Clinical Application of Three-Dimensional Quantitative Ultrasonography in Presumptive Diagnosis of Hepatic Lipidosis in Common Mynahs (Acridotheres tristis)

Hepatic disorders are important and diverse disease entities in birds with nonspecic clinical signs. However, paraclinical examinations such as analysis of hepatic enzymes and diagnostic imaging can help evaluation of the liver changes, a denitive diagnosis is possible only with the histopathological evaluation. The aim of the present study was to determine the feasibility and capability of using three-dimensional (3D) Virtual Organ Computer-aided AnaLysis (VOCAL) ultrasonography as a noninvasive method for etiologic diagnosis of hepatomegaly in common mynah (Acridotheres tristis). Result Mean gray level of liver, mean gray level of muscle, and relative proportion of mean gray level of liver and muscle were calculated using VOCAL 3D-ultrasonography. Statistical analysis of mean gray level of liver and muscle, and relative proportion of gray level of liver and muscle in relation to lipidosis, iron storage, broplasia and hepatitis revealed that calculated proportion of mean gray levels of liver and muscle was signicantly different among groups with different degrees of lipidosis. Further post-hoc analysis revealed that this proportion in the patients with no or slight lipidosis was signicantly greater than the birds which suffered from moderate and severe lipidosis.


Background
Hepatic disorders are important and diverse disease entities in birds [1,2,3]. Clinical signs of avian hepatic diseases are not speci c, so the etiologic diagnosis is challenging [3,4]. Hematological and biochemical laboratory workouts are routinely applied to asses liver damage or hepatic function in birds. Analysis of hepatic enzyme activity is a common diagnostic method for rough evaluation of hepatic diseases. Lack of standard reference ranges in different avian species, variation in individual cases, and questionable test sensitivity and speci city are limiting factors for using these biochemical tests [3,4,5].
In general, plasma activity of liver enzymes or evaluation of bile acids could be only an indication of nonspeci c hepatocyte damages or functional de ciency [1,4,5]. Valuable diagnostic information such as rough size of the liver can be achieved using radiographic evaluation, but it is incapable of diagnosis of the parenchymatous changes and the severity of hepatic lesions [5,6]. Ultrasonography (US) can help us de ning these changes in the liver parenchyma but the exact etiologic diagnosis is often not possible without additional cytological or histopathological examinations [4,5]. Postmortem pathological examination or histological evaluation of liver biopsy specimens are considered as the gold standard for de nitive diagnosis of liver lesions [1,4,5]. Taking biopsy samples from severely ill and moribund patient with impaired liver functions is always risky could be life threatening in small avian patient with possible hepatic coagulopathies [4,5]. Therefore, development of noninvasive clinical tests with strong correlation with histopathological results would be interesting for veterinarians specially avian specialists [7,8].
Hepatic lipidosis or fatty liver has been reported in many avian species as well as mammals and humans and it seems to be one of the most prevalent hepatic metabolic disorder in birds [9,10,11,12,13,14]. Hepatic lipidosis is not a speci c disease entity in birds and many different etiologies like vitamin de ciencies, high energy diets, or even infectious agents could have roles in pathogenesis of fatty liver [9,2,15,16]. While case history, clinical signs such as obesity or non-speci c signs of hepatic involvement like urate discoloration and biliverdinuria and plasma biochemical analysis could be indicative of lipidosis, but histologic evaluation of biopsy specimens is the most promising technique for de nitive diagnosis of hepatic lipidosis [3,4,5]. Repeated resampling of the liver biopsy is sometimes needed to monitor the responses to the treatment [17], and it could further put the case in the danger of biopsy complications.
Avian hepatic parenchyma has the same ultrasonographic (US) appearance as mammalian species (Nordberg et al. 2000). As any diffuse changes in normal echogenicity or echotexture could be indicative of diffuse hepatic disorders, US examination is routinely used for diagnosis of fatty liver in human and other mammals [18,19,20,21]. The application of a new US elastography technique, acoustic radiation force impulse (ARFI) imaging has been shown to be promising in diagnosis of hepatic lipidosis in chicken in an experimental design [22].
Common mynah (Acridotheres tristis) is a tropical and subtropical bird which is endemic in south and southeast of Iran. It has been also introduced into many regions of the world as an invasive species [23].
Common mynah is a popular pet bird in Iran and hepatomegaly seemed to be the second most frequently observed clinical manifestation in this bird after pododermatitis [24]. Postmortem diagnosis of hepatic lipidosis has been reported in common mynah [13], but same as other avian species antemortem diagnosis of lipidosis is challenging.
In current study the application of three-dimensional Virtual Organ Computer-aided Analysis (VOCAL) US in diagnosis of hepatic lesions especially lipidosis in common mynahs is determined and the result of US evaluation was compared with histopathologic study. The feasibility and capability of the quantitative US examination for presumptive diagnosis of hepatic lipidosis is discussed.

Methods
The study design and ethical approval was obtained by Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran (1390).

Cases
Referred common mynahs to the pet bird clinic of the Faculty of Veterinary Medicine, University of Tehran during one year with different complaints or clinical signs like anorexia, hepatomegaly diagnosed clinically by observation of the liver borders over the abdominal skin, urate discoloration and biliverdinuria suggesting the liver involvement, were selected and included in this study. After the participation in the present study, as a part of diagnostic procedure, the patients received suitable treatment and management based on their clinical conditions in the pet bird clinic.

Diagnostic imaging
Radiography was performed in two projections, lateral (L) and ventrodorsal (VD), using KODAK DIRECTVIEW Classic CR System (45 KV, 0.05 s) initially and the images were then evaluated by two nondependent national board-certi ed radiologists with more than two years of experience in avian radiology.
Detectable hepatomegaly based on deteriorated hourglass appearance associated with expansion of liver shadow from coracoacetabular axis in VD projection and expansion of liver shadow from sternum in L view was used as the radiologic criteria for hepatomegaly ( . 2C and 2D).
Subjective and objective arrangement of the cases based on 2D US obtained echogenicities were just matched at the minimum and maximum limits and no signi cant association was detected statistically.

Discussion
Diagnosis of liver diseases in bird is challenging as other animals. Valuable diagnostic information can be gathered using US. In the present study the feasibility and capability of using 3D VOCAL US for etiologic diagnosis of liver disorders in common mynahs were evaluated. Eleven mynahs with clinical and radiographic signs of hepatomegaly were evaluated by US and their liver biopsy samples were histologically investigated. Different minor to major pathologic changes were diagnosed in these birds.
MGR of two different quantitative measurements including MGL MGM was associated with moderate to severe hepatic lipidosis. On the other hand, quantitative 3D VOCAL US evaluation was not capable of diagnosis of iron storage, hepatitis, and broplasia of the liver.
The diagnosis of diffuse changes in hepatic parenchyma is conventionally based on the subjective assessment of liver echogenicity [5,25]. Evaluation of the liver by US is highly operator dependent.
Although quantitative methods for measuring tissue echogenicity have been reported in human, these methods are not yet widely applied in avian practice [26,27]. Display and formation of images in US are related to the tissue characterization, thus objective evaluation is useful to assess any changes consists in differentiating subtle changes in ultrasound speckle patterns, which may remain perceptible to human visual inspection [28]. Overall, the quanti cation is an objective evaluation of these changes and pathological state of the tissue by medical imaging that is helpful in diagnosing the disease. Research has currently focused on suggesting non-invasive methods to diagnose fatty liver as an alternative to liver biopsy. Several methods such as CT, MRI and acoustic radiation force impulse (ARFI) imaging are presented as potential methods for objective evaluation of the liver based on the changes of the tissue microstructures [22,29].
The echogenicity of some parenchymatous organs such as spleen or kidneys are usually used as reference for comparing them to the liver in mammalian species [30]. These reference organs are not easily accessible in avian species due to their special anatomic adaptation [5]. The heart or vessels were suggested in avian patient for comparison [5]. According to its good accessibility, the pectoral muscle mass was used in the present study for this purpose and the result was promising.
The liver biopsy, its histologic evaluation and also microbiological culture are often necessary for de nitive diagnosis of hepatic disorders [4,5,25]. Taking liver biopsy is a painful technique and despite the local anesthesia, the pain was retained in human patient at least 24 hours post procedure [31]. It should be considered that avian patient with hepatic disorders have less tolerance facing with stress and many birds are commonly presented in a poor condition [32]. While no complications were observed in our cases, massive internal hemorrhage could be the other adverse consequence of core needle liver biopsy in birds considering the possible coagulopathy in hepatic diseases [33,34]. The absence of commercial test for routine coagulation determination in avian species further limit the safety of biopsy taking in birds [1,4]. In this situation any alternative non-invasive diagnostic technique could be helpful in severely sick and debilitated avian patients.
As our results showed there was no correlation between brosis and US characteristics of the liver.
Interestingly it was shown that sound attenuation in human liver with connective tissue proliferation was also minimal to undetectable. In agreement with our result, more attenuation has been detected in human cases with domination of fat in the hepatic tissue [35]. In conclusion at least severe lipidosis can be presumptively diagnosed by US relying on different sound interaction in fatty liver [35]. Lu et al. (1997) showed that quantitative US is more sensitive than subjective evaluation in the detection of initial phase of diffuse liver pathologies including the accumulation of fat [36].
Water content of the liver in rats suffered from fatty liver and cirrhosis did not correlate with the sound speed. On the other hand, the nature of hepatic tissue including the fat content and cirrhosis were associated with the sound speed. Echogenicity alteration was obvious in fatty liver, but no signi cant differences were detectable among the different degrees and stages of the lipidosis in affected rats [37].
Furthermore, in another study, the quantitative US has been shown to be a feasible and e cient method for detection and follow up of the hepatic lipidosis and subjective US was a practical replacement for histopathological examination [38]. Therefore, in compliance with our study higher hepatic fat content made the liver brighter in US evaluation, while the normal appearance is the result of collagen and the water content [39].
In agreement with our result in the eld of avian practice, quantitative US was also applied as a noninvasive technique for monitoring of hepatic lipidosis in dairy cattle [40,41]. Digitalized analysis of the liver US images was shown to be a promising noninvasive screening technique for fatty liver detection in dairy herd [27].
Multiple factors including depth related attenuation of the measured area and concurrent brosis and fatty in ltration might in uence quantitative US diagnosis [42].
However, increase in liver echogenicity is expected in several vacuolar diseases; Lu et al. showed that elevation of the attenuation and backscatter coe cients in fatty liver was greater than cirrhotic liver [43].
Regarding the relatively small liver size in passerines, the impact of the chosen ROI could be minimal or even negligible in these birds.
hemosiderosis that is same as results of the present study [44]. Different signal intensity is reported in the different iron content of human liver as compared to the muscle and pigeon's hepatic iron content using magnetic resonance imaging (MRI) [45,46]. Thus, MRI seems to be prior to US for the detection of iron deposition in the liver.
Small sample size and lack of normal group in the present study was considered as limitations. These limitations are unavoidable in the eld of exotic animal research. Thus, further validation and comparison with normal livers to suggest cut-off values are needed in patients and normal population.

Conclusion
Based on qualitative and quantitative evaluation of hepatic echogenicity in the present study, quantitative

Consent for publication
The owners of the included birds were informed about and agreed to participate in this study.

Competing interests
No competing interests