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.

Molecular Mechanisms Involved in MAFLD in Cholecystectomized Patients: A Cohort Study.

Gallstone disease and metabolic dysfunction-associated fatty liver disease (MAFLD) share numerous common risk factors and progression determinants in that they both manifest as organ-specific consequences of metabolic dysfunction. Nevertheless, the precise molecular mechanisms underlying fibrosis development in cholecystectomized MAFLD patients remain inadequately defined. This study aimed to investigate the involvement of farnesoid X receptor 1 (FXR1) and fibroblast growth factor receptor 4 (FGFR4) in the progression of fibrosis in cholecystectomized MAFLD patients. A meticulously characterized cohort of 12 patients diagnosed with MAFLD, who had undergone liver biopsies during programmed cholecystectomies, participated in this study. All enrolled patients underwent a follow-up regimen at 1, 3, and 6 months post-cholecystectomy, during which metabolic biochemical markers were assessed, along with elastography, which served as indirect indicators of fibrosis. Additionally, the hepatic expression levels of FGFR4 and FXR1 were quantified using quantitative polymerase chain reaction (qPCR). Our findings revealed a robust correlation between hepatic FGFR4 expression and various histological features, including the steatosis degree (r = 0.779, p = 0.023), ballooning degeneration (r = 0.764, p = 0.027), interphase inflammation (r = 0.756, p = 0.030), and steatosis activity score (SAS) (r = 0.779, p = 0.023). Conversely, hepatic FXR1 expression did not exhibit any significant correlations with these histological features. In conclusion, our study highlights a substantial correlation between FGFR4 expression and histological liver damage, emphasizing its potential role in lipid and glucose metabolism. These findings suggest that FGFR4 may play a crucial role in the progression of fibrosis in cholecystectomized MAFLD patients. Further research is warranted to elucidate the exact mechanisms through which FGFR4 influences metabolic dysfunction and fibrosis in this patient population.

Equine Placentitis in Mares Induces the Secretion of Pro-Inflammatory Cytokine eIL-1ß and the Active Extracellular Matrix Metalloproteinase (MMP)-9.

Equine placentitis is characterized by infection and inflammation of the placenta. Different biomarkers associated with this inflammatory response have been evaluated in experimentally induced equine placentitis, but not in pregnant mares with spontaneous placentitis. The aim of the current study was to determine the concentration of eIL-1ß and the activity of proMMP-2 and proMMP-9 in the serum of healthy mares and mares with placentitis on days 240 and 320 of gestation to explore whether these biomarkers are associated with equine maternal placentitis and/or with the birth of an infected or inviable foals. Serum samples were collected from sixteen pregnant English Thoroughbred mares, retrospectively classified as follows: (1) healthy mares with full-term gestation; and (2) mares with ultrasonographic signs of placentitis. The health of each foal was examined at birth, and it was decided to classify the cases into four groups: (1) healthy mares delivering a healthy foals (HM-HF, n = 6); (2) mares with USP delivering a healthy foal (USP-HF, n = 3); (3) mares with USP delivering a live septic foal (USP-LSeF, n = 4); and (4) mares with USP delivering a dead foal (USP-DF, n = 3). eIL-1ß was quantified by ELISA, and proMMP-2 and proMMP-9 activity by gelatin zymography electrophoresis. In healthy mares, the serum concentrations of eIL-1ß underwent a significant 16.5-fold increase from day 240 to day 320 of gestation. Although similar results were found in the mares with ultrasonographic signs of placentitis that delivered a healthy foal, those delivering a live septic or nonviable foal exhibited much higher concentrations of eIL-1ß. proMMP-2 and proMMP-9 activity was not associated with maternal placentitis, foal infection, or death. Hence, the presence of placentitis severe enough to affect the health of the foal can be confirmed or discarded by determining the eIL-1ß concentration in mares that have shown ultrasonographic signs of placentitis.

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.

Assembly and Analysis of Haemonchus contortus Transcriptome as a Tool for the Knowledge of Ivermectin Resistance Mechanisms.

Haemonchus contortus (Hc) is an important parasitic nematode of small ruminants. In this study we assembled the transcriptome of Hc as a model to contribute to the knowledge about the profile of the differential gene expression between two Mexican Hc strains under different anthelmintic resistance statuses, one susceptible and the other resistant to ivermectin (IVMs and IVMr, respectively), in order to improve and/or to have new strategies of control and diagnosis. The transcript sequence reads were assembled and annotated. Overall, ~127 Mbp were assembled and distributed into 77,422 transcript sequences, and 4394 transcripts of the de novo transcriptome were matched base on at least one of the following criteria: (1) Phylum Nemathelminthes and Platyhelminthes, important for animal health care, and (2) ≥55% of sequence identity with other organisms. The gene ontology (GO) enrichment analysis (GOEA) was performed to study the level of gene regulation to IVMr and IVMs strains using Log Fold Change (LFC) filtering values ≥ 1 and ≥ 2. The upregulated-displayed genes obtained via GOEA were: 1993 (for LFC ≥ 1) and 1241 (for LFC ≥ 2) in IVMr and 1929 (for LFC ≥ 1) and 835 (for LFC ≥ 2) in IVMs. The enriched GO terms upregulated per category identified the intracellular structure, intracellular membrane-bounded organelle and integral component of the cell membrane as some principal cellular components. Meanwhile, efflux transmembrane transporter activity, ABC-type xenobiotic transporter activity and ATPase-coupled transmembrane transporter activity were associated with molecular function. Responses to nematicide activity, pharyngeal pumping and positive regulation of synaptic assembly were classified as biological processes that might be involved in events related to the anthelmintic resistance (AR) and nematode biology. The filtering analysis of both LFC values showed similar genes related to AR. This study deepens our knowledge about the mechanisms behind the processes of H. contortus in order to help in tool production and to facilitate the reduction of AR and promote the development of other control strategies, such as anthelmintic drug targets and vaccines.

Detection and genetic characterization of feline retroviruses in domestic cats with different clinical signs and hematological alterations.

Feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) are globally distributed retroviruses that infect domestic cats and cause various syndromes that can lead to death. The aim of this study was to detect and genotype feline retroviruses in Mexican domestic cats. We used PCR assays to identify proviral DNA and viral RNA in 50 domestic cats with different clinical signs and hematological alterations. Endogenous FeLV (enFeLV) was identified in the genomic DNA of all cats in the study, and we detected transcripts of the LTR region of enFeLV in 48 individuals. Exogenous FeLV (exFeLV) was found in 13 cats. Furthermore, we detected FIV proviral DNA in 10 cats. The enFeLV sequences were shown to be the most variable, while the exFeLV sequences were highly conserved and related to previously reported subgroup A sequences. Sequencing of the FIV gag gene revealed the presence of subtype B in the infected cats.

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.

Immunogenic and antigenic analysis of recombinant NSP1 and NSP11 of PRRS virus.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus in the order Nidovirales, family Arteriviridae, genus Betaarterivirus. Antibodies against nonstructural proteins (NSPs) from this virus can be found in pigs starting 4 days postinfection and they remain detectable for several months. OBJECTIVE: The goal of this study was to evaluate the immunogenicity and antigenic properties of recombinant proteins NSP1 and NSP11 expressed in Escherichia coli cells, as well as to assess the neutralization activity that they elicit. METHODS: We obtained the complete ORF-1 genes coding for NSP1 and NSP11 from PRRSV using the VR-2332 strain. Cloning was performed with the pET23a(+) vector with a histidine tag (His6), linearized by restriction enzyme digestion; the expression of the NSP1 and NSP11 clones was induced in OverExpress C41(DE3) chemically competent cells. Recombinant proteins were used to generate hyperimmune sera and we perform serological assays to confirm neutralizing antibodies. RESULTS: The expressed recombinant NSP1 and NSP11 were found to be immunogenic when injected in pigs, as well as demonstrated higher specificity in recognition of antigen in field sera from pigs positive infected with PRRSV. Furthermore, both NSP1 and NSP11 recombinant proteins elicited PRRSV neutralizing antibodies. CONCLUSIONS: In this study, we demonstrated the immune humoral response to NSP 1 and NSP11, and neutralizing-antibody response to PRRSV VR2332 strain in sera from hyperimmunized pigs.

Attachment and In Vitro Transfection Efficiency of an Anti-Rabies Chitosan-DNA Nanoparticle Vaccine.

In Mexico, urban rabies has been reduced during the last decade thanks to intensive canine control and vaccination campaigns; however, rabies transmitted by wild animals, especially by bats, has been increasing due to vampire bats feeding on livestock. Vampire bat populations has been controlled by culling with vampiricides, reducing indiscriminately other bat species. Hence, bat vaccination for rabies offers an alternative for culling. Nevertheless, available rabies vaccines are not suitable for their use in wildlife from emerging countries. This project presents an alternative for the use of plasmid vaccines using bio-nanotechnology, to create low-cost and accessible vaccines. To accomplish this goal, chitosan nanoparticles were synthesized by ionic gelation and conjugated by coacervation with a pDNA rabies vaccine to test their attachment efficiency. Also, the conjugate was functionalized with Protoporphyrin IX and Folic acid as biomarkers. The nanoparticles complex was characterized by ultraviolet visible spectroscopy, infrared spectroscopy, transmission electron microscopy, dynamic light scattering, and the Z potential was obtained. In vitro tests were performed on cell viability and transfection. The nanoparticles possessed a low polydispersity, a mean size of 118.5 ± 13.6 nm and a Z potential of 17.3 mV. The attachment efficiency was of 100% independent of pDNA added. In contrast to functionalized nanoparticles which showed a max attachment efficiency of 99.6% dependent of pDNA concentration and the method of functionalization. The conjugate did not influence the viability and they improved the transfection efficiency. Results suggest that these nanoparticles are easy to prepare, inexpensive, and exhibit potential for plasmid delivery as it improves transfection efficiency of pDNA vaccines.

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.

Effect of entomopathogenic native fungi from paddock soils against Rhipicephalus microplus larvae with different toxicological behaviors to acaricides.

The objectives of this study are to: (1) evaluate the in vitro acaricidal effect of 54 Metarhizium anisopliae strains, six Beauveria bassiana strains and one Purpureocilium lilacinum strain, against the larvae of two populations of Rhipicephalus microplus (multi-resistant and susceptible to chemical acaricides); and (2) determine the lethal concentrations required to eliminate the 50% (LC50) and 99% (LC99) of larvae through the use of entomopathogenic fungi (EF) with high acaricidal effects. The mortality percentage was evaluated by larval immersion tests at a dose of 1 × 108 conidia/mL for each fungal strain. For calculating LC50 and LC99, four doses (1 × 108, 1 × 107, 1 × 106 and 1 × 105) were used. Nine strains of M. anisopliae and the P. lilacinum strain showed a high mortality percentage in the R. microplus larvae of both populations. The best strains that showed the lowest values of LC50 and LC99 for tick elimination were MaV50 and PlV01. In conclusion, several strains of entomopathogenic fungi showed a high acaricidal effect against the R. microplus larvae of both populations, suggesting that these fungi might be a promissory adjuvant in the control of R. microplus, including those who are resistant. Finally, the discovery of a P. lilacinum strain with a high acaricidal effect is also reported.

Identification and genotyping of porcine circovirus type II (PCV2) in Mexico.

Porcine circovirus type 2 (PCV2), family Circoviridae, genus Circovirus infection in domestic pig has been associated with several pathological conditions being the most important of them the postweaning multisystemic wasting syndrome. Many studies have demonstrated the existence of three PCV2 genotypes (a, b, and c) and recently PCV3. Until now, these genotypes or subgenotypes have not been described in Mexico. We found genetic changes in ORF2 from nine strains of PCV2 obtained from samples of Jalisco, Veracruz, Estado de México, Hidalgo and Sonora states of Mexico. Our results shown the presence of two genotypes (PCV2a and PCV2b) as well as, the presence and differences between the reported subgenotypes. The subgenotype PCV2b (1A/1B, 1A) has a higher prevalence (87.5%) in comparison with PCV2a (2C) (12.5%).

Molecular characterization of the ORF2 of Torque teno sus virus 1a and Torque teno sus virus 1b detected in cases of postweaning multisystemic wasting syndrome in Mexico.

Worldwide Torque teno sus virus (TTSuV, genus Iotatorquevirus) species have been regarded as possible agents associated with porcine circovirus-associated disease. Iotatorquevirus species possess high genomic variability, suggesting that diverse genotypes are widely geographically distributed. In this study, we validated the genomic variability of Iotaroquevirus species in pigs with postweaned multisystemic wasting syndrome. Genomic DNA from nine TTSuV1a-positive tissues and 15 TTSuV1b-positive tissues was used to amplify the complete ORF2 of each species by nested PCR to perform a molecular characterization. It was found that Mexican TTSuV1a sequences belong to genotype B, sharing phylogenetic origin, high nucleic acid and amino acid sequence similarity and dominant epitope conformation with commercially linked countries, such as the United States, Canada and China, whereas the Mexican TTSuV1b sequences belong to genotype A, being more divergent among each other and displaying low nucleotide identity with worldwide genotype A sequences. In both Iotatorquevirus species, a PTPase-like signature motif was identified in the predicted amino acid sequence, being more conserved for Mexican TTSuV1b sequences than for Mexican TTSuV1a sequences, in which several substitutions were observed. These changes may influence the conformation of dominant epitopes as different arrays were determined among TTSuV1a genotypes. ORF2 variability may account for pathogenic differences by modifying viral replication and immune response, as depicted for human TTV.

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.