Phylogenetic Analysis and Comparative Genomics of Brucella abortus and Brucella melitensis Strains in Egypt.

Brucellosis is a notifiable disease induced by a facultative intracellular Brucella pathogen. In this study, eight Brucella abortus and eighteen Brucella melitensis strains from Egypt were annotated and compared with RB51 and REV1 vaccines respectively. RAST toolkit in the BV-BRC server was used for annotation, revealing genome length of 3,250,377 bp and 3,285,803 bp, 3289 and 3323 CDS, 48 and 49 tRNA genes, the same number of rRNA (3) genes, 583 and 586 hypothetical proteins, 2697 and 2726 functional proteins for B. abortus and B. melitensis respectively. B. abortus strains exhibit a similar number of candidate genes, while B. melitensis strains showed some differences, especially in the SRR19520422 Faiyum strain. Also, B. melitensis clarified differences in antimicrobial resistance genes (KatG, FabL, MtrA, MtrB, OxyR, and VanO-type) in SRR19520319 Faiyum and (Erm C and Tet K) in SRR19520422 Faiyum strain. Additionally, the whole genome phylogeny analysis proved that all B. abortus strains were related to vaccinated animals and all B. melitensis strains of Menoufia clustered together and closely related to Gharbia, Dameitta, and Kafr Elshiek. The Bowtie2 tool identified 338 (eight B. abortus) and 4271 (eighteen B. melitensis) single nucleotide polymorphisms (SNPs) along the genomes. These variants had been annotated according to type and impact. Moreover, thirty candidate genes were predicted and submitted at GenBank (24 in B. abortus) and (6 in B. melitensis). This study contributes significant insights into genetic variation, virulence factors, and vaccine-related associations of Brucella pathogens, enhancing our knowledge of brucellosis epidemiology and evolution in Egypt.

Seroprevalence and risk factors of Q-fever (C. burnetii infection) among ruminants reared in the eastern region of the Kingdom of Saudi Arabia.

Q-fever is a worldwide spread zoonotic disease associated with severe illness in humans and abortions and stillbirths in ruminants. Ruminants are major sources of human infection where subclinical carriers shed the bacteria in various secretions and excreta. The goal of the current study was to investigate the prevalence and risk factors of Coxiella burnetii infection among cattle, sheep, and goats in the eastern province of the Kingdom of Saudi Arabia (KSA). A total of 1310 serum samples were collected through a designed cross-sectional study from private farms and slaughterhouses in the study area and examined against antibodies of C. burnetii using ELISA. A multivariate logistic regression analysis was built to detect risk factors of C. burnetii infection among examined species. The prevalence of C. burnetii infection among examined animals was 9.2% (CI, 7.7-10.8)-15.6%, 9.1%, and 5.8% among goats, cattle, and sheep, respectively). The risk of getting C. burnetii infection among old animals (> 1 year old) was 23 times higher than the risk among young animals (< 1 year old) (95% CI, 10.04-53.01; P < 0.01). Goats were 2.27 (95% CI, 1.41-3.66; P < 0.01) and 3 times at higher risk than cattle and sheep, respectively, of getting C. burnetii infection. In conclusion, C. burnetii infection is widespread among different ruminant species of the eastern province of KSA which represents a high risk for environmental contamination and disseminating the infection to humans and animal species in that area. Also, our findings may reflect the disease status in other countries of the Arabian Gulf area.

Risk analysis and seroprevalence of bovine ephemeral fever virus in cattle in the Kingdom of Saudi Arabia.

Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that causes disabling clinical signs and major economic losses in cattle and water buffalo. The disease is well documented in Asia, Africa, and the Middle East; however, the seroprevalence of BEFV in different regions and bovine breeds in the Kingdom of Saudi Arabia (KSA) is unknown. The aim of this study was to analyze risk factors which affect the prevalence of antibodies against BEFV in small herds of cattle in four geographical regions of KSA. A total of 1480 serum samples from non-BEFV vaccinated small herds of cattle were collected from the Eastern, Jizan, Qasim, and Riyadh regions (370 samples per region) during the summer of 2010. Serum neutralization test was used to detect antibodies against BEFV. There was a significant effect of region, breed, sex, and age on the seroprevalence of BEFV. Seropositive ratios were 18, 18, 26, and 12 % for the Eastern, Jizan, Qasim, and Riyadh regions, respectively (P = 0.00002); 23.2 % for dairy and 13.7 % for non-dairy breeds (P = 0.00004); 24.4 % for males and 14.6 % for females (P = 0.00004); and 15.4, 29.1, and 11.4 % for animals <1 year, 1-3 years, and >3 years, respectively (P < 0.001). Risk analysis showed a significant effect of different regions of KSA on the seroprevalence of BEFV. Host risk factors (age, sex, and breed) showed also a significant effect on the seroprevalence of BEFV. This indicates active circulation of this virus in small herds of cattle. Insect control strategies and BEFV vaccination programs during the spring are recommended to reduce the spread of BEFV and minimize subsequent economic losses as this is adopted in many enzootic countries.

Inhibitory effect of allicin on the growth of Babesia and Theileria equi parasites.

Allicin is an active ingredient of garlic that has antibacterial, antifungal, antiviral, and antiprotozoal activity. However, the inhibitory effects of allicin on Babesia parasites have not yet been examined. In the present study, allicin was tested as a potent inhibitor against the in vitro growth of bovine and equine Babesia parasites and the in vivo growth of Babesia microti in a mouse model. The in vitro growth of Babesia bovis, Babesia bigemina, Babesia caballi, or Theileria equi was inhibited by allicin in a dose-dependent manner and had IC50 values of 818, 675, 470, and 742 µM, respectively. Moreover, allicin significantly inhibited (P < 0.001) invasion of B. bovis, B. bigemina, B. caballi, and T. equi into the host erythrocyte. Furthermore, mice treated with 30 mg/kg of allicin for 5 days significantly (P < 0.05) reduced the parasitemia of B. microti over the period of the study. To further examine the potential synergism of allicin with diminazene aceturate, growth inhibitory assays were performed in vitro and in vivo. Interestingly, combinations of diminazene aceturate with allicin synergistically potentiated its inhibitory effects in vitro and in vivo. These results indicate that allicin might be beneficial for the treatment of babesiosis, particularly when used in combination with diminazene aceturate.

Specific antibody to a conserved region of Babesia apical membrane antigen-1 inhibited the invasion of B. bovis into the erythrocyte.

Apical membrane antigen-1 (AMA-1) is a microneme protein that exists in all apicomplexan parasites and plays an indispensable role in the invasion into host cell. Central region of ectodomains I and II of Babesia bovis apical membrane antigen-1 (BbAMA-1P) is highly conserved with these of Babesia species and may be beneficial for vaccine development against babesiosis. In the present study, recombinant protein encoding the central region of B. bovis AMA-1 (rBbAMA-1P) was produced in Escherichia coli and its antiserum was prepared in mice for further molecular characterization. Anti-rBbAMA-1P serum specifically reacted with corresponding authentic protein of B. bovis as determined by Western blotting and IFAT. Cultured B. bovis treated with anti-rBbAMA-1P serum showed significant reduction in the in vitro growth of the parasites. Moreover, preincubated free merozoites with 1mg/ml anti-rBbAMA-1P serum inhibited their efficiency in the invasion into erythrocytes (RBCs) by 61% and 70% at 3h and 6h, respectively. Our data suggest that the central region of domains I and II of BbAMA-1 may serve as a vaccine candidate against babesiosis.

Recent progress in the genotyping of bovine tuberculosis and its rapid diagnosis via nanoparticle-based electrochemical biosensors.

Bovine tuberculosis (bTB) is considered a worldwide infectious zoonotic disease. Mycobacterium bovis causes bTB disease. It is one of the Mycobacterium tuberculosis complex (MTBC) members. MTBC is a clonal complex of close relatives with approximately 99.95% similarity. M. bovis is a spillover pathogen that can transmit from animals to humans and rarely from humans to animals with contact. Genotyping techniques are important to discriminate and differentiate between MTBC species. Spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) are widely used but they have some limitations. As an alternative, whole genome sequencing approaches have been utilized due to their high-resolution power. They are employed in typing M. bovis and explain the evolutionary and phylogenetic relationships between isolates. The control of bTB disease has attracted a large amount of attention. Rapid and proper diagnosis is necessary for monitoring the disease as an initial step for its control and treatment. Nanotechnology has a potential impact on the rapid diagnosis and treatment of bTB through the use of nanocarrier and metal nanoparticles (NPs). Special attention has been paid to voltammetric and impedimetric electrochemical strategies as facile, sensitive, and selective methods for the efficient detection of tuberculosis. The efficacy of these sensors is enhanced in the presence of NPs, which act as recognition and/or redox probes. Gold, silver, copper, cobalt, graphene, and magnetic NPs, as well as polypyrrole nanowires and multiwalled carbon nanotubes have been employed for detecting tuberculosis. Overall, NP-based electrochemical sensors represent a promising tool for the diagnosis of bTB.

Brucellae as resilient intracellular pathogens: epidemiology, host-pathogen interaction, recent genomics and proteomics approaches, and future perspectives.

Brucellosis is considered one of the most hazardous zoonotic diseases all over the world. It causes formidable economic losses in developed and developing countries. Despite the significant attempts to get rid of Brucella pathogens in many parts of the world, the disease continues to spread widely. Recently, many attempts proved to be effective for the prevention and control of highly contagious bovine brucellosis, which could be followed by others to achieve a prosperous future without rampant Brucella pathogens. In this study, the updated view for worldwide Brucella distribution, possible predisposing factors for emerging Brucella pathogens, immune response and different types of Brucella vaccines, genomics and proteomics approaches incorporated recently in the field of brucellosis, and future perspectives for prevention and control of bovine brucellosis have been discussed comprehensively. So, the current study will be used as a guide for researchers in planning their future work, which will pave the way for a new world without these highly contagious pathogens that have been infecting and threatening the health of humans and terrestrial animals.

Evaluation and Haematological Profile of an Inactivated RVF Vaccine Locally Produced in Egypt.

<b>Background and Objective:</b> The RVF virus cause diseases in newborn puppies, kittens, sheep, goats, cattle, camels, buffaloes and also humans. The RVF disease was first detected among livestock by veterinary officers. The disease causes abortions in animals. The goal of this study was to evaluate the immune response and the haematological profile associated with inactivated RFV vaccine locally produced in Egypt in young puppies and sheep. <b>Materials and Methods:</b> Through vaccination, both young puppies and sheep with local produced inactivated RVF vaccine with 2 doses with 2 weeks interval and evaluate the immune response by SNT and ELISA as well as haematological parameters at 0, 7, 14, 21 and 28 days post-vaccination. The variance between vaccinated groups and also non-vaccinated groups were compared by using a one-way Analysis of Variance (ANOVA). <b>Results:</b> The findings showed that young puppies had a strong response to antibodies after two doses of the RVF vaccine within the 2 week interval. The neutralization indices (NI) values in young puppies at different periods after RVF vaccination reported the value of 1.08±0.03, 1.23±0.04, 1.30±0.03 and 1.45±0.02 after 7, 14, 21 and 28 days post-vaccination, respectively. Parallel to this the ELISA OP values were 0.30±0.00, 0.39±0.03, 0.52±0.05 and 0.75±0.02 after 7, 14, 21 and 28 days post-vaccination, respectively. Nearly similar immune response was noticed in sheep with NI values of 1.15±0.02, 1.27±0.02, 1.42±0.05 and 1.55±0.03 at 7, 14, 21 and 28 days post-vaccination, respectively. In the same site the ELISA OP values were 0.34±0.00, 0.47±0.01, 0.68±0.00, 0.77±0.00. After 7, 14, 21 and 28 days post-vaccination respectively that are also similar to that in puppies. The haematological profile reported a significant decrease after the 1st week followed by a transient increase after booster dose at 2nd week except for the monocytes that increase after 1st week then decreases after 2nd week post-vaccination. <b>Conclusion:</b> Young puppies are similar to sheep in developing antibodies after vaccination with the RFV vaccine with no statistically significant effect within different batches. In addition, ELISA can replace the SNT for evaluation of the immune response. Young puppies are quite equal to sheep for the illustration of neutralizing antibodies for RFV vaccine. Sero-negative puppies can be easily obtained because dogs are not included in the vaccination program of RVF and so they can be used as a good model to determine the efficacy of the RVF vaccine.

Clinical, histopathological, and molecular characterization of Mycoplasma species in sheep and goats in Egypt.

BACKGROUND AND AIM: Mycoplasma infection in small ruminants is a serious problem in sheep and goat herds around the world. It is responsible for high economic losses and decreased animal productivity. This study aimed to highlight the clinical, histopathological, minimum inhibitory concentration (MIC), and molecular characterization of Mycoplasma species in sheep and goats in Menoufiya Governorate, Egypt. MATERIALS AND METHODS: A total of 234 samples were collected; 104 samples were collected from pneumonic lung tissues from the abattoir, in addition, 10 and 20 samples collected from apparently and diseased sheep, respectively, and 40 and 60 samples were collected from apparently and diseased goats, respectively, which were subjected to isolation onto pleuropneumonia-like organism medium. Polymerase chain reaction (PCR), histopathological examination, and determination of the MIC were also performed. RESULTS: Of 104 samples of lung tissues showing pneumonic lesions, 56 (53.84%) were positive for Mycoplasma isolation. The positive isolation of Mycoplasma from 10 and 20 samples from apparently and diseased sheep was 30% and 40%, respectively as well as the positive isolation of Mycoplasma was 17% and 56.66% out of 40 and 60 apparently healthy and diseased field goat's cases, respectively. All the diseased sheep and goats showed respiratory manifestations, including cough, bilateral nasal discharge, conjunctivitis, and systemic reaction. Evaluation of the MIC for Mycoplasma ovipneumoniae revealed that lincospectin and tylosin were the most effective antibiotics at 2.5 mg/mL. Histopathological examination of affected lung tissue showed extensive hemorrhagic pneumonia with extensive alveolar hemorrhage. The PCR technique proved to be a rapid, specific, and sensitive method for the detection of M. ovipneumoniae and Mycoplasma arginini at 390 and 326 bp, respectively. CONCLUSION: M. ovipneumoniae and M. arginini were the most prevalent species associated with respiratory infections in sheep and goats in the study area. Further studies are needed to investigate the role of these species in dissemination of the disease within herds of small ruminants.

Investigation of Mycobacterium paratuberculosis in Arabian dromedary camels (Camelus dromedarius).

AIM: Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants. This study aimed to investigate Mycobacterium paratuberculosis infection in clinically infected camels on the immunological, conventional bacteriological, and molecular biological basis. MATERIALS AND METHODS: A total of 30 Arabian camels (Camelus dromedarius) were examined in this study. The camels were suffering from signs ranging from mild to severe infections (that did not respond to antibiotic treatment) to chronic or intermittent diarrhea. Camels were grouped into three groups based on their age, sex, and breed. Detection of anti-MAP antibodies in camels' serum, Ziehl-Neelsen (ZN) staining technique on rectal scraps, direct recognition of MAP in stool and tissue specimens by IS900 polymerase chain reaction (PCR) assay, and finally isolation and molecular description of MAP from fecal and tissue samples were carried out. RESULTS: Five MAP isolates were recovered from these investigated camel samples giving an isolation rate of 16.6%, while eight camels were identified by PCR (26.6%). Five camels yielded MAP in their feces by ZN fecal staining (16.6%), whereas ELISA detected anti-MAP antibodies in nine camels only (30%). CONCLUSION: From the obtained results, we concluded that the gold standard for the diagnosis of MAP is the culture method despite its limitations. Molecular diagnosis (PCR) could be a useful tool in the identification of truly positive and negative camels; however, great care should be given regarding the primers specificity and sensitivity.

Serum vitamin A and E, copper, zinc and selenium concentrations and their relationship with health outcomes in dromedary hospitalized camels (Camelus dromedarius).

The goals of this study were to measure serum vitamin A (retinol) and E (α-tocopherol) and trace elements concentrations (copper, zinc and selenium) during diseases condition and to determine their association with hematological parameters and immune status of hospitalized camels. A total of 95 dromedary camels [healthy (n=65); hospitalized camels (n=30)] were included in this study. Vitamin A and E concentrations were significantly lower in hospitalized camels than apparently healthy ones (P<0.05). Hospitalized camels had lower concentrations of zinc and selenium compared to healthy camels (P<0.05). Vitamin E, copper, zinc and selenium concentrations were positively correlated with phagocytic activity in hospitalized camels (P<0.05). The likelihood of deficiency of vitamin A and E, zinc and selenium concentrations were significant in female hospitalized camels than males and in young age hospitalized camels < 6 years old compared to old ones (P<0.05). Decreased vitamin A and E and trace elements concentrations were associated with hospitalized camels' phagocytic activity and index. The prevalence of low vitamin A and E, zinc and selenium concentrations were frequent in female hospitalized camels and hospitalized camels of age < 6 years old suggesting severe oxidative stress.

Development and characterization of ORF68 negative equine herpes virus type-1, Ab4p strain.

Equine herpesvirus-1 (EHV-1) is an important pathogen, which infects horses worldwide with high morbidity but low mortality rates. The respiratory disorders and abortions are the most common indicators. Ab4p (an abortigenic and paralytic virus) is one of the most important and virulent strains. The development and functional characterization of the open reading frame-68 (ORF68) negative EHV-1 Ab4p mutants and an assessment of their roles in the infection at the cellular level were the main targets of the current study. Escherichia coli DH10ß containing the Ab4p bacterial artificial chromosome (pAb4pBAC) and Red/ET expression vector were used to develop different ORF68 mutants. Multi-step growth kinetic experiments were conducted in order to evaluate the growth properties of the constructed mutant viruses. Growth of the Ab4pΔORF68 showed the lowest titer, compared to the Ab4pΔORF68R, Ab4pΔORF68R non-sense, and the parent Ab4p viruses without any significant difference (P > 0.05). The growth of the mutant viruses was almost similar across the cell types, but viruses growth was more efficient in FHK cells as judged by the number of the obtained virus particles. The plaque size of Ab4pΔORF68 was significantly (40%) smaller than those of Ab4p (P < 0.01), Ab4pΔORF68R, and Ab4pΔORF68R non-sense viruses which confirmed the importance of ORF68 protein in the cell-to-cell transmission of EHV-1. Subcellular localization of the green fluorescent protein (GFP) ORF68 gene fusion product showed late expression with intranuclear localization of the transfected cells while immunofluorescent antibody technique (IFAT) localized it at the nucleus and nuclear membranes of the infected cells. Hence, it could be concluded that ORF68 protein may not be essential for EHV-1 Ab4p growth but plays a crucial role in virus penetration and transmission at the cellular level. Therefore, the generated EHV-1 ORF68 negative mutant could be a prospective candidate for the development of a vaccine marker.

Bovine ephemeral fever epidemics in Kingdom Saudi Arabia: clinical, epidemiological and molecular investigation.

INTRODUCTION: Bovine ephemeral fever virus (BEFV) is an arthropod borne Rhabdovirus affects cattle and water buffalo causes acute febrile disease. METHODOLOGY: The clinical picture and epidemiological pattern of BEF were described among cattle in epidemics of 2007, 2009 and 2011 in four geographical regions of Kingdom Saudi Arabia (Eastern, Jizan, Qasim, and Riyadh). Serum samples were tested using VNT. Virus isolation and molecular characterization were carried out for the first time in KSA. RESULTS: The main clinical symptoms were fever, stiffness, lameness, salivation and subcutaneous emphysema. The prevalence and the mortality rate of BEF have decreased from 70% and 4.6% in 2007 to 30% and 0.6% in 2011, respectively in the 4 studied areas. There was no region association with higher prevalence of BEF. The intracluster correlation (ICC) was estimated for the first time in KSA as 0.0034. BEFV had been isolated from 11 out of 20 samples (55%) and isolation was confirmed by VNT. The molecular detection of BEFV by RT-PCR and real- time RT-qPCR were found more sensitive for diagnosis of the disease than virus isolation; 80% and 90% for the former tests and 55% for the latter. Three isolates were sequenced, they showed 84.7% - 100% identities in between and shared 90.4%-96.5% sequence identity with a previously published sequence from Australia (KF679404). The generated sequences belonged to 3rd cluster of BEFV glycoprotein. CONCLUSIONS: BEF occurrence has cyclic nature and the efficacy of vaccines prepared from local strains has to be evaluated and considered in diseases control.

Vitamin D Metabolites and Their Association with Calcium, Phosphorus, and PTH Concentrations, Severity of Illness, and Mortality in Hospitalized Equine Neonates.

BACKGROUND: Hypocalcemia is a frequent abnormality that has been associated with disease severity and outcome in hospitalized foals. However, the pathogenesis of equine neonatal hypocalcemia is poorly understood. Hypovitaminosis D in critically ill people has been linked to hypocalcemia and mortality; however, information on vitamin D metabolites and their association with clinical findings and outcome in critically ill foals is lacking. The goal of this study was to determine the prevalence of vitamin D deficiency (hypovitaminosis D) and its association with serum calcium, phosphorus, and parathyroid hormone (PTH) concentrations, disease severity, and mortality in hospitalized newborn foals. METHODS AND RESULTS: One hundred newborn foals ≤72 hours old divided into hospitalized (n = 83; 59 septic, 24 sick non-septic [SNS]) and healthy (n = 17) groups were included. Blood samples were collected on admission to measure serum 25-hydroxyvitamin D3 [25(OH)D3], 1,25-dihydroxyvitamin D3 [1,25(OH) 2D3], and PTH concentrations. Data were analyzed by nonparametric methods and univariate logistic regression. The prevalence of hypovitaminosis D [defined as 25(OH)D3 <9.51 ng/mL] was 63% for hospitalized, 64% for septic, and 63% for SNS foals. Serum 25(OH)D3 and 1,25(OH) 2D3 concentrations were significantly lower in septic and SNS compared to healthy foals (P<0.0001; P = 0.037). Septic foals had significantly lower calcium and higher phosphorus and PTH concentrations than healthy and SNS foals (P<0.05). In hospitalized and septic foals, low 1,25(OH)2D3 concentrations were associated with increased PTH but not with calcium or phosphorus concentrations. Septic foals with 25(OH)D3 <9.51 ng/mL and 1,25(OH) 2D3 <7.09 pmol/L were more likely to die (OR=3.62; 95% CI = 1.1-12.40; OR = 5.41; 95% CI = 1.19-24.52, respectively). CONCLUSIONS: Low 25(OH)D3 and 1,25(OH)2D3 concentrations are associated with disease severity and mortality in hospitalized foals. Vitamin D deficiency may contribute to a pro-inflammatory state in equine perinatal diseases. Hypocalcemia and hyperphosphatemia together with decreased 1,25(OH)2D3 but increased PTH concentrations in septic foals indicates that PTH resistance may be associated with the development of these abnormalities.