USEFULNESS OF FINE NEEDLE ASPIRATION IN THE DIAGNOSIS OF LIVER DISEASES IN CATS

Sylwia Kajdasz, Krisztina Kungl, Agnieszka Kurosad
Department of Internal and Parasitic Diseases with Clinic for Horses, Dogs and Cats, University of Agriculture in Wrocław, Poland

ABSTRACT

20 cats of both gender, different age and breed were investigated. Animals which had clinical symptoms like: anorexia, rapid weight loss and jaundice were included in the clinical group. Additionally, hepatomegaly was confirmed in all cats by palpation. The following hematological and blood serum laboratory tests were conducted: WBC, differential white blood cells count, Ht, Hb, RBC, MCHC, MCV, serum concentrations of urea, creatinine, bilirubin, glucose and fructozamine, enzyme activities of Lipase, AlAT, AspAT, GGT, AP and electrolyte concentrations: Na⁺, K⁺, Cl^-. In animals designated blood clotting tests were performed: APTT, PT, TZ, thrombocytes and clotting time.

In the results distinct elevation of AlAT, GGT and AP activity and bilirubin concentration in serum were observed. Ultrasonographic (USG) evaluation revealed generalized hyperechocity of the liver. The above results together with clinical observations led to an initial diagnosis of primary liver disease in these animals. For specific diagnosis Fine Needle Aspiration (FNA) of the liver was performed. Histological evaluation of aspirated hepatic tissue revealed 10 cases of hepatic lipidosis, 2 cases of lymphoma and 2 cases of cholangiohepatitis. In 6 cases a definite and final diagnosis could not be reached, since either normal cytological structure of the liver or cellular atypia were found.

FNA was found to be an effective (70%), safe and easy to perform diagnostic method in liver diseases, particularly in cases of primary generalized liver disease in cats.

Key words: feline, liver, hepatic diseases, fine needle aspiration.

INTRODUCTION

Fine Needle Aspiration – FNA (Fine Needle Biopsy – FNB) is one of the best methods to obtain material for histological assessment of liver tissue [11, 14, 19, 31, 34, 42]. The significance of FNA is especially emphasized in cases of generalized pathological processes of the liver i.e. infiltrating neoplasms, poststeroidal hepatopathies, amyloidosis and fatty degeneration of the liver [4, 8, 35, 44]. Since the introduction of imaging techniques such as: Ultrasonography (USG), Computer Tomography (CTG) and Magnetic Resonance Imaging (MRI), in veterinary clinical diagnosis, the role of FNA with cytological diagnosis of focal changes in the liver parenchyma has increased [26, 28, 31, 32, 42, 43]. Currently, this is reflected in the possibility to analyze cytological biopsy specimens collected from confined changes having a diameter of 0,5 cm [12].

FNA can be performed using a normal injection needle: length 2.5-8.0 cm and diameter not exceeding 1.0 mm. The device for creating negative pressure consists of a 10 cm³ sterile syringe and a special handle, which facilitates the creation of negative pressure [6, 11, 17, 20, 29, 34]. Recently there are special biopsy “pistols” available, which allow the standardization of the volume of collected material. These pistols not only shorten the time of biopsy but also limit the appearance of post biopsy complications [29, 31].

The objective of the study was to determine the usefulness of Fine Needle Aspiration in the diagnosis of liver diseases in cats.

MATERIALS AND METHODS

A group of 20 cats of both gender and different breeds at the age of 4 to 6 years were studied. Initial clinical examination was performed according to generally accepted principles. Patients included in the experiment showed clinical symptoms such as: apathy, anorexia (lasting from 3 to 10 days) and vomiting. Additionally jaundice was observed in 13 cats. The average weight of the cats in the experiment was 4.5±1.05 kg.

In the hematological tests the following parameters were determined: red blood cell count (RBC), haemoglobin concentration (Hb), haematocrit (Ht) value, red blood cells indicators: mean corpuscular haemoglobin concentration (MCHC), mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV) as well as white blood cell count (WBC) and differential white blood cell count (leukogram). In serum the following biochemistry parameters were determined: urea, creatinine, bilirubin, bile acids, total protein, albumin, glucose, fructozamine and electrolyte (Na⁺, K⁺, Ca⁺⁺, Cl^-) concentrations and alanine aminotransferase (AlAT), asparagine aminotransferase (AspAT), alkaline phosphatase (AP), γ-glutamylotransferase (GGT) and lipase activities. Hematology and serum biochemistry analysis was performed using Cell-DynŽ 3500R automatic analyzer manufactured by ABBOTT. In order to eliminate from the study animals suspected of suffering from other than hepatic disturbances only animals having urea, creatinine, fructozamine concentrations and lipase activity within reference values were included. In these animals ultrasonographic (USG) evaluation of the abdomen was performed using EUB-525 CFA (HITACHI) ultrasonograph and sector transducer with alternating frequency of 5.0-10 MHz. Such biochemical-hematological matched profiles together with clinical symptoms and USG imaging confirmed the necessity of performing FNA of the liver.

In order to minimize the risk of post-biopsy hemorrhage the following blood clotting tests were carried out: prothrombine time (PT), thromboplastin time (TT) – Quick test, activated partial thromboplastin time (APTT), clotting time using modified Lee-White method and thrombocyte count (method of direct count). In all cats blood clotting parameters were within reference values that allowed us to perform FNA.

In four cats FNA was preceded by 12 hours of fasting, all other animals were anorectic. When necessary (in 2 cases) the animal was pre-medicated using atropine (Atropinum sulphuricum) 0.05 mg/kg body weight followed by xylazyne (Rompun®) 0.1-1 mg/kg body weight intramuscularly. In order to perform FNA cats were laid in a supine position with the chest being higher than the abdomen causing caudal displacement of the liver and making it easier to palpate the enlarged organ. After positioning the animal, the site of insertion of the needle i.e. the area of the abdomen localized near the xiphoid process, spreading between the costal arches was shaved. This area was disinfected 3 times using iodine preparation, whereas the third portion of iodine was left on the skin to dry. For the manual method of aspiration biopsy in this study needles of 0.8-0.9 mm (21-22 G) diameter and 40 mm length, Luer’s type syringes of 10 cm³ were used and also volume and vacuum controlled syringes which were part of thick liver biopsy kit Hepafix® (manufactured by BRAUN) placed in a special handle (Fig.1). The needle was inserted at an angle between 40-60° in relation to the body surface in cranio-dextral direction (Fig.2) under USG guidance (EUB-525 CFA; HITACHI). The area between the linea alba and the left costal arch, 1 cm caudally from the xiphoid process was chosen as the site of puncture. Liver tissue was aspirated from 2 to 5 times creating vacuum by pulling the piston to a volume of 3-6 cm³. After aspiration, the piston was released and the biopsy was completed. Immediately after biopsy a smear was performed on a glass slide, and fixed by drying at room temperature without using forced air circulation or commercial fixing agents containing alcohol. Afterwards the tissue specimens were sent to laboratory to obtain cytological interpretation. The total time from performing liver biopsy to cytological analysis was 3 hours. For staining smears, the following quick staining cytological methods were used: modified Wright – Giemsy method (WG) (Fig.3), Hematoxiline – Eozyne method (HE) and May Gruenwald – Giemsy method (MGG) (Fig. 4).

Fig. 1. Standard Luer type needle and syringe in a special handle for vacuum control installed for fine needle biopsy of the liver

Fig. 2. Biopsy site: the area of the xiphoid process spreading between the costal arches; insertion of needle at an angle between 40-60° in relation to body surface in cranio-dextral direction

Fig. 3. Hepatocytes showing fatty degeneration; stained by Wright – Giemsa method (W-G); magnification 400x

Fig. 4. Hepatocytes showing fatty degeneration; stained by MayGrunwald - Giemsa method (MG-G); magnification 500x

The specimens were viewed using Panavision AX70 microscope, manufactured by Olympus, equipped with Olympus camera type PM – C35 DX for electronic – standardized documentation. Analysis was performed using the following magnifications: 100x, 200x, 400x, 500x and 1000x.

RESULTS

The clinical symptoms in all cats were not pathognomic. In all animals apathy, vomiting, weight loss were observed. 16 of the cats consumed small amounts of cat food, however, the intervals between meals exceeded 24 hours. The mean fasting time in cats from the clinical group lasted 6 days. Jaundice was observed in 13 cats (75%), initially in the mucus membrane of the oral cavity and conjunctiva, later in the unpigmented skin (especially around the pinna). In two cases jaundice was not seen at first but occurred during the course of therapy. Dehydration, constipation and diarrhea was observed in 35%, 30% and 10% of cats, respectively. In the cases of diarrhea faeces was dark brown with a tinge of light yellow mucus. In clinical examination hepatomegaly of different grade was diagnosed in all cats. However, only in one case pain-reaction was observed during palpation of the abdomen in the xiphoid area.

Haematological evaluation confirmed dehydratation in 6 cats (30%) i.e. elevated Ht, Hb, RBC, electrolytes. Biochemical laboratory tests revealed marked elevation of AlAT activity in all animals compared to reference values. Increase in GGT, AP and AspAT activity levels was less marked and was stated in 70%, 65% and 35% cats in the clinical group, respectively. Concentration of bilirubine exceeded reference values in 65% cats. No other deviation of eleveted parameters from reference values observed.

In all cats the ultrasonographic scan showed generalized hyperechogenic structure of the liver (Fig.5), as compared to USG picture of the spleen or renal cortex.

Fig. 5. Ultrasonographic scan of the liver - generalized hyperechogenic structure of liver parenchyma (H) in comparison with spleen echostructure (L)

FNB of liver was conducted in all 20 cats. In 15 cases guided biopsy under the control of USG, whereas in 5 cats blind biopsy with USG scan follow up for eventual post biopsy complications was performed.

Cytological analysis revealed feline hepatic lipidosis (FHL) (Fig. 3, 4), lymphoma and cholangiohepatitis in 10, 2 and 2 cases, respecively. In 3 cats, no abnormalities in cytological examination were stated. In 3 cases both unchanged and dysplastic hepatocytes and also cellular atypia were detected, which did not allow to establish an unambiguous diagnosis of liver disease.

The only post-biopsy complication, observed during the experiment, was unsubstantial subcapsular haemorrhage with subsequent haematoma in one cat. However, it was resolved within 72 houres after biopsy according to follow-up USG examination.

Fig. 6. Macroscopic picture of fatty degeneration of the liver

In summary, performance of FNA of liver-tissue allowed to reach final diagnosis in 70% of the cases included in the experiment. After conducting the study, the animals underwent therapy for the diagnosed diseases. Euthanasia, on request of the owner, was performed in the cases of confirmed lymphoma. In one cat with confirmed cytological diagnosis of FHL death occurred on the fifth day of therapy. Postmortem examination revealed significant hepatomegaly, caused by fatty degeneration of the liver (Fig. 6).

DISCUSSION

The most common clinical symptoms, which may indicate hepatic parenchyma degeneration are anorexia, apathy, vomiting, diarrhea, polyuria, polydipsia, jaundice, coagulopathy, weight loss, dehydration, discolouring of faeces and anemia [3, 4, 13, 16, 21, 37, 40, 41].

Clinical diagnosis of feline hepatic diseases is relatively difficult, because of the substantial functional reserve of the liver. Often pathological signs appear when the function of 70 – 80% of hepatic parenchyma is compromised [37]. Additionally, symptoms of liver hypofunction can be masked by secondary disorders of other inner organs. Reverse situations are observed as well, when other primary diseases lead to secondary hepatopathy. Infectious diseases e.g. Feline Infectious Peritonitis (FIP) and Feline Leukemia Virus (FeLV) infection may also cause secondary liver disfunction and thus should be ruled out before completing the diagnosis of hepatic diseases [4, 19, 23, 24, 37, 41].

Routhuzien et al [37] ascertain that the above mentioned clinical signs, appear in multiple diseases of different pathogenesis, and thus additional diagnostic methods are essential to confirm primary hepatic disfunction.

Basic diagnostic methods (hematology, biochemical blood parameters, USG) used in the identification of hepatic and bile tract diseases are auxiliary in nature and should be interpreted in connection with anamnesis and clinical examiantion.

The clinical symptoms present, the elevated activity of liver enzymes as well as USG scan excluding focal changes constituted the prerequisites for performing FNA. The application of this diagnostic method had the purpose of specifying the changes taking place in the liver parenchyma.

Several authors point out specific indications for performing FNA of the liver. According to Day et al [11] liver biopsy may be beneficial for diagnosis when elevated concentrations of bile acids in serum, jaundice, generalized hiperechogenicity of the liver parenchyma, hepatomegaly of unknown origin, local hiperdensity and presence of multifocal abnormalities in hepatic parenchyma are observed. FNA is also indicated to determine progress of changes or as follow up examination during or after therapy. In the authors opinion that elevated activity of hepatic enzymes without clinical symptoms of disease or parenchyma lesions detected in USG scan should not be an indication to perform FNA of the liver, since transient changes in the activity of these enzymes may be present without a concurrent primary disease. However, if such elevation of transaminaze activity lasts for over 2 months FNA should be conducted.

Lefkowitch [24] proposes the introduction and use of FNA in the diagnosis of systemic disorders, such as sarcoidosis, amyloidosis and glycogen storage diseases. He concerns FNA also as the recommended method of choice in obtaining liver tissue samples for microbiological and immunocytochemical evaluation. According to Buckley et al [6], as well as Lefkowitch [24] another indication for performing FNA of the liver is prolonged fever of unknown origin.

Performance of FNA is essential to reach final diagnosis, particularly in cats with suspected hepatic lipidosis. However, Willard [42] considers that the cytologic pictures require careful interpretation, since many specific signs of FHL may occur in other hepatic diseases e.g. infiltrating neoplasms of the liver.

There are several methods of FNA proposed by different authors. Baker and Lumsden [2] suggest performing FNA from lateral insertion of the biopsy needle, while the animal is standing or lying down on its right side. When suspecting the presence of diffuse lesions in the hepatic parenchyma, they insert the biopsy needle in the 12 or 13 intercostal space on the left side of the animal. Stockhaus and Tesce [38], also suggest performing FNA on the animal`s left side when the animal is lying in abdominal position, but in the 11 intercostal space. However, Crowe et al [10] propose performing FNA on the right side of the animal between the xiphoid process and the right costal arch when the animal is lying in a supine position or on its left side. They also describe a transthoracal liver biopsy performed in half height of the rib cage in the 7 or 8 intercostal space.

Itercostal insertions of biopsy needle are usually very painful for most cats. In those cases premedication and local anaesthesia are required. In some situations such type of liver biopsy can be also life – threatening. Since the risk of lung injury and pneumothorax are very serious [5, 6, 15, 18, 25, 36]. Such technique may be prefered in wedge biopsy [9, 10].

Sometimes, fine needle biopsy without aspiration is performed in cases of very vascularized organs, like liver or thyroid gland [29, 30, 31]. However Menard et al. showed another method of liver biopsy, connecting the fine needle aspiration with fine needle biopsy without aspiration. It was named “technique 5-6-5” [29].

The choice of biopsy technique performed in this study was dictated by the anatomical structure of the liver of the cat. Placing the animal in a supine position with the chest higher than the abdomen and inserting the needle on the left side between the costal arch and the xiphoid process makes it possible to collect samples of tissue from the left lobe of the liver, which is the biggest and thickest lobe in the cat [1, 22]. At the same time relatively fewer bile ducts are found in this lobe in relation to cell mass. In this biopsy position the gall bladder is displaced to the right side which decreases the risk of puncture, particularly when it is filled up with bile [16]. For these reasons, this method of biopsy also described by Griffin [16] was selected as the most effective and minimizing the possibility of post-biopsy complications.

There are a few complications that may occur after conducting liver biopsy: trauma of liver parenchyma, puncture of gall bladder or bile ducts, haematoma and spreading infection within the line of biopsy [18, 27, 33, 39]. The results obtained in this study are consistent with others [18, 20, 31, 36, 39] that FNA as an additional diagnostic method in liver diseases is burdened with a relatively small risk of complications. Hanson et al [18] studied almost 2100 cases of fine needle aspiration, performed in dogs and cats and confirmed that the most frequent post-biopsy complication was local haemorrhage of low extent. They also showed that the most dangerous post-biopsy complications appeared rather in wedge liver biopsy [7, 20, 27, 33, 39].

CONCLUSION

Fine needle aspiration is an effective and safe method used in differential diagnosis of hepatic diseases, especially with unspecific clinical symptoms and generalized hiperechogenicity of the liver.

REFERENCES

1. Anderson W.D., Anderson B.G.: Comparative Anatomy. Veterinary gastroenterology. Lea and Febirger, Philadelphia. 1980, 127-171.

2. Baker R., Lumsden J.H.: The Gastrointestinal Tract. Colour Atlas of Cytology of The Dog and Cat. Mosby , St Louis. 2000, 177–197.

3. Barsanti J.A., Jones B.D., Spano J.S.: Prolonged anorexia associated with hepatic lipidosis in three cats. Feline Pract. 1997, 7, 52-57.

4. Blaxter A.: Diagnosis and Mangement of Hepatic Disorders. Feline Practice. London. 1991, 17-138.

5. Bravo A., Sunil G., Sheth M., Sanjiv C.: Liver Biopsy. Am. N. Engl. J. Med. 2001, 344, 495-500.

6. Buckley A., Petrunia D: Practice guadelines of Liver Biopsy. Canadian J of Gastroentero. 2000, 6 (14), 481-482.

7. Caterelli E., Giacobbe A., Facciorusso D.: Percutaneous biopsy in diffuse liver disease: increasing diagnostic yield and decreasing complication rate by routine ultrasound assesment of puncture site. J. Gasteroentero. 1996, 91, 1318-1321.

8. Center S.A.: Diagnostic procedures for Evaluation of Hepatic Disease. Small Animal Gastroenterology. W.B. Saunders comp., Philadelphia. 1996, 130–188.

9. Cole T.L., Center S.A., Flood S.N., Rowland P.H., Valentine B.A., Warner K.I.: Diagnostic comparision of needle and wedge biopsy speciments of the liver in dogs and cats. Am. J. Vet. Med Assoc. 2002, 15, 1483-1490.

10. Crow S.E., Walshaw S.O.: Visceral core biopsies (closed techniques). Manual of Clinical proceduers in the Dog, Cat and Rabbit. Lippincott – Raven, Philadelphia. 1997, 231–248.

11. Day G.D.: Indications and techniques for liver biopsy. Textbook of Veterinary Internal Medicine. W.B. Saunders comp., Philadelphia. 2000, vol 2., 1294–1298

12. De Nicola: Diagnostic cytology, collection techniques and sample handing. Proc 4^th ann Mtg ACVIM. 1997, 15–25.

13. Dimski D.S., Taboada J.: Feline idiopathic hepatic lipidosis. Vet Clin North Am Small Anim Pract. 1995, 25 (2), 357–373.

14. Fondacoro J.V., Guilpin V.O., Powers B.E: Diagnostic correlation of liver aspiration cytology with histopathology in dogs and cats with liver disease. Proc 17 th Annu Vet Med Forum, Chicago: American College of Veterinary Internal Medicine. 1999, 707-719.

15. Grant A., Neuberger J.: Guidelines on the use of liver biopsy in clinical practice. International J Gastroentero Hepato. 1999, 45, 1-24.

16. Griffin B.: Feline hepatic Lipidosis: Pathophysiology, Clinical Signs and Diagnosis. Am Small Anim Exo 2000, 9, 847–857

17. Gonciarz Z., Mazur W.: Wštroba. Gastroenterologia i hepatologia kliniczna. PZWL, Warszawa. 2001, 540–678.

18. Hanson J.A., Papageorges M., Girard E., Menard M., Herbet P.: Complication of the ultrasound – guided fine needle biopsy technique: 2100 cases. Vet. Radiol and Ultrasound. 1995, 36, 137–138.

19. Hauptman K., Tichy F., Knotek Z: Clinical diagnosis of Hepatopathies in Small Mammals: Evaluation of Importance of Individual Methods. Acta Vet Brno. 2001, 70, 297–311.

20. Hoernauf A.: Leberbiopsie: Indication, Technik, Risken. Pract Tierarzt. 1995, 223–226.

21. Hubbard B.S., Vulgamott J.C.: Feline hepatic lipidosis. Am Compend Cont Ed Pract. 1992, 14, 459–465.

22. Koening H.E, Libich H.G.: Anatomie der Haussaeugetiere. Schattauer, Stuttgard. 1999, 128-134.

23. Kraft W.: Leber. Kleintierkrankheiten, Eugen, Ulmer Stuttgart. 2000, 564–598.

24. Lefkowitch J.H.: Pathologic Diagnosis of Liver Disease. Hepathology: Textbook of Liver disease. W.B. Saunders comp., Philadelphia. 1990, 711-732.

25. Lightdale C.J., Das L.: Difficult liver biopsies: Only for radiologists? Am. J. Gastroentero. 1997, 92, 364-365.

26. Lohss E.: Abdominelle Sonographie beim Kleintier. Teil I. Tieraerztliche Praxis. 1988, 16, 423–426.

27. Mc Closkey R.V., Gold M., Weser E.: Bacteremia after liver biopsy. Arch Intern Med. 1973, 132, 213–215.

28. Menard M., Papageorges M.: Ultrasound corner: technique for ultrasound – guided fine needle biopsies. Vet radiol Ultrasound. 1994, 36, 137-138.

29. Menard M., Papageorges M.: Fine Needle Biopsy Technique, Veterinary diagnostic Imaging and Cytopathology (VDIC). Cytopathology Newsletter 2002.

30. Menard M., Papageorges M.: Fine – needle biopsies. How to increase diagnostic yield. Comp Cont Ed Pract Vet. 1997, 19, 738-740.

31. Menard M., Papageorges M.: Ultrasound – guided liver fine needle biopsies in cats: result of 307 cases. Vet Pathol 1996, 33, 570.

32. Miles K.: Imaging abdominal masses. Vet Clin Norht Am Small Anim Pract. 1997, 27, 1403–1431.

33. Piccininio F., Sagnelli E., Pasquale G.: Complications following percutaneous liver biopsy. J Hepatol. 1986, 2, 165–173.

34. Pinto M.M., Avilla N.A., Heller C.I., Criscuolo E.M.: Fine needle aspiration of the liver. Acta cytol. 1988, 32, 15-21.

35. Rebar A.H.: Diagnostic cytology as an aid to the evaluation of hepatic disease. AAHA 56^th Annual meeting proceedings St Louis. 1989, 5, 169–171.

36. Reddy K.R., Schiff E.R.: Complications of liver biopsy. Gastrointestinal emegencies. Wiliams and Wilkins, Baltimore. 1997, 959-968.

37. Rothuizen J., Meyer H.P: History, physical examination and signs of liver disease. Textbook of Veterinary Internal Medicine. W.B. Saunders comp., Philadelphia. 2000, 1272–1277.

38. Stockhaus Ch., Tesce E.: Klinische Anwendbarkeit der Leberzytologie bei Hund und Katze. Kleintierprax. 1997, 42, 687–775.

39. Stotland B.R., Lichtenstein G.R.: Liver biopsy complications and routine ultrasound. Am. J. Gastroentero. 1996, 91, 1295-1296.

40. Thornburg L.P., Simpson S., Digilio K: Fatty liver syndrom in cats. Am J Am Anim Hosp Assoc. 1982, 18, 397–400.

41. Weiss D.j., Armstrong P.J., Gagne J.: Inflammatory liver disease. Am Small Anim.1997, 12, 22–27.

42. Willard M.D., Weeks B.R., Johnson M.: Fine – needle aspirate cytology suggesting hepatic lipidosis in four cats with infiltrative hepatic disease. Am J Feline Med Surg. 1999, 1 (4), 215–220.

43. Yeager A.E.: Accuracy of ultrasonography in the detection of severe heaptic lipidosis in cats. Am J Vet Res. 1992, 53, 597-599.

44. Zawie D.A., Garvey M.S.: Feline hepatic disease. Am Vet Clin north Am Small Anim Pract. 1984, 14 (6), 1201-1230.

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Sylwia Kajdasz
Department of Internal and Parasitic Diseases with Clinic for Horses, Dogs and Cats,
University of Agriculture in Wrocław, Poland
pl. Grunwaldzki 47, 50-366 Wrocław, Poland
phone: +48 71 3205377
fax: +48 71 3205360

Krisztina Kungl
Department of Internal and Parasitic Diseases with Clinic for Horses, Dogs and Cats,
University of Agriculture in Wrocław, Poland
pl. Grunwaldzki 47, 50-366 Wrocław, Poland
phone: +48 71 3205377
fax: +48 71 3205360
email: szunyog2@ozi.ar.wroc.pl

Agnieszka Kurosad
Department of Internal and Parasitic Diseases with Clinic for Horses, Dogs and Cats,
University of Agriculture in Wrocław, Poland
pl. Grunwaldzki 47, 50-366 Wrocław, Poland
phone: +48 71 3205377
fax: +48 71 3205360

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