Induction of interleukin-8 and interleukin-12 in neonatal ovine lung following experimental inoculation of bovine respiratory syncytial virus.

This study aimed to determine the immunohistochemical expression of interleukin (IL)-1ß, tumour necrosis factor alpha (TNF)-α, interferon (IFN)-γ, IL-4, IL-6, IL-8, IL-10 and IL-12 and to measure the concentrations of these cytokines in lung tissue from lambs infected experimentally with bovine respiratory syncytial virus (BRSV). Lambs (n = 15) were inoculated at 2 days of age with 20 ml of viral inoculum (1.26 × 10(6) TCID50 per ml) or sterile medium (n = 15). Rectal temperature, pulse and respiratory rates were monitored daily in control and infected lambs. Lambs were killed and subject to necropsy examination at 1, 3, 5, 7 and 15 days post inoculation (dpi). There was a temporal association between pulmonary expression of these cytokines and lung pathology in BRSV-infected lambs. The cytokines IL-4 and IL-10 were not elevated, but there was a significant increase in IL-1ß, TNF-α, IFN-γ and IL-6 proteins and labelled cells, suggesting that these cytokines may play a role in the biological response to BRSV infection and contribute to the development of lung lesions. There was also a significant increase in the cytokine concentration and number of immunolabelled cells expressing IL-8 and IL-12 in infected lungs, suggesting that these cytokines might be used as therapeutic targets in the management of BRSV, in conjunction with measures to combat the causative pathogen and prophylactic methods aimed at preventing infection.

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

AIMS: To quantify the number of cells infected with Mannheimia haemolytica and expressing interleukin (IL)-1ß, 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 10(9) 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-1ß, 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-1ß, 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-1ß and TNFα, and 1, 3, and 5 days p.i. for IL-8. In lung tissue extracts, peak concentrations of IL-1ß (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-1ß, 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-1ß and TNFα, anti-cytokine agents directed at this mediator could be useful in the prevention and treatment of this disease.

In vitro antimicrobial susceptibility of anaerobic bacteria isolated from caprine footrot.

The in vitro antimicrobial susceptibility of Gram-negative anaerobic bacilli commonly isolated from footrot in goats was studied. A total of 97 isolates belonging to the genera Dichelobacter, Fusobacterium, Prevotella, Porphyromonas and Bacteroides, obtained from clinical cases of footrot in south-western Spain between March 2000 and May 2001, were tested against 25 antimicrobial agents comprising beta-lactams, aminoglycosides, macrolides, chloramphenicol, quinolones, lincosamides, sulphonamides and tetracyclines in order to optimise antibiotic treatment of this disease in goats. beta-lactams, tetracyclines and metronidazole displayed the highest in vitro efficacy against the species involved in the pathogenesis of footrot.

Aetiology of caprine footrot in Extremadura region, Spain.

The present study reports on the location of major foci of footrot in goats in the Extremadura region of Spain by the determination of locally occurring strictly anaerobic microorganisms involved in the pathogenesis and development of this disease. The most commonly isolated microorganisms belonged to the genera Dichelobacter, Fusobacterium, Porphyromonas and Prevotella; these were found in conjunction with other species of minor importance. The species most frequently isolated were Fusobacterium necrophorum (40%), Dichelobacter nodosus (31.7%), Porphyromonas asaccharolytica (21.1%) and Prevotella melaninogenica (12.9%). Virulence factors identified in the isolated microorganisms included haemolysins, elastases and lecithinases, which enabled the organisms involved to initiate and/or aggravate the disease. Serotyping was performed for Dichelobacter nodosus isolates, since this species is responsible for triggering the process of infection. A and C were the most frequently isolated serovarieties (representing 40.7% and 25.9% of the cases, respectively).

Comparative study of biochemical methods for the identification of Dichelobacter nodosus.

The aim of this study was to compare four identification procedures to detect Dichelobacter nodosus and develop a rapid, simple and effective method to identify D. nodosus strains isolated from cases of ovine footrot. The four methods used were: (a) the classic guidelines set down by Holdeman et al. (1977) and Summanen et al. (1993) which are based on gas liquid chromatography (GLC) and different biochemical tests, this method was considered as landmark; (b) Baron and Citron's flowchart for the rapid identification of Gram-negative rod-shaped anaerobes (1997); (c) the API rapid 32 A system (bio Mérieux), and (d) Mast ID Anaerobe ID Ring (MID8) (Mast Diagnostics). None of the four methods used allowed us to correctly identify the D. nodosus strains (neither the strains isolated from cases of ovine footrot nor those originating from type collection). Because of the difficulties encountered in obtaining a correct identification of D. nodosus, we propose a simple, rapid and effective way to achieve this task. Our flowchart will provide the means to identify this microorganism in any laboratory of general microbiology without having to use any specialised equipment.

Effectiveness of different adjuvants in stimulating Dichelobacter nodosus antibody in sheep vaccinated against ovine footrot.

This research consists of an evaluation of the effectiveness of different substances administered as adjuvants in the stimulation of humoral immune response induced by the vaccine composed of strains A1, A2 and C of Dichelobacter nodosus. To do this, a total of 120 Merino sheep were vaccinated and revaccinated. These sheep were selected from a farm located in the region of Extremadura (Spain), and they were divided into 12 groups of 10 animals each. An additional group with 10 sheep was used as control. The immune response (titre of antibodies) was determined by agglutination tests and ELISA. The most pronounced immune response was obtained by the use of Freund's incomplete adjuvant and aluminium hydroxide as adjuvants.

Minimum inhibitory concentrations for 25 selected antimicrobial agents against Dichelobacter nodosus and Fusobacterium strains isolated from footrot in sheep of Portugal and Spain.

The agar dilution method was used to determine the inhibitory activity of 25 antimicrobial agents against 69 strains of Dichelobacter nodosus and 108 strains of the genus Fusobacterium, all of which were isolated from 90 clinical cases of ovine footrot between October 1998 and November 2000. In the case of the micro-organisms belonging to the genus Fusobacterium, the six beta-lactams studied (benzyl penicillin, ampicillin, cloxacillin, cefadroxil, cefuroxime and cephalexine) proved to be, in general, the most effective antimicrobial agents. Chloramphenicol, clindamycin and doxycycline were also quite active against Fusobacterium spp. With regard to the 69 strains of D. nodosus tested, the levels of resistance remain low.

Aetiology of ovine footrot in the Portuguese region of Alto Alentejo.

In this work, we found it appropriate to carry out a study directed towards isolating and identifying the entailed microorganisms which trigger off footrot in sheep, placing special emphasis on the serotipification of the different Dichelobacter nodosus species. With this goal in mind four flocks from the Portuguese region of 'Alto Alentejo' were selected, all of them had one common feature: their main health problem was ovine footrot. We also set out to determine the elastolitic capacity of isolated strict-anaerobic bacteria, in order to be able to clarify the direct involvement of these microorganisms in the outbreak of this infectious process.

Comparison of erythromycin and oxytetracycline for the treatment of ovine footrot.

A microbiological study of 25 cases of ovine footrot was performed. Cultures belonging to Dichelobacter nodosus were isolated in 48% of the sampled animals. The sensitivity of the 99 strict anaerobic bacterial isolates to 5 antibiotics (penicillin G, amoxycillin, spiramycin, erythromycin and oxytetracycline) was studied. The percentage of resistant cultures was in all cases higher than 30%. The efficacy of erythromycin and oxytetracycline in the treatment of ovine footrot was studied. To conduct this test, an intramuscular injection was applied, of one antimicrobial or the other, at the beginning of the treatment. The tolerance of animals to the antimicrobials, the success rate of treatment and the severity of lameness were evaluated. The percentage of animals cured within 15 days was around 75%. In contrast, only 44% improvement was achieved in the lameness. No differences were found between the two antimicrobials in the above indices.

Aetiology of ovine footrot in Spain.

Four hundred and sixty strains of obligate anaerobes were isolated from 216 cases of ovine foot rot distributed throughout Spain. The predominant species was Dichelobacter nodosus, which was isolated in 168 cases (77.8 per cent). A higher proportion of the strains of D nodosus than of the other strains had elastolytic activity, 118 of the 168 strains degrading elastin. Species belonging to the genus Prevotella were isolated from 96 cases (44.4 per cent). Serotyping of the D nodosus strains showed that serovars A1, A2 and C were the most commonly isolated in Spain.

Ribotyping of Fusobacterium necrophorum strains isolated from bovine and ovine hepatic abscesses.

A microbiological study was made of 100 strains of Fusobacterium necrophorum isolated from hepatic abscesses in bovine and ovine herds. Differences between the biological activity and ribotypes within the two F. necrophorum subspecies were studied. Conventional methods identified 89 isolates as F. necrophorum subsp. necrophorum and 11 as F. necrophorum subsp. funduliforme. For ribotyping, 50 strains (35 F.n. subsp. necrophorum, 11 F.n. subsp. funduliforme and 4 reference strains) were digested with restriction endonucleases (HindIII, EcoRI and BamHI) and examined after hybridization with digoxigenin-labelled cDNA probe transcribed from a 16 and 23S rRNAs from Escherichia coli. The most discriminating restriction endonuclease enzymes for ribotyping were EcoRI and BamHI. The presence or absence of two distinct band of 5 kb (EcoRI) and 10.5 kb (BamHI) differentiated the two subspecies. This technique also revealed genetic differences between isolates which could be used in the epidemiological study of clinical processes caused by F. necrophorum.

Minimum inhibitory concentrations for selected antimicrobial agents against Fusobacterium necrophorum isolated from hepatic abscesses in cattle and sheep.

Minimum inhibitory concentrations for 35 antimicrobial agents against 100 Fusobacterium necrophorum isolates from hepatic abscesses in sheep and cattle were determined. Twelve of the thirteen beta-lactam antibiotics tested inhibited growth of 100% of strains tested. Of the remaining antimicrobial agents, extensive susceptibility was found for: spiramycin, josamycin, lincomycin, tylosin, oxytetracycline, chlortetracycline, rufloxacin, metronidazole, cotrimoxazole, sulfadimethoxine, virginiamycin and fosfomycin.

In vitro susceptibility of Escherichia coli strains isolated from diarrhoeic lambs and goat kids to 14 antimicrobial agents.

The in vitro activities of 14 anti-microbial agents were determined against 92 strains of E. coli isolated from lambs (60 strains) and kids (32 strains) affected by neonatal diarrhoea. The overall percentage of resistant strains to streptomycin, sulphadimethoxine and tetracycline was very high (above 70%). A high level of resistance (from 30% to 50%) to ampicillin, kanamycin, neomycin and chloramphenicol was also detected. The E. coli strains were highly susceptible to cephalosporins, polymyxin and quinolones. Most of the strains showed multiresistance: 77.2% of isolates were resistant to at least two antibiotics, 55.4% were resistant to at least four antibiotics and 33.7% were resistant to at least six antibiotics. A total of 34 antibiotypes could be distinguished.

In vitro activity of fifteen antimicrobial agents against methicillin-resistant and methicillin-susceptible Staphylococcus intermedius.

In this study the susceptibility of 91 methicillin-resistant and -susceptible Staphylococcus intermedius strains (MRSI and MSSI, respectively) against 15 antimicrobial agents was determined. The activity of the antimicrobial agents was studied at pH 7.2 and pH 8.5. Methicillin was more active at pH 7.2 (28 strains methicillin-resistant) than at pH 8.5 (55 strains methicillin-resistant). Gentamicin showed excellent activity, with only 3 strains resistant at pH 8.5. However, gentamicin would have to be administered parenterally. Oxytetracycline cannot be recommended for treatment of canine staphylococcal dermatitis, due to the high percentage (over 25%) of strains that were found to be resistant. Clindamycin showed little activity in inhibiting growth of the strains studied, the percent resistance at pH 7.2 was 93.4%. Rifampin behaved differently at the two pH values. However, a close relationship was noted between methicillin-resistant and rifampin-resistant strains, particularly at the lower pH. Of the fluoroquinolones, ciprofloxacin or enrofloxacin would be a good useful alternative for the treatment of methicillin-resistant strains of S. intermedius. Lastly, very high resistance to sulphamethoxypyridazine was found, as was the case with trimethoprim and a combination of trimethoprim/sulphamethoxypyridazine, against not only MRSI but also MSSI strains.

Comparative in vitro activity of 11 beta-lactam antibiotics against 91 Staphylococcus intermedius strains isolated from staphylococcal dermatitis in dogs.

A study was made of the susceptibility of 91 strains of Staphylococcus intermedius, isolated from staphylococcal dermatitis of dogs, to 11 beta-lactam antimicrobial agents. The activity of these antimicrobial agents against all strains was tested at pH 7.2 and 8.5. Finally, a comparative study was made of the susceptibility of these micro-organisms, dividing them into six groups: beta-lactamase positive and negative, methicillin resistant and susceptible and oxacillin resistant and susceptible. Only three of the 11 antimicrobial agents studied exhibited satisfactory behaviour at the two pH levels studied: mezlocillin, cefazolin and cefalotine. There was a good correlation at pH 8.5 between methicillin-resistant strains and those resistant to penicillin G, ampicillin, amoxycillin, cefamandole and cefuroxime. This correlation was not observed, however, between oxacillin-resistant strains and strains resistant to the other antimicrobial agents studied.

Efficacy of the E-test in evaluating the antimicrobial susceptibility of anaerobic bacteria of veterinary interest.

A study was made of the sensitivity of 39 clinical isolates of anaerobic bacteria to 10 antimicrobial agents. Minimal inhibitory concentrations (MICs) were calculated using a new method--the E-test (AB Biodisk, Solna, Sweden)--and compared with those obtained using the conventional agar dilution method. Agreement between the MICs obtained by the two methods with a variation of +/- 2 dilutions was 78.7%. The E-test, though less sensitive than the conventional agar dilution method, may be of value in clinical veterinary practice when rapid selection of treatment for a given infectious process is required.

In vitro activities of enoxacin, enrofloxacin, sparfloxacin, and ciprofloxacin against Escherichia coli strains isolated from diarrheic lambs and kids.

The in vitro activities of four fluoroquinolone compounds were tested against 92 Escherichia coli strains of ovine and caprine origin under aerobic and anaerobic incubation conditions. The four fluoroquinolones proved to be highly effective against the E. coli isolates tested. When bacteria were cultured anaerobically, at least fourfold increases in the MICs of enoxacin for the strains occurred and no detectable changes in enrofloxacin, sparfloxacin, and ciprofloxacin MICs occurred.

In vitro susceptibility of Actinobacillus pleuropneumoniae strains to 42 antimicrobial agents.

Minimal inhibitory concentration of 42 antimicrobial agents was determined against 57 field strains of Actinobacillus pleuropneumoniae isolated from pigs in Spain. Penicillins, aminoglycosides, and tetracyclines had irregular activity; ticarcillin, tobramycin, and doxycycline were the most active of each group, respectively. Macrolides, vancomycin, dapsone, and tiamulin, to which strains had high rate of resistance, were almost ineffective. Thiamphenicol, colistin, rifampin, fosfomycin, mupirocin, and metronidazole had good activity, with resistance ranging between 0 and 8.8%. Finally, cephalosporins (except cephalexin) and quinolones (especially ciprofloxacin, enrofloxacin, and sparfloxacin) were the most active antibiotics against A pleuropneumoniae.

Microbiological study of foot-rot in lambs: isolation, elastolytic activity and antimicrobial susceptibility.

A microbiological study was made of an outbreak of foot-rot in lambs. Elastolytic activity was detected in all 10 Bacteroides nodosus strains isolated from the hooves of 9 affected animals. Among 14 other isolated strictly anaerobic microorganisms belonging to different species or genera only 4 strains showed a low degree of elastolysis. The 14 antimicrobial agents studied effectively inhibited "in vitro" growth of B. nodosus.