Investigations of the Pathology and Chemotherapy

of Infectious Enterohepatitis (Blackhead) in T u r k e y s

Dr. László P E L L É R D Y

Veterinary Research Institute of the Hungarian Academy of Sciences, Budapest

I n 1967 considerable economic lo s s e s , of the order o f sev e r a l m i l l i o n F o r i n t s were caused by i n f e c t i o u s e n t e r o h e p a t i t i s i n t u r ­key f l o c k s . This was p a r t l y due t o inadequate knowledge of e f ­f e c t i v e c o n t r o l measures, but above a l l t o l a c k of an a f f i c i e n t s p e c i f i c drug such as has been w i d e l y a p p l i e d I n other c o u n t r i e s where turkeys are bred on a l a r g e scale. During the f i r s t h a l f of 1967 the sole a v a i l a b l e chemotherapeutic i n t h i s country was f u ­razolidone ( T i k o f u r a n ) which has been found i n our country t o have only weak antiblackhead a c t i o n .

During the second h a l f o f the year a l a r g e amount o f 2-amino-5-n i t r o t h i a z o l e , w idely used against blackhead, was imported. A f i e l d t r i a l of t h i s drug i n the form of the premix H e p a m i x * which contains i t a t a c o n c e n t r a t i o n of 10 was c a r r i e d out i n ne a r l y 300.000 tu r k e y s and provided abundant m a t e r i a l f o r i n ­v e s t i g a t i o n . This communication comprises a review of r e l e v a n t observations which both c o n f i r m and ''complement the known f a c t s about blackhead.

1. ) G r o s s l e s i o n s i n t h e l i v e r . I n most textbooks the s i t e s o f gross l e s i o n s of blackhead are given as the caeca, caecal w a l l s , peritoneum a d j o i n i n g the caeca, and l i v ­er. I n p r a c t i c e , however, e x c l u s i v e l y caecal l e s i o n s have o f t e n

* „Phylaxia" State Serum I n s t i t u t e , Budapest

been found on post-mortem examination, l i v e r l e s i o n s being e i t h e r absent or recognized only w i t h d i f f i c u l t y . I t i s , therefore, not p a r t i c u l a r l y s u r p r i s i n g t h a t because of t h e i r s i m i l a r i t y to caecal c o c c i d i o s i s of chickens, the caecal lesions have frequently been confused w i t h c o c c i d i o s i s .

F i g . 1. Turkey l i v e r w i t h lesions c h a r a c t e r i s t i c of blackhead from the m a t e r i a l designated „Tiszaroff". This l i v e r i s about 1/3 o r i g i n a l s i z e . Jellegzetes blackheades elváltozásokat feltüntető pulyka­máj a „Tiszaroff" elnevezésű anyagból. Az er e d e t i nagy­ságnak kb. az egyharamda.

The m a t e r i a l designated „ T i s z a r o f f " provides a good example of the i n e x p l i c a b l e a l t e r n a t i n g incidence of lesions I n ­v o l v i n g e i t h e r the caeca alone or both caeca and liver.The turkey f l o c k of a c o l l e c t i v e farm, t o t a l l i n g approximately 15,000 b i r d s , was housed i n two premises several kilometres apart. Conditions of management amd feeding were s i m i l a r i n the two places and me­d i c a t i o n w i t h T i k o f u r a n against blackhead was c a r r i e d out t o an i d e n t i c a l schedule.The outbreak of the disease occurred simultaneously i n both premises w i t h comparable morbi d i t y . Yet

necropsy m a t e r i a l from one place showed,in every c a s e , d i s t i n c t i v e l e s i o n s of both caeca and l i v e r , w i t h t y p i c a l c o n c e n t r i c h e p a t i c f o c i , whereas i n m a t e r i a l from the other farm c h a r a c t e r i s t i c caecal l e s i o n s , enlargement of the l i v e r w i t h i n d i s t i n c t d i s c o l o ­r a t i o n s , but no f o c a l l e s i o n s were found.

Several t r a n s i t i o n a l forms were found t o e x i s t between h e p a t i c changes t h a t macroscopically were scarcely d i s c e r n i b l e and the f o c a l l e s i o n s considered pathognomic of blackhead. Enlargement of the l i v e r was a t y p i c a l f i n d i n g i n turkeys t h a t died or were k i l l e d a t the h e i g h t of i n f e c t i o n . I n s u r v i v o r s r e g r e s s i o n of the l i v e r t o normal siz e took place over several weeks. On the enlarged l i v e r the l e a s t conspicuous l e s i o n s were p o o r l y de­marcated d i s c o l o r a t i o n s o f v a r y i n g s i z e , p a l e r i n c o l o u r than normal l i v e r t i s s u e , and seen only a t h i g h i l l u m i n a t i o n ( i n sun­shine) a f t e r the l i v e r surface had been smoothed.

I n other - supposedly more chronic - cases the considerably en­l a r g e d l i v e r showed round f o c i of c o i n size s l i g h t l y depressed beneath the surface of the organ, y e l l o w i s h or greenish i n c o l o u r and of c o n c e n t r i c p a t t e r n ( E i g . l ) . I n some cases these f o c i pe­n e t r a t e d deeply i n t o the parenchyma. Such l e s i o n s are not r e a d i l y confused w i t h those due t o other diseases.

The t h i r d type of l e s i o n was encountered i n the l i v e r s of t u r k e y s t h a t had recovered from blackhead, the c o n c e n t r i c f o c i described above having been replaced by c i c a t r i c i a l t i s s u e .

Regeneration of the n e c r o t i c areas by c i c a t r i z a t i o n produced sharply contoiired d i s c o l o u r e d spots on the surface of the l i v e r . The c i c a t r i c i a l t i s s u e was p a l e r than the surrounding h e p a t i c t i s s u e , y e t the more advanced the process of r e g e n e r a t i o n the l e s s marked was the d i f f e r e n c e . Since, however, r e g e n e r a t i o n of l i v e r c e l l s was incomplete, .the l a r g e r areas of scar t i s s u e imparted a spotted appearance t o the l i v e r throughout the l i f e t i m e of the b i r d ,

2 . ) M i c r o s c o p i c l e s i o n s . H i s t o l o g i c a l examina­t i o n s were performed on a t o t a l of 50 t u r k e y l i v e r s , p a r t l y t o

i d e n t i f y by morphological and s t a i n i n g p r o p e r t i e s the animal, (Histomonas) or p l a n t (Candida), o r i g i n of the e t i o l o g i c a l agent, and p a r t l y t o examine the h i s t o p a t h o l o g y o f l i v e r s w i t h v a r i o u s l e s i o n s . For t h i s p u r p o s e , p a r a f f i n embedded s e c t i o n s were s t a i n e d w i t h hematoxylin and eosin, M a l l o r y ' s s t a i n and p e r i o d i c a c i d -S c h i f f (PAS).

The i n i t i a l hypothesis assumed t h a t the two m o r p h o l o g i c a l l y d i s t i n c t l e s i o n s were due t o two d i f f e r e n t e t i o l o g i c a l agents of blackhead. This assumption was supported by f i n d i n g s i n the l i v e r ( d e s i g n a t i o n „Boldogasszonyfa") o f a t u r k e y which on postmortem examination showed t y p i c a l caecal l e s i o n s , but only enlargement and d i f f u s e d i s c o l o r a t i o n of the l i v e r . Microscopic examination of t h i s l i v e r revealed l a r g e numbers of conspicuous round homo­genous bodies ( P i g s . 2a, 2b), 7-lO^tc i n diameter, s t a i n i n g pale p i n k w i t h e o s i n , v i v i d r ed w i t h M a l l o r y ' s s t a i n and deep l i l a c w i t h PAS. The p a r a s i t e s , c l u s t e r e d densely t o g e t h e r , occupied the spaces between the rows of l i v e r c e l l s , sometimes g i v i n g the im­pr e s s i o n of being i n t r a c e l l u l a r i n l o c a t i o n . A s i m i l a r i n t r a c e l ­l u l a r p o s i t i o n was p r e v i o u s l y described by CHESTER and ROBIN (1900) although l a t e r TYZZER (1920) advocated the i n t e r c e l l u l a r l o c a t i o n o f the p a r a s i t e s . I n the p a r t i c u l a r case being described only a few p a r a s i t e s o t h e r than those mentioned above were found; they also s t a i n e d pale pink but showed an i n t e r n a l s t r u c t u r e .

P i g . 2a. Causative agents o f blackhead i n a s e c t i o n from t u r k e y l i v e r , a: The round s t r u c t u r e l e s s bodies resemble f u n g i . Hematoxylin and eosin. X 300 Blackhead-kóroközók pulyka májában, a: A kerek,struktúra nélküli képletek gombára emlékeztetnek. Hem-eozin 300 X

Pi g . 2b. Mag n i f i e d p a r t o f F i g . 2. PAS. X 1000 Uaz, mint 2a. PAS. 1000 X

F i g . 3a. Causative agents of blackhead i n t u r k e y l i v e r , a: Bodies resembling t o Histomonas. Hematoxylin and eosin. X 300 Blackhead-kóroközók pulyka májában, a: histomonasnak meg­felelő képletek. Hem-eozin. 3OO X

Pig. 3b. Magnified p a r t o f F i g . 3a. Hematoxylin and eosin. x 800 7az, mint 3a. Hem-eozin. 800 X

T h e i r shape was g e n e r a l l y round, hut o f t e n ovoid or ameboid.

I n l a t e r s t u d i e s on both types of l i v e r l e s i o n one or other pa­r a s i t e was noted w i t h o u t apparent r e l a t i o n s h i p t o the type of l e s i o n .

The macroscopically d i s t i n g u i s h a b l e l i v e r l e s i o n s showed no cor­responding v a r i a t i o n of microscopic s t r u c t u r e . I n the l i v e r pa­renchyma n e c r o t i c f o c i w i t h k a r y o l y s i s and subacute inflammation of the a d j o i n i n g t i s s u e were found. The f o c i d i d not become en­capsulated by f i b r o u s t i s s u e ; o n l y a s l i g h t increase of connective t i s s u e being noted i n inflammatory areas around the blood vessels and b i l i a r y ducts. Besides i n f i l t r a t i n g lymphocytes and mono­nucle a r c e l l s these inflamed areas contained many e o s i n o p h i l i c and pseudoeosinophilic c e l l s . Granulocytes and numerous g i a n t c e l l s were also found. I n the a f f e c t e d areas the lumina of blood vessels and sinusoids were distended w i t h red blood c e l l s ( F i g . 5 ) .

I n the present examinations the p a r a s i t e s considered to be the e t i o l o g i c a l agent of blackhead were of two m o r p h o l o g i c a l l y d i s t i n c t t y p e s . They occurred e i t h e r s i n g l y or i n groups ( n e s t s ) , most of them, i n t ere v - l l u l a r l y . One type,a round s t r u c t u r e l e s s body of 7-11 s* diameter, s t a i n e d v i v i d r e d w i t h M a l l o r y ' s s t a i n . S t a i n i n g p o s i t i v e l y w i t h PAS, i t was very l i k e l y a fungus. The other type, s t a i n i n g pale p i n k w i t h eosine, appeared as s i n g l e or m u l t i p e r o u n d i s h bodies surrounded by a c l e a r r i n g (Pigs.3a,3b, 4 ) . M a l l o r y ' s s t a i n imparted t o them an u n c e r t a i n g r e y i s h hue. They were l a r g e r than the f u n g u s - l i k e organisms,-of s l i g h t l y ovoid shape and some had diameters above 40 Occasionally they were seen i n an apparently i n t r a c e l l u l a r p o s i t i o n , a d j a c e n t t o the enlarged and f l a t t e n e d nucleus of the host c e l l . Some of the pa­r a s i t e s showed one or more t i n y b r i g h t ruby-red granules, sur­rounded by a c l e a r halo-, s i t u a t e d close to the nucleus. The occurrence of such granules seemed t o be associated w i t h d i v i s i o n of the p a r a s i t e . Single granules were u s u a l l y seen close t o the nucleus, whereas when s e v e r a l granules were present they were d i s t r i b u t e d i n the cytoplasm,sometines w i t h obvious signs of c e l l d i v i s i o n around them. I n l a r g e r p a r a s i t e s the c l e a r halo was mere

d i s t i n c t and a t h i n , f i l a m e n t was seen t o connect i t s border w i t h the body of the p a r a s i t e . The morphological appearance and p a r t i ­c u l a r l y t he f i l a m e n t suggested the p a r a s i t e t o be a Histomonas m e l e a g r i d i s

I n healed l e s i o n s the l i v e r c e l l s were arranged more densely and t h e i r n u c l e i s t a i n e d darker w i t h hematoxylin than d i d h e a l t h y l i v e r t i s s u e ( P i g . 6 ) . I n a d d i t i o n , a few r e s i d u a l granulocytes and s t r u c t u r e s resembling the p a r a s i t e s were encountered i n p a l e r areas where the l i v e r t rabeculae had been replaced by d i s o r d e r l y aggregates o f l i v e r c e l l s .

Thus h i s t o l o g i c a l examination f a i l e d t o provide evidence as t o the p r e c i s e nature of the e t i o l o g i c a l agent o f blackhead. The PAS-positive s t r u c t u r e l e s s organisms were probably f u n g i , whereas the pale p i n k s t r u c t u r e d organisms c l o s e l y resembled H. m e l e a g r i - d l s . I t seems f e a s i b l e , t h e r e f o r e , t h a t both organisms and, ac­cor d i n g t o more recent f i n d i n g s , even c e r t a i n b a c t e r i a , p l a y a r o l e i n the causation o f blackhead (TAIL and PRAKKER, 1963).

3 - ) A c t i o n o f H e p a m i x . Mixed i n the d i e t o f t u r k e y f l o c k s s e v e r e l y a f f e c t e d w i t h blackhead (10-20 $> l o s s e s ) , 1 $> H e p a m i x feed a d d i t i v e stopped the outbreak i n 5 days. l a t e r deaths were mostly due t o i n t e r c u r r e n t diseases.

Given i n the i n i t i a l stage o f the outbreak, treatment w i t h H e-p a m I X minimized l o s s e s . N a t u r a l l y , e a r l y medication r e q u i r e s e a r l y c o r r e c t d i a g n o s i s . The experienced v e t e r i n a r i a n , and even t e c h n i c a l a s s i s t a n t s can recognize blackhead by the c l i n i c a l symtomps p r i o r t o occurrence o f l o s s e s . Diseased b i r d s lose

Pi g . 4. Histomonas-like p a r a s i t e s - i n the l i v e r o f a t u r k e y a f ­f e c t e d w i t h blackhead. Adjacent t o the nucleus r u b i n g r a ­nules are apparent. Oilimmersion. X 1500 Histomonas-szerü élősködők blackheades pulyka májában. A mag m e l l e t t a rubinszemcsék i s feltűnnek. Immerziós f e l ­vétel. 1500 X

a p p e t i t e , are sleepy, l a g behind the r e s t and have diarrhoea, the y e l l o w i s h pasty droppings of s i c k t u r k e y s beiny immediately n o t i c e d . I n such circumstances blackhead i s e a s i l y diagnosed by postmortem examination of the most a i l i n g b i r d s

Delayed treatment f o r blackhead I s o f t e n due t o erroneous diagno­s i s . Considerable d i s t e n t i o n of the ceaca of a f f e c t e d b i r d s and c a s e o u s - f i b r i n o u s plugs i n the caecal lumina s u p e r f i c i a l l y r e ­semble l e s i o n s encountered i n the caecal c o c c i d i o s i s of chickens. I n f a c t , l i k e most domesticated animals, the t u r k e y has several c o c c i d i a n p a r a s i t e s . Yet oocysts are always demonstrable i n the droppings, o f t e n even i n the pasty s t o o l seen d u r i n g the e a r l y phase of blackhead. Although the presence of oocysts apparently supports the diagnosis of caecal c o c c i d i o s i s no t u r k e y coccidium i s known t o cause such extensive caecal l e s i o n s .

Good durable t h e r a p e u t i c r e s u l t s can be obtained i f 1 $> H e p a ­m i x i s given i n the d i e t f o r 7-10 days. Subsequently treatment must not be stopped but continued a t h a l f the dose r a t e f o r a t l e a s t as l o n g a t i m e . As i n d i c a t e d i n the l i t e r a t u r e , the disease i s l i a b l e t o r e c u r even then, i n d i c a t i n g t h a t the immune response to blackhead i s very low ( f o r ref.see BIESTER and SCHWARTE,1965).

4 . ) P r o p h y l a x i s o f b l a c k h e a d . I n the spread of blackhead the most important r o l e has been a t t r i b u t e d t o the eggs of H e t e r a k i s g a l l i n a r u m . The e t i o l o g i c a l agents of blackhead are c a r r i e d e i t h e r by these eggs, or by the l a r v a e hatched from them, through the w a l l of the caecum and invade the host. There­f o r e , t u r k e y s should be housed and managed so t h a t they remain f r e e of i n f e c t i o n w i t h Heterakis eggs. I n t h i s r e s p e c t , Heterakis eggs shed w i t h chicken droppings seem t o be even more dangerous than those excreted by the t u r k e y s themselves. I t i s known t h a t blackhead also occurs i n g a l l i n a c e o u s b i r d s , b u t a f f e c t s them only s l i g h t l y or net a t all» Nevertheless,the Heterakis eggs shed w i t h the droppings o f g a l l i n a c e o u s b i r d s may i n f e c t r e l a t i v e l y l a r g e areas f o r l o n g periods of time.

The f i r s t and most important r u l e o f blackhead p r o p h y l a x i s i s t o prevent a l l c o n t a c t of the t u r k e y s w i t h Heterakis ova. These pa-

r a s i t e egga may remain v i a b l e i n the s o i l f o r aa l o n g as 2 years although the ovum-transmitted agent o f blackhead d e t e r i o r a t e s i n a few months. Therefore, care should be taken t o place the t u r k e y f l o c k i n „virgin" premises, where n e i t h e r t u r k e y s nor chickena have been kept f o r the preceding two years, or a t l e a s t one. Ex­perience w i t h m a t e r i a l designated „Orosháza" emphasise the impor­tance o f t h i s p a r t i c u l a r requirement. W i t h i n a giv e n area, con­t a i n i n g farms not f a r d i s t a n t from each ot h e r , blackhead occurred o n l y where chickens and tu r k e y s had been kept d u r i n g the preced­i n g year. Farms where t h i s was not the case remained f r e e of blackhead.

Another method o f p r e v e n t i n g blackhead i s t o r e a r t u r k e y p o u l t s up t o 3-4 weeks' age i n i s o l a t i o n on a deep l i t t e r n ot i n f e c t e d w i t h H e t e r a k i s egga.

I f subsequent exposure i s i n e v i t a b l e , losses can be minimized by s t a r t i n g p r e v e n t i v e medication a t the proper t i m e .

S u m m a r y

A f i e l d t r i a l o f the feed a d d i t i v e H e p.a m 1 x, c o n t a i n i n g 2-a m i n o - 5 - n i t r o t h i a z o l e a t a c o n c e n t r a t i o n o f 10 was c a r r i e d out

Ei g . 5. Microscopic l e s i o n s i n the l i v e r o f a t u r k e y a f f e c t e d w i t h blackhead. Inflammatory f o c i c o n t a i n i n g d e t r i t u s , granu­l o c y t e s and f u n g u s - l i k e p a r a s i t e s . Hematoxylin and eosin. X 150 Kórszövettani elváltozások blackheades pulyka májában. Gyulladásos gócok a májban, törmelékkel, granulocytákkal és gombaszerü élősködőkkel. Hem-eozin. 150 X

Ei g . 6. Microscopic p i c t u r e of l i v e r i n the convalescent stage a f t e r blackhead. Note h e a l t h y l i v e r i n the c e n t r e . Hema­t o x y l i n and eos i n . X 150 A blackhead gyógyuló esete, a középen az érintetlen máj­szövet. Hem-eozin. 150 X

on n e a r l y 300,000 t u r k e y s . D e t a i l e d examination o f gross and mic­r o s c o p i c l e s i o n s o f the l i v e r i n d i c a t e t h a t i n the e t i o l o g y of blackhead both presumed causative agents,viz. Histomonas melea g r i - d i s and Candida a l b i c a n s , are i m p l i c a t e d . H e p a m i x proved t o be very o f f i c i e n t a g a i n s t blackhead; when mixed i n the d i e t a t a c o n c e n t r a t i o n of 1 i t stopped the outbreak i n 3-5 days. P r a c t i c a l experience suggests t h a t planned p r e v e n t i o n of b l a c k ­head remains t o be e s t a b l i s h e d .

P E L L É R D Y , L . : Patológiás és terápiás vizsgálatok

fertőző enterohepatitisben beteg (blackheades)

pulykaállom ányokban

A blackhead Magyarországon 1967-ben több millió Pt kárt o k o z o t t . Ennek az v o l t az oka, hogy egyrészt nem rendelkeztünk kellő t a ­p a s z t a l a t t a l a betegségnek időben történő felismeréséhez, más­részt pedig nem v o l t b i r t o k u n k b a n blackhead e l l e n b i z t o s hatású gyógyszer. Amikor 2 - a m i n o - 5 - n i t r o t h i a z o l - t nagyobb mennyiségben importáltunk és azt a P h y l a x i a H e p a m i x nevű takarmány-gyógykeverékben elkészítette,alkalmam n y i l o t t kórbonctani és kór­szövettani megfigyelések végzésére.

A kórbonctani elváltozások számos esetben csak a vakbelekben v o l ­t a k feltűnők, azonban a máj mindig megnagyobbodott v o l t és azon erős megvilágítással (napon) a máj lesimitása után f o l t o s szinkü-lönbséget l e h e t e t t találni az egyes területek között. A black-headre t i p i k u s májelváltozásokon ( 1 . kép) kivül a gyógyuló ese­tekben a májénál világosabb szinü gócos elváltozásokat találtam, amelyek mindenek s z e r i n t regenerálódó hegszcvetnek f e l e l t e k meg. Ezt. a szövettani vizsgálatok i s igazolták. Jóllehet a blackhead két feltételezett kórokozójának,a Histomonas meieagridisnek és a Candida albicansnak a -májból való kitenyésztése csak néhány eset­ben sikerült, a szövettani vizsgálatok alapján a r r a az eredményre k e l l e t t jutnom, hogy a blackhead előidézésében a C. albicansnak (2a, 2b. kép) és a H. meieagridisnek (3a, 3b, 4.kép) egyaránt sze­repe l e h e t . A betegség okozójának megállapítására további vizsgá­l a t o k vannak folyamatban.

A H e p a m i x kitűnő hatásn gyógykeverék. Közel 300 ,000 p u l y ­kára kiterjedő megfigyelésem megerősítette,hogy a H e p a m i x -nak 1 ̂ -ban több napig történő etetése után a blackhead 3-5 napon belül megszűnik. A b i z t o s s i k e r a l a p j a a gyógyszer alkalmazásánál a mielőbbi és helyes diagnózis. Pulykában a vakbél-elváltozások nem téveszthetők össze a csirkék vakbélcoccidiosisa során észlel­hető elváltozásokkal, de egyébként sincs a pulykának olyan c o c c i -diuma, amely az Eimeria t e n e l l a - h o z hasonló patogén hatású volna. A blackhead e l l e n i védekezés és a gyógyszer s i k e r e s alkalmazásá­nak előfeltétele az, hogy kellő időben avatkozzunk be és hogy a tervszerű megelőzésre nagy súlyt helyezzünk. Közismert tény, hogy a blackheadet a vakbélférgek (He t e r a k i s g a l l i n a r u m ) t e r j e s z t i k o l y módon, hogy petéiket a pulykák f e l s z e d i k . A Heterakis-ok f e r -főzött pulykából származhatnak, de a tyúkban élő Heterakis-ok i s a blackhead közvetitŐi lehetnek. Fontos tehát, hogy az állatokat b i z t o s a n Heterakis-tól mentes környezetben neveljük.

R e f e r e n c e s

CHESTER,P.D. - ROBIKBA.: E n t e r o - h e p a t i t i s or blackhead of f o w l s . Rep.Delaware A g r i c . Exp. Sta. 6 6 - 7 0 . 1 9 0 0 .

BIESTER, H.E. - SCHWARTE, H.L.: Diseases of p o u l t r y . 5 t h ed» lova State Univ. Press. 1 9 6 5 . (p. 1105-1107)

TAIL, J.P. - FRANKER, C.K.: Experimental Histomoniasis i n gnoto-b i o t i c t u r k e y s . I . I n f e c t i o n and h i s t o p a t h o l o g y of the bacte­r i a - f r e e h o s t . J. P a r a s i t . 4 9 . 4 1 1 - 4 1 4 . 1963 .

TYZZER,E.E.: The f l a g e l l a t e character and r e c l a s s i f i c a t i o n of the p a r a s i t e producing „blackhead" i n turkeys-Histomonas (gen.noy.) m e l e a g r i d i s ( S m i t h ) . J , P a r a s i t . 6 . 1 2 4 - 1 3 1 . 1 9 2 0 .

Received: 23 .5 .196 -8 . Dr. L. PEL1ÉRDY V e t e r i n a r y Research I n s t i t u t e of the Hungo Acad, o f Sei. Budapest XIV. Hungária k r t . 2 1 .