-

Zbl. Vet. Med. B, 22, 205 219 (1975) @ 1975 Verlag Paul Parey, Berlin und Hamburg ISSN 0044-42941ASTM-Coden: ZVRBAZ

Veterinary Research Institute, Brno, Czechoslovakia

Incidence and Evaluation of the Microbial Flora in the Lungs of Pigs with Enzootic Pneumonia BY M. Go13, F. SISAK, F. KUKSAand M. SOVADINA With 6 tables (Received for publication June 12, 1974)

Introduction Chronic diseases of the respiratory tract are very frequent in pigs reared in large-capacity piglet houses and fattened in large feeder pig operations. The syndrome is generally referred to as enzootic pneumonia (E. P.). In our opinion, it is caused by a number of microbial agents, although many writers BETTS and (MARB and SWITZER,1965; GOODWINet al., 1965; HODGES, JENNINGS, 1969; L’ECUYER, 1969) have ascribed a decisive role in its development to Mycoplasma hyopneumoniae (M.suipneumoniae). There is evidence to suggest that an activity similar to that of M . hyopneumoniae can be developed in the pig lung also by M. hyorhinis (MARTINet al., 1968; Go13 and VAL~CEK, 1968; GOISet al., 1968, 1971; FRIIS,1971). In pigs affected naturally with E. P., search has generally been made only for some organisms of major aetiological importance. To date, the syndrome has not been studied as an entity of complex aetiology. This study presents the results of microbiological examination of the lungs of slaughter pigs affected with E. P. The specimens were examined for mycoplasmas and aerobic bacterial flora by standard microbiological techniques. The isolates were evaluated with respect to their prevalence and the extent of lung lesions and some were also examined for their pathogenic activity in gnotobiotic piglets. Materials and Methods Collection and Processing of Specimens The lungs were obtained from pigs kept in a large-capacity fattening unit which was supplied by 18 farms with piglets approximately 25 kg. in weight and it produced about 20,000 slaughter pigs per year. The animals were fed in air-conditioned fattening houses equipped with self-feeders. Each building housed 1,OOO-I,2OO pigs kept in iron-bar pens in groups of 15-20 animals. Frequent and severe coughing of many pigs suggested a severe form

206

GOIS,SISAK, KUKSAand SOVAIXNA

of E. P. which was confirmed by extensive lung lesions in the majority of the specimens. T o evaluate the lung lesions, the following 0-3 scale was employed: 0 = no or healed lesions; 1 = slight lesions up to 2 sq. cm. in size, occurring in one or more lobes; 2 = well-marked lesions up to 5 sq. cm. in size in 1-2 lobes; 3 = extensive lesions in 3 or more lobes. Portions of the affected lungs, mostly of the apical and cardiac lobes, were collected aseptically from pigs approximately IOO kg. in weight. The appearance of the lung lesions generally suggested a process of long duration. “Fresh”, plum-coloured lesions, sharply demarcated from adjacent normal tissue, changed in the distal parts to lesions grey t o greyish in colour and of rather solid consistency showing a nodular surface and fissures. “Fresh” lesions, where present, were collected for examination. Portions were fixed in chilled acetone immediately after removal of the lung while others were processed as 10 per cent suspensions in Hanks’ solution about 4 hours later and examined for mycoplasmas. The remainder of each sample was examined bacteriologically under conditions of aerobic cultivation.

Isolation and Identification of Mycoplasmas Suspensions of 10 per cent tissue homogenates from lung specimens were inoculated in 0.1-0.2 ml. amounts into tubes of mycoplasma growth fluid medium of the composition described previously (Go16 et al., 1968) except that the proportion of inactivated horse serum was reduced from 20 to 10 per cent and the remaining 10 per cent were replaced by pig serum from specificpathogen-free (SPF) pigs. The medium contained either 1 per cent glucose or 0.1 per cent L-arginine HCl. Each suspension was inoculated into a t least two tubes of glucose- or arginine-containing medium. The cultures were incubated at 37 O C and passaged, for the most part, a t 48- to 96-hour intervals. The majority of samples suspended in glucose-containing medium were passaged in addition a t weekly intervals. This applied particularly to those materials which were suspected of containing M . b y orhinis and M . hyopneumoniae. After 5-7 weekly passages M . b y orhinis was generally dead, whereas M . hyopneumoniae continued to multiply. If M . hyorhinis was not eliminated by passage a t these weekly intervals, the samples were diluted or supplemented with hyperimmune rabbit serum against M . hyorhinis and subjected to further passage. The growth of mycoplasmas in fluid media was verified by inoculation onto 1 per cent agar plates containing the same components as the fluid media. The agar plates were incubated aerobically and anaerobically (nitrogen 5 per cent carbon dioxide, v/v). For the identification of mycoplasmas, the growth-inhibition test (CLYDE,1964) was employed in all cases. Mycoplasma arginini strains were identified in addition by the metabolic-inhibition test (PURCELL et al., 1966), using the prototype strain G 230 (Dr. Tully, NIAID, National Institutes of Health, Bethesda, Maryland, U. S . A.). Moreover, mycoplasmas were also identified directly in tissue sections using the indirect immunofluorescence technique.

+

Isolation and Identification of Bacteria Excisions taken from the lung lesions after surface cauterization were smeared over the surface of Endo and blood agar plates, and the applied material was spread with a bacteriological loop. The identification of bacteria was made by standard microbiological methods (COWANand STEEL, 1965; BAILEYand SCOTT,1970). Growth properties of some strains were followed in addition on MacConkey agar (BBL), Koser medium containing sodium citrate and in gelatine. To detect haemophili, use was made of the phenomenon of

Incidence and Evaluation of the Microbial Flora in the Lungs of Pigs

207

satellitism round the growth of Staphylococcus aureus on blood agar and PPLO agar (Difco) supplemented with 5 per cent horse serum, 0.1 per cent dextrose and 2.5 per cent yeast extract. Differential diagnostic tests were employed particularly for demonstration of Pasteurella (MRAz et al., 1971), Neisseria, Moraxella, Achromobacter et al., 1968; and similar Gram-negative coccobacillary organisms (BAUMANN WILCOX,1970). To differentiate Diplococcus pneumoniae from alpha-haemolytic streptococci, the optochin test and the assay of pathogenicity for mice (MERRILL et al., 1973) were employed.

Infection of Gnotobiotic Piglets Piglets were obtained by hysterectomy of SPF Large White sows and 1963). were reared artificially on a sterilized diet (HOLUB, The techniques used for bacteriological control of the piglets have been described previously (Go16 et al., 1971). A t 3-5 days of age, the piglets were infected intranasally with 0.5 ml. of well-grown cultures of some microbial species as given in the sections to follow. They were killed at various intervals over a period of 34 days after infection and examined particularly for changes in the respiratory tract; the organs were subjected to bacteriological examinations. Results Microbiological Findings A total of 79 lungs examined yielded 19 microbial species. The numbers of isolations are presented in Table 1. It can be seen that the most frequently demonstrated organism was M . hyorhinis which was found in 51 (i. e. 64 per cent) lung specimens, followed in descending order of frequency by M . hyopneumoniae ( M . suipneumoniae), Streptococcus spp., and Pasteurella multocida demonstrated in 39, 33 and 28 (i. e. 49, 41 and 35 per cent) lung specimens, respectively. The numbers of individual organisms isolated from the lungs were related to the extent of lung lesions (Table 1). This relation seemed worthy of consideration, particularly for those organisms whose prevalence in the lung was rather high. It can be seen, however, that none of these organisms proved so highly pathogenic that its incidence would be found exclusively in extensive lung lesions. Nevertheless, it is worthy of note that M . hyorhinis occurred in almost 90 per cent of the lesions. O n the other hand, it can be seen that organisms with a high frequency of occurrence were demonstrated also in the lungs without gross changes or showing only healed lesions. Table 2 shows the relation between the number of microbial species and the extent of lesions in the lungs. It can be seen that the more extensive the lung lesions, the larger the number of the microbial species involved. Five microbial species represented the maximum number isolated from one lung. Large numbers of M . b y orhinis and M . hyopneumoniae strains demonstrated in the lungs are due to the use of two methods for mycoplasma detection. Unlike bacteria, which were demonstrated only by cultural evidence, mycoplasmas were detected in addition by immunofluorescence owing to the difficulties of their cultivation. The use of two methods for mycoplasma demonstration is therefore to be regarded as a necessity rather than an advantage. As can be seen from Table 3, the findings obtained by the two methods are far from being in agreement. A surprising result was the relatively large number of mycoplasma strains detected only by immunofluorescence.

GOIS, SISAK, KUKSAand SOVADINA

2C8

Table

1

Relation between number of organisms isolated from the lung and extent of lung lesions in 79 pigs Organisms isolated

ier cent

lo. solations

healed

slight

*

wellmarked

Fxtensive

4

28

+

9

16

M. arginini

8+

4

4

M. hyosynoviae

3'

1

M. hyorhinis

51

M. hyopneumoniae

39

I 16

Sfreptococcus spp.

33

41

Pasteurella rnulfocida

28

35

12

Slreptomyces spp.

19

24

I

Klebsiella aerogen. and Klebsiella spp.

16

20

4

Micrococcus spp.

12

15

L 1

Acinefobacter anifraturn

I

Escherichia coli

4

1

Diplococcus pneurnoniae

3

3 2

Neisseria cafarrhalis

3

Staphylococcus aureus

2

Corynebacferium pyogenes

2

Bacillus spp.

2

Bordetello bronchiseptica

1

Haemophilus parasuis

1

1

Proteus vulgaris

1

1

2

Total number of organisms Total number of lungs

*

41 I 14

104 I 31

Including demonstrations of mycoplasmas by immunofluorescence

Table 2 Relation between lung lesions and number of microbial species isolated from the lung microbial species isolated

'> Figures denote numbers of the lungs

Twenty selected lung samples were also examined for the concentration of three mycoplasma species. To determine the titres, the lung suspensions were passaged in serial tenfold dilutions. Details of the incubation time for M . hyorhinis and M . suipneumoniae are given in Materials and Methods. The resultant titres of the three mycoplasma species are presented in Table 4. I t can be seen that the highest concentrations were shown by M . hyorhinis. Our and HURREL (1970) findings are different from the observations of GOODWIN

Incidence and Evaluation of the Microbial Flora in the Lungs of Pigs

209

Table 3 No. of rnycoplasrna demonstrations

Mycoplasma

only by cultural evidence

only by imrnunofluorescence

by both cultural evidence and irnrnunofluorescence

Total

M. hyorhinis

31

I

13

51

M. hyopneumoniae

18

11

10

39

M. arginini

6

2

8

M. hyosynoviae

1

2

3

who found that the numbers of passageable particles of M . suipneumoniae generally exceeded those of M . hyorhinis. Streptococci were isolated from the lungs rather frequently. Judged by their morphological and some biochemical properties, they did not appear to constitute a species-homogeneous group. Eleven of these isolates were sent for typing to the WHO Streptococcus Reference Laboratory of the Institute of Hygiene and Epidemiology, Prague, Czechoslovakia. Attempts to classify our strains by the microprecipitation test with streptococcal extracts prepared from these strains according to Fuller, using antistreptococcal sera of Groups A-U, failed. Three of these strains were later identified as Diplococcus pneumoniae. They gave a positive reaction in the optochin test; when cultivated in Todd-Hewitt broth, one of them showed poor growth while the remaining two completely failed to grow. All three strains killed mice within 48 hours of i/p infection. Table 4 Mycoplasma titres in the lungs of slaughter pigs Titres of mycoplasrna isolations . . Lung No.

Mycoplasma hyorhinis

30 L 31 L

103 106

32 P

104

33 P 34 L 35 L 31 P 38 P 39 L 40 P

to'

41 L 42 P

104

Mycoplasma pncomoniae

Mycopbsma arginini

10'

102 10

'

106 104 102 103 103

102 103

104

43 P

lo5

44 L 45 L 46 L L1 L 40 L 49 P 50 L

- = negative

103 105 101 105 105

104

lo5

102

finding

Zbl. Vet. Med., Reihe B, Bd. 22, Heft 3

15

GoiS, hXhK, KUKSA and SOVADINA

210

Table I BioQemical differentiation of coccobacillary Gram-negative bacteria isolated from pneumonic lesions of pigs

0 0 28 28 0 0 23 0 0 0 28 0 12 (14) 0 9 I121 19 0 9 (13)

MacConkey agar Motility Hydrogen sulphide Nitrates Urea Koser citrate lndol Gelat ine Methyl red test Voges Proskauer test Glucose Lactose Saccharose Maltose Mannitol Sor bitol Salicin Cytochrome oxidase

-

+ = positive

tion;

+ --

+

lest employed

1

Acinetobacter anitraturn 7

Pasteurella No. strains tested

28 28 0 0 28 28 5 28 28 28 0 28 2 28 7 9 28 6

3

+

7

0

0 0 0 0 7 0 0 0

7 7 7 7 0 7 7 7 7 0 7 0 7 7 7 7 0

0 7

0 (4) 0 0 0 0 2t

Neisseria ca1ar;halis

0 0 0 0 0 0 0 0

0 0 0 0

0 0 0 0 0 3

Bordetella bronchisept ica 1

+ -3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 0

reaction; (+) = delayed or moderately positive reaction; reaction

- = negative

1

0 0 1 0 0

1

0

0 0 0

1

1

1 0

1

1 1 1 1 1 1

0

0

0 0 0

1

0

1 1 1 0

0 0

1

f = dubious reac-

The relatively high proportion of Pasteurella organisms isolated from the lung suggests their affinity to the respiratory tract. The strains isolated showed biochemical properties which identified them as Pasteurella multocida. In addition to these, Acinobacter anitraturn, Neirseria catarrhalis and Bordetella bronchireptica were found (Table 5). Table 6 Experimental infection of gnotobiotic piglets with some microbial strains isolated Infection of gnotobiotic piglets

Days after

No. piglets

Age (in days)

infection when piglets killed

M. hyorhinis H l 48

3

6

M. hyopneurnoniae H l L7

3

6

7,14, 21

M. arginini H l 42

5

5

5,6,9,11,15

M. hyosynoviae

3

6

7, 14.21

Dipl. pneurnoniae 774 b

3

5

3,6,10

Streptococcus spp. 853 b

3

5

3,649

Pasteurella rnultocida 861 a

5

3.6.9.12

fasleurella mullocida 75 318

4 4

5

3,6,

Bordetella bronchiseptica 595 b

L

5

5, 16, 22, 34

Haemophilus p a r a w i s

3

3

1.2.2

3

1,488

7.1L.21

~~

-

Klebsiella aerogenes

I

3

1

17,23

Incidence and Evaluation of the Microbial Flora in the Lungs of Pigs

21 1

Tests o f 10 Microbial Species for Pathogenic Activity in Gnotobiotic Piglets. Results of experimental infections are summarized in Table 6 . The piglets were killed a t various times after infection in view of the progress of clinical disease or the presumed pathological process. Clinical disease was developed only by piglets infected with M . b y orhinis, Haemophilus parasuis and Borde-

tella bronchiseptica. Of three piglets infected M . hyorhinis, two became clinically ill; one had polyserositis and polyarthritis, another had pleuritis and pericarditis and the remaining one showed pneumonic foci a t post-mortem. The concentrations of mycoplasmas in the affected portions of the lung amounted to 106-107. As in our previous experiments (GOISet al., 1971; Go19 et al., 1974), M . hyorhinis showed a tendency to persist in the lung in high concentrations for a considerable length of time. Of three piglets infected with M . hyopneumoniae, two had pneumonic foci and their mycoplasma titres reached 102; the third piglet was negative on culture. The potential pathogenicity for pigs of M . arginini seemed of interest in view of the fact that the findings in the present study represent practically the first isolations of these organisms from the pig in this country. M . arginini as well as M . hyosynowiae were therefore included among organisms tested in gnotobiotic piglets, even though their incidence in the lungs of pigs in the field was rare. M . arginini colonized, for the most part, merely the tonsils, although in one case it was isolated also from the lung (in a titre of lo4), spleen and blood of a piglet showing slight pneumonic lesions when killed 9 days after infection. Similar results were obtained with M . hyosynowiae which was recovered from the tonsils and, on one occasion, from grossly normal lungs (in a titre of lo1). Using the immunofluorescence technique, both M . arginini and M . hyosynowiae were detected a t the bronchiolar epithelial surface; one piglet infected with M . arginini had in addition marked positive fluorescence in the pleura a t the sites adjacent to pathologically changed portions of the lung. Diplococcus pneumoniae produced pneumonia in all three piglets, but gave rise to marked pneumonic foci in only two of them. The pneumonic lesions were located particularly in the apical and cardiac lobes. The lungs of Table 6 from the lungs of naturally infected pigs Macroscoplcal changes

I

I

Re-isolation of organisms

- = negative isolation attempts 15'

212

Got$ SISAK, KUKSAand SOVADINA

all these animals, including the third killed 10 days after infection, yielded diplococci in relatively large numbers. Histological changes suggested catarrhal bronchopneumonia. Alpha-haemolytic Streptococcus spp. (strain 853 b) which could not be classified within any of the serogroups A-U were found to colonize only the tonsil and nasal mucosa. Pneumonic lesions were not revealed in any of the piglets. Neither of two strains of Pasteurella multocida growing on meat-peptone agar in the smooth phase gave rise to marked pneumonic lesions in experimentally infected piglets. Both infecting strains produced moderate gross lesions only in animals killed within 3 days of infection; histological examination suggested catarrhal bronchopneumonia. The lungs of piglets killed 6 or more days after infection showed no gross pathological changes and were negative on culture. In one piglet infected with strain 861 a the organisms were isolated also from the kidney and liver. Both strains multiplied massively in the nasal cavities and tonsils. The mucous membranes of the nasal conchae and septa were slightly hyperaemic. N o other changes were observed. Of four piglets infected with Bordetella bronchiseptica, two had a cough for about 14 days and all the animals sneezed from time to time. Post-mortem examination of piglets killed 5 and 16 days after infection revealed slight pneumonic lesicns approximately 1 sq. cm. in size. An animal killed 22 days after infection showed only post-pneumonic scarring of the lung. The fourth piglet, killed 34 days after infection, had prominent healed and “fresh” lesions. Bordetella bronchiseptica organisms were present in the mucosae of the nasal conchae and septa of all piglets in massive amounts. Macroscopically, the mucous membranes were considerably swollen and covered with some mucus. Haemophilus parasuis was demonstrated only once in the field lungs examined in this study. When inoculated into gnotobiotic piglets, however, it proved to be a markedly pathogenic agent as it produced severe peracute septicaemic states in all three piglets within 24 hours of infection. One piglet died 30 hours after infection and the remaining two animals were killed at the point of death within 48 hours of infection. Haemophilus parasuis organisms were present in all the organs examined. Post-mortem examination revealed marked haemorrhagic exudates in the thoracic cavity and fibrinous peritonitis suggestive of sepsis. One cannot rule out the possibility that Haemophilus parasuis organisms might have been isolated more frequently if additional selective media had been used. Klebsiella aerogenes exerted no apparent patho enic effects on piglets during 8 days incubation. The animals were clinical y healthy and had no gross pathological changes in the lung a t post-mortem. Histologically, however, the lungs showed local bronchopneumonic foci and marked hyperaemia. Klebsiella organisms were isolated from the upper respiratory tract of all three piglets and from the lower respiratory tract, liver, spleen and kidney of two animals.

P

Discussion The finding in the present study of a remarkably broad spectrum of microbial species in the lungs of piglets with E. P. can be explained presumably by a consideration of the following two factors involved in the present animal husbandry practices: (1) Large-capacity fattening houses are supplied continually with weanling pigs coming from a large number of small herds. Most of these piglets arrive in the feedlot after their colostrum-derived anti-

Incidence and Evaluation of the Microbial Flora in the Lungs of Pigs

213

bodies have practically waned and with each group of new arrivals endowed with the specific microflora of the original herd. (2) The density of animal populations in large-capacity fattening houses is very high since one building can house as many as 1,000 pigs. This gives rise to a permanently highly infected environment where infection spreads from pig to pig particularly by contact and is responsible for a high prevalence of pneumonia. I n contrast, 107 lungs of control pigs from a SPF herd examined in the framework of another study were bacteriologically and macroscopically negative, an observation which suggests that a high incidence of organisms in the porcine lung is an anomalous phenomenon. The number of microbial species demonstrated in the lung increased with the extent of lung lesions. This finding suggests summation of the pathogenic activities, although it is not possible to estimate the contribution to the process by each species. Microbial synergism as a prerequisite for the development of extensive pathological processes finds support in extensive experimental work (1968). In a number reviewed by LOOSLI(1968) and by DEGRBand GLASGOW of studies, viruses have been incriminated as initiators of pathological processes potentiated by superimposed bacterial infection. Of interest in this connexion is also the observation of LIU et al. (1970) who found that pneumococcal bacteraemia of experimentally infected hamsters was more severe and frequent if the animals were infected previously with Mycoplasma pneumoniae. Similarly, well-marked lung lesions were produced in SPF and gnotobiotic piglets by experimental infection with mycoplasmas in combination with Pasteurella organisms (Dzu et al., 1971; SMITHet al., 1973 b). The phenomenon of qynergism was also observed in the infections of organ cultures with Myxovirus influenzae and M . hyorhinis (REED,1971). Moreover, a number of writers concerned with epidemiological studies described simultaneous outbreaks of two aetiologically different processes - viral and bac1967; YOUNGet al., 1972) or two viral infecterial (NICHOLand CHERRY, et al., 1966). tions (LEFKOWITZ and JACKSON, 1966; PORTNOY To determine which of the organisms isolated in the present study may play a decisive role in the development of pneumonic lesions, consideration was given to their prevalence in the lung and to the results of their pathogenic activity in colostrum-deprived piglets. Both approaches are subject to certain limitations. The former is limited by the possibility that some other agents - in particular viruses - may come into play. It can be assumed that viruses are involved in the given chronic processes as preparatory, contributory, provoking and possibly also relatively non-specific factors (Fox, 1968) which attenuate antibacterial activity of the lung (KASSet al., 1966; GREEN,1968). Assessment of the pathogenic activity of organisms in gnotobiotic piglets is also subject to certain limitations since the intensity of infection, clinical disease and pathological changes would not be so high in conventionally reared animals (BRENNAN et al., 1969; GOODWINand WHITLESTONE,1971; GOIS and KUKSA,1974). Nevertheless, the manifestations of the pathogenic activity in gnotobiotic animals are to be regarded as a useful source of information. This applies particularly to those cases where the organism produced marked reactions in the lung and persisted there for a long time. A point to bear in mind in evaluating both the prevalence and pathogenicity of organisms in experimental piglets is that the results may be substantially different from corresponding processes in naturally infected animals. We do not know, for example, to what degree the organisms were attenuated in virulence by passage on artificial nutrient media and we are in ignorance as to the virulence of the microbial populations and the extent to which the development of the

214

GOIS,SISAK, KUKSAand SOVADINA

process was related to the number of organisms and to the mode of their administration. From the evaluation of the prevalence and pathogenicity of the organisms it appears that the most effective part in the development of the chronic respiratory syndrome in this study is to be ascribed to M . hyorhinis. This, however, does not necessarily imply that in conventionally reared piglets the organism acts alone without requiring a triggering mechanism or co-operation of other agents. N o satisfactory explanation can be offered at present for the discrepancies between the mycoplasma findings obtained by cultural evidence and immunofluorescence. Negative fluorescence findings in cases where mycoplasmas were demonstrated on culture could be accounted for by actively produced antibodies as suggested by MEYLING (1 971). Conversely, positive fluorescence findings and negative isolation attempts could be explained by et al.,. 1969). mycoplasmacidal activity of some lung suspensions (KAKLAMANIS Recently, ENNISet al. (1972) reported persistent M . hyorhinis antigen in joints of pigs affected with chronic mycoplasmal arthritis, although the organism could not be cultured from the joint. M . hyopneumoniae appeared to have a role similar to that of M . hyorhinis, although the frequency of its incidence as demonstrated by cultural evidence and immunofluorescence was lower. A number of writers, however, have put forward the view that M . hyopneumoniae is the only causative agent 1965; GOODWIN et al., 1965; L'ECUYER,1969). of E. P. (MARBand SWITZER, In the present study, the titres of M . hyopneumoniae in the lung of experimentally and naturally piglets were lower than those reported by GOODWIN and HURREL (1970). Our infecting strain, which was administered to piglets intranasally at its seventh passage, produced generally slight lesions. Practically the same results were obtained in repeated experiments with the first passage of mycoplasmas from a suspension of lung specimens from gnotobiotic piglets. The findings of pneumonia in piglets infected with Diplococcus pneumoniae warrant further study. The data currently available on similar (1971), RILEYet al. (1973), observations in recent years are meagre. AGARWAL SCHNELER et al. (1973), using cultural methods and the direct immunofluorescence technique, have recently demonstrated the persistence in the piglet tonsils of group E streptococci which colonized the upper respiratory tract. Of the Pasteurella multocida strains employed in this study, none gave rise to regular development of lung lesions. Another strain isolated from pigs with E. P. and tested for pathogenicity outside the framework of the present experiments gave similar results. These findings are rather surprising in view of the relatively high prevalence of the strains in the pneumonic lesions. Similar results were reported by SMITHet al. (1973 a) and other writers who found that some of colostrum-deprived piglets infected experimentally with Pasteurella septica had severe intrathoracic lesions or generalized infection, an observation which suggests that some strains of this species ma occasionally be infective. In animals with naturally occurring E. P., the dif erences in the susceptibility of individual animals and in the virulence of strains are presumably equalized by the existence of other infectious processes in the respiratory tract. Combined experimental infection of gnotobiotic piglets with M . hyopneumoniae and Pasteurella organisms was reported to produce lung lesions considerably more severe than those reported in the present study (SMITHet al., 1973 b).

Y

Incidence and Evaluation of the Microbial Flora in the Lungs of Pigs

215

Our findings in piglets infected experimentally with Bordetella bronchiseptica are similar to those reported by other writers (DUNNEand KRADEL, 1961; L’ECUYERet al., 1961). However, in view of the solitary finding of

Bordetella bronchiseptica in the lungs of slaughter pigs these organisms are unlikely to play any major role in the development of the chronic respiratory syndrome in our conditions. The same applies apparently also to Haemophilus spp. which include causative agents of septicaemic diseases of piglets. The (University of strain identified as Haemophilus parasuis by Dr. J. NICOLET Bern, Switzerland) produced peracute septicaemia, the course of which was similar to that described in piglets infected naturally with Haemophilus parahemolyticus (NICOLET et al., 1969) that is presumably responsible also for outbreaks of E. P. A relatively high proportion (24 per cent) of organisms isolated from the lungs belonged to the species-rich family Streptomyces. No data are available concerning their pathogenicity for pigs. Monospora vulgaris and Thermomycetes have been incriminated as causes of hypersensitivity of the lung in man (HUGHES,1971). From the results reported here it appears that a number of microbial species may co-operate in producing pathological processes in the lung. The number of different species involved was related, to a certain extent, to the extent of lung lesions and suggests that the defence mechanisms were impaired and eventually overcome by multiple infections. Under these conditions the organisms could apparently not be suppressed effectively in the lung and gave rise to a chronic process. Of ten microbial species tested in gnotobiotic piglets, seven were responsible for the development of lung lesions to a less or greater extent.Their co-operation and co-existence are presumably a prerequisite for their successful activity and survival in the lung. Although the actual contribution to the process by each organism remains undisclosed, the present findings suggest summation of the individual pathogenic activities and can be regarded as the first steps to a study of the pathogenesis of E. P. of pigs as a disease of complex aetiology.

Summary Nineteen microbial species were isolated from the lungs of 79 slaughter pigs affected with enzootic pneumonia and the findings were related to the extent of lung lesions. The most frequently isolated organism was Mycoplasma hyorhinis (isolated from 64 per cent of the lung specimens) followed in descending order of frequency by M . hyopneumoniae, Streptococcus spp. and Pasteurella multocida (isolated from 49, 41 and 35 per cent of the lungs, respectively) and by a number of other organisms with a lower prevalence in the lung. The mycoplasma findings included in addition a few strains of M . arginini and M . hyosynoviae. Demonstration of mycoplasmas on the basis of cultural evidence proved more reliable than that by immunofluorescent staining. Strains of ten microbial species were employed for intranasal infection of gnotobiotic piglets aged 3-6 days. Clinical disease was produced only by infection with M . hyorhinis, Bordetella bronchiseptica and Haemophilus parasuis, the last-named of which was responsible for development of peracute sepsis. Infection with M . hyopneumoniae produced only slight pneumonic lesions. Two strains of Pasteurella multocida, M . arginini and M . hyosynoviae colonized predominantly the upper respiratory tract and were only occasionally recovered from other organs. Diplococcus pneumoniae and Bordetella bronchiseptica activated the lung tissue to gross pathological changes.

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Klebsiella aerogenes colonized the respiratory tract and produced bacteriaemia without gross pathological changes in the organs. It is suggested that enzootic pneumonia of pigs is an entity of complex aetiology. Zusammenfassung Vorkommen und Bedeutung der mikrobiellen Flora in Lungen von Schweinen rnit Enzootischer Pneumonie Neunzehn verschiedene Agentien wurden aus Lungen von 79 Schlachtschweinen rnit Enzootischer Pneumonie isoliert. Die Ergebnisse wurden in Beziehung zu den in der Lunge gefundenen pathologischen Veranderungen gesetzt. Am haufigsten wurde Mycoplasma b y orhinis (aus 64 O / o aller Lungen) isoliert, gefolgt von M . hyopneumoniae Streptococcus sp. und Pasteurella multocida (isoliert aus 49, 41 und 35 O/o der Lungen). Bei Mycoplasmen wurden zusatzlich noch einige Stamme vom M . argini und M. hyosynoviae isoliert. Der Nachweis von Mycoplasmen in der Kultur war sicherer als rnit Hilfe der Immunofluoreszenz-Technik. 10 Stamme der isolierten Agentien wurden fur die intranasale Infektion von 3-5 Tage alten gnotobiotischen Ferkeln verwendet. Klinische Erscheinungen traten nur nach Infektion mit M . hyorhinis, Bordetella bronchiseptica und Haemophilus parasuis auf, das zu einer perakuten Sepsis fiihrte. Die Infektion mit M . hyopneumoniae induzierte nur leichte pneumonische Erscheinungen. Zwei Stamme von Pasteurella multocida, M . argini und M . hyosynoviae siedelten sich vorwiegend im oberen Respirationstrakt an und wurden nur gelegentlich von anderen Organen isoliert. Diplococcus pneumoniae und Bordetella bronchiseptica fiihrten zu makroskopisch sichtbaren Lungenveranderungen. Klebsiella aerogenes vermehrte sich im Respirationstrakt und rief eine Bakteramie hervor ohne nachfolgende pathologische Veranderungen in den Organen. Es wird vorgeschlagen, dai3 die Enzootische Pneumonie der Schweine eine Faktorenerkrankung mit komplexer Atiologie ist. Resume Presence et signification de la flore microbienne dans les poumons de porcs atteints de pneumonie enzootique 19 agents diffkrents ont ktk isolks A partir des poumons de 79 porcs d’abattage atteints de pneumonie enzootique. Les rksultats sont donnks en fonction des lesions pathologiques trouvkes dans les poumons. Mycoplasma hyorhinis fut le plus frkquemment isolk (64 O / o de tous les poumons), suivi par M . hyopneumoniae, Streptococcus sp. et Pasteurella multocida (49, 41 et 35 O / o ) . O n a isolk en plus quelques souches de M . arginini et M . hyosynoviae. La recherche des mycoplasmes fut plus sOre avec les cultures qu’avec la technique de l’immunofluorescence. 10 souches parmi les agents isolks ont ktk utiliskes pour des infections intranasales chez des porcelets gnotobiotiques 9gks de 3-6 jours. Des symptbmes cliniques sont apparus aprks une infection avec M . hyorhinis, Bordetella bronchiseptica et Haemophilus parasuis se terminant par une septickmie suraigue. L’infection avec M . hyopneumoniae ne provoqua que de lkgkres lksiones pulmonaires. Deux souches de Pasteurella multocida, M . arginini et M . hyosynoviae se localiskrent avant tout dans les voies respiratoires supkrieures et furent isolks parfois dans d’autres organes.

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Diplococcus pneumoniae et Bordetella bronchiseptica provoqukrent des lksions pulmonaires macroscopiques visibles. Klebsiella aerogenes se multiplia dans l’appareil respiratoire, provoqua une bactkrikmie sans lksions pathologiques des organes. Les auteurs estiment que la pneumonie enzootique du porc est une maladie facteurs dont l’ktiologie est complexe. Resumen Incidencia y significacih de la flora microbiana en 10s pulmones de cerdos con pneumonia enzo6tica Diezinueve agentes diferentes se aislaron de pulmones de 79 cerdos de matadero con pneumonia enzobtica. Los resultados se relacionaron con las modificaciones histopatolbgicas halladas en 10s pulmones. Con frecuencia mayor se aislb Mycoplasma hyorhinis (del 64 O / o de todos 10s pulmones), seguido por M . hyopneumoniae, Streptococcus spp. y Pasteurella multocida (aislados del49, 41 y 35 O/o de 10s pulmones). Entre 10s micoplasmas se aislaron tambiCn algunas cepas de M . argini y M . hyosynoviae. La identificacibn de micoplasmas en el cultivo era mis segura que con ayuda de la tkcnica de inmunofluorescencia. 10 cepas de 10s agentes aislados se utilizaron para infectar por via intranasal lechones gnotobibticos de 3-6 dias de edad. Solo aparecieron manifestaciones clinicas tras infeccibn con M . b y orhinis, Bordetella bronchiseptica y Haemophilus parasuis, las cuales condujeron a una septicemia peraguda. La infecci6n con M . hyopneumoniae no inducia mis que ligeras manifestaciones pneumbnicas. Dos cepas de Pasteurella multocida, M . argini y M . hyosynoviae arraigaron sobre todo en el tracto respiratorio superior y solo se aislaron muy contadas veces de otros brganos. Diplococcus pneumoniae y Bordetella bronchiseptica conducian a modificaciones pulmonares visibles macroscbpicamente. Klebsiella aerogenes se multiplicaba en el tracto respiratorio y originaba una bacteriemia sin modificaciones patolbgicas subsecuentes en 10s brganos. Se propone admitir que la pneumonia enzobtica de 10s cerdos es una enfermedad factorial con etiologia compleja. References AGARWAL. K. K.. 1971 : Distribution of antimeumococcal antibodies in normal swine serum. Indian J. Anim. Sci., 41, 298-300. BAILEY,W. H., and E. G. SCOTT,1970: Diagnostic microbiology. 3 ed. The C. V. Mosby Co., St. Louis, 342. BAUMAN,P., M. DOUDOROFF and R. Y. STANIER,1968: Study of the Moraxella group. I. Genus Moraxella and the Neisseria catarrhalis group. J . Bact., 95, 58-73. BRENNAN, P. T. E. FRITZ and R. J. FLYNN,1969: Role of Pasteurella pneumotropica and Mycopjasma pulmonis in murine pneumonia. J. Bact., 97, 337-349. CLYDE,W. A., 1964: Mycoplasma species identification based upon growth inhibition by specific antisera. J. Immunol., 92, 958-965. COWAN,S. T., and K. J. STEEL,1965: Identification of medical bacteria. Cambridge Univ. Press, London, 217. DEGRB, M., and L. A. GLASGOW,1968 : Synergistic effect in viral-bacterial infection. I. Combined infection of the respiratory tract in mice with parainfluenza virus and Hemophilus influenza. J. inf. Dis., 118, 449-462. DUNNE,H. W., and D. C. KRADEL,1961 : Bordetella bronchiseptica (Brucella bronchiseptica) in pneumonia in young pig. Am. J. vet. med. Ass., 139, 897-899. Dzu, N. M., A. PUSTOVAR, W. BARTHKE, TH. HUBRIG, H. KRAUSSE und D. SCHIMMEL, 1971: Zur Ktiologie und Diagnose der Mykoplasmose der Schweine. I. Experimentelle Infektionen mit Mykoplasmen und Pasteurellen. Monatsh. Vet. Med., 26, 169-181. ENNIS,R. S., J. S. JOHNSON and J. L. DECKER, 1972: Persistent Mycoplasma hyorhinis (MH) antigen in chronic Mycoplasma arthritis of swine. Arthr. & Rheum., 15, 108-115. A

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GOIS, M., and F. KUKSA,1974: Intranasal infection of gnotobiotic piglets with Mycoplasma hyorhinis: Difference in virulence of the strains and influence of age on the development of infection. Zbl. Vet. Med., B, 21, 352-361. GOODWIN, R. F. W., A. P. POMERY and P. WHITTLESTONE, 1965: Production of enzootic pneumonia in pigs with Mycoplasma. Vet. Rec., 77, 1247-1249. GOODWIN, R. F., and J. M. HURRELL, 1970: Further observations on the problem of isolating Mycoplasma suipneumoniae from field cases of enzootic pneumonia in pigs. J. Hyg. Camb., 68,313-325. GOODWIN,R. F. W., and P. WHITTLESTONE, 1971: The comparative susceptibility of hysterectomy-produced, colostrum-deprived pigs and naturally born, enzootic-pneumonia-free pigs to enzootic pneumonia. J. Hyg., Camb., 69, 391-397. GREEN,G. M., 1968: Pulmonary antibacterial mechanisms and the pathogenesis of pulmonary disease. Yale J. Biol. Med., 40, 414-428. HODGES, R. T., A. 0. BETTSand A. R. JENNINGS, 1969: Production of pneumonia in gnotobiotic pigs with pure cultures of Mycoplasma hyopneumoniae. Vet. Rec., 84, 268-273. HOLUB, A., 1963: Semisynteticki vysokokaloricki dieta pro selata umrle odchovivanh od druhbho dne Fivota. Vet. Med., Praha, 2, 427-430. HUGHES, W . F., 1971: Hypersensitivity pneumonitis. N. Y. State J. Med., 71, 2851-2852. KAKLAMANIS, E., L. THOMAS, K. STAVROPOULOS, I. BORMANand C. BOSHWITZ,1969: Mycoplasmacidal action of normal tissue extracts. Nature, 221, 860-862. KAS, E. H., G. M. GREENand E. GOLDSTEIN, 1966: Mechanisms of antibacterial action in the respiratory system. Bact. Rev., 30, 488-496. L'ECUYER,C., E. D. ROBERTSand W. P. SWITZER,1961: An outbreak of Bordetella bronchiseptica pneumonia in swine. Vet. Med., 16, 421-424. L'ECUYER,C., 1969: Enzootic pneumonia in pigs: Propagation of a causative myoplasma in cell cultures and in artificial medium. Can. J. comp. Med., 33, 10-19. LEFKOWITZ, L. B., and G. G. JACKSON, 1966: Dual respiratory infection with parainfluenza and rhinovirus. The pathogenesis of transmitted infection in volunteers. Am. Rev. resp. Dis., 93, 519-526. LIU, CH., P. JAYANETRA and D. W. VOTH, 1970: Effect of combined Mycoplasma pneumoniae and pneumococcal infections in hamsters. Ann. N . Y. Acad. Scien., 174, 828-834. LOOSLI,C. G., 1968: Synergism between respiratory viruses and bacteria. Yale J. Biol. Med., 40, 523-539.

MAR&,C. J., and W. P. SWITZER,1965: Mycoplasma hyopneumoniae, a causative agent of virus pig pneumonia. Vet. Med., 60, 841-846. MARTIN,J., D. SCHIMMEL, H. KRAUSEund TH. HUBRIG, 1968: Zur Bedeutung von Mykoplasmen fur das Schwein. Mh. Vet. Med., 23, 652-658. MERRIL,C. W., J. M. GWALTNEY, J. 0. HENDLEY and M. A. SANDE,197: Rapid identification of pneumococci. Gram stain vs. the quellung reaction. The New England Journal of Medicine, 288, 510-512. MEYLING, A., 1971 : Mycoplasma suipneumoniae and Mycoplasma hyorhinis demonstrated in pneumonic pig lungs by the fluorescent antibody technique. Acta vet. Scand., 12, 137-141.

MRAz, O., F. MASSALEMA and M. SOVADINA, 1971: Diagnostika suspektnich kmeni Pasteurella multocida Vet. Med., Praha, 16, 19-28. NICHOL,K. P., and J. D. CHERRY, 1967: Bacterial-viral interrelations in respiratory infections of children. New. Engl. J. Med., 277, 667-672. NICOLET, J., H. KONIG und E. SCHOLL,1969: Zur Hamophilus-Pleuropneumoniae beim Schweine. Schweitz. Arch. Tierheil., I 1 1, 166-174. PORTNOY, B., H. L. ECKERT,H. L. HANES and M. A. SALVATORE, 1966: Multiple respiratory virus infectious in hospitalized children. Am. J. Epidemiol., 82, 262-272.

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PURCELL,R. H., D. TAYLOR-ROBINSON and R. M. CHANOCK,1966: Color test for the measurement of antibody to T-strain mycoplasmas. J. Bact., 92, 6-11. REED,S. E., 1971: The interaction of mycoplasmas and influenza viruses in tracheal organ cultures. J. inf. Dis., 124, 18-25. RILEY,M. C. I., L. G. MOREHOUSE and L. D. OLSON, 1973: Detection of tonsillar and nasal colonization of group E Streptococcus in swine. Am. J. vet. Res., 34, 1167-1169. SCHNELER, R. L., L. G. MOREHOUSE and L. D. OLSON,1973: A direct fluorescent antibody test for identification of group E streptococci. Can. J. comp. Med., 37, 327-329. SMITH,I. M., A. 0. BETTS,R. G. WATT and A. H. S. HAYWARD, 1973 a: Experimental infections with Pasteurella sePtica (sero-erouo A) and an adeno- or enterovirus in enotobiotic piglets. J. comp. Pith., 8j, 1-r2. SMITH.I. M.. R. T. HODGES. A. 0. BETTSand A. H. S. HAYWARD. 1973 b: Exoerimental infections of gnotobiotic piglets with Pasteurella septica (sero-group A) aloie or with Mycoplasma hyopnerrmoniae. J. comp. Path., 83, 307-321. WILCOX,G . E., 1970: An examination of Moraxella and related genera commonly isolated from the bovine eye. J. comp. Path., 80, 65-74. YOUNG,L. S., F. M. LAFORCE, J. J. HEAD,J. C. FEELEY and J. BENNETT,1972: A simultaneous outbreak of meningococcal and influenza infections. J. Med., 287, 5-9. *

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Author’s addresses: Dr. M. GOIS, Forschungsanstalt f. Veterinarmedizin, 62132 Brno/ Tschechoslowakei.