Genetic diversity and phylogenetic relationships of Clostridium perfringens strains isolated from mastitis and enteritis in Egyptian dairy farms.

BACKGROUND: Clostridium perfringens, a common environmental bacterium, is responsible for a variety of serious illnesses including food poisoning, digestive disorders, and soft tissue infections. Mastitis in lactating cattle and sudden death losses in baby calves are major problems for producers raising calves on dairy farms. The pathogenicity of this bacterium is largely mediated by its production of various toxins. RESULTS: The study revealed that Among the examined lactating animals with a history of mastitis, diarrheal baby calves, and acute sudden death cases in calves, C. perfringens was isolated in 23.5% (93/395) of the total tested samples. Eighteen isolates were obtained from mastitic milk, 59 from rectal swabs, and 16 from the intestinal contents of dead calves. Most of the recovered C. perfringens isolates (95.6%) were identified as type A by molecular toxinotyping, except for four isolates from sudden death cases (type C). Notably, C. perfringens was recovered in 100% of sudden death cases compared with 32.9% of rectal swabs and 9% of milk samples. This study analyzed the phylogeny of C. perfringens using the plc region and identified the plc region in five Egyptian bovine isolates (milk and fecal origins). Importantly, this finding expands the known data on C. perfringens phospholipase C beyond reference strains in GenBank from various animal and environmental sources. CONCLUSION: Phylogenetic analyses of nucleotide sequence data differentiated between strains of different origins. The plc sequences of Egyptian C. perfringens strains acquired in the present study differed from those reported globally and constituted a distinct genetic ancestor.

Development, preparation, and evaluation of a novel non-adjuvanted polyvalent dermatophytes vaccine.

Ringworm is a worldwide distributed contagious disease infecting both man and animals that constitute an economic, zoonotic, and health problem concern all over the world. During the last decade, attention has been directed to vaccination as an ideal approach to the control of such diseases. In the present study, non-adjuvanted polyvalent vaccines were prepared from locally isolated hot and virulent dermatophyte species, namely Trichophyton verrucosum (T. verrucosum), Trichophyton mentagrophytes (T. mentagrophytes), and Microsporum canis (M. canis) were immunologically evaluated. The prepared vaccine evaluation was focused on the aspects of immunogenicity and protective efficacy using guinea pigs. Both in its living or inactivated forms, the vaccine-induced significant humoral and cell-mediated immune responses and achieve proper protection of guinea pigs against challenging infections with homologous and heterologous dermatophyte strains. On the other hand, investigations on dermatophyte exo-keratinases showed that it was better produced and more expressed in a mineral-based medium containing pure keratin (3 g/L) than in the same medium with human hair supplementation (2.6 g/L). The maximum dermatophyte productivity of exo-keratinases was found to be between 18 and 21 days post-incubation. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two fractions with molecular weights of 40 kDa (fraction I) and 28 kDa (fraction II) have been identified in the culture filtrate of the three involved dermatophyte species. Both fractions demonstrated keratinolytic activity. The specific activity of the isolated keratinases (number of Keratinase units (KU)/mg protein) was stronger in fraction I, where it reached 18.75, 15.38, and 14 KU/mg protein as compared to 12.9, 8.74, and 12 KU/mg protein in fraction II of T. verrucosum, T. mentagrophytes, and M. canis, respectively. The dermatophyte exo-keratinases proved to be immunogenic as they stimulated high keratinase-specific antibody titers and induced strong delayed skin hypersensitivity reactions in vaccinated animals. Anti-keratinase-specific IgG was detected in sera of guinea pigs immunized with the inactivated or living polyvalent dermatophyte vaccines by a homemade enzyme-linked immunosorbent assay (ELISA) using dermatophyte exo-keratinases as coating antigen. The intradermal injection of dermatophyte exo-keratinases induced specific delayed skin reactions in guinea pigs immunized with the inactivated or the living polyvalent dermatophyte vaccines. The intradermal injection of dermatophyte exo-keratinases in the control non-sensitized guinea pigs was associated with itching, swelling, and bloody scar formation, however, no skin indurations were formed. The development of those post-exo-keratinases injection reactions in the control non-sensitized apparently healthy guinea pigs group, suggests an exo-keratinases possible role in the pathogenesis of dermatophytosis.

Development, preparation, and evaluation of a novel dotted lateral flow immunochromatographic kit for rapid diagnosis of dermatophytosis.

Dermatophytosis is a widely spread contagious zoonotic disease, affecting both man (tinea) and animals (ringworm). This disease is caused by a group of closely related keratinophilic fungi known collectively as the dermatophytes group. Although the wide distribution of dermatophytosis cases throughout the whole world and its adverse clinical effect on human health, economical effect on productive animals, and pet animal welfare, there is no rapid accurate diagnostic tool for such disease. The current conducted study tries to accomplish the difficult equation by achieving an accurate, sensitive, specific, user-friendly, rapid, robust, device-less, deliverable to end-users, and economic cost for the development and production of diagnostic kits. Through the development of a rapid diagnostic kit based on immunochromatographic assay with three major affordable reproducible production stages; preliminary stage, developmental and standardization stage, and evaluation stage. Obtaining dermatophytes-specific polyclonal antibodies against criteria-based selected dermatophytes strains associating proper gold nanoparticle preparation, characterization, and conjugation, with proper loading of the different bio-reactants on the efficiently laminated and fabricated lateral flow strips were the main challenge and control points through the whole process. Also, as a result of examining 100 animal samples using the new kit, the κ coefficients of the kit with the direct microscopy while the kit with the culture were 0.44 and 0.76, respectively. Therefore, the newly designated and developed kit showed a very promising competitive diagnostic result within 5-7 min through easy-to-be-performed three steps.

Recent trends in rapid diagnostic techniques for dermatophytosis.

Dermatophytosis is a common contagious disease of both humans and animals. It is caused by a group of filamentous fungi known as dermatophytes, including several genera and various species. An accurate diagnosis of dermatophytes as a causative agent of a skin lesion requires up to one month of conventional laboratory diagnostics. The conventional gold standard diagnostic method is a direct microscopic examination followed by 3 to 4 weeks of Sabouraud's dextrose agar (SDA) culturing, and it may require further post-culturing identification through biochemical tests or microculture technique application. The laborious, exhaustive, and time-consuming gold standard method was a real challenge facing all dermatologists to achieve a rapid, accurate dermatophytosis diagnosis. Various studies developed more rapid, accurate, reliable, sensitive, and specific diagnostic tools. All developed techniques showed more rapidity than the classical method but variable specificities and sensitivities. An extensive bibliography is included and discussed through this review, showing recent variable dermatophytes diagnostic categories with an illustration of weaknesses, strengths, and prospects.