Gamma Irradiated Pasteurella multocida Vaccine induces strong humoral immunity and protects rabbits from disease.

Pasteurella multocida is affecting a multitude of animals and severely affects livestock production. Existing vaccines are mostly chemically inactivated and do not lead to wide protection. Irradiated vaccines are enjoying a renaissance and the concept of "replication defficient but metabolically active" vaccines was recently evaluated in several vaccine trials. P. multocida was isolated from the nasal swab, blood, and lung swab samples from infected rabbits. Gamma irradiation of P. multocida for inhibition of replication was evaluated at an optimized irradiation dose of 10 Kgy established. Four groups of rabbits were (mock) vaccinated with a commercial P. multocida vaccine and three irradiated formulations as liquid, lyophilized formulations with added Trehalose and lyophilized-Trehalose with an "activation" culturing the irradiated bacteria for 24 in broth. Evaluation of humoral immune response by ELISA showed that all three irradiated vaccines produced an effective, protective, and continued IgG serum level after vaccination and bacterial challenge. The IFN-γ expression is maintained at a normal level, within each individual group however, the lyophilized trehalose irradiated vaccine showed peak mean of IFN-γ titer at one week after booster dose (day 21) which was statistically significant. Cumulatively, the results of this study show that gamma-irradiated P. multocida vaccines are safe and protect rabbits against disease. Moreover, Rabbits' immunization with the three irradiated formulations avoided adverse side effects as compared to commercial polyvalent vaccine, the body weight gain for the irradiated vaccine groups indicates less stress compared to the commercial polyvalent vaccine.

Evaluation of room temperature (30°C to 35°C) lyophilized vaccine with radio inactivated Mannheimia haemolytica whole cells isolated from infected sheep.

Background and Aim: Vaccines are one of the important tools for fighting diseases and limiting their spread. The development of vaccines with high efficacy against diseases is essential. Ionizing radiation is the method used for the preparation of the irradiated gamma Mannheimia haemolytica vaccine. The study aimed to measure the metabolic activity and electron microscopic examination of the irradiated bacterial cells and immunological efficiency of different preparations of the irradiated M. haemolytica vaccine. Materials and Methods: The irradiated vaccines were prepared in three forms at a dose of 2×109 colony-forming unit (CFU) (irradiated M. haemolytica, trehalose irradiated M. haemolytica, and trehalose lyophilized irradiated M. haemolytica). The formalin-killed vaccine was prepared at a dose of 2×109 CFU. Scanning electron microscopy was used to determine the difference between the non-irradiated bacterial cells and the bacterial cells exposed to gamma radiation. The metabolic activity of the irradiated bacterial cells was measured using the Alamar blue technique. Rabbits were divided into five groups (control, vaccinated groups with the formalin-killed vaccine, irradiated bacterial cells without trehalose, trehalose irradiated bacteria, and trehalose lyophilized irradiated bacterial cells). The rabbits were subcutaneously inoculated twice in 2-week intervals. Enzyme-linked immunosorbent assay, interferon-gamma (IFNγ), and interleukin 4 (IL4) assays were used to evaluate the vaccines' immunological efficiency in rabbits. Results: The metabolic activity tests showed that the bacterial cells exposed to gamma radiation at the lowest lethal dose have metabolic activity. The difference in the metabolic activity between preparations of the irradiated bacterial cells varied according to the cell concentration and incubation time. The highest level of metabolic activity was 8 h after incubation in the nutrient broth medium compared with 4 and 18 h. The scanning electron microscopy of irradiated bacterial cells showed a cavity at the bacterial cell center without rupture of the surrounding cell membrane compared to the non-irradiated bacterial cells. The antibody level in the groups vaccinated with the different preparations of the irradiated bacterial cells was high compared with the control and formalin-killed vaccine groups. The level of the IFNγ showed an increase after the second dose in the group vaccinated with irradiated bacterial cells without trehalose compared with the other groups. The IL4 level in the vaccinated groups with the irradiated bacterial cells without trehalose, irradiated bacterial cells with trehalose, and trehalose lyophilized irradiated bacterial cells were at a high level when compared with the formalin-killed vaccinated group and control group after the second inoculation. Conclusion: The irradiated M. haemolytica vaccine provides a wide range of humoral and cellular immunity. This study showed high immunological efficiency in rabbits inoculated with the irradiated M. haemolytica vaccine that was shown in the high levels of antibodies (IFNγ and IL4) compared with the group treated with the formalin-killed vaccine. The second dose of irradiated M. haemolytica vaccine is an immune booster that gives the irradiated vaccine a long-acting immunological efficiency.

Electron microscopy and phylogenetic analysis of Bovine papillomavirus infection in cattle from four Egyptian governorates.

Cutaneous warts are the common clinical feature of infection with Bovine papillomavirus (BPV), and it is commonly known as bovine papillomatosis. It causes significant economic losses, especially in the dairy sector. The aim of this study was surveillance of the circulating strains of BPV in four Egyptian governorates and characterization by electron microscopy. Warts skin lesions and whole blood from seventy-eight native breed cattle were obtained. Molecular detection using two different sets of primers, phylogenetic analysis, and electron microscopy were carried out. The obtained results showed that using FAP59/FAP64 primer set is more sensitive than the MY09/My11 primer set in the detection of the papilloma L1 gene either in the blood or in the skin lesion. Sequence analysis of the partially amplified L1 gene revealed 4 different strains belonging to Deltapapillomavirus 4. Only Alfayoum_Deltapapillomavirus_2018 (accession no: MW018705) was found to be closely related to the strain previously isolated in different Egyptian governorates in 2017, and 2 strains were closely related to an isolate of equine origin. Electron microscopy examination of the skin lesions showed the presence of negatively stained rounded, non-enveloped virus particles with a size of 60 nm in diameter. In conclusion, continuous surveillance and characterization of the circulating strains using multiple sets of primers are important. Efficient biosecurity measures must be applied to decrease transmission of papillomavirus between the different animal species, especially in the mixed management system.

Presence of bluetongue and epizootic hemorrhagic disease viruses in Egypt in 2016 and 2017.

BTV and EHDV are closely-related orbiviruses that are transmitted between domestic and wild ruminants via the bites of hematophagous midges. Previous studies have reported seropositivity against BTV antibodies in sheep and goats in two Egyptian governorates (Beni Suef and Menoufia). However, no recent data are available on the BTV serotype(s) circulating in Egypt and the likely presence of EHDV has never been explored. This study investigated the presence of BTV and EHDV among cattle which had been found BTV-seropositive by ELISA method. These cattle living in proximity to sheep and goats previously found BTV-seropositive. These cattle displayed no clinical signs of BT but reproductive problems had been reported in herds. A total of 227 cattle blood samples were therefore collected in 2016 and 2017. Ninety-four of the 227 animals tested by a BTV ELISA were positive for BTV antibodies (41.4%). Of these 94 ELISA-positive cattle, only 83 EDTA-blood samples were available and therefore tested for BTV and EHDV genome detection by RT-PCR and sequencing. Of the cattle sampled in 2016, results revealed that two were RT-PCR-positive for BTV and seven for EHDV. Sequencing showed the presence of EHDV-1 and BTV-3 genome sequences. EHDV-1 S2 shared 99.5% homology with an EHDV-1 S2 from a strain isolated in 2016 in Israel. BTV-3 S2 and S8 sequences shared >99.8% nucleotide similarity with the BTV-3 Zarzis S2 and S8 sequences (Tunisian BTV, also detected in 2016). Of the 66 blood samples tested following their collection in 2017, they were all EHDV-negative by RT-qPCR while five were BTV- positive by RT-qPCR. However, attempts to identify the BTV serotype of these five samples were unsuccessful. Only part of BTV S8 was sequenced and it showed 79% nucleotide similarity with S8 of atypical BTV serotypes (particularly with BTV-26 and another BTV serotype strain isolated from a sheep pox vaccine). Overall, these findings demonstrate that both BTV and EHDV were circulating in Egypt in 2016 and 2017.

Molecular detection and immunopathological examination of Deltapapillomavirus 4 in skin and udder of Egyptian cattle.

AIM: Bovine papillomaviruses (BPVs) are the main cause of bovine papillomatosis resulting in cutaneous and/or mucosal benign tumors that could be transformed to malignant ones with marked economic importance, especially in the dairy farms. Molecular, pathological, and immunohistochemical (IHC) diagnosis of bovine papillomatosis cases was conducted to identify and characterize the circulating BPV genotype in some Egyptian governorates. MATERIALS AND METHODS: Wart-like lesions in skin, udder, and teats were collected from 123 infected cases in Giza, Beni Suef, and El Menoufia Governorates, Egypt, during 2016-2017. Pathological and IHC characterization, molecular identification, genotyping, and phylogenetic analysis based on the conserved late (L1) gene of the all samples were carried out. RESULTS: 89 of the 123 collected samples (72.3%) were positively detected by polymerase chain reaction (PCR). The sequence analysis of the obtained PCR amplicons was identical revealing identification and genotyping of only one type (Deltapapillomavirus 4 isolate EGY 2017) with accession number (MG547343) which found to be closely related to the recently detected Deltapapillomavirus 4 isolate 04_asi_UK (accession no. MF384288.1) and isolate Deltapapillomavirus 4 isolate 25_equ_CH (accession no. MF384286.1) with 99% nucleotide sequence identity. Histopathological examination revealed severe hyperkeratosis in stratum corneum and acanthosis in most of the cases. These tissue changes were confirmed by the presence of golden brown stained proliferating cell nuclear antigen which was localized intranuclear and perinuclear in other cells using IHC Technique. CONCLUSION: It is the first time to detect and genotype the BPVs in these areas with no record of previous genotyping in the whole country. The obtained results will highlight the importance of this disease.

Interspecies comparison of probiotics isolated from different animals.

AIM: The aim of the current study was to isolate and identify naturally occurring probiotic Lactobacillus species in different animals with the different environmental background including fish, and farm animals to investigate interspecies differences in probiotics on the species level. MATERIALS AND METHODS: A total of 44 fecal and milk samples were collected under aseptic conditions from cattle, buffalo, camel, sheep, goats, and fish. The samples were cultured, and the isolated strains were confirmed biochemically and molecularly using 16S rRNA multiplex polymerase chain reaction (PCR) analysis following DNA extraction from the bacterial isolates. RESULTS: A total of 31 isolates identified as lactobacilli were isolated from cattle milk, goat feces, sheep feces, fish feces, buffalo milk, camel milk, and goats' milk. Lactobacillus species were identified based on the size of the PCR product. The results showed that different species were different in their lactobacilli content. At the same time, there were some differences between individuals of the same species. CONCLUSION: The diversity of probiotic strains isolated from different animal species implies different types of benefits to the host. Although it would be both money - and time-consuming research, discovering the benefit of each of these strains may provide very important information for the health of both human and animal. Furthermore, transferring these beneficial effects either to individuals within the same species or between different species would be of great importance.

Serological investigation of some important RNA viruses affecting sheep and goats in Giza and Beni-Suef governorates in Egypt.

AIM: The aim of this study was to investigate the seroprevalence of antibodies against foot and mouth disease (FMD), Peste des Petits ruminants (PPR), and bluetongue (BT) in sheep and goats within Giza and Beni-Suef governorates at the second half of 2016. MATERIALS AND METHODS: A total of 300 animals (sheep and goats) randomly selected from small stocks with no history of previous vaccination against FMD virus (FMDV), PPR, or BT viruses (BTV) and examined with competitive enzyme-linked immunosorbent assay for detection of FMD-non-structural protein, PPR, and BT antibodies. RESULTS: Seroprevalence analysis revealed that antibodies against FMDV were 40.8% and 37.1% at Giza governorate, while at Beni-Suef governorate, the percent was 36.7% and 50% in sheep and goat, respectively. Antibodies against PPR were 63.8% in sheep and 45.7% in goats at Giza governorate, whereas the results for Beni-Suef governorate were 71.7% in sheep and 45% in goats. Antibodies against BT were 45% and 37% in sheep and goats, respectively, in Giza governorate, whereas the results for Beni-Suef governorate were 80% and 55% in sheep and goats, respectively. The average of BTV antibody prevalence was significantly higher in sheep (45% and 80%) than in goats (37% and 55%) in Giza and Beni-Suef, respectively. Statistical analysis for the three viruses showed the high relation between the two governorates in case of sheep (r=0.85) and in case of goats (r=0.87). In general, a strong positive correlation was observed between the governorates (r=0.93). CONCLUSION: Giza and Beni-Suef governorates are endemic with FMDV, PPR, and BTV. Regional plan for characterization and combating FMD, PPR, and BT is recommended to help in the achievement of the most suitable combination of the vaccine regimen.