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.

Genome Variability of Infectious Bronchitis Virus in Mexico: High Lineage Diversity and Recurrent Recombination.

The avian infectious bronchitis virus (IBV) is a coronavirus that mutates frequently, leading to a contagious and acute disease that results in economic losses to the global poultry industry. Due to its genetic and serological diversity, IBV poses a challenge in preventing and controlling the pathogen. The full-length S1 sequence analysis identifies seven main genotypes (GI-GVII) comprising 35 viral lineages. In addition to the previously described lineage, a new GI lineage (GI-30) and two lineages from novel genotypes (GVIII-1 and GIX-1) have been described in Mexico. To prevent the spread of IBV outbreaks in a specific geographic location and select the suitable vaccine, it is helpful to genetically identify the circulating IBV types. Moreover, sequencing genomes can provide essential insights into virus evolution and significantly enhance our understanding of IBV variability. However, only genomes of previously described lineages (GI-1, GI-9, GI-13, and GI-17) have been reported for Mexican strains. Here, we sequenced new genomes from Mexican lineages, including the indigenous GI-30, GVIII-1, and GIX-1 lineages. Comparative genomics reveals that Mexico has relatively homogenous lineages (i.e., GI-13), some with greater variability (i.e., GI-1 and GI-9), and others extremely divergent (GI-30, GVIII-1, and GIX-1). The circulating lineages and intra-lineage variability support the unique diversity and dynamic of Mexican IBV.

Research Note: High genetic diversity of infectious bronchitis virus from Mexico.

The avian infectious bronchitis virus (IBV) is a highly mutable coronavirus that causes an acute and highly contagious disease responsible for economic losses to the poultry industry worldwide. Preventing and controlling bronchitis disease is difficulted by the numerous IBV circulating types with limited antigenic cross-protection that hamper the prevention and control by heterologous vaccines. The coding region of the variable spike S1 receptor-attachment domain is used to classify IBV in 7 genotypes (GI-GVII) comprising 35 viral lineages (1-35). Knowledge of the circulating IBV types causing outbreaks in a specific geographic region is beneficial to select better the appropriate vaccine(s) and contribute to disease control. In the study, 17 avian infectious bronchitis virus strains were obtained from chickens showing signs of illness in Mexico from 2007 to 2021. We detected 4 lineages within genotype I, three already known (GI-3, GI-9, GI-13) and one newly described (GI-30). In addition, we identified 2 divergent monophyletic groups that are tentatively described as lineages of new genotypes (GVIII-1 and GIX-1). Our findings revealed that Mexico's high genetic IBV diversity results from the co-circulation of divergent lineages belonging to different genotypes. Mexican IBV lineages differ significantly from Massachusetts and Connecticut vaccine strains, indicating that the currently used vaccines may need to be updated.

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.

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.

Mitochondrial DNA sequence analysis of the Mexican Creole sheep (Ovis aries) reveals a narrow Iberian maternal origin.

The Creole sheep in America is supposed to have originated specifically from the Iberian Peninsula and introduced by the Spaniards during the colonization. However, it is not clear their genetic relationship with Iberian breeds. The genetic origin and diversity of the Mexican Creole sheep (MCS) were investigated by mitochondrial DNA control region nucleotide sequences. DNA sequence from 33 MCS samples from three regions of México revealed 21 different haplotypes. Phylogenetic analysis including European and Iberian sheep haplotypes showed that the MCS population belongs to a differentiated and defined genetic lineage. This finding suggests that the MCS populations may be the result of a founder effect originating from a discrete Iberian population. MCS haplotypes were related to haplotypes found in the Churro Trunk and the Entrefino Trunk groups of Iberian breeds, supporting historical reports. In the Mexican genetic branch, there were also haplotypes reported from Lacaune and Awassi sheep breeds. Although it is uncertain whether a particular breed was involved as a founder of the MCS, these populations have a common phylogenetic origin.

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.

Association between major histocompatibility complex microsatellites, fecal egg count, blood packed cell volume and blood eosinophilia in Pelibuey sheep infected with Haemonchus contortus.

The objective of this study was to assess the correlation among traits associated with resistance or susceptibility to Haemonchus contortus infestation and to evaluate the participation of the ovine major histocompatibility complex (MHC) in Pelibuey sheep, a prevalent breed in tropical and sub-tropical regions in Mexico and elsewhere. Association among the fecal egg count (FEC), blood packed cell volume (PCV), antibody (AB) levels, serum proteins (SP) and blood eosinophil count (EOS) was assessed in 52 lambs experimentally infected with H. contortus, and the participation of the MHC was evaluated using polymorphisms in three microsatellites, located at the class I (OMHC1) and class II (OLADRB1, OLADRB2) regions of the MHC. Spearman correlation analysis among the traits showed a negative association (P<0.01) between FEC and PCV (-0.35), EOS (-0.50), SP (-0.30) and AB (-0.57), and a positive correlation of antibodies with EOS (0.50). The homozygotes for the OMHC1-188 and OLADRB2-282 alleles were associated with a reduction in FEC (-813 and -551, respectively). Conversely, the OMHC1-200 and OMHC1-206 alleles were associated with an increase in FEC (1704 and 1008, respectively). Furthermore, the OLADRB1-482 allele was associated with an increase of 163 EOS by allele copy, while the OMHC1-200 allele showed a reduction of 95 EOS in homozygotes. The associations among microsatellite MHC loci and the remaining variables were not significant. These results reinforce the evidence that MHC polymorphisms have an important role in parasite resistance or susceptibility in Pelibuey sheep and could be used as genetic markers to assist selection and improve parasite resistance to H. contortus.

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.

Genetic polymorphism of the alpha s1-casein locus in five populations of goats from Mexico

With the objective of estimating allele frequencies, and testing for population divergence for the CSN1S1 locus, genotypes of animals from five goat populations; Saanen (n = 97), Alpine (n = 81) Toggenburg (n = 92), local goats with external appearance similar to the Murciana-Granadina breed from Central Mexico (n = 26) and heterogeneous local animals denominated Mosaico Lagunero (n = 30), from Northern Mexico, were identified using PCR and Xmn1 PCR-RFLP methodology. For Saanen, Alpine and Toggenburg, the sum of E and F alleles had the largest frequencies (from 0.468 to 0.789), while for the groups local Murciana-Granadina and Mosaico Lagunero the sum of the most frequent allelic groups (A and B), were 0.385 and 0.533 respectively. Both local Murciana-Granadina and Mosaico Lagunero populations showed heterozygote excess (P < 0.08). The percentage of the total genetic variation (F ST) explained by population differences was 5.16. There was genetic differentiation for most pair comparisons between populations (P < 0.05), excepting for Alpine versus Toggenburg, and Toggenburg versus Mosaico Lagunero (P > 0.05). For Saanen and Alpine the frequencies of alleles E and F were similar to the same breeds previously analyzed in Europe. Therefore there are opportunities of increasing the frequency of the strong alleles for protein content Gene Assisted Selection (GAS) in these two breeds. For Toggenburg the most frequent allelic groups were F (0.32) and B (0.21). Results indicate differentiation between most populations for this locus. Moreover, heterozygote excess in local populations indicated breed admixture.