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.

Molecular epidemiology of certain vector-borne bacterial microorganisms in domestic animals and their ectoparasites in Egypt.

Vector-borne bacterial diseases (VBBD) are a diverse group of tropical and subtropical zoonotic diseases. This study investigated the possibility of domestic animals to carry certain vector-borne bacterial microorganisms (VBBMs), as well as the presence of these targeted DNAs in their ectoparasites in different localities of Egypt using molecular analyses. For this study, 234 animal hosts (112 cattle, 38 sheep, 28 goats, 26 buffaloes, 22 donkeys, and 8 horses) in addition to 115 ectoparasites (95 ticks and 20 lice) were investigated for the molecular detection of Bartonella spp., Borrelia spp., and Rickettsia spp., targeting 16S-23S rRNAITS, 16S rRNA, and gltA genes, respectively. The results indicated that the overall prevalence of VBBD was observed in 17 animals (7.26%), of which 16 (6.84%) were positive for Bartonella spp. and one (0.43%) was positive for Borrelia theileri. All blood samples were negative for the DNA of Rickettsia spp. In addition, the results demonstrated that all ectoparasites were free from VBBDNA. Furthermore, of the animals examined for ectoparasite infestation, 28 (11.97%) and 5 (2.14%) represented Rhipicephalus annulatus ticks and Haematopinus tuberculatus lice, respectively, which infested animals. Analysis of epidemiological factors revealed that gender, age, and ectoparasitic infestation of animals had a significant effect on Bartonella infection, whereas no significant difference between animal species was observed. Hence, we report a potential novel Bartonella sp. from cattle and buffaloes, including a new genotype of Bo. theileri from cattle, in Egypt.

Perspective vaccines for emerging viral diseases in farm animals.

The world has watched the emergence of numerous animal viruses that may threaten animal health which were added to the perpetual growing list of animal pathogens. This emergence drew the attention of the experts and animal health groups to the fact that it has become necessary to work on vaccine development. The current review aims to explore the perspective vaccines for emerging viral diseases in farm animals. This aim was fulfilled by focusing on modern technologies as well as next generation vaccines that have been introduced in the field of vaccines, either in clinical developments pending approval, or have already come to light and have been applied to animals with acceptable results such as viral-vectored vaccines, virus-like particles, and messenger RNA-based platforms. Besides, it shed the light on the importance of differentiation of infected from vaccinated animals technology in eradication programs of emerging viral diseases. The new science of nanomaterials was explored to elucidate its role in vaccinology. Finally, the role of Bioinformatics or Vaccinomics and its assist in vaccine designing and developments were discussed. The reviewing of the published manuscripts concluded that the use of conventional vaccines is considered an out-of-date approach in eliminating emerging diseases. However, these types of vaccines are considered the suitable plan especially in countries with few resources and capabilities. Piloted vaccines that rely on genetic-based technologies with continuous analyses of current viruses should be the aim of future vaccinology. Smart genomics of emerging viruses will be the gateway to choosing appropriate vaccines, regardless of the evolutionary rates of viruses.

Clinical and Immunological Impacts of Latent Toxoplasmosis on COVID-19 Patients.

Background Parasites are well-known immune-modulators. They inhibit some aspects of the immune system to ensure persistence inside the host for a long time; meanwhile, they stimulate other immune aspects to assure the survival of the host. Wide variations in the severity of coronavirus disease 2019 (COVID-19) among developed and developing countries were reported during the COVID-19 pandemic. Parasitic infections, including Toxoplasma gondii (T. gondii), were claimed to contribute to such variations. Methods To explore a possible relationship between latent toxoplasmosis and COVID-19 severity, our study included 44 blood samples from moderate/severe COVID-19 patients, who were admitted to Mansoura University Hospitals, Egypt, during the pandemic. Patients' sera were screened for Toxoplasma IgG antibodies using ELISA (Roche Diagnostics, Indianapolis, USA), and the gene expression of important immune markers (iNOS, arginase-1, IFN-γ, TNF-α, IL-6, IL-10, and TGF-ß) was checked using real-time quantitative PCR. Clinical and laboratory data were obtained from the patients' medical records. Results Toxoplasma IgG antibodies were detected in 33 (75%) of patients. None of the studied clinical or laboratory parameters showed any significant changes in relation to toxoplasmosis seroprevalence. Further classification of the patients according to COVID-19 severity and Toxoplasma seroprevalence did not reveal any changes related to toxoplasmosis as well. Conclusion Our study indicates that latent toxoplasmosis has no effect on the severity of COVID-19.

Phenotyping and genotyping studies on extended-spectrum ß-lactamase-producing Escherichia coli isolates from mastitic cows on dairy farms in Egypt.

Background and Aim: Extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae have become a serious public health hazard worldwide. This importance is derived from the increase of new variants, particularly blaTEM, blaSHV, and blaCTX-M genes. This study aimed to examine ESBL-producing Escherichia coli isolated from different governorates in Egypt from dairy cows infected with subclinical and clinical mastitis. Materials and Methods: This study examined 207 milk samples for the resistance of isolates against 14 different antibiotics and ran serological identification of ESBL-producing E. coli isolates with complete antibiotic resistance. Genotypic and sequencing analyses of several resistance genes were conducted using a polymerase chain reaction. Results: E. coli was identified in cases with subclinical mastitis (80.5%) and clinical mastitis (85.7%). ESBL-producing E. coli was isolated from 38.2% of subclinical mastitic milk compared to 39.3% in clinical cases, where O26:k60, O125:k70, and O25:k11 were the serotypes with complete resistance to antibiotics. ESBL-producing E. coli isolates were resistant to cefotaxime, amoxicillin, cloxacillin, oxacillin, rifampicin, and penicillin in 100% but susceptible to amoxicillin and clavulanic acid in 82.5% of the cases. Results also revealed that 51.25%, 52.5%, 66.25%, 77.5% and 60% of ESBL-producing E. coli isolates were responsive to ciprofloxacin, ofloxacin, norfloxacin, levofloxacin, and gentamycin, respectively. The detected genes were registered in GenBank as MW345819.1 and MW345820.1 for the E. coli blaTEM gene and MW295407 for the E. coli blaSHV gene. Conclusion: This study found ESBL-producing E. coli in mastitic milk samples from Egyptian dairy farms and confirmed the occurrence and circulation of the main antibiotic genes (blaTEM and blaSHV) in the samples. Regular and thorough surveillance of ESBL-producing E. coli and subsequent preventive actions are essential for preventing the spread of these resistance genes in the future, which could pose serious and catastrophic health risks. Authorities should cling to the concept of One Health to minimize the risk of new varieties.

Molecular Epidemiological Investigation of Piroplasms and Anaplasmataceae Bacteria in Egyptian Domestic Animals and Associated Ticks.

Piroplasmosis and anaplasmosis are serious tick-borne diseases (TBDs) that are concerning for the public and animal health. This study aimed to detect the molecular prevalence and epidemiological risk factors of Piroplasma and Anaplasma species in animal hosts and their associated ticks in Egypt. A total of 234 blood samples and 95 adult ticks were collected from animal hosts (112 cattle, 38 sheep, 28 goats, 26 buffaloes, 22 donkeys, and 8 horses) from six provinces of Egypt (AL-Faiyum, AL-Giza, Beni-Suef, Al-Minufia, Al-Beheira, and Matruh). Blood and tick samples were investigated by polymerase chain reaction coupled with sequencing targeting 18S and 16S RNA genes for Piroplasma and anaplasmataceae, respectively. Statistical analysis was conducted on the potential epidemiological factors. Of the 234 animals examined, 54 (23.08%) were positive for pathogens DNA distributed among the six provinces, where 10 (4.27%) were positive for Piroplasma, 44 (18.80%) for anaplasmataceae, and 5 (2.14%) were co-infected. Co-infections were observed only in cattle as Theileria annulata and Anaplasma marginale plus Babesia bigemina, A. marginale plus B. bigemina, and T. annulata plus B. bigemina. Piroplasmosis was recorded in cattle, with significant differences between their prevalence in their tick infestation factors. Animal species, age, and tick infestation were the potential risk factors for anaplasmosis. All ticks were free from piroplasms, but they revealed high prevalence rates of 72.63% (69/95) with anaplasmataceae. We identified T. annulata, B. bigemina, and A. marginale in cattle; A. platys in buffaloes; A. marginale and A. ovis in sheep; for the first time, A. ovis in goats; and Ehrlichia sp. in Rhipicephalus annulatus ticks. Our findings confirm the significant prevalence of piroplasmosis and anaplasmosis among subclinical and carrier animals in Egypt, highlighting the importance of the government developing policies to improve animal and public health security.

Preparation and field study of combined vaccine against Clostridium perfringens type A and bovine viral diarrhea virus in camels.

PURPOSE: The key objective of this study was to formulate a local combined inactivated gel adjuvanted vaccine containing bovine viral diarrhea virus (BVDV)-1, BVDV-2 viruses and Clostridium perfringens type A toxoid. The study evaluated its ability to enhance protective active immune response in camels' calves against these infectious pathogens under field conditions. MATERIALS AND METHODS: The local BVDV cytopathic strains and a local strain of toxigenic C. perfringens type A were used in vaccines formulation. Vaccines A and B were monovalent vaccines against C. perfringens and both strains of BVDVs, respectively. While the vaccine C was the combined vaccine used against the three agents. All vaccines were adjuvanted with Montanide gel. Sterility, safety, and potency tests were applied on the formulated vaccines. Virus neutralization and toxin anti-toxin neutralization tests were used to evaluate the immune responses. RESULTS: Both monovalent (vaccine A) and combined vaccines (vaccine C) showed a protective level (4.5 and 3 IU/mL, respectively) against C. perfringens from the 2nd-week post-vaccination. The titer declined to 3 and 2 IU/mL, respectively at the 5th-month post-vaccination. The titer against BVDV, the monovalent vaccine (vaccine B) reached the beak (1.95 IU/mL) at the 1st-month post-vaccination and lasted till 6th-month post-vaccination (0.92 and 0.94 IU/mL) for BVDV-1a and BVDV-2, respectively. CONCLUSION: Vaccination of camels with the combined vaccine adjuvanted by Montanide gel containing C. perfringens type A toxoid and BVDV strains with 6-month intervals is recommended to protect camels safely and efficiently against such infections in the field.

Systemic Lupus Erythematosus with Crohn's Disease: A Rare Combination and Unusual Presentation.

The coexistence of systemic lupus erythematosus (SLE) and Crohn's disease (CD) is very rare. The usual sequence of occurrence is CD followed by SLE, where CD treatment with anti-tumour necrosis factor (anti-TNF) induces the latter. Here, we present a case of this rare combination but with sequence reversal. The patient was unresponsive to steroids and we achieved remission with infliximab. LEARNING POINTS: Crohn's disease complicating stable systemic lupus erythematosus is extremely rare.Although it may delay time to diagnosis, it is important to rule out other common causes such as infections and medication-induced colitis.If the patient is steroid unresponsive, infliximab might be a reasonable therapeutic alternative.

Molecular characterization of the 2018 outbreak of lumpy skin disease in cattle in Upper Egypt.

BACKGROUND AND AIM: Lumpy skin disease (LSD), an infectious disease of cattle, is characterized by raised nodules on the skin. Although the morbidity rate of LSD is low, it has a considerable fatality rate. Despite the annual mass vaccination of livestock with sheep pox vaccine (Veterinary Serum and Vaccine Research Institute, Egypt) enforced by Egyptian authorities, the LSD virus (LSDV) continues to circulate almost every summer. The present study aimed to discover the cause of cows naturally infected with LSDV circulating in Upper Egypt during the summer of 2018 using polymerase chain reaction (PCR) assay and to analyze their phylogenetics against reference genome sequences. MATERIALS AND METHODS: We cultured LSDV in specific pathogen-free embryonated chicken eggs (SPF-ECE) and used conventional PCR to identify fusion and P32 genes, previously deposited in GenBank (MN694826, MN694827, and MN954664). Sequencing and phylogenetic analyses were performed on these two highly conserved viral genes. RESULTS: LSDV infection of SPF-ECE resulted in characteristic white pock lesions. PCR products were identified on 1.5% agarose gel after electrophoresis at the expected positions for the fusion and P32 genes at 472 and 587 bp, respectively. CONCLUSION: The present study revealed that the two viral genes were identified from the Beni Suef and Sohag Governorates in all clinical cases and confirmed the circulation of LSDV in this outbreak. After sequencing, these genes were identical to those of the LSDV that had been identified and recorded in GenBank for the past 3 years.

Milk amyloid A as a biomarker for diagnosis of subclinical mastitis in cattle.

BACKGROUND AND AIM: Mastitis is one of the most vital noteworthy monetary risks to dairy ranchers and affects reproductive performance in dairy cattle. However, subclinical mastitis (SCM) negatively affects milk quality and quantity and associated with economic losses as clinical mastitis. It is recognizable only by additional testing. Somatic cell count (SCC) is currently used worldwide for the screening of intramammary infection (IMI) infections. However, somatic cells (SC) are affected by numerous factors and not always correlate with infection of the udder. Therefore, the aim of the present study was to evaluate the milk amyloid A (MAA) in the milk of normal and SCM cows and compare the sensitivity of both MAA secretion and SCC in response to mammary gland bacterial infection. MATERIALS AND METHODS: A total of 272 quarter milk samples collected from 68 Friesian cows after clinical examination for detection of clinical mastitis were employed in this study. All quarter milk samples (272) were subjected to bacteriological examination, while SCs were assessed in samples (220). Following SCC estimation and bacteriological examination, the apparently normal quarter milk samples were categorized into 7 groups and MAA concentration was estimated in normal and subclinical mastitic milk samples. RESULTS: Prevalence of clinical mastitis was 19.12 % (52 quarters), while 80.88 % (220 quarters) were clinically healthy with normal milk secretion. Of those 220 clinically healthy quarter milk samples, 72 (32.73%) showed SCM as detected by SCC (SCC ≥500,000 cells/ml). The most prevalent bacteria detected in this study were streptococci (48.53%), Staphylococcus aureus (29.41%), Escherichia coli (36.76%), and coagulase-negative staphylococci (11.76%). Results of MAA estimation revealed a strong correlation between MAA secretion level and SCC in agreement with the bacteriological examination. Interestingly, there was a prompt increase in MAA concentration in Group III (G III) (group of milk samples had SCC ≤200,000 cells/ml and bacteriologically positive) than Group I (G I) (group of milk samples with SCC ≤500,000 cells/ml and bacteriologically negative), as MAA concentration in G III was about 4 times its concentration in G I. CONCLUSION: Our study provides a strong evidence for the significance of MAA measurement in milk during SCM, and MAA is more sensitive to IMI than SCC. This can be attributed to rapid and sensitive marker of inflammation. The advantage of MAA over other diagnostic markers of SCM is attributed the minute or even undetectable level of MAA in the milk of healthy animals, it is not influenced by factors other than mastitis, and could be estimated in preserved samples. Therefore, we recommend that estimation of MAA concentration in milk is a more useful diagnostic tool than SCC to detect SCM and to monitor the udder health in dairy cattle.

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.