Aerosol challenge of calves with Haemophilus somnus and Mycoplasma dispar.

The aim of the study was to examine the ability of Haemophilus somnus and Mycoplasma dispar to induce pneumonia in healthy calves under conditions closely resembling the supposed natural way of infection, viz. by inhalation of aerosol droplets containing the microorganisms. The infections were investigated by recording clinical data, cytokine expression of peripheral blood cells and pathology. Twelve calves were included in the study: Three animals were exposed to H. somnus only, and two to M. dispar only, whereas five were challenged to M. dispar followed by exposure to H. somnus 11-14 days later. Also, one calf was exposed to M. dispar followed by exposure to a sterile saline solution 11 days later, and one calf was only exposed to a sterile saline solution. Just one animal, only challenged with H. somnus, developed a focal necrotizing pneumonia, from which H. somnus was isolated. Thus, the ability of H. somnus and M. dispar to act as primary pathogens under these conditions were minimal and inconsistent.However, a transient rise in body temperature, a marked granulocytosis and increased levels of interleukin-8 in peripheral blood after inoculation with H. somnus indicated a clear systemic response, probably as a consequence of the natural non-specific local and systemic defence mechanisms acting in healthy calves.

Introduction

Diseases of the respiratory tract are one of the primary sources of losses in the Danish beef and dairy cattle industries. It represents a complex problem as the development and severity of the disease are influenced by a number of infectious agents including bacteria, virus and mycoplasmas, as well as factors related to host and environmental conditions, such as immunity, stress, stocking rate, etc. Though intensively studied, the relative significance of several of the infectious agents by themselves remains to be demonstrated conclusively. In Denmark, Haemophilus somnus is one of the most common bacteria isolated from fatal cases of calf pneumonia (Tegtmeier et al., 1999b). Therefore, the present study was focused on H. somnus in order to clarify its potential for inducing pneumonia in healthy unstressed animals under experimental conditions closely resembling the presumed natural route of infection, viz. by aerosol exposure. Mycoplasma dispar is another microorganism often isolated from pneumonic calf lungs (Tegtmeier et al., 1999b), and this microorganism was used for studying concomitant infection with M. dispar and H. somnus.

Most previous experimental studies of H. somnus pneumonia have been based on intratracheal or intrabronchial deposition of bacterial suspensions (Krogh et al., 1986, Gogolewski et al., 1987, Jackson et al., 1987, Potgieter et al., 1988, Tegtmeier et al., 1999a). This way of introducing the bacterium to the respiratory tract has yielded valuable information on the pathogenesis of the disease. It is, however, not a design that closely mimics the natural route of infection. One can speculate that the introduction of such a huge number of bacteria in a viscous suspension might overload the physical barriers leading to a vicious circle of exacerbated cytokine release, thereby, compromizing the natural defence mechanisms of the lung to a degree uncompatible with the natural pathogenesis of the disease. In that case, the lung lesions might partly be explained as an unbalanced reaction independent of the viability of H. somnus organisms, that is, the lesions could be induced by inoculation of dead bacteria or bacterial components in itself. In line with this, Whiteley et al. (1991) found that inoculation of killed Mannheimia (Pasteurella) haemolytica bacteria were capable of causing fibrin exudation, platelet aggregation and alveolar epithelial damage in a degree similar to live bacteria (The name Pasteurella haemolytica has been changed to Mannheimia haemolytica, Angen et al., 1999).

Only a limited number of papers report on calves that have been exposed to aerosols of H. somnus. Nayar et al. (1977) aerosol-inoculated three calves with H. somnus. Two of these calves developed clinical signs of respiratory disease 24 hrs post inoculation and H. somnus was cultured from blood samples. However, none of these animals were necropsied, thus no information on pulmonary pathology or concomitant infection with other pathogens was available. Krogh et al. (1986) inoculated four calves with aerosols containing H. somnus and/or M. dispar through the nostrils. When H. somnus was inoculated alone (one animal), no pulmonary lesions were present at necropsy, whereas inoculation with M. dispar prior to the H. somnus inoculation (three animals) lead to scanty to more extensive consolidation in the lungs.

In pigs, Actinobacillus pleuropneumoniae is a common cause of pneumonia and an experimental infection model has recently been developed for aerosol inoculation of pigs with A. pleuropneumoniae (Jacobsen et al., 1996). In this model, pigs are placed in a closed chamber and subjected to bacterial aerosols generated by a nebulizer. This model was used in the present study to expose calves to aerosols containing M. dispar and/or H. somnus.

The aim of the present study was (1) to examine the ability of viable H. somnus bacteria to act as the primary causative organism in development of calf pneumonia in healthy animals under conditions closely resembling the proposed natural way of infection, that is, by inhalation of aerosol droplets containing H. somnus, and (2) to examine the possible effect of infection with M. dispar prior to inoculation with H. somnus. The infections were investigated by recording clinical data, post-mortem pathology and changes in the composition of and cytokine expression of peripheral blood cells.

Materials and methods

Animals

Twelve calves (No. 1–12) were purchased from a closed Red Danish Dairy herd free of Salmonella and bovine viral diarrhea virus, as monitored by the Danish Veterinary Laboratory (DVL). The calves were removed immediately after birth without any contact to the mother cow or the surroundings, transported to DVL and placed two and two in isolation units. The calves were fed with milk substitute and in addition, each calf was given 100 ml bovine hyperimmune serum orally three times a day for the first 8 days of life. The serum was obtained from a donor cow that had been kept in isolation its entire life without episodes of respiratory disease. The cow had previously been vaccinated and boosted with Trivacton 6 (Rhône Merieux), a vaccine containing antigens against Escherichia coli, rota- and coronavirus. Also, intramuscular antibiotic treatment (Ampivet, Boehringer Ingelheim), 10 mg/kg was given twice a day for the first 8 days of life.

The calves were challenged when approximately 4 weeks old. During the whole life span, the health status was monitored by clinical examination.

To exclude the presence of pneumonic lesions before challenge, all calves were radiographed approximately 1 week prior to the first aerosol exposure as described elsewhere (Tegtmeier and Arnbjerg, 1999).

Heparin stabilized peripheral blood samples were collected from the jugular vein before and after challenge of animal number 6, 7, 8 and 11 for determination of cytokine expression and cellular parameters in the peripheral blood cells.

As described elsewhere (Grell et al., 2000), cytokine expression (interleukin-8 (IL-8)) was determined by reverse transcribed polymerase chain reaction with specific primers. Briefly, total RNA was extracted by guanidine thiocyanate (Promega) and dissolved in RNAse-free water (diethylpyrocarbonate treated). Contaminating DNA was digested with DNAse. A standardized amount of total RNA (measured by absorbance at 260 nm) was reverse transcribed to cDNA using oligo dT as a primer and reverse transcriptase from GibcoBRL (Superscript II). Specific primers for bovine IL-8 were: TTC ACA GCA CTC GGA ATC CTG and ATG ACT TCC AAA CTG GCT GTT. The identity of the RT-PCR product was verified by sequencing.

Two housekeeping genes (β-actin and GAPDH) visualized after PCR by agarose gele electrophoresis were used to calibrate samples. The relative concentration of specific cytokine transcripts in the samples was estimated by the amount of RT-PCR product obtained, as judged from product band density after agarose gel electrophoresis. All cytokine mRNA levels were estimated relative to the levels observed after the M. dispar challenge but before the H. somnus aerosol challenge.

Differential leukocyte counts were performed by a combination of automated leukocyte counting (AUTOCounter AC 900) and FACS analysis (FACScan, Becton Dickinson).

Bacterial and mycoplasma strains

A Danish β-haemolytic field strain of H. somnus (DVL 939/90), originally isolated from a case of bovine pneumonia, was used throughout the study. This strain has previously been used as inoculum for experimental pulmonary infection of calves (Tegtmeier et al., 1999a). Also, the strain has earlier been characterized by biotyping, plasmid profiling, REA-patterns and ribotyping, and represents the dominant group of Danish H. somnus strains isolated from cases of calf pneumonia (Fussing and Wegener, 1993).

Four Danish M. dispar strains isolated from cases of calf pneumonia (MK 332, MK 341, MK 358 and MK 372) were used for inoculation. A pool of four isolates was used, in an attempt to ensure an optimal potentiating effect, as heterogenesity of M. dispar has previously been described (Friis, 1978).

Preparation of inoculum

The H. somnus strain, kept at −80°C, was resuscitated on 5% bovine blood agar plates (Columbia agar base, Oxoid), incubated overnight in an atmosphere of 10% CO2 and 90% air at 37°C, subcultivated and re-incubated for 16 h prior to use. Immediately before use, the bacteria were harvested by washing each agar plate with 1 ml of a 37°C sterile 0.9% NaCl solution, giving an inoculum with approximately 109 colony forming units per ml (CFU/ml). The purity of the inoculum was verified by bacteriological examination (as described under 2.5).

The M. dispar strains were all kept at −80°C until use. The inoculum was prepared as a pool of broth cultures of the four different strains which had been filtered through a 0.45 μm membrane and cloned once from solid medium. The cultures were used in the 5th or 6th passage in artificial medium, representing a 1012–1015 dilution of the original lung tissue. A medium described by Kobisch and Friis (1996) was used for cultivation, and identification was performed by the disc growth inhibition test by using antiserum for the type strain NCTC 10125 (462/2). The titer was estimated to 108–109 viable units per ml in the M. dispar containing medium, used for inoculation.

Aerosol exposure

Equipment for an aerosol infection model previously developed for inoculation of pigs with A. pleuropneumoniae (Jacobsen et al., 1996) was used in the present study, where calves were exposed to M. dispar and/or H. somnus. Briefly, the equipment consisted of a closed wooden chamber into which aerosols were introduced by an ultrasonic nebulizer (Model 99, DeVilbriss Company, Somerset, PA).

The 12 calves were exposed for 15 min to aerosol droplets of H. somnus and/or M. dispar and/or a sterile saline solution (Table 1 ). Three calves were exposed to H. somnus only (No. 1–3), two animals were exposed to M. dispar only (No. 4–5), five animals were exposed to M. dispar followed by exposure to H. somnus 11–14 days later (No. 6–10). One animal was exposed to M. dispar followed by exposure to a sterile saline solution 11 days later (No. 11), and one animal was only exposed to a sterile saline solution (No. 12). All calves were placed singly in the aerosol chamber and exposed to approximately 25 ml of the inoculum. For determination of airborne viable H. somnus or M. dispar organisms in the chamber, a two step Andersen Sampler (Andersen, 1958), was connected. The Andersen Sampler allows 23 l air per minute to pass, and the lowest step of sampler only allows the respirable fraction, that is, particles with an aerodynamic diameter <5 μm to pass (Jacobsen et al., 1996). During inoculation, blood agar plates were placed in the Andersen Sampler for periods of 5 min, followed by incubation as described under post-mortem examination (2.5). Animals exposed to H. somnus or sterile saline (No. 1–3 and 6–12) were euthanized 4 days after inoculation, whereas the two calves only exposed to M. dispar (No. 4 and 5) were euthanized 11 days after inoculation.

Table 1

Results of necropsy and microbiological examinations of lungs of calves inoculated with Mycoplasma dispar and Haemophilus somnus

	Inoculation witha			Euthanasia	Necropsy findingsb	Microbiology
Calf No.	M. dispar	H. somnus	Sterile saline			Bacterial-, viral-, & mycoplasma examination
						Lungc	Nasal svabd
1	–	Day 0	–	Day 4	Acute focal necrotizing pneumonia	H. somnus	H. somnus
2	–	Day 0	–	Day 4	None	None	H. somnus
3	–	Day 0	–	Day 4	None	None	H. somnus
4	Day −11	–	–	Day 0	None	M. dispar	Negative
5	Day −11	–	–	Day 0	None	M. dispar	Negative
6	Day −11	Day 0	–	Day 4	None	M. dispar	H. somnus
7	Day −11	Day 0	–	Day 4	None	M. dispar	H. somnus
8	Day −11	Day 0	–	Day 4	None	M. dispar	H. somnus
9	Day −14	Day 0	–	Day 4	None	M. dispar	H. somnus
10	Day −14	Day 0	–	Day 4	None	M. dispar Pasteurella spp.	H. somnus
11	Day −11	–	Day 0	Day 4	None	M. dispar	Negative
12	–	–	Day 0	Day 4	None	None	Negative

The time of challenge with H. somnus was considered as Day 0.

In some animals, a few atelectatic lobules were present at necropsy, these are not mentioned in the table.

Lung tissue was examined for the presence of pathogenic bacteria, virus and mycoplasmas.

Nasal svabs were examined only for bacteria.

Post-mortem examination

The calves were euthanized with an overdose of sodium pentobarbital and necropsied.

Samples from 5 to 10 areas of the lung, and the brain, liver, spleen and jejunum were examined bacteriologically. Cultivation attempts for bacteria were performed as described elsewhere (Tegtmeier et al., 1999b). In brief, samples were cultured on two 5% bovine blood agar plates of which one plate was incubated in a normal atmosphere at 37°C and the other in an 37°C atmosphere consisting of 10% CO2 and 90% air. All plates were inspected for growth after 16 h and 40 h. The identification of H. somnus was based on growth of tiny white or yellow-white colonies on plates incubated with 10% CO2 and no or feeble growth in normal air, by being Gram-negative, oxidase positive, catalase negative and by production of indol and fermentation of glucose.

Other bacterial pathogens were identified according to standard laboratory procedures.

The H. somnus isolates were ribotyped in order to compare and verify that the isolate was identical to the strain used for inoculation. Ribotyping was performed by DNA digestion by HindIII and hybridization at 56°C as described by Fussing and Wegener, (1993).

In addition, nasal swabs and 4–6 lung samples were examined for H. somnus by a species specific PCR test based on the 16s rDNA gene (Angen et al., 1998). DNA was obtained from the nasal svabs by chloroform/phenol extraction, precipitation with 96% ethanol and washing twice in 70% ethanol and dissolved in dH2O. 2 μl of the solution was used for PCR. Lung samples were incubated on agar plates as described above and each plate was washed with 2 ml of dH2O. After lysis, 2 μl was used for PCR.

Mycoplasma examination of lungs was performed by cultivation as described elsewhere (Tegtmeier et al., 1999b). Furthermore, lung tissue was examined for relevant respiratory virus (bovine respiratory syncytial virus (BRS virus), parainfluenza-3 virus (PI-3 virus), bovine coronavirus, bovine virus diarrhea virus) according to (Tegtmeier et al., 1999b).

Results

All calves had normal rectal temperature, normal respiratory rate and were without clinical signs of disease before challenge. The body weight varied from 37–63 kg at the time of euthanazia (mean 47.5 kg).

By radiography, an approximate 2 cm × 4 cm area of consolidated tissue was detected in the cranio-ventral lung region of calf No. 4. No radiographic abnormalities were determined in the other animals.

After exposure, most animals inoculated with H. somnus showed a transient rise in rectal temperature within the first 24 hrs (data not shown), whereas no rise in temperature was observed in the animals when inoculated with M. dispar or sterile saline.

Before inoculation, leukocyte counts were 4–9 × 106/ml but increased up to 3–4 times 2–3 days post inoculation (p.i.) with H. somnus (No. 6–10). For the control calf (No. 11) only inoculated with M. dispar, the leukocyte counts were 4–9 × 106/ml throughout the experiment. In those calves inoculated with H. somnus, a pronounced increase in granulocytes accounted for the distinct increase in peripheral leukocytes (Fig. 1 ). No significant differences between control and inoculated animals were found for monocytes and lymphocytes.

Fig. 1

Leukocyte counts of peripheral blood in calves infected with Haemophilus somnus. All calves were aerosol inoculated with H. somnus at Day 0. ■: Mean ±SEM of inoculated calves (No. 6–10), (the latter two were only sampled at −1 h, 3 h, 24 h p.i.). □: One calf (No. 11) infected with M. dispar on Day −11 and saline on Day 0. Measurements were performed in duplicate.

Semiquantitative RT-PCR on PBMC showed an increase in IL-8 expression as early as 3 h after challenge, compared to pre-challenge levels; there was, however, individual differences in the rapidity of the IL-8 response after challenge. Among a number of other cytokines investigated (IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IFNγ, TNFα and GM-CSF) no consistent changes were found before and after challenge with H. somnus.

The estimation of viable airborne microorganisms during the inoculation process revealed an uncountable number (>103  CFU) of H. somnus on both detection plates of the Andersen Sampler and also widespread growth of M. dispar. In all samplings, a small number (5–50 CFU) of non-pathogenic bacteria were identified by cultivation.

The results of necropsy and microbiology are summarized in Table 1.

An approximate 1 cm × 2 cm acute focal necrotizing pneumonic lesion surrounded by a rim of red consolidated tissue, was present in the right cranial lobe of calf No. 1 inoculated with H. somnus. In this animal, H. somnus was isolated from the pneumonic lesion in pure culture, whereas no other pathogens were determined. The ribotype of the isolate was identical to that of the inoculation strain (data not shown). A few areas of atelectasis scattered in a lobular, primarily subpleural pattern, were observed in some animals (including calf No. 4 where pre-inoculation radiography revealed a small area of consolidation). All such atelectatic areas were bacteriologically negative by cultivation and PCR, in addition to the 5–10 other investigated areas of the lung.

H. somnus was only recovered from calf No. 1, whereas M. dispar was isolated in pure culture from the lungs of all calves inoculated with this microorganism. A few Pasteurella spp. were isolated from one area of the lungs in calf No. 10 (inoculated with both M. dispar and H. somnus), where no lesions were present. Virus was not detected in any case.

Pathological and microbiological examinations of other organs revealed no significant findings.

PCR examination of lung tissue for detection of H. somnus was negative, except for the necrotic area in calf No. 1. At necropsy, all nasal swabs from calves inoculated with H. somnus were postive in the PCR test, whereas nasal swabs from calves inoculated with only M. dispar or sterile saline were negative.

Discussion

The presumed natural route of pulmonary infection was imitated by an aerosol inhalation model with the purpose to evaluate, whether H. somnus was capable of inducing pneumonia under such conditions in healthy animals. The study revealed that the pathogenic potential of H. somnus under these circumstances was minimal and inconsistent, as only one (No. 1) out of the eight calves developed a focal necrotizing pneumonic lesion, too small to induce clinical signs of respiratory disease.

Five animals were inoculated with M. dispar prior to H. somnus, as it was speculated that a previous challenge with M. dispar could induce pulmonary lesions, thereby compromizing the respiratory defence mechanisms to a degree severe enough to facilitate the establishment of H. somnus within the lung. In a previous experimental study of aerosol-induced M. dispar-associated calf pneumonia, a few small lung lesions were present at necropsy (Friis, 1980). Also, Krogh et al. (1986) observed lesions when calves were aerosol-inoculated with M. dispar prior to H. somnus. However, in the present study, the previous challenge with M. dispar did not enhance the ability of H. somnus to induce pneumonia, nor did M. dispar in itself induce macroscopic pneumonia. Furthermore, the calf (No. 1) that developed the pneumonic lesion had only been challenged with H. somnus.

In contrast to H. somnus, M. dispar became established in the lower respiratory tract, as it was re-isolated from all animals inoculated with this microorganism. The few scattered areas of atelectasis observed in some animals might in part have been induced by M. dispar, which is capable of inducing bronchiolitis with secondary atelectasis of the surrounding parenchyma (Friis, 1980). However, such atelectatic aeras were also observed in animals not challenged with M. dispar.

The reasons for the discrepancy between our results and the results previously presented by Krogh et al. (1986) might be several. In both studies, the animals were placed in isolation barns immediately after birth and challenged when approximately 1 month old and in the present study, radiography was performed prior to challenge in order to ensure that no lung lesions were present before inoculation. However, differences in the immune status of the animals, differences in inoculation technique and differences in the virulence of inoculation strains might contribute to the conflicting observations.

In calves, much research has been focused on the presumed pathogenesis of pneumonia caused by M. haemolytica. This microorganism and associated diseases have been studied intensively and was reviewed by Frank (1989). According to that review, healthy calves carry small numbers of M. haemolytica serotype 1 in the nasopharynx and when, for example, concomitant stress or viral infection compromise the defence mechanisms, the animals get highly susceptible to pneumonic pasteurellosis as the bacteria are able to multiply rapidly in the nasopharynx. Thus, high numbers of bacteria may subsequently be inhaled in aerosol droplets to the lower parts of the respiratory system where a rapid replication in the lungs will result in development of pneumonia. A similar pathogenesis for H. somnus induced pneumonia is a natural assumption, although the ability of H. somnus to develop disease in the present study was minimal. Even though direct inhalation of high numbers H. somnus into the lung parenchyma as well as multiplication of the bacteria on the nasal mucosa were possible (as H. somnus was detected from nasal svabs by PCR in all animals inoculated with H. somnus), no pneumonic lesions developed in the majority of the cases. This indicates that H. somnus should not be considered as a primary pulmonary pathogen capable of inducing pneumonia alone.

Previous studies on aerosol induction of bacterial calf pneumonia have focused on M. haemolytica and Pasteurella multocida. Jericho and Langford, 1978, Jericho et al., 1982, Yates et al., 1983 studied the effects of combinations of aerosols of bovine herpes virus-1 (BHV-1), PI-3 virus and M. haemolytica. In these studies, exposure of calves to aerosols containing M. haemolytica failed to produce respiratory disease, whereas calves previously exposed to BHV-1 or PI-3 virus developed pneumonia when subsequently exposed to M. haemolytica. However, Jericho and Carter (1985) reported that pneumonia could be induced by aerosol exposure to P. multocida alone.

Thus, most previous studies show that aerosol exposure to bacteria alone is not capable of inducing pneumonia. These observations strongly indicate that stress due to, for example, transportation, concomitant infection, damage of the respiratory tract, and/or immunosuppresion is necessary for establishment of a bacterial pneumonia. This is in accordance with our results which furthermore revealed that infection with M. dispar alone was not sufficient as predisposing factor. Therefore, one should consider other predisposing factors. In Denmark, BRS virus is a common pathogen detected in pneumonic lungs (Tegtmeier et al., 1999b) and this virus might, as BHV-1, be able to play the triggering role in development of pneumonia.

Another reason for the inconsistent results in the present experiments could be that the particular H. somnus strain used throughout the study might represent a low-virulent variety of the species. However, this H. somnus strain has previously induced severe pulmonic lesions in calves when inoculated intrabronchially (Tegtmeier et al., 1999a).

Even though no major lesions were observed at necropsy in the present study, except the focal necrotizing pneumonia in one calf and the few small areas of atelectasis, the results of blood cell and cytokine analysis clearly indicate that a systemic response was provoked after inoculation with H. somnus as does the rise in rectal temperature within the first day after inoculation. This response is most probably due to the inhalation of bacteria into the lungs where resident macrophages have been reported, at least in the pig to be particularly sensitive to bacterial LPS (Lin et al., 1994). Peripheral blood cell changes occurred 2–3 days after inoculation with H. somnus. The granulocytes were active as shown by phagocytosis of propidium iodide-labelled Staphylococcus aureus measured by FACS analysis (data not shown). The changes in peripheral blood cells clearly indicated that a significant expansion of granulocytes took place immediately after challenge and peaked at 2 days after challenge whereas the lymphocyte population remained remarkably constant (Fig. 1). This is consistent with the induction of mRNA for IL-8 peaking from 3 h to between 12 and 72 h after exposure in different animals, all indicating that bacteria were indeed introduced into the lungs giving rise to a systemic granulocyte reponse.

Conclusion

In conclusion, the most likely explanation for the results obtained in the present study is that the calves were able to eliminate the inhaled H. somnus bacteria by the innate non-specific local and systemic defence responses operating in healthy animals. The amount of bacteria used for inoculation is most probably higher than what is found under natural conditions. Nevertheless, the granulocytosis and the induction of mRNA for IL-8 presumably mimic the response occurring when calves are exposed to H. somnus under natural conditions, thus giving valuable information on the cellular mechanisms involved in defeating the development of pneumonia.