Aspiration pneumonia in gnotobiotic piglets

J. C.OMP.

PATH.

196%

VOL.

ASPIRATION

237

79.

PNEUMONIA PIGLETS

IN

GNOTOBIOTIC

BY

I. M.

and A. H. S.

SMITH

Dqbrbnmt of Path&~,

HAYWARD

Royal Vetcrimy Collqe, London

INTRODUCTION

Aspiration pneumonia has been recorded in all speciesof domestic animal at any age, although in its diffuse form it arises perhaps most frequently among pail-fed calves (Jubb and Kennedy, 1963). In normal animals the pathological effects of aspirated material are believed to depend in part on the nature, consistency and volume of the material. The pulmonary reaction that follows is likely in normal animals to be complicated by the activities of a variety of micro-organisms which find suitable conditions for growth in tissue already heavily damaged and with enfeebled defensive powers. In gnotobiotic animals, however, the nature of

the pulmonary reaction might be simpler. This paper records some observations on piglets of known microbiological status which suffered from accidental aspiration pneumonia. MATERIALS

AND

METHODS

Animals. The piglets were pure Large White which were obtained, managed and monitored as described by Trexler (personal communication). Piglets that died were removed with aseptic precautions from their isolators as soon as observed. Animals killed for examination were given a lethal dose of sterile sodium pentobarbitone by intraperitoneal injection and removed aseptically. All animals were examined immediately after death. Histological examinations. Tissueswere fixed in neutral 10 per cent. form01 saline, embedded in paraffin, and routinely stained by haematoxylin and eosin, Gram’s method, von Kossa’smethod, periodic acid Schiff, and for iron by Perls’ Prussianblue reaction. Microbiological examinations. In addition to the routine system for the detection of contamination carried out during life (Trexler, personal communication), bacteriological cultures were made from each piglet from the followin organs and tissues: axillary skin, nose, throat (tonsillar region), turbinates, trac a ea, bronchi of two pulmonary lobes, and the bronchial, presternal and prescapular lymph nodes; the parietal pleura, pericardial fluid and heart blood; the contents of the jejunum and colon and faeces; the liver, lung, spleen, kidney and the peritoneal fluid. In each case, liberal loopfuls of material were taken from each site or the surface was rubbed vigorously with a dry swab. A pair of chocolate yeast agar plates and a tube of nutrient and thioglycollate broth were inoculated with material from each site. One set of plates was incubated aerobically, the other anaerobically at 37OC. for 14 days. All cultures were inspected daily for growth and on the 4th and 14th day inoculations were made from each broth culture onto chocolate yeast agar plates. The plates inoculated from the nutrient broths were incubated aerobically and those from the thioglycollate medium anaerobically : they were inspected daily for 14 davs for evidence of microbial growth. Small pieces (abuut 0.05 to 0.20 g.) of both diseasedand apparently normal areas of each lung were removed aseptically and disintegrated in one ml. of phosphate

238

ASPIRATION

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IN

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PIGLETS

buffer (pH 7.2) in a Mickle disintegrator. Samples from these preparations were included in the bacteriological examinations already described. From lung specimens that had been stored at about -65OC. for approximately three months, pieces weighing between about 0.1 to 0.3 g. were ground to prepare 20 per cent. suspensions in Earle’s and mycoplasma medium respectively. The latter medium was that of Goodwin, Pomeroy and Whittlestone (1965) as modified by Hodges (personal communication). These suspensions were passed through membranes of 450 rnp A.P.D.” and the filtrates inoculated into mycoplasma medium, and onto cultures of primary pig kidney cells and a swine kidney cell line. Three serial, blind passages of these cultures were made in a search for viruses and mycoplasmas. The tissue cultures were examined for cytopathic effects and haemadsorption tests were made by application of suspensions of human and bovine erythrocytes to these cultures. Agar gel diffusion. Antiserum was prepared by the intravenous injection of an adult rabbit on three occasionsat 4day intervals with one, two and two ml. of the sterile diet fed to the piglets made up of 50 per cent. milk and 50 per cent. glucose supplemented with a mixture of minerals. Gel diffusion tests were carried out by standard methods using the milk+ as antigen in a central well. RESULTS

Clinical 0 bservations The piglets of the first group (Nos. 1, 2 and 3) were survivors of a litter removed from the uterus 13 days before normal parturition was expected (Table 1). The other members of the litter died within 24 hours. The survivors were fed three times daily by trickling a few ml. of sterile milk, containing added glucose and a TABLE ASPIRATION

Piglet

No. days survived

NO.

from

PNEUMONIA

1

IN FIVE

CNOTOBIOTIC

Severity

delivery : 3

died killed 3,

4 17

20

: 4

4 5

died ,3

3 9

2 4

- = absent or virtually absent. +, + +, + + + = slight, extensive,

PIGLETS

of abnormality

Micro-organisms

present

Macroscopic

Microscopic

++ -

+ -t + +

none detected 1. ,I >> >,

+++ +-t+

Stajh epidcrmidis I, >,

+++ + + fvery

only a>

extensive.

solution of inorganic salts, including 1 per cent. FeS04, into the mouth from a dropper. During the first few days the piglets were more or less continuously recumbent and one (No. 1) died four days after delivery. The others (Nos. 2 and 3) became active physically on the fifth day and were then able to drink from troughs; no hand feeding was practised from that day. They were killed for examination 17 to 20 days respectively after birth, and both were healthy in the clinical sense. In the second litter some deaths were recorded within 48 hours of surgical removal, and hand feeding, as described above, was attempted for two particularly feeble piglets. One (No. 4) died on the third day and the other (No. 5) on the ninth day after delivery (Table 1). * Millipore t Carnation

Filter Milk:

Corporation. Carnation

Food

Co., Ltd.

I.

Microbiological

M.

SMITH

AND

A.

H.

239

S. HAYWARD

Findings

No evidence of the presence of viruses, mycoplasmas, bacteria or fungi was found in any material from piglets 1, 2 or 3. Piglets 4 and 5 were mono-contaminated with a bacterium with the characters of Staphylococcus epidermidis (Cowan and Steel, 1965; Branson, 1968). This coagulase and mannitol-negative organism was found on the skin, in the throat, gut and faeces, and in the upper respiratory passages. The numbers in the lungs were much greater in macroscopically abnormal than in apparently normal tissue. The organism was not recovered from any other site examined, including the bronchial lymph nodes Macroscopic

Findings

Of the five pigs, three (1, 4 and 5) showed gross pulmonary changes (Table l), the extent of which was not wholly evident on the uncut lungs. Abnormality was greatest in the hilar region of the lungs of each animal and was most severe in the medial aspects of the cardiac and diaphragmatic lobes, although .all lobes were involved to some extent (Figs. 1 and 2). Both lungs were approximately equally affected. The macroscopic lesions in the anterior lobes were of two types both of which were essentially areas of consolidation. The first consisted of dark red areas about two mm. across and up to about two cm. in length : they were sharply demarcated from apparently normal tissue and the consolidation was essentially peribronchial. The other lesion consisted of very deep red, more or less circular areas with irregular, diffuse margins. In piglet 1, discrete, circular, white areas about one to two mm. in diameter were also prominent superficially in

Dark-red Consolidation

peribronchial

consolidation

and congestion

with lobular

Discrete circular white areas l-2mm. apparently accumulations of milk Fig.

1.

Piglet

5-Schematic (a) dorsal

extent of macroscopic (b) ventral.

pulmonary

distribution

diameter,

damage

as determined

by dissection

240

ASPIRATION

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PIGLETS

the diaphragmatic lobes. They appeared to be accumulations of milk that was not provoking any reaction and may have represented part of the last feed. In piglets 2 and 3 no macroscopic abnormality was observed except for a possible distortion of the right cardiac lobe in 3. With two exceptions, no other organ or tissue in any of the piglets was obviously abnormal. Piglet 2 was severely lipaemic and the cut surface of several organs, notably the liver, exuded a milky fluid. Piglet 4 had small areas of a dry epidermal necrosis, about 8 x 3 mm. in size, involving the tip of the tail and the underside of the chin. Histological

Findings

The principal change in all pig lungs occurred in the alveoli and terminal bronchioles as scattered and often massive lobular damage. In piglet 1 (Table 1) severe haemorrhage was present in some lobules. In other lobules the alveolar pattern was seriously disrupted and the interstitial tissue was heavily infiltrated predominantly with macrophages and lymphocytes (Fig. 3). Almost all lobules in areas observed as macroscopically abnormal either showed this interstitial pneumonitis of varying severity or were emphysematous. In many alveoli and terminal bronchioles in these areas, amorphous, densely-staining masses were present (Fig. 3). This basophilic material was regarded as the diet fed (which contained 1 per cent. FeSOe) because it was negative for calcium by von Kossa’s method, and gave a positive P.A.S. reaction and a positive Prussian Blue reaction for iron. The material was sometimes infiltrated with macrophages and small round cells (Fig. 3). It was not detected in the lymphatic channels. The smaller bronchioles also contained this material and its presence was accompanied by some epithelial desquamation, plugs of cellular exudate and cellular infiltration of the bronchiolar walls. The bronchi and trachea were virtually free from exudate and appeared normal apart from one slight haemorrhage in the trachea. In piglets 2 and 3 (Table 1) the lesions described above were resolving and comparatively few alveoli in affected lobules contained the amorphous material. The interstitial pneumonitis was less marked although, in comparison with normal germ-free piglet lung (Fig. 4), some lobules had thickened alveolar walls (Fig. 5) as a result of cellular infiltration. A less dense, rather stringy material believed to be the remnants of the aspirated material was, however, still present in some alveoli and bronchioles (Fig. 5), and, whereas in piglet 1 the positive Perls’ reaction was given by amorphous material that filled many alveoli and bronchioles, in piglets 2 and 3 the reaction was mainly confined to the cytoplasm of macrophages that were free in the lumina and in the walls of the small air spaces. Thus, 15 days after damage could last have arisen from hand feeding, some lobules were still abnormal and in a few alveoli, free alveolar macrophages were unusually prominent. These various changes were much more obvious in piglet 2 than in 3 although no abnormalities were seen in either the bronchi or trachea. In piglets 4 and 5, the basic pattern of damage was similar to that seen in piglet 1 (Table 1). However, the basophilic material was much more sparse, and alveolitis and bronchiolitis were comparatively greater and accompanied by a change in the cellular pattern of infiltration, neutroph& being readily observed

I.

M.

SI’dXTH

AND

A.

IX.

S. HAYWARD

241

in damaged tissue (Fig. 6). Moreover, more free alveolar macrophages and neutrophils were present in the alveoli and bronchioles, and the degree of desquamation of bronchiolar epithelium was much more extensive (Fig. 6) than in piglet 1. These differences were probably induced by the presence of Staph. epidermidis. About 5 x 10’ organisms per gramme were recovered from these abnormal areas, and in sections the bacteria were readily detected in the exudate composed of free alveolar macrophages and neutrophils which was present not only in affected alveoli and terminal bronchioles but also in the walls of these structures (Fig. 7). Staph. efidermidis was also recovered in small numbers from apparently normal areas of the lungs of piglets 4 and 5. In these areas, in some lobules that appeared otherwise virtually normal histologically, cocci had, however, penetrated alveolar walls to produce small haemorrhages which were the first sign of pathological change and probably explained the general impression of a greater amount of haemorrhage in the lungs of piglets 4 and 5 compared with 1. The continued multiplication of the cocci in the alveolar and bronchiolar walls presumably led to the cellular responses already mentioned. In these two piglets patchy desquamation of epithelium in the bronchi and trachea was also detected. In piglets 1 and 4, changes in the bronchial lymph nodes were not obvious in comparison with the same lymph node from a normal germ-free piglet, but in piglets 2, 3 and 5 a relative lymphoid hyperplasia with the emergence of active germinal centres had occurred. In addition, in piglet 5 the sinusoids were packed with macrophages. Thus, while there was a clear response in the nodes, the peribronchial and perivascular lymphocytic cuffing, which is found regularly in the lungs of normal and pneumonic pigs (Jericho, 1966), was notably absent in the lungs of any of the piglets described here. Gnotobiotic piglets only a few days old are known to be immunologically competent (Watson, Kim and Bradley, 1968) but, despite the appearance of activity in the bronchial lymph nodes of piglets 2 and 3, their sera did not contain any detectable amount of precipitating antibody against milk constituents when compared with the positive rabbit an-. DISCUSSION

The staining reactions of the amorphous material in the lungs suggest very strongly that it originated from the diet on which the piglets were fed. Although the damage produced was not lethal in each instance, caution is obviously necessary in the management of gnotobiotic piglets to avoid accidental production of pulmonary lesions for, as shown, this damage can persist for at least about a fortnight even in the absence of micro-organisms. The unsuspected presence of such damage might lead to unwarranted assumptions about either the pathogenicity or lack of pathogenicity of experimentally inoculated agents because the lung was already undergoing an inflammatory r=ponse. However, in other work in this Department, no evidence has been detected of pulmonary damage among comparable gnotobiotic piglets which had fed naturally by drinking from the time of delivery onwards. This evidence has been derived so far mainly from piglets aged about 10 to 21 days; at the latter age, minor lung damage from aspiration might be hard to detect. The histological changes in affected lungs when Staph. epidermidis was also

242

ASPIRATION

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PIGLETS

present were striking. This coccus is generally considered to be non-pathogenic in normal animals (Evans, 1957) although it is known to persist in mouse kidneys after intravenous inoculation (Smith and Dubos, 1956). It may be capable of producing low grade disease in the human urinary tract and the bovine mammary gland, and of producing severe illness in debilitated persons (Morse, 1965 ; Branson, 1968). Whilst it seems probable that the presence of milk in the lungs favoured the multiplication of the staphylococcus it is impossible to be certain that the organism, per se, initiated pneumonitis. However, once established in the lung, possibly in devitalised areas, the organism could apparently damage alveoli and bronchioles in lobules that did not show any histological signs of previous abnormality. Since the coccus was capable of attacking such tissues, it would appear that very young gnotobiotic piglets in isolators are unusually susceptible to at least some infectious agents, for the piglets were apparently incapable of controlling Staph. epidermidis which is very readily destroyed by phagocytosis (Tompsett, 1954). The fact, therefore, that a given agent is pathogenic for such piglets may not be relevant to the situation in piglets in their natural state. This view receives some confirmation from the occurrence in one piglet of skin lesions also in which the coccus was multiplying. When first observed, the possibility that this piglet might have had vegetative dermatosis (HjZ-re, 1953; Done, Loosmore and Saunders, 1967) was considered, but feet lesions were absent, no giant cells were detected in the lungs and no crossbreeding with Landrace pigs was known to have occurred. As in the lung, it is not possible to say whether these skin lesions arose in devitalised tissue or whether the coccus actively initiated lesions in healthy tissue. However, other piglets of similar ages to those described in this paper have, on occasion, been detected as contaminated by Staph. epidermidis without lesions of any kind being found. In general, therefore, it may be presumed that the coccus is capable of invasion only in the debilitated animal. SUMMARY

Five feeble gnotobiotic piglets were fed through droppers with sterile bovine milk, containing glucose and added mineral salts, for up to five days after they were delivered by caesarian section. Two died within four days, one on the ninth day and two were killed 17 and 20 days respectively after delivery. All three that died had macroscopic pneumonia at post-mortem examination. Histologically, the lesions were those of severe haemorrhage and interstitial pneumonitis with the presence of amorphous material, presumably milk, in many alveoli and the bronchioles of many lobules, particularly in the hilar regions. This material was infiltrated with lymphocytes and macrophages as were the alveolar and terminal bronchiolar walls. The severity of these changes seemed to be accentuated when Staph. epidermidis was present in the lung as it was in two piglets. The organism multiplied and stimulated a bronchopneumonia and also seemed to be capable of attacking alveoli in apparently normal lobules, initiating small haemorrhages that were followed by a reaction of alveolar macrophages and by neutrophilic infiltration. In two piglets in which forced feeding ceased on the fifth day after delivery, macroscopic pneumonia was absent when they were killed 17 and 20 days, respectively, after delivery, but some interstitial pneumonitis in the form of infiltra-

I.

tion with remnants present.

M.

SMITH

AND

A.

H.

S. HAYWARD

243

small round cells and cellular reaction by macrophages around the of aspirated material in the alveoli and terminal bronchioles was still

ACKNOWLEDCMENTS

The work, part of which is described in this paper, is supported by the Pig Industry Development Authority and thanks are recorded for its financial assistance. Gratitude is expressed to Messrs. R. T. Hodges and R. Watt, Department of Pathology, Royal Veterinary College for valued help with the examinations for mycoplasmas and virtue+ and to Professor A. 0. Betts, Dr. E. C. A pleby and Dr. K. Jericho for constructive criticisms of the presentation and for o t.Ker suggestions. REFERENCES

Branson, 0. (1968). Appl. Microbial., 16, 906. Cowan, S. T., and Steel, K. J. (1965). M anual for the Identification of Medical Bacteria, University Press; Cambridge. Done, J. T., Loosmore, R. M., and Saunders, C. N. (1967). Vet. Rec., 80,292. Evans, J. B. (1957). In M anual of Determinative Bacteriology, Bergey, 7th Ed., Bailliere, Tindall & Cox; London. Goodwin, R. F. W., Pomeroy, A. P., and Whittlestone, P. (1965). Vet. Rec., 77, 1247. HjHrre, A. (1953). Dtsch. tieriirztl. Wschr., 60, 106. Jericho, K. W. F. (1966). Vet. Bull., 36, 687. Jubb, K. V. F., and Kennedy, P. C. (1963). Pathology of Domestic Animals, 1, p. 155, Academic Press Inc. Ltd.; London. Morse, S. I. (1965). In Bacterial and Mycotic Infections of Man, 4th Ed., Pitman Medical Publishing Co. Ltd.; London. Smith, J. M., and Dubos, R. J. (1956). 1. exp. Med., 103, 87. Tompsett, R. (1954). Bull. N.Y. Acad. Med., 30, 480. Watson, P. W., Kim, Y. B., and Bradley, S. G. (1968). Advances in Germ-free Research and Gnotobiology, Ed. Miyakawe and Luckey, IlifFe Books Ltd.; London. [Received for publication,

September 28th, 19681

244

ASPIRATION

PNEUMONIA

LEGENDS

GNOTOBIOTIC

TO

PLATES

Piglet

5-Macroscopic

Fig. 3.

Piglet

l-Accumulations of aspirated material (basophilic, iron and PAS positive and von Kossa negative) in alveoli and bronchioles infiltrated by macrophages and lymphocytes and interstitial pneumonitis. H & E x 120.

Fig. 4.

Normal

Fig. 5.

Piglet

3-Remnants

Fig. 6.

Piglet

5-Masses of macrophages, neutrophils and desquamation of bronchiolar

Fig.

Piglet

5-Cellular

7.

from

uninoculated, of aspirated

exudate

of consolidation

in anterior

PIGLETS

Fig. 2.

lung

lesions

IN

lobes

germ-free

piglet

aged 15 days.

material

in lung.

H & E

and staphylococcal

of right

x

120.

and staphylococci in alveoli epithelium. H & E x 120.

and

clumps

x

H & E

lung.

120.

in alveoli.

Gram.

x

900.

bronchioles