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This study aimed to evaluate the effect of various heating temperatures on the antioxidant activities of camel milk caseins. The samples were processed with three different heat treatments: Pasteurization at low and high temperatures and boiling. Fresh camel milk (unheated) was used as a control. Camel milk caseins were separated by fast ion exchange liquid chromatography (FPLC) and identified by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS page). The antioxidant activities of caseins were measu- red by three different in vitro methods: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2, 2'-azino-bis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) radical scavenging activity and ferric reducing power assay (FRAP). The antioxidant activity evaluated by the DPPH assay decreased significantly (p0.05) with the increase in heat treatment of caseins. However, there was no significant difference in ABTS radical scavenging activity and Ferric Reducing Antioxidant Power assay (FRAP) of heat-treated camel caseins compared to unheated onesStill, a decrease was observed in those activities by the increase of temperature in the different casein concentrations. Besides, whatever the concentration tested and the methods applied, the antioxidant activity of beta-casein (β-CN) was more pronounced than the alpha-casein (α-CN). Therefore, camel milk casein could be used as a natural source of antioxidants which may have a potential application in the food and nutraceutical industries. Throughout the different heat treatments applied, pasteurization at low temperature could be the most suitable alternative to preserve the antioxidant properties of camel milk.

Bacterial diseases cause tremendous economic losses due to high morbidity and mortality in livestock animals. F17A protein, the major subunit of F17 fimbriae, is one of the most prevalent and crucial virulence factors among the pathogenic Escherichia coli (E. coli) isolated from diarrheic and septicemic animals of various species. Purification and detection of this protein is regarded as an interesting field of investigation due to its important role as a therapeutic target, such as vaccines, and as a diagnostic tool. In this context, polyclonal rabbit antibodies recognizing F17A protein (anti−F17A antibody) were developed and used for its detection. In fact, sandwich biosensor using anti−F17A/gold nanoparticles conjugates as capture probe and anti−F17A antibody labelled with horseradish peroxidase as signal amplification probe was developed for electrochemical and fluorescent detection of purified F17A protein and live F17–positive E. coli bacteria. Good specificity and sensitivity for detection of F17–positive E. coli strains were obtained. The dynamic range for the biosensor varies from 1 × 102 to 1 × 109 CFU·mL−1 (R2 = 0.998) and the detection limit (LOD) and the IC50 value were estimated to be 37 CFU·mL−1 and 75 CFU·mL−1, respectively.

Background: Diarrhea caused by Escherichia coli is a major cause of morbidity and mortality in young animals. Few treatment options are available, mainly antibiotic therapy increasingly limited by resistance to commonly used drugs. Objectives: The aim of this work was to develop immunotherapy based on the use of camel VHH antibody fragments, or nanobodies, to target pathogenic E. coli surface antigens. Material and methods: We immunized a camel with a killed strain we had previously isolated from a diarrheic camel calf and identified as expressing the F17 fimbriae antigen Results: The immunized animal developed an anti-E.coli immune response including heavy-chain antibodies. Lymphocytes from this animal were purified and RNA isolated to create a VHH library by phage display with a size of about 109 individual transformants. Panning on live E. coli cells resulted in the isolation of VHH fragments specific to the cell surface antigens. Conclusion: The identification of these antigens can lead to the development of new diagnostic and therapeutic tools against diarrhea.

An impedance-based DNA multiplexed biosensor was designed to simultaneously detect Escherichia coli (yaiO gene) and its virulent f17 variant. The thiolated DNA dual probe was self-assembled onto the surface of the gold nanoparticle-modified screen-printed carbon electrode (AuNPs/SPCE) to recognize selected sequences from yaiO and f17 genes. The optimal conditions to prepare the bioelectrode were determined based on the monitoring of the impedimetric response fitted to an equivalent electrical circuit model. The charge transfer resistance of the bioelectrode increased by recognizing the target DNA sequences. The limit of detection was 0.8 fM and 1.0 fM for yaiO and f17 target DNA, respectively, and the linearity ranged from 1 × 10−15 to 1 × 10−7 M with a linear regression coefficient R ≥ 0.995. The nanodevice provided a novel strategy for simultaneous detection of E. coli and its virulence f17 gene with excellent discrimination with a single-base mismatch, two-base mismatch, and non-complementary sequences. Moreover, genomic DNA extracted from E. coli bacteria isolated from diarrheic camel calves and control animals in Tunisia was successfully detected using the as-prepared biosensor with minimal treatment of the extracted DNA samples. Graphical abstract

In this paper, we focus on the camelid nanobodies as a revolutionary therapy that can guide efforts to discover new drugs for Coronavirus disease (COVID-19). The small size property makes nanobodies capable of penetrating efficiently into tissues and recognizing cryptic antigens. Strong antigen affinity and stability in the gastrointestinal tract allow them to be used via oral administration. In fact, the use of nanobodies as inhalant can be directly delivered to the target organ, conferring high pulmonary drug concentrations and low systemic drug concentrations and minimal systemic side effects. For that, nanobodies are referred as a class of next-generation antibodies. Nanobodies permit the construction of multivalent formats that may achieve ultra-high neutralization potency and then may prevent mutational escape and can neutralize a wide range of SARS-CoV-2 variants. Due to their distinctive characteristics, nanobodies can be of great use in the development of promising treatment or preventive strategies against SARS-CoV-2 infection. In this review, the state-of-the-art of camel nanobodies design strategies against the virus including SARS-CoV-2 are critically summarized. The application of general nanotechnology was also discussed to mitigate and control emerging SARS-CoV-2 infection.

Healthy animals can constitute a reservoir for

This study was conducted to determine the prevalence of virulence genes, serogroups, antimicrobial resistance and phylogenetic groups of Escherichia coli strains isolated from diarrheic and healthy camel calves in Tunisia. From 120 fecal samples (62 healthy and 58 diarrheic camel calves aged less than 3 months), 70 E. coli isolates (53 from diarrheic herds and 17 from healthy herds) were examined by PCR for detection of the virulence genes associated with pathogenic E. coli in animals. A significantly greater frequency of the f17 gene was observed in individual camels and in herds with diarrhea, this gene being found in 44.7% and 41.5% of isolates from camels and herds with diarrhea versus 22.5% and 11.7% in camels (p=0.05) and herds without diarrhea (p=0.02). The aida, cnf1/2, f18, stx2 and paa genes were found only in isolates from camels with diarrhea, although at a low prevalence, 1.8%, 3.7%, 1.8%, 3.7% and 11.3%, respectively. Prevalence of afa8, cdtB, eae, east1, iroN, iss, kpsMTII, paa, sfa, tsh and papC genes did not differ significantly between herds with or without diarrhea. Genes coding for faeG, fanC, f41, estI, estII, CS31a and eltA were not detected in any isolates. All isolates were sensitive to amikacin, chloramphenicol, ciprofloxacin, gentamicin and ceftiofur and the highest frequency of resistance was observed to tetracycline, and ampicillin (52.8% and 37.1% respectively). The phylogenetic groups were identified by conventional triplex PCR. Results showed that E. coli strains segregated mainly in phylogenetic group B1, 52.8% in diarrheic herds and 52.9% in healthy herds.

In many mammalian species, newborns are agammaglobulinemic; thus, colostrum and milk are the main sources of early protective antibodies. These antibodies are produced in the mother's serum and transferred to mammalian glands a few days before parturition. Here, we have studied the transfer of immunity from a she-camel immunized with human serum albumin (HSA) to her calf via colostrum and milk. Our results show that HSA-specific antibodies are produced in the mother's serum and are subsequently transferred to her colostrum. These specific antibodies are then transferred by suckling to the calf. The calf serum did not contain HSA-reactive antibodies at parturition and before the first feed, after suckling, a rise in reactivity was observed peaking at 24 h postpartum. The involvement of heavy chain antibodies (HCAbs) in the process of immunity transfer was also examined, and it was found that they were also transferred from the colostrum to the calf serum like conventional antibodies.

The aim of this work was to assess maternal and neonatal changes in plasma proteins, glucose and cortisol and to quantify the colostral immunoglobulin G (IgG) transfer in the peri-partum period in D'man sheep, a prolific breed, taking into account the parity of the ewe. The concentrations of proteins and glucose were high in the ewes on day 7 and at lambing before decreasing. Likewise, cortisol plasma concentration was maximal during the 6 h following lambing and dropped at 12 h. Protein and glucose concentrations were low in lambs at 1 h of birth after which they increased. By contrast, cortisol level was the highest during the first 12 h of birth and then decreased. The colostral IgG level was high at lambing and dropped by over 87 % from 1 to 48 h post-partum. In the newborn, the plasma IgG concentration was lowest at birth and increased rapidly during the first 24 h of birth. Parity influenced maternal physiology with multiparous ewes having the lowest concentrations of proteins, glucose, IgG and cortisol, but the highest colostrum IgG level. Accordingly, lambs born from primiparous ewes had lower protein, glucose and plasma IgG concentrations than lambs born from multiparous ewes. The main outcome of this study was that lambs born from primiparous ewes are characterized by the lowest physiological indices and this may influence their survival chance.

The survival and replication of Brucella in macrophages is initially triggered by a low intraphagosomal pH. In order to identify proteins released by Brucella during this early acidification step, we analyzed Brucella suis conditioned medium at various pH levels. No significant proteins were released at pH 4.0 in minimal medium or citrate buffer, whereas in acetate buffer, B. suis released a substantial amount of soluble proteins. Comparison of 13 N-terminal amino acid sequences determined by Edman degradation with their corresponding genomic sequences revealed that all of these proteins possessed a signal peptide indicative of their periplasmic location. Ten proteins are putative substrate binding proteins, including a homologue of the nopaline binding protein of Agrobacterium tumefaciens . The absence of this homologue in Brucella melitensis was due to the deletion of a 7.7-kb DNA fragment in its genome. We also characterized for the first time a hypothetical 9.8-kDa basic protein composed of five amino acid repeats. In B. suis , this protein contained 9 repeats, while 12 were present in the B. melitensis orthologue. B. suis in acetate buffer depended on neither the virB type IV secretory system nor the omp31 gene product. However, the integrity of the omp25 gene was required for release at acidic pH, while the absence of omp25b or omp25c displayed smaller effects. Together, these results suggest that Omp25 is involved in the membrane permeability of Brucella in acidic medium.

The aim of this study was to compare the physicochemical parameters of milk samples of five different species: cow, goat, donkey, camel and human. Also the analysis of whey protein profile in different milk samples was performed by anion-exchange fast protein liquid chromatography (FPLC) while polyacrylamide gel electrophoresis was used to identify a single fraction. Camel milk was the most acid (pH 6.460±0.005) and the richest in total proteins (3.41±0.31 %) and ash (0.750±0.102 %), whereas donkey milk had a neutral pH (7.03±0.02) and characterised by low proteins (1.12±0.40 %) and fat (0.97±0.03 %) content, being very close to human milk. Proteomic analysis of cow, goat, donkey, camel and human milk highlighted significant interspecies differences. Camel milk was similar to human milk in lacking of β-lactoglobulin and richness of α-lactalbumin. The knowledge gained from the proteomic comparison of the milk samples analysed within this study might be of relevance, both, in terms of identifying sources of hypoallergenic alternatives to bovine milk and detection of adulteration of milk samples and products., Cilj ovog istraživanja bio je usporediti fizikalno-kemijska svojstva 5 različitih vrsta mlijeka - kravljeg, kozjeg, magarećeg, devinog i majčinog. Primjenom ionske izmjene i brzo-proteinske tekućinske kromatografije (FPLC) provedena je i analiza profila proteina sirutke, dok je pomoću poliakrilamid gel elektroforeze identificirana svaka pojedina frakcija. Devino mlijeko imalo je najveću kiselost (pH 6,460±0,005) te najviše udjele proteina (3,41±0,31 %) i mineralnih tvari (0,750±0,102 %). S druge strane, magareće mlijeko imalo je pH vrijednost u neutralnom području (7,03±0,02) te niske udjele proteina (1,12±0,40 %) i mliječne masti (0,97±0,03 %) uslijed čega je bilo najsličnije majčinom mlijeku. Proteomička analiza uzoraka kravljeg, magarećeg, devinog i majčinog mlijeka rezultirala je značajnim razlikama ovisno o vrsti mlijeka. Devino mlijeko pokazalo se vrlo slično majčinom zbog odsutnosti frakcije β-laktoglobulina te visokog udjela α-laktalbumina. Rezultati dobiveni proteomičkom analizom različitih vrsta mlijeka mogu poslužiti za pronalaženje hipoalargenih alternativa kravljem mlijeku, ali i kao pomoć pri određivanju patvorenja mlijeka i mliječnih proizvoda.

International audience; Ovarian cancer has the highest mortality rate among gynecologic malignancies. The monoclonal antibody 12G4 specifically recognizes the human Mullerian inhibiting substance type II receptor (MISRII) that is strongly expressed in human granulosa cell tumors (GCT) and in the majority of human epithelial ovarian cancers (EOC). To determine whether MISRII represents an attractive target for antibody-based tumor therapy, we first confirmed by immunohistochemistry with 12G4 its expression in all tested GCT samples (4/4) and all, but one, EOC human tissue specimens (13/14). We then demonstrated in vitro the internalization of 12G4 in MISRII(high)COV434 cells after binding to MISRII and its ability to increase the apoptosis rate (FACS, DNA fragmentation) in MISRII(high)COV434 (GCT) and MISRII(medium)NIH-OVCAR-3 (EOC) cells that express different levels of MISRII. A standard (51)Cr release assay showed that 12G4 mediates antibody-dependent cell-meditated cytotoxicity. Finally, in vivo assessment of 12G4 anti-tumor effects showed a significant reduction of tumor growth and an increase of the median survival time in mice xenografted with MISRII(high)COV434 or MISRII(medium)NIH-OVCAR-3 cells and treated with 12G4 in comparison to controls treated with an irrelevant antibody. Altogether, our data indicate that MISRII is a new promising target for the control of ovarian GCTs and EOCs. A humanized version of the 12G4 antibody, named 3C23K, is in development for the targeted therapy of MISRII-positive gynecologic cancers.

The main objective of this study was to demonstrate the effect of seminal plasma ovulation inducing factor (OIF) on ovulation in female camels. Seminal plasma was fractionated using gel filtration chromatography, and two protein peaks were obtained and analysed by western blotting. The effect of the bioactive protein fraction (OIF/β-NGF) was tested by intramuscular injection (1 ml) in synchronized females with the following treatments: PBS (negative control group, n = 3); 20 µg Buserelin (GnRH analogue, positive control group; n = 3); and purified OIF with doses of 1 and 2 mg (n = 5, each treatment). Blood samples were collected every two days from day 0 until 14 days post-treatment, and the progesterone concentration was assessed. The obtained results showed that the OIF is highly present in the seminal plasma of dromedary camels as a protein with a molecular mass of approximately 14 kDa. It was detected as a beta-Nerve Growth Factor, named Cam-β-NGF. The effect of this molecule on ovulation was clearly demonstrated by the significant increase of the plasma progesterone concentration in the treated female groups (1 and 2 mg of Cam-β-NGF) as observed in Buserelin group. In summary, intramuscular injection of β-NGF isolated from dromedary camel seminal plasma induces ovulation in females with similar rate to Buserelin treatment.

International audience; Natural killer (NK) cells are at the crossroad between innate and adaptive immunity and play a major role in cancer immunosurveillance. NK cell stimulation depends on a balance between inhibitory and activating receptors, such as the stimulatory lectin-like receptor NKG2D. To redirect NK cells against tumor cells, we designed bifunctional proteins able to specifically bind tumor cells and to induce their lysis by NK cells, after NKG2D engagement. To this aim, we used the 'knob into hole' heterodimerization strategy, in which 'knob' and 'hole' variants were generated by directed mutagenesis within the CH3 domain of human IgG1 Fc fragments fused to an anti-CEA or anti-HER2 scFv or to the H60 murine ligand of NKG2D, respectively. We demonstrated the capacity of the bifunctional proteins produced to specifically coat tumor cells surface with H60 ligand. Most importantly, we demonstrated that these bifunctional proteins were able to induce an NKG2D-dependent and antibody-specific tumor cell lysis by murine NK cells. Overall, the results show the possibility to redirect NK cytotoxicity to tumor cells by a new format of recombinant bispecific antibody, opening the way of potential NK cell-based cancer immunotherapies by specific activation of the NKG2D receptor at the tumor site.

Anti-Müllerian hormone (AMH) [also called Müllerian inhibiting substance (MIS)] is a member of the transforming growth factor-β family. AMH and its type II receptor (AMHR-II) are involved in the regression of the Müllerian ducts in the male embryo, and in gonadal functions in the adult. AMH is also known to be a marker of granulosa and Sertoli cell tumours. We selected a high-affinity monoclonal antibody, mAb 12G4, specific for human AMHR-II (hAMHR-II), by FACS analysis, Western blotting and immunohistochemical staining of a hAMHR-II-transfected CHO (Chinese hamster ovary) cell line, normal adult testicular tissue and granulosa cell tumours. Using peptide array screening, we identified the binding sequences of mAb 12G4 and AMH on the receptor. Identification of Asp53 and Ala55 as critical residues in the DRAQVEM minimal epitopic sequence of mAb 12G4 definitively accounted for the lack of cross-reactivity with the murine receptor, in which there is a glycine residue in place of an aspartic acid residue. In a structural model, the AMH-binding interface was mapped to the concave side of hAMHR-II, whereas the mAb 12G4-binding site was located on the convex side. mAb 12G4, the first mAb to be raised against hAMHR-II, therefore has unique properties that could make it a valuable tool for the immunotargeting of tumours expressing this receptor.

Impairment of the omp25 gene in Brucella spp. leads to attenuated strains and confers protection to the host. Omp25 and Omp31, whose functions remain unknown, were the first characterized members of group 3 outer membrane proteins (Omps) (25 to 34 kDa). Recently, genomic and proteomic approaches identified five new putative members of this family, some of which are produced in B. melitensis or B. abortus . In the present study, using protein microsequencing, we identified new members of group 3 Omps proteins produced in B. suis . Since several monoclonal antibodies (MAbs) against Omp25 cross-reacted with other members of group 3 Omps, we also performed Western immunoblotting to compare wild-type B. suis with mutants systematically having B. suis omp25 -related genes knocked out. We demonstrate the production of three paralogs of Omp31 and/or Omp25 in B. suis , and the existence of a common site of signal peptide cleavage (AXAAD), which is very similar to that present in the five homologous Omps of Bartonella quintana . The seven group 3 Omps were classified in four-subgroups on the basis of percentage amino acid sequence identities: Omp25 alone, the Omp25b-Omp25c-Omp25d cluster, the Omp31/31b subgroup, and the less related Omp22 protein (also called Omp3b). Together with previous data, our results demonstrate that all new members of group 3 Omps are produced in B. suis or in other Brucella species and we propose a nomenclature that integrates all of these proteins to facilitate the understanding of future Brucella interspecies study results.

Often diagnosed at an advanced stage, ovarian cancer is responsible for the highest mortality rate among patients with gynecologic malignancies. Due to the limitations of the current therapeutic approaches, there is a strong need for novel, more efficient, therapies, among which targeted immunotherapy. The monoclonal antibody 12G4 specifically recognizes the human Müllerian Inhibiting Substance type II Receptor (MISRII). Expressed at high levels on most human Granulosa Cell Tumors (GCT) as well as on a majority of human epithelial ovarian cancer (EOC) cells, MISRII represents a good target for antibody-based tumor targeting. Characterization of murine antibody 12G4 has been previously reported, in particular epitope mapping and in vitro assays showing that 12G4 binding does not affect MIS interaction with MISRII. In the present study we further assess 12G4 activity in vitro and in vivo. 12G4 was shown to induce 37% and 30% apoptosis in vitro (FACS, DNA fragmentation) on the two MISRII expressing cell lines cov434-MISRII and OVCAR3, respectively. Internalization of the antibody-MISRII complex was also observed in vitro on cov434-MISRII. Using 12G4 antibody we confirmed by immunohistochemistry the expression of the MISRII target in all GTC samples and all but one EOC tissue sections (13/14). Furthermore, we have developed several immunodeficient Nude mouse models of ovarian tumor xenografts expressing human MISRII. In these models the efficacy of 12G4 could be clearly demonstrated with a significant effect on tumor growth. According to our data, MISRII, expressed on a majority of EOCs appears as a good target for ovarian cancer immunotherapy. Furthermore, based on the good efficacy observed in vivo for 12G4, the development of a humanized anti-MISRII antibody, derived from 12G4, has been initiated and represents a new promising targeted therapeutic approach. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4568. doi:10.1158/1538-7445.AM2011-4568