Plasma endotoxin activity in kangaroos with oral necrobacillosis (lumpy jaw disease) using an automated handheld testing system.

The aim of the present study was to evaluate the reliability and effectiveness of directly determining endotoxin activity in plasma samples from kangaroos with lumpy jaw disease (LJD, n=15) and healthy controls (n=12). Prior to the present study, the ability of the commercially available automated handheld portable test system (PTS(TM)) to detect endotoxin activity in kangaroo plasma was compared with that of the traditional LAL-kinetic turbidimetric (KT) assay. Plasma samples, which were obtained from endotoxin-challenged cattle, were diluted 1:20 in endotoxin-free water and heated to 80°C for 10 min. The performance of the PTS(TM) was not significantly different from that of the traditional LAL-based assay. The data obtained using PTS(TM) correlated with those using KT (r(2)=0.963, P<0.001). These findings indicated that the PTS(TM) is applicable as a simplified system to assess endotoxin activity in macropods. In the present study, we demonstrated the diagnostic value of plasma endotoxin activity in kangaroos with systemic inflammation caused by oral necrobacillosis and identified plasma endotoxin activity as a sensitive marker of systemic inflammation in kangaroos with LJD. Based on ROC curves, we proposed a diagnostic cut-off point for endotoxin activity of >0.22 EU/ml for the identification of LJD. Our results indicate that the assessment of plasma endotoxin activity is a promising diagnostic tool for determining the outcome of LJD in captive macropods.

Fusobacterium species infections: clinical spectrum and outcomes at a district general hospital.

PURPOSE: Fusobacterium species infections are rare. Recently, however, this potentially deadly pathogen has been attracting interest, and efforts are being made to characterise its epidemiology and clinical spectrum of disease. The aim of our study is to provide further evidence towards this cause, in what is, to date, the largest study of its kind from the UK. METHOD: A 22-year, retrospective, descriptive study was performed at Royal Hampshire County Hospital. An electronic database was used to identify patients with microbiologically confirmed infection with Fusobacterium, and clinical records were examined to provide further information on the presentation, source, treatment and outcome. RESULTS: Fusobacterium species infections were identified in 18 patients during the study period, which is an incidence of 0.76 cases/100,000/year. The overall death rate was 29 %. Half of these patients had Fusobacterium necrophorum infections and were a predominantly young, fit and uniquely male population who had excellent outcomes. Among the remaining patients with Fusobacterium species infections, 22 % had infection with F. varium and 11 % with F. nucleatum. These patients were an older cohort who tended to have co-morbidities and unsurprisingly worse outcomes. We identified a number of Fusobacterium bacteraemias likely to have resulted from pressure ulcers, a presentation that has been rarely reported. Interestingly, we also identified a case of neonatal F. nucleatum bacteraemia that was not associated with premature nor stillborn birth. CONCLUSION: As work continues to depict the spectrum of disease caused by this enigmatic bacterium, it is hoped that improved clinical suspicion will result in better outcomes and management.

Biochemical tests cannot differentiate between tonsillar and middle ear-derived infections.

INTRODUCTION: Infection markers are appreciated supplements in the clinical diagnosis of ear, nose and throat (ENT) infections. We aimed to examine the differential diagnostic usefulness of C-reactive protein (CRP), white blood cell count (WBC) and absolute neutrophil count (ANC) according to severity of middle ear and tonsillar infections. MATERIAL AND METHODS: This was a retrospective study including all patients admitted to the ENT Department, Aarhus University Hospital, from January 2001 to December 2008 and diagnosed with acute otitis media, mastoidismus, acute mastoiditis, acute tonsillitis, peritonsillar abscess, parapharyngeal abscess and necrotizing fasciitis. RESULTS: A total of 1,773 patients were included. Between the tonsil subgroups, significant differences were found in CRP (p < 0.001), WBC (p < 0.001) and ANC (p < 0.001) levels. However, sensitivities and specificities related to differential diagnostics were low. In the middle ear group, no differences in CRP (p = 0.84), WBC (p = 0.46), and ANC (p = 0.72) levels were found. The number of CRP levels above the reference value was significantly higher than the corresponding number of WBC and ANC levels. A trend (non-significant) was found towards lower parameter levels in acute tonsillitis and peritonsillar abscess patients who grew Staphylococcus aureus compared with patients infected with other bacteria. CONCLUSION: CRP and ANC levels were related to severity of tonsillar-derived infections, but no such relation was found in infections with middle ear origin. None of the infection markers studied were useful for differential diagnostics. FUNDING: not relevant. TRIAL REGISTRATION: not relevant.

Unusual presentation of Lemierre's syndrome: two cases and a review.

Lemierre's syndrome is a potentially fatal disease that usually presents with oropharyngeal infection, followed by sepsis, thrombosis of the internal jugular vein and septic emboli. Most cases are caused by the Gram-negative, anaerobic Fusobacterium necrophorum. We present two patients with an atypical presentation of Lemierre's syndrome and a review. These cases illustrate that a positive blood culture for F. necrophorum, even without the presence of clinical symptoms pointing towards thrombosis of the internal jugular vein, justifies further radiological testing for thrombophlebitis of the internal jugular vein.

[Case report: positive signals from automated blood-culture system with negative direct Gram examination]. / A propos d'hémocultures détectées positives en système automatisé mais dont l'examen direct est négatif.

Detection of positive haemoculture is usually managed by an automated system. When a bottle is detected positive but that the Gram coloration does not reveal germs by direct examination, transfer onto chocolate blood agar generally allows to confirm or infirm bacteraemia. In light of a case of Fusobacterium nucleatum bacteraemia, we discuss the opportunity of pairing it with an enrichment broth. M. N, hospitalized in the hepatogastroenterology department, runs a fever of undetermined origin. Three pairs of blood samples are collected on May 7th, 2004, another pair on May 9th, 2004 and a last pair on May 10th, 2004. They are incubated in a Bactec 9120 analyzer. A positive signal is detected in the two last anaerobic haemocultures pairs after four days of incubation, but in both cases, the Gram coloration does not bring germs to light. A systematic transfer of the broth onto chocolate blood agar with incubation under CO2 enriched atmosphere and anaerobiosis is carried out. After 24 hours, the solid media remain sterile. The samples found positive by the Bactec(TM) are then transferred onto Schaedler broth in order to favour a potential growth of fastidious germs. The culture will prove to be positive only in this enrichment medium, allowing the identification of F. nucleatum. An hepatic abscess will then be revealed in the patient. It thus appears judicious to associate an enrichment medium with transplanted solid medium when the context is evocative of a real infection (clinic, positivity delays...).

Molecular diagnosis of Fusobacterium necrophorum infection (Lemierre's syndrome).

Presented here is the case of a 27-year-old male with atypical features of Lemierre's syndrome in which a definitive diagnosis was achieved using molecular methods. While routine investigations, including bacterial cultures, were unhelpful, two real-time PCR assays demonstrated Fusobacterium necrophorum-specific DNA in aspirates from brain and renal abscesses. This is the first report demonstrating that a laboratory diagnosis can be made using molecular methods in suspected cases of Lemierre's syndrome. Use of these methods can thus resolve diagnostic confusion, prevent unnecessary investigation, and direct specific antimicrobial treatment.

Serum antibodies of periodontitis patients compared to the lipopolysaccharides of Porphyromonas gingivalis and Fusobacterium nucleatum.

Serum antibody titers against the lipopolysaccharides (LPSs) of Porphyromonas gingivalis and Fusobacterium nucleatum were compared between 9 periodontitis patients and 24 healthy persons. The IgG titers against the LPSs of P. gingivalis ATCC 33277(T) and W50 were clearly higher in the patients than in the healthy persons. However, IgM titers against the LPSs of P. gingivalis strains were relatively low, and no significant difference was observed between the patients and healthy persons. On the other hand, IgG and IgM titers against the LPS of Fusobacterium nucleatum JCM 8532(T) in some patients were significantly higher than those in the healthy persons, although the difference in IgG titers was not large compared to that of the LPS of P. gingivalis. These results suggest that the antibody measurement of patients' sera against the LPS of periodontal bacteria can be applied for the diagnosis of periodontitis.

Effect of Fusobacterium nucleatum on the T and B cell responses to Porphyromonas gingivalis in a mouse model.

T cell cytokine profiles and specific serum antibody levels in five groups of BALB/c mice immunized with saline alone, viable Fusobacterium nucleatum ATCC 25586, viable Porphyromonas gingivalis ATCC 33277, F. nucleatum followed by P. gingivalis and P. gingivalis followed by F. nucleatum were determined. Splenic CD4 and CD8 cells were examined for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma and IL-10 by dual colour flow cytometry and the levels of serum anti-F. nucleatum and anti-P. gingivalis antibodies determined by an ELISA. Both Th1 and Th2 responses were demonstrated by all groups, and while there were slightly lower percentages of cytokine positive T cells in mice injected with F. nucleatum alone compared with the other groups immunized with bacteria, F. nucleatum had no effect on the T cell production of cytokines induced by P. gingivalis in the two groups immunized with both organisms. However, the percentages of cytokine positive CD8 cells were generally significantly higher than those of the CD4 cells. Mice immunized with F. nucleatum alone had high levels of serum anti-F. nucleatum antibodies with very low levels of P. gingivalis antibodies, whereas mice injected with P. gingivalis alone produced anti-P. gingivalis antibodies predominantly. Although the levels of anti-F. nucleatum antibodies in mice injected with F. nucleatum followed by P. gingivalis were the same as in mice immunized with F. nucleatum alone, antibody levels to P. gingivalis were very low. In contrast, mice injected with P. gingivalis followed by F. nucleatum produced equal levels of both anti-P. gingivalis and anti-F. nucleatum antibodies, although at lower levels than the other three groups immunized with bacteria, respectively. Anti-Actinobacillus actinomycetemcomitans, Bacteroides forsythus and Prevotella intermedia serum antibody levels were also determined and found to be negligible. In conclusion, F. nucleatum immunization does not affect the splenic T cell cytokine response to P. gingivalis. However, F. nucleatum immunization prior to that of P. gingivalis almost completely inhibited the production of anti-P. gingivalis antibodies while P. gingivalis injection before F. nucleatum demonstrated a partial inhibitory effect by P. gingivalis on antibody production to F. nucleatum. The significance of these results with respect to human periodontal disease is difficult to determine. However, they may explain in part differing responses to P. gingivalis in different individuals who may or may not have had prior exposure to F. nucleatum. Finally, the results suggested that P. gingivalis and F. nucleatum do not induce the production of cross-reactive antibodies to other oral microorganisms.

Correlation of serum concentration of alpha 1-acid glycoprotein with lymphocyte blastogenesis and development of experimentally induced or naturally acquired hepatic abscesses in cattle.

Changes in serum alpha 1-acid glycoprotein (alpha 1AG) concentration in cattle with hepatic abscesses were observed, and function of alpha 1AG was evaluated, particularly its influence on cellular immune response. Test cattle (n = 4) were inoculated with Fusobacterium necrophorum, control cattle (n = 2) were inoculated with inactivated bacteria, and naturally affected cattle (n = 11) were found in a slaughterhouse. Determination of alpha 1AG was made by use of a single radial immunodiffusion method. The action on lymphocyte blastogenesis was determined by [3H]thymidine incorporation. Cultured lymphocytes from healthy cattle were treated with variable concentrations of alpha 1AG purified from serum obtained from cattle with hepatic abscesses and suppression of blastogenesis stimulated by each of 3 mitogens was measured. In cattle with experimentally induced abscesses, serum alpha 1AG concentration increased for 7 to 10 days after F necrophorum inoculation, its change being parallel to that of sialic acid. High concentration of alpha 1AG was found in naturally affected cattle and was highly correlated to sialic acid concentration. Suppression of lymphocyte blastogenesis in cattle with experimentally induced hepatic abscesses was highly correlated to serum alpha 1-AG concentration.

Hepatic ultrasonography and blood changes in cattle with experimentally induced hepatic abscesses.

Hepatic abscesses were induced experimentally in 5 steers by inoculating Fusobacterium necrophorum via ultrasonography-guided, percutaneous catheterization of the portal vein. Hepatic ultrasonography was performed to determine the onset and progression of abscessation. Blood samples were collected before and after inoculation for performing leukocyte counts and hepatic function tests. Ultrasonographic evidence of liver abscesses was observed as early as 3 days after inoculation. Abscesses appeared as hyperechoic centers (cellular debris and pus) surrounded by hypoechoic or anechoic areas (fluid). Increases in rectal temperature, leukocyte counts, fibrinogen, globulin, bilirubin, gamma-glutamyltransferase, and sorbitol dehydrogenase concentrations were detected. Hepatic dysfunction was evidenced by decrease in serum albumin concentration and low sulfobromophthalein clearance. The ultrasonographic diagnosis of abscesses correlated well with necropsy findings.

Fusobacterium necrophorum and Actinomyces pyogenes associated facial and mandibular abscesses in blue duiker.

Anaerobic and aerobic cultures of facial and mandibular abscesses were made from 12 blue duiker (Cephalophus monticola fusicolor) housed at the Deer and Duiker Research Facility of the Pennsylvania State University (USA). Increases in concentrations of total protein and serum globulin occurred in all cases. Actinomyces pyogenes was isolated from nine animals. Fusobacterium necrophorum was present in eight and Bacteroides sp. was found in seven animals; other genera of isolated bacteria included: Streptococcus (from two animals), Lactobacillus (one), Staphylococcus (one) and Actinomyces (two). Eight (67%) of affected animals were less than or equal to 2 yr of age. Facial soft tissues and mandibles were the tissues most often affected. Tissues within the oral cavity were not affected at the time of presentation. A common finding, not reported in other host species with necrobacillosis, was the presence of nondestructive mandibular proliferation.

Plasma prolidase activity as a possible diagnostic index of chronic hepatic abscess in cattle.

The usefulness of prolidase as a biochemical parameter to represent the chronic state of hepatic abscess was discussed in eight cattle experimentally inoculated with Fusobacterium necrophorum and 18 spontaneously affected cattle. Blood was daily collected to measure the plasma prolidase activity and sialic acid level from the experimental cattle for 90 days after inoculation. In three out of four cattle affected with hepatic abscess, prolidase activity began to rise about 40 days after inoculation, and maintained high activity till 90 days. In the same cattle the sialic acid concentration increased from 7 to 10 days after inoculation, and gradually returned to the normal value 50 days after it. Another cow showed a similar change in early stage of experiment, but prolidase activity decreased after 70 days and sialic acid concentration maintained high level till 90 days. In two cattle, which showed scars but no abscess on autopsy, the prolidase activity increased temporarily from 40 to 55 days after inoculation. In the control cattle inoculated with an inactivated bacterial suspension, neither the sialic acid level nor the prolidase activity showed any large variation in the experimental period. Among the spontaneously affected cattle, those with a high sialic acid level revealed normal prolidase activity and those with a normal sialic acid level had high prolidase activity.

Aggregation of platelets by Fusobacterium necrophorum.

Broth cultures and washed cells of 13 of 24 bovine isolates of Fusobacterium necrophorum aggregated human platelets in platelet-rich plasma. The cell-free culture fluid was inactive. Bacteria stored at 4 degrees C in saline remained active for at least 3 months, but they did not release activity into the storage solution. Aggregation typically began within 1 min after the addition of 10(3) bacteria to 10(3) platelets was complete within 5.5 min. Assays for cytosolic lactic dehydrogenase revealed that platelet lysis did not occur. The release of [14C]serotonin from platelets preincubated with this amine accompanied aggregation, indicating that this was a typical aggregation-degranulation reaction. Platelet aggregation was inhibited by EDTA (88% at 2.0 mM), aspirin (75% inhibition at 1.0 mM), and quinacrine (80% inhibition at 0.25 mM). Thus the reaction was an ion-dependent, cyclooxygenase-sensitive event. Gel-filtered platelets were less sensitive to aggregation than were platelets in plasma, but this sensitivity was fully restored by the addition of plasma and partially restored with fibrinogen. Biotyping of the cultures revealed that none of the avirulent, B-type strains of F. necrophorum could aggregate platelets, whereas 13 of 16 virulent A type strains were positive. These results suggest that platelet aggregation by F. necrophorum is related to the virulence of this organism.

Haematological findings in red-necked wallabies (Protemnodon rufogrisea) with necrobacillosis.

Results of blood counts on 17 red-necked wallabies (Protemnodon rufogrisea) with confirmed necrobacillosis have been compared with those of 36 clinically normal animals. The most common abnormality in infected animals was a raised fibrinogen level, found in all of the cases tested. Neutrophilia occurred in 10 individuals and neutrophil morphology was abnormal in 15. Some infected animals showed monocytosis, raised erythrocyte sedimentation rates, high platelet counts and changes in their red cells. The findings provide useful information about the response of the blood to severe bacterial infections in red-necked wallabies and demonstrate the potential diagnostic use of clinical haematology in this species.

Fusobacteremia in a calf with necrotic stomatitis, enteritis and granulocytopenia.

This case of fusobacteremia appears to be identical to an interesting and unusual syndrome previously reported. We wish to bring the syndrome to the attention of others who may be able to elucidate the etiology further. Because hematological examinations are frequently not done on calves, this condition may be more common than reports suggest. Perhaps others who observe this syndrome in calves may be able to investigate the role of other agents such as viruses or mycotoxins. Experimental work may be able to establish whether or not the exotoxins of Fusobacterium necrophorum can suppress granulopoiesis.