Goat pasteurellosis: serological analysis of circulating Pasteurella serotypes in Tanqua Aberegelle and Kola Tembien Districts, Northern Ethiopia.

OBJECTIVES: A cross-sectional study was employed with the aim to explore the serological status of goats; we evaluated the presence of serum antibodies of the circulating serotypes of the genus Pasteurella. A total of 124 serum samples were collected from randomly selected goats and subsequently serotyped using indirect haemagglutination test. RESULTS: In the current study, the overall prevalence of pasteurellosis in goats was 31.4%. Additionally, a total of eight serotypes of Pasteurella were serotyped. It is evident that 25% out of 124 sampled animals were found infected by four or more circulating serotypes and 6.4% animals were also found positive for all serotypes. Accordingly, the prevalence of Pasteurella multocida serotype A were 16.9%, Mannheimia haemolytica serotype A1 26.6%, M. haemolytica serotype A2 18.5%, M. haemolytica serotype A7 16.1%, Bibersteinia trehalosi serotype T3 20.9%, B. trehalosi serotype T4 21.7%, B. trehalosi serotype T10 27.4%, and B. trehalosi serotype T15 was 25.8%. Therefore, although there has been vaccination campaign with monovalent vaccine P. multocida type A, the diseases still exerts negative impacts through death of goats to smallholder farmers. Therefore, to control the disease the government should provide multivalent vaccine of the above serotypes.

Small ruminant pasteurellosis in Tigray region, Ethiopia: marked serotype diversity may affect vaccine efficacy.

The aim of this study was to investigate the prevalent Bibersteinia, Mannheimia and Pasteurella serotypes, risk factors and degree of serotype co-infections in sheep and goats in the Tigray region of Ethiopia. Serum was collected from 384 sheep and goats from the Tanqua-Abergelle district of Tigray region using cross-sectional random sampling. An indirect haemagglutination test was used for serotyping. Risk factors for infections were evaluated by logistic regression. Potential clustering of multiple serotypes within individual animals due to common risk factors was evaluated by redundancy analysis. Eight serotypes were identified: all studied animals were serologically positive for at least one serotype. Overall, 355 (92·45%) of the animals were infected by four or more serotypes. Of the five risk factors studied, peasant association (PA), animal species, age (serotype A1), and bodyweight (serotype T15) were significantly associated with infection, but sex was not significant. Only PA explained a significant proportion of the variation (adjusted R 2 = 0·16) in the serological responses. After the effect of PA was accounted for, T3 and T4; A7 and Pasteurella multocida A; and A7 and T10 were positively correlated for co-infection, while T4 and T10 were less likely to be found within the same animal. Diverse serotypes were circulating in the Tigray region and could be a challenge in selecting serotypes for vaccine.

Selection of Vaccine Candidates for Fish Pasteurellosis Using Reverse Vaccinology and an In Vitro Screening Approach.

The advent of new technologies in recent years has revolutionized the methods by which pathogens are studied and at the same time it has provided new tools to design vaccines against infections for which vaccine development has so far been unsuccessful. The availability of genomic data provides the basis for the reverse vaccinology approach, a biotechnological strategy that uses bioinformatics analysis of microbial genome data for the in silico selection of potential vaccine candidates for the development of protein-based vaccines. The antigens selected by reverse vaccinology can be produced as recombinant proteins and subjected to further in vitro screening assays before in vivo experiments to assess immunogenicity and protection. The reverse vaccinology approach has been applied to several pathogens affecting human health, but also to marine bacteria, including Photobacterium damselae subsp. piscicida causing significant harm in marine aquaculture.

Regulating effects of porcine interleukin-6 gene and CpG motifs on immune responses to porcine trivalent vaccines in mice.

In order to develop novel immunoadjuvants to boost immune response of conventional vaccines, experiments were conducted to investigate the regulating effects of porcine interleukin-6 gene and CpG motifs as the molecular adjuvants on immune responses of mice that were co-inoculated with trivalent vaccines against Swine fever, the Pasteurellosis and Erysipelas suis. Synthetic oligodeoxynuleotides containing CpG motifs were ligated into pUC18, forming recombinant pUC18-CpG plasmid. Eukaryotic plasmid expressing porcine interleukin-6 (VPIL-6) were also constructed as molecular adjuvants in an attempt to enhance levels of immune responses of mice co-administered with the trivalent vaccines in this paper. The cellular and humoral immune responses of mice were systematically analysed, and the experimental results were observed that the number of white blood cells, monocytes, granuloytes and lymphocytes significantly increased, respectively, in the mice immunized with VPIL-6, compared with those of the control; the IgG content and titre of specific antibodies to the trivalent vaccine mounted remarkably in the sera from the VPIL-6 vaccinated mice; the proliferation of lymphocytes and induced IL-2 activities were significantly increased in the vaccinated groups. The above-mentioned immune responses of mice co-inoculated with pUC18-CpG plasmid were significantly stronger than those of co-inoculated with pUC18 plasmid, suggesting that the immunostimulatory effect of oligodeoxynuleotides CpG is closely connected with the number of CpG motifs. These results suggest that the porcine IL-6 gene and CpG motifs could be employed as effective immunoadjuvants to elevate immunity to conventional vaccines.

Characterization of two lipoproteins in Pasteurella multocida.

An in vivo expression technology (IVET) system was previously developed and used to identify Pasteurella multocida genes, which are upregulated during infection of the host. Of the many genes identified, two encoded products which showed similarity to the Haemophilus influenzae lipoproteins, protein D and PCP, which have been shown to stimulate heterologous immunity against infection with H. influenzae. Therefore, the lipoprotein homologues in P. multocida, designated GlpQ and PCP, were investigated. GlpQ and PCP were shown to be lipoproteins by demonstrating that post-translational processing of the proteins was inhibited by globomycin. The P. multocida GlpQ homologue showed glycerophosphodiester phosphodiesterase enzyme activity, indicating that it is a functional homologue of other characterized GlpQ enzymes. Using surface immunoprecipitation, PCP was found to be surface exposed, but GlpQ was not. Non-lipidated forms of GlpQ and PCP were expressed and purified from Escherichia coli and used to vaccinate mice. However, mice were not protected from challenge with live P. multocida. The lipoproteins were then expressed in E. coli in the lipidated form and used to vaccinate mice and chickens. Protection against challenge with live P. multocida was not observed.

Detection and elimination of contaminating microorganisms in transplantable tumors and cell lines.

As a quarantine of biological materials, we tested 96 transplantable tumors and cell lines for contamination with microorganisms in a mouse antibody production (MAP) test, enzymatic assay and microbiological culture. Contamination with lactic dehydrogenase elevating virus (LDV), mycoplasmas and Pasteurella pneumotropica was detected. A considerable difference in the contamination rate was observed between in vivo- and in vitro- propagated tumors. LDV in the tumors could be eliminated by both in vitro subculture and subpassage in nude rats. Mycoplasmas were eliminated by means of the mycoplasma-removal agent and P. pneumotropica by subpassage in mice. These results suggest that there is still a high risk of contamination in transplantable tumors and emphasizes the importance of adequate microbiological quality control.

Dual infection with Pneumocystis carinii and Pasteurella pneumotropica in B cell-deficient mice: diagnosis and therapy.

BACKGROUND AND PURPOSE: The clinical presentation, diagnosis, histopathologic findings, and elimination of dual respiratory tract infection with Pasteurella pneumotropica and Pneumocystis carinii were studied in 100 adult barrier-reared C.B17 and MRL- lpr mice homozygous for a targeted mutation of the JH region of the immunoglobulin heavy chain. METHODS: Necropsy, aerobic bacteriologic culture of hematogenous and pulmonary tissues, histochemical staining of pulmonary tissues, polymerase chain reaction analysis of pulmonary tissues and feces, and viral serologic testing were performed on 19 clinically affected mice and 8 clinically normal mice, then later on antibiotic-treated and caesarian re-derived mice. Therapeutic strategies included sequential administration of trimethoprim/ sulfamethoxazole and enrofloxacin or enrofloxacin administration and caesarian rederivation. RESULTS: Clinically affected mice had diffuse, nonsuppurative, interstitial pneumonia with superimposed pyogranulomatous lobar pneumonia that was detected microscopically. Affected lung tissue yielded pure culture of P. pneumotropica. Aged-matched, clinically normal mice of both genotypes had interstitial histiocytic pneumonia without lobar pneumonia, and P. pneumotropica was not isolated. Histochemical staining of lung tissues from normal and clinically affected mice revealed scattered cysts consistent with P. carinii, principally in the interstitium. Treatment with sulfamethoxazole/trimethoprim and enrofloxacin eliminated bacteriologic detection of P. pneumotropica, decreased mortality from 50% to 6%, and improved breeding performance. CONCLUSION: A successful antibiotic therapy and rederivation approach, incorporating enrofloxacin, cesarian section, and isolator rearing, was developed for B cell-deficient mice with opportunistic infections.

Production and evaluation of a panel of monoclonal antibodies against Haemophilus paragallinarum.

We report on the production and characterisation of monoclonal antibodies (MAbs) against Haemophilus paragallinarum, the causative agent of infectious coryza. A bank of 8 MAbs were produced by traditional techniques - four against the reference strain for Page serovar A (0083) and four against the reference strain for Page serovar C (Modesto). Seven of the eight MAbs were shown to be IgG(1) with one being nontypable. None of the MAbs had HI activity and none gave any detectable reaction when examined by Western blotting. None of the MAbs gave a positive reaction in the indirect ELISA with any of the eight type strains of Pasteurella species or sub-species. None of our 8 MAbs gave serovar specific reactions when used in an indirect ELISA format. There was a trend for the serovar A MAbs to give a higher titre with serovar A isolates/strains and a similar trend for the serovar C MAbs to give higher titres with the serovar C isolates/strains.

A critical role of natural immunoglobulin M in immediate defense against systemic bacterial infection.

To evaluate the role of natural immunoglobulin (Ig)M in the immediate response against microbial infection, we tested mutant mice that are deficient in secreted (s)IgM in an acute peritonitis model induced by cecal ligation and puncture (CLP). 20% of wild-type mice died within 32 h of CLP, whereas 70% of sIgM-deficient mice died within the same time period. The increased susceptibility was associated with a reduced level of tumor necrosis factor (TNF)-alpha, a decreased neutrophil recruitment and an increased bacterial load in the peritoneum, and elevated levels of endotoxin and proinflammatory cytokines in the circulation. Resistance to CLP by sIgM-deficient mice was restored by reconstitution with polyclonal IgM from normal mouse serum. Reconstitution with a monoclonal IgM specific to phosphatidylcholine, a conserved cell membrane component, has a modest effect but a monoclonal IgM specific to phosphocholine is not protective. These findings demonstrate a critical role of natural IgM in the immediate defense against severe bacterial infection.

Proliferative responses of peripheral blood leucocytes of sheep infected with Trypanosoma evansi.

The effects of Trypanosoma evansi on the proliferative responses of ovine peripheral blood leucocytes (PBL) were examined in in vitro cell culture systems. Sheep were vaccinated against pneumonic pasteurellosis with a monovalent Pasteurella haemolytica vaccine and then infected with T. evansi TREU 2143. From 1 week post-infection (p.i.), the PBL were separated and stimulated in cultures with either Concanavalin A (Con A), bacterial lipopolysaccharide (LPS), pasteurella antigen (P.ag), or homologous trypanosome antigen (T.ag). The proliferative responses of the cells to Con A and LPS were significantly (P < 0.001) suppressed by the infection. This suppression was associated with active infection, as treatment of the sheep with a trypanocide restored the proliferative ability of the cells to both mitogens. Similarly, active infection significantly (P < 0.001) suppressed specific responses to P.ag and T.ag but although treatment resulted in full specific proliferative responsiveness to the homologous trypanosome antigen, the same was not true of P.ag, in which the responsiveness of cells from uninfected vaccinated sheep to it were still significantly higher (P < 0.001) than those of cells from infected sheep.

Divergent activity and function of superoxide dismutases in Pasteurella haemolytica serotypes A1 and A2 and Pasteurella trehalosi serotype T10.

Representative strains of Pasteurella haemolytica serotypes A1 and A2 and Pasteurella trehalosi serotype T10 were examined for the presence of superoxide dismutase. Visualisation of superoxide dismutase enzyme activity on polyacrylamide gels, and specific inhibition with potassium cyanide verified a copper/zinc (Cu/Zn) superoxide dismutase only in serotype A2 whereas serotypes A1 and T10 showed other superoxide dismutase activity. Using a simple freeze-thaw method the cellular location of superoxide dismutase enzyme activity was determined in all three serotypes. In serotypes A1 and A2 but not T10 superoxide dismutases were located in the periplasm. The viability of serotypes A2 and T10 cells in the presence of exogenous superoxide was unchanged over a 30 min period, whereas serotype A1 cells declined in viability between 15 and 30 min. Purified immunoglobulin from sheep convalescent serum did not reduce superoxide dismutase activity in the serotypes in an in vitro assay. The presence of this enzyme within the pasteurellae suggests a supportive role in the virulence of this major pathogen of ruminants.

Response of eosinophilic granule cells of gilthead seabream (Sparus aurata, Teleostei) to bacteria and bacterial products.

Eosinophilic granule cells in the gills and peritoneal exudate of gilthead seabream (Sparus aurata L.) are characterized by the presence of prominent eosinophilic granules in their cytoplasm and are here described for the first time. The oval granules of these cells contain an electron-dense inclusion surrounded by a less dense filamentous matrix and are peroxidase- and acid phosphatase-negative. Unlike other granulocytes of gilthead seabream, eosinophilic granule cells do not ingest bacteria in vivo. The intraperitoneal injection of extracellular products of Pasteurella piscicida induces mobilization of eosinophilic granule cells to the blood and other tissues and causes changes in their structure. Shortly after injection, the granules of eosinophilic granule cells become swollen and some fuse with the cell membrane. From 7 h post-injection, many eosinophilic granule cells in the gills degenerate and are then phagocytosed by macrophages, which are especially abundant after 24 h. From 24 h to 72 h, eosinophilic granule cells from the gills contain abundant autolysosomes together with granules of a normal morphology.

Phenotype and serotype of Pasteurella multocida isolates from diseases of dogs and cats in Zimbabwe.

A variety of disease manifestations, comprising skin bite wounds, pyothorax, respiratory and genitourinary tract infections, in 202 dogs and cats presented to the University Clinic, were investigated for the presence of Pasteurella multocida. Of these, 25-42% of various cases (69) were found to be infected with P. multocida. P. multocida-associated respiratory tract infections were more common than bite wounds or genitourinary tract infections. The regimen of treatment consisted of those antibiotics, sensitivity to which had been confirmed in vitro. Following detailed characterization of the isolates of P. multocida, in order to assign them to the reclassified taxa of Pasteurella, a preponderance of P. multocida subspecies multocida and septica were recorded. There did not appear to be a correlation between the reclassified taxa and their serotypes. Certain strains of different species or subspecies belonged to a common serotype and vice versa. However, the strains which were serotyped belonged to capsular type A, except for a solitary isolate from a cat which was capsular type D. Type D is known to cause atrophic rhinitis and does not appear to have been isolated either from a dog or a cat. Two strains, one from a dog and another from a cat, were identified as group EF-4 bacteria. This group of organisms has been incriminated in human wounds resulting from dog/cat bites, and has so far not been reported in Africa. Three different species, P. stomatis, P. dagmatis and P. multocida subspecies multocida were simultaneously isolated from a case of chronic bronchitis in a dog. There was no evidence of any relationship between disease manifestation in a host and the isolation of a particular taxon of Pasteurella, except that P. canis and Pasteurella taxon 16 were only isolated from dogs.

Immunization with bacterial antigens: pasteurellosis.

Pasteurella piscicida is the aetiological agent of pasteurellosis or pseudotuberculosis, one of the most threatening diseases of wild and cultured marine fish. This bacterium has been reported from many geographical areas including USA, Japan, and the Mediterranean countries. In this review, the biochemical, serological, and molecular characteristics of the pathogen are described. In addition, its main virulence mechanisms, such as the presence of capsule, the iron uptake system, and the phospholipase activity, as well as their putative role in the pathogenicity of P. piscicida are also discussed. Finally, a detailed survey of the strategies for controlling the disease is performed, with a special emphasis on the vaccination programmes and the most effective protective antigens to be included in the vaccine formulations.

Environment, incidence, aetiology, epizootiology and immunoprophylaxis of soil-borne diseases in north-east Mexico.

Within the framework of an extensive research programme, the socio-economic and environmental conditions which influence the emergence of soil-borne diseases in north-eastern Mexico were analysed. Furthermore, specimens collected from carcasses in the field were bacteriologically examined and the causal organisms of soil-borne diseases differentiated by means of gas chromatographic analysis of their metabolic products and the long-chained fatty acids contained in the cell. With experimental clostridial vaccines prepared with the Goettingen Bioreactor Technique, trials to protect cattle and guinea-pigs against gas gangrene were carried out. It was found that the farm structure and the dry climate as well as the specific soil conditions and plant cover favour the emergence of soil-borne diseases. Causal organisms B. anthracis, C. perfringens, C. sordellii, C. haemolyticum, C. chauvoei/septicum, C. novyi A, C. botulinum and site-specific field strains of clostridia were detected. Experimental site-specific vaccines proved to be highly efficient in protecting cattle and guinea pigs.

Mycoplasma hyorhinis infection levels in lungs of piglets with porcine reproductive and respiratory syndrome (PRRS).

The infection levels of Mycoplasma hyorhinis, M. hyopneumoniae and M. hyosynoviae in the lung of piglets were examined in relation to porcine reproductive and respiratory syndrome (PRRS). These animals consisted of 43 PRRS piglets with PRRS, 2 piglets infected with PRRS virus but symptom-free, and 10 control piglets free of PRRS virus and its antibody. M. hyorhinis was isolated from 40 of the 43 PRRS piglets, from 1 of the 2 latent infected piglets and from 3 of the 10 control piglets. The number of M. hyorhinis isolated from the lungs of PRRS piglets was more than 10(5) CFU/g, but those isolated from the latent infected piglets and the control piglets were less than 10(3) CFU/g. In addition to this, Haemophilus parasuis and Pasteurella spp. were frequently isolated from the piglets with PRRS (51.2% and 25.6%, respectively). On the other hand, M. hyopneumoniae was isolated from only 4 of 55 piglets tested, and M. hyosynoviae was not isolated. M. hyorhinis was also detected directly in the lung emulsion samples from almost all the PRRS piglets using a polymerase chain reaction-based method.