Br. vet. J. (1991) . 147, 3 52
ISOLATION OF PASTEURELLA HAEMOLYTICA FROM THE NASAL CAVITY OF GOATS
S . JASNI, M . ZAMRI-SAAD*, A . R. MUTALIB and A . R. SHEIKH-OMAR
Faculty of Veterinary Medicine and Animal Science, Universiti Pertanian Malaysia, 43400 Serdang, Selangor, Malaysia
SUMMARY
Twenty transport-stressed goats were divided into two groups . The first group was further stressed with steroid . Pasteurella haemolytica was found at various sites in the nasal cavity of goats in this group as early as 2 weeks post-transportation . The successful isolations continued consistently with more goats having pure growth of P. haemolytica at later stages. Mild catarrh rhinitis, loss of epithelial cilia and erosions were the main lesions observed in the nasal cavity. Goats in the second group that were not given steroid injections had inconsistent bacterial isolation and less severe pathological lesions .
INTRODUCTION
Pneumonic pasteurollosis is one of the most important diseases of sheep and goats throughout the world . Both Pasteurella haemolytica and P. multocida have been associated with the disease although P. haemolytica is more frequently isolated from such cases (Davies, 1985) . The organisms have been recognized as part of the normal flora of the nasopharynx and mouth of various animals, and stress factors such as shipping, climatic changes and the damaging effects of viral infection have been incriminated as important predisposing factors for the proliferation of these organisms in the nasal cavity before the lung lesions are produced (Jubb et al, 1985) . The present study reports on the isolation of P. haemolytica from the nasal cavity of stressed goats and their effect on the nasal mucosa .
MATERIALS AND METHODS
Twenty clinically healthy indigenous goats between 5 and 9 months old were transported by road over a distance of about 150 km to the animal experimental house . Immediately upon arrival, they were divided into two equal groups and kept in two separate rooms . The goats in the first group were injected with 4 mg of dexamethasone intramuscularly for 3 consecutive days to stress the goats further . Cut grass and drinking water ad libitum were provided daily. *Author for correspondence .
ISOLATION OF P. HAEMOLYTICA FROM GOATS
353
Nasal swabs for bacterial isolation were collected prior to transportation, upon arrival and at weekly intervals after arrival . At 2-weekly intervals post-transportation, two goats from each group were euthanized by barbiturate overdose. The heads were split longitudinally into two halves before swabs from six different sites of the nasal cavity, submandibular and retropharyngeal lymph nodes and lung samples were taken for bacterial isolation . Samples were cultured on blood agar and incubated at 37 ° C for 24 h before colonies suspected to be of P. haemolytica were recultured on blood and MacConkey agars . Part of the newly formed pure cultures were subjected to Gram staining and biochemical tests consisting of triple sugar ion, oxidase, urease and motility tests to confirm P. haemolytica (Lennette et al., 1974) . Sections of nasal mucosa from the same sites were fixed in 10% buffered formalin and in 2 .5% glutaraldehyde in 0 .1 M sodium cacodylate for histopathology and scanning electron microscopy (SEM) respectively . Samples for histopathological examination were embedded in paraffin, sectioned at 4-6,um and stained with haematoxylin and eosin whereas samples for SEM were dehydrated in a graded alcohol series, transferred into amyl acetate, critical point dried in liquid CO 2 and coated with gold .
RESULTS
P. haemolytica was the most prominent organism isolated from the nasal cavity of the goats in both groups . Other frequently isolated organisms included Streptococcus pyogenes, Staphylococcus aureus, Pseudomonas aeruginosa and Pasteurella multocida. Most isolations of P. haemolytica were made from the anterior half of the nasal cavity . The first isolations of P. haemolytica from goats in both groups were made in week 2 after arrival . An average of seven (70%) goats in the first group and two (20%) goats in the second group showed consistent P. haemolytica isolations from all sites of the nasal cavity in the subsequent five swab samplings, but none had the organism in their lungs . Initially, goats in the first group had mixed growth of P . haemolytica, but with increasing time more goats had pure growth of P. haemolytica in their nasal cavity . In contrast, most goats in the second group had mixed and inconsistent P. haemolytica growth . Twenty-five per cent of the goats in both groups had P. haemolytica in either one or both submandibular and retropharyngeal lymph nodes regardless of whether they had the organism in the nasal cavity or not . However, 15% of the goats had the organism in the lymph node without the organism in nasal cavity while 10% had the organism both in lymph nodes and nasal cavity . Five goats in the first group and one goat in the second group had mucoid nasal discharge . The nasal mucosa of most goats in the first group harbouring P. haemolytica were grossly congested while some had excessive mucus cover . Histologically, the affected mucosa was severely congested and had scattered infiltrations of mononuclear leucocytes, mostly lymphocytes and occasionally macrophages and plasma cells in the subepithelial layer (Fig . 1) . In many areas, there were numerous goblet cells that produced excessive amount of mucus to cover the epithelium (Fig . 2) . Cilia were not observed histologically but examination by SEM showed that cilia had collapsed onto the mucosa surface . Erosions with desquamated epithelium were observed in some areas . The lesions were observed to be most severe in goats harbouring pure growth of P. haemolytica, especially those that had died in later stages of the study . Similar lesions were less obvious in goats of the second group .
35 4
BRITISH VETERINARY JOURNAL, 147, 4
Fig. 1 . Catarrhal rhinitis in a goat with consistent isolation of P . haemolytica. The subepithelial layer is being infiltrated by numerous mononuclear cells .
Fig . 2. Scanning electron micrograph showing excessive mucus production by goblet cells in the nasal mucosa of a goat with rhinitis due to P . haemolytica .
ISOLATION OF P. HAEMOLrTICA FROM GOATS
3 55
DISCUSSION The high frequency of the isolation of P. haemolytica from lymph nodes and from various sites in the nasal cavity of transport-stressed goats suggests the potential of these organs as proliferation sites for this organism . The fact that it was not isolated immediately upon arrival indicates that time is required for active proliferation to take place (Zamri-Saad et al., 1989) . Additional stresses such as concurrent disease process, poor nutrition or poor ventilation provide better environment for the proliferation of this organism especially in the nasal cavity . This was evident in this study using dexamethasone which has been known to produce an immunosuppressive effect on animals by suppressing neutrophil function and antimicrobial activity of alveolar macrophages (Kaeberle & Roth, 1984 ; Schaffner, 1985) . Thus, dexamethasone-treated animals produce a much more severe pneumonia than those without dexamethasone treatment (Chiang et al ., 1990) . It is believed that, like dexamethasone, stresses somehow compromise the defence mechanism of the respiratory tract, allowing proliferation and persistence of P. haemolytica in the nasal cavity, causing the loss of epithelial cilia, erosion and rhinitis . These lesions have been recognized as key factors in the pathogenesis of respiratory tract infection in animals (Baskerville, 1981) . Hence animals exposed to several stress factors are more likely to develop the disease .
ACKNOWLEDGEMENTS Thanks to Basri Kassim, Kamaruzaman Ahmad and Nurida Abu Bakar for the bacterial isolations, Md Noh Manaf and Abdullah Misron for histological preparations and Aminah Yusuff for technical assistance in electron microscopy . Financial assistance from IRPA grant no . 50245 is gratefully acknowledged .
REFERENCES BASKERVILLE, A . (1981). New Zealand Veterinary journal 29, 235 . CHIANG, Y . W ., ROTH, J . A. & ANDREWS, J . J . (1990) . American Journal of Veterinary Research 51, 759 . DAVIES, D . H . (1985) . In Progress in Veterinary Microbiology and Immunology, Vol . 1, p . 229 . Basel :
Karger. K. V . F ., KENNEDY, P . C . & PALMER, N . (1985) . In Pathology of Domestic Animals, Vol . 2, p . 487 . London : Academic Press . KAEBERLE, M . L . & ROTH, J . A . (1984). Immunopharmacology 8, 129. LENNETFE, E . H., SPAULDING, E . H . & TRUANT, J . P . (1974) . Manual of Clinical Microbiology, p . 246 . Washington : American Society for Microbiology . SCHAFFNER, A . (1985) . Journal of Clinical Investigation 76, 1755 . ZAMRI-SAAD, M ., SHARIF, H . & BASRI, K. (1989) . Veterinary Record 124, 171 .
JUBB,
(Accepted for publication 12November 1990)
ISOLATION OF PASTEURELLA HAEMOLYTICA FROM THE NASAL CAVITY OF GOATS
S . JASNI, M . ZAMRI-SAAD*, A . R. MUTALIB and A . R. SHEIKH-OMAR
Faculty of Veterinary Medicine and Animal Science, Universiti Pertanian Malaysia, 43400 Serdang, Selangor, Malaysia
SUMMARY
Twenty transport-stressed goats were divided into two groups . The first group was further stressed with steroid . Pasteurella haemolytica was found at various sites in the nasal cavity of goats in this group as early as 2 weeks post-transportation . The successful isolations continued consistently with more goats having pure growth of P. haemolytica at later stages. Mild catarrh rhinitis, loss of epithelial cilia and erosions were the main lesions observed in the nasal cavity. Goats in the second group that were not given steroid injections had inconsistent bacterial isolation and less severe pathological lesions .
INTRODUCTION
Pneumonic pasteurollosis is one of the most important diseases of sheep and goats throughout the world . Both Pasteurella haemolytica and P. multocida have been associated with the disease although P. haemolytica is more frequently isolated from such cases (Davies, 1985) . The organisms have been recognized as part of the normal flora of the nasopharynx and mouth of various animals, and stress factors such as shipping, climatic changes and the damaging effects of viral infection have been incriminated as important predisposing factors for the proliferation of these organisms in the nasal cavity before the lung lesions are produced (Jubb et al, 1985) . The present study reports on the isolation of P. haemolytica from the nasal cavity of stressed goats and their effect on the nasal mucosa .
MATERIALS AND METHODS
Twenty clinically healthy indigenous goats between 5 and 9 months old were transported by road over a distance of about 150 km to the animal experimental house . Immediately upon arrival, they were divided into two equal groups and kept in two separate rooms . The goats in the first group were injected with 4 mg of dexamethasone intramuscularly for 3 consecutive days to stress the goats further . Cut grass and drinking water ad libitum were provided daily. *Author for correspondence .
ISOLATION OF P. HAEMOLYTICA FROM GOATS
353
Nasal swabs for bacterial isolation were collected prior to transportation, upon arrival and at weekly intervals after arrival . At 2-weekly intervals post-transportation, two goats from each group were euthanized by barbiturate overdose. The heads were split longitudinally into two halves before swabs from six different sites of the nasal cavity, submandibular and retropharyngeal lymph nodes and lung samples were taken for bacterial isolation . Samples were cultured on blood agar and incubated at 37 ° C for 24 h before colonies suspected to be of P. haemolytica were recultured on blood and MacConkey agars . Part of the newly formed pure cultures were subjected to Gram staining and biochemical tests consisting of triple sugar ion, oxidase, urease and motility tests to confirm P. haemolytica (Lennette et al., 1974) . Sections of nasal mucosa from the same sites were fixed in 10% buffered formalin and in 2 .5% glutaraldehyde in 0 .1 M sodium cacodylate for histopathology and scanning electron microscopy (SEM) respectively . Samples for histopathological examination were embedded in paraffin, sectioned at 4-6,um and stained with haematoxylin and eosin whereas samples for SEM were dehydrated in a graded alcohol series, transferred into amyl acetate, critical point dried in liquid CO 2 and coated with gold .
RESULTS
P. haemolytica was the most prominent organism isolated from the nasal cavity of the goats in both groups . Other frequently isolated organisms included Streptococcus pyogenes, Staphylococcus aureus, Pseudomonas aeruginosa and Pasteurella multocida. Most isolations of P. haemolytica were made from the anterior half of the nasal cavity . The first isolations of P. haemolytica from goats in both groups were made in week 2 after arrival . An average of seven (70%) goats in the first group and two (20%) goats in the second group showed consistent P. haemolytica isolations from all sites of the nasal cavity in the subsequent five swab samplings, but none had the organism in their lungs . Initially, goats in the first group had mixed growth of P . haemolytica, but with increasing time more goats had pure growth of P. haemolytica in their nasal cavity . In contrast, most goats in the second group had mixed and inconsistent P. haemolytica growth . Twenty-five per cent of the goats in both groups had P. haemolytica in either one or both submandibular and retropharyngeal lymph nodes regardless of whether they had the organism in the nasal cavity or not . However, 15% of the goats had the organism in the lymph node without the organism in nasal cavity while 10% had the organism both in lymph nodes and nasal cavity . Five goats in the first group and one goat in the second group had mucoid nasal discharge . The nasal mucosa of most goats in the first group harbouring P. haemolytica were grossly congested while some had excessive mucus cover . Histologically, the affected mucosa was severely congested and had scattered infiltrations of mononuclear leucocytes, mostly lymphocytes and occasionally macrophages and plasma cells in the subepithelial layer (Fig . 1) . In many areas, there were numerous goblet cells that produced excessive amount of mucus to cover the epithelium (Fig . 2) . Cilia were not observed histologically but examination by SEM showed that cilia had collapsed onto the mucosa surface . Erosions with desquamated epithelium were observed in some areas . The lesions were observed to be most severe in goats harbouring pure growth of P. haemolytica, especially those that had died in later stages of the study . Similar lesions were less obvious in goats of the second group .
35 4
BRITISH VETERINARY JOURNAL, 147, 4
Fig. 1 . Catarrhal rhinitis in a goat with consistent isolation of P . haemolytica. The subepithelial layer is being infiltrated by numerous mononuclear cells .
Fig . 2. Scanning electron micrograph showing excessive mucus production by goblet cells in the nasal mucosa of a goat with rhinitis due to P . haemolytica .
ISOLATION OF P. HAEMOLrTICA FROM GOATS
3 55
DISCUSSION The high frequency of the isolation of P. haemolytica from lymph nodes and from various sites in the nasal cavity of transport-stressed goats suggests the potential of these organs as proliferation sites for this organism . The fact that it was not isolated immediately upon arrival indicates that time is required for active proliferation to take place (Zamri-Saad et al., 1989) . Additional stresses such as concurrent disease process, poor nutrition or poor ventilation provide better environment for the proliferation of this organism especially in the nasal cavity . This was evident in this study using dexamethasone which has been known to produce an immunosuppressive effect on animals by suppressing neutrophil function and antimicrobial activity of alveolar macrophages (Kaeberle & Roth, 1984 ; Schaffner, 1985) . Thus, dexamethasone-treated animals produce a much more severe pneumonia than those without dexamethasone treatment (Chiang et al ., 1990) . It is believed that, like dexamethasone, stresses somehow compromise the defence mechanism of the respiratory tract, allowing proliferation and persistence of P. haemolytica in the nasal cavity, causing the loss of epithelial cilia, erosion and rhinitis . These lesions have been recognized as key factors in the pathogenesis of respiratory tract infection in animals (Baskerville, 1981) . Hence animals exposed to several stress factors are more likely to develop the disease .
ACKNOWLEDGEMENTS Thanks to Basri Kassim, Kamaruzaman Ahmad and Nurida Abu Bakar for the bacterial isolations, Md Noh Manaf and Abdullah Misron for histological preparations and Aminah Yusuff for technical assistance in electron microscopy . Financial assistance from IRPA grant no . 50245 is gratefully acknowledged .
REFERENCES BASKERVILLE, A . (1981). New Zealand Veterinary journal 29, 235 . CHIANG, Y . W ., ROTH, J . A. & ANDREWS, J . J . (1990) . American Journal of Veterinary Research 51, 759 . DAVIES, D . H . (1985) . In Progress in Veterinary Microbiology and Immunology, Vol . 1, p . 229 . Basel :
Karger. K. V . F ., KENNEDY, P . C . & PALMER, N . (1985) . In Pathology of Domestic Animals, Vol . 2, p . 487 . London : Academic Press . KAEBERLE, M . L . & ROTH, J . A . (1984). Immunopharmacology 8, 129. LENNETFE, E . H., SPAULDING, E . H . & TRUANT, J . P . (1974) . Manual of Clinical Microbiology, p . 246 . Washington : American Society for Microbiology . SCHAFFNER, A . (1985) . Journal of Clinical Investigation 76, 1755 . ZAMRI-SAAD, M ., SHARIF, H . & BASRI, K. (1989) . Veterinary Record 124, 171 .
JUBB,
(Accepted for publication 12November 1990)
