Nasr-Eldin M. Aref

Design and characterization of highly immunogenic heat-stable enterotoxin of enterotoxigenic Escherichia coli K99+

In this study, STa peptide of enterotoxigenic Escherichia coli K99(+) was purified and successfully covalently cross-linked to modified bovine serum albumin after thorough evaluation of three different hapten-carrier conjugation protocols. Dimethyformamide (DMF) based STa-conjugation protocol demonstrated higher biological activity (10×10(6) STa Total Mouse Units [MU]) and 100% conjugation efficiency. A range of conjugation ratio of 4-12 STa molecules per one molecule of BSA was achieved and confirmed by matrix-assisted laser desorption ionization-time of flight/mass spectroscopy (MALDI-TOF/MS). This conjugate was used for immunization of ten rabbits for STa antibody production. A high antibody binding titer (10(6)) against STa was obtained with a neutralization capacity of 3×10(4) STa MUs/ml serum. These levels of high STa binding and neutralizing antibodies titers propose the potential use of this conjugate for the development of immunotherapeutic reagents and/or STa-based vaccine against ETEC K99(+).

Generation of high-titer of neutralizing polyclonal antibodies against heat-stable enterotoxin (STa) of enterotoxigenic Escherichia coli

In this study, polyclonal antibodies with high titer and avidity to native heat-stable enterotoxin (STa) of enterotoxigenic Escherichia coli (ETEC) have been generated and evaluated for their neutralizing effect in STa-induced enterotoxic animal model. Native STa was purified to homogeneity and coupled to modified bovine serum albumin (MBSA) using dimethylformamide (DMF)-based conjugation protocol. STa conjugate was used for immunization of female New Zealand white rabbits. The humoral immune response of the rabbits against native STa was monitored and evaluated for its antibody binding and neutralization capacity by ELISA and suckling mouse assay, respectively. After three subsequent boosts by STa conjugate, the animals were capable of eliciting high levels of STa-antibody binding titer (10(6)) and STa-neutralizing antibody capacity (3×10(4) mouse units of STa/ml serum). STa antibody maturation (avidity) was improved dramatically after multiple boosters with the STa conjugate. Comparison of the avidity of STa antibodies demonstrated that the strength in the STa antibody avidity developed in time corresponding to the development of the STa-neutralizing and binding titers. High avid STa antibodies (48.21% avidity index) were demonstrated 24 weeks post immunization (PI). However, differences in the onset of STa antibody production were noticed among animals and may need further investigation.

Novel Heat-Stable Enterotoxin (STa) Immunogen Based on Cationic Nanoliposomes: Preparation, Characterization and Immunization

The novelties encountered in the field of formulation have provided promising solutions for the problematic vaccine development of hapten molecules. In the current study, the novel preparation of a cationic nanoliposomal immunogen of the heat stable enterotoxin (STa) was reported. STa was produced from clinically ETEC isolate of diarrheic neonatal calves and purified using RP-HPLC. STa was loaded into the cationic vesicles which were characterized for their particle size, surface charge, morphology, STa loading, and stability. The STa loaded cationic nanoliposome was used for mice immunization and the generation of STa antibody was monitored using ELISA. Results displayed the spherical nature of the STa loaded vesicles, their suitable size and homogeneity represented by a particle size of 228.1 nm and a PDI of 0.202. The surface charge of the STa nanoliposomes was +29.9, demonstrating sufficient stability during refrigeration storage. The STa loaded cationic nanoliposome was able to elicit specific STa antibody response, and to confer effective protection against STa challenge in mice. The STa antibody binding titer and neutralization capacity were 105 and 104 mouse units/ml serum, respectively. The developed system is a one-step procedure, which overcomes the disadvantage of the complexity of generation of the hapten-carrier conjugate. In conclusion, the developed STa-cationic nanoliposomal immunogen is feasible and has the potential to improve effectiveness against ETEC, suggesting its applicability in preventing the harmful effects of ETEC infection in neonatal calves.

An enhanced protocol for expression and purification of heat‐stable enterotoxin of enterotoxigenic Escherichia coli

We present an improved protocol for expression and purification of heat‐stable enterotoxin (STa) of enterotoxigenic Escherichia coli (ETEC). In this protocol, controlled growth conditions at different pHs (7.4, 8.0, and 8.6) were adopted using a bioreactor. In addition, specific adsorbent resins, methacrylate, were used for STa purification. The bioreactor provided optimal ETEC growth at pH 7.4 with high STa production. Furthermore, methacrylate bounded specifically to STa and dramatically enhanced the purification process of STa. The STa‐specific activity was high (8.9 × 106 units/mg protein), and the minimal effective dose of STa required for production of gut weight to remaining body weight ratio ≥ 0.083 was recorded as less than 0.2 ng in 2–3 days old suckling mice. The protocol presented, produces highly purified STa as documented by matrix‐assisted laser desorption ionization‐time of flight mass spectroscopy/. Also, as compared with the traditional methods, this procedure is trouble‐free and practical for scale‐up production and purification of STa peptides.

Construction and immunogenicity analysis of nanoparticulated conjugate of heat-stable enterotoxin (STa) of enterotoxigenic Escherichia coli

The ultimate goal of this research was to overcome the low immunogenicity of the biological macromolecule (heat stable enterotoxin STa) via its conjugation to biodegradable PLGA nanoparticles (NP). STa was first isolated from Enterotoxigenic Escherichia coli (ETEC), purified and identified using reported HPLC procedures. Optimized homogenous PLGA NP, prepared using the nanoprecipitation technique were used for conjugating STa using the carbodiimide synthesis. Covalent binding of STa to PLGA NP was confirmed via FTIR and 1HNMR analysis. Safety and tolerability of the developed nanoparticulated STa-PLGA conjugate were confirmed by MTT assay on A549 lung cancer cells. After subcutaneous immunization, STA-PLGA NP conjugate induced a significant immune response in mice showing a strong binding and neutralizing antibody titer. The developed novel STa-PLGA NP conjugate is expected to provide promising protection against enterotoxigenic Escherichia coli (ETEC).

Clinical and ultrasonographic observations of functional and mechanical intestinal obstruction in buffaloes (Bubalus bubalis)

Aim: This study was designed for clinical and laboratory evaluation of intestinal obstruction (IO) in buffaloes (Bubalus bubalis) with special emphasis on the diagnostic value of ultrasonographic findings. Materials and Methods: A total number of 30 buffaloes were included in the study and divided into 2 groups: Healthy (n=10) and diseased group (n=20). Diseased buffaloes were admitted to the Veterinary Teaching Hospital at Assiut University, Egypt, with a history of anorexia, abdominal pain, various degrees of abdominal distention, and absence or presence of scanty mucoid faces. These animals were subjected to clinical and ultrasonographic as well as laboratory examinations. Results: Based on ultrasonographic findings, various forms of IO were diagnosed. Functional obstruction, paralytic ileus, was diagnosed in 17 cases (85%) while mechanical IO was diagnosed only in 3 cases (15%). Out of 17 cases of paralytic ileus, both proximal and distal ileuses were successfully imaged in 8 and 9 cases, respectively. Proximal ileus was imaged from the right dorsal flank region as a single dilated loop of diameter >6 cm, while distal ileus was imaged as multiple dilated loops of diameter <6 cm. Mechanical obstruction due to duodenal intussusception was visualized as two concentric rings with outer echogenic wall and hypoechoic lumen. All cases of IO showed leukocytosis, hypoproteinemia, and increased activity of alkaline phosphatase and aspartate aminotransferase. Conclusion: Ultrasonography proved to be an essential tool for diagnosis and differential diagnosis of various forms of IO in buffaloes.

Hepatobiliary diseases in buffalo (Bubalus bubalis): clinical, laboratory, and ultrasonographic findings

This study describes ultrasonographic observations of five hepatobiliary diseases in buffalo (Bubalus bubalis). Fifty buffalo, including 20 clinically normal and 30 hepatobiliary diseased buffalo were enrolled in the study. Complete clinical, radiographic and ultrasonographic examinations and laboratory analyses were conducted. Focal parenchymal lesions including liver abscess (n = 12) and hepatic cyst (n = 6), diffuse parenchymal lesion (hepatobiliary cirrhosis, n = 5) and obstruction of hepatobiliary passages including cholestasis (n = 4), and hepatocholelithiasis (n = 3) were successfully imaged by ultrasonography. Hepatic abscess imaged as a hypoechoic to echogenic circumscribed mass of various diameters with a distinct echogenic capsule. Hepatic cyst imaged as a pear-shaped sac with a bright echogenic margin, anechoic content, and distal acoustic enhancement. In hepatobiliary fibrosis, the liver showed linear bands of increasing echogenicity with less distinct imaging of the portal vasculature. Cholestasis was imaged as dilatation of the gallbladder (GB) with wall thickening and homogeneous or heterogeneous contents. Hepatocholelithiasis imaged as an echoic structure within the hepatic parenchyma, or within and around the GB and bile duct, with more echogenicity of the hepatic parenchyma than normal. Ultrasonography can be an efficient rapid, noninvasive tool for screening of common hepatobiliary diseases in buffalo under field conditions.

New insights on ill-thriftiness in early-weaned buffalo calves.

Aim: The present study was designed to: (1) Investigate the effect of weaning time on various metabolic indices and growth pattern in buffalo calves compared to cow calves under field condition and (2) Shed light on the potential relationship between early weaning, growth metabolites, and suboptimal growth (ill-thrift) in buffalo calves. Materials and Methods: A total number of 18 neonatal calves of both sexes and species (cattle and buffalo) were included in the study. Animals were divided into three groups according to their age at weaning as following: Cow calves (n=8) weaned at 4.5 months, buffalo calves (n=6) weaned at 3.5 months (early-weaned), and buffalo calves (n=4) weaned at 5.5 months (late-weaned). Morphological traits, growth metabolites, and hormonal profile were measured at monthly interval over the period of the study and around the time of weaning (2 weeks pre- and post-weaning). Results: The obtained results showed that the trend of growth pattern was significantly increased in a linear pattern in cow calves and late-weaned buffalo calves, whereas early-weaned buffalo calves showed sharp decline in their body weight (BW) post-weaning. By the end of the study, early-weaned buffalo calves showed the lowest BW gain (ill-thrift). There is a positive association between the morphological traits and various growth metabolites and hormonal indices. A significant decrease (p<0.05) in the concentrations of growth hormones (insulin-like growth factor-1 [IGF-1] and insulin) and other metabolites were reported in early-weaned buffalo calves compared to other animals. There is no association between stress indices (cortisol level and neutrophil to lymphocyte ratio) and growth rate. Conclusion: Suboptimal growth rate (ill-thriftiness) is common in early-weaned buffalo calves and is attributed to low blood levels of growth metabolites, in particularly, IGF-1. In addition, the strong positive associations between concentrations of IGF-1 and morphological characters of growth suggest that IGF-1 is a reliable indicator for assessing metabolic status of individual calves.