Multocida Organisms Research Articles

Simultaneous infection with rabbit hemorrhagic disease virus and Pasteurella multocida in rabbits

Rabbit infectious hemorrhagic fever has been reported in rabbits worldwide. The disease is also frequently reported on Korean rabbit farms, and the pathological study of 9 rabbits on such disease-occurring farms was attempted to identify the pathogen. Clinical signs were torticollis and ear ulceration. Most rabbit died with bloody nasal discharges. At necropsy, multiple hemorrhages and inflammation were observed in heart, lung, liver and uterus. The main histopathologic features were hemorrhagic suppurative meningoencephalitis, fibrinous bronchointerstitial pneumonia, bacteremia, liver cell necrosis, multifocal hemorrhages in kidney and disseminated intravascular coagulopathy. The viral VP60 gene of RHDV was identified by Reverse Transcriptase PCR. Pasteurella multocida organisms were cultured, identified by biochemical test and serotyped as A by multiplex capsular typing PCR. In conclusion, the fatal hemorrhagic disease was due to combined infection with both RHDV and P. multocida in rabbits. To our knowledge, this is the first case report about co-infection with both RHDV and P. multocida in rabbits in Korea.

Isolation, Identification and Antimicrobial susceptibility patterns of Pasteurella multocida Isolated from Diseased Rabbits

Pasteurella multocida is a widespread pathogen associated with major animal diseases of considerable economic significance to livestock sector in Egypt. Pasteurella species (spp.) isolated from rabbits are poorly characterized. Therefore, this study was conducted to isolate and study the phenotypic characteristics and antimicrobial susceptibility profiles among P. multocida organisms recovered from diseased rabbits. Isolation of P. multocida was attempted from the collected nasal swabs and tissue samples of rabbits. A total of 16 P. multocida isolates (8%) were recovered from lung tissues and nasal swabs with incidence rates of 6.5 and 1.5%, respectively. The pure cultures were identified on the basis of their cultural, morphological and biochemical properties, pathogenicity studies in rabbits and molecular recognition by a species specific PCR assay. The in vitro antibiotic sensitivity results revealed the occurrence of antimicrobial resistance among all the isolates. The resistance was more frequent to doxycycline, tetracycline and colistin (100% each), followed by erythromycin, cefotaxime, chloramphenicol, ciprofloxacin and trimethoprim/sulfamethoxazole (93.8% each). Meanwhile, low levels of susceptibility were found; with the greatest level of sensitivity to nitrofurantoin (75%). All the examined strains demonstrated remarkably multi-drug resistant (MDR) patterns with resistance to at least 8 drugs. The results of this investigation provide useful information on the diagnosis and antimicrobial resistance patterns of P. multocida for selection of appropriate antimicrobial agents.

Isolation and molecular characterization of Pasteurella multocida isolates of buffaloes

Pasteurella multocida is a Gram negative bacterial pathogen associated with Haemorrhagic septicemia (HS) in cattle and buffaloes. A total of 196 samples were collected from buffaloes over a period of one year in Krishna district of Andhra Pradesh for the purpose of isolation of Pasteurella multocida. Among the 196 samples 23 were found positive by molecular test, where as only 16 isolates of P. multocida were recovered and identified by using cultural, biochemical and molecular tests. Capsular typing of these isolates by multiplex PCR revealed two capsular types (A and B). Among these Cap A was common (87.5%) and was isolated from tonsil samples of apparently healthy animals, whereas the Cap B isolates (12.5%) were obtained from HS suspected cases. The results of this study implied the application of molecular tests in the diagnosis as well as typing of P. multocida organisms.

Aqueous Humor Antimicrobial Activity: In Vitro Analysis after Topical 0.5% Chloramphenicol Application

ABSTRACTPurpose: To assess aqueous humor antimicrobial activity in vitro after topical 0.5% chloramphenicol application.Materials and methods: This investigation included 63 eyes from 65 cataract surgery patients. The study group of 48 eyes received preoperatively four topical applications of 0.5% chloramphenicol. The control group of 15 eyes was given no topical applications. Aqueous humor samples were collected for in vitro antimicrobial analysis using Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Pasteurella multocida organisms by means of disk diffusion test.Results: No inhibition halo was observed around all aqueous humor samples from all chloramphenicol-treated patients, irrespective of the sample quantity added to the paper disks, with no significant difference from aqueous humor from untreated control patients.Conclusions: Aqueous humor displayed no bactericidal effect against any of the microorganisms evaluated after topical 0.5% chloramphenicol application.

Isolation of Pasteurella multocida from chickens, preparation of formalin killed fowl cholera vaccine, and determination of efficacy in experimental chickens

Objectives: The objectives of this study were to isolate and identify Pasteurella multocida from fowl cholera (FC) suspected chicken, and to prepare and efficacy determination of formalin killed fowl cholera vaccine using the isolated P. multocida strain. Materials and methods: A total of five suspected dead chickens were collected from Brothers Poultry Farm located at Gazipur district, Bangladesh. The samples were processed and the P. multocida was isolated through conventional bacteriological techniques, were finally confirmed by polymerase chain reaction using P. multocida specific primers targeting cap gene. The P. multocida isolate was used to develop a formalin killed fowl cholera vaccine. The efficacy of the newly prepared vaccine was determined in Starcross-579 chickens (n=30) aging 15 weeks either by injecting 1 mL (group-A; n=10) or 0.5 mL (group-B; n=10) vaccine containing approximately 3.2x108 CFU/mL P. multocida organism; 10 birds were kept as unvaccinated control. The sera from the vaccinated and control birds were collected and were subjected for antibody titre determination by enzyme-linked immunosorbent assay (ELISA). Finally the vaccinated birds were challenged using virulent strains of P. multocida to confer the protection against FC. Results: P. multocida could be isolated from both the samples. The formalin killed vaccine prepared from the isolated bacteria was subjected for the determination of antibody titre in chicken, and found that the antibody titres in the birds of group A and group B were 4.513 and 4.07 respectively after primary vaccination, and 4.893 and 4.37 respectively after booster vaccination. Most of the vaccinated birds were found to be survived after challenging with virulent strain of P. multocida. Conclusion: It is concluded that the causal agent of FC (P. multocida) was successfully isolated from FC affected dead chickens. The prepared formalin killed fowl cholera vaccine induces protective immune response and conferred protection against challenge infection caused by the virulent strain of P. multocida.

Isolation and molecular detection of Pasteurella multocida Type A from naturally infected chickens, and their histopathological evaluation in artificially infected chickens in Bangladesh

Pasteurella multocida type A is the etiologic agent of fowl cholera, a highly contagious and fatal disease of chickens. The present research work was performed for the isolation, identification and molecular detection of P. multocida Type A from chickens. Liver, heart and spleen of suspected dead chicken (n=35) were collected from Gazipur and Pabna districts in Bangladesh. The targeted bacteria from the samples were isolated, identified and characterized based on their morphology, staining, cultural, biochemical characters, pathogenicity test, histopathological study and Polymerase Chain Reaction (PCR). The P. multocida organism was isolated from 11.42% (n=4/35) samples. The organisms were gram negative, non-spore forming rod, non-motile, occurring singly or pairs in Gram staining, whereas in Leishman’s stain, bipolar shaped organisms were observed. All the isolates were found positive for oxidase and catalase tests, produced indole, and fermented glucose, mannitol and sucrose. Necrotic foci in liver and congestion with hemorrhages in heart were found on necropsy. After pathogenicity test, the pathological changes were reconfirmed by histopathology depicting congestion, hemorrhage and lymphocyte infiltration in heart, liver and spleen tissues. In type specific PCR reaction, the organisms were confirmed as P. multocida Type A. In conclusion, P. multocida type A is prevalent among poultry in the studied regions; thus, care must be taken to control of the disease. http://dx.doi.org/10.5455/javar.2015.b104

Occurrence of Pasteurellosis and Newcastle Disease in indigenous chickens in Sirajgonj district

A study was carried out on indigenous layer chickens to know occurrence of Avian Pasteurellosis (AP) and Newcastle disease (ND) in Sirajgonj during the period from January/2012 to December/2013. The clinical signs showed before death was recorded by taking history and the birds were subjected to post mortem examination. In addition to the clinical and necropsy findings, ND was detected by Anigen® rapid antigen detection kit. The AP was confirmed by isolation and identification of Pasteurella (P.) multocida from liver, spleen and heart samples. The P. multocida was found to grow on nutrient broth, nutrient agar, blood agar and Eosin Methylene Blue agar where it produced whitish, opaque, round, flat, translucent colonies. It produced turbidity on nutrient broth. The organism was not found to grow on MacConkey agar media and did not cause hemolysis on blood agar media. The impression smear of liver and heart blood were stained by Gram’s staining, Leishman staining and Methylene blue staining techniques to detect P. multocida. P. multocida organism was also found to fermnt dextrose, lactose and mannitol with the production of only acid but did not ferment maltose and lactose. P. mutocida was found non-motile, indole positive and urease negative. On triple sugar iron test it produced H2S and fermented only glucose. It was found negative to both methyl red test and Voges Proskauer test. Out of 360 birds tested 59.72% (215) was AP positive and 40.28% was ND positive. Age, Sex, season and mortality due to other diseases were not considered.DOI: http://dx.doi.org/10.3329/bjvm.v11i2.19123 Bangl. J. Vet. Med. (2013). 11 (2): 97-105

Concurrent outbreak of pasteurellosis and aflatoxicosis in Kuttanad ducks

One hundred and forty eight ducks of age group of 12–14 months out of a total population of two hundred and fifty ducks died of concurrent outbreak of pasteurellosis and aflatoxicosis in an organized farm. Tissue samples were collected for histopathology and bacterial isolation. Hepatic tumours were observed in six ducks. Heart blood smear and impression smears from various visceral organs were taken and bipolar stained Pasteurella organisms were detected by Leishman staining. Feed samples were collected for aflatoxin estimation and found to be 500 ppb. Pasteurella multocida organism was isolated from representative tissue samples collected. Characteristic pathological alterations corresponding to aflatoxicosis and pasteurellosis were found in different visceral organs.

Prevalent Serotypes of Pasteurella multocida Isolated from Different Animal and Avian Species in India

Identification and estimation of the prevalence of Pasteurella multocida organisms in different animal and avian species in India during November 2000 to July 2003 was carried out. Out of 418 samples collected from different outbreaks suspected to be caused by P. multocida, a total of 206 bacterial cultures were identified as P. multocida on the basis of cultural, morphological and biochemical characteristics. All the 206 cultures were isolated from different domestic animal species (cattle, buffalo, sheep, goat, pig and rabbit), avian species (chicken, duck, quail, turkey, goose) and wild animals such as leopard and deer. Serotyping of P. multocida cultures revealed the presence of various serotypes (A:1, A:3, A:1,3, A:4, B:2, D:1 and -:1) among the livestock population. P. multocida polymerase chain reaction (PCR) assay applied on different forms of bacterial cultures (bacterial culture lysate, direct bacterial colony and mixed bacterial culture lysate) yielded an amplified product of approximately 460 bp specific for P. multocida. The results of PCR assay correlated well with conventional methods of identification. The present investigation revealed the presence of varied serotypes among livestock and PCR assay was found to be useful for rapid, sensitive and specific diagnosis of pasteurellosis in animals and avian species.

Variation in resistance to Pasteurella multocida among turkey lines

Previous research has shown that a line (F) of turkeys selected long-term for increased 16-wk body weight was more susceptible to challenge with washed Pasteurella multocida than a randombred control line (RBC2), the base population of F. A previous study also indicated that the mortality of the F line following challenge with P. multocida was similar to that of sire lines from two of the three major primary breeders. The purpose of the present study was to compare the resistance of the sire line from the third major primary turkey breeder (C) not previously studied with that of the F and RBC2 lines to determine whether there is variation in resistance among the sire lines from three major primary breeders. The sire lines from all three primary breeders were used in the production of commercial turkeys. Body weights of the F line were greater than those of the C line at the time of challenge with P. multocida. Both the C and F lines were heavier than the RBC2 line. The birds were challenged at 6 wk of age with a field isolate of washed P. multocida (1.2 x 10(7) organism per bird of capsular serogroup A and somatic serotype 3,4). Mortality was recorded daily for 14 d. Mortality following challenge with P. multocida was higher in the F line than in the C line, and both large-bodied lines had higher mortality than the RBC2 line. Based on the present results and those published in the literature, there may be variation in resistance among commercial sire lines from the three major primary breeders.

Effects of ammonia inhalation and acetic acid pretreatment on colonization kinetics of toxigenic Pasteurella multocida within upper respiratory tracts of swine.

Pigs reared in intensive production systems are continuously exposed to ammonia released by the microbial degradation of their excrement. Exposure to this gas has been shown to increase the severity of the disease progressive atrophic rhinitis by facilitating colonization of the pig's upper respiratory tract by Pasteurella multocida. The etiological mechanism responsible for this synergy was investigated by studying the colonization kinetics of P. multocida enhanced by ammonia and comparing them with those evoked by an established disease model. Three-week-old Large White piglets were weaned and allocated to five experimental groups (groups A to E). Pigs in groups A and B were exposed continuously to ammonia at 20 ppm for the first 2 weeks of the study. Pigs in group C were pretreated with 0.5 ml of 1% acetic acid per nostril on days -2 and -1 of the study. On day 0 all the pigs in groups A, C, and D were inoculated with 1.4 x 10(8) toxigenic P. multocida organisms given by the intranasal route. The kinetics of P. multocida colonization were established by testing samples obtained at weekly intervals throughout the study. The study was terminated on day 37, and the extent of turbinate atrophy was determined by using a morphometric index. The results of the study showed that exposure to aerial ammonia for a limited period had a marked effect on the colonization of toxigenic P. multocida in the nasal cavities of pigs, which resulted in the almost total exclusion of commensal flora. In contrast, ammonia had only a limited effect on P. multocida colonization at the tonsil. The exacerbation of P. multocida colonization by ammonia was restricted to the period of ammonia exposure, and the number of P. multocida organisms colonizing the upper respiratory tract declined rapidly upon the cessation of exposure to ammonia. During the exposure period, the ammonia levels in mucus recovered from the nasal cavity and tonsil were found to be 7- and 3.5-fold higher, respectively, than the levels in samples taken from unexposed controls. Acetic acid pretreatment also induced marked colonization of the nasal cavity which, in contrast to that induced by ammonia, persisted throughout the time course of the study. Furthermore, acetic acid pretreatment induced marked but transient colonization of the tonsil. These findings suggest that the synergistic effect of ammonia acts through an etiological mechanism different from that evoked by acetic acid pretreatment. A strong correlation was found between the numbers of P. multocida organisms isolated from the nasal cavity and the severity of clinical lesions, as determined by using a morphometric index. The data presented in the paper highlight the potential importance of ammonia as an exacerbating factor in respiratory disease of intensively reared livestock.

Role of outer membrane protein H (OmpH)- and OmpA-specific monoclonal antibodies from hybridoma tumors in protection of mice against Pasteurella multocida.

Two major outer membrane proteins of Pasteurella multocida, designated OmpH and OmpA, were characterized and shown to be related to the families of porin and heat-modifiable proteins, respectively. The backpack hybridoma tumor system in BALB/c mice was used to continuously deliver immunoglobulin G2b (IgG2b) monoclonal antibodies (MAbs) specific for OmpH (MAb MT1) and OmpA (MAb MT4.1). MAbs were detected in serum and peritoneal lavage samples of mice bearing hybridoma tumors by an enzyme-linked immunosorbent assay and an immunoblot assay. Highly significant protection was observed in mice bearing MT1 hybridoma tumors against both intraperitoneal and intranasal challenge infections with homologous nontoxigenic P. multocida strains possessing MAb MT1-reacting epitopes, whereas the mice bearing MT4.1 hybridoma tumors were not protected. The numbers of P. multocida organisms in the lungs of mice bearing MT1 hybridoma tumors were significantly less than those in lungs of mice bearing MT4.1 hybridoma tumors at 48 h postchallenge. These results indicate that the OmpH-specific MAb inhibited proliferation of P. multocida in the lungs. MAb MT1 was unable to kill P. multocida in vitro in the presence of complement. However, an enhanced phagocytosis by polymorphonuclear cells (PMNs) was observed in mice bearing MT1 hybridoma tumors. P. multocida induced a more extensive and rapid influx of PMNs into the peritoneal cavity of mice bearing MT1 hybridoma tumors than of mice bearing MT4.1 hybridoma tumors. The results of this study demonstrate for the first time that IgG MAbs against OmpH of P. multocida are involved in the protection of mice against lethal challenge infection by means of opsonization and inhibition of proliferation of P. multocida as a result of increased influx of PMNs into the infection site.

Detection of Pasteurella multocida-Specific DNA in Turkey Flocks by Use of the Polymerase Chain Reaction

A polymerase chain reaction (PCR)-based assay using primers constructed to amplify the gene (psl) encoding the P6-like protein (Psl) of Pasteurella multocida was developed. After Southern blotting and hybridization with psl, the assay (PCR-H) was found to be specific (it did not detect a variety of other avian bacterial pathogens) and sensitive (detected > or = 10 P. multocida organisms or > or = 24 femtograms of extracted P. multocida DNA). Samples were collected from the oropharynx of randomly selected birds housed on premises that had recently experienced an outbreak of avian cholera (outbreak farms) or from birds housed on premises that had not reported an outbreak of this disease during the preceding 12 mo (control farms). The PCR-H assay detected 11 infected turkeys out of a total of 178 sampled on six outbreak farms as compared with isolation of P. multocida from 23 turkeys by using mouse inoculation. Neither method detected P. multocida in samples collected from 174 turkeys sampled on six control farms. Statistical analysis using the Kappa test demonstrated that the results of the two tests showed poor agreement from five outbreak flocks (K = 0, 0, 0, 0.35, 0.47) and strong agreement from one outbreak flock (K = 0.89). Combined results from the outbreak flocks showed poor agreement (K = 0.49) between the two methods.

Synergistic role of gaseous ammonia in etiology of Pasteurella multocida-induced atrophic rhinitis in swine.

One-week-old Large White piglets were weaned and allocated to 14 experimental groups, each composed of five animals. Each group was housed in a separate Rochester exposure chamber and exposed continuously to gaseous ammonia at either 0, 5, 10, 15, 25, 35, or 50 ppm (two groups per exposure level). One week after ammonia exposure commenced, the pigs from one group at each exposure level were inoculated intranasally with 9 x 10(7) CFU of Pasteurella multocida type D. After a further 4 weeks of exposure, all the pigs were euthanized and the extent of turbinate degeneration was assessed by using a morphometric index (J.T. Done, D. H. Upcott, D. C. Frewin, and C. N. Hebert, Vet. Rec. 114:33-35, 1984) and a subjective scoring system (Ministry of Agriculture, Fisheries and Food, Atrophic Rhinitis: a System of Snout Grading, 1978). Exposure to ammonia at a concentration of 5 ppm or greater resulted in a significant increase in the severity of turbinate atrophy induced by P. multocida compared with that occurring in pigs kept in 0 ppm of ammonia. This effect was maximal at 10 ppm but decreased progressively at concentrations above 25 ppm. Regression analysis revealed a significant relationship between the severity of turbinate degeneration and the number of P. multocida organisms isolated from the nasal epithelium at the end of the experiment (R2 = 0.86). These findings suggest that exposure to ammonia facilitates the growth and/or survival of P. multocida within the upper respiratory tract of the pig, thereby contributing to the severity of the clinical disease atrophic rhinitis. Furthermore, exposure of pigs to ammonia at 10 ppm or greater, in the absence of either P. multocida or Bordetella bronchiseptica, induced a mild but statistically significant degree of turbinate atrophy. The findings of this study demonstrate that exposure to ammonia, at concentrations within the range encountered commonly in commercial piggeries, contributes to the severity of clinical lesions associated with atrophic rhinitis.

Cellular Defense of the Avian Respiratory System: Dose-Response Relationship and Duration of Response in Intratracheal Stimulation of Avian Respiratory Phagocytes by a Pasteurella multocida Bacterin

In experiments analyzing dose-response, intratracheal inoculation of chickens with 10(8) and 10(9) avirulent Pasteurella multocida organisms induced the migration within 24 hr of large numbers of respiratory lavage cells (RLC) with increased phagocytic proportions and phagocytic capacity. Doses from 10(4) to 10(7) organisms per bird resulted in elevated numbers of RLCs that were not significantly higher (P > 0.05) than values of uninoculated or mock-inoculated control chickens. When analyzing the duration of response, we found that inoculation with 10(9) organisms resulted in significantly higher (P < 0.05) numbers of RLCs for 63 to 89 hr without significant elevation in phagocytic proportion and capacity. The numbers of RLCs were elevated, although not significantly (P > 0.05), up to 11 days after inoculation. These results indicate that RLCs migrate to the respiratory tract only in response to a relatively high number of stimulating bacterial organisms and that the duration of response is relatively short. Although there were elevated numbers of RLCs beyond 89 hours after stimulation, the question remains as to whether these cells would enhance nonspecific defense of the respiratory system of chickens.

Chemical Ions Affect Survival of Avian Cholera Organisms in Pondwater

Avian cholera (Pasteurella multocida) is a major disease of wild waterfowl, but its epizootiology remains little understood. Consequently, we examined whether chemical ions affected survival of avian cholera organisms in water collected from the Nebraska Rainwater Basin where avian cholera is enzootic. We tested the response of P. multocida to ammonium (NH 4 ), calcium (Ca), magnesium (Mg), nitrate (NO 3 ), and ortho-phosphate (PO 4 ) ions individually and in combination using a fractional factorial design divided into 4 blocks. High concentrations of Ca and Mg, singly or in combination, increased survival of P. multocida organisms (P<0.001). We developed a survival index to predict whether or not specific ponds could be problem or nonproblem avian cholera sites based on concentrations of these ions in the water

Antibodies to outer membrane proteins but not to lipopolysaccharide inhibit pulmonary proliferation of Pasteurella multocida in mice

The role of rabbit antibodies against Pasteurella multocida outer membrane proteins and lipopolysaccharides (LPS) in resistance remains unknown. Pooled immune sera against P. multocida outer membranes were prepared from specific-pathogen-free rabbits immunized with sucrose gradient-purified P. multocida outer membranes. Western immunoblotting showed that purified outer membrane protein antibodies reacted strongly against the outer membrane proteins but not the purified LPS. Affinity-purified LPS antibodies exhibited strong reactivity against purified LPS and very little reactivity against outer membrane vesicles. Mice were inoculated intranasally with immune serum or normal rabbit serum, challenged intranasally with 10(6) CFU of P. multocida, and euthanatized 48 h later to determine the number of P. multocida organisms in the lungs. Mice inoculated with pooled immune serum had a 3,300-fold reduction (P less than 0.001) in the numbers of P. multocida in the lungs as compared with the controls. Similarly, mice inoculated with purified outer membrane protein antibodies had a 201-fold reduction (P less than 0.001) in the numbers of P. multocida. Conversely, mice inoculated with affinity-purified LPS antibodies had a 1.1-fold reduction (P greater than 0.50) in the numbers of P. multocida. These results show that antibodies against the outer membrane proteins but not the LPS are the components of rabbit immune sera which inhibit P. multocida proliferation in mouse lungs.

Rapid identification of Pasteurella multocida organisms responsible for haemorrhagic septicaemia using an enzyme-linked immunosorbent assay

Haemorrhagic septicaemia ( hs) is caused by specific serotypes of Pasteurella multocida and is one of the major economic diseases of cattle and buffalo in South East Asia. Definitive diagnosis of the disease-causing organism with the available methods is labour intensive and not totally reliable, consequently, an elisa system to identify P multocida organisms which cause Hs was developed. One hundred and twenty-four P multocida isolates were tested, 58 were type strains and 66 were field isolates. Analysis of these strains indicated the assay had a specificity of 99 per cent and sensitivity of at least 86 per cent. The sensitivity could be an underestimate, as five isolates assumed to be false negative reactions may not all be HS-causing strains. The Hs ELISA provides a rapid, simple, accurate and inexpensive diagnostic assay for identification of Hs causing organisms but does not represent a new typing system for P multocida. This assay will also enable countries to assess the impact of hs more accurately.

Comparison of Enzyme-Linked Immunosorbent Assay with Dot Immunobinding Assay for Detection of Antibodies against Pasteurella multocida in Turkeys

A dot immunobinding assay (DIA) was developed to detect antibodies against Pasteurella multocida in turkey serum. Five coating antigens, namely, whole-cell (WC) antigen, sonicated cell lysate (SCL), crude capsular extract (CE), formalin extract (FE), and heat-stable antigen (HSA), were compared by enzyme-linked immunosorbent assay (ELISA) and DIA using reference antisera against P. multocida organisms. WC and SCL antigens showed higher sensitivity, whereas FE and HSA antigens were more specific coating antigens in both assays. The specificity of DIA was greater than ELISA by comparing the P/N ratios of HSA against serum prepared from heterologous serotype of P. multocida. The DIA had also several distinct advantages over the ELISA, which included reduction of the manipulation time and more uniform binding of coating antigens onto the nitrocellulose membranes compared with binding of coating antigens to microtiter plates for ELISA.

Effect of Polymyxin B Sulfate on Endotoxin Activity in a Gram-Negative Septicemia Model

The antiendotoxin effect of polymyxin B was investigated in experimentally induced septicemia in rabbits. The Pasteurella multocida organisms were sensitive to the antibacterial action of penicillin but not to polymyxin B. Animals pre-treated with polymyxin showed positive blood cultures and significantly reduced plasma endotoxin levels (Limulus test) with normal white blood cell and platelet counts when analyzed 6 hr after the injection of live organisms. Polymyxin therapy given after the animals had established septicemia-endotoxemia reduced the plasma endotoxin levels and improved the survival, but had no effect on the leukopenia and thrombocytopenia. The best survival data were obtained in rabbits who were treated with both penicillin and polymyxin. The data suggest that polymyxin is effective in neutralizing the endotoxic effects from live organisms and that the timing and perhaps duration of the polymyxin treatment is of critical importance. In addition, a modified Limulus lysate method was developed which showed that quantitative plasma endotoxin determination could be made more sensitive by prior heating of the plasma to remove the natural inhibitors.