Ultrastructure and molecular characterization of Fusobacterium necrophorum biovars.

The ultrastructural features and molecular components of 18 strains of Fusobacterium necrophorum biovars A, AB and B, isolated from animal and human infections, were examined by electron microscopy, multilocus enzyme electrophoresis (MEE) and by sodium dodecyl sulfate-gradient polyacrylamide gel electrophoresis (SDS-PAGE). High resolution scanning electron microscopy revealed that the strains possessed a convoluted surface pattern. Transmission electron microscopy showed that all strains possessed a cell wall structure typical of gram-negative bacteria. Bleb formation was not uncommon. Numerous extracellular materials, resembling lipopolysaccharide (LPS) fragments, surrounded cells of both human strains and biovar B animal strains. Biovar A field strains revealed capsules as stained by ruthenium red whereas a stock culture strain showed the capsule only when immunostabilized with hyperimmune serum. Starch gel electrophoresis showed all strains to possess adenyl kinase, glutamate dehydrogenases and lactate dehydrogenase; each enzyme migrated uniformly (monomorphic) among the strains and represented an electrotype. However, SDS-PAGE indicated differences in the protein profiles between all of the strains; the most distinctly different was a human isolate (FN 606). Silver staining to detect LPS showed extensive "ladder" patterns among the majority of biovar A strains but not in the animal biovar B strains. Immunoblotting of LPS with a rabbit antiserum prepared against phenol extracted LPS from a biovar A animal isolate (LA 19) suggested marked variability in the LPS antigens among the isolates studied.     

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.     

Development of a new PCR assay to identify Brucella abortus biovars 5, 6 and 9 and the new subgroup 3b of biovar 3

One hundred twenty-nine Brucella field strains isolated from cattle in Cantabria, Spain, from March 1999 to February 2003, were analysed by using the AMOS-ERY PCR assay and by Southern blot hybridisation with a probe from insertion sequence IS711. Most of the field isolates produced only the ery band in the AMOS-ERY assay and showed a hybridisation pattern identical to that exhibited by reference strains of biovars 5, 6 and 9 of Brucella abortus, but different from strain Tulya, belonging to biovar 3 of B. abortus. However, typing of these strains by standard methods demonstrated that they belonged to biovar 3 of B. abortus. These results indicated that B. abortus biovar 3 was not genetically homogeneous and at least could be divided in two. In one class, that we called biovar 3a, would be the Tulya strain, while the local field strains would belong to biovar 3b. Cloning and nucleotide sequencing of a DNA fragment containing an IS711 copy exclusive of the B. abortus field strains from biovar 3b and reference strains from biovars 5, 6 and 9, revealed the existence of a 5.4 kb deletion close to an IS711 copy. Based on these data, we designed a new primer, which together with the IS711 AMOS primer produced a PCR fragment of 1.7 kb only from the isolates of biovars 3b, 5, 6 and 9 of B. abortus. No amplification products were produced with these primers from strains of the rest of species and biovars of Brucella and from bacteria phylogenetically close to Brucella analysed in this work. Addition of this primer to the AMOS-ERY PCR primer cocktail allows the positive distinction of B. abortus biovars 3b, 5, 6 and 9 from the rest of Brucella species and biovars.     

Infection trials in pigs with a human isolate of Brucella ( isolate 02/611 'marine mammal type' )

AIM: To determine if pigs could support infection of a human Brucella isolate (Brucella 02/611) from New Zealand, and to study seroconversion to this isolate using a competitive ELISA. METHODS: Ten weaner piglets were challenged with 4.8 x 10(8) cfu of organisms by the oral and ocular routes. Culture was attempted on blood samples taken prior to challenge, and 4, 7, 9, 11, 14, 21 and 28 days post-challenge, and on tissue samples taken at the termination of the trial, 1 month after challenge. Sera were analysed for antibody using an ELISA. For reference comparison, similar trials were conducted in two pigs using an isolate of Brucella suis biovar 1, and two pigs using an isolate of B. suis biovar 3. RESULTS: Brucella 02/611 organisms were re-isolated from one lymph node each from three pigs; all other samples were negative. Low and transient antibody titres were detected using a competitive ELISA in three pigs, two of which were culture negative. Organisms of B. suis reference strains were re-isolated from multiple samples from each of the four animals. CONCLUSION: Brucella 02/611 does not seem to replicate readily in pigs. It is unlikely that pigs were the original maintenance hosts for Brucella 02/611.     

Studies on a suitable antibiotic therapy for treating swine brucellosis

The aim of this work was developing effective treatments against Brucella suis biovar 2, responsible for swine brucellosis in Europe. MICs for antibiotics used classically in brucellosis and two new macrolides (tulathromycin and tildipirosin) were determined for 33 B. suis biovar 2 field and B. suis reference strains. MIC₉₀ values ranged from 0.01 to 0.25 μg/mL. The best candidates, given alone or combined, were then evaluated in mice. Ten groups (n = 7) of BALB/c mice were inoculated (1 × 10⁵ CFU/mouse) with a virulent B. suis biovar 2 field strain. All groups, excepting untreated control, were treated for 14 days with, respectively, doxycycline, dihydrostreptomycin, tulathromycin (one or two doses), or tildipirosin (one or two doses) given alone, and doxycycline combined with dihydrostreptomycin, tulathromycin, or tildipirosin. Combined tildipirosin treatment was the most effective, then selected for pig studies. Sixteen B. suis biovar 2 naturally infected sows were treated with oxytetracycline (20 mg/kg BW/daily) for 21 days. The half of these received also tildipirosin (4 mg/kg BW) in two doses with a 10‐day interval. An extensive bacteriological study conducted ten days after ceasing treatments proved the efficacy of this combined oxytetracycline/tildipirosin treatment.     

Brucella suis identification and biovar typing by real-time PCR

Fast and accurate identification of Brucella suis at the biovar level is an important issue for public health laboratories because some of the biovars that infect suidae (boars and pigs) are pathogenic for humans while others are not. Since classical biovar typing methods are often time-consuming, hard to standardize and require high-level biosafety containment, methodological improvements are desirable. This article describes new single nucleotide polymorphism (SNP) signatures for the rapid identification and biovar characterization of B. suis. These SNPs were included together with previously described ones in real-time PCR assays applicable to low-biosafety conditions. Allelic profiles unique for each B. suis biovar were defined and the most relevant signatures were determined on a collection of 137 field strains of worldwide origin characterized previously. Biovars assigned with both present and classical methods were globally consistent except for some biovar 3 field strains which matched the allelic profile of biovar 1.     

Comparative behaviour of Brucella abortus strains 19 and RB51 in the pregnant mouse.

Pregnant BALB/c mice received various doses of either Brucella abortus strain 19, a smooth vaccine strain, or B abortus strain RB51, a stable rough organism, intraperitoneally on day 9 of gestation to compare the relative pathogenicity of the two attenuated strains. Nine days after inoculation, spleens and placentas were collected for bacteriological and histopathological examination. A dose of 10(7.5) and strain 19 organisms produced a severe necrosuppurative placentitis occasionally accompanied by fetal death. This dose resulted in a 10-fold higher level of splenic infection than did a dose of 10(9.5) strain RB51 organisms, which produced only mild to minimal placentitis not associated with fetal death. Strain 19 infected mice showed seroconversion in the standard tube agglutination test in contrast to the seronegative titre of strain RB51 infected mice. The results of this study corroborate previous investigations on the relative pathogenicity and the serological response of the non-pregnant mouse to strain RB51.     

Aggregation of bovine platelets by Fusobacterium necrophorum.

Washed cell suspensions of biovar A strains of Fusobacterium necrophorum aggregated cattle platelets, but similar suspensions of biovar B strains did not. Platelets were also aggregated by heat-treated bacterial cells or the lipopolysaccharide of biovar A. No platelet aggregation occurred in the presence of the cell-free culture supernatant of biovar A and of all samples prepared from biovar B. Scanning electron microscopy revealed that aggregated platelets were not damaged. Platelet aggregation was inhibited by EDTA, aspirin and quinacrine, and lag time was retarded by these inhibitors, indicating the reaction was a Ca(2+)-dependent, cyclo-oxygenase sensitive event. Platelet aggregation may be a virulence marker, probably mediated by the lipopolysaccharide of F. necrophorum biovar A strains.     

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.     

Prevention of experimental haemorrhagic septicaemia with a live vaccine

Pasteurella multocida serotype B:3,4 isolated from a fallow deer in England was used as a vaccine to prevent haemorrhagic septicaemia. The deer strain was less virulent for calves than typical serotype B:2 of haemorrhagic septicaemia strains. It elicited antibodies in cattle that protected mice against serotype B:2 infection. The live deer vaccine containing 2 X 10(7) viable organisms per dose was used to immunise calves. Six months after vaccination, five of six calves were protected against serotype B:2 challenge. Two calves challenged nine months after vaccination survived the same challenge. The live vaccine was more efficacious than an alum precipitated vaccine in protecting calves against B:2 challenge.     

Experimental infection of rabbits (Oryctolagus cuniculus) with Brucella suis biovar 1 isolated from wild hares (Lepus europaeus)

Brucella suis biovar 1 is the causative agent of brucellosis in several domestic and wild animals and it is a common agent of human brucellosis. European hares (Lepus europaeus) have been shown to be infected by B. suis biovar 1 and the transmission to other animals has been suggested. In this work, experimental rabbits (Cuniculus orictolagus) were infected with B. suis biovar 1 isolated from wild hares. Infected rabbits showed high serological response in 2 weeks after discharge and typical granulomatous lesions (2mm diameter) were found in liver, spleen and kidneys after 50 days. B. suis biovar 1 was cultured from the lesion of the organs mentioned above as well as from urine, placenta and fetuses. These data suggest that hares are a potential source for horizontal transmission of B. suis biovar 1 to other mammalians.     

Immunostimulating effect of culture filtrates of Yersinia enterocolitica

Application of sterile culturing supernatant of Yersinia (Y.) enterocolitica (tested serovars were 03 and 08) caused significantly accelerated transplant rejection in mice of various inbreeding strains. Action correlated with dosage (r = -0.92). C57B16 mice were tested for their pregnancy rates in response to the same filtrate (serovar 03), with 5.5 live births per mating being recorded from 47 control matings but only 4.4 from 45 experimental matings (alpha less than 0.0025). The mean gestation period of experimental animals was extended by five percent over that of simultaneous controls (alpha = 0.25). Particular reference is made in this paper to Vesikari et al. (1987) who found Y. enterocolitica to function as interleukin-1 inductor via lipopolysaccharide. The active substance tested in this context, however, proved to be thermolabile, with 30 minutes of heating to 56 degrees C eliminating the action tested before. Y. enterocolitica infections were frequently found to coincide with rheumatoid arthritis, and evidence has been produced to the unspecific stimulating effect of Y. enterocolitica culture filtrates (testing being applied to serovar 03, biovar 4 and serovar 08, biovar 2). It is against the background of these aspects that chronic and other infections by Y. enterocolitica are considered to be of substantive relevance to the etiopathogenesis of autoimmune diseases, above all rheumatoid arthritis.     

Antigenic analysis of Pasteurella haemolytica serovars 1 through 15 by crossed immunoelectrophoresis

Pasteurella haemolytica serovars 1 through 12, grown in broth and on agar plates, and 2 field isolates (types A1 and T10) were used to develop polyvalent crossed immunoelectrophoresis (XIE) reference systems. The maximal number of antigens was revealed by XIE when sonicates of agar plate-grown organisms were used as the immunogen (to produce antibodies) and as the soluble antigen for XIE. Antigens produced from agar plate-grown organisms were less contaminated (by antigenic components of the medium) than were those produced from organisms grown in broth. Seventy-two antigens were detected in sonicated preparations of agar plate-grown P haemolytica. The common antigen of gram-negative bacteria was identified in the P haemolytica XIE reference system; precipitation was observed with rabbit antiserum to the common antigen of gram-negative bacteria isolated from Escherichia coli, as well as with rabbit immunoglobulins (obtained from unvaccinated rabbits). Most preimmune sera from our vaccinated rabbits also precipitated the common antigen. Serovar-specific antigens in the P haemolytica XIE reference system were defined and presumptively identified as part of the bacterial lipopolysaccharide complex by use of the limulus amebocyte lysate test. Partial cross-reactions were found between serovar-specific antigens within each biovar (A and T). Pasteurella haemolytica biovar A-specific and biovar T-specific antigens were defined by crossed-line immunoelectrophoresis. When serovars A13, A14, and T15 were tested in the P haemolytica XIE reference system, they gave high matching coefficient values of 0.98, 0.98, and 0.87, respectively. The proposal to separate P haemolytica biovars A and T into 2 different species was supported by immunotaxonomic data obtained from crossed immunoelectrophoresis, but more extensive studies will be necessary to establish the appropriate taxonomic position of these 2 groups of organisms.     

Mycobacterium avium infection through the alimentary tract in mice

Intestinal infection by Mycobacterium avium was investigated in C57BL/6 and BALB/c mouse strains. Single intragastric administration of a massive dose (10(8] or multiple administration of a lower dose (10(7), 10 times) established infection principally in the mesenteric lymph-node (MLN); a continuous or intermittent fecal excretion of the bacilli was detected by 6-8 weeks after the administration. Based on three criteria--isolation of the organisms from the MLN and from feces, and detection of acid-fast bacilli in sections of the MLN--germ-free (GF) BALB/c mice exhibited clearer dose-effect relations than the flora-bearing (FB) counterparts. After intragastric administration, the organisms were probably trapped in the Peyer's patch and then transferred to the MLN at an early period (by 4-7 days), persistent infection thus being established in the MLN. Systemic involvement evolved both in athymic and euthymic mice after a prolonged period of time (more than 40 weeks) showing far more severe involvement in the former regardless of the presence of floral organisms.     

Human brucellosis at a pig slaughterhouse

Seventeen workers in a pig slaughterhouse with signs and symptoms compatible with brucellosis were clinically examined at the outpatient service of different health institutions and studied by serological tests during the period 2005–2011. Eleven blood cultures were taken and six Brucella suis strains were isolated, three biovar 1 and three with atypical characteristics. In order to confirm that these cases had no common source, a variable number of tandem repeat (VNTR) analyses were performed on 5 of the 6 strains whose results showed substantial heterogeneity in the genotypes, thereby demonstrating that the immediate origin was not the same. Two hundred adult pigs admitted for slaughter at the plant were sampled by convenience and tested by buffered antigen plate test (BPAT), serum agglutination test (SAT) and 2-mercapto-ethanol test (MET). Seven of 62 males (11%) and 25/138 (18%) females tested positive. The study results contribute information on risk scenarios for packing plant workers and underscore the need to improve plant workers’ education on appropriate containment measures and to actively screen animals for swine brucellosis.     

The opsonic activity of bovine milk whey for the phagocytosis and killing by neutrophils of encapsulated and non-encapsulated Escherichia coli

Encapsulated strains of Escherichia coli were found to be more resistant to phagocytosis and killing by bovine neutrophils; requiring in the order of 100 times more serum opsonins than non-encapsulated strains. Mid-lactation pooled whey from cows with no history of mastitis was opsonic for non-encapsulated strains, but had no effect on encapsulated organisms. In contrast, early lactation pooled whey (5-10 days post-partum) was opsonic for all strains of E. coli. It is concluded that since early lactation milk contains sufficient opsonins, severe E. coli mastitis at this stage of lactation is not due to opsonic deficiency.     

广西部分地区猪链球菌的分离鉴定及小鼠致病性研究

对来自广西边境地区部分发病猪场及屠宰场的370份猪组织进行细菌分离鉴定,共分离到16株链球菌,对这16株链球菌进行了形态学观察、PCR鉴定及小鼠致病性试验。结果表明,其形态、染色、生化特性均符合猪链球菌的特点,通过GDH鉴定与测序,证实这16株菌株均为猪链球菌,分别命名为GX1~GX16。通过特异性引物PCR分型（35个血清型）,其中有1株为1型,1株为2型,1株为5型,1株为29型,5株为8型,7株未分出型。用这16株猪链球菌对昆明小鼠和BALB/c小鼠进行动物致病性试验,结果发现,菌株GX10对昆明小鼠和BALB/c小鼠致死率均为100%;菌株GX11对BALB/c小鼠致死率为80%;其他菌株对昆明小鼠和BALB/c小鼠均无致病性。菌株GX10、GX11对BALB/c小鼠的LD₅₀分别为4.5×10⁵和3.6×10⁹ CFU。本试验结果表明,广西边境地区存在致病性猪链球菌8型。BALB/c小鼠对猪链球菌的敏感性高于昆明小鼠,更适合作为猪链球菌致病性研究的动物模型。     

Isolation,Identification and Pathogenicity on Mice of Streptococcus suis in Pig in Guangxi Border Area

16 strains of Streptococcus were isolated and identified from 370 pig tissues of clinically morbidity farms and slaughter houses in Guangxi border area.Morphological observation, PCR identification and pathogenicity test were carried out. The results showed that the morphology,dyeing,biochemical characteristics were coincidence with the characteristics of Streptococcus suis,confirmed that 16 strains were Streptococcus suis,and named as GX1 to GX16. Further by divided type identification (35 serotypes) by GDH,1 strains was type 1,1 strains was type 2,1 strains was type 5,1 strain was type 29,5 strains were type 8 and 7 strains could not differentiate types. The pathogenicity of 16 strains Streptococcus suis were detected by Kunming mice and BALB/c mice. The results showed that the letal rate of GX10 for Kunming mice and BALB/c mice was 100%;GX11 for BALB/c mice was 80%;Other strains were apathogenicity to the Kunming mice and BALB/c mice. The LD₅₀ of GX10 and GX11 was 4.5×10⁵ and 3.6×10⁹ CFU for BALB/c mice,respectively. The results showed that pathogenic Streptococcus suis type 8 occured in Guangxi border area. The sensitivity of BALB/c mice to Streptococcus suis was higher than Kunming mice, BALB/c mice was more suitable for the pathogenic study of Streptococcus suis.     

From the discovery of the Malta fever's agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a re-emerging zoonosis

Brucellosis is not a sustainable disease in humans. The source of human infection always resides in domestic or wild animal reservoirs. The routes of infection are multiple: food-borne, occupational or recreational, linked to travel and even to bioterrorism. New Brucella strains or species may emerge and existing Brucella species adapt to changing social, cultural, travel and agricultural environment. Brucella melitensis is the most important zoonotic agent, followed by Brucella abortus and Brucella suis. This correlates with the fact that worldwide, the control of bovine brucellosis (due to B. abortus) has been achieved to a greater extent than the control of sheep and goat brucellosis (due to B. melitensis), these latter species being the most important domestic animals in many developing countries. The long duration and high cost of treatment of human brucellosis reduces the efficacy of the therapy. There is no human vaccine for brucellosis and the occurrence of brucellosis is directly linked to the status of animal brucellosis in a region. In this context, the Word Health Organization has defined the development of a human vaccine, besides the implementation of control and eradication programs in animals, as a high priority. The pathogenicity for humans of B. suis biovars 1, 3 and 4 is well established, whereas B. suis biovar 2 seems to be less pathogenic. Indeed, although hunters and pig farmers have repeatably experienced infectious contact with B. suis biovar 2 (found in wild boar and outdoor-rearing pigs in Europe), isolation of B. suis biovar 2 from human samples have only been seldom reported. Marine mammal brucellosis, due to two new proposed Brucella species i.e. B. cetaceae and B. pinnipediae, represents a new zoonotic threat but the pathogenicity for humans of the different Brucella species found in cetaceans and pinnipeds still has to be clearly established.     

Specificity in response of vaccinated swine and mice to challenge exposure with strains of Erysipelothrix rhusiopathiae of various serotypes.

Swine and mice were vaccinated with standard erysipelas adsorbate bacterins made from Erysipelothrix rhusiopathiae of serotype 2 and were subsequently exposed to pathogenic strains of E rhusiopathiae, serotypes 1, 2, 4, 9, 10, and 11. Response to challenge of immunity in swine was determined by presence of urticarial lesions at the sites of intradermal injection of culture; response in mice was determined by the quantal (live-dead) method. After vaccination with standard bacterins, swine and mice were significantly more susceptible (P less than of equal to 0.01) to infection with strains of serotypes 9 and 10 than with strains of serotypes 1, 2, 4, or 11. An adsorbate bacterin made from the challenge strain of serotype 10 induced specific immunity to homologous challenge exposure in swine but not in mice. Bacterins made from the other challenge strains induced little or no immunity.