Development of gold nanoparticles-lateral flow test as a novel field diagnostic assay for detecting foot-and-mouth disease and lumpy skin disease viruses.

Background and Objectives: Rapid diagnosis is a cornerstone for controlling and preventing viral disease outbreaks. The present study is aimed to develop a rapid field diagnostic test based on gold nanoparticles for the detection of lumpy skin diseases (LSD), and foot and mouth diseases (FMD) in animals with high sensitivity and specificity. Materials and Methods: FMD and LSD vaccines were used as a source of viruses' antigens for preparing monoclonal antibodies and conjugated with gold nanoparticles that characterized using various techniques such as UV-visible spectrometry, and transmission electron microscopy (TEM). Monoclonal antibodies (mAbs) for each serotype produced in experimental rats and used to capture antibodies for FMDV and/or LSDV. ELISA was used to screen 469 milk samples and 1165 serum samples from naturally infected cattle, buffaloes, sheep, and goats for validation of the lateral flow test (LFT). LSDV DNA was extracted from 117 blood and skin biopsy samples collected from naturally infected cattle during the 2019 outbreak. Results: The specificity and sensitivity of GNP-LFT were evaluated and compared to Ag-ELISA, Western blot tests (WB), and PCR. A total of 95 FMDV positives out of 469 (20.25%) milk samples and 268 FMDV positives out of 1165 (23.3%) serum samples from natural infected cattle, buffaloes, sheep, and goats examined by ELISA to valid GNPS-LFT Viral LSDV DNA was detected in 60/117 (51.5%) and 31/60 (52.9%). While the GNPS-LFT assay results were 49/117 (41.9%) and 29/60 (48.3%) blood and skin biopsy samples, respectively. The diagnostic sensitivity and specificity of the GNP-LFT test were 72% and 82%, respectively. All vesicular fluid and epithelium samples collected from infected animals were identified as positive by the GNP-LFT and Ag-ELISA. Ag-ELISA, on the other hand, was 90% and 100%. While the developed GNP-LFT used LSDV polyclonal antibodies were similar to ELISA and IgG-WB with a sensitivity of 72.8% and a specificity of 88.8%, respectively. Conclusion: The GNPS-LFT is a novel immunoassay based on mono or polyclonal antibodies conjugated with gold nanoparticles that provides an accurate, rapid, specific, and sensitive tool for field rapid diagnosis of FMDV and LSDV.

Detection of lumpy skin disease virus in cattle using real-time polymerase chain reaction and serological diagnostic assays in different governorates in Egypt in 2017.

BACKGROUND AND AIM: Lumpy skin disease (LSD), is a highly infectious viral disease of cattle, caused by LSD virus (LSDV) which belongs to the genus Capripoxvirus of family Poxviridae. In the summer of 2017, skin lesions suggestive of LSD were observed in cattle at several governorates in Egypt. This study aimed to detect LSDV in cattle specimens using rapid serological and molecular diagnostic assays. MATERIALS AND METHODS: A total of 46 skin biopsies and uncoagulated blood samples were collected from cattle with LSD suggestive clinical signs, as well as 290 coagulated whole blood samples from cattle without skin lesion in different governorates in Egypt during the summer of 2017. Skin biopsies were used for virus isolation from the chorioallantoic membrane of 11-day-old specific pathogen-free embryonated chicken eggs (SPF-ECEs). LSDV was identified using conventional polymerase chain reaction (PCR), real-time PCR (RT-PCR), and fluorescent antibody technique (FAT) with specific hyperimmune serum against LSDV. Cattle sera were examined using indirect FAT (IFAT) and indirect enzyme-linked immunosorbent assay (ELISA). RESULTS: Skin nodules and sitfast lesions were significant clinical signs observed in all LSD suspect cattle. SPF-ECEs, from which positive isolations were made and it showed characteristic inflammatory and focal white pock lesions. The isolated viruses were identified as LSDV by FAT, conventional gel-based PCR, and RT-PCR. Among the skin biopsies and corresponding blood samples, LSDV-positive samples percentage were 39.13 and 36.95 by RT-PCR, followed 34.78 and 28.26 by conventional PCR and then 32.6 and 26.8 by FAT, respectively. The total positive percentage of LSDV antibody detected in cattle serum samples were 17.93 and 14.48 by indirect ELISA and IFAT. CONCLUSION: LSDV was detected and identified in skin biopsies and corresponding blood samples of naturally infected cattle, more LSDV-positive samples were detected by RT-PCR, followed by conventional PCR and then FAT. The indirect ELISA detected more antibody-positive samples than the IFAT from cattle serum samples. The RT-PCR assay is simple, sensitive, rapid, and reliable for the detection of LSDV in blood and skin nodule biopsies of suspected cattle.

Isolation and identification of bacteria causing mastitis in small ruminants and their susceptibility to antibiotics, honey, essential oils, and plant extracts.

AIM: The present work aims to isolate and identify bacteria that cause mastitis in small ruminants and evaluates the antibacterial activity of some antibiotics, honey, essential oils, and plant extracts. MATERIALS AND METHODS: A total of 289 milk samples were collected from udder secretions of sheep (n=189) and goat (n=100) from El-Fayoum, Beni-Suef, and Giza governorates. Screening subclinical mastitis (SCM) was done using California Mastitis Test (CMT); identification of the isolates was achieved using Gram's staining, hemolytic pattern, colony morphology, and biochemical tests using Analytical Profile Index. RESULTS: On clinical examination, the incidence of clinical mastitis (CM) was found to be 5.88% and 7% in sheep and goat, respectively. On CMT, SCM was found to be 25 (13.23%) and 11 (10%) in sheep and goat, respectively. Bacteriological examination of all milk samples found the presence of Staphylococcus aureus (SA) (31.1%), coagulase-negative staphylococci (CNS) (19.5%), Escherichia coli (EC) (8.3%), Streptococcus spp. (5.6%), Klebsiella spp. (3.77%), and Pseudomonas spp. (1.89%), while no bacteria were cultured from 81.66% of the samples. Identification of 9 isolates of CNS was achieved by using API staph test to Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus cohnii, and Staphylococcus saprophyticus. The highest bacterial resistance was found in EC (67.14%) followed by Kp (45.28%) and SA (26.57%). CONCLUSION: Onion and black cumin essential oils followed by Egyptian honey showed strong antibacterial effects against multidrug-resistant bacteria. Finally, our study proved that Egyptian honey, onion, and black cumin essential oils have a marked strong antibacterial effect against bacteria isolated from small ruminant mastitis, but still further extensive studies are needed to discover the therapeutic properties of these plant extracts and honey.