Bacteriologic and pathologic studies of hepatic lesions in sheep

At an abattoir, lesion specimens from 140 condemned sheep livers were collected for bacteriologic culture and for pathologic examination. Grossly, 23 lesions were abscesses; from 9 of which, Fusobacterium necrophorum biovar A (3 in pure culture and 6 in mixed culture) was isolated and from 14 of which, biovar B (6 in pure culture and 8 in mixed culture) was isolated. Escherichia coli was the predominant facultative anaerobic bacterium and Clostridium perfringens was the predominant obligate anaerobic bacterium isolated from the 14 lesions with mixed bacterial infection. Histologically, these lesions had a core of coagulation necrosis, encircled by a zone of necrotic phagocytic cells and bacteria with cellular characteristics of F necrophorum biovars A or B, and a connective tissue capsule. Of the 117 lesions without F necrophorum, 49 were culture-positive (for other organisms) and 69 were culture-negative. These 117 lesions were fibrous and were smaller than the 23 abscesses. A variety of gram-positive and gram-negative facultative anaerobic and obligate anaerobic bacteria was isolated from the culture-positive lesions, but always in low numbers. Eleven culture-negative and 18 culture-positive lesions were examined and had histologic characteristics of parasite-induced granulomas, with numerous eosinophils and epithelioid giant cells. Results of the study indicated that the histologic appearance of ovine hepatic lesions with F necrophorum was similar to bovine liver abscesses caused by F necrophorum, but unlike bovine liver abscesses, F necrophorum biovar B was isolated more frequently than was biovar A and often in pure culture. Most of the lesions in the condemned livers were parasite-induced granulomas.     

Occurrence of anaerobic bacteria in diseases of the dog and cat.

A survey for anaerobic bacteria was conducted in 314 clinical specimens from dogs and cats. A total of 187 anaerobic isolates in pure and mixed culture were isolated from 111 of the specimens that contained anaerobic bacteria. Common isolated included Actinomyces (9.1%), Clostridium perfringens (19.3%), other Clostridium spp (11.2%), Peptostreptococcus anaerobius (7.5%), Bacteroides melaninogenicus (13.4%), other Bacteroides spp (17.6%), and Fusobacterium necrophorum (5.3%). Anaerobic bacteria were involved in serious lesions that often were life threatening to the animals. Antibiotic susceptibility data indicated that the lincomycin family, the penicillin family, chloramphenicol, and cephaloridine are preferred drugs for treatment of anaerobic infections. Data from the survey were used in formulation of a table to aid practitioners in clinical diagnosis of disease caused by anaerobes. Clostridium perfringens was isolated in large numbers from five of six dogs with a clinical diagnosis of canine hemorrhagic gastroenteritis and from one cat with hemorrhagic diarrhea. Experimental infections were induced in rats, using caine feces as inoculum. Induced lesions contained aerobic and anaerobic bacteria similar to those bacteria isolated in the clinical survey, indicating that feces may serve as a major source of these bacteria in clinical infections of the dog.     

Bacterial flora of liver abscesses in feedlot cattle fed tylosin or no tylosin.

Bacterial flora of liver abscesses from cattle fed tylosin or no tylosin and susceptibilities of the predominant bacterial isolates to tylosin and other antimicrobial compounds were determined. Abscessed livers were collected at slaughter from cattle originating from feedlots that had fed tylosin (n = 36) or no tylosin (n = 41) for at least 2 yr, and segments of livers with one or two intact abscesses were transported to the laboratory. Abscesses were cultured for anaerobic and facultative bacteria. Fusobacterium necrophorum, either as single culture or mixed with other bacteria, was isolated from all abscesses. The incidence of subsp. necrophorum, as part of the mixed infection, was lower (P < .05) in the tylosin group than in the no-tylosin group (33 vs 61%). However, the incidence of Actinomyces pyogenes was higher (P < .01) in the tylosin group than in the no-tylosin group (53 vs 10%). Totals of 119 F. necrophorum and 21 A. pyogenes isolates were used for determinations of susceptibilities to bacitracin, oxytetracycline, chlortetracycline, lasalocid, monensin, tylosin, tilmicosin, and virginiamycin. The minimum inhibitory concentrations (MIC) of antibiotics were determined with a broth microdilution method. The mean MIC of tylosin for F. necrophorum and A. pyogenes were not different between isolates from tylosin and no-tylosin groups. We concluded that continuous feeding of tylosin did not induce resistance in F. necrophorum or A. pyogenes. Also, the higher incidence of mixed infection of F. necrophorum and A. pyogenes in liver abscesses of tylosin-fed cattle suggests a potential synergistic interaction between the two organisms in causing liver abscesses.     

奶牛腐蹄病坏死杆菌分离鉴定及白细胞毒素基因lktA1序列分析

为分离纯化奶牛腐蹄病坏死杆菌,分析其与其他菌株的亲缘关系,本研究利用坏死杆菌白细胞毒素特异性引物,对奶牛腐蹄病病牛蹄部拭子样品进行了PCR检测,利用厌氧培养基对PCR检测阳性样品进行了坏死杆菌的分离培养,以分离的坏死杆菌基因组DNA为模板,对白细胞毒素基因进行了克隆和序列分析。结果显示,9份奶牛腐蹄病病牛蹄部拭子样品PCR检测结果均为阳性,对其中一份样品中的坏死杆菌进行分离培养,获得了纯培养物,命名为bFR13-1。坏死杆菌bFR13-1菌株白细胞毒素基因测序结果显示,与GenBank已发表的H05、A25和B35菌株的白细胞毒素基因在核苷酸水平的同源性分别为98.40%、98.35%和90.79%,推导氨基酸的同源性分别为97.7%、97.6%和89.0%。进化树分析结果显示,坏死杆菌bFR13-1菌株白细胞毒素与H05菌株的同源性最高,bFR13-1菌株与H05菌株和A25菌株呈较近亲缘关系。结果表明,不同坏死杆菌分离株的白细胞毒素呈现一定的变异性,这种变化是否与坏死杆菌致病性相关,值得深入研究。     

Anaerobic bacterial infections and response to treatment in dogs and cats: 36 cases (1983-1985)

Anaerobic bacteria have been increasingly implicated as important pathogens in animals. To determine the prevalence of anaerobic bacterial infection, the results of anaerobic bacteriologic culture of 599 specimens obtained from dogs and cats hospitalized at the Colorado State University Veterinary Teaching Hospital were reviewed. Obligate anaerobic bacteria were isolated from 35% of properly submitted specimens; Bacteroides spp and Fusobacterium spp were the organisms most commonly isolated. Infections most often containing anaerobes were abscesses, pleuropulmonary infections, and abdominal infections. A complication rate of 28% was observed with anaerobic bacterial infections; failure to initially treat with an effective antibiotic increased the rate of recurrence of infection.     

Isolation,Identification and Sequence Analysis of Leukotoxin Gene lktA1 of Fusobacterium necrophorum of Cow Footrot

For separation and purification of Fusobacterium necrophorum of cow footrot, and analysis of genetic relationship with other strains, the hoof ministry swab samples were detected by PCR based on specific primers of leukotoxin gene, and genomic DNA were isolated from PCR positive samples of Fusobacterium necrophorum culturing in anaerobic medium.The genes of leukotoxin were cloned and sequenced.The results showed that nine of hoof ministry swab samples were all PCR positive samples, and we obtained Fusobacterium necrophorum pure culture from one of the samples which named bFR13-1.The gene sequencing results indicated that the homologies of leukotoxin gene nucleotide sequence of bFR13-1 strain compared with H05, A25 and B35 strains from GenBank were 98.40%, 98.35% and 90.79%, respectively, and the homologies of deduced amino acid sequence were 97.7%, 97.6% and 89.0%, respectively.Phylogenetic tree analysis results showed that leukotoxin gene of Fusobacterium necrophorum bFR13-1 and H05 had high homology and bFR13-1, H05 and A25 showed a close genetic relationship.The result indicated that leukotoxin showed variability between different Fusobacterium necrophorum isolated strains, and it was worth to study whether this change and pathogenicity of Fusobacterium necrophorum were related.     

The two major subspecies of Fusobacterium necrophorum have distinct leukotoxin operon promoter regions

Fusobacterium necrophorum, a gram-negative, non-spore-forming anaerobe, is a normal inhabitant of the alimentary tract of animals and humans. Two types of F. necrophorum, subspecies necrophorum (biotype A) and funduliforme (biotype B), have been recognized, which differ morphologically, biochemically and biologically. The organism is an opportunistic pathogen that causes numerous necrotic conditions (necrobacillosis) such as bovine hepatic abscesses and ruminant foot abscesses. Subspecies necrophorum strains are considered to be more virulent for cattle and have been shown to produce greater amounts of leukotoxin than subspecies funduliforme strains. The leukotoxin operon of F. necrophorum consists of three genes (lktBAC) of which the leukotoxin structural gene (lktA) is the second gene in the operon. In this study, the promoter regions of the leukotoxin operons from the two subspecies were identified and their nucleotide sequence compared. The promoter regions were found to differ in sequence, in length of the sequence between the upstream determinant (oppF) and the first gene of the leukotoxin operon (lktB), and in promoter strength as assayed in Escherichia coli host cells.     

Summer mastitis in heifers: a bacteriological examination of secretions from clinical cases of summer mastitis in Denmark

A total of 166 samples of secretions collected from Danish heifers suffering from clinically diagnosed summer mastitis were examined bacteriologically. One hundred and sixty three samples yielded positive findings whereas no growth was obtained from 3 specimens. The majority of samples revealed a mixed flora in which the predominant components were Actinomyces pyogenes, Peptostreptococcus indolicus, a microaerophilic coccus (Stuart-Schwan coccus), Fusobacterium necrophorum, Bacteriodes melaninogenicus and Streptococcus dysgalactiae. Pure cultures were recovered in only 7% of cases. P. indolicus was isolated from 875 of the cases, a microaerophilic coccus from 84%, A. pyogenes from 72%, Fusobacterium necrophorum from 52%, Str. dysgalactiae from 37%, B. melaninogenicus from 33% and various unidentified obligate anaerobic Gram-negative rods from 31%. The results confirm the complex bacterial aetiology of summer mastitis.     

The obligate and facultatively anaerobic bacterial flora of the normal feline gingival margin

Samples from the gingival margins of 14 cats considered normal on clinical examination were cultured for facultative and obligate anaerobic bacteria. All mouths were free from any gingival marginal inflammation and tartar build-up; all cats were between 6 and 12 months of age. A mixed growth was obtained from all samples. The mean number of bacterial species per sample was 10.7 with a range of 7-16 isolates. Of the 150 isolates processed, 109 (72.66%) were obligate anaerobes. Of the facultatively anaerobic species, Actinomyces (including A. viscosus, A. hordeovulneris and A. denticolens) comprised 12%, Pasteurella multocida 9.33% of isolates and Propionibacterium species 6% of all isolates. Gram-negative bacilli belonging to the genera Bacteroides and Fusobacterium were isolated from 12 of the 14 samples, and comprised 77% of the obligate anaerobes isolated. Clostridium villosum comprised 10.1% of obligately anaerobic isolates, Wolinella species made up 6.42%, while 4.58% were Peptostreptococcus anaerobius. The most commonly isolated obligately anaerobic species was C. villosum and the most commonly isolated facultatively anaerobic species was P. multocida. These findings show a bacterial flora of the normal feline mouth which is very similar in composition to that of cat fight abscesses and feline pyothorax.     

Fusobacterium equinum possesses a leukotoxin gene and exhibits leukotoxin activity

Fusobacterium equinum, a gram negative, rod-shaped and an obligate anaerobic bacterium is a newly described species. The organism is associated with necrotic infections of the respiratory tract in horses that include necrotizing pneumonia, pleuritis and paraoral infections. The species is closely related to F. necrophorum that causes liver abscesses in cattle and sheep, calf-diphtheria in cattle, and foot-rot in sheep and cattle. Leukotoxin, an exotoxin, is an important virulence factor in bovine strains of F. necrophorum. Our objective was to examine strains (n=10) of F. equinum for leukotoxin (lktA) gene and its toxic effects on equine leukocytes. Southern hybridization and partial DNA sequencing revealed that all the 10 strains had the lktA gene with greater similarities to F. necrophorum subsp. necrophorum. The secreted leukotoxin was detected in the culture supernatant and its biological activity was determined by viability assays with equine polymorphonuclear cells (PMNs) using flow cytometry. While culture supernatants of four strains (E1, E7, E9, and E10) were highly toxic to equine PMNs; strain E5 was moderately toxic and the remaining strains (E2, E3, E4, E6, and E8) were only mildly toxic. Our data indicated that F. equinum isolates had lktA gene and its product was toxic to equine leukocytes. Therefore, leukotoxin may be an important virulence factor in F. equinum infections.     

Anaerobic Bacteria Isolated from Osteomyelitis in Dogs and Cats

Anaerobic bacteria were isolated from 18 of 28 animals (64%) with osteomyelitis. The bones most commonly infected with anaerobic bacteria were radius and ulna, mandible, and tympanic bulla. Fights or abscesses commonly preceded the osteomyelitis. Seven anaerobic genera were isolated. Mixed infections of anaerobic and aerobic organisms occurred in 16 animals. Staphylococci were isolated in only one such mixed infection, but they were isolated commonly when there were aerobic bacteria only. Staphylococcal infections were often single.     

Comparative biological features of a rat liver abscess model induced with three Fusobacterium necrophorum strains

Several biological features were compared in a rat liver abscess model, using intraportal inoculations with 3 bovine strains of Fusobacterium necrophorum which varied in virulence. Serum alanine aminotransferase activities were increased significantly (P less than 0.05) in rats inoculated with F necrophorum 2101 by postinoculation hours 6, 12, and 24. Thereafter, alanine aminotransferase values returned to base line for the remainder of the experiment. Also, rats inoculated with F necrophorum 2101 had a significantly greater (P less than 0.05) weight loss than did the control rats during the first 5 postinoculation days and developed leukocytosis characterized by a neutrophilia with a left shift. The duration of the bacteremia was related directly to the virulence of the F necrophorum strain. Fusobacterium necrophorum 2101, a biotype A which was the most virulent, induced the most persistent bacteremia; F necrophorum 2035, a biotype B which was the least virulent, produced the shortest bacteremia; and F necrophorum 2030, a biotype AB which was of intermediate virulence, led to bacteremia of intermediate duration. Plasma endotoxin was demonstrated intermittently during the first 24 hours, but did not correlate with the bacteremia.     

Isolation and partial characterisation of bacteria recovered from abscesses of normally slaughtered pigs

One hundred and one pig abscesses of different localisation were examined. Bacteria were re-covered from all abscesses. In 6’8 abscesses a mixed aerobic and anaerobic flora was found. In 22 abscesses solely aerobes and in 11 solely anaerobes were found. Gorynebaoterium pyogenes and Fuso-bacterium necrophorum were found in 61 and 45 abscesses, respectively. Hemagglutination litres of G. pyogenes strains were higher with pig erythrocytes than with sheep erythrocytes. Three of 4 isolates of Staphylococcus aureus produced enterotoxin type A.Key words: pig abscess, Corynebacterium pyogenes, Fuso-bacterium necrophorum, hemagglutination     

The detection of obligate anaerobic bacteria in udder secretions of dry cattle with mastitis during summer: a comparison between gas-liquid chromatography and bacteriological culturing methods

Udder secretions sampled during the summer in 1984 and 1985 from mastitic quarters of 51 non-lactating cattle, mainly heifers less than 2 years of age, were examined bacteriologically for the presence of (facultative) aerobic and obligate anaerobic bacteria (OAB) and by gas-liquid chromatography (GLC) in order to detect volatile fatty acids (VFA), metabolic end-products of OAB. Forty-nine samples yielded positive cultures and in 20 cases these were mixtures of (facultative) aerobes and OAB. Only two specimens appeared to be sterile and from one specimen only were OAB cultured. Corynebacterium pyogenes was isolated from 35% of the cases and Peptococcus indolicus and Fusobacterium necrophorum from 31 and 22%, respectively. In most specimens (19/21) which yielded OAB after culturing, VFA (C3-C6) could be detected by GLC. Detection of VFA in summer mastitis secretions appeared to be a useful technique to evaluate the importance and association of OAB with summer mastitis. Because samples can be easily collected and stored at -20 degrees C, this is especially advantageous in situations where adequate facilities for the isolation of OAB are not readily available.     

Pathogenicity of Fusobacterium necrophorum biovar C in mice by intraperitoneal and intraportal injections

Three strains of Fusobacterium necrophorum biovar C were injected into mice intraperitoneally and intraportally. All the mice survived. In one mouse out of 15 mice injected intraperitoneally, a few focal abscesses were formed in the liver. The microorganisms were recovered from the liver abscess and the tissue of liver with abscess. No changes were observed in the organs of other 14 mice and no bacteria were recovered from them. In the 15 mice injected intraportally, no liver abscesses and no macroscopic changes in the organs were formed. However, the inoculated bacteria were recovered from the liver of four mice. The pathogenicity of F. necrophorum biovar C was weaker than that of other two biovars.     

A microaerophilic coccus in pyogenic infections of ruminants

Pyogenic infections of cattle, sheep and goats were examined for the presence of a Gram positive bacterium that has been designated "microaerophilic coccus" by other workers. The bacterium was found to be involved in a range of disease processes, including foot and soft tissue abscesses, mastitis, pericarditis and pyometra in cattle, joint and foot abscesses in sheep and foot abscesses in goats. The characteristic feature of the bacterium was its satellitic growth around colonies of other organisms. The microaerophilic coccus was usually part of a mixed flora, which included Corynebacterium pyogenes, Fusobacterium necrophorum, Peptostreptococcus indolicus and Bacteroides sp.     

Bacteria in enteric lesions of cattle

Thirty-nine species of bacteria were isolated from or demonstrated in the abomasal and small and large intestinal mucosa of 23 adult cattle and 41 calves and identified. The bacteria isolated were related to the gross and microscopical lesions. Campylobacters, Clostridium perfringens type A, C sordellii, Actinobacillus lignieresii, Fusobacterium necrophorum, Mycobacterium paratuberculosis, Aeromonas hydrophila and Escherichia coli were all associated with specific lesions. The relationship of other bacteria such as Bacillus licheniformis, Branhamella catarrhalis and Bacteroides vulgatus to lesions in which they were found was discussed. It was concluded that some of the bacteria could be responsible for the lesions in which they were found. However, proof of this supposition could only be obtained by experimental infections of non-immune cattle with pure cultures.     

Minimum inhibitory concentrations of some antimicrobial drugs against bacteria causing uterine infections in cattle

The minimum inhibitory concentrations (MICs) of oxytetracycline, cephapirin, cephapirin/mecillinam, cefquinome, ceftiofur and enrofloxacin, candidate antibiotics for the principal bacteria associated with uterine infections: Escherichia coli, Arcanobacterium pyogenes and the anaerobic bacteria Fusobacterium necrophorum and Prevotella melaninogenicus, were determined by the agar dilution method. The bacteria were isolated from animals with clinical metritis and/or endometritis. For E coli, cefquinome and enrofloxacin had the lowest MIC90 and MIC50 values (< 0.06 microg/ml), and oxytetracycline and cephapirin had the highest values. For A pyogenes, oxytetracycline had the highest MIC50 value (16 microg/ml), but all the cephalosporins had values below 0.06 microg/ml. For the anaerobic bacteria, enrofloxacin and oxytetracycline had the highest MIC50 values but all the cephalosporins had values of 0.06 microg/ml or below.     

Jaw disease in macropod marsupials: Bacterial flora isolated from lesions and from the mouths of affected animals

Bacterial infections of the jaws are a common cause of death in macropods. Lesions and oral cavities from 50 affected animals yielded wide ranges of aerobic and anaerobic organisms. The most frequent isolate from lesions (81%) was Fusobacterium necrophorum, generally combined with other bacteria, but in 5 lesions, in pure culture. It was also isolated from 61% of mouths and this was the chief difference between the oral flora of affected and normal macropods. Other groups of organisms isolated from over 50% of lesions were: Gram-negative aerobic and anaerobic rods, streptococci, and anaerobic Gram-positive cocci. Actinomycetes were isolated from 29% of lesions and from one lesion in pure culture. Differences in the flora were detected between lesions in bone and soft tissue and between closed and open lesions. Antibiotics were given to 22 animals, but without significant differences in frequencies of isolation of organisms between treated and untreated groups, and with no permanent elimination of infection. It was concluded that, while different organisms might be present in the complex of “jaw disease”, the pathogenic agent in the majority of cases was F. necrophorum. Actinomycetes were capable of producing lesions in bone, but their role in “jaw disease” remains undefined.