Clinical and hematological evaluation in dogs with myoclonus derived from canine distemper supplemented with vitamin D3

In dogs, the synthesis of vitamin D in the skin is considered inefficient, making dietary supplementation the main source of this vitamin for these animals. In humans, there are established values for 25-hydroxyvitamin D (25(OH)D) deficiency, insufficiency and sufficiency levels, however in dogs, the serum concentrations of these values are not well established. The purposes of this study were to evaluate the 25(OH)D serum levels in dogs carrying myoclonus as sequelae of distemper, to evaluate the response to vitamin D levels on oral supplementation, to evaluate PTH, calcium, phosphorus, alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood count and leukogram levels, in addition to conduct clinical observations of myoclonus. Venous blood samples were collected from nine dogs carrying myoclonus derived from distemper, however with no other clinical or laboratorial change, of varied breeds and same age group (1 8 years old). Screening laboratory tests were performed to attest to the health of the animals in a 30-day period and the collections were divided into three periods: days 0, 15 and 30. After the initiation of treatment, the animals underwent physical and laboratorial evaluations every 15 days, for 90 days, completing a total of 120 days. The dose used for oral supplementation of vitamin D3 was 1000IU/kg administered every day, once a day, during the entire experimental period. For clinical evaluation, parameters of anatomical distribution, speed and rhythm, and distribution of myoclonic changes over time were observed. The laboratory results were subjected to analysis of variance and, when significant (P<0.05), submitted to regression analysis. Descriptive statistics were used to analyze clinical results. There was a significant difference in blood concentrations of 25(OH)D, PTH, calcium and phosphorus, however there was no significant effect of vitamin D on the other parameters evaluated. It was possible to conclude that the dose of vitamin D3 used was sufficient to increase 25(OH)D serum levels in the blood, to levels of sufficiency, having influence on PTH, phosphorus and calcium levels, not changing the other hematological and clinical parameters evaluated. However, the dose and duration of the treatment used did not change the myoclonus derived from distemper in dogs.


Introduction
Vitamin D, currently defined as an important hormone, has been known for its role in osteomineral physiology, mainly for calcium and phosphorus regulation, promoting the absorption of those minerals by the intestinal mucosa (Davies et al., 2012). In human medicine, the search for the benefits of vitamin D for the extraskeletal health has become a frequent and expanding theme and its antineoplastic and immunomodulator effects have been studied (Weidner & Verbrugghe, 2017).
Although the development of research in this area for veterinary medicine is only beginning, there has been strong evidence that vitamin D has effects besides the osteomineral metabolism (Lips, 2006), as it is known that there is interaction of vitamin D receptors in more than 40 tissues acting in almost all body systems (Norman, 2012).
In humans, sun exposure remains the main source of vitamin D, both for most of the children and for adults (Holick, 2016;Holick, 2017). During sun exposure, 7-dehydrocholesterol, the immediate precursor in the biosynthetic pathway of cholesterol, absorbs ultraviolet B radiation (290 -315nm), resulting in breakage of the link between carbon 9-carbon 10 to produce pre-vitamin D3. Once formed, this unstable steroid suffers a rearrangement of its triene system to form the thermodynamically stable vitamin D3 (cholecalciferol). Following, it is led to the liver, where it is converted into 25hydroxyvitamin D. Then, this metabolite goes to the kidneys, where it is converted to the active form 1.25-didihydroxyvitamin D (Holick, 2007;Wacker & Holick, 2013).
In dogs, however, evidence suggests that the production of vitamin D in the skin, mediated by ultraviolet exposure, is essentially insignificant (Laws et al., 2018). High amounts of cholesterol have been found in lipid extracts of dog's skin, however no intermediate product of cholesterol synthesis, such as the precursor of vitamin D, 7-dehydrocholesterol (Wheatley & Sher, 1961). The absence of 7-dehydrocholesterol has formed the basis of the hypothesis that dogs have lost the ability to produce vitamin D, becoming dependent on its dietary intake (Weidner & Verbrugghe, 2017).
There is still no consensus on the acceptable blood concentrations of vitamin D for dogs, therefore, reference values remain undefined. However, citations for the optimal serum concentration of 25(OH)D in dogs are often found, such as the one from the Diagnostic Center for Population and Animal Health at the Michigan State University, which defined the interval between 43.6 -169.2ng/mL (Nachreiner et al., 2014), and others suggesting the interval between 100 -120ng/mL (Selting et al., 2016;Weidner & Verbrugghe, 2017).
Following medicine trends, there has also being interest in the extraskeletal effects of vitamin D and the role of the vitamin for health and disease conditions in companion dogs, since researchers have reported associations between low serum concentrations of 25(OH)D and canine mastocitoma tumors (Wakshlag et al., 2011), chronic kidney disease (Gerbe et al., 2003), congestive heart failure (Kraus et al., 2014) and inflammatory bowel disease (Gow et al., 2011).
The infection by the canine distemper virus may cause demyelination in different central nervous system regions (Orsini & Bondan, 2008), corresponding to the removal process of previously formed myelin sheaths (Santos, 2016).
Demyelinating lesions are responsible for permanent sequelae, including myoclonus and the decreased quality of life of affected dogs. Thus, researchers have invested in alternative therapies with the purpose of optimizing the repair of these lesions in the central nervous systemet (Nunes, 2016;Santos, 2016).
A suggestive treatment for those lesions would be the use of vitamin D. Studies with the drug report the improvement of neurological lesions in autoimmune diseases and its probable benefit for the treatment of demyelination caused by the distemper virus in the neurological phase (Embry, Snowdon & Vieth, 2000).
According to Zhang and Ko (2009), vitamin D3 stimulates the neutrophins responsible for regulating axonal growth and myelination, in addition to regulating neural death. Through that, the neurotrophic factors for the development and regeneration of the nervous system have an important role in pathological situations (Nunes, 2016).
The hypothesis of the study considers that daily supplementation with a dose of 1000IU/kg of vitamin D3 (cholecalciferol) could decrease the myoclonus derived from distemper in dogs, evaluating the effective increase in vitamin D, PTH, total calcium, phosphorus, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, urea, blood count and leukogram levels and the clinical evaluation. Therefore, the purposes of this study were to identify the 25(OH)D levels in dogs carrying myoclonus derived from canine distemper and to evaluate the response to the levels on oral supplementation.

Methodology
The ethical approval for the study was obtained from the Ethics Committee on the Use of Animals/CEUA -Jataí (protocol 022/2017).
Nine adult male or female dogs carrying myoclonus derived from canine distemper, feeding normally with commercial balanced ration, examined at the Clínica São Francisco Vet Center in Rio Verde -GO, were selected to be included in the study. The informed consent for using the animals and the clinical blood samples for research purposes was obtained on admission for each dog selected. For selection, patients with a history of pharmacological vitamin D or calcium supplementation in the previous six months were excluded, as well as pregnant and breastfeeding patients, and those receiving medications that affect vitamin D metabolism, or with a diagnosis of any disease that changes calcium or vitamin D metabolism, such as bone, parathyroid, renal and hepatic diseases.
Age, gender, breed and body weight were recorded for each dog. Blood samples were extracted from the jugular vein after antisepsis, in the period from day 0 to day 120, with an interval of 15 days between each collection, totaling nine samples.
Analyses on days 0, 15 and 30 were performed to establish the health of the animals. The following clinical information was measured for each patient: parathormone, 25-hydroxyvitamin D, red blood cells, hematocrit, hemoglobin, MCV, MCH, MCHC, plasma proteins, platelets, total leukocytes, metamyelocytes, rod cells, eosinophil and basophil, lymphocytes, monocytes, ALT, creatinine, urea, AST, phosphorus and calcium.
For the measurement of paratormonium and 25-difroxyvitamin D, 2mL of blood were collected in a serum clot activator tube, according to the automated modular method Cobas 6000 (Roche). The hematological variables were measured in an Automated System SDH-3 VET. The biochemical parameters (ALT, AST, creatinine, urea, phosphorus and calcium) were collected in serum clot activator tubes and measured in a semiautomatic biochemical analyzer Spectrum -Celer.
Myoclonus was classified and measured, for each animal, according to anatomical distribution, frequency and rhythm intensity. According to distribution, it was defined as focal (1), segmental (2) or myoclonic twitch (3). According to the frequency of contractions, it was classified as intermittent (1), grouped and permanent (2) or continuous and permanent (3). According to speed or rhythm, it was classified as slow (0.05 -1 contraction per second) (1), fast (2 -8 contractions per second) (2), arrhythmic (3) or oscillatory (4) (Almeida, 1995). The evaluations of all animals were performed by the same professional, in the period from day 0 to day 120, with an interval of 15 days between each evaluation, totaling nine samplings.
The manipulation of vitamin was made in oily medium at the Laboratório de Manipulação Artesanal in Rio Verde -GO, determining 1000IU/drop in a total of 30mL per vial. The dose established per animal was 1 drop/kg per day, from day 31 to day 120 of the experiment.
For statistical analysis, the software Sisvar was used to obtain the analysis of variance (ANOVA) and the software Sigmaplot was used for regression graphs.

Results and Discussion
Nine dogs fulfilled the inclusion criteria, but only five were selected for statistical analysis by homogeneity based on their standard deviation.
According to the F Test, it was possible to observe that vitamin D3 did not influence the ALP, hemoglobin, AST, ALT, urea, creatinine and hematocrit variables.  The only research to address the concentration in dogs, using the chemiluminescence assay to measure the serum 25(OH)D, suggested as sufficiency 25(OH)D levels >100ng/mL, insufficiency between 25 and 100ng/mL and deficiency in concentrations <25ng/mL (Selting et al., 2016). Considering this classification, it was observed that in the period from day 0 to day 30, in which the animals were not supplemented, the 25(OH)D serum levels found were considered insufficient. At days 90 and 120, 25(OH)D serum concentrations with adequate sufficiency level were observed. These results imply that more research should be performed to establish hematological sufficiency, deficiency and insufficiency parameters in dogs and to establish a safe vitamin D3 dose for daily supplementation, at health and disease conditions. The 25(OH)D serum concentrations, found before the beginning of supplementation, are consistent with the findings by Young & Backus (2016), where 71.7% of the apparently healthy dogs evaluated had 25(OH)D serum concentration below 100ng/mL and consistent with the findings by Sharp et al., (2015) who, collecting samples from 320 dogs, observed that many apparently healthy dogs are insufficient in vitamin D while some others are deficient. Research, Society andDevelopment, v. 10, n. 3, e57310313607, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i3.13607 6 The reason for 25(OH)D being below the levels considered optimal for the species has not been defined yet, however it may be connected to deficiency in the synthesis of vitamin D by sun exposure, its dietary insufficiency and the fact that it is not supplemented.
Although the initial results were consistent, after the initiation of treatment different results were observed when compared to Young & Backus (2016), who, using a dose of 2.3g/kg of vitamin D3 did not observe an increase in the concentration levels reported considered as sufficiency. It should be emphasized that the methods and tests to measure the 25(OH)D serum concentration are not standardized, which may result in different interpretations of results.
Regarding the parathormone, more consistent results were observed in the application of quadratic regression (Y=7.28-0.147X+0.001x2; r2=0.9787*), in which most of the values remained close to the overall mean of the group (Figure 2), confirming that the dogs responded similarly to the treatment proposed. It was possible to observe that the serum levels of PTH decreased with increased concentrations of 25(OH)D.
Observing the graphs from day 45, when the blood concentration of 25(OH)D began to increase, PTH levels began to decline and remained within the mean between 0 -2ng/mL not seeming to stabilize and showing an increase in day 120 (2 -4ng/mL).
The same results were obtained by Selting et al., (2016), in which PTH continued to decrease with the increased concentrations of 25(OH)D.
In cases of vitamin D deficiency, there is a compensatory increase in PTH secretion that will stimulate the kidneys to produce the 1.25OH2D3 (Marques et al., 2010). In contrast, with the increased vitamin D concentration, PTH levels decrease, therefore corresponding to the results obtained through PTH analysis.
In the calcium analysis, it was possible to observe, by quadratic regression, that means on days 0, 15, 30, 45 and 60 remained closer to the overall mean (Figure 3). It was also observed that calcium serum levels increased during treatment. This result is explained as PTH, together with activated vitamin D, stimulates bone resorption by osteoclasts, thus increasing calcium serum concentrations (Barral et al., 2007). In the kidneys, vitamin D acts on the distal tubules promoting calcium reabsorption and, in the intestine, it acts on the endothelial cells stimulating the active calcium absorption in the duodenum and the passive absorption in the jejunum (Castro, 2011). This could explain the increased calcium serum levels observed in this study.
Between days 0 and 90, the mean of the total calcium serum concentration remained between 4.0 -6.0mg/dL, increasing on day 105 (8.0 -10.0mg/dL). According to Schenck (2008), the reference value of total calcium for dogs is 9.0 -11.5mg/dL and of ionizable calcium is 5.0 -6.0mg/dL. Therefore, it could be concluded that the mean obtained on day 105 was the closest to the optimal levels of total calcium.
Regarding the phosphorus serum levels found in this study with application of simple linear regression, it was observed that both means were not close to the overall mean. When analyzing the graph of phosphorus concentration over time, it can be concluded that the serum levels increased linearly with supplementation of vitamin D3 (Fig. 4). This linear increase is explained by the greater absorption of this ion in the intestine, stimulated by calcitriol (Abrita, 2015).
The role of vitamin D in maintaining the balance between calcium and phosphorus is already well established. In this study, it was observed that the 25(OH)D influenced the serum concentration of calcium and phosphorus. In contrast, there are studies that did not obtain the same observations. Fonseca (2017), for example, did not observe the direct correlation between the serum concentrations of total calcium and phosphorus and the levels of 25(OH)D. Similar results were also noted by Selting et al., (2016), who did not observe correlation.
When observing the results obtained in the clinical analysis, it was possible to confirm that, regardless of the time of use of vitamin D3, the animals visually evaluated as to the anatomical distribution obtained mean, median and mode 2, most of them presenting segmental myoclonus, i.e., the myoclonic twitch affected two or more muscle groups in a specific body segment (Table 1).
Analyzing speed and rhythm results, it could be observed that 60% of the animals evaluated had slow rhythmic myoclonus from 0.5 to 1 contraction/second during the entire experimental period ( Table 2). The rhythmic myoclonus has a specific, regular and uniform rhythm.   For distribution over time, the mean, median and mode had value 3, and the animals visually evaluated were classified with continuous and permanent myoclonic twitches, happening continuously, periodically and for extended periods. Regarding the evaluation of the anatomical distribution in the speed and rhythm and the distribution of myoclonus over time, no differences were observed between the experimental periods for each animal evaluated (Table 3).
The results obtained from the clinical evaluations showed that there was no influence of vitamin D3 on the evaluated parameters of speed and rhythm and distribution over time, that is, the animals did not show significant visual improvement to the treatment proposed. It is believed that this result was influenced by the experimental period, since the animals only had sufficiency levels as of the evaluation on day 90. We can suggest that the time for vitamin D3 levels to present satisfactory concentrations for the treatment of myoclonic lesions was not sufficient.

Conclusion
Dogs not supplemented with vitamin D do not have sufficient levels of this vitamin, being dependent on oral supplementation. This occurs due to the absence of 7-dihydrocholesterol in the vitamin D metabolization chain. The dose used in this study, of 1000IU/kg of vitamin D3 (cholecalciferol) orally, had the potential to increase it to levels considered sufficient for dogs, changing, as expected, the serum levels of PTH, phosphorus and calcium, not changing red blood cells, ALP, hemoglobin, AST, ALT, urea, creatinine and hematocrit. It can also be concluded that the dose used did not have a toxic effect for the dogs supplemented, being considered safe to be used in dogs. However, the dose and duration of the treatment used did not change the myoclonus derived from distemper in dogs.
It is suggested, for the future, to perform studies with longer treatment periods with vitamin D3, using different doses, with neuronal myelination evaluation methods, such as magnetic resonance imaging and evaluation of vitamin D concentration levels in the diet.