Search

Lambs harboring the Hb-AA β-globin haplotype present improved cell-mediated responses and increased resistance against Haemonchus contortus infection. The aim of the present study was to compare the effect of sex and β-globin haplotypes on specific humoral responses and phenotypes of resistance during H. contortus infection in Morada Nova sheep. As expected, females displayed stronger resistance during the first and second experimental challenges. Differential systemic humoral immune responses were observed comparing sex groups, in which higher levels of specific antibodies targeting 24 kDa excretory-secretory (ES24) protein of H. contortus of IgG and IgM antibodies were respectively observed as predominant isotypes in males and females. The IgM levels were significantly correlated with phenotypes of resistance, evaluated by packed cell volume and fecal egg counts. To our knowledge this is the first study reporting divergent humoral responses profiles to H. contortus infection between male and female sheep. The impact of β-globin haplotypes was less pronounced in females compared to males. Notably, only males showed significant weight differences across haplotypes, with Hb-AA lambs being the heaviest. Additionally, Hb-AA males had significantly higher PCV (indicating better red blood cell health) and lower FEC (indicating lower parasite burden). These findings suggest a more pronounced effect of β-globin polymorphisms on H. contortus infection in males, potentially due to their generally weaker resistance compared to females. This study highlights the importance of sex and β-globin haplotypes in shaping immune responses to H. contortus infection. Specifically, IgM antibodies targeting the ES24 protein appear to play a crucial role in host-parasite interactions and may hold promise for therapeutic development., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: The integration of molecular data from hosts, parasites, and microbiota can enhance our understanding of the complex biological interactions underlying the resistance of hosts to parasites. Haemonchus contortus, the predominant sheep gastrointestinal parasite species in the tropics, causes significant production and economic losses, which are further compounded by the diminishing efficiency of chemical control owing to anthelmintic resistance. Knowledge of how the host responds to infection and how the parasite, in combination with microbiota, modulates host immunity can guide selection decisions to breed animals with improved parasite resistance. This understanding will help refine management practices and advance the development of new therapeutics for long-term helminth control., Methods: Eggs per gram (EPG) of feces were obtained from Morada Nova sheep subjected to two artificial infections with H. contortus and used as a proxy to select animals with high resistance or susceptibility for transcriptome sequencing (RNA-seq) of the abomasum and 50 K single-nucleotide genotyping. Additionally, RNA-seq data for H. contortus were generated, and amplicon sequence variants (ASV) were obtained using polymerase chain reaction amplification and sequencing of bacterial and archaeal 16S ribosomal RNA genes from sheep feces and rumen content., Results: The heritability estimate for EPG was 0.12. GAST, GNLY, IL13, MGRN1, FGF14, and RORC genes and transcripts were differentially expressed between resistant and susceptible animals. A genome-wide association study identified regions on chromosomes 2 and 11 that harbor candidate genes for resistance, immune response, body weight, and adaptation. Trans-expression quantitative trait loci were found between significant variants and differentially expressed transcripts. Functional co-expression modules based on sheep genes and ASVs correlated with resistance to H. contortus, showing enrichment in pathways of response to bacteria, immune and inflammatory responses, and hub features of the Christensenellaceae, Bacteroides, and Methanobrevibacter genera; Prevotellaceae family; and Verrucomicrobiota phylum. In H. contortus, some mitochondrial, collagen-, and cuticle-related genes were expressed only in parasites isolated from susceptible sheep., Conclusions: The present study identified chromosome regions, genes, transcripts, and pathways involved in the elaborate interactions between the sheep host, its gastrointestinal microbiota, and the H. contortus parasite. These findings will assist in the development of animal selection strategies for parasite resistance and interdisciplinary approaches to control H. contortus infection in sheep., (© 2024. The Author(s).)

Haemonchus contortus is a constraint to sheep production. Seeking to reduce the use of hosts and produce parasitic stages in large-scale, a 42-day in vitro culture protocol of H. contortus third-stage larvae was optimized using Dulbecco's modified Eagle's medium (DMEM). In cell-free culture, larvae were maintained at 39.6°C, in acidic media (pH 6.1) for 3 or 6 days with Δ4-dafachronic acid followed by DMEM pH 7.4 supplemented or not with Fildes' reagent. In DMEM pH 7.4 at 37°C, supplementation with Caco-2 cells was compared to Fildes. On Day 14, fourth-stage larvae (L4) development rates in acidic media supplemented (86.8-88.4%) or not (74.4-77.8%) with Fildes and in Caco-2 cell co-culture (92.6%) were similar, and superior to DMEM pH 7.4 with Fildes (0.0%). On Day 21, Caco-2 cell co-culture resulted in higher larvae differentiation (25.0%) and lower degeneration (13.9%) compared to acidic media (1.5-8.1% and 48.6-69.9%, respectively). This is the first report of prolonged in vitro culture of H. contortus larvae using commercial media in co-culture with Caco-2 cells. Although no progression to the adult stage, Caco-2 cell co-culture resulted in morphological differentiation of H. contortus L4 and larval viability for up to 28 days.

The objective of the study was to evaluate energy or energy/protein supplementation in the ewe diet, in the last third of gestation, on maternal placental and endocrine characteristics, as well as its effects on the behavior of neonatal lambs and productive performance until weaning. A total of 128 ewes were used, and the experimental diet was fed from 100 days gestation until lambing, with the birth of 172 lambs. The ewes were distributed into three treatments: CTL (control, n = 43) with metabolizable energy (ME) and crude protein (CP) intake according to NRC (1985); ME (energy supplementation, n = 44) plus 21% ME; and MECP (energy/protein supplementation, n = 41) plus 26% ME and CP. Body weight, body condition score, serum hormone concentrations, placental characteristics, lamb performance and behavior, and production efficiency of the ewe from lambing to weaning were measured. ME and MECP ewes were heavier before (p = 0.006) and just after lambing (p = 0.002) and had higher serum triiodothyronine (p = 0.001) and cortisol (p = 0.004) concentrations on the day of lambing. ME ewes had higher placental efficiency (p = 0.036) and lower total cotyledon weight (p = 0.011). ME and MECP diets increased both lamb birth weight (p = 0.015) and weaning weight (p = 0.009). Production efficiency at birth and at weaning was not influenced (p > 0.05) by treatments. Lamb behavior was influenced by the ME and MECP diets, reducing the time to kneel (p ≤ 0.05), to try to stand (p ≤ 0.05), and the latency to stand (p ≤ 0.005). It is concluded that overnutrition in the last third of gestation was positive for the ovine production system, with improved production rates, hormonal profile, placental characteristics, and neonatal behavior.

This study evaluated the economic impact of gastrointestinal nematode (GIN) infection in Morada Nova lambs under different parasite chemical control conditions. For this, 246 lambs, in the rainy and dry season, were randomized into groups according to their anthelmintic treatment with levamisole: control (CT: no treatment); routine treatment (RT: treated every 42 days); and targeted selective treatment (TST: treated according to the average daily weight gain, DWG). From 63 days of age (D63) to D210, the lambs were weighed and monitored for GIN infection parameters. Spending on anthelmintics in the production system was 1.3% of the total economic result. The economic result per animal (R$ 5.00 = US$ 1.00) was higher in the RT group, amounting to US$ 6.60 in the rainy and US$ 5.69 in the dry season, due to higher DWG. Thus, RT presented economic results 14.4% and 10.9% higher than CT, and 7.2% and 1.9% higher than TST, in the rainy and dry season, respectively. However, fast development of resistance made RT unfeasible. Here, the economic impact of GIN infection on a national scale is discussed, demonstrating its importance and the impossibility of profitable and sustainable sheep production without adequate control.

Among the gastrointestinal nematodes affecting sheep, Haemonchus contortus is the most prevalent and virulent, resulting in health problems and production losses. Therefore, selecting sheep resistant to H. contortus is a suitable and sustainable strategy for controlling endoparasites in flocks. Here, 287 lambs of the native Brazilian Morada Nova hair sheep breed were subjected to two consecutive artificial infections with H. contortus and assessed for fecal egg count (FEC), packed cell volume (PCV), and live weight (LW). Forty-four animals ranked as having extreme resistance phenotypes were genotyped using the Illumina OvineSNP50v3 chip. A case−control genome-wide association study (GWAS) detected 37 significant (p < 0.001) markers in 12 ovine chromosomes in regions harboring quantitative trait loci (QTL) for FEC, Trichostrongylus spp. adults and larvae, weight, and fat; and candidate genes for immune responses, mucins, hematological parameters, homeostasis, and growth. Four single-nucleotide polymorphisms (SNP; OAR1&#95;rs427671974, OAR2&#95;rs419988472, OAR5&#95;rs424070217, and OAR17&#95;rs401006318) genotyped by qPCR followed by high-resolution melting (HRM) were associated with FEC and LW. Therefore, molecular markers detected by GWAS for H. contortus resistance in Morada Nova sheep may support animal selection programs aimed at controlling gastrointestinal nematode infections in flocks. Furthermore, genotyping of candidate genes using HRM qPCR may provide a rapid and efficient tool for animal identification.

This study assessed the feasibility of in vivo embryo production and nonsurgical embryo recovery (NSER) in Morada Nova ewes (an endangered native Brazilian breed of sheep) subjected to different estrus synchronization and/or superovulation protocols. Ewes received intravaginal sponges soaked with 60 mg medroxyprogesterone acetate (MAP), which were kept in place for six (G6; n  = 12), nine (G9; n  = 12), or 12 (G12; n  = 12) days. Half of the ewes in each group remained estrus synchronized only ( SYNCH ) and the other half was superovulated ( SOV ) with 133 mg porcine follicle-stimulating hormone (pFSH). There were no differences ( p  > 0.05) in antral follicle counts determined with ultrasonography 60 hours before MAP sponge removal (or at the time of the first pFSH dose) among G6 (6.4 ± 0.9), G9 (6.2 ± 0.7), and G12 (5.5 ± 0.6). Estrus responses and NSER success rates did not vary ( p  > 0.05) among the three progestin-treatment groups of ewes for either estrus-induced or superovulated animals. The onset of estrus occurred 10-12 hours later ( p  < 0.01) in G9 SYNCH ewes compared with G6 SYNCH and G12 SYNCH, and the duration of estrus was ∼19 hours greater ( p  < 0.01) in G9 SOV than in G6 SOV . The average duration of the NSER procedure was 32.6 ± 1.3 minutes. At least one structure was recovered in 85.7% of synchronized and in 87.5% of superovulated ewes. Viable embryo recovery rates were also similar ( p  > 0.05) for G6 (1.0 ± 0.3 and 2.5 ± 1.5), G9 (1.3 ± 0.5 and 4.8 ± 2.0), and G12 groups (1.0 ± 0.3 and 4.8 ± 2.3; estrus-synchronized and superovulated ewes, respectively). In conclusion, progestogen pretreatment of different durations and NSER can be employed in Morada Nova ewes, resulting in reasonable viable embryo recovery rates in both estrus-synchronized and superovulated animals. Therefore, both techniques are suitable for use in commercial settings as well as small ruminant conservation programs.

This study evaluated the resistance status of Haemonchus contortus from sheep flocks in the state of São Paulo, Brazil, through comparison between the fecal egg count reduction test (FECRT) and the larval development test (LDT). For the FECRT, 35 sheep were selected in each of five flocks and divided into groups treated with: benzimidazole, levamisole, ivermectin, monepantel and control. Feces were collected for EPG and fecal cultures. The LDT was performed using thiabendazole (TBZ), levamisole (LEV), ivermectin aglycone (IVM-A) and Zolvix (ZLV). Resistance to all drugs was detected using FECRT in 100% of the flocks, except in relation to ZLV (40% resistant and 20% suspected of resistance). LDT indicated resistance to TBZ and IVM-A in all flocks, to LEV in 80% of flocks and to ZLV in 10%. Total agreement was obtained between the two tests for TBZ and IVM (k = 1.0), while for LEV (k = 0.8) and ZLV (k = 0.9), substantial and almost perfect agreement were obtained, respectively. The concordance between the tests was significant, thus showing that it is possible to use the outcome of the LDT to predict the FECRT, and hence validating the former as a fast diagnostic test for use by sheep farmers in Brazil.

The immune response against Haemonchus contortus infections is primarily associated with the Th2 profile. However, the exact mechanisms associated with increased sheep resistance against this parasite remains poorly elucidated. The present study is aimed at evaluating mediators from the innate immune response in lambs of the Morada Nova Brazilian breed with contrasting H . contortus resistance phenotypes. Briefly, 287 lambs were characterized through fecal egg counts (FEC) and packed cell volume (PCV) after two independent experimental parasitic challenges with 4,000 H. contortus L 3 . 20 extreme resistance phenotypes (10 most resistant and 10 most susceptible) were selected, subjected to a third artificial infection with 4,000 L 3 , and euthanized 7 days later. Tissue samples were collected from abomasal fundic and pyloric mucosa and abomasal lymph nodes. Blood samples were collected at days 0 and 7 of the third parasitic challenge. RNA was extracted from tissue and blood samples for relative quantification of innate immune-related genes by RT-qPCR. For the abomasal fundic mucosa, increased TNFα and IL1β expression levels ( P < 0.05) were found in the susceptible animals, while resistant animals had IL33 superiorly expressed ( P < 0.05). Higher levels ( P < 0.05) of TLR2 and CFI were found in the abomasal pyloric mucosa of resistant animals. TNFα was at higher levels ( P < 0.05) in the blood of susceptible lambs, at day 0 of the third artificial infection. The exacerbated proinflammatory response observed in susceptible animals, at both local and systemic levels, may be a consequence of high H . contortus parasitism. This hypothesis is corroborated by the higher blood levels of TNFα before the onset of infection, which probably remained elevated from the previous parasitic challenges. On the other hand, resistant lambs had an enhanced response mediated by TLR recognition and complement activation. Nevertheless, this is the first study to directly associate sheep parasitic resistance with IL33, an innate trigger of the Th2-polarized response., Competing Interests: The authors have no conflict of interest., (Copyright © 2019 João Henrique Barbosa Toscano et al.)

Morada Nova is a Brazilian hair sheep breed that is well adapted to the country's mainly tropical climate and has good potential for meat and leather production. This breed is reported to be resistant to Haemonchus contortus infection, a highly desired characteristic due to the large impact of this parasite on sheep farming. Therefore, the present study aimed to characterize 287 recently weaned Morada Nova lambs and 123 ewes in relation to their resistance against H. contortus . The animals were dewormed and 15 days later artificially infected with 4000 H. contortus L 3 (D0). They were individually monitored by periodic assessment of fecal egg count (FEC), packed cell volume (PCV), and live weight (LW). On D42, the sheep were again dewormed and submitted to a new parasitic challenge, following the same scheme. The animals of each category (lambs and ewes) were ranked according to individual mean FEC values, and classified as resistant (R, 20%), intermediate (I, 60%), or susceptible (S, 20%) to H. contortus infection. At weaning, high FEC were observed in all three phenotypes (P > 0.05). After the artificial infections, there was a significant difference (P < 0.05) among the three lamb phenotypes for the mean FEC (R < I < S), PCV (R > I > S), and LW (R = I > S). The infection levels (FEC) were negatively correlated with PCV (r = -0.66; P < 0.001), and LW (r = -0.30; P < 0.001). Despite this, the lambs were resilient, since more than 88% of these animals maintained the PCV above 24%, even when heavily infected. The importance of selective parasite control before weaning to reduce the negative impact on slaughter weight was evidenced, taking into account the high positive correlation between LW at weaning and final LW (r = 0.73; P < 0.001). The ewes, in turn, were strongly resistant to the parasite. Despite highly significant differences (P < 0.001) for mean FEC between phenotypes (R < I < S), 98% of the ewes maintained FEC below 4000 EPG. Their health was not affected, since PCV and LW did not differ between phenotypes, and these parameters were not significantly correlated with FEC (P > 0.05). With the phenotypic characterization performed here, it is possible to introduce procedures for parasite control in Morada Nova flocks, facilitating the target-selective treatment approach. The results of this study can also support improvement of meat production by the Morada Nova breed., (© 2019 The Authors.)

Background: Haemonchus contortus, a gastrointestinal nematode parasite of sheep, is mainly controlled by anthelmintics; the occurrence of anthelmintic resistance leads to treatment failures and increases economic burden. Because molecular mechanisms involved in drug resistance can be elucidated by genomic studies, an extreme quantitative trait locus (X-QTL) mapping approach was used to identify co-segregation of the resistance phenotype with genetic markers to detect the genome-wide variants associated with monepantel resistance in H. contortus., Methods: A cross between H. contortus isolates using parental susceptible (Par-S) males and monepantel resistant (Par-R) females resulted in SR progeny, while reciprocal cross resulted in RS progeny. Pools (n = 30,000) of infective larvae (L3) recovered from Par-R, and from SR and RS populations in the F3 generation, collected both before (unselected group) and 7 days after (selected group) selection with monepantel treatment in sheep hosts, were subjected to genome sequencing (Pool-Seq). Pairwise comparisons of allele frequencies between unselected and selected groups were performed for each population by Fisher's exact test (FET) and for both populations combined by a Cochran-Mantel-Haenszel (CMH) test., Results: Mapping rates varied from 80.29 to 81.77% at a 90.4X mean coverage of aligned reads. After correction for multiple testing, significant (P < 0.05) changes in allele frequencies were detected by FET for 6 and 57 single nucleotide polymorphisms (SNPs) in the SR and RS populations, respectively, and by the CMH test for 124 SNPs in both populations. The significant variants located on chromosome 2 generated a selection signal in a genomic region harboring the mptl-1, deg-3 and des-2 genes, previously reported as candidates for monepantel resistance. In addition, three new variants were identified in the mptl-1 gene., Conclusions: This study expands knowledge on genome-wide molecular events underlying H. contortus resistance to monepantel. The identification of a genome region harboring major genes previously associated with monepantel resistance supports the results of the employed X-QTL approach. In addition, a deletion in exon 11 of the mptl-1 gene should be further investigated as the putative causal mutation leading to monepantel resistance.

The excessive use of anthelmintics to control nematodes has resulted in anthelminthic resistance. Essential oils (EOs) are a rich source of bioactive molecules that can be assessed for their ability to control resistant parasite populations. The aims of this study were to screen EOs from 10 plant species in vitro for anthelmintic activity against Haemonchus contortus , evaluate the cytotoxicity of those EOs in a human immortalized keratinocyte cell line (HaCaT), and test the most promising EO candidate in vivo in Santa Inês sheep. The efficacy was investigated in vitro using an egg hatch test (EHT) and a larval development test (LDT). EO cytotoxicity was evaluated with the sulforhodamine-B assay. In the in vivo experiment, 28 Santa Inês sheep naturally infected were distributed into groups: G1- Mentha arvensis (EO 5 ), 200 mg kg -1 ; G2-menthol, 160 mg kg -1 ; G3-negative control; and G4-positive control (monepantel). EO 5 , from M. arvensis (86.7% menthol), had the lowest LC 50 and LC 90 values in the EHT (0.10, 0.27 mg mL -1 , respectively), good performance in the LDT (0.015, 0.072 mg mL -1 , respectively), and the lowest cytotoxicity (190.9 µg mL -1 ) in HaCaT cells. In the in vivo test, a single dose of the EO 5 (200 mg kg -1 BW) had an efficacy of approximately 50% on days 1, 14, and 21; however, values were not significantly from day 0. Conversely, pure menthol at a dose of 160 mg kg -1 BW showed no in vivo efficacy. This can be attributed to key factors related to bioavailability and pharmacology of terpenes in the host organism, as well as to the fact that menthol is mainly excreted as glucuronides in urine. Thus, further studies should be conducted with formulation systems that deliver bioactives directly to the abomasum, focusing on terpenes, whose excretion route is mainly via faeces., Competing Interests: The authors declare that they have no competing interests.All experimental protocols were approved by the CPPSE Animal Care and Use Committee (No. 02/2014). Animals were under the care of a veterinary medical assistant during all 21 days of the experiment. The number of eggs per gram of faeces of the donor animal and the animals of the in vivo anthelmintic test was counted to monitor the level of infection during the experiment. After treatment, the animals were also observed for 1 h to monitor any toxicity symptoms caused by the essential oils, such as signs of discomfort, apathy, lethargy and/or drowsiness. None died or was in poor physical condition.

This study aimed to assess the capacity of Morada Nova (MN) and Santa Inês (SIN) rams to maintain body and testicular homeothermy under thermal challenge. For 5 days in the summer, 16 males (SIN = 7 and MN = 9) underwent a heat tolerance test, i.e., period 1-animals maintained in the shade (11 to 12 h); period 2-animals exposed to sunlight (12 to 13 h); and period 3-animals returned to the shade (13 to 14 h). The respiratory rate, heart rate, rectal temperature, and infrared surface temperatures (IRT) of the trunk, back, eyeball, and testicles were assessed in each period. The index of capacity of tolerance to insolation (ICTI), which indicates the animals' level of adaptability, was calculated for each animal. Semen quality and testicular parenchyma integrity were assessed before and after the thermal challenge. Statistical analyses were performed at 5% significance. In period 1, the variables had baseline values for both genotypes. In period 2, the variables involved in thermolysis significantly increased (P < 0.05), which matches a thermal discomfort situation. In period 3, the variables returned to baseline values and some values were lower than those in period 1. Semen quality and testicular parenchyma integrity suffered no negative effects with the thermal challenge. IRT ocular and IRT testicular were positively correlated (P < 0.05). It is concluded that MN and SIN rams had efficient thermolytic mechanisms that favor preserving gonadal functionality. The animals were considered resilient to a thermal challenge. In addition, infrared thermography was an efficient tool to verify body and testicular thermoregulation.

The anthelminthic activity of the essential oil (EO) of Piper aduncum L. was tested in vitro on eggs and larvae of resistant (Embrapa2010) and susceptible (McMaster) isolates of Haemonchus contortus. The EO was obtained by steam distillation and its components identified by chromatography. EO concentrations of 12.5 to 0.02 mg/mL were used in the egg hatch test (EHT) and concentrations of 3.12 to 0.01 mg/mL in the larval development test (LDT). Inhibition concentrations (IC) were determined by the SAS Probit procedure, and significant differences assessed by ANOVA followed by Tukey's test. In the EHT, the IC50 for the susceptible isolate was 5.72 mg/mL. In the LDT, the IC50 and IC90 were, respectively, 0.10 mg/mL and 0.34 mg/mL for the susceptible isolate, and 0.22 mg/mL and 0.51 mg/mL for the resistant isolate. The EO (dillapiole 76.2%) was highly efficacious on phase L1. Due to the higher ICs obtained for the resistant isolate, it was raised the hypothesis that dillapiole may have a mechanism of action that resembles those of other anthelmintic compounds. We further review and discuss studies, especially those conducted in Brazil, that quantified the major constituents of P. aduncum-derived EO.

This report describes the naturally occurring atypical neuropathological manifestation of systemic canine distemper virus (CDV) infection in two 16-day-old Pit Bull pups. CDV-induced changes affected the gray and white matter of the forebrain while sparing the hindbrain. Histologically, there was necrosis with destruction of the nervous parenchyma due to an influx of inflammatory and reactive cells associated with eosinophilic intranuclear inclusion bodies within glial cells. Positive immunoreactivity against CDV antigens was predominantly observed within astrocytes and neurons. RT-PCR was used to amplify CDV-specific amplicons from brain fragments. These findings suggest the participation of CDV in the etiopathogenesis of these lesions.

Gastrointestinal nematode infections were evaluated in Santa Inês crossbreed sheep (Santa Inês predominance) in a rotational grazing system and in lambs kept indoors, born from the breeding of these females with purebred Santa Ines, Dorper and Suffolk males. Fecal egg counts (FEC), fecal cultures, packed cell volume (PCV) and weights were evaluated during two years. Climate data were registered. Only animals that presented FEC higher than 4,000 and/or PCV lower than 21% were drenched with anthelmintic treatment and this rational use controlled the infection in the flock satisfactorily. An adequate diet during the rainy season and supplementation in dry period were important to increase relative resistance and resilience to parasites, since Haemonchus contortus was detected year-round in this region. The peripartum physiological condition influenced gastrointestinal nematode infections significantly. Crossbred (1/2) Santa Inês + (1/2) Dorper lambs did not show a significant difference in FEC when compared with other breeds, but they had greater live weight and reached slaughter weight earlier.