A study on ovine pneumonic pasteurellosis: isolation and identification of Pasteurellae and their antibiogram susceptibility pattern in Haramaya District, Eastern Hararghe, Ethiopia.

BACKGROUND: Sheep constitute the second major component of livestock in Ethiopia. However, efficient utilization of this potential resource is hampered by combination of health problems, poor management and feed shortage. Haramaya district is one of the remote settings in Ethiopia where information about the livestock disease is not well documented. Hence this study was conducted to determine the causative agents and their antimicrobial susceptibility pattern of bacterial Pasteurella isolates among pneumonic ovine in Haramaya district, Eastern Hararghe, Ethiopia. RESULTS: Out of 256 samples examined, Pasteurella was isolated in 64 (25%), of which 38 (59.4%) were from lungs and 26 (40.6%) were from nasal cavities. 87.5% of the isolates were Mannheimia haemolytica and 12.5% were Pasteurella multocida. All of the isolates from the lungs were Mannheimia haemolytica whereas 69% of the isolates from nasals cavities were Mannheimia haemolytica. Age and body temperature were significantly associated with Pasteurella isolates from clinic (P < 0.05). Despite diverse in the site of origins, the isolates exhibited uniformity in sensitivity to a majority of the antibacterial agents. The most effective drug was Cholramphenicol (100%) followed by Sulfamethoxazole (89.1%) and Tetracycline (84.4%). Both species were completely resistant to Gentamycin and Vancomycin. CONCLUSION: Mannheimia haemolytica is the most common cause of ovine pneumonic pasteurellosis in the study area. The isolates were susceptible to limited antimicrobial agents. Therefore, the antimicrobial susceptibility test should be conducted before treatment, except for critical cases.

Ceftaroline versus isolates from animal bite wounds: comparative in vitro activities against 243 isolates, including 156 Pasteurella species isolates.

More than 5 million Americans are bitten by animals, usually dogs, annually. Bite patients comprise ∼1% of all patients who visit emergency departments (300,000/year), and approximately 10,000 require hospitalization and intravenous antibiotics. Ceftaroline is the bioactive component of the prodrug ceftaroline fosamil, which is FDA approved for the treatment of acute bacterial skin and skin structure infections (ABSSSIs), including those containing methicillin-resistant Staphylococcus aureus (MRSA). There are no in vitro data about the activity of ceftaroline against Pasteurella multocida subsp. multocida and Pasteurella multocida subsp. septica, other Pasteurella spp., or other bite wound isolates. We therefore studied the in vitro activity of ceftaroline against 243 animal bite isolates. MICs were determined using the broth microdilution method according to CLSI guidelines. Comparator drugs included cefazolin, ceftriaxone, ertapenem, ampicillin-sulbactam, azithromycin, doxycycline, and sulfamethoxazole-trimethoprim (SMX-TMP). Ceftaroline was the most active agent against all 5 Pasteurella species, including P. multocida subsp. multocida and P. multocida subsp. septica, with a maximum MIC of ≤0.008 µg/ml; more active than ceftriaxone and ertapenem (MIC(90)s, ≤0.015 µg/ml); and more active than cefazolin (MIC(90), 0.5 µg/ml) doxycycline (MIC(90), 0.125 µg/ml), azithromycin (MIC(90), 0.5 µg/ml), ampicillin-sulbactam (MIC(90), 0.125 µg/ml), and SMX-TMP (MIC(90), 0.125 µg/ml). Ceftaroline was also very active against all S. aureus isolates (MIC(90), 0.125 µg/ml) and other Staphylococcus and Streptococcus species, with a maximum MIC of 0.125 µg/ml against all bite isolates tested. Ceftaroline has potential clinical utility against infections involving P. multocida, other Pasteurella species, and aerobic Gram-positive isolates, including S. aureus.

Development and in vitro evaluation of biopolymers as a delivery system against periodontopathogen microorganisms.

Periodontal disease is the major cause of tooth loss in adults. Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are considered key pathogens in periodontitis. The treatment consists of oral hygiene education, instrumentation for removal of calculus (scaling), chemotherapy and periodontal surgery. Several agents are commercially available; these chemicals can alter oral microbiota and have undesirable side-effects such as vomiting, diarrhea and tooth staining. Hence, the search for alternative products continues and natural phytochemicals isolated from plants used as traditional medicine and the use of biomaterials are considered good alternatives. Chitosan and pullulan are polymers that have been proposed due to their favorable properties such as biocompatibility, biodegradability, and adhesion ability. They can be used as local delivery systems of active principles of plant extracts. Thymus vulgaris, Matricaria chamomilla, Croton lechleri, Calendula officinalis L. and Juliana adstringens Schl. are known to have medicinal activity, and they are used in Mexican traditional medicine. Their extracts were tested in vitro for antimicrobial activity against P. gingivalis and A. actinomycetemcomitans, using agar diffusion and microdilution methods. The antimicrobial activity of films from biopolymers with plant extracts was evaluated by measuring the zones of inhibition against the tested organisms. The aim of this study was to develop bioadhesive films from chitosan and pullulan with added plant extracts and determine the antimicrobial activity of films against periodontal pathogens.

Development and in vitro evaluation of biopolymers as a delivery system against periodontopathogen microorganisms

Periodontal disease is the major cause of tooth loss in adults. Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are considered key pathogens in periodontitis. The treatment consists of oral hygiene education, instrumentation for removal of calculus (scaling), chemotherapy and periodontal surgery. Several agents are commercially available; these chemicals can alter oral microbiota and have undesirable sideeffects such as vomiting, diarrhea and tooth staining. Hence, the search for alternative products continues and natural phytochemicals isolated from plants used as traditional medicine and the use of biomaterials are considered good alternatives. Chitosan and pullulan are polymers that have been proposed due to their favorable properties such as biocompatibility, biodegradability, and adhesion ability. They can be used as local delivery systems of active principles of plant extracts. Thymus vulgaris, Matricaria chamomilla, Croton lechleri, Calendula officinalis L. and Juliana adstringens Schl. are known to have medicinal activity, and they are used in Mexican traditional medicine. Their extracts were tested in vitro for antimicrobial activity against P. gingivalis and A. actinomycetemcomitans, using agar diffusion and microdilution methods. The antimicrobial activity of films from biopolymers with plant extracts was evaluated by measuring the zones of inhibition against the tested organisms. The aim of this study was to develop bioadhesive films from chitosan and pullulan with added plant extracts and determine the antimicrobial activity of films against periodontal pathogens.

La enfermedad periodontal es la principal causa de perdida de dientes en los adultos. Los agentes causales comunmente identificados con la enfermedad son Porphyromonas gingivalis y Aggregatibacter actinomycetemcomitans. El tratamiento de la enfermedad consiste en educacion sobre higiene oral, remocion de calculos por medio de instrumentacion (raspado y alisado de la raiz), la administracion de medicamentos y cirugia. Hay multiples agentes quimicos disponibles comercialmente; estos pueden alterar la microflora oral y tener efectos secundarios indeseables como vomito, diarrea y pigmentacion dental. Por lo tanto, los productos naturales como los fitoquimicos aislados de plantas que son usadas como medicinas tradicionales y los biomateriales, son considerados buenas alternativas. El quitosan y el pululan son polimeros que han sido propuestos debido a sus propiedades de biocompatibilidad, biodegradabilidad, habilidad de adhesion y que pueden ser usados como sistemas de liberacion de los principios activos de extractos de plantas. Los extractos de Thymus vulgaris, Matricaria chamomilla, Croton lechleri, Calendula officinalis L. y Juliana adstringens Schl. son conocidos por tener actividad medicinal y se usan en la medicina tradicional Mexicana. La actividad antimicrobiana de sus extractos fue probada in vitro contra P. gingivalis y A. actinomycetemcomitans usando los metodos de difusion en agar y de microdilucion. La actividad antimicrobiana de peliculas a base de biopolimeros con extractos de plantas fue evaluada midiendo las zonas de inhibicion de crecimiento de los organismos probados. El proposito de este estudio fue desarrollar peliculas bioadhesivas de quitosan y pululan adicionadas con extractos de plantas y evaluar su actividad antimicrobiana contra periodontopatogenos.

Identification and antimicrobial susceptibility patterns of bacteria causing otitis externa in dogs.

Bacterial agents are considered important pathogens causing external otitis in dogs. It is essential to carry out bacterial culture and antimicrobial susceptibility test in the case of otitis externa, particularly for chronic or recurring cases. Sterile swab samples were obtained from terminal part of vertical ear canals of 74 dogs with otitis externa for cytology, bacterial culture and antimicrobial susceptibility test. Cytologic smears were stained using Gram and Giemsa staining methods. Aerobic bacterial culture performed on blood agar and MacConkey agar. Among total number of 92 isolated bacteria, 68 were Staphylococcus intermedius. Other isolated bacteria included: Pseudomonas aeruginosa, Proteus mirabilis, Escherichia coli, Pasteurella canis, and six other species of coagulase-negative Staphylococcus. Antimicrobial susceptibility test were performed for all isolated bacteria using 14 antibiotics. Based on the results of this study, all isolated Staphylococcus spp. were sensitive to amikacin, enrofloxacin, and rifampin, and had low resistance to gentamicin, cephalothin and ceftriaxone. More than half of gram-positive isolates were resistant to penicillin and ampicillin. Generally, all isolated gram-negative bacteria, were sensitive to amikacin and enrofloxacin, and had low resistance to ceftriaxone and gentamicin. They were highly resistant to penicillin, eythromycin, and cephalothin. Regarding the results of this study, in cases of uncomplicated otitis externa, it is possible to select antimicrobial drugs merely based on cytology, but it is recommended to perform bacterial culture and antimicrobial susceptibility test. However, in complicated or refractory cases, antimicrobials should be selected based on bacterial culture and antimicrobial susceptibility test.

Effectiveness and kinetic behaviour of tilmicosin in the treatment of respiratory infections in sheep.

Nineteen sheep which were anorexic, pyrexic, coughing, dyspnoeic and had a nasal discharge and symptomatic thoracic sounds on auscultation, received a single subcutaneous dose of 10 mg/kg bodyweight of tilmicosin. The clinical signs were eliminated within four to six days. The kinetic profiles of the drug after a single subcutaneous injection were compared in five healthy sheep and five infected sheep. More of the drug was absorbed by the infected animals and its concentration remained higher for significantly longer. The drug was well tolerated and no local or systemic side effects were observed.

Antibiotic resistance in bacterial isolates from laboratory animal colonies naive to antibiotic treatment.

Antiobiogrammes were made of a number of isolates of Staphylococcus aureus, Escherichia coli and Pasteurella pneumotropica derived from rodent, rabbit or minipig colonies never treated with antibiotics. For S. aureus no differences between rats and mice were found in the percentage of resistant isolates. Gentamicin and erythromycin were found to be the most efficient, while the highest percentages of resistance were found to be against penicillins and sulphonamides. In general, the results from antibiogrammes on E. coli were rather uniform, with only slight differences between isolates from different species, except that more vancomycin and tetracycline-resistant minipig isolates were found. In almost all isolates of E. coli, resistance was shown against penicillin, fucidin, macrolides, lincosamides and tiamulin. For a number of antibiotics, mouse isolates of P. pneumotropica were more frequently found to be sensitive than rat isolates. The resistance patterns of E. coli from the minipigs were quite similar to resistance patterns found in farm pigs, but apart from this, the resistance patterns of the bacterial species tested did not resemble human or farm animal patterns in any of the animal species, and, therefore, these studies do not support the theory that S. aureus and E. coli in laboratory animal colonies derive from the normal flora of the human caretakers. The fact that rodent species of E. coli, in contrast to human and farm animal species, are sensitive to ampicillin, tetracyclines, and the combination of sulphonamides and trimethoprim, might be due to the fact that these antibiotics are not used in rodent populations.

Dual infection with Pneumocystis carinii and Pasteurella pneumotropica in B cell-deficient mice: diagnosis and therapy.

BACKGROUND AND PURPOSE: The clinical presentation, diagnosis, histopathologic findings, and elimination of dual respiratory tract infection with Pasteurella pneumotropica and Pneumocystis carinii were studied in 100 adult barrier-reared C.B17 and MRL- lpr mice homozygous for a targeted mutation of the JH region of the immunoglobulin heavy chain. METHODS: Necropsy, aerobic bacteriologic culture of hematogenous and pulmonary tissues, histochemical staining of pulmonary tissues, polymerase chain reaction analysis of pulmonary tissues and feces, and viral serologic testing were performed on 19 clinically affected mice and 8 clinically normal mice, then later on antibiotic-treated and caesarian re-derived mice. Therapeutic strategies included sequential administration of trimethoprim/ sulfamethoxazole and enrofloxacin or enrofloxacin administration and caesarian rederivation. RESULTS: Clinically affected mice had diffuse, nonsuppurative, interstitial pneumonia with superimposed pyogranulomatous lobar pneumonia that was detected microscopically. Affected lung tissue yielded pure culture of P. pneumotropica. Aged-matched, clinically normal mice of both genotypes had interstitial histiocytic pneumonia without lobar pneumonia, and P. pneumotropica was not isolated. Histochemical staining of lung tissues from normal and clinically affected mice revealed scattered cysts consistent with P. carinii, principally in the interstitium. Treatment with sulfamethoxazole/trimethoprim and enrofloxacin eliminated bacteriologic detection of P. pneumotropica, decreased mortality from 50% to 6%, and improved breeding performance. CONCLUSION: A successful antibiotic therapy and rederivation approach, incorporating enrofloxacin, cesarian section, and isolator rearing, was developed for B cell-deficient mice with opportunistic infections.

Effect of local application of the antimicrobial peptide IB-367 on the incidence and severity of oral mucositis in hamsters.

OBJECTIVE: The purpose of this animal study was to determine whether IB-367, an antimicrobial peptide, is able to ameliorate oral mucositis by reducing microflora densities on the mucosal surfaces of the mouth. STUDY DESIGN: Oral mucositis was induced in hamsters by intraperitoneal injection of 5-fluorouracil followed by superficial abrasion of the buccal mucosa. A test formulation was applied topically to the buccal mucosa 5 or 6 times per day starting 6 to 8 hours before abrasion. RESULTS: Mucositis scores were significantly lower (P < .05) in hamsters given formulations containing 0.5 or 2.0 mg/mL of IB-367 than in placebo-treated controls. Treatment with IB-367 produced a more than 100-fold reduction in oral microflora densities. In a second experiment, treatment of hamsters with a formulation containing IB-367 at 0.12, 0.5 or 2.0 mg/mL resulted in a dose-dependent reduction in mucositis severity. CONCLUSION: The results indicate that reduction of local microflora densities through use of IB-367 may improve clinical outcomes in patients at risk for the development of oral mucositis.

In vitro assessment of the efficacy of erythromycin in combination with oxytetracycline or spectinomycin against Pasteurella haemolytica.

The effects of combining erythromycin (Ery) with oxytetracycline (Oxy) or spectinomycin (Sp) on Pasteurella haemolytica were evaluated in vitro using the chessboard (checkerboard) technique. These combinations were selected because all are drugs widely used in bovine respiratory disease treatment, and they represent possible sequential or complementary mechanisms of action. Using the recommended breakpoints of greater than 4 micrograms/ml for Ery, 16 micrograms/ml for Oxy, and 32 micrograms/ml for Sp, of the 33 P. haemolytica isolates, 32 were resistant to Oxy, 27 to Sp, and 14 to Ery. Based on the fractional inhibitory concentration index, Ery and Oxy in combination were synergistic or additive against 32 of 33 isolates. The combination of Ery and Sp was synergistic or additive against 27 of 33 isolates. No instances of antagonism were seen. When the effects were considered within the context of therapeutically achievable serum/tissue concentrations, the effects of Ery and Oxy in combination were only marginal. Thus, against P. haemolytica isolates, Ery and Sp appeared to represent an effective antimicrobial combination, whereas Ery and Oxy were only of marginal efficacy as a combination.

In vitro antimicrobial activity of sulfonamides against some porcine pathogens.

The minimal inhibitory concentrations (MIC) of sulfonamides were determined against Bordetella bronchiseptica (n = 10), Pasteurella multocida (n = 10), Haemophilus pleuropneumoniae (n = 20), and Streptococcus suis (n = 10) strains isolated from pigs with atrophic rhinitis, pneumonia, or meningitis. Sulfonamides tested in an agar dilution method were sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfamethazine, sulfadoxine, sulfisoxazole, sulfamerazine, sulfamethoxazole, sulfamethoxypyridazine, sulfanilamide, sulfatroxazole, and sulfisomidine. Results indicated that monotherapy of S suis infections with sulfonamides should not be encouraged because the MIC50 of all sulfonamides investigated was greater than 32 micrograms/ml. The MIC50 of the sulfonamides against B bronchiseptica ranged from 0.5 to 8 micrograms/ml, against P multocida from 2 to 32 micrograms/ml, and against H pleuropneumoniae from 8 to 64 micrograms/ml. The MIC50 of sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfamerazine, and sulfamethoxazole for the gram-negative bacteria did not exceed 16 micrograms/ml. Among these compounds, sulfamethoxazole had the highest activity. The frequently prescribed sulfamethazine had an overall low antimicrobial activity.

[Selective media for Pasteurella pneumotropica].

Bacteria from the subcutaneous abscesses which appeared in a laboratory colony of DS mouse since October of 1977 were identified as Pasteurella pneumotropica by various biological examinations. The abscess formation was limited to multiparous female mice over 100 day-age, but virgin females were free from the disease. The MIC of various antibacterial substances showed that potassium tellurite, kanamycin and bacitracin were effective to isolate the organism selectively from various infection sites harboring many other species of bacteria. A novel NKBT medium was prepared by adding these antibacterial substances to the heart infusion agar (HIA) supplemented by 10% Fildes digested blood. A fluid culture medium, TGN broth was prepared for multiplication of the organism by adding 10% Fildes digested blood and potassium tellurite to GN broth. To isolate the organism from the pharyngo-larynx a direct application of mucus wiped off the infection site onto the culture medium was sufficient, but pre-multiplication in the TGN broth was required for isolation of the organism from gut contents before inoculation onto the NKBT medium. The pre-cultivation in the TGN broth vastly improved the recovery of the organism especially from feces. Thereby we could easily detect the latent infection of this bacterium without sacrificing animals.

Cytotoxin (leukotoxin) production by Pasteurella haemolytica: requirement for an iron-containing compound.

In studies of Pasteurella haemolytica type 1 cytotoxin, filter-sterilized culture supernatants from organisms grown in RPMI-1640 tissue culture medium generally have been used. Supplementation of the medium with 7% bovine fetal serum was shown to be necessary for maximal cytotoxin production, as measured by percentage of bovine peripheral blood leukocytes that were killed. The serum-induced increase in cytotoxicity could not be explained simply by a greater percentage of increase in the number of viable organisms produced in the enriched medium. There also was no correlation between encapsulation of the organisms and cytotoxin production. Several natural iron-containing proteins including transferrin, lactoferrin, conalbumin, and hemoglobin stimulated cytotoxin production in lieu of bovine fetal serum, leading to the conclusion that one function of serum supplementation may be to increase the medium's iron concentration. A number of additional iron-containing and iron-chelating compounds were tested, with the conclusion that the iron concentration of the growth medium, as well as the presence of a suitable carrier molecule, may be critical for efficient cytotoxin production by P haemolytica.

Differentiation among closely related organisms of the Actinobacillus-Haemophilus-Pasteurella group by means of lysozyme and EDTA.

Bacteriolysis in Tris-maleate buffer (0.005 M, pH 7.2) supplemented with EDTA (0.01 M) and hen egg white lysozyme (HEWL, 1.0 microgram/ml) was set up to assist differentiation between the taxonomically closely related Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus. A. actinomycetemcomitans was more sensitive to lysis in this system than H. aphrophilus. The standard method for bacteriolysis separated the 10 tested strains of A. actinomycetemcomitans into two groups (I and II) based on their lysis patterns, whereas the 7 strains of H. aphrophilus examined were homogeneous. In group I of A. actinomycetemcomitans, EDTA displayed a considerable lytic effect, which was not increased by supplementation with HEWL. In group II, the lytic effect of EDTA was much less, but HEWL had a considerable supplementary lytic effect. When the turbidity of A. actinomycetemcomitans (ATCC 29522) or H. aphrophilus (ATCC 33389) suspended in Tris buffer was monitored at close pH intervals (0.2) from pH 5.2 to 9.2, maximal lysis of ATCC 29522 occurred with EDTA at pH 8.0 and with EDTA-HEWL at pH 7.6, while ATCC 33389 lysed with EDTA at pH 9.0 and with EDTA-HEWL at pH 9.2. When other members of the family Pasteurellaceae (Haemophilus influenzae type b, Haemophilus paraphrophilus, Pasteurella multocida, Pasteurella haemolytica, and Pasteurella ureae) were included for comparison, the group I strains of A. actinomycetemcomitans were the most rapidly lysed by EDTA. H. paraphrophilus was the least sensitive of the gram-negative strains tested, but not as resistant as Micrococcus luteus (control). M. luteus was the organism most sensitive to lysozyme, followed by P. ureae and the group II strains of A. actinomycetemcomitans, while the group I strains of A. actinomycetemcomitans, H. paraphrophilus, and P. haemolytica were the least sensitive organisms.

Lysates of turkey-grown Pasteurella multocida: protection against homologous and heterologous serotype challenge exposures.

Pastereulla multocida organisms were separated from the blood of experimentally infected turkeys by differential centrifugation. An average of 92% of the residual host-cell contamination was removed from the pasteurellas by density gradient centrifugation in sucrose. Sucrose suspensions of the turkey-grown pasteurellas partially lysed after freezing and thawing. Treatment of freeze-thawed suspensions with DNAse, hyaluronidase, lysozyme, EDTA, and Triton X-100 did not influence their ability to induce protection against homologous and heterologous serotype challenge exposures. Lysozyme, EDTA, and Triton X-100 completely lysed the pasteurellas and rendered the cross-protection factor(s) filterable. Addition of adjuvant to completely lysed P multocida did not appear to enhance protection in turkeys against heterologous serotype challenge exposure. Adjuvant added to the pellet or supernatant fraction of centrifuged complete lysate enhanced protection in turkeys. Vaccines prepared from different serotypes of turkey-grown P multocida protected chickens and mice against homologous and heterologous serotype challenge exposures.

Effects of antipyresis on bacterial numbers in infected rabbits.

A previous investigation demonstrated that infusion of an antipyretic drug into the preoptic anterior hypothalamus (PO/AH) of rabbits reduced the fever usually seen during the initial stages of infection. This was followed by an increased fever and an increased mortality rate [32]. The work reported here investigated the hypothesis that the increased mortality was the result of decreased killing and/or increased multiplication of bacteria during the initial, attenuated phase of the febrile course in the antipyretic-treated rabbits. Rabbits were injected intravenously with Pasteurella multocida and either sodium salicylate or a control solution was infused directly into the PO/AH. Infusion of sodium salicylate reduced the mean fever 4 hours after injection of bacteria from 2.07 +/- 0.28 degrees C (S.E.M.) to 0.62 +/- 0.43 degrees C. Rabbits with reduced fevers had decreased blood leucocyte counts and greater numbers of bacteria in lung and liver samples. No differences were seen in reticuloendothelial clearance of carbon, hematocrit, or intracellular viability of bacteria when antipyretics were administered. This increase in bacterial numbers corresponds well to the increased mortality found in previous studies in animals with reduced fevers.