Clinical and cardiologic assessment of Anatolian shepherd dogs with asymptomatic degenerative mitral valve disease

Degenerative mitral valve disease (DMVD) is the leading cause of cardiac disease and heart failure in the dog. Advanced age, breed and male gender are well-known risk factors for DMVD. The incidence of the disease in German Shepherds seems to be noteworthy. Early diagnosis of DMVD is related to the identification of a left apical systolic murmur, characteristic of MR in a dog. Dogs with DMVD had a low frequency of arrhythmias compared to other cardiac conditions. The goal of the study was (i) to evaluate the age and gender incidences of the asymptomatic Anatolian Shepherd Dogs (ASHs) with DMVD and, (ii) to investigate the importance of its clinical, radiological, electrocardiographic (ECG) findings and the correlations of those with some echo cardio logical measurements. 35 healthy ASHs (control group) and 38 ASHs with DMVD (experimental group) were used as the materials. The severity of cardiac disease was classified according to the American College of Veterinary Internal Medicine (ACVIM) consensus statement. Thirty two dogs (84.2%) were males and 6 dogs (15.8%) were females in the experimental group. The median age, the intensity of heart murmur and the severity of mitral regurgitation (MR) of the B2 dogs were bigger (p <0.05) than that of the B1 dogs. There was a positive correlation (P<0.05) between age and mitral valve lesions (MVLs). The clinical examination assessed by cardiac auscultation (murmur) was not correlated to MVLs, VHS, ECG findings and ARJ/LAA (P>0.05). The intensity of murmur was correlated to left ventricle to aorta ratio (LA/Ao) and it was not correlated (P>0.05) to MVLs, vertebral heart scale (VHS), ECG findings and regurgitant jet area to LA area ratio (ARJ/LAA). The correlations between ECG findings and VHS, along with, LA/Ao and ARJ/LAA were positive (P <0.05). In conclusion, aging and male gender may have a significant impact on DMVD progression in ASHs. Assessment of higher murmur in group B2 might be related to the progressive severity of the illnesses. The prevalence of arrhythmia was low in asymptomatic ASHs with DMVD. P-mitrale was noteworthy.


*Anatolian
Shepherd dog, which is a popular breed in Turkey, is a large, rugged and powerful livestock guardian. DMVD is the leading cause of cardiac disease in dogs throughout the world (Bonagura and Schober, 2009;Borgarelli et al., 2012;Terzo et al., 2009). Its prevalence has been found to be associated with breed, age and body size (Bernay et al., 2010;Borgarelli et al., 2004;Petric, 2015). Older small-breed dogs are predisposed (Petric, 2015). However, less is known about the disease prevalence in large breeds. The incidence of the disease in German Shepherds seems to be noteworthy (de Madron, 1992;1998). Advanced age and male gender are well known risk factors for DMVD (Chetboul and Tissier, 2012;Connell et al., 2012;Garncarz et al., 2013;Terzo et al., 2009). Jones and Zook (1965) reported that DMVD is an age dependent disease. Males predominated in most of the studies (Pedersen and Häggström, 2000;Serfass et al., 2006;Thrusfield et al., 1985;Petric, 2015).
The disease progresses through various stages (A to D) in dogs. Stage B is the non-symptomatic period of DMVD which is characterized by stage B1 and stage B2 (Turgut, 2017). There is no evidence of cardiac enlargement in stage B1 dogs, however stage B2 dogs have cardiac enlargement to compensate for the volume load (Bonagura and Schober, 2009;Häggström et al., 2009;McGinley et al., 2007). Nevertheless, even at the asymptomatic compensated stage (stage B), DMVD may still cause problems by complicating elective surgical interventions, such as dental care, and by causing anxiety for the owner (Häggström et al., 2009).
The clinical, radiological, ECG findings along with the changes evident by integrated echocardiographic studies should be considered in the diagnosis and assessing the severity of DMVD, even in the asymptomatic compensated stage of the disease. Cardiac murmur, cardiomegaly, supraventricular and ventricular arrhythmias and, MVLs and MR are common findings in the disease (Borgarelli and Buchanan, 2012;Borgarelli et al., 2004;Buchanan and Bücheler, 1995;Connell et al., 2012;Ljungvall et al., 2014).
Studies focused on a particular breed can give some additional information about the diseases. There is no information about DMVD and its clinical importance in the Anatolian Shepherd dogs (ASHs). This study was aimed (i) to evaluate age and gender incidences of the disease and, (ii) to assess the importance of the clinical, radiological, ECG findings and the correlations of those with some echo cardio logical measurements in ASHs with a symptomatic DMVD.

Animals
The medical records of ASHs which were brought to the Small Animal Hospital of the Faculty of Veterinary Medicine of Selçuk University between June 2015 and December 2017 were reviewed. A total of 38 ASHs affected by DMVD was used as the experimental group. The control group was consisted of 35 healthy ASHs. All the ASHs had been presented for a cardiology consultation both for the identification of a heart murmur and for the evaluation of the cardiovascular system or for the regular checkup purpose.

Study design
The severity of cardiac disease was classified according to the ACVIM consensus statement (Matos and Glaus, 2010). VHS and echocardiographic LA/Ao ratio were the criteria to differentiate between class B1 and B2. Eighteen dogs from the experimental group which had both VHS scores >10.5 and LA/Ao ratios >1.7 were classified as stage B2 (enlarged atrium and ventricle), whereas 20 dogs from the experimental group which had both VHS scores ≤10.5 and LA/Ao ratios ≤1.7 were classified as stage B1 (normal cardiac size). Inclusion criteria in the experimental group were the recognition of mitral valve leaflet thickening and/or mitral valve prolapse (MVP) and the determination of mitral regurgitation (MR) in echo graphic examination. The control dogs were normal according to the all cardiologic examinations findings. Exclusion criteria in the control group were the detection of concomitant non-cardiac diseases or cardiac diseases other than DMVD. A physical examination, ECG study, chest radiography and blood pressure (BP) measurements were performed in each ASHs of both the experimental and the control groups.

Clinical and cardiologic examination
The intensity of cardiac murmurs was evaluated according to Levine's classification (Ware, 2011). The systolic and diastolic BPs of each dog was measured with an oscillometric technique (Ware, 2011). All the radiographs were examined for the cardiac silhouette, pulmonary parenchyma, and vessels. The VHS of each dogs were measured and the grade of cardiac enlargement was classified according to the VHS method (Buchanan and Bücheler, 1995). The ECGs were performed using a standard 6 lead ECG (Vet ECG Electrocardiograph VE-300 Vega Group), with the dog in right lateral recumbence, and the ECG recordings were analyzed (Martin, 2007). Electrocardiographic variables based on the followings: sinus arrhythmia, as a RR interval >180% longer than the previous RR interval; Pmitrale as a P duration> 0.04 sn (Turgut, 2017).
Each dog had standard 2-D, M-mode, and color flow Doppler echo graphic examinations. Transducer arrays of 4-7 MHz were used (SUIU, CZXL-43C). The examinations were performed in conscious and unsedated dogs. Right parasternal and left apical echocardiographic examinations were performed in accordance with techniques described by Turgut (2017). The presence of MVL and MVP was evaluated by 2-D examination. Mitral valve lesions were graded as (1) fibrotic, (2) fibrotic+nodular, (3) fibrotic+nodular+chordate tendinea involvement and (4) MVP associated with mitral valve leaflet thickening. The LA/Ao was obtained from the right parasternal short-axis view in 2-D mode (Rishniw and Erb, 2000). The mitral valve insufficiency jet (%) was evaluated, and its severity was subjectively assessed using the left apical four-chamber view. The MR was classified as mild, moderate or severe if the maximal ratio of the ARJ/LAA; <%20, %20-40, >%50, respectively (Turgut, 2017).

Statistical methods
Apart from the age, all values are reported as the mean±SE. Data (control, B1 and B2 groups) was evaluated with ANOVA and Tukey. Date for the age was evaluated with Kruskal-Wallis and Mann-Whitney U test. Independent sample t-test was used to compare the auscultation findings, the echocardiographic grading of mitral valve lesions and the qualitative echocardiographic grading of mitral regurgitation (ARJ/LAA) from the two groups (Group B1 and Group B2). To examine univariate associations between the parameters, Pearson correlation test was used (SPSS 19.0). Statistical significance level was set P<0.05.

Clinical and cardiologic examination findings
Thirty-two dogs (84.2%) were males and 6 dogs (15.8%) were females in the experimental group. 23 dogs (65,7%) were males and 12 dogs (34,3%) were females in the control group (Fig. 1). There was no statistically significant difference among the B1 dogs, the B2 dogs and the control dogs concerning with the body weight or heart rate (HR) ( Table 1). The median age in the control dogs (median and interquartile: 3.94 and 2.35; range 1-7 years) was significantly lower (p<0.05) than the B1 dogs (median and interquartile: 5.67 and 4.57; range 3-9 years) and the B2 dogs (median and interquartile: 7.42 and 6.43; range 4-8.5). The median age of the B2 dogs was bigger (p <0.05) than that of the median age of the B1 dogs (Table 1 and Fig. 1). On the physical examination, sixteen (80%) of the B1 dogs had left-apical holosystolic murmurs (13 with grade I-II/VI; 3 with grade III-IV/VI) and 4 (20%) had no an auscultable murmur. Seventeen (94.4 %) of the B2 dogs had left-apical holosystolic murmurs (10 with grade I-II/VI; 7 with grade III-IV/VI) and 1 (5.6%) had no an auscultable murmur (Table 1). The B2 dogs presented more often with a detectable heart murmur and more frequently with a high intensity heart murmur (grade III-IV/VI), when it was compared with the B1 dogs (p < 0.05) (Table 1 and Fig. 2). The control dogs were in sinus rhythm. 19 of the B1 dogs (95%) had a normal sinus rhythm on a screening ECG while 1 of them (5%) had a sinus arrhythmia. 10 of the B2 dogs (56%) had a normal sinus rhythm while the other 2 of the dogs (11%)

Fig. 3:
Proportion of ECG findings (sinus rhythm, sinus arrhytmia and P mitrale) of the control dogs, theB1 dogs and theB2 dogs Important differences (p< 0.05) was found between the B2 dogs and the B1 dogs concerning with the frequency of ECG findings (sinus rhythm, sinus arrhythmia and P-mitrale; Table 1). The pulmonary parenchyma and vessels were all normal in thoracic radiographies in the both groups of dogs (B1, B2). However, VHS was significantly bigger (p < 0.05) in B2 group of dogs compared with B1 dogs and healthy control dogs (mean 10.9 for B2 group, 10.3 for B1 group and 9.74 for control dogs; Table 1).
Systolic and diastolic BPs determined by oscillometric method of all the ASD used in this study, were within normal reference values (systolic BP <180 mm Hg and diastolic BP >60 mm Hg). There was no statistically significant difference among the B1 dogs, the B2 dogs and the control dogs concerning with BP (Table 1).

Echocardiographic findings
According to the ranging of MVLs, there was no difference (p >0.05) between the B2 dogs and the B1 dogs (Table 1). The median LA/Ao was significantly higher (p < 0.05) in B2 dogs compared with B1 dogs and Control dogs (1.80, 1.44 and 1.20 respectively). The difference between B2 Group and B1 Group (Table 1) was also significant (p < 0.05). On the color flow Doppler examination, the B1 dogs consisting of 20 dogs (100 %) had mild MR (< 20%), whereas in the B2 dogs consisting of18 dogs; 6 had mild, 11 had moderate and 1 had severe MR. Therefore, the severity of MR was higher (p <0.001) in the B2 dogs than the B1 dogs (Table 1).

Bivariate analyses
There was a positive correlation (P<0.05) between the age and MVLs ( Table 2).
The clinical examination assessed by cardiac auscultation (murmur; Table 3) was not correlated to MVLs, VHS, ECG findings and ARJ/LAA (P>0.05).  The cardiac auscultation (murmur) was correlated to LA/Ao. The correlations between ECG findings and VHS, along with, LA/Ao and ARJ/LAA (Table 4) were positive (P <0.05).

Discussion
In dogs, the prevalence and severity of the DMVD have been reported to be closely age dependent (Bernay et al., 2010;Borgarelli and Buchanan, 2012;Buchanan, 1977). Two comprehensive studies, carried out by Whitney (1974) and Kogure (1980), have demonstrated that the structural changes in canine mitral valves were an age dependent process. In the present study, it was determined that the median age of the B2 dogs was bigger (P<0.05) than that of the B1 dogs (Table 1 and Fig. 1). This age distribution in ASHs suggested that the structural changes in mitral valves might be an age dependent process because MVLs were more severe in the B2 group (Table 1). There was a positive correlation between MVLs and age (Table 2). However, the sequence of events and the time course should be investigated with longitudinal studies in ASHs with DMVD.
Risk factors such as male gender are also important in the development of DMVD in dogs (Hyun, 2005). The diseases in males develop at a younger age and progress rapidly from mild to severe (Pedersen and Häggström, 2000;Pedersen et al., 1999a;Serfass et al., 2006;Thrusfield et al., 1985). In this study, the gender distribution in the experimental group shows an imbalance favor to the male (Table 1 and Fig. 1). For these reasons, DMVD in ASH may be interpreted as a gender dependent disease.
Early diagnosis of DMVD is related to the identification of a left apical systolic murmur, characteristic of MR in a dog (Gordon et al., 2017). In the early stage of the DMVD, the patients may have no detectable clinical signs. Nevertheless, progression of the MVLs causes more insufficiency in the coaptation of the leaflets, more regurgitation of blood back into the LA, and more dilation of the LV and mitral annulus. As a result of these, mitral systolic murmurs occur (Connell et al., 2012;Häggström et al., 2009). On physical examination in this study, 16 (80 %) of the B1 dogs had left-apical holosystolic murmurs (13 with grade I-II/VI; 3 with grade III-IV/VI) and 4 (20%) had no an auscultable murmur. 17 of the B2 dogs (94,4%) had left-apical holosystolic murmurs (10 with grade I-II/VI; 7 with grade III-IV/VI) and 1 (5,6%) had no an auscultable murmur (Table 1 and Fig. 2). So, 5 ASHs (4 in Group B1, 1 in Group B2) with echocardiographic evidence of DMVD did not have murmurs. Although the finding of systolic murmur is a good indicator of the disease, it has been stated that the dogs with mild disease might not have a murmur, despite the echocardiographic evidence of MVLs and MR (Garncarz et al., 2013;Pedersen et al., 1999a). In a study on Norfolk terriers with DMVD (32), 44% of the dogs did not have an auscultable murmur, which is higher than previous reports in which 23% of CKCS with a greater than 10% MR jet area did not have a murmur on physical examination (Pedersen et al., 1999b;Trafny et al., 2012). Many factors, such as background noise or heart rate, stress level and chest conformation may be the reason of this. Assessment of higher murmur in group B2 might be related to the progressive severity of the illnesses. It has been reported that the severity of DMVD in dogs was well associated with lower serum serotonin concentrations and higher LA/Ao which causes low serotonin concentrations (Ljungvall et al., 2014;Petric, 2015). The positive correlation between the cardiac auscultation (murmur) and LA/Ao (Table 3) can explain this result. Lopez-Alvarez et al. (2014) stated that dogs with DMVD had a low frequency of arrhythmias compared to other cardiac conditions, with 19% of dogs showing some form of ectopic activity but only 1.6% of dogs developing atrial fibrillation in the course of their disease. This confirms the general suspicion that DMVD is a disease with a low prevalence of arrhythmia occurrence. In this study, 19 of B1 dogs (95%) had a normal sinus rhythm on a screening ECG, while 1 of the dogs (5%) had a sinus arrhythmia. However, 10 of the B2 dogs (56%) had a normal sinus rhythm on a screening ECG while the other 2 of the dogs (11%) had sinus arrhythmia, and remaining 6 dogs (33%) had P-mitrale. Significant difference (p<0.05) was found between the B2 dogs and the B1dogs concerning the frequency of ECG findings (sinus rhythm, sinus arrhythmia and Pmitrale; Table 1 and Fig. 3). Sinus arrhythmia is common in healthy dogs without any clinical significance. Sinus arrhythmia results from cardiac vagal function reflecting respiratory-circulatory interactions. It is also possible to observe sinus arrhythmia in dogs with MR (Turgut, 2017). It has been reported that in early stages of DMVD, sinus arrhythmia was often present, but during progression to CHF, tachycardia usually developed and the sinus arrhythmia ceased because of compensatory increase in sympathetic tone (Rasmussen et al., 2011;. Determination of sinus arrhythmia in 3 dogs of the experimental group (1 in the B1, 2 in the B2 groups) may also indicate the early stages of DMVD. Six dogs (33%) in the B2 dogs had P-mitrale (prolonged P wave >0.04 sec) in this study (Fig. 3). LA is affected directly by increased ventricular filling pressure and resistance across the mitral valve, or volume overload due to MR (Chirife et al., 1975;Ware, 2011). The dogs having P-mitrale in this study had also LA enlargement (LA/Ao>1.7), and ECG findings were positively correlated (p<0.05) to VHS, LA/Ao and ARJ/LAA (Table 4). These results can support the development of P-mitrale in the B2 dogs.

Conclusion
In conclusion, the results of the present study showed that aging and male gender might have a significant impact on DMVD progression in ASHs. Assessment of higher murmur in group B2 might be related to the progressive severity of the illnesses. The prevalence of arrhythmia was low in asymptomatic ASHs with DMVD. P-mitrale was noteworthy.