Pasteurella haemolytica in the Tracheal Air of Calves

Pasteurella haemolytica was shown to be present in the tracheal air of calves and was likely transported in droplet nuclei formed in the nasal passages. The number of colonies of P. haemolytica found in the tracheal air of the calves ranged from 1.9 to 12.5 colonies per cu ft of air. As long as P. haemolytica colonized the nasal passage in numbers detectable in nasal swabs it could be found in the tracheal air but there was no direct correlation between the numbers in the nasal flora and the numbers found in the tracheal air. Of the P. haemolytica which travel via the tracheal air 47.8% were in droplets of the aerodynamic size of from less than one to five microns, the size range which is considered hazardous for lung penetration in man.

The technique used demonstrated the presence of P. haemolytica in the tracheal air of calves and provides a useful tool for monitoring and determining the phase in the colonization of the respiratory tract in which the majority of the potential pathogen P. haemolytica pass from the nose to the tracheal air and presumably to the lung.

     

Pasteurella haemolytica of Cattle: Serotype, Production of Bete-galactosidase and Antibacterial Sensitivity

Two hundred and three isolates of Pasteurella haemolytica from cattle were studied. They originated from the nasal cavity of cattle in housed herds; the nasal cavity and pneumonic lungs of experimental feedlot calves and from pneumonic bovine lungs submitted for bacteriological diagnosis.

To determine whether a single characteristic or combination of characteristics might be a feature of isolates collected from animals with pneumonic pasteurellosis (Shipping Fever), the following tests were made. Cultures were serotyped by indirect haemagglutination; the ability to produce beta-galactosidase was examined in the ortho-nitrophenyl-beta-D-galactopyranoside (ONPG) test and antibacterial sensitivity tests were done. None of these factors could be directly related to the role of P. haemolytica in “Shipping Fever”.

     

Regional Histological Variations of the Nasal Mucosa of Cattle

Aspects of the histology of the nasal mucosa of calves from four to six months of age have been described.

The functional significance of the type of epithelium, its thickness, the degree of neutrophil invasion, and its relationship to the numbers of Pasteurella haemolytica in the nasal cavity have been discussed. In addition, the arrangement and depth of glands in the lamina propria and the presence of lymph follicles have been described.

     

Epizootiologic Studies of Shipping Fever of Cattle: I. The Microbial Agents Isolated

Nineteen strains of Pasteurella spp., but no viruses cytopathogenic for bovine embryonic kidney cells were isolated from pneumonic lesions present in “normal” veal calves at slaughter.

In studies on two herds of native cattle and six lots of western feeder calves, Pasteurella spp. were isolated from nasal swabs from healthy cattle and those with shipping fever. Viruses of the psittacosis-lymphogranuloma group were isolated from nasal swabs from animals in five groups. Viruses provisionally identified as bovine enteroviruses were isolated from nasal swabs of calves in two lots.

There was serologic evidence of a temporal association of myxovirus para-influenza 3 (PI3) with shipping fever in three lots of calves. From two of these three lots, strains of PI3 were isolated from ten animals, four of which had clinical shipping fever at the time of virus isolation.

     

Diversity of Mannheimia haemolytica and Pasteurella trehalosi Serotypes from Apparently Healthy Sheep and Abattoir Specimens in the Highlands of Wollo,North East Ethiopia

The prevalence and serotypic diversity of Mannheimia [Pasteurella] haemolytica and Pasteurella trehalosi from nasal swabs, sera and abattoir specimens from sheep in the highlands of Wollo, North East Ethiopia was investigated. Prevalence rates of 83% and 75% of these microorganisms were found in the serum samples and nasal swabs, respectively, from apparently healthy sheep. In a local abattoir, 205 lungs were investigated, 34% of which showed pneumonia, from which samples were collected from 51 lungs and the same number of corresponding tonsils. Mannheimia and Pasteurella species were isolated from 59% of these pneumonic lungs and 69% of the respective tonsils. M. haemolytica serotypes accounted for 41 (59%) and P. trehalosi for 11 (32%) of the isolates from the abattoir specimens. The majority (67%) of isolates from nasal swabs were P. trehalosi, M. haemolytica being isolated f rom 4 (13%) of the swabs. M. glucosida was isolated only from the tonsils. The predominant serotypes of the isolates from both the nasal swabs and the abattoir specimens were M. haemolytica A1 (17%) and P. trehalosi T4 (16%) and T3 (13%). P. trehalosi T15 was less commonly encountered, while M. haemolytica A9 and A13 were not isolated. Studies on sera from 100 sheep indicated that antibodies against M. haemolytica serotype A1 (14%) were most common, followed by A5 and A8 (each 10%) and A9 and P. trehalosi T3 (each 9%) and T4 (8%). Antibodies against M. glucosida or serotype A11 occurred in 2% of the sera. Multiple serotypes were common in all types of samples. The importance of including in vaccines the most prevalent serotypes involved in the pneumonia of sheep in the area is discussed.     

Diversity of bacterial species in the nasal cavity of sheep in the highlands of Ethiopia and first report of Histophilus somni in the country

A study was conducted to isolate bacterial species/pathogens from the nasal cavity of apparently healthy and pneumonic sheep. Nasal swabs were collected aseptically, transported in tryptose soya broth and incubated for 24 h. Then, each swab was streaked onto chocolate and blood agar for culture. Bacterial species were identified following standard bacteriological procedures. Accordingly, a total of 1,556 bacteria were isolated from 960 nasal swabs collected from three different highland areas of Ethiopia, namely Debre Berhan, Asella, and Gimba. In Debre Berhan, 140 Mannheimia haemolytica, 81 Histophilus somni, 57 Staphylococcus species, and 52 Bibersteinia trehalosi were isolated. While from Gimba M. haemolytica, Staphylococcus, Streptococcus, and H. somni were isolated at rates of 25.2, 15.9, 11.4, and 5.9 %, respectively, of the total 647 bacterial species. In Asella from 352 bacterial species isolated, 93 (26.4 %) were M. haemolytica, 48 (13.6 %) were Staphylococcus species, 26 (7.4 %) were B. trehalosi, and 17 (4.8 %) H. somni were recognized. Further identification and characterization using BIOLOG identification system Enterococcus avium and Sphingomonas sanguinis were identified at 100 % probability, while, H. somni and Actinobacillus lignerisii were suggested by the system. The study showed that a variety of bacterial species colonize the nasal cavity of the Ethiopian highland sheep with variable proportion between healthy and pneumonic ones. To our knowledge, this is the first report on isolation of H. somni, an important pathogen in cattle, from the respiratory tract of a ruminant species in the country.     

Cytotoxic effect of Pasteurella haemolytica on ovine bronchoalveolar macrophages in vitro

Live Pasteurella haemolytica A1 was shown to have a cytotoxic effect on suspensions of sheep bronchoalveolar macrophages. Cytotoxic activity was also demonstrable in bacteria-free supernatants from suspensions containing P. haemolytica. Heat-killed and ultraviolet killed organisms of P. haemolytica and live Staphylococcus aureus were not toxic to sheep BAM. These results suggest that a bacterial cell-free cytotoxin is produced by metabolically active P. haemolytica. Guinea-pig peritoneal macrophages, McCoy and pig kidney epithelial cell suspensions were unaffected by live P. haemolytica and supernatant from P. haemolytica cultures, indicating that the cytotoxin may only affect phagocytic cells of ovine or bovine origin.     

Subclinical pneumonia in lambs

Groups of lambs were either slaughtered at weekly intervals between November and June, or regularly sampled over this period. The number of isolations of Pasteurella haemolytica and Mycoplasma sp. from the lungs increased during the later part of two seasons, as did the incidence of lesions typical of enzootic pneumonia. P. haemolytica was frequently isolated after February from nasal swabs in spite of the presence of serum antibody titres against this organism. Serum antibody titre to two adenoviruses were found to fall to a low level, one in January, the other in May, and then to rise again. Virus isolations were made at the time the homologous antibody titres were lowest.     

Oat cells in the pathology of ovine pneumonia-pleurisy

Light microscope observations on oat cells in the ovine pneumonia-pleurisy complex are presented. This study is based on the experimental production of the disease by viruses and Pasteurella haemolytica. Oat cells appeared only in necrotic lesions associated with large numbers of P. haemolytica in thi pneumonic lung. It is suggested that oat cells originate from blood monocytes, which transform into the oat shape when developing in the necrotic, hypoxic environment created by P. haemolytica. They were not, however, observed to be phagocytic. Oat cells are characteristic of pneumonic pasteurellosis but are not pathognomonic because they can also be found in extrapulmonary locations and in other pathological conditions of the lungs.     

The Effect of Medicated Feed on the Nasal Microflora and Weight Gain of Pigs

Antimicrobial agents were added to the feed of swine for three weeks to determine the interrelationships of potentially pathogenic agents in the nasal tract, turbinate atrophy and weight gains.

Bordetella bronchiseptica was not isolated from the groups fed the combination of chlortetracycline, penicillin and sulfamethazine. B. bronchiseptica was found in some pigs after the feeding trail, but this organism was not significantly associated with turbinate atrophy at the time of slaughter.

Mycoplasma hyorhinis was not found in the nasal passages of the pigs that received feed containing high concentration chlortetracycline but was found in pigs that received other diets. Hemophilus suis was not significantly reduced by any of the treatments used.

The organisms studied in the pigs were not isolated from the personnel handling the pigs.

     

The Pulmonary Clearance of Bacteria by Calves and Mice

Using a modified aerosol generator, white mice and calves were exposed to aerosols of viable Staphylococcus aureus and Pasteurella haemolytica and the clearance of the inhaled organisms by the lungs of the experimental animals was measured. Fifty-seven percent of inhaled S. aureus were cleared in two hours by the mouse lungs, 79% were cleared in four hours and 93% were cleared in eight hours. Fifty-six percent of inhaled P. haemolytica were cleared in two hours by the mouse lungs, 76% were cleared in four hours and 93% were cleared in eight hours. Seventy percent of inhaled S. aureus were cleared in two hours by the calf lungs, 90% were cleared in four hours and 95% were cleared in eight hours. Seventy-five percent of inhaled P.haemolytica were cleared in two hours by the calf lungs, 90% were cleared in four hours and 92% were cleared in eight hours.     

Solid-phase enzyme immunoassay of bovine antibody responses following immunization against and natural infection with Pasteurella haemolytica

A solid-phase enzyme immunoassay (EIA) was developed in order to monitor bovine antibody responses following immunization against and natural infection with $\text{Pasteurella haemolytica}$ serotype A:1. Non-ionic surfactants, used in many antibody EIAs to reduce non-specific immunoglobulin binding, had to be avoided because they inhibited specific binding of bovine antibodies to $\text{P. haemolytica}$ antigens. Calves were immunized with a KSCN extract of $\text{P. haemolytica}$. Subcutaneously immunized animals developed a significantly higher humoral antibody response than did intranasally vaccinated animals. Intranasally immunized calves developed a slightly, but not significantly higher nasal antibody response than did calves vaccinated subcutaneously. Field study results based on bacterial isolation and EIA detection of antibodies to $\text{P. haemolytica}$ indicate that cattle can generate carrier states where bacteria are present in the upper respiratory tract, yet no humoral antibody response is induced. The converse was also found where cattle were free from $\text{P. haemolytica}$ in the upper respiratory tract, yet possessed a good humoral antibody response to $\text{P. haemolytica}$.     

Experimental Bovine Pneumonic Pasteurellosis II. Genesis and Prevention

Two experiments were conducted. In the first, 16 crossbred Hereford calves were divided into two equal groups. The first group was vaccinated intranasally with a commercial vaccine against bovid herpesvirus 1 and the second group was unvaccinated. The calves were later exposed to an aerosol of bovid herpesvirus 1 (strain 108) for five minutes. Four calves from each group were subjected to transportation and four calves from each group were kept in an environmental chamber for four days. Four days after viral aerosol all calves were exposed to an aerosol of Pasteurella haemolytica and the same subgroups were again transported or held in the chamber for a further four days.

The calves that did not die from pneumonia were necropsied ten days after the final day of transport. Pulmonary lesions were present in both vaccinated and control animals but were less extensive in the vaccinated calves. Six of eight vaccinated but none of the eight control calves survived.

In the second experiment, eight crossbred Hereford calves were divided into two equal groups. One group was vaccinated with bovid herpesvirus 1 (strain 108) and the other acted as controls. Four weeks later all calves were sequentially exposed to aerosols of bovid herpesvirus 1 (strain 108) and P. haemolytica four days apart. Three of the four controls but none of the vaccinates died from pneumonia. Every lobe of the lungs in all the controls was affected by pneumonia while no pulmonary lesions were found in the vaccinated calves. The differences in efficacy of the modes of vaccination and the possible role of transport stress are discussed.

     

Transmission dynamics of Mannheimia haemolytica in newly-received beef bulls at fattening operations

The primary objective of this study was to determine, at the lung level, whether single or multiple clones of Mannheimia haemolytica are present within a pen during a bovine respiratory disease (BRD) episode. A secondary objective was to assess whether M. haemolytica isolates obtained from nasal swabs (NS) are identical to those isolated deeper within the respiratory tract. Sixteen BRD episodes that naturally occurred in 12 pens of eight to 12 bulls (n = 112) newly-received at three fattening operations were investigated. One hundred and seventy five M. haemolytica isolates were collected from 239 pairs of trans-tracheal aspirations (TTA) and NS performed during these 16 BRD episodes. M. haemolytica isolates were characterized by pulsed-field gel electrophoresis (PFGE). PFGE types obtained from NS and TTA were then compared. M. haemolytica was isolated during 14 BRD episodes. Two to three different clones of M. haemolytica were recovered during 10 episodes whereas only one clone was recovered in four episodes. A moderate agreement (kappa = 0.50) between NS and TTA for M. haemolytica isolation was observed. Identical PFGE types were only observed in 77% of matched NS-TTA pairs. The significant within-pen diversity of M. haemolytica during BRD episodes indicates that the disease is not primarily due to the spread of a single virulent clone among cattle and highlights the importance of predisposing factors that enable the resident flora to overcome the cattle's immune system. The results also demonstrate that isolates recovered from NS are not always representative of the isolates present deeper within the respiratory tract.     

Antimicrobial Use in Feedlot Calves: Its Association with Culture Rates and Antimicrobial Susceptibility

Data were collected on anti-microbial usage and health problems, in beef feedlot calves. Although the association between them was not significant, many feedlot owners did not use recommended dosages or duration of treatment and this may have led to a reduced recovery rate.

Injectable tetracyclines were the most frequent antimicrobial for primary treatment of sick calves; whereas chloramphenicol was selected most frequently when the primary treatment appeared to be ineffective. Treatment with anti-microbials reduced the likelihood of isolating both Pasteurella multocida and Haemophilus somnus. Chloramphenicol therapy reduced the likelihood of isolating Pasteurella haemolytica.

Therapy with a particular antimicrobial, in the week prior to death, increased the level of resistance in P. haemolytica to that antimicrobial. Treatment with other antimicrobials also increased the level of resistance to that antimicrobial; although to a lesser degree. The lowest levels of resistance were observed in Pasteurella isolated from nontreated cattle. After adjustment for antimicrobial exposure, resistance to penicillin, tetracyclines and chloramphenicol occurred together more frequently than expected by chance alone.

     

The prevalence and microbiology of pneumonia in a flock of lambs

A flock of New Zealand Romney lambs on a property in Hawkes Bay was examined from August 1978 to June 1979. In November, the lambs in the flock were allocated to groups as follows: 600 lambs from which groups were selected for slaughter by commercial criteria each month (selected groups); 700 lambs which were subdivided into 8 groups (random groups), one of which was sent for slaughter each month; and 25 lambs to monitor serological responses to viral infections (surveillance group). At slaughter, lesions in the anterior lobes of the lungs were classified into small, large or mottled lesions while those in the posterior regions of the diaphragmatic lobes comprised a separate group. The prevalence of the pneumonic and pleural lesions was recorded in all groups of lambs. Pneumonic lesions in lambs from the random groups were further classified after microscopic examination. The prevalence of infections with parainfluenza virus type 3, adenoviruses, Mycoplasma spp., and Pasteurella haemolytica was also recorded in the random groups. Average carcase weights of all groups of lambs were also recorded.

The prevalence of large lesions in the anterior lobes, pleural lesions and devaluation of carcases due to pleural lesions was significantly lower in the selected than in the random groups.

In the random groups, the prevalence of small lesions in the anterior lobes of the lungs was high and was not always associated with infections with the viruses, P. haemolytica or Mycoplasma spp. Microscopically, these lesions could he divided into four types as previously described. The prevalence of large lesions in the anterior lobes was low in November but increased to peak levels in February and March and then declined to intermediate levels. The prevalence of large lesions was associated with that of infections with the micro-organisms and significantly more large lesions contained P. haemolytica and Mycoplasma spp. than did small lesions or normal lungs. The prevalence of large lesions also appeared to be inversely related to the average carcase weights of the lambs. Most large lesions were similar on microscopy to those of “enzootic” or “atypical” pneumonia but were divided into four types on the basis of exudative and proliferative features. The prevalence of mottled lesions in the anterior lobes was low throughout the period of observation but peaks were observed in January and May. The peaks were associated with adenovirus infection and elevated prevalence of infection with P. haemolytica and Mycoplasma spp. Significantly more mottled lesions contained P. haemolytica than did small lesions or normal lungs. Microscopically, most mottled lesions were similar to exudative large lesions.

Lesions in the posterior diaphragmatic lobes were most numerous in November and from April to June. Their prevalence appeared to be related to anthelminthic treatment and environmental conditions. Microscopically, these lesions were typical of those following infection with Dictyocaulus filaria.     

Dominant expression of interleukin-8 vs interleukin-1β and tumour necrosis factor alpha in lungs of lambs experimentally infected with Mannheimia haemolytica

Abstract

AIMS: To quantify the number of cells infected with Mannheimia haemolytica and expressing interleukin (IL)-1β, tumour necrosis factor alpha (TNFα) and IL-8 using immunohistochemistry, and to measure the immunoreactivity of cytokines in pulmonary tissue extracts using ELISA, in the lung of lambs experimentally infected with M. haemolytica, and to compare the patterns of expression of cytokines in airways at different times post-infection (p.i.).

METHODS: Twenty 3-month-old lambs of both sexes were randomly assigned to two groups, viz infected (n=15), and uninfected controls (n=5). Each lamb in the infected group was inoculated with 1.5 x 10⁹ cfu M. haemolytica in 5 mL sterile nutrient broth, control lambs were inoculated with 5 mL sterile nutrient broth and clinical signs were monitored. Infected and control animals were killed at 1, 3, 5, 7, and 15 days p.i. Histopathology and immunohistochemistry were conducted to determine the number of immunolabelled cells in pneumonic lungs, and study the pattern of expression of IL-1β, TNFα and IL-8 in lung extracts using ELISA.

RESULTS: Lesions in bronchi and bronchioles ranged from epithelial desquamation to bronchiolitis obliterans and necrosis. The alveoli had areas of seroproteinaceous fluid, fibrin and bacterial aggregates that evolved to foci of pyogranulomatous inflammation with clustered inflammatory cells, referred to as ‘oat cells’. M. haemolytica antigen was observed in the cytoplasm of inflammatory cells. Labelling of IL-1β, TNFα and IL-8 was observed in bronchial and bronchiolar epithelial cells, alveolar exudate, and in interstitial inflammatory infiltrate, with increased expression on 1 and 3 days p.i. for IL-1β and TNFα, and 1, 3, and 5 days p.i. for IL-8. In lung tissue extracts, peak concentrations of IL-1β (55 (SD 5) ng/mL), TNFα (92 (SD 6) pg/mL) and IL-8 (8 [SD 2] μg/mL) occurred at 3 days p.i.

CONCLUSIONS: The results of this study suggested that the inflammatory cytokines IL-1β, TNFα and IL-8 may play an important role in enhancing the biological response to M. haemolytica, and contribute to the development of lesions in the lung in pulmonary pasteurellosis in sheep. Given that the expression of IL-8 in lung was much greater than that of IL-1β and TNFα, anti-cytokine agents directed at this mediator could be useful in the prevention and treatment of this disease.     

Comparison of Pathogenicity of Various Isolates of Bordetella Bronchiseptica in Young Pigs

Eight groups of 12-to 24-hour-old pigs were procured from a respiratory disease-free herd of swine and reared in isolation using a box-rearing procedure. They were inoculated intranasally at 3 days of age with different isolates of Bordetella bronchiseptica.

It was found at necropsy 4 weeks post-inoculation that 4 isolates of swine origin, an isolate of rabbit origin and an isolate of cat origin caused mild to moderate turbinate atrophy in 22 of 24 pigs. An isolate of rat origin caused mild turbinate atrophy in 1 of 4 pigs and an isolate of dog origin caused no turbinate atrophy. Pneumonia was present in most of the pigs inoculated with the swine, cat and rabbit isolates.

Bordetella bronchiseptica was recovered in heavy growth from the nasal and tracheal exudate collected at necropsy from pigs inoculated with the 4 isolates of swine origin and the isolate of cat origin. Fewer organisms were isolated from nasal exudate collected from pigs inoculated with the rat, dog and rabbit isolates.

     

Experimental Atrophic Rhinitis in Gnotobiotic Pigs

Twenty-nine caesarian derived colostrum deprived germfree pigs were reared in isolators in groups of three to four per isolator. At seven days of age each group was inoculated intranasally with one of four strains of Bordetella bronchiseptica (designated B, J, L and 55B), or Pseudomonas aeruginosa or a mucoid strain of Escherichia coli, all previously isolated from nasal mucus of pigs affected with clinical atrophic rhinitis. Another group was inoculated simultaneously with B. bronchiseptica B and Pasteurella multocida. The animals were observed for clinical signs of atrophic rhinitis and monitored bacteriologically at weekly intervals for seven weeks. Then they were bled for serology and killed and their respiratory organs examined for gross and histopathological lesions.

All of the pigs inoculated with the Bordetellae had inflammation of the nasal mucosa and developed positive serum antibody titers against all four of the Bordetella strains used in this study. Strain J caused sneezing and turbinate atrophy in three of four pigs. One of the three pigs inoculated with strain L died in ten days from bronchopneumonia and pericarditis and had turbinate atrophy. Strains B and B55 caused no turbinate atrophy, but two out of three pigs inoculated with both B. bronchiseptica B and P. multocida had turbinate atrophy. No nasal lesions were observed in the pigs inoculated with E. coli or P. aeruginosa or in the noninoculated germfree controls.

The results indicate a variation in the ability of different strains of B. bronchiseptica to cause turbinate atrophy in pigs and demonstrate that nasal infections by these organisms stimulate serum antibody response. Presence of P. multocida appears to increase the severity of the lesions. As the E. coli and Pseudomonas failed to produce atrophic rhinitis, they are probably of no significance as primary etiological agents in the atrophic rhinitis syndrome in swine.

     

Ultrastructural Observation of Nasal and Pulmonary Intracellular Pasteurella multocida A:3 in Rabbits

Sixteen 8- to 9-week-old Pasteurella multocida-free rabbits were divided into two equal groups. Eight rabbits in one group were inoculated intranasally with P. multocida type A:3. The other eight were inoculated intranasally with phosphate-buffered saline and used as controls. Nasal swabs taken before and after inoculation were cultured for bacterial isolation. Post-mortem nasal swabs and lung samples were cultured for bacteriological isolation. Nasal mucosa and lung samples were collected and processed for transmission electron microscopy. Pasteurella multocida was isolated from the nasal cavity of all infected rabbits and from the lungs of four infected rabbits. Degenerative ultrastructural changes in epithelial cells and endothelial cells were seen in the infected rabbits. Deciliation of the cilated epithelium and hyperplasia of the goblet cells in the nasal mucosa were noted. Thickening of the alveolar septa due to hyperplasia of type II pneumocytes, swelling of the endothelial lining of capillaries and infiltration of inflammatory cells were also observed. Intracellular invasion of the nasal epithelial cells and of type II pneumocytes by the organism was observed. Coccobacilli were observed in membrane-bound vacuoles in the cytoplasm of these cells. The vacuoles were adjacent to the host-cell mitochondria and some of these vacuoles appeared to be fused to the mitochondrial membrane. Some type I pneumocytes with intracellular membrane-bound vacuoles containing bacterial cells showed protrusions, which appeared to detach into the alveolar lumina. These results indicated that P. multocida serotype A:3 in rabbits can invade the epithelial cell and cause structural changes in the interstitium, epithelium and endothelium. Heterophils and macrophages appear to play important roles in tissue injury.