Toxigenic and non-toxigenic Pasteurella multocida genotypes, based on capsular, LPS, and virulence profile typing, associated with pneumonic pasteurellosis in Iran.

Pasteurella multocida is an important cause of pneumonic pasteurellosis in small ruminants. Its prevalence was investigated in 349 pneumonic lungs from sheep (n = 197) and goats (n = 152), and genotypes of isolates were determined by capsular and lipopolysaccharide (LPS) typing as well as by virulotyping based on the detection of 12 virulence-associated genes. P. multocida was isolated from 29.4 % of sheep lungs and 13.8 % of goat lungs. A (78.5 %) and D (21.5 %) capsular types, as well as L3 (41.8 %) and L6 (57.0 %) LPS genotypes, were detected, with the A:L6 genotype being the most prevalent in both sheep (59.6 %) and goat (52.4 %) isolates. A total of 19 virulence profiles (VP) were detected, seven non-toxigenic and 12 toxigenic, which correlated with the capsular-LPS genotype. All isolates of each VP belonged to the same LPS and capsular genotype, except for one isolate of VP1. The diversity in VP was higher among toxigenic (0.29) than non-toxigenic (0.18) isolates. Moreover, the toxigenic VPs showed more diversity in their capsular-LPS genotypes, with the two main toxigenic VPs belonging to genotypes D:L3 (VP2) and A:L3 (VP3). Therefore, the abundance of toxigenic isolates among sheep and goat isolates does not seem to correspond to the expansion of a more virulent lineage associated with pneumonic pasteurellosis in small ruminants. The most prevalent genotypes among sheep isolates were the non-toxigenic VP1:A:L6 (41.4 %) and the toxigenic VP3:A:L3 (17.2 %) genotypes, whereas the most prevalent among goat isolates were the toxigenic VP2:D:L3 (33.3 %) and the non-toxigenic VP1:A:L6 (14.3 %) and VP4:A:L6 (14.3 %) genotypes. These prevalent toxigenic and non-toxigenic genotypes seem to be epidemiologically relevant in pneumonic pasteurellosis of small ruminants.

Molecular characterization of Mannheimia haemolytica associated with ovine and caprine pneumonic lung lesions.

This study investigated via polymerase chain reaction (PCR) three main serotypes (A1, A2, and A6) and nine virulence-associated genes in 71 ovine and caprine Mannheimia haemolytica isolates obtained from lungs (n = 349) with pneumonic lesions from a slaughterhouse in Iran. The lung specimens were collected from sheep (n = 197) and goats (n = 152) between December 2018 and January 2020. A total of 71 M. haemolytica isolates were identified in sheep (37/197; 18.8%) and goat (34/152; 22.4%) pneumonic lungs. Serotypes A2 (30/71; 42.3%) and A6 (29/71; 40.9%) were the most frequently detected, whereas the A1 serotype was detected with a frequency of less than 10% (7/71; 9.9%) and five isolates remained unknown. The virulence genes lkt, pomA, and nanH were present in all the isolates. The detection rates for the remaining virulence-associated genes were: gcp (95.8%), lpsA (93%), fhaC (90%), irp (70.4%), hf (57.7%), and sodC (21%). The sodC gene was exclusively detected among A2 isolates (50%), while the irp gene was more prevalent among A2 isolates and the hf gene among A1 and A6 isolates. These data may be useful for the typing of isolates in epidemiological studies. This study provides information about the main serotypes and the prevalence of virulence-associated genes among M. haemolytica ovine and caprine isolates in Iran.

Investigation of iron uptake and virulence gene factors (fur, tonB, exbD, exbB, hgbA, hgbB1, hgbB2 and tbpA) among isolates of Pasteurella multocida from Iran.

BACKGROUND AND OBJECTIVES: Iron is an essential compound in metabolic pathway of wide range of organisms. Because of limited free iron supply in mammalian and avian hosts, bacteria have applied various ways to acquire iron. MATERIALS AND METHODS: In this study, the frequency of 8 iron acquisition factors was examined among 63 avian and ovine Pasteurella multocida field isolates and their vaccine strains using PCR method. RESULTS: Five candidate genes (fur, tonB, exbD, exbB and hgbA) were identified among all isolates. For the first time, 2 loci (hgbB1 and hgbB2) of the hgbB gene were identified, which were previously reported as 1 gene. Also, it was found that 5 ovine and 1 avian isolates possessed all the virulence factors, which could also be considered for evaluating the frequency of other virulence factors. CONCLUSION: More studies need to be conducted on the frequency of all other virulence factors among these isolates, which can provide basic information for improvement or substitution of current vaccinal strains. Overall, as the new designed sets of primers showed more potential in detecting the corresponded genes, researchers can consider them in further studies.

"In-house" production of DNA size marker from a vaccinal Bacillus anthracis strain.

BACKGROUND AND OBJECTIVES: DNA molecular weight marker is widely used in molecular biology experiments incurring considerable costs on low-budget settings. MATERIALS AND METHODS: Here a PCR-supported procedure is described that uses 10 primer pairs targeting chromosomal DNA from the harmless vaccinal Bacillus anthracis Sterne 34F2 strain as template. A single PCR protocol is used to reproduce all the 10 fragments of a 100 bp DNA size marker. RESULTS AND CONCLUSION: The unpurified amalgam of 10 PCR products can be directly loaded to agarose gels. This work was intended to develop a reasonably cost-effective DNA ladder that is useful for researchers in laboratories with limited funding.

Construction of a DNA Vaccine Encoding Mtb32C and HBHA Genes of Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis (TB) is a contagious disease caused by Mycobacterium tuberculosis. Development of a new vaccine for tuberculosis requires immunogenic antigens capable of inducing suitable and long-lasting T cell immunity. The emergence of multidrugs and extensively drug-resistant strains of M. tuberculosis has made it a global public health concern. OBJECTIVES: DNA vaccine is a straightforward method to introduce antigens to the host. In the present study, two immunodominant mycobacterial antigens (Mtb32C and HBHA) were isolated and cloned into pcDNA3.1 (+) to design and construct a new DNA vaccine. This vector is capable of producing new potent chimeric protein. MATERIALS AND METHODS: Mtb32C (Rv0125) and heparin-binding haemagglutinin adhesion (HBHA) genes were amplified using polymerase chain reaction (PCR) of M. tuberculosis H37Rv genome and ligated into pcDNA3.1 (+). Colony-PCR and restriction enzyme analysis were used to confirm the accuracy of the cloning procedure. RESULTS: In the current study, recombinant pcDNA3.1 (+) vector containing Mtb32C and HBHA genes was successfully constructed. The desired size of DNA fragment was observed using single and double digestion methods. CONCLUSIONS: Mtb32C and HBHA genes were successfully isolated from H37Rv genome and cloned into an eukaryotic expression vector. This vector can be considered as a vaccine to evaluate immune responses in animal models.