- Email: [email protected]
Comparative pathology in experimental infection of pigs and calves with larvae of Angiostrongylus cantonensis
J. Chap.
PATH.
1968.
VOL.
78.
371
COMPARATIVE PATHOLOGY IN EXPERIMENTAL INFECTION OF PIGS AND CALVES WITH LARVAE ANGlOSTRONGYLUS CANTONENSIS*
OF
BY K. JINDRAK
and J. E. ALICATA
Pamsi&logy Lubaratoy, Dqkzrtment of Animal Sciem, Univmi& of Hawaii, Honahlu, Hawaii INTRODUCTION
During the last few years, the rat lungworm, Angfostrong$us cuntonensis, has received much attention because it has been suspected as the most probable cause of human eosinophilic meningitis in the Pacific islands and Southeast Asia. This theory proposed by Alicata (1961, 1962) has been supported by the findings of this parasite in man in the cerebrospinal fluid (Nomura and Lin, 1945), in the brain (Rosen, Chappell, Laqueur, Wallace and Weinstein, 1962; Jindrak and Alicata, 1965 ; Tangchai, Nye and Beaver, 1967), and in the anterior chamber of the eye (Prommindaroj, Leelawongs and Pradatsundarasar, 1962; Ketsuwan and Pradatsundarasar, 1966). Recent epidemiological and parasitological investigations in various areas of the tropical belt (Ahcata, 1962, 1963, 1965a; Lii, Ow-Yang and Lie, 1962; Alicata and McCarthy, 1964 ; Kuntz and Myers, 1964; Punyagupta, 1965) have confirmed the presence of this parasite in places where eosinophilic meningitis has been reported. Alicata (1963, 1965a, 1965b) and Alicata and Brown (1962) found several carrier hosts of the infective larvae of this parasite as possible sources of human infection, including land planarians, freshwater prawns, crabs, cattle and pigs. Among these carrier hosts, pigs and calves occupy an important place since in preliminary observations (Alicata, 1965b) third-stage larvae were found in the visceral organs of these animals several days after the infection. The present study was carried out to obtain more information on the presence and survival of third-stage larvae of A. cantonensis in various organs of cattle and pigs and to determine the pathological changes which occurred in them. The results are useful in (a) identifying similar changes which may be found in cattle and pigs in endemic areas, and (b) obtaining a better understanding of the pathology in man, who is also an abnormal host. MATERIALS
AND
METHODS
Animals. Six pigs and 5 calves were used. The pigs were reared on a concrete floor at the University of Hawaii farm and received dry feed. The calves were obtained from a dairy farm and kept in a small dry paddock under strict sanitary conditions. All the animals were l-month-old when placed on the experiment. Larvae. The infective third-stage larvae of A. cantonensis were obtained by artificial * This investigation Diseases and Blindnes-9, Agricultural Experiment
WBS supported by grant No. NB-CM965 from NIH, USPHS. Published with the approval Station as Technical Paper No. 937.
the Institute of the Director
of Neurological of the Hawaii
372
INFECTION
OF
PIGS
AND
CALVES
WITH
A.
CU?ltOnt%SiS
digestion of experimentally infected giant African snails, Achatina fulic~. The soft parts of the snail were. minced in a small meat grinder, digested in a solution of 1 per cent. pepsin and 1 per cent HCl for 2 hours at 37OC. after which the suspension was strained through a fine metal mesh into a sedimentation cone and allowed to stand for 2 hours. The number of larvae in the sediment was estimated by the usual dilution counting method. The larvae were administered with the aid of a dose syringe. The calves received approximately 70,000 larvae each and 5 of the 6 pigs received 20,000 larvae each. The sixth pig was left uninfected and served as a control. No control calf was available. One pig and one calf were killed at 3, 7, 14, 28 and 56 days after infection. The control pig was killed on the 14th day of the experiment. Post-mortem examination. At necropsy, the stomach wall, lung, liver, heart, spleen, kidney, brain, spinal cord, diaphragm, tongue and masseter muscle were examined from each animal for larvae by mincing 15 g. of each tissue and standing in a Baermann apparatus for 2 hours. In addition, brain and other tissue from each animal were examined microscopically as press preparations. The pleural and peritoneal cavities were washed with saline solution and the sediment of the washings examined with a dissecting microscope for Angiostrongylus larvae. Histology. Tissue blocks for histological examination were taken from the following organs : liver, kidney, lungs, heart, spleen, pancreas, adrenals, thymus, thyroid gland, submaxillary salivary gland, gonads, diaphragm, intercostal muscles,massetermuscle, musclesfrom the right thigh, the eye bulb with the optic nerve and oculomotor muscles, stomach (in calves from each part of it), small and large intestines with corresponding mesenteric lymph nodes, gastrosplenic lymph nodes, urinary bladder, mediastinal lymph nodeswith parts of the trachea, oesophagus and aorta, omentum, parietal pleura from the cupula pleurae. Blocks were taken from other organs whenever necessary. The brain and the spinal cord, after removal of portions required for parasitological examination, were preserved. The tissues were fixed in Bouin solution, embedded in paraffin and sectioned at 8 microns. From the musclesof pigs 1 and 2 and calves 1, 2 and 3, several hundred sectionswere prepared and sthed with Harris’ haematoxylin and eosin. The P.A.S. technique, Gomori’s method for precollagen fibres, Van Gieson’s technique for collagen fibres, and Fast-Blue Luxol stain for myelin were used in individual sectionsas appropriate. RESULTS
Clinical
Observations
None of the pigs showed any neurological symptoms, whereas calves 2, 3 and 4 showed an uncertain gait on the day of slaughter, which was 1, 2 and 4 weeks, respectively, following infection.
Distribution
of Larvae
Details of the larvae found in the organs of pigs and calves are summarized in Table 1. Undeveloped third-stage larvae were found in the stomach wall, liver, spleen, and peritoneal cavity of pig 1 and the liver of pig 2. Similar larvae were found in the stomach wall, liver, lungs, heart, kidney, mesenteric and mediastinal lymph nodes, peritoneal and thoracic cavity of calf 1, the peritoneal cavity of calf 2, and the lung-sof calves 2, 3 and 4 (Fig. 1). In most instances, these larvae appeared normal, except that about one-half of those in the lungs of the calves showed large vacuoles. White rats fed larvae recovered from the stomach of pig 1, and from the lungs and the peritoneal and thoracic cavities of calf 1, all showed developing A. cantonensis in their brains 20 days after infection. None
*
and muscle
cavity cavity
nodes
8 z x
2: 4:B 0 8
:t. 1-P
8
16-B0 4-w 0
7
3
Pig 2
8
8 0
8
8
l!P
I4
Pig 3
RECOVERJZD
FROY
I
8
8
::
0”
8 0
:
8
8
56
Pig 5
OF ORGANS
1-i 0”
0 8-B**
ii 0
25P,
‘ilW
z
3-B 3-B 40-B* 26-W 125-w 2-B 76-B 3-B
7
Calf 2
3
calf1
EXAMINED
without further development W = Washings P = Press preparation
:It
x
28
Pig 4
PORTIONS
TABLE
* = Larvae isolated from a mediastinal lymph node l = Advanced third- or fourth-stage larvae; all others, third-stage larvae t = Calcified larva in wall of the cavity B = Baermann extraction
LunsS Heart Spleen Kidney Diaphragm masseter Brain
Peritoneal peyic ‘V
Stomach walllymph Mesenteric
Days of infection
Pig 1
LARVAE
2-B:*
: 0
8 10-B
0
8
8 0
-i 4-B
0
8
28
I4 0 0
calf4
calf3
-ii
8
8 0 0
0
E
56
Calf5
2:
W
A
Liver
Brain
A= A = B = C = D = E = F = G = H = I =
ABCDEG +++
Mediastinal lymph nodes
Lungs
ABCDEG if
Mesenteric lymph nodes
DEG ++
G ++
+
---
GI i-
E ++
ACDEG +++
A
A
EG
Pig 2 7
---
+
-I-E+
AEG +++
GI
AEG ++
G ++
AEG iA
OF THE
Pig 3 I4
Larvae (several of them morphologically intact) Larvae (disintegrating) Oedema Haemorrhage Tissue destruction (larval tracks. necrosis) Infiltrations contaihing many edsinophili Newly formed lymphatic follicles Granulomas (with or without larvae) Fibrosis, scar Mononuclear-cell infiltrations
-
GI i-
CDE +
DEG ++
A
Intestines
EG +
A
Pig 1 3
Stomach
Days of infection
SUMMARY
+
AEFG +++-I-
GHI +
+
+
+
-
EH
F
F
---
F
-t
TABLE
-t
---
---
-
--
EFH ++
FH -t
FH +
---
2
I
A
ABC ii++ C
ABCDI ++++
ABCDI
B
f
:
+
I
-t
-
3
I
+E
i-f++
I +
2
++
a i. = Significant
I
3
EG +++
lesions
AC
AC GHI ++++
+
ACDEG ++++
EG +
+
E
---
Calf14
lesions of increasing
or minimal
changes:
ACE
ABC GI ++++
- = No lesion % = Insignificant 4-l
J
f
*
EG +
I
I
Calf 7
ABCDEG ++++
A
FINDINGS
Degree of pathological
-
---
calf
HISTOLOGICAL
Pig 5 56
IMPORTANT
Pig 4 28
MOST
F FG ++
f
:
-
severity
AE G +++
F + -1
HI + A
A
---
calf428
H
---
---
f
+ I +
-
FH
FH *
I
-
5
+
calf56
K. JINDtiK
AND
J. E. ALXATA
375
of the rats, however, which received from 4 to 15 larvae from the lungs of calves 2,3 and 4 showed parasites in the brain. Dead and partially calcified larvae were found in press preparations from small cyst-lie nodules on the surface of the liver, on the ligamentum pulmonale (Fig. 2) and on the omentum (Fig. 9) of pig 2. In addition, similar isolated nodules containing calcified larvae were noted on the capsule of a mesenteric lymph node of pig 3 and on the peritoneal surface of the diaphragm of pig 4. The larvae found in the brain tissue of calf 2 were still in the third stage, but showed increased growth. One measured 578 microns long and another was 826 microns long. From the brain of calf 3, two fourth-stage larvae were isolated. They measured 1,32 1 and 1,65 1 microns in length and 50 to 54 microns in width, respectively (Fig. 3). Pathological
Observations
The most important histological findings are summarized in Table 2. No pathological changes were observed in pig 6. Stomach and intestines. Rust-coloured sulxrosal and intramucosal foci were found in the stomach of pigs 1, 2, 3 and 4 (3, 7, 14 and 28 days after infection, respectively), on the muccsa of the ileum of pig 1 and in the rectum and colon of pigs 2 and 3, calves 1, 2 and 3. In pig 1, there was an ulcer, 3 mm. in diameter, in the mucosa on the greater curvature. Microscopically, in pigs 1,2 and 3, focal infiltrations of eosinophils, thrombosis of blood vessels, and small granulomas consisting of epithelioid cells and eosinaphils were present in the submucosa. Solitary foreign-body giant cells were found inside the submucous lymphatics. Larval tracks, surrounded by epithelioid cells and eosinophils and f&xl with cellular debris, were scattered in the Peyer’s plaques. The granulomas contained morphologically intact larvae in pig 1 and fragments of larvae in pigs 2 and 3 (Fig. 4). Only focal lymphocytic infiltrations and lymphatic follicles were found in the gastric submucosa of pig 4. In calves, the microscopical changes were restricted to the intestines, predominantly to the jejunum, and no larvae were found. The intestinal villi in calf 1 showed dilation of the axial lymphatics and infiltration with lymphocytes. In calf 2, the inflammatory cells permeated the whole thickness of the mucosa. In calf 3, many eosinophils appeared in the infiltrations. In calves 4 and 5, the infiltration was less marked and consisted predominantly of lymphocytes. Lymph nodes and lymphatic vessels. Striking differences related to the number of larvae and lesions were noted between the lymph nodes of the pigs and those of calves. In general, the lesions in the mesenteric lymph nodes were scarce and considerable differences in the degree of the involvement of individual nodes were noted. A single mediastmal node usually contained more larvae and showed more pathological lesions than any one of the mesenteric nodes of the same animal. In each of two animals, one pig and one calf, killed on the same day after infection, the mesenteric nodes of the pig contained many more larvae than the corresponding nodes of the calf. A reverse relationship applied to the mediastinal nodes : those of the pig contained significantly fewer larvae than ,those of the calf.
376
INFECTION
OF PIGS
AND
CALVES
WITH
A.
cantOnenSiS
The invasion of the lymph nodes by the larvae was marked by congestion, oedema and haemorrhage. Several nodes of the calves showed areas of haemorrhagic necrosis. The larvae were located in the a&rent lymphatics, subcapsular sinuses and in the capsules. In the lymphatic tissues, many larval tracks were filled with cellular debris and fibrinous exudate, and larvae were found. The inflammatory infiltration in the form of a perilymphadenitis was especially marked on the afferent and efferent lymphatics. As the infection progressed, the larvae were surrounded by granulomas containing foreign-body giant cells, epithelioid cells, lymphocytes and eosinophils; the larvae eventually distintegrated and disappeared. Eventually the inflammatory reaction subsided and finally only focal accumulations of lymphocytes and lymphatic follicles were found in places where in earlier stages larvae and granulomas were present. In pigs, many larvae together with giant cells were present in the sinuses rimming the trabeculae of the lymph nodes (Fig. 5). The trabeculae were oedematous and infiltrated with eosinophils; their lymphatics were partially thrombosed and showed small tears in their walls. Many eosinophils were present in the inflammatory infiltrations as early as 3 days after infection (pig 1); in addition, disintegration and calcification of some of the larvae and the formation of granulomas with epithelioid cells was evident at the same time. In pig 4, at 24 days after infection, only accumulations of histocytes in the subcapsular sinuses were noted; larvae, eosinophils and granulomas were not found. In calves, the larvae were especially numerous in the subcapsular sinuses of the mediastinal lymph nodes and in their afferent lymphatics which were dilated and partially thrombosed (Fig. 6). The eosinophilic and granulomatous reactions were first seen in calf 2 at 7 days after the infection, and were most pronounced in calf 3 at 14 days after infection. In the latter, some of the larvae were disintegrated and surrounded by foreign-body giant cells, eosinophils, plasmocytes and lymphocytes (Figs. 7, 8). In calf 4 at 28 days after infection, the granulomas simulated lymphatic follicles with fragmented or apparently morphologically intact larvae in their centres. No larvae were found in these follicles in calf 5 at 56 days after infection. In all the animals, small foci of inflammatory infiltration or granulomas were scattered through the mediastinal loose connective tissue and even in other mediastinal organs such as the oesophagus. These lesions corresponded in appearance with those in the lymph nodes. In calf 2, a granuloma with a larva was seen in the perineurium of the vagus nerve. With the exception of pig 1, varying numbers of small nodules, 0.5 to 3 mm. in diameter, were scattered in the visceral and parietal peritoneum and pleura, in the retroperitoneal and mediastinal connective tissue, and often along arteries. This distribution is summarized in Table 3. Some of the nodules were colourleq small, thin-walled cysts filled with a clear fluid (Fig. 9) while others were pink or red. In a few of them, live or calcified larvae were found. They originated from the lymphatics. All stages were observed microscopically, from a simple partially thrombosed lymphangiectasis, to dilated lymphatics containing a loose meshwork of fibroblasts populated by lymphocytes, and finally the assumption of the shape of a small lymph node. In general, the mesenteric and mediastinal lymphatics were more severely injured in calves than in pigs.
K. JINDRiiK
AND
377
J. E. ALICATA
;
c
I
8
Y hl
9 m
1
I
I
I
I I I
I I I
I I I
I , I
Y’: mm
I I I
1 I
9 -
I I I
I I
I I I
I I I
I I I
0 24
0 e;
I I I
I I I
ij
I I I
x &
378
INFECTION
OF PIGS
AND
CALVES
WITH
A. cantonensis
Liver. No gross lesions occurred in the liver of the calves. Histologically, a few small, focal infiltrations of mononuclear cells, mainly lymphocytes, were disseminated in the liver parenchyma irregularly in calves 1, 2, 3 and 4. In 4, some of the foci were transformed into minute scars. No pathological lesions were found in the liver of calf 5. In pigs, disseminated white spots from 2 to 7 mm. in diameter were present under the capsule and on the cut surface. Only small numbers were seen in pigs 1, 2, 4 and 5, but were more numerous in pig 3, in which they were confluent in places (Fig. 10). The lobular pattern of the hepatic parenchyma in the spots was distinct. In pigs 4 and 5, the foci were firm with a rigid prominent white nodule in their centre. Microscopically, a few larvae in the porto-bilary spaces (Fig. 11) and several small foci of necrosis and haemorrhage were found in the hepatic parenchyma of pig 1. Small groups of mononuclear cells and eosinophils were present at the periphery of hepatic lobules in pigs 1, 2 and 3. In the areas of the macroscopically visible white spots, the interlobular septa were oedematous, dilated, and infiltrated with mononuclear cells and eosinophils. In pig 3, the inflammatory cells had penetrated a considerable distance inside the hepatic lobules where the parenchymatous cells had undergone atrophy (Fig. 12). In pigs 4 and 5, the inflammatory reaction subsided, but thickened fibrous interlobular septa remained (Fig. 13). In some of the persisting white foci stellate scars with calcified centres and sometimes with conglomerates of lymphatic follicles were found (Fig. 14). Lungs. Several foci of consolidation in the diaphragmatic lobe of the right lung were noted macroscopically in pig 5. Histologically, a few granulomas formed by multinucleate giant cells and rimmed with lymphocytes were found in pigs 1, 2 and 3. Several small lymphocytic infiltrates were seen in the adventitia of small arteries and in the interlobular septa. In pig 4, the granulomas contained epithelioid cells and showed fibrous rims, the lymphocytes being almost completely absent. In pig 5, foci of non-specific purulent bronchopneumonia in one diaphragmatic lobe were observed. In calves 1 and 2, the lungs were oedematous, with numerous haemorrhagic points on the surface and inside the parenchyma. In calves 3 and 4, small clear cysts about l/4 mm. in diameter were dispersed over the surface of the lungs. The lungs of calf 5 appeared morphologically normal. Histologically, the interlobular septa and pleura were oedematous with dilated and partially thrombosed lymphatics in calves 1 and 2. A few larvae were found in the alveoli, many in the interlobular septa and in the lymphatics and their walls where they were surrounded by inflammatory cells (Fig. 15). One larva was found in a small parietal coagulum in a branch of the pulmonary artery (Fig. 16). Small arterioles in the lung parenchyma were encircled by cuffs of blood which filled the periarteriolar lymphatic vessels (Fig. 17). In calf 2, the inflammatory infiltration of the interlobular septa and pleura, and thrombosis of the lymphatics were more pronounced (Fig. 19). Organization of the thrombi produced a form of verrucose granulomatous endo-1ymphangid.s (Fig. 18). Segmental inflammatory infiltration was noted in the walls of the interlobular and subpleural blood vessels with proliferation of the i&ma and obliteration of some of these vessels. Several lobules showed partial collapse and pneumonic infiltration. In calves 3 and 4, oedema was absent and the endo-lyhphatic thrombi were organized and contained a few giant cells. In calf 3, the larvae found in the
K. JINDdK
AND
J. E. ALICATA
379
granulomas were morphologically intact. In calf 4, morphologically intact larvae and some of their remnants, were enclosed in newly formed lymphatic follicles (Figs. 20,2 1). The cysts observed macroscopically were parts of dilated lymphatics. Brain. Of the 5 infected pigs, the brain of one only (pig 4) showed pathological changes. Solitary granulomas with epithelioid cells and foreign-body giant cells containing disintegrated parasites were found in the pons, hypothalamus and in the pia mater of the sulci of the parietal and occipital lobes. A layer of granulomatous tissue with eosinophils and multinuclear giant cells was present under the arachnoid at the base of the third ventricle. The choroid plexus of the fourth ventricle contained focal areas of infiltrations of lymphocytes and eosinophils. In calves 1, 2, 3 and 4, larvae were found in the white matter just under the cortex of the cerebral hemispheres and in the cerebellar cortex. In their vicinity, minute haemorrhages were present in calves 1 and 2. Perivascular cuffs consisting of lymphocytes were seen in the pons of calf 2. In calves 3 and 4, eosinophils appeared in the perivascular cuffs; the latter were more abundant in areas where intact larvae or granulomas containing dead larvae were present. The pia mater of the cerebellum was slightly infiltrated with mononuclear cells in calf 3 and also with eosinophils in calf 4. In the latter, one morphologically intact worm, 80 microns in diameter, was found under the arachnoid of a sulcus. In calf 5, minute perivascular lymphocytic cuffs were scattered in the white matter, but neither parasites nor granulomas were found. Spinal cord. In calves 2, 3 and 4, parasites, perivascular cufhng and granulomas were found in the white and grey substance. In these animals, epidural, intradural, and in several instances, subdural inflammatory infiltrations with eosinophils and mononuclear cells surrounded some of the spinal nerve roots. Their appearance was associated with parasites under the adjoining arachnoid or pia mater. In pig 4, a worm, 50 microns in diameter, was found under the dura mater of the cervical spinal cord. A detailed study of these findings has been prepared as a separate publication (Jindr&k, 1968). Miscellaneous organs. In the masseter muscle of pig 4 and in the diaphragm and intercostal muscles of calf 2 a few foci showing destruction of muscle fibres associated with a few lymphocytes in the interstice were detected. In the musculature of the auricles of calves 1 and 2, a few foci of lymphocytic and eosinophilic infiltration were noted. In calf 2, granulomas were found in the i&ma of the iliac artery. In one kidney of calf 5, a granuloma with a few foreign-body giant cells was present in the cortex. DISCUSSION
In considering the possible role of calves and pigs as sources of human infection, the results of the present study largely confirm reports of Alicata (1965b) that calves and pigs ha&our third-stage larvae of A. cantonensis in the visceral organs in the first few days of infection. In the present study, the larvae recovered 3 days after infection from pigs and calves were numerous and infectious as determined by animal feeding experiments. Beyond that period, pigs are probably of little importance in the transmission of the larvae since within a few days after infection the latter start to disintegrate and to calcify. In the case of calves, on the other’
380
INFECTION
OF
PIGS
AND
CALVES
WITH
A.
Ca?Zk?m??Z.&
hand, the period of transmissibility may be longer. The few larvae recovered from the lungs of calves 7 to 28 days after infection were not infective to rats although some of these larvae were motile and appeared normal even at 28 days after infection. Since only 4 to 25 larvae were fed to the rats, the results of the rodent feeding experiments cannot be considered altogether conclusive. Future studies should include the feeding of suspected tissues to susceptible mammals such as cats since the methods of obtaining Angiostrongylus larvae from tissues of large animals by use of the Baermann apparatus or by artificial digestive methods are often difficult. As to the role of calves as epidemiologically important carriers of the larvae, it is important to establish whether natural infection in calves exists. The migration of the third-stage larvae in the body of the mammalian host is important. Mackerras and Sandars (1955) found several third-stage larvae in the mesenteric lymph nodes of rats a few hours after infection, but other findings in their experiment supported the view that the larvae migrated by the blood stream. In the present study, most of the larvae as well as pathological changes were found in the lymphatic system. The mesenteric lymph nodes were probably invaded by the larvae on their migration from the digestive canal and represented an important trap for the larvae in pigs. In calves, the larvae injured the mesenteric lymph nodes, but were not stopped there. Most of them were trapped in the lung lymphatics and mediastinal lymph nodes, probably after circulation in the blood. The intralymphatic periarteriolar haemorrhages in the lungs probably indicated the area where the larvae had left the blood stream. In pigs, the mediastinal lymph nodes also contained many larvae, but the lung lymphatics did not. Anderson and Strelive (1967), in their experjments with Pneumostrongylus tenuis infection in white-tailed deer, did not find any infective larvae in the lymph nodes. This parasite, which is similar to Angiostrongylus cantonensis, undergoes an intracerebral migration in its life cycle. The negative findings could be due to species differences or to the fact that the mediastinal nodes were not examined. The larval migration did not leave many traces in the livers of calves, whereas in pigs larvae were found 3 days after infection and later a form of interstitial hepatitis developed. These lesions, first observed by Alicata (1965b), resemble those following the migration of larvae of Ascaris suum or Toxocara canis and known under the term “milk-spots” (Jubb and Kennedy, 1963), and also the lesions in the pig liver after infection with the larvae of Stephanurus dentatus (Alicata, 1953). The findings now reported indicate that a considerable number ‘of larvae must have migrated through the liver at least in the pigs. The larvae may well take more than one route to get from the digestive tract to the brain and the routes used in abnormal hosts may differ from those in the normal host. The rare and minute lesions in the musculature together with granulomas on the endothelium of the iliac artery, and in the kidney of some of the animals could have resulted from larvae brought into these tissues through the blood stream. It seems that pigs, unlike calves, ~OS.WSS additional resistance to infection with larvae of A. cantonensis. Thus, although the difference in pathology of the liver and Iymph nodes in these two species may be due, in part, to the increased fibrous tissue in the pig, it is likely that these animals also p-s m increased immunity, even at the beginning of the infection. In pigs, many of the larvae
K. JINDRk
AND
J. E. ALICATA
381
were surrounded by granulomas and had been destroyed within the wall of the digestive tract and in the lymph nodes and lungs as early as 3 days after infection. In calves, on the other hand, the larvae were not stopped in the wall of the digestive tube or in the mesenteric lymph nodes; in addition the granulomatous reaction was not well marked until 14 days after infection. In the visceral organs of pigs, the larvae were quickly destroyed, but they persisted for as long as 4 weeks in the lung lymphatics of the calves. Interstitial hepatitis was found in pigs, but was absent in calves. In some other nematode infections this kind of hepatitis is believed to be due to an allergic reaction probably against the excretory products which the larvae leave in their wake (Jubb and Kennedy, 1963). The higher resistance of pigs against the infection seems to account for the larvae reaching the brain in only one pig in the experiment. In spite of this higher resistance, however, there was little morphological difference between the lesions found in the central nervous system of one calf and those in one pig; furthermore there was no difference in the size of the parasites found in these two animals. SUMMARY
Angiostrongylus cantonensis infection was produced experimentally in pigs and calves by the oral administration of third-stage larvae. Living and normal thirdstage larvae, without evidence of further development, were isolated from the visceral organs of a pig at 3 days and of calves at 3, 7, 14 and 28 days after dosing. Larvae recovered 3 days after infection from the stomach of a pig, and from the lungs, peritoneal and thoracic cavities of a calf, produced cerebral infection when fed to rats. A few which appeared normal and were recovered from the lungs of calves 7 to 28 days after infection failed to produce cerebral infection when fed to rats in small numbers. It is not known to what extent these results were due to the comparatively few larvae which were fed to the rats. In pigs, larvae, in processof disintegration, were found enclosed in granulomas in the wall of the digestive tract and in mesenteric and mediastinal lymph nodes. A characteristic form of interstitial focal hepatitis also developed during the second week of infection. In calves, the larvae were found mostly in the pulmonary lymphatics and in mediastinal lymph nodes. The granulomatous reaction was delayed and several larvae, apparently morphologically intact, were found in the lungs as late as 28 days after infection. Unlike the pigs, no interstitial focal hepatitis developed in any of the calves. In one pig and in all five calves, some of the larvae migrated and developed to the fourth-stage in the central nervous system. The lesions are described. REFERENCES
Alicata, J. E. (1953). Amer. J. vet. Res., 53, 563; (1961). Hawaii Farm Bureau J. (Spec. Publ.) Dec., 1; (1962). Canad. J. Zool., 40, 5; (1963). S. Pacific Comm. Tech. Paper, 139; (1965a). Canad. J. Zool., 43, 667; (196513).In Advances in Parasitology, 3; p. 223, Academic Press; London and New York. Alicata, J. E., and Brown, R. W. (1962). Canad. 1. Zool., 40, 755. Alicata, J. E., and McCarthy, D. D. (1964). Ibid., 42, 605.
382
INFECTION
Anderson, Jindr&k, JindrAk,
OF
PIGS
AND
CALVES
WITH
A.
CfZ?ZtO?lt??ZSiS
R. C., and Strelive, U. R. (1967). Ibid., 45, 285. Exp. Neural., (in press). K. (1968). J. N europath. K., and Alicata, J. E. (1965). Ann. trap. Med. Parasit.,
Jubb, K. V. F., and Kennedy, Academic
Press;
New
P. C. (1963). Pathology York
and
of Domestic
59, 294. Animals,
2, p. 140,
London.
Ketsuwan, P., and Pradatsundarasar, A. (1966). Amer. J. trop. Med. H?,g., 15, 50. Kuntz, R. E., and Myers, B. J. (1964). Ibid., 13, 686. Lim, B. L., Ow-Yang, C. K., and Lie, K. J. (1962). Med. f. Malaya, 17, 89. Mackerras, M. J., and Sandars, D. F. (1955). Austr. J. Zool., 3, 1. Nomura, S., and Lin, P. H. (1945). Taiwan No Ikai, 3, 589. Prommindaroj, K., Leelawongs, N., and Pradatsundarasar, A. (1962). Arncy;. J. trap. Med. Hyg., 11, 759. Punyagupta, S. (1965). Ibid., 14, 370. Rosen, L., Chappell, R., Laqueur, G. L., Wallace, G. D., and Weinstein, P. P. (1962). J. Amer. med. Assoc., 179, 620. Tangchai, P., Nye, S. W., and Beaver, P .C.. (1967). Amer. J. trol,. MC (1. Hyg.,
16, 464.
[Received
Fig.
1.
Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. Fig. Fig. Fig. Fig.
8. 9. 10. 11. 12.
Fig. 13. Fig. 14. Fig. 15. Fig. Fig. Fig. Fig.
16. 17. 18. 19.
Fig. 20. Fig. 21.
for
publication,
Drccmber
27th,
19671
Morphologically intact, live third-stage larva from the lungs of calf‘ 4. 28 da)-s after infection. x zoo. Calcified larvae in a nodule on the ligamenturn pulmonale of pig 2, 7 days after infection. 35. Fourth-stage larvae from the brain of calf 3, 14 days after infection. Larva surrounded by a granuloma in the submucosa of the intestine of pig 1. 3 days after infection. H. & E. x 100. Coiled larva and several foreign-body giant cells in the sinus rimming a fibrous trabecula in a mediastinal lymph node of pig 1. H. & E. x 50. Numerous larvae in a dilated afferent lymphatic to an oedematous haemorrhagic mediastinal lymph node of calf 1. H. & E. x 25. Larvae in granulomas filling an afferent lymphatic to a mediastinal lymph-node of calf 3. H. & E. 30. Detail from fig 7, showing larvae and foreign-body giant cells. H. & E. :t 75. A small cyst-like nodule which contained a partially calcified larva. Omentum of pig 2. ;c 2. Liver of pig 3, showing numerous white spots on its surface. x 2/3. A larva in the porto-biliary space in the liver of pig 1. H. & E. ‘G 75. Dilation and inflammation of the interlobular septa in a white spot in the liver of piq 3. H. B E. I. 20. Thickened interlobular septa in the liver of pig 4. H. & E. i; 50. Focus of lymphoid tissue in the liver of pig 5. H. & E. x 20. A larva in the wall of a thrombosed lung lymphatic. Note oedema and inflammatory infiltration. Calf 1. H. & E. x 75. Larva in a parietal thrombus of a small branch of the pulmonary artery. Calf 1. H. Sr E. 75. Periarteriolar haemorrhage into the lymphatic vessels in the lung of calf 1. H. & E. 7.5. Granulomatous verrucose endo-lymphangitis [arrow) in the lung of calf 2. H. & E. 25. $ila;ly2andHth;;bos;d2\ymphatics in the dilated interlobuIar septa and pleura of the lung . . .~ A nodule containing a lar;a in the pleura of the lung of calf 5. Collapse of a lobule of lung parenchyma. H. & E. x 20. Detail of fig. 20, with the larva and several foreign-body giant cells. H. Bt E. ill 7.5.
K. JINDtiK
AND J. E. ALICATA
INFECTIONOF
PIGSAND~ALVES
WITH
~.cu~fonensis
K. JINDdK
AND J. E. ALICATA
PATH.
1968.
VOL.
78.
371
COMPARATIVE PATHOLOGY IN EXPERIMENTAL INFECTION OF PIGS AND CALVES WITH LARVAE ANGlOSTRONGYLUS CANTONENSIS*
OF
BY K. JINDRAK
and J. E. ALICATA
Pamsi&logy Lubaratoy, Dqkzrtment of Animal Sciem, Univmi& of Hawaii, Honahlu, Hawaii INTRODUCTION
During the last few years, the rat lungworm, Angfostrong$us cuntonensis, has received much attention because it has been suspected as the most probable cause of human eosinophilic meningitis in the Pacific islands and Southeast Asia. This theory proposed by Alicata (1961, 1962) has been supported by the findings of this parasite in man in the cerebrospinal fluid (Nomura and Lin, 1945), in the brain (Rosen, Chappell, Laqueur, Wallace and Weinstein, 1962; Jindrak and Alicata, 1965 ; Tangchai, Nye and Beaver, 1967), and in the anterior chamber of the eye (Prommindaroj, Leelawongs and Pradatsundarasar, 1962; Ketsuwan and Pradatsundarasar, 1966). Recent epidemiological and parasitological investigations in various areas of the tropical belt (Ahcata, 1962, 1963, 1965a; Lii, Ow-Yang and Lie, 1962; Alicata and McCarthy, 1964 ; Kuntz and Myers, 1964; Punyagupta, 1965) have confirmed the presence of this parasite in places where eosinophilic meningitis has been reported. Alicata (1963, 1965a, 1965b) and Alicata and Brown (1962) found several carrier hosts of the infective larvae of this parasite as possible sources of human infection, including land planarians, freshwater prawns, crabs, cattle and pigs. Among these carrier hosts, pigs and calves occupy an important place since in preliminary observations (Alicata, 1965b) third-stage larvae were found in the visceral organs of these animals several days after the infection. The present study was carried out to obtain more information on the presence and survival of third-stage larvae of A. cantonensis in various organs of cattle and pigs and to determine the pathological changes which occurred in them. The results are useful in (a) identifying similar changes which may be found in cattle and pigs in endemic areas, and (b) obtaining a better understanding of the pathology in man, who is also an abnormal host. MATERIALS
AND
METHODS
Animals. Six pigs and 5 calves were used. The pigs were reared on a concrete floor at the University of Hawaii farm and received dry feed. The calves were obtained from a dairy farm and kept in a small dry paddock under strict sanitary conditions. All the animals were l-month-old when placed on the experiment. Larvae. The infective third-stage larvae of A. cantonensis were obtained by artificial * This investigation Diseases and Blindnes-9, Agricultural Experiment
WBS supported by grant No. NB-CM965 from NIH, USPHS. Published with the approval Station as Technical Paper No. 937.
the Institute of the Director
of Neurological of the Hawaii
372
INFECTION
OF
PIGS
AND
CALVES
WITH
A.
CU?ltOnt%SiS
digestion of experimentally infected giant African snails, Achatina fulic~. The soft parts of the snail were. minced in a small meat grinder, digested in a solution of 1 per cent. pepsin and 1 per cent HCl for 2 hours at 37OC. after which the suspension was strained through a fine metal mesh into a sedimentation cone and allowed to stand for 2 hours. The number of larvae in the sediment was estimated by the usual dilution counting method. The larvae were administered with the aid of a dose syringe. The calves received approximately 70,000 larvae each and 5 of the 6 pigs received 20,000 larvae each. The sixth pig was left uninfected and served as a control. No control calf was available. One pig and one calf were killed at 3, 7, 14, 28 and 56 days after infection. The control pig was killed on the 14th day of the experiment. Post-mortem examination. At necropsy, the stomach wall, lung, liver, heart, spleen, kidney, brain, spinal cord, diaphragm, tongue and masseter muscle were examined from each animal for larvae by mincing 15 g. of each tissue and standing in a Baermann apparatus for 2 hours. In addition, brain and other tissue from each animal were examined microscopically as press preparations. The pleural and peritoneal cavities were washed with saline solution and the sediment of the washings examined with a dissecting microscope for Angiostrongylus larvae. Histology. Tissue blocks for histological examination were taken from the following organs : liver, kidney, lungs, heart, spleen, pancreas, adrenals, thymus, thyroid gland, submaxillary salivary gland, gonads, diaphragm, intercostal muscles,massetermuscle, musclesfrom the right thigh, the eye bulb with the optic nerve and oculomotor muscles, stomach (in calves from each part of it), small and large intestines with corresponding mesenteric lymph nodes, gastrosplenic lymph nodes, urinary bladder, mediastinal lymph nodeswith parts of the trachea, oesophagus and aorta, omentum, parietal pleura from the cupula pleurae. Blocks were taken from other organs whenever necessary. The brain and the spinal cord, after removal of portions required for parasitological examination, were preserved. The tissues were fixed in Bouin solution, embedded in paraffin and sectioned at 8 microns. From the musclesof pigs 1 and 2 and calves 1, 2 and 3, several hundred sectionswere prepared and sthed with Harris’ haematoxylin and eosin. The P.A.S. technique, Gomori’s method for precollagen fibres, Van Gieson’s technique for collagen fibres, and Fast-Blue Luxol stain for myelin were used in individual sectionsas appropriate. RESULTS
Clinical
Observations
None of the pigs showed any neurological symptoms, whereas calves 2, 3 and 4 showed an uncertain gait on the day of slaughter, which was 1, 2 and 4 weeks, respectively, following infection.
Distribution
of Larvae
Details of the larvae found in the organs of pigs and calves are summarized in Table 1. Undeveloped third-stage larvae were found in the stomach wall, liver, spleen, and peritoneal cavity of pig 1 and the liver of pig 2. Similar larvae were found in the stomach wall, liver, lungs, heart, kidney, mesenteric and mediastinal lymph nodes, peritoneal and thoracic cavity of calf 1, the peritoneal cavity of calf 2, and the lung-sof calves 2, 3 and 4 (Fig. 1). In most instances, these larvae appeared normal, except that about one-half of those in the lungs of the calves showed large vacuoles. White rats fed larvae recovered from the stomach of pig 1, and from the lungs and the peritoneal and thoracic cavities of calf 1, all showed developing A. cantonensis in their brains 20 days after infection. None
*
and muscle
cavity cavity
nodes
8 z x
2: 4:B 0 8
:t. 1-P
8
16-B0 4-w 0
7
3
Pig 2
8
8 0
8
8
l!P
I4
Pig 3
RECOVERJZD
FROY
I
8
8
::
0”
8 0
:
8
8
56
Pig 5
OF ORGANS
1-i 0”
0 8-B**
ii 0
25P,
‘ilW
z
3-B 3-B 40-B* 26-W 125-w 2-B 76-B 3-B
7
Calf 2
3
calf1
EXAMINED
without further development W = Washings P = Press preparation
:It
x
28
Pig 4
PORTIONS
TABLE
* = Larvae isolated from a mediastinal lymph node l = Advanced third- or fourth-stage larvae; all others, third-stage larvae t = Calcified larva in wall of the cavity B = Baermann extraction
LunsS Heart Spleen Kidney Diaphragm masseter Brain
Peritoneal peyic ‘V
Stomach walllymph Mesenteric
Days of infection
Pig 1
LARVAE
2-B:*
: 0
8 10-B
0
8
8 0
-i 4-B
0
8
28
I4 0 0
calf4
calf3
-ii
8
8 0 0
0
E
56
Calf5
2:
W
A
Liver
Brain
A= A = B = C = D = E = F = G = H = I =
ABCDEG +++
Mediastinal lymph nodes
Lungs
ABCDEG if
Mesenteric lymph nodes
DEG ++
G ++
+
---
GI i-
E ++
ACDEG +++
A
A
EG
Pig 2 7
---
+
-I-E+
AEG +++
GI
AEG ++
G ++
AEG iA
OF THE
Pig 3 I4
Larvae (several of them morphologically intact) Larvae (disintegrating) Oedema Haemorrhage Tissue destruction (larval tracks. necrosis) Infiltrations contaihing many edsinophili Newly formed lymphatic follicles Granulomas (with or without larvae) Fibrosis, scar Mononuclear-cell infiltrations
-
GI i-
CDE +
DEG ++
A
Intestines
EG +
A
Pig 1 3
Stomach
Days of infection
SUMMARY
+
AEFG +++-I-
GHI +
+
+
+
-
EH
F
F
---
F
-t
TABLE
-t
---
---
-
--
EFH ++
FH -t
FH +
---
2
I
A
ABC ii++ C
ABCDI ++++
ABCDI
B
f
:
+
I
-t
-
3
I
+E
i-f++
I +
2
++
a i. = Significant
I
3
EG +++
lesions
AC
AC GHI ++++
+
ACDEG ++++
EG +
+
E
---
Calf14
lesions of increasing
or minimal
changes:
ACE
ABC GI ++++
- = No lesion % = Insignificant 4-l
J
f
*
EG +
I
I
Calf 7
ABCDEG ++++
A
FINDINGS
Degree of pathological
-
---
calf
HISTOLOGICAL
Pig 5 56
IMPORTANT
Pig 4 28
MOST
F FG ++
f
:
-
severity
AE G +++
F + -1
HI + A
A
---
calf428
H
---
---
f
+ I +
-
FH
FH *
I
-
5
+
calf56
K. JINDtiK
AND
J. E. ALXATA
375
of the rats, however, which received from 4 to 15 larvae from the lungs of calves 2,3 and 4 showed parasites in the brain. Dead and partially calcified larvae were found in press preparations from small cyst-lie nodules on the surface of the liver, on the ligamentum pulmonale (Fig. 2) and on the omentum (Fig. 9) of pig 2. In addition, similar isolated nodules containing calcified larvae were noted on the capsule of a mesenteric lymph node of pig 3 and on the peritoneal surface of the diaphragm of pig 4. The larvae found in the brain tissue of calf 2 were still in the third stage, but showed increased growth. One measured 578 microns long and another was 826 microns long. From the brain of calf 3, two fourth-stage larvae were isolated. They measured 1,32 1 and 1,65 1 microns in length and 50 to 54 microns in width, respectively (Fig. 3). Pathological
Observations
The most important histological findings are summarized in Table 2. No pathological changes were observed in pig 6. Stomach and intestines. Rust-coloured sulxrosal and intramucosal foci were found in the stomach of pigs 1, 2, 3 and 4 (3, 7, 14 and 28 days after infection, respectively), on the muccsa of the ileum of pig 1 and in the rectum and colon of pigs 2 and 3, calves 1, 2 and 3. In pig 1, there was an ulcer, 3 mm. in diameter, in the mucosa on the greater curvature. Microscopically, in pigs 1,2 and 3, focal infiltrations of eosinophils, thrombosis of blood vessels, and small granulomas consisting of epithelioid cells and eosinaphils were present in the submucosa. Solitary foreign-body giant cells were found inside the submucous lymphatics. Larval tracks, surrounded by epithelioid cells and eosinophils and f&xl with cellular debris, were scattered in the Peyer’s plaques. The granulomas contained morphologically intact larvae in pig 1 and fragments of larvae in pigs 2 and 3 (Fig. 4). Only focal lymphocytic infiltrations and lymphatic follicles were found in the gastric submucosa of pig 4. In calves, the microscopical changes were restricted to the intestines, predominantly to the jejunum, and no larvae were found. The intestinal villi in calf 1 showed dilation of the axial lymphatics and infiltration with lymphocytes. In calf 2, the inflammatory cells permeated the whole thickness of the mucosa. In calf 3, many eosinophils appeared in the infiltrations. In calves 4 and 5, the infiltration was less marked and consisted predominantly of lymphocytes. Lymph nodes and lymphatic vessels. Striking differences related to the number of larvae and lesions were noted between the lymph nodes of the pigs and those of calves. In general, the lesions in the mesenteric lymph nodes were scarce and considerable differences in the degree of the involvement of individual nodes were noted. A single mediastmal node usually contained more larvae and showed more pathological lesions than any one of the mesenteric nodes of the same animal. In each of two animals, one pig and one calf, killed on the same day after infection, the mesenteric nodes of the pig contained many more larvae than the corresponding nodes of the calf. A reverse relationship applied to the mediastinal nodes : those of the pig contained significantly fewer larvae than ,those of the calf.
376
INFECTION
OF PIGS
AND
CALVES
WITH
A.
cantOnenSiS
The invasion of the lymph nodes by the larvae was marked by congestion, oedema and haemorrhage. Several nodes of the calves showed areas of haemorrhagic necrosis. The larvae were located in the a&rent lymphatics, subcapsular sinuses and in the capsules. In the lymphatic tissues, many larval tracks were filled with cellular debris and fibrinous exudate, and larvae were found. The inflammatory infiltration in the form of a perilymphadenitis was especially marked on the afferent and efferent lymphatics. As the infection progressed, the larvae were surrounded by granulomas containing foreign-body giant cells, epithelioid cells, lymphocytes and eosinophils; the larvae eventually distintegrated and disappeared. Eventually the inflammatory reaction subsided and finally only focal accumulations of lymphocytes and lymphatic follicles were found in places where in earlier stages larvae and granulomas were present. In pigs, many larvae together with giant cells were present in the sinuses rimming the trabeculae of the lymph nodes (Fig. 5). The trabeculae were oedematous and infiltrated with eosinophils; their lymphatics were partially thrombosed and showed small tears in their walls. Many eosinophils were present in the inflammatory infiltrations as early as 3 days after infection (pig 1); in addition, disintegration and calcification of some of the larvae and the formation of granulomas with epithelioid cells was evident at the same time. In pig 4, at 24 days after infection, only accumulations of histocytes in the subcapsular sinuses were noted; larvae, eosinophils and granulomas were not found. In calves, the larvae were especially numerous in the subcapsular sinuses of the mediastinal lymph nodes and in their afferent lymphatics which were dilated and partially thrombosed (Fig. 6). The eosinophilic and granulomatous reactions were first seen in calf 2 at 7 days after the infection, and were most pronounced in calf 3 at 14 days after infection. In the latter, some of the larvae were disintegrated and surrounded by foreign-body giant cells, eosinophils, plasmocytes and lymphocytes (Figs. 7, 8). In calf 4 at 28 days after infection, the granulomas simulated lymphatic follicles with fragmented or apparently morphologically intact larvae in their centres. No larvae were found in these follicles in calf 5 at 56 days after infection. In all the animals, small foci of inflammatory infiltration or granulomas were scattered through the mediastinal loose connective tissue and even in other mediastinal organs such as the oesophagus. These lesions corresponded in appearance with those in the lymph nodes. In calf 2, a granuloma with a larva was seen in the perineurium of the vagus nerve. With the exception of pig 1, varying numbers of small nodules, 0.5 to 3 mm. in diameter, were scattered in the visceral and parietal peritoneum and pleura, in the retroperitoneal and mediastinal connective tissue, and often along arteries. This distribution is summarized in Table 3. Some of the nodules were colourleq small, thin-walled cysts filled with a clear fluid (Fig. 9) while others were pink or red. In a few of them, live or calcified larvae were found. They originated from the lymphatics. All stages were observed microscopically, from a simple partially thrombosed lymphangiectasis, to dilated lymphatics containing a loose meshwork of fibroblasts populated by lymphocytes, and finally the assumption of the shape of a small lymph node. In general, the mesenteric and mediastinal lymphatics were more severely injured in calves than in pigs.
K. JINDRiiK
AND
377
J. E. ALICATA
;
c
I
8
Y hl
9 m
1
I
I
I
I I I
I I I
I I I
I , I
Y’: mm
I I I
1 I
9 -
I I I
I I
I I I
I I I
I I I
0 24
0 e;
I I I
I I I
ij
I I I
x &
378
INFECTION
OF PIGS
AND
CALVES
WITH
A. cantonensis
Liver. No gross lesions occurred in the liver of the calves. Histologically, a few small, focal infiltrations of mononuclear cells, mainly lymphocytes, were disseminated in the liver parenchyma irregularly in calves 1, 2, 3 and 4. In 4, some of the foci were transformed into minute scars. No pathological lesions were found in the liver of calf 5. In pigs, disseminated white spots from 2 to 7 mm. in diameter were present under the capsule and on the cut surface. Only small numbers were seen in pigs 1, 2, 4 and 5, but were more numerous in pig 3, in which they were confluent in places (Fig. 10). The lobular pattern of the hepatic parenchyma in the spots was distinct. In pigs 4 and 5, the foci were firm with a rigid prominent white nodule in their centre. Microscopically, a few larvae in the porto-bilary spaces (Fig. 11) and several small foci of necrosis and haemorrhage were found in the hepatic parenchyma of pig 1. Small groups of mononuclear cells and eosinophils were present at the periphery of hepatic lobules in pigs 1, 2 and 3. In the areas of the macroscopically visible white spots, the interlobular septa were oedematous, dilated, and infiltrated with mononuclear cells and eosinophils. In pig 3, the inflammatory cells had penetrated a considerable distance inside the hepatic lobules where the parenchymatous cells had undergone atrophy (Fig. 12). In pigs 4 and 5, the inflammatory reaction subsided, but thickened fibrous interlobular septa remained (Fig. 13). In some of the persisting white foci stellate scars with calcified centres and sometimes with conglomerates of lymphatic follicles were found (Fig. 14). Lungs. Several foci of consolidation in the diaphragmatic lobe of the right lung were noted macroscopically in pig 5. Histologically, a few granulomas formed by multinucleate giant cells and rimmed with lymphocytes were found in pigs 1, 2 and 3. Several small lymphocytic infiltrates were seen in the adventitia of small arteries and in the interlobular septa. In pig 4, the granulomas contained epithelioid cells and showed fibrous rims, the lymphocytes being almost completely absent. In pig 5, foci of non-specific purulent bronchopneumonia in one diaphragmatic lobe were observed. In calves 1 and 2, the lungs were oedematous, with numerous haemorrhagic points on the surface and inside the parenchyma. In calves 3 and 4, small clear cysts about l/4 mm. in diameter were dispersed over the surface of the lungs. The lungs of calf 5 appeared morphologically normal. Histologically, the interlobular septa and pleura were oedematous with dilated and partially thrombosed lymphatics in calves 1 and 2. A few larvae were found in the alveoli, many in the interlobular septa and in the lymphatics and their walls where they were surrounded by inflammatory cells (Fig. 15). One larva was found in a small parietal coagulum in a branch of the pulmonary artery (Fig. 16). Small arterioles in the lung parenchyma were encircled by cuffs of blood which filled the periarteriolar lymphatic vessels (Fig. 17). In calf 2, the inflammatory infiltration of the interlobular septa and pleura, and thrombosis of the lymphatics were more pronounced (Fig. 19). Organization of the thrombi produced a form of verrucose granulomatous endo-1ymphangid.s (Fig. 18). Segmental inflammatory infiltration was noted in the walls of the interlobular and subpleural blood vessels with proliferation of the i&ma and obliteration of some of these vessels. Several lobules showed partial collapse and pneumonic infiltration. In calves 3 and 4, oedema was absent and the endo-lyhphatic thrombi were organized and contained a few giant cells. In calf 3, the larvae found in the
K. JINDdK
AND
J. E. ALICATA
379
granulomas were morphologically intact. In calf 4, morphologically intact larvae and some of their remnants, were enclosed in newly formed lymphatic follicles (Figs. 20,2 1). The cysts observed macroscopically were parts of dilated lymphatics. Brain. Of the 5 infected pigs, the brain of one only (pig 4) showed pathological changes. Solitary granulomas with epithelioid cells and foreign-body giant cells containing disintegrated parasites were found in the pons, hypothalamus and in the pia mater of the sulci of the parietal and occipital lobes. A layer of granulomatous tissue with eosinophils and multinuclear giant cells was present under the arachnoid at the base of the third ventricle. The choroid plexus of the fourth ventricle contained focal areas of infiltrations of lymphocytes and eosinophils. In calves 1, 2, 3 and 4, larvae were found in the white matter just under the cortex of the cerebral hemispheres and in the cerebellar cortex. In their vicinity, minute haemorrhages were present in calves 1 and 2. Perivascular cuffs consisting of lymphocytes were seen in the pons of calf 2. In calves 3 and 4, eosinophils appeared in the perivascular cuffs; the latter were more abundant in areas where intact larvae or granulomas containing dead larvae were present. The pia mater of the cerebellum was slightly infiltrated with mononuclear cells in calf 3 and also with eosinophils in calf 4. In the latter, one morphologically intact worm, 80 microns in diameter, was found under the arachnoid of a sulcus. In calf 5, minute perivascular lymphocytic cuffs were scattered in the white matter, but neither parasites nor granulomas were found. Spinal cord. In calves 2, 3 and 4, parasites, perivascular cufhng and granulomas were found in the white and grey substance. In these animals, epidural, intradural, and in several instances, subdural inflammatory infiltrations with eosinophils and mononuclear cells surrounded some of the spinal nerve roots. Their appearance was associated with parasites under the adjoining arachnoid or pia mater. In pig 4, a worm, 50 microns in diameter, was found under the dura mater of the cervical spinal cord. A detailed study of these findings has been prepared as a separate publication (Jindr&k, 1968). Miscellaneous organs. In the masseter muscle of pig 4 and in the diaphragm and intercostal muscles of calf 2 a few foci showing destruction of muscle fibres associated with a few lymphocytes in the interstice were detected. In the musculature of the auricles of calves 1 and 2, a few foci of lymphocytic and eosinophilic infiltration were noted. In calf 2, granulomas were found in the i&ma of the iliac artery. In one kidney of calf 5, a granuloma with a few foreign-body giant cells was present in the cortex. DISCUSSION
In considering the possible role of calves and pigs as sources of human infection, the results of the present study largely confirm reports of Alicata (1965b) that calves and pigs ha&our third-stage larvae of A. cantonensis in the visceral organs in the first few days of infection. In the present study, the larvae recovered 3 days after infection from pigs and calves were numerous and infectious as determined by animal feeding experiments. Beyond that period, pigs are probably of little importance in the transmission of the larvae since within a few days after infection the latter start to disintegrate and to calcify. In the case of calves, on the other’
380
INFECTION
OF
PIGS
AND
CALVES
WITH
A.
Ca?Zk?m??Z.&
hand, the period of transmissibility may be longer. The few larvae recovered from the lungs of calves 7 to 28 days after infection were not infective to rats although some of these larvae were motile and appeared normal even at 28 days after infection. Since only 4 to 25 larvae were fed to the rats, the results of the rodent feeding experiments cannot be considered altogether conclusive. Future studies should include the feeding of suspected tissues to susceptible mammals such as cats since the methods of obtaining Angiostrongylus larvae from tissues of large animals by use of the Baermann apparatus or by artificial digestive methods are often difficult. As to the role of calves as epidemiologically important carriers of the larvae, it is important to establish whether natural infection in calves exists. The migration of the third-stage larvae in the body of the mammalian host is important. Mackerras and Sandars (1955) found several third-stage larvae in the mesenteric lymph nodes of rats a few hours after infection, but other findings in their experiment supported the view that the larvae migrated by the blood stream. In the present study, most of the larvae as well as pathological changes were found in the lymphatic system. The mesenteric lymph nodes were probably invaded by the larvae on their migration from the digestive canal and represented an important trap for the larvae in pigs. In calves, the larvae injured the mesenteric lymph nodes, but were not stopped there. Most of them were trapped in the lung lymphatics and mediastinal lymph nodes, probably after circulation in the blood. The intralymphatic periarteriolar haemorrhages in the lungs probably indicated the area where the larvae had left the blood stream. In pigs, the mediastinal lymph nodes also contained many larvae, but the lung lymphatics did not. Anderson and Strelive (1967), in their experjments with Pneumostrongylus tenuis infection in white-tailed deer, did not find any infective larvae in the lymph nodes. This parasite, which is similar to Angiostrongylus cantonensis, undergoes an intracerebral migration in its life cycle. The negative findings could be due to species differences or to the fact that the mediastinal nodes were not examined. The larval migration did not leave many traces in the livers of calves, whereas in pigs larvae were found 3 days after infection and later a form of interstitial hepatitis developed. These lesions, first observed by Alicata (1965b), resemble those following the migration of larvae of Ascaris suum or Toxocara canis and known under the term “milk-spots” (Jubb and Kennedy, 1963), and also the lesions in the pig liver after infection with the larvae of Stephanurus dentatus (Alicata, 1953). The findings now reported indicate that a considerable number ‘of larvae must have migrated through the liver at least in the pigs. The larvae may well take more than one route to get from the digestive tract to the brain and the routes used in abnormal hosts may differ from those in the normal host. The rare and minute lesions in the musculature together with granulomas on the endothelium of the iliac artery, and in the kidney of some of the animals could have resulted from larvae brought into these tissues through the blood stream. It seems that pigs, unlike calves, ~OS.WSS additional resistance to infection with larvae of A. cantonensis. Thus, although the difference in pathology of the liver and Iymph nodes in these two species may be due, in part, to the increased fibrous tissue in the pig, it is likely that these animals also p-s m increased immunity, even at the beginning of the infection. In pigs, many of the larvae
K. JINDRk
AND
J. E. ALICATA
381
were surrounded by granulomas and had been destroyed within the wall of the digestive tract and in the lymph nodes and lungs as early as 3 days after infection. In calves, on the other hand, the larvae were not stopped in the wall of the digestive tube or in the mesenteric lymph nodes; in addition the granulomatous reaction was not well marked until 14 days after infection. In the visceral organs of pigs, the larvae were quickly destroyed, but they persisted for as long as 4 weeks in the lung lymphatics of the calves. Interstitial hepatitis was found in pigs, but was absent in calves. In some other nematode infections this kind of hepatitis is believed to be due to an allergic reaction probably against the excretory products which the larvae leave in their wake (Jubb and Kennedy, 1963). The higher resistance of pigs against the infection seems to account for the larvae reaching the brain in only one pig in the experiment. In spite of this higher resistance, however, there was little morphological difference between the lesions found in the central nervous system of one calf and those in one pig; furthermore there was no difference in the size of the parasites found in these two animals. SUMMARY
Angiostrongylus cantonensis infection was produced experimentally in pigs and calves by the oral administration of third-stage larvae. Living and normal thirdstage larvae, without evidence of further development, were isolated from the visceral organs of a pig at 3 days and of calves at 3, 7, 14 and 28 days after dosing. Larvae recovered 3 days after infection from the stomach of a pig, and from the lungs, peritoneal and thoracic cavities of a calf, produced cerebral infection when fed to rats. A few which appeared normal and were recovered from the lungs of calves 7 to 28 days after infection failed to produce cerebral infection when fed to rats in small numbers. It is not known to what extent these results were due to the comparatively few larvae which were fed to the rats. In pigs, larvae, in processof disintegration, were found enclosed in granulomas in the wall of the digestive tract and in mesenteric and mediastinal lymph nodes. A characteristic form of interstitial focal hepatitis also developed during the second week of infection. In calves, the larvae were found mostly in the pulmonary lymphatics and in mediastinal lymph nodes. The granulomatous reaction was delayed and several larvae, apparently morphologically intact, were found in the lungs as late as 28 days after infection. Unlike the pigs, no interstitial focal hepatitis developed in any of the calves. In one pig and in all five calves, some of the larvae migrated and developed to the fourth-stage in the central nervous system. The lesions are described. REFERENCES
Alicata, J. E. (1953). Amer. J. vet. Res., 53, 563; (1961). Hawaii Farm Bureau J. (Spec. Publ.) Dec., 1; (1962). Canad. J. Zool., 40, 5; (1963). S. Pacific Comm. Tech. Paper, 139; (1965a). Canad. J. Zool., 43, 667; (196513).In Advances in Parasitology, 3; p. 223, Academic Press; London and New York. Alicata, J. E., and Brown, R. W. (1962). Canad. 1. Zool., 40, 755. Alicata, J. E., and McCarthy, D. D. (1964). Ibid., 42, 605.
382
INFECTION
Anderson, Jindr&k, JindrAk,
OF
PIGS
AND
CALVES
WITH
A.
CfZ?ZtO?lt??ZSiS
R. C., and Strelive, U. R. (1967). Ibid., 45, 285. Exp. Neural., (in press). K. (1968). J. N europath. K., and Alicata, J. E. (1965). Ann. trap. Med. Parasit.,
Jubb, K. V. F., and Kennedy, Academic
Press;
New
P. C. (1963). Pathology York
and
of Domestic
59, 294. Animals,
2, p. 140,
London.
Ketsuwan, P., and Pradatsundarasar, A. (1966). Amer. J. trop. Med. H?,g., 15, 50. Kuntz, R. E., and Myers, B. J. (1964). Ibid., 13, 686. Lim, B. L., Ow-Yang, C. K., and Lie, K. J. (1962). Med. f. Malaya, 17, 89. Mackerras, M. J., and Sandars, D. F. (1955). Austr. J. Zool., 3, 1. Nomura, S., and Lin, P. H. (1945). Taiwan No Ikai, 3, 589. Prommindaroj, K., Leelawongs, N., and Pradatsundarasar, A. (1962). Arncy;. J. trap. Med. Hyg., 11, 759. Punyagupta, S. (1965). Ibid., 14, 370. Rosen, L., Chappell, R., Laqueur, G. L., Wallace, G. D., and Weinstein, P. P. (1962). J. Amer. med. Assoc., 179, 620. Tangchai, P., Nye, S. W., and Beaver, P .C.. (1967). Amer. J. trol,. MC (1. Hyg.,
16, 464.
[Received
Fig.
1.
Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. Fig. Fig. Fig. Fig.
8. 9. 10. 11. 12.
Fig. 13. Fig. 14. Fig. 15. Fig. Fig. Fig. Fig.
16. 17. 18. 19.
Fig. 20. Fig. 21.
for
publication,
Drccmber
27th,
19671
Morphologically intact, live third-stage larva from the lungs of calf‘ 4. 28 da)-s after infection. x zoo. Calcified larvae in a nodule on the ligamenturn pulmonale of pig 2, 7 days after infection. 35. Fourth-stage larvae from the brain of calf 3, 14 days after infection. Larva surrounded by a granuloma in the submucosa of the intestine of pig 1. 3 days after infection. H. & E. x 100. Coiled larva and several foreign-body giant cells in the sinus rimming a fibrous trabecula in a mediastinal lymph node of pig 1. H. & E. x 50. Numerous larvae in a dilated afferent lymphatic to an oedematous haemorrhagic mediastinal lymph node of calf 1. H. & E. x 25. Larvae in granulomas filling an afferent lymphatic to a mediastinal lymph-node of calf 3. H. & E. 30. Detail from fig 7, showing larvae and foreign-body giant cells. H. & E. :t 75. A small cyst-like nodule which contained a partially calcified larva. Omentum of pig 2. ;c 2. Liver of pig 3, showing numerous white spots on its surface. x 2/3. A larva in the porto-biliary space in the liver of pig 1. H. & E. ‘G 75. Dilation and inflammation of the interlobular septa in a white spot in the liver of piq 3. H. B E. I. 20. Thickened interlobular septa in the liver of pig 4. H. & E. i; 50. Focus of lymphoid tissue in the liver of pig 5. H. & E. x 20. A larva in the wall of a thrombosed lung lymphatic. Note oedema and inflammatory infiltration. Calf 1. H. & E. x 75. Larva in a parietal thrombus of a small branch of the pulmonary artery. Calf 1. H. Sr E. 75. Periarteriolar haemorrhage into the lymphatic vessels in the lung of calf 1. H. & E. 7.5. Granulomatous verrucose endo-lymphangitis [arrow) in the lung of calf 2. H. & E. 25. $ila;ly2andHth;;bos;d2\ymphatics in the dilated interlobuIar septa and pleura of the lung . . .~ A nodule containing a lar;a in the pleura of the lung of calf 5. Collapse of a lobule of lung parenchyma. H. & E. x 20. Detail of fig. 20, with the larva and several foreign-body giant cells. H. Bt E. ill 7.5.
K. JINDtiK
AND J. E. ALICATA
INFECTIONOF
PIGSAND~ALVES
WITH
~.cu~fonensis
K. JINDdK
AND J. E. ALICATA