysfunction. Urine specific gravity typically changes when there is damage to the proximal tubules or resistance to vasopressin in the distal tubules; however, such changes generally appear only after approximately 70% of nephrons are damaged, as seen in advanced chronic kidney disease (CKD) (CHEW et al., 2011).

Serum creatinine concentrations were significantly lower in the DG. Although, previous studies also noted reduced creatinine levels in dogs with early-stage VL, these differences were not always statistically significant compared to healthy animals (CORTADELLAS et al., 2009; OLIVEIRA et al., 2020). This reduction may be explained by the higher frequency of thin or cachectic animals in the DG since creatinine originates from muscle metabolism and is directly related to muscle mass (STOCKHAM & SCOTT, 2011).

Serum phosphorus levels did not differ between groups, which was expected, as phosphorus tends to increase only in advanced stages of CKD or acute kidney injury (CHEW et al., 2011). Its concentration often remains within the reference range due to compensatory actions of hormones such as FGF-23 and parathyroid hormone (PTH) (WOLF, 2010; GALVÃO et al., 2013; BAR et al., 2019; OLIVEIRA et al., 2020).

CONCLUSION

In conclusion, hypovitaminosis D is more frequent in dogs with visceral leishmaniasis, even in the early stages of the disease; however, no significant correlation was found between vitamin D levels and commonly used inflammatory markers in dogs.

ACKNOWLEDGMENTS

This study was supported by the Universidade Federal de Mato Grosso do Sul (UFMS) and was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasil - Finance code 001. The Vetanalisa^® laboratory contributed to the development of this study by performing the hormonal and CRP analyses.

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BIOETHICS AND BIOSECURITY COMMITTEE APPROVAL

• CR-2024-0481.R2
• This study was approved by the Animal Use Ethics Committee of the Universidade Federal do Mato Grosso do Sul (CEUA/UFMS, protocol no. 1192/2021) and the Animal Use Ethics Committee of the Centro Universitário da Grande Dourados (CEUA/UNIGRAN, protocol no. 014/22).
• DATA AVAILABILITY STATEMENT
  Not applicable.
• DECLARATION OF USE OF ARTIFICIAL INTELLIGENCE
  The authors used AI only to review the text, supervising the work to avoid changes in results or scientific interpretation.

Edited by

• ASSOCIATE EDITOR:
  Rudi Weiblen (0000-0002-1737-9817)

• SCIENTIFIC EDITOR:
  José Valter Joaquim Silva Júnior (0000-0001-5932-0971)

Data availability

Not applicable.

Publication Dates

• Publication in this collection
  17 Nov 2025
• Date of issue
  2026

History

• Received
  11 Sept 2024
• Accepted
  28 May 2025
• Reviewed
  19 Sept 2025