Fasciola hepatica and Fasciola gigantica: comparison of cellular response to experimental infection in sheep

Cellular responses to Fasciola gigantica and to Fasciola hepatica infection in sheep were compared. Eosinophil numbers increased more quickly and strongly in F. gigantica-infected sheep than in F. hepatica-infected sheep. In both groups, peripheral blood mononuclear cell (PBMC) proliferation in response to the parasitic excretory-secretory products (ESP) showed similar kinetics. Interferon-gamma (IFN-gamma) production by ESP-stimulated PBMC was early and showed similar kinetics in both groups. Interleukin-10 (IL-10) production by FhESP-stimulated PBMC was very high throughout infection even at 0 weeks post-infection (WPI) in F. hepatica-infected sheep, while in F. gigantica-infected sheep, IL-10 production by FgESP-stimulated PBMC increased between 1 and 4 WPI. IL-10 production in F. gigantica-infected sheep was significantly lower than in F. hepatica-infected sheep during infection. The lower susceptibility to F. gigantica infection in sheep could be explained by the more intense cellular response induced by the parasite and the weaker capacity of F. gigantica to evade the immune response.     

Comparison of humoral response in sheep to Fasciola hepatica and Fasciola gigantica experimental infection

Humoral response of sheep to F. gigantica was compared with the well known humoral response to F. hepatica, in order to explain the difference of susceptibility of sheep to these two parasites. In this work, a lesser susceptibility of sheep to F. gigantica than to F. hepatica infection was confirmed. Humoral response to F. hepatica infection is similar to that previously described by several authors. IgG level of F. gigantica infected sheep increased from week 2 post-infection (2WPI) and displayed a peak at 13WPI. F. gigantica excretory-secretory products (FgESP) analyzed by SDS-PAGE showed at least 31 bands from 12.0 to 127.6 kDa in FgESP. Western blot indicated that F. gigantica infected sheep sera recognized, in FgESP, at least 30 antigens from 7.8 to 119.2 kDa of which 12 major bands recognized after OWPI. In FhESP and FgESP, F. hepatica infected sheep serum reacted only with the lower molecular mass antigens, while F. gigantica infected sheep serum reacted with the lower and the higher molecular mass antigens. These differences of antigenic recognition might be associated with the difference of susceptibility of sheep. Further investigation must be done to study the mechanism of resistance between the sheep infected with F. hepatica or F. gigantica.     

Distribution of R-r-i-blood group system in Australian sheep

The distribution of the R-r-i blood group system was studied in eight breeds of Australian sheep. The recessive i gene was found to be absent from Merinos and, consequently, the i phenotype was absent in Merino crosses. Distribution of the groups in Dorset Horns and Ryelands was found to be strikingly different from that of other breeds. Two animals were found to have anti-r antibodies in serum.     

绵羊X染色体59383635位点多态性与脂尾性状的相关性分析

脂尾(臀)性状是绵羊逆境生存的必要性状, 其脂肪在尾臀部大量沉积的遗传特性与分子机制仍不明晰。为此, 文章以筛选的X染色体59383635位点SNP为候选分子标记, 利用PCR-SSCP技术检测该位点在我国尾型极端差异的阿勒泰羊、小尾寒羊、湖羊、中国美利奴细毛羊以及引入品种萨福克羊群体中的多态性, 并采用模型分析其与尾(臀)性状的相关性。结果表明, X染色体59383635位点T等位基因高频出现在表型分值较高的阿勒泰群体中, 而C等位基因则在瘦尾型绵羊品种中高频出现; 等位基因频率T/C的比值与尾臀表型分值相关性模型表明T/C比值随着尾臀表型分值增加呈指数倍增长。以上结果提示, 绵羊X染色体59383635位点多态性在脂尾(臀)与瘦尾绵羊群体中分布存在较大差异, 该SNP可作为一个理想的分子标记应用于高、低脂绵羊品种选育, 但其生物功能仍有待进一步深入研究。     

Merino and Merino-derived sheep breeds: a genome-wide intercontinental study

Background

Merino and Merino-derived sheep breeds have been widely distributed across the world, both as purebred and admixed populations. They represent an economically and historically important genetic resource which over time has been used as the basis for the development of new breeds. In order to examine the genetic influence of Merino in the context of a global collection of domestic sheep breeds, we analyzed genotype data that were obtained with the OvineSNP50 BeadChip (Illumina) for 671 individuals from 37 populations, including a subset of breeds from the Sheep HapMap dataset.

Results

Based on a multi-dimensional scaling analysis, we highlighted four main clusters in this dataset, which corresponded to wild sheep, mouflon, primitive North European breeds and modern sheep (including Merino), respectively. The neighbor-network analysis further differentiated North-European and Mediterranean domestic breeds, with subclusters of Merino and Merino-derived breeds, other Spanish breeds and other Italian breeds. Model-based clustering, migration analysis and haplotype sharing indicated that genetic exchange occurred between archaic populations and also that a more recent Merino-mediated gene flow to several Merino-derived populations around the world took place. The close relationship between Spanish Merino and other Spanish breeds was consistent with an Iberian origin for the Merino breed, with possible earlier contributions from other Mediterranean stocks. The Merino populations from Australia, New Zealand and China were clearly separated from their European ancestors. We observed a genetic substructuring in the Spanish Merino population, which reflects recent herd management practices.

Conclusions

Our data suggest that intensive gene flow, founder effects and geographic isolation are the main factors that determined the genetic makeup of current Merino and Merino-derived breeds. To explain how the current Merino and Merino-derived breeds were obtained, we propose a scenario that includes several consecutive migrations of sheep populations that may serve as working hypotheses for subsequent studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0139-z) contains supplementary material, which is available to authorized users.     

优良的BMPR-IB基因型可提高绵羊冷冻胚胎的受胎效果

以控制BooroolaMerino羊高繁殖力的BMPR-IB基因为候选基因,以小尾寒羊及其杂交羊、东北半细毛羊、澳洲美利奴羊、德国肉用美利奴羊、萨福克羊、特克塞尔羊、夏洛莱羊为试验对象,采用PCR-限制性片段长度多态性(PCR-RFLP)方法进行基因单核苷酸多态性(SNP)检测和基因型分析,同时研究基因对高繁殖力的影响.研究结果表明:小尾寒羊及其杂交羊、东北半细毛羊和夏洛莱羊群体中发现了与BooroolaMerino羊相同的A746G碱基突变,而小尾寒羊及其杂交羊群体的B等位基因频率明显高于其他2个品种.另外4个品种中未发现此突变.携带B等位基因的群体较非携带B等位基因群体排出更多的卵子,排卵后黄体直径较小.移植入冷冻胚胎后, 、B 和BB3种基因型群体的妊娠率分别为38.78%、45.71%和66.67%.由此推断,BMPR-IB基因突变很有可能从增加卵巢排卵数和提高胚胎着床及妊娠建立效率两个方面同时影响绵羊高繁殖力性状.所得BB型群体冻胚移植妊娠率明显高于 和B 型群体,已接近鲜胚移植水平,通过PCR-RFLP方法进行基因型分析,选用合适基因型群体作为胚胎移植受体,有可能为提高绵羊胚胎移植受胎率提供新的方向.     

The growth of sheep scab lesions in relation to sheep breed and time of the year

Numerically equal groups of between five and ten young Merino and Dorper sheep were artificially infested with sheep scab mites, Psoroptes ovis. Separate groups of sheep were infested during the early winter of two successive years as well as during the early summer of the second year. Lesion growth was measured at two-weekly intervals for a period of six weeks during the first winter and for eight weeks during the second winter and in the summer. The rate of lesion growth and mean lesion size on Merino sheep was significantly greater than that on Dorper sheep at each two-weekly recording during the winter of both years. The rate of growth of the lesions did not differ significantly between the two sheep breeds during summer, although, with the exception of eight weeks post-infestation, the mean lesion size on Merino sheep was significantly larger than that on Dorper sheep at each assessment. This revised version was published online in July 2006 with corrections to the Cover Date.     

Fasciola gigantica fatty acid binding protein (FABP) as a prophylactic agent against Schistosoma mansoni infection in CD1 mice

Although schistosomicidal drugs and other control measures exist, the advent of an efficacious vaccine remains the most potentially powerful means for controlling this disease. In this study, native fatty acid binding protein (FABP) from Fasciola gigantica was purified from the adult worm's crude extract by saturation with ammonium sulphate followed by separation on DEAE-Sephadex A-50 anion exchange chromatography and gel filtration using Sephacryl HR-100, respectively. CD1 mice were immunized with the purified, native F. gigantica FABP in Freund's adjuvant and challenged subcutaneously with 120 Schistosoma mansoni cercariae. Immunization of CD1 mice with F. gigantica FABP has induced heterologous protection against S. mansoni, evidenced by the significant reduction in mean worm burden (72.3%), liver and intestinal egg counts (81.3% and 80.8%, respectively), and hepatic granuloma counts (42%). Also, it elicited mixed IgG(1)/IgG(2b) immune responses with predominant IgG1 isotype, suggesting that native F. gigantica FABP is mediated by a mixed Th1/Th2 response. However, it failed to induce any significant differences in the oogram pattern or in the mean granuloma diameter. This indicated that native F. gigantica FABP could be a promising vaccine candidate against S. mansoni infection.     

Resistance to experimental infection with Fasciola hepatica in exotic and domestic breeds of sheep

Significant breed differences were detected in fecal egg counts and fluke counts following experimental infection of five breeds of sheep with metacercariae of F. hepatica. Two experiments compared the responses of Barbados Blackbelly, St. Croix, Florida Native, Finn X Rambouillet and Targhee sheep to primary and challenge infections (Expt. 1) and to multiple infections (Expt. 2). Barbados Blackbelly sheep were more susceptible to infection than the other breeds in both experiments. St. Croix and Florida Native sheep were the most resistant breeds in Expt. 1, while Targhee and Florida Native were the most resistant in Expt. 2. There was no evidence of acquired immunity in any breed in either experiment.     

Fasciola gigantica cathepsin L proteinase-based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis

Sheep fasciolosis is a devastating burden for the livestock industry. We herein report on immunodiagnosis of fasciolosis, and significant protection of sheep against challenge infection with Fasciola gigantica following immunization with a peptide based on the H-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH (Fas14p) sequence of F. gigantica cathepsin L-cysteine proteinase. This sequence was synthesized in three different forms: as N(alpha) acetylated (Ac-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAc14p), bearing at the amino-terminus an N(alpha) acetylated cystein (Ac-Cys-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAcCys14p), and conjugated to sequential oligopeptide carrier Ac-[Lys-Aib-Gly](4)-OH (Ac-SOC(4)) through an amide bond formed between Val(123) carboxylic group of the epitope and the lysine N(epsilon) groups of the carrier (Ac-[Lys(Fas14p)-Aib-Gly](4)-OH). Ac-[Lys(Fas14p)-Aib-Gly](4)-OH failed to readily discriminate between na?ve and infected sheep. In contrast, the free peptides reproducibly differentiated between parasite-free sheep, sheep infected with parasites other than Fasciola, and experimentally Fasciola-infected sheep. The data together indicated that the peptides might be of considerable use for discriminating between early and late, and low and high burden, sheep infection with F. gigantica. FasAc14p was chosen to determine whether a peptide based on a critical enzymatic site of cathepsin L proteinase may induce protection against challenge infection. Sheep immunization with FasAc14p peptide induced significant expression of interleukin-4 mRNA, and humoral antibodies that bound to molecule(s) on the intact surface membrane of newly excysted juvenile worms, and mediated their attrition. The immune responses were associated with significant (P < 0.02) decrease of 23.1% in worm recovery, but with no decrease in the size or maturation of worms recovered.     

Boophilus microplus: the pattern of bovine immunoglobulin isotype responses to high and low tick infestations

Cattle present variable levels of resistance to ticks and the immune correlates of these heritable phenotypes must be known in order to develop effective vaccines. The antibody responses to tick salivary antigens were examined in cattle of tick-susceptible (Holstein) and tick-resistant (Nelore) breeds. After heavy infestations, levels of IgG1 and IgG2 antibodies decreased in Holsteins and remained the same in Nelores. Conversely, levels of IgE antibodies increased in Holsteins. Different sizes of tick burdens modulated the IgG1 antibody response in a susceptible breed (Aberdeen): levels were higher than in controls in heavily infested animals, but not in those undergoing intermediary or minimal infestations. The three experimental groups presented similar levels of IgG2 antibodies. Levels of IgE antibodies were higher only in animals undergoing intermediate infestations. These results indicate that tick infestations suppress the IgG antibody response in susceptible breeds, that IgE antibodies are not protective, and that the dose of tick saliva modulates the isotype of host antibody responses.     

Polymorphisms of coding region of BMPR-IB gene and their relationship with litter size in sheep

The bone morphogenetic protein receptor IB (BMPR-IB) was studied as a candidate gene for the prolificacy of sheep. Nine pairs of primers (P1-P9) were designed to detect single nucleotide polymorphisms (SNPs) of exons 1-4 and 6-10 of the BMPR-IB gene in both high (Small Tail Han and Hu sheep) and low prolificacy breeds (Texel and Chinese Merino sheep) by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP). Only the products amplified by primers P2, P5, P6, P7, P8 and P9 displayed polymorphisms. The present study identified 22 SNPs in partial coding regions of ovine BMPR-IB, in which 20 SNPs were reported for the first time. In total of the 22 mutations, 18 DNA variations were originated from the Hu breed, three were found in the Small Tail Han breed (two of them were found in other sheep breeds), three in the Chinese Merino breed, and none in the Texel breed. These results preliminarily demonstrated that BMPR-IB is a major gene affecting the hyperprolificacy in Small Tail Han and Hu sheep, and could be used as a molecular genetic marker for early auxiliary selection for hyperprolificacy in sheep.     

Studies of stage-specific immunity against Trichostrongylus colubriformis in sheep: immunization with adult parasites.

Merino sheep immunized by the adoptive transfer of adult T. colubriformis for 8 weeks were significantly protected against a challenge infection of 20,000 larvae. Two additional groups of sheep received a primary infection of 9000 adult worms which were allowed to persist for 14 weeks before one group was drenched. When both groups were challenged 10 days later with 30,000 larvae, serial necropsies of these and naive sheep revealed that worm rejection did not occur until 7-10 days after challenge. By comparison with the rapid rejection of larval challenges from sheep immunized with normal primary infections, the results suggest that the antigens which elicited rejection in these experiments are stage-specific and were only present or synthesized in sufficient quantities when parasites had developed for 1 week.     

Protective role of purified cysteine proteinases against Fasciola gigantica infection in experimental animals

Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG(1), and IgG(2) (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-γ, and TNF-α, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-β, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships.     

Genetic polymorphism in sheep plasma detected using antibodies to human plasminogen

Protein variation was identified in sheep when Western blots of polyacrylamide gels (routinely used to resolve transferrin polymorphism) were stained using antibodies to human plasminogen. The affinity of the antibodies to ovine plasma was less than 7% that of a human standard but they bound specifically to a single polymorphic protein. In 146 lambs and their parents the inheritance of the ovine plasminogen antigen polymorphism was consistent with four autosomal alleles segregating codominantly. However, an additional two lambs had types which were incompatible with their putative parents. The pedigrees of these lambs were tested by DNA fingerprinting and shown to have been incorrectly recorded. The genetic polymorphism detected by human plasminogen antiserum provided a probability of sire exclusion (PE) ranging from 0.04 to 0.32 and a polymorphic information content (PIC) of 0.08 to 0.50 in flocks of five sheep breeds: Perendale, Romney, Merino, Texel and Coopworth (in order of increasing genetic variation in this locus). Significant differences in allele frequency were observed between breeds but sampling did not assess the variation among flocks within a breed.     

B cell responses to paramyosin. Isotypic analysis and epitope mapping of filarial paramyosin in patients with onchocerciasis.

To examine the fine specificity of the human immune response to filarial paramyosin, the antigenicity of an expressed rcDNA (2.55 kb) of Dirofilaria immitis paramyosin was detailed by ELISA. Using sera from patients infected with Onchocerca volvulus, we analyzed both the entire paramyosin molecule and six subcloned fragments for their IgG, IgG subclasses, and IgE responses. Patients from both Guatemala (64% positive) and Ghana (100% positive) reacted to paramyosin with specific IgG levels above normal controls. Although there was no anti-paramyosin subclass restriction common to all patients, the IgG3 response in the Ghananians was significantly greater than that of Guatemalans (p less than 0.001). IgE anti-paramyosin responses showed positive correlations with IgG2 (p less than 0.001), IgG4 (p less than 0.002), and IgG1 (p less than 0.04) responses. Epitope mapping using the IgG response to the six subclones demonstrated preferential recognition of the amino terminal end of the molecule (nucleotides 1 to 360). IgG2 reactivity was clearly localized to the most amino-terminal 120 amino acids, and the IgG4 antibodies recognized amino acids immediately adjacent to this fragment. These studies examining the fine specificity of anti-filarial immune reactions should provide a method for understanding how parasites either evade or induce host immune responses.     

Genetic relationships among Spanish sheep using microsatellites

Five indigenous Spanish breeds of sheep, Churra, Latxa, Manchega, Rasa–Aragonesa and Merino, with Awassi sheep as a reference breed were genotyped for 19 DNA microsatellites. Allele frequencies and mean heterozygosities revealed the greatest genetic variation in Merino sheep and the lowest in Awassis. Differences in variability were not great in the other breeds studied. The dendrograms obtained based on genetic distances showed a large differentiation between Awassi sheep and the Spanish breeds, as was to be expected from their distinct genetic origin. Merinos appeared separated from the other four breeds, of which, according to a classification based on the fleece characteristics, Churra and Latxa belong to the churro type and Manchega and Rasa–Aragonesa to the so called entrefino type, though no clear separation was evident between the two types. These results suggest that morphological data alone are insufficient for determining relationships between breeds and that studies involving genetic markers may be of great assistance.