Parasitol Res (1992) 78 : 525 528

Parasitnlegy Research 9 Springer-Verlag 1992

Interaction between Ascaris suum and Pasteurella multocida in the lungs of mice Kirsten Tjornehoj 1, Lis Eriksen z, Bent Aalbvek i, and Peter Nansen 1 Department of Veterinary Microbiology and 2 Department of Clinical Studies, The Royal Veterinary and Agricultural University, 13, Bfilowsvej, DK-1870 Frederiksberg C, Denmark Accepted April 15, 1992

Abstract. In an experiment including 8 g r o u p s o f 15 mice, the effect o f migrating Ascaris suum larvae in the lungs on the establishment and pathogenicity o f aerosol exposure to Pasteurella multocida was investigated. Following aerosol exposure to P. multocida, mice with migrating A. s u u m in their lungs developed m o r e severe p n e u m o n i a a n d septicaemia t h a n did parasite-free mice. The parasite-induced effect on bacterial pathogenicity was m o r e m a r k e d for a n o n - t o x i n - p r o d u c i n g P. multocida as c o m p a r e d with a t o x i n - p r o d u c i n g strain o f P. multocida, possibly due to the higher s p o n t a n e o u s p a t h o g e n icity o f the non-toxigenic strain o f P. multocida. The present results should encourage controlled experiments on possible interactions between A. suum and various airborne microbial infections in pigs.

da in mice in the presence or absence of pulmonary migratory Ascaris suum larvae. In all, 4 groups (2, 4, 5 and 6) of mice were inoculated with 3800 infective A. suum eggs on day 0. On day 8 after inoculation with Ascaris, which is consistent with peak levels of larvae in the lungs (Eriksen 1981), groups 1 and 2 were exposed to an aerosol of a toxin-producing strain of P. multocida and groups 3 and 4 were exposed to an aerosol of a non-toxin-producing strain of P. multocida. Groups 7 and 8 served as uninoculated controls. Clinical examinations of the mice were carried out daily during the experiment.

Mice

Female BALB/c mice aged 7 weeks were separated into 8 groups of 15 animals each for the present experiments.

A. suum procedures Ascaris suum infections in swine m a y lead to reduced feed conversion (Eriksen et al. 1992), decreased g r o w t h (Andersen 1976; Stephenson et al. 1980; F o r s u m et al. 1981; Hale et al. 1985), gastro-intestinal disturbances a n d c o n d e m n a t i o n o f the liver at slaughter due to liver fibrosis (white spots; R o n e u s 1966). However, the impact o f the m i g r a t o r y phase o f the parasite in the lungs has thus far received little attention (Lloyd a n d Soulsby 1985), a l t h o u g h it has been s h o w n that this m a y p r o m o t e the establishment o f microbial p n e u m o n i a ( U n d e r d a h l and Kelley 1957). The present study was designed to investigate the possible effect o f migrating A. suum larvae in the lungs o f mice on the establishment a n d pathogenicity o f Pasteurella multocida, an i m p o r t a n t s e c o n d a r y p a t h o g e n o f respiratory diseases in swine (Gois et al. 1980).

Embryonation of A. suum eggs was performed as described by Eriksen (1981). Briefly, adult female worms were collected from baconers at slaughter. The eggs were subsequently obtained by dissection of worm uteri and were then decoated by stirring in a 0.5% sodium hypochloritc solution. After repeated washing and sedimentation to remove the hypochlorite solution, the eggs were suspended in 0.1 N sulphuric acid at a concentration of approximately 25 eggs/gl and then placed in closed Ehrlenmeyer flasks to a depth of 1-2 cm. The eggs were maintained in this acid at room temperature and were aerated by mechanical stirring two to three times per week. A recent investigation has shown that the infectivity to pigs of such treated eggs is comparable with that of naturally deposited and developed eggs (Oksanen et al. 1990). Each mouse was inoculated with 3800 infective A. suum eggs by stomach intubation using a blunt needle. Lung larval recovery was performed according to the Baermann technique previously described by Eriksen (1981).

Materials and methods

P. multocida procedures

E x p e r i m e n t a l protocol

The experiment was designed to study the pathogenicity of toxinproducing and non-toxin-producing strains of Pasteurella multociCorrespondence to : L. Eriksen

Two different strains of P. multocida multocida, capsular type A, were used: a toxigenic strain (number 2330, isolated from a pneumonic swine lung at the National Veterinary Laboratory, Copenhagen) and a non-toxigenic strain (number 420, isolated from the nasal mucous membrane of a specific-pathogen-free pig at the Na-

526 tional Veterinary Laboratory, Copenhagen). The strains were tested for toxin production as previously described by Foged et al. (1988). The strains were grown for 24 h in tryptic soy broth (Difco 0370-01-1). The culture fluid was aerosolised by a nebuliser (Inhalator, distributed by Kruuse Ltd., Odense, Denmark), the diameter of the particles in the aerosol ranging from 1 to 6/am (90% < 4.2/am). The mice were exposed groupwise in a 0.06-m3 closed chamber to a total amount of 7 ml broth culture containing approximately 2 x 108 bacterial cells/ml. The nebuliser was kept on for 15 rain to fill the chamber. The mice were then placed in the chamber and the nebuliser was kept running for 15 min with the chamber closed. Finally, the nebuliser was switched off and the mice were left in the chamber for a subsequent period of 15 min. The infection rate was determined by cultivation from organ impressions of the left lung and of the liver and spleen. Cultivations were carried out at 37~ for 24 h on tryptose blood-agar base (Difco 0232-01-9) supplemented with 5% bovine blood. Subcultures were identified as P. multocida on the basis of colony morphology similar to that of the inoculation strains (greyish, mucoid colonies with a diameter of 1-3 ram, a complete margin and a characteristic smell), cell morphology (Gram-negative rods, pleomorphism), and the following character set: catalase positivity, non-motility, urease-negativity, inability to grow on MacConkey agar, sucrose positivity, dulcitol negativity, mannitol positivity and sorbitol positivity. Motility was determined in stab cultures in heart infusion broth (Difco, 0038-01) supplemented with 0.25% agar (Difco, 0140-01). Catalase activity was determined using 3% hydrogen peroxide (Cowan 1974). The ability to produce urease was determined according to Cowan (1974). The ability to grow on MacConkey agar was observed on Merck medium (Darmstadt, FRG). Fermentation of carbohydrates was tested in meat extract-peptone broth [extract of dry meat (Merck, 3979), 1%; bacto peptone (Difco, 0118-01), 1%; NaC1, 0.3%; NazHPO4, 12 H20 (Merck, 6579), 0.2%; bromothymol blue (Merck, 3026), 0.024% ; pH 7.4) supplemented with 0.5% of the following carbohydrates/sugar alcohols added as filtersterilised solutions: sucrose, dulcitol, D(-)-mannitol and D(--)sorbitol.

P o s t - m o r t e m examination

Mice exposed to aerosols of the P. multocida strains were killed after 5 days (groups 1-4), and mice mono-inoculated with A. suurn were killed on days 8 and 12 post-inoculation (p.i.) (groups 5 and 6) together with uninoculated controls (groups 7 and 8). The animals were killed by intraperitoneal injection of pentobarbital followed by cutting of the vessels of the forelimb. Mice killed at the aforementioned intervals and those that died during the study were autopsied aseptically. Macroscopical lesions were recorded, i.e. liver lesions caused by migrating A. suum larvae, granuloma formation in the peritoneal cavity and lung lesions, including lung hemorrhages and pneumonia. Bacteriological cultures were recorded as showing negative growth, non-specific growth or growth of P. multocida from the lungs, liver or spleen, For each individual mouse, lung pathology was graded into three categories, i.e. normal lungs, pneumonic lungs showing negative or non-specific bacterial growth or pneumonic lungs from which P. multoeida could be isolated. The extent and distribution of lung lesions were compared with the results of bacteriological examinations.

Statistical methods

The results were compared by chi-square analysis.

Results Numbers of sick and dead mice are shown in Table 1. The non-inoculated controls (groups 7 and 8) were clinically normal and exhibited no pathological lesion at autopsy on days 8 (group 7) and 12 (group 8). Neither Pasteurella multocida nor Ascaris suum were demonstrated. N o n e of the mice mono-inoculated with A. suum (groups 5 and 6) showed any clinical signs. At autopsy, larval migratory lesions were noted in the liver, peritoneum and lung. On day 8 p.i. (group 5), several hemorrhages measuring 0.5-1 m m were found in the lungs and one mouse had developed focal aseptic pneumonia. The average n u m b e r of A. suum larvae recovered from the lung was 25. On day 12 p.i. (group 6), the lung hemorrhages had developed into diffuse areas of lung consolidation. The average n u m b e r of A. suum larvae recovered declined to 1.6. In 7 of the 30 mice in groups 5 and 6, non-specific bacterial growth was detected in the lungs. Mono-inoculation with toxigenic P. multocida (group 1) caused neither sickness nor death during the experimental period (Table 1). One-third o f these animals (i.e. five mice) developed focal lung consolidation; two cultures were negative, and three showed non-specific bacterial growth (Tables 2, 3). Inoculation with A . suum prior to aerosol exposure to toxigenic P. multocida resulted in severe sickness in 11 of the t5 mice in group 2, and 7 mice died during the experimental period (Table 1). Diffuse p n e u m o n i a and septicaemia were demonstrated, and P. multocida were recovered from 13 o f the double-inoculated mice (Table 2). Focal lung consolidation and negative bacteriological results were obtained in two of the mice in group 2 that showed no clinical symptoms (Table 3). The non-toxigenic strain of P. multocida given as a mono-inoculation produced clinical sickness and death in 7 of the 15 mice in group 3 (Table 1). These mice were found to have widespread pneumonic lesions and Pasteurella septicaemia (Tables 2, 3). A m o n g the animals that showed no clinical symptoms, one developed nonspecific pneumonia and one, a slight case of Pasteurella p n e u m o n i a . The remainder of this group was unaffected and did not suffer from pneumonia, and cultures of the lungs showed scanty, non-specific bacterial growth. All of the mice pre-inoculated with A. suum in group 4 developed Pasteurella septicaemia and died during the experiment (Table 1). Widespread Pasteurella p n e u m o n i a was demonstrated in 11 mice, whereas the remainder showed only focal p n e u m o n i a (Tables 2 and 3). A m o n g the mice exposed to the toxigenic strain of P. multocida, b o t h the n u m b e r of macroscopically demonstrable cases of p n e u m o n i a and the n u m b e r of P. multocida-positive cultures were significantly higher for the double-inoculated animals (group 2) than for the mono-inoculated mice (group 1; P < 0.001, Tables 2, 3). As the non-toxigenic strain of P. multocida caused severe sickness in 50% of the mice that had not been inoculated with A. suum (group 3), the results of our statistical analyses are somewhat less clear (Tables 2 and 3). Nevertheless, both the number of pathological lung

527

Discussion

Table 1. Number of sick and dead mice on day 12

Group

Inoculation

1

Pro+

2 3 4 5 6 7 8

A+Pm+ PmA+PmA A None None

Number of sick mice

Number of dead mice

0

0

11 7 15 0 0 0 0

7 7 15 0 0 0 0

A, Inoculation with embryonated Ascaris suurn eggs per os; Pm + , aerosol exposure to toxigenic Pasteurella rnultocida; P r o - , aerosol exposure to non-toxigenic P. multocida

Table 2. Results and bacteriological examination Group

Inoculation

Negative

Non-specific

Pasteurella multocida

reisolated 1

Pm+

10

5

0

2 3 4 5 6 7 8

A+Pm+ Pm-A+PmA A None None

2 0 0 11 12 15 15

0 4 0 4 3 0 0

13 7 15 0 0 0 0

A, Inoculation with embryonated Ascaris suum eggs per os; Pro+, aerosol exposure to toxigenic P. multocida; P r o - , aerosol exposure to non-toxigenic P. multocida

Table 3. Distribution of pathological lung lesions

Group

1

2 3 4 5 6 7 8

Inoculation

Pm+ A+Pm+ Pm -A+Pm-A A None None

Normal lungs

10 0 6 0 15 0 15 15

Lung consolidations Without

With

Pasteurella multocida

Pasteurella multocida

5

0

2 1 0 0 15 0 0

13 8 15 0 0 0 0

A, Inoculation with embryonated Ascaris suum eggs per os; Pm + , aerosol exposure to toxigenic P. multocida; P m - , aerosol exposure to non-toxigenic P. multocida

lesions (pneumonia) and the number of P. multocidapositive cultures were significantly higher (0.01 < P < 0.05) for the double-inoculated animals (group 4) than for the mono-inoculated mice (group 3).

The present study clearly demonstrates that in the period during which Ascaris suum larvae are migrating in the lungs, mice are more susceptible to pneumonia and septicaemia resulting from aerosol exposure to Pasteurella multocida of both toxigenic and non-toxigenic strains. In the present mouse experiment, we could not demonstrate increased lung pathology due to the production of toxin by P. multocida. This observation is in accordance with previous findings in swine (B~ekbo 1989). The course of the A. suum infection followed the pattern described by Eriksen (1981). The widespread lung lesions detected on day 12 were considered to represent a resolution stage of the parasite infection in the absence of bacteria. Future interaction experiments should also incorporate exposure of mice to aerosols of P. multocida on day 12 after A. suum inoculation, at which time the lung pathology reaches maximal levels, as compared with day 8, at which point the numbers of live larvae reach their maximum. Possible explanations for the deleterious effect of A. suum larvae in the lungs might include a direct effect on the alveolar epithelium or a result of decreased pulmonary bacterial clearance such as that previously shown for aerogen exposure to Escherichia colt in A. suum-inoculated mice (Curtis et al. 1987). Another possibility may involve an immunosuppressive effect of A. suum, as has been suggested by Lloyd and Soulsby (1985). In the experiments described above, A. suum was inoculated at rather high doses, but it is likely that even lower doses might aggrevate Pasteurella and other microbial infections. It would thus be relevant to compare the number of live third-stage larvae in the lungs per kilogram of body weight in mice and swine (Oksanen et al. 1990). Exposure to respiratory pathogens is common in modern pig production, and Ascaris larvae in the lung may enhance the susceptibility of the host to airborne pathogens. The result of the present investigation in mice should encourage controlled experiments in pigs on the possible interaction between migrating Ascaris larvae and airborne infections. References Andersen S (1976) The influence of Ascaris suum upon growth rates in pigs. Nord Vet Med 28:322-330 B~ekbo P (1989) Luftkvalitet og sundhed i svinestalde. Under sogelser af luftkvaliteten i danske svinestalde og patogenitetsstudier ved lungeinfektion med Pasteurella multocida. Ph.D. Thesis, Royal Veterinary and Agricultural University, Copenhagen Cowan ST (1974) Cowan and Steel's manual for the identification of medical bacteria, 2nd edn. Cambridge University Press, Cambridge Curtis SE, Tisch DA, Todd KS, Simon J (1986) Pulmonary bacterial deposition and clearance during ascarid larval migration in weanling pigs. Can J Vet Res 51 : 525-527 Eriksen L (1981) Host parasite relations in Ascaris suum infection in pigs and mice. Thesis, Royal Veterinary and Agricultural University, Copenhagen

528 Eriksen L, Nansen P, Roepstorff A, Lind P, Nilsson O (1992) Response to repeated inoculations with Ascaris s u u m eggs in pigs during the fattening period: I. Studies on worm kinetics. Parasitot Res (in press) Foged NT, Nielsen JP, Pedersen KB (1988) Differentiation of toxigenie from nontoxigenic isolates of Pasteurella multocida by enzyme-linked immunosorbent assay. J Clin Microsc 26:14191420 Forsum EM, Nesheim C, Crompton DWT (1981) Nutritional aspects of Ascaris infection in young protein deficient pigs. Parasitology 83:497-512 Gois M, Kusa F, Sisak F (1980) Microbiological findings in the lungs of slaughter pigs. Proceedings, 6th Congress of the International Pig Veterinary Society, Copenhagen June 30 July 3 Hale OM, Stewart TB, Marti OG (1985) Influence of an experimental infection of Asearis s u u m on performance of pigs. J Anita Sci 60: 220-225

Lloyd S, Soulsby EJL (1985) Ascariasis in animals. In: Crompton DWT, Nesheim MC, Pawlowski ZS (eds) Ascariasis and its public health significance 1st edn. Taylor & Francis, London Philadelphia, pp 25 36 Oksanen A, Eriksen L, Roepstorff A, Ilsoe B, Nansen P, Lind P (1990) Embryonation and infectivity of Ascaris suum. A comparison of eggs collected from worm uteri with eggs isolated from pig faeces. Acta Vet Scand 31:393-398 Roneus O (1966) Studies on the aethiology and pathogenesis of white spots in the liver of pigs (thesis). Acta Vet Scand 7 [Suppl 16] : 112 Stephenson LS, Pond WG, Nesheim MC, Krook LP, Crompton DWT (1980) Ascaris suum: nutrient absorption, growth and intestinal pathology in growing pigs experimentally infected with 15 day old larvae. Exp Parasitol 49:15-25 Underdahl NR, Kelley GW (1957) The enhancement of virus pneumonia of pigs by the migration of Ascaris s u u m larvae. J Am Vet Med Assoc 130 : 173-176

Interaction between Ascaris suum and Pasteurella multocida in the lungs of mice.

In an experiment including 8 groups of 15 mice, the effect of migrating Ascaris suum larvae in the lungs on the establishment and pathogenicity of aer...
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