Pasteurella Multocida Serotype Research Articles

Mass Die-Off of Saiga Antelopes, Kazakhstan, 2015.

In 2015, a mass die-off of ≈200,000 saiga antelopes in central Kazakhstan was caused by hemorrhagic septicemia attributable to the bacterium Pasteurella multocida serotype B. Previous analyses have indicated that environmental triggers associated with weather conditions, specifically air moisture and temperature in the region of the saiga antelope calving during the 10-day period running up to the event, were critical to the proliferation of latent bacteria and were comparable to conditions accompanying historically similar die-offs in the same areas. We investigated whether additional viral or bacterial pathogens could be detected in samples from affected animals using 3 different high-throughput sequencing approaches. We did not identify pathogens associated with commensal bacterial opportunisms in blood, kidney, or lung samples and thus concluded that P. multocida serotype B was the primary cause of the disease.

The ABA392/pET30a protein of Pasteurella multocida provoked mucosal immunity against HS disease in a rat model

Haemorrhagic septicaemia (HS) is a well-known high fatality septicaemic disease happening among bovines. The disease is caused by the Pasteurella multocida serotype B:2 bacteria. P. multocida B:2 has high mortality and morbidity rates and is spread through the intranasal and oral routes in bovines. In this study, our aim was to investigate the efficacy of the recombinant protein vaccine, ABA392/pET30a via intranasal inoculation by targeting the mucosal immunity. The constructed recombinant protein vaccine ABA392/pET30a was subjected to an animal study using Sprague Dawley rats. The study was divided into two parts: active and passive immunization studies. Both studies were carried out through the determination of immunogenicity (using Total White Blood Cell (TWBC) Count with Indirect ELISA) and histopathogenicity, analyzing (Bronchus Associated Lymphoid Tissue (BALT) formation) in lungs. As a result, the IgA and IgG development of both tested groups: group 1 (50μg/mL protein vaccine) and group 2 (100μg/mL protein vaccine) showed equivalent with the positive control group 4 (formalin-killed P. multocida B:2). However, there was a significant difference when compared with the negative control group 3 (normal saline). These results demonstrate that both the protein vaccine at the concentration 50μg/mL and 100μg/mL have the same efficacy as the commercially available positive control vaccine. From the studies, higher concentration of protein vaccine at 100μg/mL showed higher development of both IgA and IgG compared to 50μg/mL protein vaccine. Higher and rapid development of IgA compared to IgG showed that mucosal immunity has been induced through the intranasal administration of the protein vaccine. In addition, leucocytosis was observed at each dose of vaccination showed that the protein vaccine is capable to induce the immune responses of the host. Histopathogenicity studies of the vaccinated groups showed more BALT formation and no severe lesions after challenge compared to the negative control group. Besides, no inflammatory onsite or anaphylactic responses were observed after the intranasal inoculation which proved to be safer and provided longer lasting immunity. Therefore, recombinant protein vaccine ABA392/pET30a could be a potential candidate for intranasal administration which can provoke mucosal immunity against HS disease.

A new pleuromutilin candidate with potent antibacterial activity against Pasteurella multocida

This study assessed the antimicrobial activity of 14-O-[(4,6-Diaminopyrimidine-2-yl) thioacetyl] mutilin (DPTM), a novel pleuromutilin candidate with a substituted pyrimidine moiety, against Pasteurella multocida. Minimum Inhibitory Concentration (MIC), Oxford Cup assay, and time-kill experiments were used to measure the activity of DPTM against P. multocida serotype A in vitro. We observed that DPTM was potent against Pasteurella multocida serotype A with the MIC value of 0.781 μg/mL. The mean inhibition-zone diameters of DPTM (50, 25, and 12.5 μg/mL) were 29.4, 24.2 and 20.1 mm, respectively. Time-kill experiments showed that the drug caused a rapid decline in the number of bacteria compared with the initial inoculum at 4 h and killed 94.6% of the bacteria during 24 h. Furthermore, DPTM activity was also assessed in a lung infection model challenged with 4.0 × 109 CFU/mL P. multocida serotype A. The results showed that DPTM significantly reduced mortality rate and bacterial load, and alleviated the pathological changes of lung. The antibacterial effect of DPTM found in this study suggested that it was useful in the prevention or control of pneumonia caused by P. multocida.

Influence of amino acids and vitamins on the growth of gdhA derivative Pasteurella multocida B:2 for use as an animal vaccine.

Pasteurella multocida serotype B:2 is the causative agent of haemorrhagic septicaemia, a fatal disease in cattle and buffaloes. For use as a vaccine in the treatment of HS disease, an efficient cultivation of attenuated gdhA derivative P. multocida B:2 (mutant) for mass production of viable cells is required. In this study, the role of amino acids and vitamins on the growth of this particular bacterium was investigated. Initially, three basal media (Brain-heart infusion, Terrific broth, and defined medium YDB) were assessed in terms of growth performance of P. multocida B:2. YDB medium was selected and redesigned to take into account the effects of amino acids (glutamic acid, cysteine, glycine, methionine, lysine, tyrosine, and histidine) and vitamins (vitamin B1, nicotinic acid, riboflavin, pyridoxine, pantothenic acid, and biotin). High viable cell number was largely affected by the availability of micronutrient components and macronutrients. Histidine was essential for the growth whereby a traceable amount (20mM) was found to greatly enhance the growth of gdhA derivative P. multocida B:2 mutant (6.6×109cfu/mL) by about 19 times as compared to control culture (3.5×108cfu/mL). In addition, amongst the vitamins added, riboflavin exhibited the highest impact on the viability of gdhA derivative P. multocida B:2 mutant (5.3×109cfu/mL). Though the combined histidine and riboflavin in the culture eventually did not promote the stacking impact on cell growth and cell viability, nonetheless, they were still essential and important in either growth medium or production medium.

Suppression of cellular immunity factors by toxic fraction of supernatant of broth culture Pasteurella haemolytica

In the etiology of many infectious animal diseases, Pasteurella haemolytica belongs to a specific place. An important factor in the pathogenicity of this microorganism, like the Pasteurella multocida serotype D, is the thermostable exotoxin. It can be obtained from bacterial-purified culture fluid. Although the study of toxin formation among microorganisms is quite popular, however, the features of toxin formation in hemolytic pasteurals, depending on the virulence of the pathogen, the nature of the toxic effects of these objects in vivo remain unclear. Materials for research were 16 isolates of Pasteurella haemolytica, isolated from pathological and biological material obtained during the outbreaks of respiratory pathology in farm animals. Initially, the nature of the research concerned the establishment of the potential for toxin formation in the isolates obtained. The method provided for a comparative analysis of the DNA nucleotide sequences of each of the investigated isolates P. haemolytica and information obtained from the international database. Another part of the work concerned the actual allocation of the major groups of toxic components of Pasteurella haemolytica by extraction to determine their biochemical nature. Exotoxin isolation was carried out from the Pasteurella spp. The components of the sediment and supernatant were separated by ion exchange chromatography on TSK gels. In order to detect the harmful effects of toxin hemolytic pasteurals on the body, they used the method of determining the opsonic index (the ratio of the phagocytic number in the mixture without the products of toxic fractions to the mixture with the toxin-containing fraction). It has been established that an important factor of the pathogenetic effect in Pasteurella haemolytica is the toxic fraction. Electrophoregram analysis of the results of DNA amplification in a comparative aspect with the data of standard samples helped to determine the presence of elements of the genome, which indicate the potential for toxin formation in isolated hemolytic pasteurized isolates from the test material. Toxic fractions isolated from Pasteurella haemolytica broth culture supernatant are substances of protein-carbohydrate nature. The isolated peak toxicogenic fractions of dialysate of a bacterial culture sieve contained protein and carbohydrates within the limits of 12.5–20 μg / ml and 0–20 μg/ml, respectively. In the dialysate of the broth culture supernatant, where 5 groups of toxigenic fractions were identified, the content of protein and carbohydrates in them varied, respectively, in the range from 20 to 95 μg/ml and from 3.3 to 26.62 μg/ml. At reproduction of opsono-phagocytic reaction with participation of toxigenic fractions of hemolytic pasteurel, a sufficiently expressed immunosuppressive effect of these complexes on the body of warm-blooded substances with an opsonic index of 3 ± 0.03 was established. During further research it is planned to determine the dermal necrotic and lethal effects of the isolated toxicogenic fractions of hemolytic pasteuride on the body of warm-blooded ones.

Pastereloz of crafts and its prophylaxis

Pasteurleozy is a factor disease. The manifestation of pathogenic action of pasteurals occurs under conditions of influence on a susceptible organism of a number of negative factors, which are conventionally divided into contributing and adversarial. In the etiology of pasteurial infection, they play a decisive role. The problem of the struggle against rabbit pasterelosis in our country is paid insufficient attention. The analysis of the documents of the state veterinary report gives grounds to assert that the tension of the epizootic situation with regard to rabies pasteurse in the country remains unexplored. So, in official data on outbreaks of rabies pasterelosis during the last 10 years there are separate reports, from which it is impossible to make a comprehensive assessment of the epizootic process of this infection in Ukraine. There is no data on the characteristics of circulating serotypes of Pasteurella multocida in rabbit farms. There are only brief information about the spread of the disease in the rabbits in certain regions, in particular, the Luhansk, Donetsk and Zaporizhzhya oblasts. These reports indicate a poor condition for the maintenance of rabbits and an insufficient level of antiepizootic measures, which, according to the authors, is the main reason for the spread of pasteurellosis. Given these data, it can be assumed that pasteurellosis is a rather common disease of rabbits, not only in the east of Ukraine, but also in the whole country. In the system of antiepirosis measures of rabies pasteurellosis, the leading role is given to active immunization of the susceptible animal population. The article presents the results of the analysis of the domestic and world market of veterinary immunobiological means for the specific prevention of this infection. It is shown that only one vaccine against pasterelosis of rabbits of foreign production and no domestic vaccine has been registered in Ukraine. A comparative analysis of the main components of foreign vaccines and vaccines developed by our scientists was carried out. The most common inactivator in pasteurial vaccines is formaldehyde, despite the fact that it destroys the capsule, which plays an important role in the formation of the immune response and immunity tension. All foreign vaccines as a preservative contain thiomersal – an organic derivative of mercury. As an adjuvant mineral oil and aluminum hydroxide are used, both substances are reactogenic for the rabbit organism. It has been shown that the immunogenic component of pasteurial vaccines includes several serotypes of P. multocida.

STANDARDIZATION OF EFFECTIVE DOSE OF FOWL CHOLERA VACCINE IN PIGEON IN BANGLADESH

An experiment was conducted to determine the effective dose of formalin killed (FK) fowl cholera (FC) vaccines prepared with virulent avian Pasteurella multocida (PM 38) serotype 1 (X-73) collected from the laboratory of the Department of Microbiology and Hygiene, BAU, Mymensingh. To determine the effective dose of vaccine, 7 weeks old 30 pigeons were immunized and each group consists of 5 birds. The groups are represented by A, B, C, D, E and F. The birds belonging to groups (A-E) were vaccinated with different doses of vaccine, after two weeks of first, second immunization and challenge experiment, blood was collected from all vaccinated birds, and serum was analyzed to determine antibody titer against P. multocida by passive hemagglutination test (PHA). The PHA titer after two weeks of first vaccination were 16±3.92, 17.6±3.92, 25.6±3.92, 32±8.76, 35.2±7.84 of group A,B,C,D and E, respectively at the dose of 0.2ml (0.26×108 CFU)/birds, 0.4ml (0.5×108 CFU)/birds, 0.8 ml (1.04×108 CFU)/birds, 1ml (1.3×108 CFU)/birds, respectively. The PHA titer of prevaccination and control birds was <4. The PHA titer after 2 weeks of second vaccination or boostering were 32±8.76, 35.2±7.84, 44.8±7.84, 57.6±18.66, 70.4±15.68, of group A,B,C,D and E, respectively. After 2 weeks of challenge infection, the mean PHA titer were 44.8±7.84, 51.2±7.84, 70.4±15.68, 102.4±15.68 and 140.8±31.34 of group A,B,C,D and E, respectively. In this experiment, the antibody titer of the vaccinated pigeons with 0.4, 0.6, 0.8 and 1ml per bird via intramuscular route were higher than that of the pigeons vaccinated with 0.4ml/bird, 0.6ml/bird, 0.8ml/bird and 1ml/bird were satisfactory in terms of protective potential against P. multocida. For prevention and control of avian pasteurellosis 0.4ml to o.6ml (0.52×108 CFU to 0.78×108 CFU)/birds of vaccine may be used instead of 1ml (1.3×108 CFU)/birds for better immunization of pigeon against fowl cholera infection.

In vitro treatment of lipopolysaccharide increases invasion of Pasteurella multocida serotype B:2 into bovine aortic endothelial cells.

Pasteurella multocida serotype B:2 causes hemorrhagic septicemia in cattle and buffalo. The invasion mechanism of the bacterium when invading the bloodstream is unclear. This study aimed to characterize the effects of immunomodulatory molecules, namely dexamethasone and lipopolysaccharide, on the invasion efficiency of P. multocida serotype B:2 toward bovine aortic endothelial cells (BAECs) and the involvement of actin microfilaments in the invasion mechanism. The results imply that treatment of BAECs with lipopolysaccharide at 100 ng/mL for 24 h significantly increases the intracellular bacteria number per cell (p < 0.01) compared with those in untreated and dexamethasone-treated cells. The lipopolysaccharide-treated cells showed a significant decrease in F-actin expression and an increase in G-actin expression (p < 0.001), indicating actin depolymerization of BAECs. However, no significant differences were detected in the invasion efficiency and actin filament reorganization between the dexamethasone-treated and untreated cells. Transmission electron microscopy showed that P. multocida B:2 resided in a vacuolar compartment of dexamethasone-treated and untreated cells, whereas the bacteria resided in cellular membrane of lipopolysaccharide-treated cells. The results suggest that lipopolysaccharide destabilizes the actin filaments of BAECs, which could facilitate the invasion of P. multocida B:2 into BAECs.

Complete Genome Sequence of Pasteurella multocida Serotype A Strain PMTB2.1 Isolated from Buffaloes That Died of Septicemia in Malaysia.

ABSTRACTPasteurella multocida causes pneumonic pasteurellosis and hemorrhagic septicemia (HS) in large ruminants. In this study, we determined the complete genome sequence of P. multocida strain PMTB2.1 capsular serotype A isolated from buffaloes that died of septicemia.

Ultrastructural pathology Associated with Attachment of Pasteurella multocida serotype B: 2 to Tracheal and Pulmonary Endothelial Cells and Tracheal cilia of Mice

Ultrastructural pathology Associated with Attachment of Pasteurella multocida serotype B: 2 to Tracheal and Pulmonary Endothelial Cells and Tracheal cilia of Mice

Ultrastructural pathology Associated with Attachment of Pasteurella multocida serotype B: 2 to Tracheal and Pulmonary Endothelial Cells and Tracheal cilia of Mice

Ultrastructural pathology Associated with Attachment of Pasteurella multocida serotype B: 2 to Tracheal and Pulmonary Endothelial Cells and Tracheal cilia of Mice

Pathological Changes Associated with Natural Outbreak of Swine Pasteurellosis

Swine pasteurellosis is an acute infectious disease and is considered to be one of the most economically important diseases in Asia particularly in South and South East Asia leading to huge economic loss in livestock industry. A study was conducted to analyze the histopathological alterations caused by cytotoxins released by Pasteurella multocida serotype B, which caused Swine pasteurellosis in descript as well as nondescript pigs of Chhattisgarh, India. Affected animals exhibited pyrexia, dullness, staggering gait, anorexia, serous nasal discharge and dyspnoea. Typical lesions of oedematous swellings noted in the pharyngeal region. Case fatality rate was 95% in adult animals and 100% in piglets. The causative organism, Pasteurella multocida was isolated and characterized by biochemical tests and the detection of the KMT1, CapB, toxA, and ompH genes was performed using Polymerase Chain Reaction (PCR). Gross lesions included severe pneumonia and haemorrhages in lungs, petechial haemorrhages on serous membranes and other visceral organs. Lymph nodes were enlarged, oedematous and haemorrhagic. The histopathological lesions developed in lungs were typical fibrinous bronchopneumonia, multifocal suppuration and severe thickening of pleura. Heart showed presence of thrombi, haemorrhages and necrosis of myocardium. Hepatic architecture showed server haemorrhages and necrosis while kidneys and spleen showed degenerative and necrotic changes.

Improved cultivation of gdhA derivative Pasteurella multocida B:2 for high density of viable cells through in situ ammonium removal using cation-exchange resin for use as animal vaccine

Abstract Pasteurella multocida serotype B:2 is the causative agent of hemorrhagic septicemia, a fatal disease of cattle and buffaloes. A live attenuated vaccine, gdhA derivative P. multocida B:2 mutant, was created to defeat the disease. During the cultivation of P. multocida B:2 mutant, substantial amount of ammonium was accumulated in the culture, which greatly inhibited the growth of this bacterium. The feasibility of using integrated cultivation with in situ removal of ammonium by cation-exchange resin for the improvement of growth and viability of P. multocida cells was investigated. The ability of various cation-exchange resins, which include Amberlite IRC86, Amberlite IR120 H, and Dowex DRG8 H, to selectively adsorbed ammonium was first investigated using sorption isotherm experiments. Amberlite IRC86 has the highest ability for ammonium adsorption. The incorporation of 10 g/L of Amberlite IRC86 resin into the shake flask culture (100 mL) of P. multocida B:2 mutant, improved the final viable cell concentration (7.2 × 1010 cfu/mL) by about 13-fold compared to that obtained in cultivation without resin (5.5 × 109 cfu/mL). In cultivation with Amberlite IRC86 resin, approximately 41% of the ammonium accumulated in the culture was removed.

Prevalence of different OmpH-types among Pasteurella multocida isolated from lungs of calves with respiratory problems.

Pasteurella multocida is among the most important respiratory pathogens of cattle. Outer-membrane protein (OmpH) constitutes an essential bacterial antigen and is well studied in avian bacterial strains. Studies on isolates from cattle with signs of respiratory disease caused by Pasteurella multocida serotypes A and D have not yet been covered in the literature. The objective of this study was a comparative analysis of the ompH gene sequences from 83 isolates and four Russian reference strains of P.multocida to assign them to the allelic variants of the gene (OmpH-types). In addition, the above P.multocida strains have been characterized on the basis of capsular serotypes and virulence-associated genes. The isolates were classified into the OmpH -types based on allele specific PCR and gene fragment sequencing. The isolates of capsular serotype A have been subdivided into 6 OmpH -types, of which the most common types identified were A1 and A2. All capsular serotype D isolates belong to the same OmpH-type (D1). On 16 of a total of 23 farms all isolates belong to only one OmpH-type, on 4 farms - to 2, and on 3 farms - to three OmpH-types. The tbpA and pfhA genes were found more often in the isolates of capsular group А as compared to capsular group D (p≤0.05). OmpH-types of serogroup А differ significantly (p≤0.05) among themselves by the prevalence of the pfhA and hgbB genes.

Hematology of layers chickens vaccinated with fowl cholera vaccine and experimentally inoculated with virulent Pasteurella multocida serotypes in Zaria, Nigeria

Hematology of layers chickens vaccinated with fowl cholera vaccine and experimentally inoculated with virulent Pasteurella multocida serotypes in Zaria, Nigeria

Experimental pathogenicity and complete genome characterization of a pig origin Pasteurella multocida serogroup F isolate HN07

Pasteurella multocida serotype F isolates are predominately prevalent in avian hosts, but rarely seen in pigs. However, we isolated several strains of P. multocida serotype F from clinical samples of pigs in China. To understand the pathogenicity of these strains, one of the serotype F isolates designated HN07, was used to challenge experimental chickens, as P. multocida of this serotype is predominately prevalent in avian hosts. However, strain HN07 could not resulted in significant clinical signs in experimental chickens even at an infective dose of ∼109 CFU, suggesting the isolate was avirulent to chickens and therefore raising the possibility that the porcine serotype F isolate is not transmitted by chickens. We then used HN07 to challenge experimental pigs, as this strain was isolated from pigs. As expected, the strain led to the clinical signs and the pathological lesions in experimental pigs that are similar to the pasteurellosis disease. We then determined the complete genome sequence of the pig origin serogroup F isolate HN07 for the first time. Genome comparison between HN07 and the avian serotype F P. multocida Pm70 identified a novel integrative conjugative element (ICE) ICEpmcn07 which was likely to harbor a series of genes responsible for a putative type IV secretion system (T4SS) in HN07. This is the first time that we determined an ICE carrying a T4SS in P. multocida. Besides, comparative analysis also defined a number of virulence-associated genes in HN07 but absent in Pm70 which may have a contribution to the pathogenicity of the strain. This is the first report of the pathogenicity and genome characterization of a pig origin Pasteurella multocida serogroup F isolate. The pathogenic and genomic definition of the pig origin P. multocida serogroup F in our study would have significance on the pathogenesis and genetic diversity and virulence variability of P. multocida.

Molecular detection of Pasteurella multocida Type B causing haemorrhagic septicemia in cattle and buffaloes of Bangladesh

Hemorrhagic septicemia (HS) is an acute septicemic disease that primarily affects cattle and buffaloes. The disease is caused by Pasteurella multocida sero types B:2 and E:2. The objective of this study was to isolate P. multocida from clinical cases and to confirm its identity using polymerase chain reaction (PCR) based approach. Clinical samples of two suspected cases of haemorrhagic septicemia of cattle and buffalo from Mymensingh and Rajshahi districts respectively were collected. Two isolates were isolated from these suspected cases and primarily identified as P. multocida based on morphological study, staining properties, and cultural and biochemical characteristics. The isolates were confirmed initially as P. multocida at genus level by PCR using genus specific primers. Later, the isolates were confirmed as P. multocida type B, the causal agent of haemorrhagic septicemia, by PCR with primers specific for P. multocida type B. These isolated organisms can be used as vaccine candidate for the production of effective vaccine against haemorrhagic septicemia.Progressive Agriculture 27 (2): 175-179, 2016

Differences in Virulence Between Bovine-Derived Clinical Isolates of Pasteurella multocida Serotype A from the UK and the USA in a Model of Bovine Pneumonic Pasteurellosis

The time of onset and subsequent degree and progression of clinical signs, bacterial colonization and tissue pathology during experimental disease induced by intratracheal inoculation of either a UK or USA isolate of Pasteurella multocida serotype A recovered from clinical cases of bovine pneumonia were determined. Calves aged 8 weeks were challenged with 300ml phosphate buffered saline (PBS) alone (group 1, n=3, negative control) or containing 7.1×10(8) colony forming units (cfu) of UK isolate (group 2, n=8) or 5.8×10(8)cfu of USA isolate (group 3, n=8). Bronchoalveolar lavage (BAL) at 0, 1 and 4 days post challenge (dpc) and at the time of necropsy examination (7-8 dpc) showed no significant differences between groups 2 and 3 in bacterial numbers recovered. No P. multocida were recovered from group 1 animals. No clinical disease was present in group 1 calves and in group 3 was limited to scour in 1 calf at 1 dpc. All calves in group 2 had reduced food intake at 4-5 dpc, five had periods of dullness, three a mild nasal discharge at 1 dpc, four had mild to substantial respiratory stridor and one was killed at 6 dpc for humane reasons. Rectal temperatures remained about 39°C in group 1 calves, but increased in P. multocida-challenged calves to 40-41°C within 8-12h of challenge. Significantly (P=0.01) greater percentages of lung surface area were consolidated in group 2 (mean±SD, 21±10.1) compared with group 3 (7±8.6) calves. Significantly more extensive and severe histological lesions were present in the lung lobes (P=0.006) and lymph nodes (P=0.02) of group 2 compared with group 3 calves. Pleurisy was present in group 2 calves only and no pathology was present in group 1. Pulsed-field gel electrophoresis (PFGE) produced 11 (group 2, UK isolate) or 10 (group 3, USA isolate) bands with differences in banding patterns. Results overall showed that two isolates, distinct geographically and genetically (by PFGE), caused pneumonic pasteurellosis in a single host with significantly different severity of pathology. This information is relevant to the development of novel vaccine control and interpretation of diagnostic results.

Involvement of the nervous system following experimental infection with Pasteurella multocida B:2 in buffalo (Bubalus bubalis): A clinicopathological study

Haemorrhagic septicaemia (HS) is an acute, fatal, septicaemic disease of cattle and buffaloes caused by one of two specific serotypes of Pasteurella multocida B:2 and E:2 in Asian and African, respectively. It is well known that HS affect mainly the respiratory and digestive tracts. However, involvement of the nervous system in pathogenesis of HS has been reported in previous studies without details. In this study, nine buffalo calves of 8 months old were distributed into three groups. Animals of Group 1 and 2 were inoculated orally and subcutaneously with 10 ml of 1 × 1012 cfu/ml of P. multocida B:2, respectively, while animals of Group 3 were inoculated orally with 10 ml of phosphate buffer saline as a control. All calves in Group 1 and Group 3 were euthanised after 504 h (21 day) post-infection, while calves in Group 2 had to euthanise after 12 h post-infection as they develop sever clinical signs of HS. Significant differences were found in Group 2 in the mean scores of clinical signs, gross and histopathological changes which mainly affect different anatomic regions of the nervous system. In addition, successful bacterial isolation of P. multocida B:2 were obtained from different sites of the nervous system. On the other hand, less sever, clinical, gross and histopathological changes were found in Group 1. These results provide for the first time strong evidence of involving of the nervous system in pathogenesis of HS, especially in the peracute stage of the disease.

Comparison of Phenotypic and Genotypic Identification Methods of Pasteurella multocida Serotypes Isolated from Pigs

Background: Pasteurella multocida serotypes A and D are commonly associated with pneumonia and pleuritis in pigs. Different phenotypic techniques, such as hyaluronidase and acriflavine tests, and genotyping techniques, such as PCR, are used to distinguish between these serotypes. The objective of this study was to compare the capsular identification methods of type A and type D P. multocida isolated from pigs using both phenotypic (hyaluronidase and acriflavine tests) and genotypic (multiplex PCR) techniques.Materials, Methods &amp; Results: A total of 44 lyophilized P. multocida isolates, obtained between 1981 and 1997 from pig farms at Rio Grande do Sul State, Brazil, were analyzed. The isolates were reactivated in Brain Heart Infusion (BHI) broth and cultured in BHI broth and blood agar supplemented with 5% sheep blood. Colony identity was further confirmed by evaluating colony morphology in blood agar and confirming the absence of growth on MacConkey agar. Bacteria in Tryptone Soy Agar (TSA) were used for the Triple Sugar Iron (TSI), Sulfide-Indole-Motility (SIM), and nitrate, glucose, lactose, sucrose and mannitol fermentation tests. For hyaluronidase test, P. multocida colonies were streaked transversally across the entire plate, approximately 3-5mm apart, in order to observe their lines of growth. Following this, a hyaluronidase producing strain of Staphylococcus aureus was heavily streaked at right angles to the P. multocida lines and the plates were incubated at 37°C for 24 h. Type A isolates were then identified as those with smaller colonies in the region adjacent to the Staphylococcus aureus streak (negative satellitism). For acriflavine test, the isolates were inoculated into tubes containing 2 mL of BHI, incubated at 37°C for 18-24 h, centrifuged (500 g for 15 min) and 1.5 mL of the supernatant was discarded. A 1:1000 solution of acriflavine neutral (0.5 mL) was then added to the residual broth containing bacteria and kept at room temperature. Solutions of acriflavine were freshly prepared each week and stored protected from light. Type D strains were identified by the appearance of a heavy flocculent precipitate within 5 min. DNA extraction by heat shock was performed prior to multiplex PCR for the detection of capsular genes hyaD-hyaC (capsular typing A) and dcbF (capsular typing D). Test of symmetry and a weighted kappa coefficient were used to evaluate correlations and to assess agreement of the results between the identification methods, respectively. Phenotypic tests showed that two isolates were type D (4.55%), 40 were type A (90.9%) and two (4.55%) were untypable isolates (4.55%) while PCR showed that 38 isolates were type A (86.36%) and six were type D (13.64%). The correlation analysis between the phenotypic and genotypic tests showed that 90.9% of the strains were identified as belonging to the same serotype by both tests and the weighted kappa coefficient (K = 0.633) indicates a substantial agreement between the two tests.Discussion: There was a disagreement between the phenotypic and genotypic results in four of the isolates (9.09%). The phenotypically untypable isolates were classified as type D by multiplex PCR. Nonetheless, we conclude that PCR testing is a more reliable method to differentiate between P. multocida serotypes A and D.