Photomicrographs of Skin from Zinc-Deficient Calves

TE CttNIC'AL NOTES

1285

TABLE 2 Comparison of methods ~ in measuring numbers of bacteria and leucocytes in 20 samples of raw milk Membrane microscopic count

Directmicroscopic count

Sample no.

Standard plate count

1 3 4 7 15

10,000 orfewer 10 3 1.6 3.2 8.4 10,001-50,000

10 24 0 0 10

310 750 1~0 440 270

0 10 0 O 0

450 66.0' 260 380 570

2

50

51

5 10 14 18

4.0 26 13 44 50,001 ormore 160 1,100 270 330 210 64 10'0 170 60 310

44 18 100 160

1,500 1,000 140 1,600 1,800

20 20 18 83 23

1,700 750 50 1,600 1,900

6 8 9 11 12 13 16 17 19 20

Bacteria

Leucocytes

Bacteria

Leucocytes

(thousands/ml)

17

~00

50

850

750

900

340 270 370 78 300 92 150 900

510 1,400 310 580 310 2,000

120 120 10 120 280 96

470 690 670 2,00'0 220 3'80 709 1,900

510

110

530

140

450

140

Bacteria were counted microscopically as clumps; leucocytes as individuals. Count of Microorganisms Directly on Membrane Filters. J. Bacteriol., 70: 265. ]955. (4) LgvowI~z, D. An Inquiry into the Usefulness of the Standard Methods Direct Microscopic Count. J. Milk Food Tcchnol., 20:288. 1957. (5) RICHARD'S, O. W., AND KR4kBEIK, W. B. Visi-

PHOTOMICROGRAPHS

OF

SKIN

A zinc deficiency in calves was e x p e r i m e n t a l l y produced, studied, a n d described i n p r e v i o u s p u b l i c a t i o n s (3, 4). Of the lesions p r o d u c e d in the zinc-deficient calves, the m o s t d i s t i n g u i s h i n g c h a r a c t e r i s t i c was in the skin. This condition, which is called p a r a k e r a t o s i s , is v e r y s i m i l a r i n gross a p p e a r a n c e to h y p e r k e r a t o s i s (1). P a r a keratosis a n d h y p e r k e r a t o s i s can be readily dist i n g u i s h e d b y histological sections of the skin

(L 2). W h e n the d e s c r i p t i o n of t h e zinc deficiency was p u b l i s h e d no p h o t o m i c r o g r a p h s of the affected skin sections were included (3, 4). Since t h a t time requests have been received f r o m p a t h o l o g i s t s a n d others f o r slides or photom i c r o g r a p h s of the sections. These are needed to aid in d e t e r m i n i n g w h e t h e r a n a n i m a l m i g h t be suffering f r o m a zinc deficiency, or p a r a keratosis. Thus, the p u r p o s e of this note is to p u b l i s h p h o t o m i c r o g r a p h s f r o m animals which were m a d e zinc-deficient in the p r e v i o u s s t u d y (3).

bilizing Microorganisms on Membrane Filter Surfaces. J. Bacterio]., 67:613 *. 1954. (6) SttANAt~A~ A. J-., FORNWAL~), R. E., CHAP]~AN, D. M., AND BEACItLEI¥, A. N. Visualizing Bacterial Cells for Direct Microscopic Counting on the Millipore Filter, Hydrosol Type. 3. Bacteriol., 71 : 4,99. 1956.

FROM

ZINC-DEFICIENT

CALVES

Details of the d e v e l o p m e n t of the zinc deficiency h a v e been described p r e v i o u s l y (3). A t necropsy, sections of the skin were t a k e n f r o m the neck, which is one of the most severely affected p a r t s of the animals. A t t h a t time t h e a n i m a l s were 19 wk of age a n d suffering f r o m severe zinc deficiency. The slides were p r e p a r e d as follows: fixed in 1 0 % f o r m a l i n f o r 48 hr, embedded in paraffin, sectioned a t 5 ~, a n d stained w i t h h e m a t o x y l i n a n d eosin in the usual way. The microscopic section of the skin indicated t h a t : there was excessive k e r a t i n f o r m a tion ( F i g u r e 1) ; a l a r g e n u m b e r of nuclei were r e t a i n e d i n the s t r a t u m c o r n e u m ( F i g u r e s 1, 2 ) ; a n d the n u m b e r of cells i n the s t r a t u m g r a n u l o s u m was reduced ( F i g u r e 4). T h e r e was also acanthosis w i t h elongation of the rete n e t (pegs) ( F i g u r e 3). N o n e of these abn o r m a l changes were observed in skin sections f r o m n o r m a l calves. F o r a more complete deseriptiou of' the o t h e r

1286

JOURNAL

OF D A I R Y S C I E N C E

~<

~x

x

~gx~

gg~ •

=

N~N

changes which occur in zinc deficiency, the reader is referred to previous publications (3, 4). The histological condition of the affected calf skin was v e r y similar to that observed in swine with parakeratosis (1).

N

ACKNO~VLEDG~cIENTS

The authors thank Dr. Dennis Sikes for the necropsy work, the histopathological description, preparation of the slides, and diagnosis of the tissue. They are indebted to Dr. S. C. Schmittle for making the photonlicrographs and for advice

T E C H N I C A L NOTES

and assistance in the preparation of the manuscript. W. J. M I L I ~ A~D J. K. MILLER ~ Dairy Department University of Georgia, Athens ~Present address: UT-AEC Agricultural Research Laboratory, Oak Ridge, Tennessee.

1287

REFERENCES

(1) K~R~KaMr, H. C. H., ANn FEaai~z, E. F. Parakeratosis in Swine. J. Am. Vet. Med. Assoc., 123: 217. ]953. (2) LE~R, W. F. Histepathology of the Skin. Lippincott, Philadelphia. 1949. (3) MmL~R, J. K., ANn MmL~R, W. J. Development of Zinc Deficiency in Holstein Calves Fed" a Purified Diet. J. Dairy Sci., 43: 1854. 1960. (4) MILL.~R, J. K., AND MILLFa% W. J. Experimental Zinc Deficiency and Recovery of Calves. J. Nutrition, 76: 4~7. 1962.

RESIDUE' STUDIES WITH 2,4-DICHLOROPHENOXYACETIC ACID HERBICIDE IN THE DAIRY COW AND IN A NATURAL AND ARTIFICIAL RUMEN E'lectron affinity gas chromatography was used to follow the disappearance of 4-(2,4-dichlorophenoxybutyric) acid [4-(2,4-DB)] herbicide in the dairy cow (1). In the work reported, this method has been used to study the fate of 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide in the dairy cow. ~XPERIlVIENTAL PROCIgDURE

A Jersey cow was fed 5 . p p m (based on a daily ratioa of 50 lb) of 2,4-D for five days. The pure herbicide in ethyl alcohol was mixed with the grain. Grain containing alcohol with no herbicide was fed daily to a second-check Jersey cow. Morning and evening samples of milk were taken from each animal, prior to feeding, daily throughout the feeding period, and f o r two days thereafter. Feces samples were also collected daily. Analyses of the milk a n d feces samples were made immediately. The method of analysis consisted of blending 25 g of milk or feces with 70 ml of acetone and 1 ml of 85% phosphoric acid. The solids were filtered off and the filtrate was made to a total volume of 100 ml with acetone. One milliliter of this solution was evaporated in a 10-ml volumetric flask and the contents esterifled with 2 ml of BF3-methanol reagent (2). One milliliter of hexane was added to the flask and it was made to volume with 2% sodium sulfate and mixed. Up to ten td of the resulting hexane solution of the methyl ester of 2,4-D was then ehromatographed. A Barber-Colman Model 10 gas chromatograph was used with a battery-operated 6 cm ~ electron affinity detector "containing 56 t~e of Radium 226. The column was U-shaped, made of 9-ram OD borosliicate glass, and was 6 ft long. I t contained 5% Dow Coming highvacuum silicone grease (the ethyl acetatesoluble fraction) on 80-100-mesh acid-washed Chromosorb W. The operating temperatures for the column, flash heater, and detector were

200, 265, and 235 C, respectively, and nitrogen (60 ec per minute) was the carrier gas. The retention time for 2,4-D methyl ester was about 3 rain. RtSSULTS AND DISCUSSION No 2,4-D was detected in the milk or feces throughout the period of study. An instrument gain setting of 3,000 was used which would have permitted detection of about 0.1 ppm of 2,4-D. Since 4-(2,4-DB) largely disappeared in the rumen within 5 hr (1), it seemed advisable to study this aspect with 2,4-D. To follow its fate in the rumen, 5 ppm (based on a 50-1b daily ration) of 2,4-D mixed in 3 lb of grain was fed to a fistulated Holstein heifer. Eleven samples of manually well-mixed rumen contents were taken over a 23-hr period immediately following the feeding. A sample of rumen contents was taken prior to feeding the grain, to serve as check material. Results of the analysis of these samples are depicted in Figure 1. Figure 2 shows typical chromatograms of rumen contents containing 2,4-D. The recovery of 0.5 ppm of the herbicide added to milk, feces, and rumen contents ranged from 4

"

,

3 B o

22

o

4

HOURS

2'o

24

FIG. l. Disappearance of 2,4-D ill the rumen of a fistulated Holstein heifer.