Development and validation of a pentaplex assay for the identification of antibodies against common viral diseases in cattle.

Animal welfare and economic implications of infectious diseases in cattle demand an efficient surveillance as the foundation for control and eradication programmes. Bovine respiratory syncytial virus (BRSV), Parainfluenza virus type 3 (PI3V), Bovine herpes virus-1 (BoHV-1), Bovine viral diarrhoea virus (BVDV), and Enzootic bovine leukosis virus (EBLV) cause common and often underdiagnosed diseases in cattle that are endemic in most countries [1]. A hallmark of individual exposure to a viral pathogen is the presence of antibodies directed towards that virus. The aim of this study was to develop and validate a pentaplex assay to simultaneously detect and quantify antibodies against BRSV, PI3V, BoHV-1, BVDV and EBLV in serum, as an efficient tool to yield epidemiological data. Monoplex assays were initially developed using either complete BRSV or BoHV-1 viral lysates, or recombinant proteins for BVDV, EBLV or PI3V as capture antigens. In addition, 125 serum samples from unvaccinated cattle, which were classified as positive or negative for each of the viruses by commercial ELISA kits, were used for validation. Conditions established for the Luminex monoplex assays were adopted for the pentaplex assay. The accuracy, determined by the area under the ROC curve, was greater than 0.97, and assay diagnostic sensitivities and specificities were over 95 and 90%, respectively, for all antigens. Intra (r) and interassay (R) coefficients of variation were under 10 and 20 %, respectively. Selectivity towards target viruses was shown by binding inhibition assays where unbound viruses reduced fluorescence intensities. Diagnostic agreement for samples analysed simultaneously in the monoplex and multiplex assays was almost perfect. In conclusion, a highly sensitive pentaplex assay was validated for the simultaneous identification of antibodies directed against BVDV, BoHV-1, PI3V, BRSV and EBLV in serum. The developed pentaplex assay complies with performance characteristics established by international guidelines for diagnostic tests and may be used as a tool for the implementation of epidemiological surveillance.

Identification of Eimeria tenella sporozoite immunodominant mimotopes by random phage-display peptide libraries-a proof of concept study.

Introduction: Coccidiosis, caused by parasites of numerous Eimeria species, has long been recognized as an economically significant disease in the chicken industry worldwide. The rise of anti-coccidian resistance has driven a search for other parasite management techniques. Recombinant antigen vaccination presents a highly feasible alternative. Properly identifying antigens that might trigger a potent immune response is one of the major obstacles to creating a viable genetically modified vaccine. Methods: This study evaluated a reverse immunology approach for the identification of B-cell epitopes. Antisera from rabbits and hens inoculated with whole-sporozoites of E. tenella were used to identify Western blot antigens. The rabbit IgG fraction from the anti-sporozoite serum exhibited the highest reactogenicity; consequently, it was purified and utilized to screen two random Phage-display peptide libraries (12 mer and c7c mer). After three panning rounds, 20 clones from each library were randomly selected, their nucleotide sequences acquired, and their reactivity to anti-sporozoite E. tenella serum assessed. The selected peptide clones inferred amino acid sequences matched numerous E. tenella proteins. Results and Conclusions: The extracellular domain of the epidermal growth factor-like (EGF-like) repeats, and the thrombospondin type-I (TSP-1) repeats of E. tenella micronemal protein 4 (EtMIC4) matched with the c7c mer selected clones CNTGSPYEC (2/20) and CMSTGLSSC (1/20) respectively. The clone CSISSLTHC that matched with a conserved hypothetical protein of E. tenella was widely selected (3/20). Selected clones from the 12-mer phage display library AGHTTQFNSKTT (7/20), GPNSAFWAGSER (2/20) and HFAYWWNGVRGP (8/20) showed similarities with a cullin homolog, elongation factor-2 and beta-dynein chain a putative E. tenella protein, respectively. Four immunodominant clones were previously selected and used to immunize rabbits. By ELISA and Western blot, all rabbit anti-clone serums detected E. tenella native antigens. Discussion: Thus, selected phagotopes contained recombinant E. tenella antigen peptides. Using antibodies against E. tenella sporozoites, this study demonstrated the feasibility of screening Phage-display random peptide libraries for true immunotopes. In addition, this study looked at an approach for finding novel candidates that could be used as an E. tenella recombinant epitope-based vaccine.

Combined Subcutaneous-Intranasal Immunization With Epitope-Based Antigens Elicits Binding and Neutralizing Antibody Responses in Serum and Mucosae Against PRRSV-2 and SARS-CoV-2.

New vaccine design approaches, platforms, and immunization strategies might foster antiviral mucosal effector and memory responses to reduce asymptomatic infection and transmission in vaccinated individuals. Here, we investigated a combined parenteral and mucosal immunization scheme to induce local and serum antibody responses, employing the epitope-based antigens 3BT and NG19m. These antigens target the important emerging and re-emerging viruses PRRSV-2 and SARS-CoV-2, respectively. We assessed two versions of the 3BT protein, which contains conserved epitopes from the GP5 envelope protein of PRRSV-2: soluble and expressed by the recombinant baculovirus BacDual-3BT. On the other hand, NG19m, comprising the receptor-binding motif of the S protein of SARS-CoV-2, was evaluated as a soluble recombinant protein only. Vietnamese mini-pigs were immunized employing different inoculation routes: subcutaneous, intranasal, or a combination of both (s.c.-i.n.). Animals produced antigen-binding and neut1ralizing antibodies in serum and mucosal fluids, with varying patterns of concentration and activity, depending on the antigen and the immunization schedule. Soluble 3BT was a potent immunogen to elicit binding and neutralizing antibodies in serum, nasal mucus, and vaginal swabs. The vectored immunogen BacDual-3BT induced binding antibodies in serum and mucosae, but PRRSV-2 neutralizing activity was found in nasal mucus exclusively when administered intranasally. NG19m promoted serum and mucosal binding antibodies, which showed differing neutralizing activity. Only serum samples from subcutaneously immunized animals inhibited RBD-ACE2 interaction, while mini-pigs inoculated intranasally or via the combined s.c.-i.n. scheme produced subtle neutralizing humoral responses in the upper and lower respiratory mucosae. Our results show that intranasal immunization, alone or combined with subcutaneous delivery of epitope-based antigens, generates local and systemic binding and neutralizing antibodies. Further investigation is needed to evaluate the capability of the induced responses to prevent infection and reduce transmission.

Immunotherapy With Egg Yolk Eimeria sp.-Specific Immunoglobulins in SPF Leghorn Chicks Elicits Successful Protection Against Eimeria tenella Infection.

Avian coccidiosis is the first to most economically important parasite disease affecting poultry industries worldwide. Current prevention measures are largely based upon prophylactic chemotherapy supplemented by the application of live attenuated or wild-type parasite vaccines. However, the rising appearance of drug resistance, consumer's concern for antibiotics use in poultry production and higher manufacturing cost of live vaccines has driven to adopt new technologies aimed at increasing animal health and production efficiency. Supplementing chickens with egg yolk Eimeria sp.-specific immunoglobulins can be a viable alternative to avoid severe outbreaks of the disease. Twelve-week-old SPF White Leghorn chickens were experimentally infected with a large dose of E. tenella. During the prepatent period, the birds were supplemented by oral gavage with 60 or 120 mg/bird of hyperimmune egg yolk Eimeria species-specific immunoglobulins Y (Supracox®, SC) on a daily basis. The animals were euthanized 7 days post-infection (PI) and their passive immune protection was evaluated. Birds treated with 120 mg/bird of SC showed more viability, increased body weight gain (BWG), a normal hematocrit level (HCT), reduced oocyst output per gram of feces (OPG) or cecal tissue (OPGC), and fewer cecal lesions compared to the untreated infected (UI) control group. Birds supplemented with 60 mg/bird of SC did not show any significant difference on BWG, HCT, OPG, OPGC, and cecal lesion score when compared with the UI group. An ELISA test of the SC showed a weak cross-reactivity of IgY toward two asexual zoite stages of E. tenella. Western blot analysis of the sporozoite with SC showed few antigens barely recognized, while more stained bands were detected in the merozoite (≈82, ≈60, ≈54, ≈40, ≈38, ≈27.5, and ≈13 kDa). Oral immunotherapy using egg yolk polyclonal IgYs against Eimeria sp. represents an effective and natural resource against severe E. tenella infection favoring the gradual withdrawal of the anticoccidial drugs and antibiotics.

Protective Immunity Induced by an Eimeria tenella Whole Sporozoite Vaccine Elicits Specific B-Cell Antigens.

This study investigated protection against Eimeria tenella following the vaccination of chicks with 5.3 × 106 E. tenella whole-sporozoites emulsified in the nanoparticle adjuvant IMS 1313 N VG Montanide™ (EtSz-IMS1313). One-day-old specific pathogen-free (SPF) chicks were subcutaneously injected in the neck with EtSz-IMS1313 on the 1st and 10th days of age. Acquired immunity was assayed through a challenge with 3 × 104 homologous sporulated oocysts at 21 days of age. The anticoccidial index (ACI) calculated for every group showed the effectiveness of EtSz-IMS1313 as a vaccine with an ACI of 186; the mock-injected control showed an ACI of 18 and the unimmunized, challenged control showed an ACI of -28. In a comparison assay, antibodies from rabbits and SPF birds immunized with EtSz-IMS1313 recognized almost the same polypeptides in the blotting of E. tenella sporozoites and merozoites. However, rabbit antisera showed the clearest recognition pattern. Polypeptides of 120, 105, 94, 70, 38, and 19 kDa from both E. tenella life cycle stages were the most strongly recognized by both animal species. The E. tenella zoite-specific IgG antibodies from the rabbits demonstrated the feasibility for successful B cell antigen identification.

Kappa-casein genotyping in tropical milking Criollo and its association to milk production and composition.

Caseins are abundant proteins in milk and found in four types (αS1, αS2, ß, and κ). There is substantial variation in the allelic and genotypic frequencies of the κ-casein gene in different cattle breeds, although the tropical milking Criollo (TMC) has not yet been investigated. The aim was to determine the allelic and genotypic frequencies in the κ-casein gene for two alleles (A and B) in TMC and further investigate its association to milk production and composition. A total of 180 TMC females were genotyped from blood samples. κ-Casein genotyping was performed using restriction fragment length polymorphism (RFLP) after polymerase chain reaction (PCR)-based amplification of genomic DNA. Allele frequencies were 0.39 for A-allele and 0.61 for B-allele (P < 0.05). Genotype frequencies were 0.09 (AA), 0.60 (AB), and 0.31 (BB) (P < 0.05). The κ-casein genotype in TMC cows did not affect milk yield or composition. In sum, the TMC has high frequencies of the B-allele and AB/BB genotypes, although there are no association of such genotypes and milk traits.

Comparative study of transcription profiles of the P-glycoprotein transporters of two Haemonchus contortus isolates: Susceptible and resistant to ivermectin.

The objective of this study was to analyze the mRNA transcription levels of ten functional genes of P-glycoproteins (P-gp) in free life stages, eggs and infective larvae (L3) and in endoparasitic stages, fourth larval stage (L4) and adult males of two native isolates of Haemonchus contortus: resistant and susceptible to IVM. The IVM resistant isolate was obtained from sheep naturally infected with H. contortus, and the susceptible isolate (with no pressure to IVM) conserved since 1990. The lethal effect of IVM was evaluated under in vitro conditions, which showed significant differences between susceptible and resistant H. contortus L3 isolates (P < 0.01). The IVM susceptible isolate revealed a lethal effect of 79.22% at 11.42 mM, whereas that resistant isolate showed no lethal effect at any of the four assessed concentrations (1.43, 2.85, 5.71 and 11.42 mM) of IVM. The expression levels of ten Hco-pgp genes (1, 2, 3, 4, 9, 10, 11, 12, 14, and 16) were evaluated in the resistant isolate of H. contortus and compared to the susceptible isolate (as control), using two constitutive genes (GAPDH and ß-tubulin). Up-regulation at two statistical significant values (P ≤ 0.05, 0.1) was the criterion to associate IVM resistance with the free life and endoparasitic stages of H. contortus. The expression levels in H. contortus adult nematodes showed 5.64 to 127.56-fold increase for Hco-pgp genes 1, 9, 12, 14, and 16, followed by an increase for Hco-pgp-2 (49.75-fold) and Hco-pgp-10 (106.40-fold) in L4, and for Hco-pgp-16 (2.90-fold) in eggs (P ≤ 0.05). In addition, high expression levels with P < 0.1 were detected in H. contortus L3, L4, and adults for Hco-pgp genes 1, 4, 11, 12, and 16, with changes ranging from 2.17 to 29.72-fold. In conclusion, the highest expression was observed in the adult stage of H. contortus, and the most frequent gene with a significant P-value was Hco-pgp-16, revealing it plays an important role in IVM resistance.

The presence of VEGF and Notch2 during preantral-antral follicular transition in infantile rats: Anatomical evidence and its implications.

Folliculogenesis is a process that depends on angiogenesis, in which VEGF and Notch signaling pathway members are involved. Although this pathway is present in preantral and antral follicular structures during the second stage of folliculogenesis, this association has not been described. Therefore, this study aimed to identify VEGF and Notch2 in ovary structures of infantile rats after induction of follicular development with a gonadotropin stimulus. In order to explore this possibility we analyzed rat ovary morphology from days 10-25 after birth; subsequently, the transition from preantral follicle to an antral stage was analyzed by the induction of follicular development with equine chorionic gonadotropin (eCG) and VEGF and Notch were identified in the rat ovary by fluorescence. The histological analysis revealed that the ovary of a 10-day-old rat has the highest percentage of preantral follicles and based on this a 10IU eCG dose promoted an increase in the number of antral follicles, as well as a decrease in the number of preantral follicles, related to which there was an increase in ovary weight and size. In addition, a higher concentration of circulating estradiol was observed, proliferation of granulosa cells in both follicle groups was stimulated, and the accumulation of VEGF in granulosa and theca cells and in the antral follicle oocyte was increased (p<0.05), whereas the presence of Notch2 was limited to mural granulosa cells, in granulosa cells that formed the cumulus oophorus and in the oocyte of both groups of follicles. The multiple correspondence analysis allowed us to support an association between VEGF and Notch2 during the transition from preantral to antral follicles in the ovary of an infantile rat.

Additive and dominance effects of the α(s1)-casein locus on milk yield and composition traits in dairy goats.

The objective of this study was to evaluate the effects of the CSN1S1 locus polymorphism on 305-d records of milk, fat, protein, lactose and total solids yields, fat, protein, lactose and total solids contents in Mexican dairy goats. A total of 514 lactation records belonging to Alpine (n=60), Saanen (n=105) and Toggenburg (n=74) goats, born from 2003 to 2006 in three herds were used. Discrimination between alleles E, F, N, A* (CSN1S1 A, G, H, I, O1 and O2) and B* (CSN1S1 B1, B2, B3, B4, C and L) were made by amplification of fragments of the gene CSN1S1 and digestion with the restriction endonuclease XmnI. In order to estimate additive and dominance effects, data sets including (1) all genotypes, and (2) only homozygote genotypes, were analysed using linear mixed models. The allele A*, had significant additive effects for protein content (0·21±0·07%; P=0·002) and total solids content (0·66±0·23%; P=0·005) when compared with allele F. An unfavourable additive effect of allele A* on milk yield was found in the Alpine breed (-81·4±40·2; P=0·046) when compared with allele F. Favourable dominance effects were found for some genotypes (P<0·05) for milk yield (A*N and B*N), fat yield (A*N and B*E), protein yield (A*N and B*E), lactose yield (A*N) and total solids yield (A*N). Also, unfavourable dominance effects were found (P<0·05) for protein content (A*B* and A*N) and total solids content (A*B*, A*N, and A*F). Allele A* was the only one with a positive effect for protein content. Significant allele-year interaction effects were also observed. The presence of significant dominance effects, estimated between specific pairs of alleles, challenged the purely additive nature of the genetic effect at the CSN1S1 locus. Implications from use of CSN1S1 effects in goat breeding programmes are presented.

Immunogenic peptides from phage display libraries with potential of protecting mice against the Pseudorabies virus.

Phage display selection of combinatorial peptide libraries has demonstrated its almost unlimited potential in identifying binding ligands for many targets. The method shows promise for selection of immunogenic peptides against pathogens by antibodies. We have undertaken a study designed to select such mimics for one of the representatives of Herpesviridae, the Pseudorabies virus (PrV), infecting pigs and causing severe neurological complications known as Aujeszky's disease. By screening a 12mer linear and a 7mer cysteine-constrained libraries with immunoglobulins of a rabbit immunized with the virus, a family of 10 antigenic and immunogenic peptides was derived sharing a sequence motif K(L/P/V)GDP(R/K/L). Groups of six C57BL/6 mice were immunized with bacteriophages expressing peptides with this motif sequences. Some of the mice were found to be positive in seroneutralization assay; in a challenge setting, all but two immunized mice survived, albeit presenting some disease symptoms. We discuss the perspectives and limits of generating peptide leads by library screening with immune polyclonal antiserum for designing pure epitope-based vaccines to PrV in the future.

La lipofección incrementa la eficiencia de mutagénesis dirigida en células troncoembrionarias de ratón E14 TG2a / Lipofection improves gene targeting efficiency in E14 TG2a mouse embryonic stem cells

Electroporation has been the method of election for transfection of murine embryonic stem cells for over 15 years; however, it is a time consuming protocol because it requires large amounts of DNA and cells, as well as expensive and delicate equipment. Lipofection is a transfection method that requires lower amounts of cells and DNA than electroporation, and has proven to be efficient in a large number of cell lines. It has been shown that after lipofection, mouse embryonic stem cells remain pluripotent, capable of forming germ line chimeras and can be transfected with greater efficiency than with electroporation; however, gene targeting of mouse embryonic stem cells by lipofection has not been reported. The objective of this work was to find out if lipofection can be used as efficiently as electroporation for regular gene targeting protocols. This context compares gene targeting efficiency between these techniques in mouse embryonic stem cells E14TG2a, using a gene replacement type vector. No differences were found in gene targeting efficiency between groups; however, lipofection was three times more efficient than electroporation in transfection efficiency, which makes lipofection a less expensive alternative method to produce gene targeting in mouse embryonic stem cells.

Durante los últimos 15 años se ha demostrado que la electroporación representa el método ideal para la transfección de células troncoembrionarias de ratón; sin embargo, demanda grandes cantidades de ADN y células, así como equipo caro y delicado, ello hace que este proceso sea costoso y laborioso. La lipofección es un método de transfección que requiere menos de células y ADN que la electroporación; asimismo, ha probado ser eficiente en gran número de líneas celulares. Se ha demostrado que después de lipofectar células troncoembrionarias de ratón, éstas mantienen su pluripotencia y son capaces de formar quimeras de línea germinal y se transfectan con mayor eficiencia que con electroporación, pero no se ha notificado la mutagénesis dirigida mediante la lipofección de células troncoembrionarias de ratón. El objetivo del presente trabajo fue saber si la lipofección puede ser utilizada con la misma o mayor eficiencia que la electroporación para los protocolos regulares de mutagénesis dirigida; en este contexto, se compara la eficiencia en mutagénesis dirigida entre estas técnicas en células troncoembrionarias de ratón E14TG2a, utilizando un vector de reemplazo. Entre las células transfectadas no se hallan diferencias en la eficiencia en mutagénesis dirigida entre grupos; sin embargo, los resultados que aquí se ofrecen muestran que la lipofección es tres veces más eficiente en la transfección, lo cual indica que la lipofección es un método alternativo menos costoso para obtener mutagénesis dirigida en células troncoembrionarias de ratón.

Polymorphism of locus DRB3.2 in populations of Creole Cattle from Northern Mexico

The polymorphism of locus BoLA-DRB3.2 of the Major Histocompatibility Complex was evaluated in two northern Mexican Creole cattle populations, Chihuahua (n = 47) and Tamaulipas (n = 51). The BoLA-DRB3.2 locus was typed by amplification and digestion with restriction endonuclease enzymes (PCR-RFLP). Fifty-two alleles were detected (28 previously reported and 24 new ones). In the Chihuahua population, 18 alleles and 5.5 effective alleles were found, while in the Tamaulipas population there were 34 and 10.8, respectively. The allele frequencies ranged from 0.011 to 0.383 in Chihuahua and from 0.010 to 0.206 in Tamaulipas. The frequencies of the new alleles in both cattle populations were low (0.010 to 0.053). The expected heterozygosity was 0.827 and 0.916, respectively, for the Chihuahua and Tamaulipas populations. Both populations presented a heterozygote deficit: [Chihuahua FIS = 0.1 (p = 0.019) and Tamaulipas FIS = 0.317 (p < 0.001)]. In conclusion, this study showed that the Mexican Creole cattle have many low-frequency alleles, several of which are exclusive to these populations. Genetic distances obtained show that the Mexican Creole cattle population is composed of independent populations, far apart from other South American Creole populations.