568 VERMA, SEHGAL, CHAKRA VARTI AND CHHUTTANI

INTESTINAL VILLI IN THE DOG AND THE EFFECT OF

ANK YLOSTOMA CANINUM INFESTATION

S. VERMA,A. K. SEHGAL, R. N. CHAKRAVARTI AND P. N. C H H U ~ A N I

Departments of’Medicine and Experimental Medicine, Postgraduate
Institute of Medical Education and Research, Chandigarh, India

PLATECC
REPORTS concerning the damaging effect of ankylostomiasis on the structure and
function of the intestinal mucosa are conflicting. Several authors like Sheehy
et al. (1962), Salem and Truelove (1964) and Tandon et al. (1966) have demonstrated
mucosal abnormalities and malabsorption in association with hookworm infesta-
tion; others (Abdalla et al.. 1963; Banwell, Hutt and Tunnicliffe, 1964; Chaud-
huri and Saha, 1964; Layrisse and Roche, 1964) failed to confirm these findings.
The dog is commonly infested with the hookworm Ankylostoma caninum and
provides a suitable model to study the effects of the parasite on the host’s intestinal
mucosa. The present study was undertaken to see whether A . caninum caused
any structural changes in the mucosa of the small intestine.

MATERIALS AND METHODS

One hundred stray adult mongrel dogs were procured from the dogcatching
squad of the Municipal Corporation, New Delhi. They were fasted overnight,
anaesthetised by intraperitoneal injection of sodium pentothal and killed by
injection of air into the heart. The small intestine was dissected out immediately
after death and was slit open for inspection of the internal surface. The hookworms
were picked up with forceps and counted. The gut was then washed gently and
at least one piece from duodenum, jejunum and ileum was studied with the dis-
section stereomicroscope. The tissues were fixed in 4 per cent. buffered formal-
dehyde in saline, and parallin sections of blocks from different segments of the gut
of 50 animals were stained with haematoxylin and eosin. To study the site of
penetration, extra blocks were taken containing the hookworm attached to the
mucosa.
TABLEI
Intensity of intestinal worm load

Category 1
i
No. of worms per gut
i NO. of animals

Uninfested . . . 0 7
Mild infestation . 1-5 9
Moderate infestation . . 6-50 22
Severe infestation . . 5 1-250 62

RESULTS
Macroscopic examination
The lumen of the small intestine contained thick, tenacious and often bile-
stained mucus which bore no direct relation to the worm load. The mucosa
was occasionally congested and was smeared with blood wherever hookworms
were present. The parasites appeared as thread-like structures that were either
anchored to the mucosa or moved sluggishly in the mucus. The animals were
grouped into 4 categories on the basis of worm load in the gut (table I).
VERMA,SEHGAL, AND C H H U ~ A N I
CHAKRAVARTI PLATECC

INTESTINAL
VILLI I N ANKYLOSTOMIASIS

FIG. 1-Tongue-shaped duodenal villi. FIG. 2.-Long and flattened villi in the
Dissection microscopy. X 86. transition zone between duodenum and
jejunum. Dissection microscopy. X 86.

FIG. 3.-Long and tubular jejunal villi. F1~.4.-Jejunumshowing micro-haemorrhage

Dissection microscopy. X 86. and inflammatory reaction in submucosa
around head of hookworm. Haematoxylin
and eosin. X 95.
 INTESTINAL VILLI I N ANK YLOSTOMIASIS 569

Dissection microscopy
Uninfested animals. Jn general the duodenum shows tongueshaped or leaf-
like villi (fig. 1) under the dissection microscope. The transition zone between
duodenum and jejunum shows mostly long flattened finger-like villi (fig. 2). Less
frequently these villi have apices wider than the base; tongue-shaped villi are
also seen. The pattern gradually changes into long thin tubular finger-like
villi, which are the predominant pattern in the distal part of the jejunum (fig. 3).
These villi occasionally become club-shaped at the free ends. The transition of
the villi from the jejunum to the ileum is gradual. The distal ileum shows a mixture
of various types of villi though they are mainly relatively short, stubby and finger-
like; short and thick tongue-shaped and leaf-like forms are also present.
The morphological picture is characterised by the marked variation in villus
types and by the uniformity of segmental distribution in every animal. Apart
from the finger, tongue and leaf-like villi, a large number of transitional types
are also present.
Infested animals. There is no difference between the villus pattern in dogs
with mild, moderate or severe infestation and that in uninfested animals.

Histology
Uninfested animals. Usually the villi are covered with columnar epithelial
cells that show basal nuclei and a brush border along the free margin. The
distribution of goblet cells is variable in different parts of the gut. The corium of
the villi contains a moderate number of lymphocytes and mononuclear cells;
eosinophils and plasma cells are seen in a few sections. The mean height of the
villi (table 11) is greater in the duodenum (741 p) than in the jejunum (669 p) and
ileum (602 p). Crypt to villus ratio ranges between 1 to 1-4 and 1 to 1.7.
TABLEI1
Mean values of villus height and crypt length
I I

Segment of intestine Villus height Crypt length

(mean rt standard error) &) (mean & standard error) (p)

Duodenum.
Jejunum .
.
.
. 1 741 &24.7(37)
669&42-7(30)
429+20(33)
403f 17*6(29)
Ileum . . 429116.2(39)

Figures in parentheses denote number of observations, which were unequal because the
villus and crypt size could not be measured accurately in all the sections.

To assess the shape of the villi, more reliance is placed on dissection microscopy
than on histological appearances. This is because the former method provides a
three-dimensional view whereas the findings with the latter method vary with the
plane of the section.
Infested animals. In a number of sections the hookworm penetrates the entire
thickness of the mucosa to reach the submucosa. There is only a mild mucosal
and submucosal inflammatory reaction around the worm, but the neighbouring
villi appear more or less unaffected except for localised pressure distortion.
Occasionally micro-haemorrhages are seen at the point of burrowing, but there
is no evidence of extensive haemorrhage (fig. 4). The mild inflammatory reaction
and the micro-haemorrhages do not extend beyond the area of 2-3 villi around
the worm. These differences are insigniticant in extent when compared with the
whole mucosal area, and there are no other structural abnormalities in the infested
group of animals as compared with the uninfested group.
1. PATH. BACT.-VOL. 95 (1968) 2N2
570 VERMA, SEHGAL, CHAKRA VARTI A N D CHHUTTANI

DWXISSION
Descriptions of the small intestinal villus pattern in the dog are few and brief.
Sisson and Grossman (1953) reported long thin villi in the small intestine of the
dog. Titkemeyer and Calhoun (1955) stated that the average height of the villi
was 645 p ; they may branch and their ends are usually expanded or club-shaped.
The present study has shown that the villi are tongue- or leaf-like in the duodenum
and become finger-like in the jejunum. They then change into stubby finger-like
forms or frequently tongue- or leaf-like forms in the ileum. The longest villi are
seen in the duodenum (741 p) and the shortest in the ileum (602 p). However,
there was little variation in the crypt length in the 3 main parts of the small
intestine.
We have also demonstrated that the mere harbouring of hookworms, irrespec-
tive of the number, does not cause significant structural abnormalities of the small
intestinal mucosa in dogs. Grossly, the villus configuration in infested animals
was similar to that seen in the dogs free from hookworms. Microscopically also,
there was no evidence to suggest villus destruction, atrophy or inflammatory
changes away from the site of penetration by the worm. Occasional micro-
haemorrhages around the site of penetration were of limited extent. The haemor-
rhages seen frequently on naked-eye examination were not due to vascular damage
in the mucosa or submucosa, but were due to blood smeared over the mucosa of
the intestine, perhaps as the result of erythrocytes passed by the worm through
its anus getting smeared on the surface of the mucosa (Leichtensterno, 1886,
1887, cited by Sheehy and Floch, 1964; Wells, 1930-31).
The presence of partial villus atrophy in human ankylostomiasis as reported
by Sheehy et al. (1962), Salem and Truelove (1964) and Tandon et af. (1966) was
not confirmed by us in the dog. Tbe lack of significant morphological changes
in this animal may have been due to development of immunity or to short duration
of infection, both of which were unknown factors in the present study.
An extended study is needed, to assess the intestinal effects of varying degrees
of infestation with A . cuninum over different periods of time in pups devoid of
acquired immunity; the systemic effects of the parasite on the host, such as anaemia
and hypoproteinaemia, should also be investigated.

SUMMARY
Dissection stereomicroscopic examination of the mucosa of the duodenum,
jejunum and ileum of 100 stray mongrel dogs failed to reveal any difference in the
morphology of the villi between the infested and the uninfested animals. Histo-
logical examination of 50 animals showed only a minor inflammatory reaction
at the site of the mucosal penetration by the hookworm. The height, configuration
and villus height:crypt length ratio of the neighbouring villi were normal. Although
the intestinal mucosa was smeared macroscopically with blood in the regions of
parasitic infestation, there was no microscopical evidence of significant vascular
damage or'tissue haemorrhage.
We are grateful to Dr H. K. Chhuttani, Additional Professor of Medicine,
Maulana Azad Medical College, Delhi, for providing some facilities to conduct
this study. Thanks are also due to Mr G. S. Sekhon for preparing the photographs.

REFERENCES
ABDALLA,A., GAD-ELMAWLA,N., 1963. J. Egypt. Med. Assoc., 46,544.
EL-ROOBY,A., SHAKER,M., AND
GALIL,N.
BANWELL,J. G., H m , M. S. R., 1964. E. Afr. Med. J., 41, 46.
AND TUNNICLIFFE,R.
 ADRENAL CORTICOSTEROIDS AND MAST CELLS 571

CHAUDHURI,
R. N., AND SAW,T. K. 1964. Amer. J . Trop. Med. Hyg., 13,
410.
LAYRISSE, M., AND ROCHE,M. . . 1964. Arner. J . Dig. Dis., ns. 9, 776.
SALEM,S . N., AND TRUELOVE, S. C.. 1964. Br. Med. J., 1, 1074.
SHEEHY, T. W., AND FLOCH, M. H.. 1964. The small intestine, New York.
SHEEHY T. W.,, MERONEY,W. H., 1962. Gastroenterology, 42, 148.
COX, R. S., JR, AND S O U R , J. E.
SISSON,S.,AND GROSSMAN, J. D. . 1953. The anatomy of the domestic
animals, 4th ed., Philadelphia
and London, p. 508.
TANDON, B. N., DAS,B. C., SARAYA,1966. Br. Med. J., 1, 714.
A. K.,AND DEO,M. G.
TITKEMEYER, C. W., AND CALHOUN,1955. Amer. J . Vet. Res., 16, 152.
M. LOIS
WELLS,H. S . . . . . .
. , 193&31 J . Parasit., 17, 167.

MAST CELLS 1N RATS GIVEN ADRENAL CORTICOSTEROIDS

W. K. BLENKINSOPP
Department of Pathology, St Mary's Hospital Medicnl School, London

THEaction of glucocorticoids on mast cells has been variously reported to be

no change, degranulation with an apparent decrease in the number of cells, de-
granulation with a real decrease in the number of cells or an increase in the
number of cells (Selye, 1965~). Most workers have concluded that rnineralo-
corticoids have no effect on mast cells (Selye, 19656), but Csaba, Tor0 and Bodoky
(1963) reported a decrease in number after administration of deoxycorticosterone.
In the present work, the effect of prednisolone or aldosterone on the number
and rate of proliferation of mast cells was studied in the tongue in rats.

MATERIAL AND METHODS

Dosage of corticosteroids. The normal output of glucocorticoid in the rat has

been estimated to be equivalent to less than 200 pg. cortisone per 100 g . rat per
day (Blenkinsopp and Blenkinsopp, 1967). A dose. of 300 pg. prednisolone
(equivalent to 1500 pg. cortisone) per 100 g . rat per day was chosen for one group
of rats. Singer (1959-60) found a mean rate of secretion of aldosterone of 0.0415 pg.
per adrenal per 100 g. rat per hr in anaesthetised, surgically stressed rats, which
is 2.0 pg. per 100 g. rat per day. A dose of 5 pg. aldosterone per 100 g. rat per
day was chosen for another group of rats.
Mast cell proliferation. Continuous intraperitoneal infusion of tritiated (3H-)
thymidine (Foot, 1963) was used to label cells synthesising deoxyribonucleic acid,
so that all cells formed during the period of infusion were labelled. The proportion
of mast cell nuclei labelled was ascertained and used to compare the rate of pro-
liferation of the mast cells in the various groups of rats.
3H-thymidine (specific activity 5.0 c per mM) was infused intraperitoneally
into each of 8 adult male inbred black-hooded PVG/C rats maintained on a normal
41B cubed diet and weighing 180-210 g., for 3 days at a dose rate of 1 pc per g.
body weight per day. Four controls (no. 1-4) were not given steroid. Two rats
(no. 5 and 6 ) were given a daily intraperitoneal injection of 600 pg. prednisolone
for 6 days before 3H-thymidine infusion, then 600 pg. prednisolone daily for 3