Development of ELISA-based diagnostic methods for the detection of haemorrhagic septicaemia in animals.

Haemorrhagic septicaemia (HS) is an acute infection of cattle and buffaloes caused by the B:2 serotype of Pasteurella multocida. This disease is highly endemic in South Asia. In some peracute cases, there is 100% mortality in infected animals within a few hours of infection. Therefore, timely diagnosis of infection may contribute to its treatment and control to minimize economic losses. The current work reported the development of ELISA-based assays for the detection of anti-P. multocida antibodies and pathogen i.e. P. multocida. Owing to high immunogenicity, membrane proteins (MPs) extracted from local isolates of P. multocida serotype B:2 (PM1, PM2, and PM3) were employed as a potential diagnostic antigen for the development of indirect ELISA (i-ELISA) to detect HS antibodies in animals. MPs extracted from PM1, PM2 and PM3 isolates showed very low heterogeneity; hence MPs from the PM3 isolate were selected for the development of i-ELISA. The concentration of MPs (as coating antigen) of 3.13 µg/well and test sera dilution 1:100 was found to be optimal to perform i-ELISA. The developed method was validated through the detection of anti-P. multocida antibodies in sera of mice, immunized with MPs and formalin killed cells from the three local isolates (PM1, PM2 and PM3) of P. multocida. The significantly higher antibody titer in immunized mice was determined compared to unimmunized mice with the cut off value of 0.139. To detect P. multocida directly from the blood of infected animals, whole cell-based ELISA (cb-ELISA) assay was developed. A better detection signal was observed in the assay where bacterial cells were directly adsorbed on plate wells as compared to poly L-lysine (PLL) assisted attachment at a cell concentration of 106 CFU and 107 CFU respectively. The developed assays can be scaled up and potentially be used for the rapid detection of HS antibodies to gauge the immune status of the animal as well as vaccination efficacy and pathogen detection.

Comparison of two indirect ELISA coating antigens for the detection of dairy cow antibodies against Pasteurella multocida.

The ELISA is recognized as an efficient diagnostic tool for antibody detection, but there is no standard ELISA assay for detection of antibodies against hemorrhagic septicemia (HS) in cattle. The present study reports on an indirect ELISA assay for antibody detection of HS in dairy cows, and evaluates the sensitivity (Se) and specificity (Sp) of the method using a Bayesian approach. An indirect ELISA was developed with two types of heat extract antigens, Pasteurella multocida strains P-1256 and M-1404, as coating antigens. A checkerboard titration was employed using dairy cow sera immunized with P. multocida bacterin and colostrum-deprived calf sera. The concentrations of heat extract antigen (160µg/mL), sample serum (1:100) and goat anti-bovine immunoglobulin G labeled with horseradish peroxidase (1:2000) were optimal for the assay. The cut-off values were 0.147 and 0.128 for P-1256 and M-1404 coating antigens, and there were no differences in the results of tests with positive and negative sera (p<0.05). The characteristics of three diagnostic tests were evaluated using a one-population Bayesian model, assuming conditional dependence between two types of coating antigen-based ELISAs and indirect hemagglutination assay (IHA). A total of 415 sera samples from dairy cows without HS vaccination and no history of disease were tested. The Se and Sp of the P-1256 and M-1404 ELISAs were higher than those of the IHA. The Se and Sp of the P-1256 ELISA were 90.3% and 90.1%, while the Se and Sp of the M-1404 ELISA were 92.1% and 71.9%. The median values of Se and Sp from the IHA were 36.0% and 58.2%.

Epidemiological features and financial loss due to clinically diagnosed Haemorrhagic Septicemia in bovines in Karnataka, India.

The epidemiological features and financial losses due to Haemorrhagic Septicemia (HS) in bovines were studied in Karnataka state using the primary data collected from 133 clinically diagnosed HS affected farms. The various losses due to HS and the Benefit- Cost of the vaccination programme in cattle and water buffaloes were studied using mathematical models. The number of HS outbreaks were higher during the year 2002 and peaked during 2005 and thereafter declined due to targeted vaccination against HS. The morbidity and mortality risks were lower in large farms than medium and small farms, and lower in indigenous cattle compared to high yielding crossbred cattle and water buffaloes. The disease occurrence was more in in-milk animals causing serious economic loss to the farmers. Most outbreaks were observed during monsoon season, though the disease was prevalent throughout the year. The mean milk loss per animal was $2, $11 and $50 in indigenous cattle, water buffaloes and crossbred cattle, respectively. In the case of draught animals, the average effective draught power was unavailable for 1.2days/outbreak resulting in a loss of $5 per affected oxen. The treatment and extra labor expenses incurred per animal were $24 and $7, respectively. The average loss per animal due to mortality loss was $275, $284 and $415 in case of indigenous cattle, water buffaloes and crossbred cattle, respectively. The projected loss for the state of Karnataka were $23.89, $17.92 and $11.95 million under high, medium and low HS incidence scenarios, respectively. The Benefit Cost Analysis (BCA) of the vaccination against HS has been estimated at 5.97:1, 4.48:1 and 2.98:1 under high, medium and low incidence scenarios, respectively. The results highlight the important epidemiological features and financial losses to the affected households and the state of Karnataka.

Development of loop-mediated isothermal amplification-based diagnostic assays for detection of Pasteurella multocida and hemorrhagic septicemia-associated P multocida serotype B:2.

OBJECTIVE To develop 2 rapid loop-mediated isothermal amplification (LAMP) assays for detection of Pasteurella multocida DNA (Pm-LAMP assay) and P multocida DNA from strains associated with hemorrhagic septicemia (HS) in cattle and buffalo (HS-LAMP assay). SAMPLE Solutions containing 16 P multocida strains and 9 other bacterial species at various concentrations. PROCEDURES Optimal conditions were determined for running the Pm-LAMP and HS-LAMP assays. The assays were then used to detect DNA of the test organisms. Results of LAMP assays were validated against conventional PCR assays designed for specific detection of P multocida and the B:2 serotype of HS-associated strains. RESULTS Following incubation of sample reaction mixtures for 27 minutes, specificity and sensitivity of the HS-LAMP assay at template DNA amounts as low as 5 pg were 93% and 97%, respectively. When duplicates of each sample were incubated for 28 minutes (a positive result defined as positive results for both reactions of a given sample), specificity and sensitivity of the HS-LAMP assay in the same conditions increased to 100%. The best specificity and sensitivity of Pm-LAMP single (93% and 91%) and duplicate (97% and 98%) reactions at template DNA amounts as low as 10 pg were achieved at 33 and 34 minutes, respectively. CONCLUSIONS AND CLINICAL RELEVANCE These preliminary findings suggested the developed HS-LAMP assay had high sensitivity and specificity for detection of HS-associated P multocida. Additional research is needed to determine the accuracy of the assay for use on clinical specimens obtained in HS-endemic countries such as Pakistan and Thailand.

Evaluation of an In-house indirect ELISA for detection of antibody against haemorrhagic septicemia in Asian elephants.

Pasteurella multocida causes haemorrhagic septicemia in livestock and wild animals, including elephants. The disease has been reported in Asian elephants in India and Sri Lanka, but to date there have been no reported cases in Thailand. ELISA or indirect hemagglutination assays (IHA) have been demonstrated to be able to detect the antibody against the disease in cattle, but no data are available for elephants. The present study reports a novel in-house indirect ELISA for antibody detection of haemorrhagic septicemia in Asian elephants, and evaluates the sensitivity and specificity of the method using a Bayesian approach. The characteristics of ELISA and IHA were analyzed using a one population Bayesian model assuming conditional dependence between these two diagnostic tests. The IHA was performed as recommended by the World Organization for Animal Health (OIE) manual for haemorrhagic septicemia. An in-house indirect ELISA was developed with a heat extract antigen of P. multocida strain M-1404 (serovar B:2) as a coating antigen and rabbit anti-immunoglobulin G conjugated with horseradish peroxidase (eIgG-HRP). The checkerboard titration method was done using elephant sera immunized with P. multocida bacterin and negative sera from colostrum-deprived elephant calves. The concentrations of heat extract antigen (160µg/ml), sample serum (1:100), and eIgG-HRP (1:1000) were optimal for the assay. The calculated cut-off value was 0.103. Of the elephant sera, 50.59% (43/85) were considered seropositive by ELISA. The sensitivity of the ELISA test was higher than that of the IHA test [median=86.5%, 95% posterior probability interval (PPI)=52.5-98.9%] while the specificity was lower (median=54.1%, PPI=43.6-64.7%). The median sensitivity and specificity of IHA were 80.5% (PPI=43.8-98.0%) and 78.4% (PPI=69.0-87.0%), respectively. These findings suggest that our in-house indirect ELISA can be used as a tool to detect the antibody against haemorrhagic septicemia in Asian elephants.

Septicemia y celulitis por Vibrio vulnificus en paciente cirrótico / Sepsis and cellulitis by Vibrio vulnificus in cirrhotic patient

No disponible

Neonatal Campylobacter coli hemorrhagic enteritis and bacteraemia

Um caso de campylobacteriose neonatal com enterite hemorrágica e bacteremia produzido por Campylobacter coli é apresentado. A mãe, proveniente de uma região rural, apresentou durante a gravidez, três episódios de diarréia autolimitada. A infeccão no recém nascido provavelmente foi adqüirida durante o parto. Os altos níveis séricos de immunoglobulinas específicas poderiam explicar a escassa sintomatologia, apesar da demorada prescricão do tratamento com gentamicina.

Haemorrhagic septicaemia by Aeromonas salmonicida subsp. salmonicida in a black-tip reef shark (Carcharhinus melanopterus).

One black-tip reef shark (Carcharhinus melanopterus) was found dead without previous signs of disease. Major lesions consisted in cutaneous erythema, mainly at the base of the fins, focal to diffuse inflammatory lesions in gills and intestinal wall, and discrete haemorrhages in the same organs, liver and kidneys. Microcolonies of Gram-negative rods were observed in the lamina propia of gills, underneath the intestinal mucosa and randomly distributed in the renal and hepatic parenchymas. Also, emboli containing Gram-negative rods were observed in capillaries of these organs. Aeromonas salmonicida subsp. salmonicida was isolated in pure culture from gills, liver and intestine. Specific immunostaining confirmed the relationship between the isolate and lesion-associated bacteria. No previous reports on this infection in sharks have been found in the literature.

Development of a PCR test based on a gene region associated with the pathogenicity of Pasteurella multocida serotype B:2, the causal agent of haemorrhagic septicaemia in Asia.

We have developed a PCR assay to detect Pasteurella multocida serotype B:2, the causal agent of Haemorrhagic Septicaemia (HS) in Asia. Nucleotide sequence determination of a 16S rRNA-23S rRNA PCR product unique to B:2 strains was shown to share amino acid sequence homology with a bacteriophage Mu protein. Primers designed from this sequence when tested against a panel of isolates recovered from a wide geographical area and representing a large range of bacterial genera and species, were found to specifically amplify DNA from P. multocida, serotype B:2. Southern hybridisation confirmed the presence of this sequence in only the B:2 serotype of P. multocida, suggesting an association between bacterial virulence and the presence of bacteriophage genes in the bacterial genome. The results of this study demonstrate the potential application of PCR to the diagnosis of HS in cattle and buffalo in Asia. Application of PCR to support diagnosis of HS will greatly improve accuracy, laboratory response time, and will facilitate rational deployment of resources for controlling this disease.

Absceso hepático piogeno. A propósito un caso / Hepatic abces. report of a case

Los abscesos hepáticos se relacionan a 2 grupos diferentes: Bacterias Piogenas y Entomoeba Histolitica. Con diferentes manifestaciones clínicas y tratamiento. El absceso hepático tiene una frecuencia de 0, 36 por ciento de Necropsia y ocurre en 6t§ y 7mo. decenios, sin predilección de sexo o raza. Los reportes residentes indican que la colangitis secundaria a cálculos o carcinoma, Septicemia generalizada y apendicitis aguda perforada son las causas más frecuentes. El 80 por ciento de los cultivos son positivos a Scherichia Coli, Staphilococus Aureus, Streptococos Hemolitico, Proteus, Klepsiella Bacteriodes y Anaerobios, estos últimos son cada ves más frecuentes

Evidence of phenotypic dichotomy within an individual Pasteurella multocida type strain and among some haemorrhagic septicaemia-causing field isolates.

Haemorrhagic septicaemia-causing strains of Pasteurella multocida were identified by a disease-specific ELISA. Some strains, however, were of the same serotype as those which cause haemorrhagic septicaemia (HS) but were negative when tested in the disease specific ELISA. The suspect false negative isolates were passaged in mice and retested in the HS ELISA with the same result. Immunoelectron microscopy was used to examine further these suspect HS-causing strains. Monoclonal antibodies and protein A-gold showed that the suspect negative organisms were a mixture of phenotypes with less than 10 per cent, and usually less than 2 per cent, of the population expressing HS-associated epitopes. The degree of staining on the organisms expressing the HS-epitopes was of the same intensity as the positive control organism. Expression of the HS-associated epitopes is presumably too low to allow detection in the current HS ELISA.

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.

[Diagnosis and differential diagnosis of rinderpest]. / Zur Diagnose und Differentialdiagnose der Rinderpest

Starting from the clinical symptoms and the pathological-anatomical changes hints are given on the diagnosis of the rinderpest and how to distinguish it from other diseases. The paper discusses the differential diagnosis of the rinderpest with respect to mucosal disease, malignent catarrhal fever, Nairobi sheep disease, salmonellosis, pasteurellosis, and coccidiosis.