Molecular characterization of MHC class II region in guinea fowl

1. The MHC class II gene was amplified, cloned and sequenced in guinea fowl. 2. The NumeMHC II sequence of 754 nucleotides included complete exon 1 (91 nt), exon 2 (270 nt), exon 3 (282 nt) and exon 4 (110 nt). 3. The size of β(1) and β(2), domains were 89 and 93 amino acids, respectively in guinea fowl. 4. High amino acid variability (38·2%) was observed within guinea fowl in β(1) domain, while in β(2) domain, amino acid variability (6·3%) was low. 5. Among poultry species, the percent amino acid identity between guinea fowl and chicken, quail, pheasant and duck was 38·8, 42·2, 44·4 and 58·8 in β(1) domain; and 13·8, 17·0, 13·8 and 27·6 in β(2) domain, respectively. 6. Sequence alignment with mammalian and avian MHC showed that many of the conserved features of MHC class II glycoprotein was conserved in guinea fowl. 7. Within-species genetic distances (Poisson correction) based on cumulative amino acid variability in β(1) domain and β(2) domains was 0·141 in guinea fowl. 8. Guinea fowl showed low and similar genetic distances with all the poultry species (0·255-0·268) except duck (0·456). 9. Guinea fowl made separate branch within the major cluster having chicken, quail and pheasant, showing equal distance from these poultry species, whereas duck MHC II clustered separately.     

Monoclonal antibodies to sheep MHC class I and class II molecules: biochemical characterization of three class I gene products and four distinct subpopulations of class II molecules

The availability of a panel of monoclonal antibodies to sheep MHC class I and class II gene products has allowed for the first time an assessment of the relative complexity of the sheep MHC. By using four monoclonal antibodies to MHC class I, and seven monoclonal antibodies to MHC class II molecules together with one-dimensional SDS-PAGE, sequential immunoprecipitation and 2-dimensional gel analysis, three class I gene products and four distinct subsets of class II molecules have been identified. Sheep class I molecules showed heterogeneity on 2-dimensional gels and as in mouse and man, represented the products of at least three different non-allelic class I genes. Interestingly, the sheep beta 2 microglobulin molecule also displayed heterogeneity, consistent with either two primary gene products or allelic variation. Four sheep class II monoclonal antibodies identified distinct, non-overlapping subsets of sheep class II molecules of Mr 32-36 K (alpha chain) and 25-28 K (beta chain). These class II molecules were co-expressed on sheep B lymphocytes and represented the primary products of different sheep MHC class II genes. The class II molecules within three of these subsets displayed allelic polymorphism essentially restricted to their beta polypeptides, while the fourth subset of class II molecules showed allelic variation in both their alpha and beta polypeptides. The results of this study represent the first evidence for gene duplication and heterogeneity within the sheep MHC. The identification of three primary class I gene products and four distinct subsets of class II molecules suggests three class I loci and up to four distinct class II subregions within the sheep MHC. Potentially large numbers of allelic variants of these different gene products may be expressed in normal sheep.     

泰州地区猪肠道寄生虫感染情况调查

为了解泰州地区猪肠道寄生虫感染情况,共采集泰州3个地区(泰兴、姜堰、兴化)1206份猪新鲜粪便样品,通过饱和食盐水漂浮法、饱和蔗糖液漂浮法进行检测。结果:阳性粪便数为327份,占总调查粪便样品数的27.1%;小型猪场感染率为50.1%(236/471),规模化猪场感染率为12.4%(91/735)。泰州地区共检测到7种肠道寄生虫,分别是蛔虫、毛尾线虫、球虫、小袋纤毛虫、隐孢子虫、食道口线虫、类圆线虫,其中,泰兴地区7种寄生虫感染率分别是6.2%、2.7%、6.2%、7.2%、8.7%、2.5%、1.0%;姜堰地区7种寄生虫感染率分别是8.3%、0.7%、5.6%、7.8%、5.4%、1.5%、0.5%;兴化地区7种寄生虫感染率分别是2.8%、0.5%、8.8%、9.1%、3.3%、1.0%、0%。结果表明:小型猪场感染率比规模化猪场高;小型猪场蛔虫、球虫、小袋纤毛虫、隐孢子虫感染率较高;规模化猪场球虫、小袋纤毛虫、隐孢子虫等原虫感染率较高。     

Cytokine regulation of resistance and susceptibility to intestinal nematode infection - from host to parasite

Intestinal nematode infection is one of the most common forms of parasite infection worldwide. Both man and domestic stock suffer considerable morbidity from these infectious agents. The majority of our current understanding of the host parasite relationship to gut dwelling nematodes comes from well-defined laboratory models. One of the most informative over recent years has been study of whipworm infection in the mouse, Trichuris muris (T. muris). Infection in inbred strains of mouse shows a spectrum of response phenotypes reflecting the variation observed under natural conditions in the wild. Resistance and worm expulsion is mediated by CD4+ T helper two cells with a dominant role for interleukin (IL)-13. The effector mechanisms responsible for worm expulsion remain undefined but new evidence suggests a role for tumour necrosis factor alpha (TNF-alpha). Susceptibility to chronic infection is mediated through a T helper 1 (Th1) response characterised through the secretion of interferon gamma (IFN-gamma). A major new role for IL-18 has been defined in induction of a Th1 response through a novel down-regulation of IL-13. Moreover, progression to chronic infection may involve the parasite itself. T. muris secretes a protein that shares epitopes with host IFN-gamma, which may interfere with host protective cytokine, mediated protection and thus, promotes its own survival.     

Detection of porcine MHC class II antigens in different immunoassays

A monoclonal antibody (Mab), Mab 4-24-11, to human class II major histocompatibility antigens has been tested in commonly used immunoassays for detection of porcine class II major histocompatibility antigens (SLA-D). In a radioimmunoprecipitation assay, Mab 4-24-11 reacted with proteins from mitogen-stimulated porcine mononuclear cells (MNC). The molecular weights of the precipitated proteins were approximately 34 and 28 Kd. In indirect immunofluorescence, approximately 25% of porcine MNC in suspension reacted with Mab 4-24-11. This percentage diminished to 14% when Ig bearing MNC were removed, while it increased to 36% when adherent MNC were enriched. Thus, it was concluded that Mab 4-24-11 cross-reacts with SLA-D. In a immunohistochemical study, Mab 4-24-11 reacted with cells in acetone fixed cryostat sections from the gastric mucosa and endometrium. These properties of Mab 4-24-11 make it useful as a tool for further studies on the porcine immune system.     

MHC class II expression in the bovine mammary gland.

The distribution of major histocompatibility complex (MHC) class II positive cells within the connective tissue and the epithelium of the involuted bovine mammary gland has been determined. The effect of intramammary administration of the antigens ovalbumin and formalin killed Streptococcus uberis on the distribution pattern has also been investigated. Infusion of formalin killed S. uberis increased cellular expression of class II antigens when compared with quarters either infused with ovalbumin, not infused at all, or from which minor pathogens had been isolated. The increased expression occurred particularly in the area of the gland cistern-secretory tissue junction.     

Benzimidazole resistance in Trichostrongylus axei in sheep: long-term monitoring of affected sheep and genotypic evaluation of the parasite

This is the first report of benzimidazole (BZ) resistance in the nematode Trichostrongylus axei in sheep. Trichostrongylus axei infects several species of herbivores including sheep, cattle and horses, and the emergence of anthelmintic resistance could lead to significant problems in its control. Benzimidazole resistance in two sheep flocks in central France was detected by post-treatment worm counts. The sequencing of a central region of the isotype 1 beta-tubulin gene from adult T. axei recovered post-mortem revealed only one, non-synonymous single nucleotide polymorphism at position 200 (Phe200Tyr), which had already been reported for other nematodes. Seven years after BZ treatment ceased, T. axei helminths present were still resistant to BZ suggesting these parasites do not revert to susceptibility to this anthelmintic, even when the selection pressure had been removed for many years. The findings also highlight major changes in the make-up of the nematode burden in sheep flocks that accompanies the emergence of BZ resistance.     

Requirement for MHC class II positive accessory cells in an antigen specific bovine T cell response

Purified populations of bovine antigen presenting cells (APCs) and T cells have been isolated from peripheral blood and characterised using various monoclonal antibodies (mAbs) for cell surface markers. Bovine APCs were found in an adherent cell fraction and were non-specific esterase positive, phagocytic and expressed bovine major histocompatibility complex (MHC) class II determinants, all of which are typical macrophage characteristics. T cells were rigorously depleted of accessory cell function before being used in an antigen presenting cell assay. The generation of T helper cells in response to the soluble antigen, ovalbumin, was entirely dependent upon a critical number of APCs. Further the proliferative response was inhibited by several mAbs to bovine MHC class II molecules. Thus the interaction between bovine APCs and helper/inducer T lymphocytes (TH/I) appears to be similar to that in other species.     

Survey of anthelmintic resistance in Western Australian sheep flocks 2. Relationship with sheep management and parasite control practices

Owners of 116 farms, whose flocks had been tested for anthelmintic resistance, were interviewed to determine their use of various sheep management and parasite control practices and their knowledge and adoption of recommended procedures for the prevention and control of resistance. Farmers knowledge of current recommendations related mainly to changing drenches and drench groups. Other aspects of the recommended program including reduction of drenching frequency and the use of alternative management strategies were not considered as important by farmers. For most questions a high proportion of farmers (greater than 20%) had no opinion. Associations between various strategies for nematode control and resistance of Trichostrongylus and Teladorsagia to thiabendazole and levamisole were examined. These relationships differed between anthelmintics and nematode genera. A number of factors were related to resistance of one or both nematode genera to one or both anthelmintic groups. These factors included flock size, percentage of ewes in the flock, cattle number, main sheep production activity, grazing strategy, frequency of drenching, changes in the frequency of drenching, number of summer drenches and the method of estimating dose rates. It was concluded that the methods employed to control anthelmintic resistance may vary with the nematode, its resistance status and the anthelmintic to which it is exposed. Modifications to the previously recommended program have been proposed which incorporate selection of the anthelmintic to be used following a test for anthelmintic resistance.     

Further studies on anthelmintic resistance in sheep at an organised farm in arid region of Rajasthan

Faecal egg count reduction test (FECRT) and in vitro egg hatch assay (EHA) was performed on Indian Karakul (3/4 crosses of Karakul with Malpura, Marwari and Sonadi) transported from arid region campus, Bikaner, where resistance was reported in 1996. FECRT revealed that fenbendazole and levamisole were 100% effective in reducing the egg counts. LC50 value on EHA was 0.074 +/- 0.015 microg thiabendazole ml(-1). The faecal culture examination revealed the presence of Haemonchus contortus only. It was concluded that H. contortus was fully susceptible to both benzimidazole and levamisole.     

寄生虫的抗药性管理

抗药性在畜禽寄生虫中普遍存在，且具有一些共同的特征。了解这些共同特征将会为寄生虫的抗药性管理提供一些新观点、新思路。本文着重讨论了寄生虫的抗药性问题，并对其共同特征做了描述；同时也论述了寄生虫综合管理方案，旨在通过总结一种寄生虫的综合管理方案，为其他寄生虫病的控制开辟一条新途径。     

MHC class II expression by follicular keratinocytes in canine demodicosis--an immunohistochemical study

MHC class II proteins present fragments of extra cellular antigen to stimulate CD4(+) T lymphocytes. Aim of this study was the detection of MHC class II antigens on different cutaneous cells in canine demodicosis. Histopathological and immunohistochemical examination of skin biopsies from 44 dogs with demodicosis is reported. The control group consisted of skin biopsies taken from 10 necropsied dogs without obvious skin lesions. The immunohistological assessment of the MHC class II expression revealed MHC class II proteins on different cell types of infiltrating inflammatory cells, i.e. APCs (antigen-presenting cells), macrophages, T lymphocytes and B lymphocytes. The plasma cells, however, only showed expression in 32 (73%) of 44 cases. Generally it was noticeable that most plasma cells but never all of them expressed MHC class II. Neutrophils, mast cells and eosinophils were MHC class II negative. Furthermore, in 39 biopsies (89%) from dogs with demodicosis MHC class II positive follicular keratinocytes were found. The control group did not show MHC class II expression on epithelial cells. Concerning the endothelial cells, a total of 25 biopsies (57%) showed MHC class II expression in which different vascular plexuses were affected by staining. This examination shows that MHC class II expression in the skin of dogs suffering form demodicosis is elevated. Especially the MHC class II expression by follicular keratinocytes seems to be conspicuous. We hypothesize that this is in association with the development and the maintenance of follicular inflammation.     

Disease resistance in Merino sheep. II. RFLPs in Class IIMHC and their association with resistance to footrot

SUMMARY: Restriction fragment length polymorphism (RFLP) within the Major Histocompatibility Complex (MHC) Class II region was examined in 83 sheep. Two restriction enzymes (TaqI and PvuII) and two human cDNA probes (DQα-folke and DRβ-malta) were used to probe Southern blots from all sheep. In addition, a second human DRβ probe (signe) and the restriction enzyme, EcoRI, were used for 15 sheep to match previously published polymorphism in sheep using the same probe/enzyme combination. All sheep were part of an experiment in which footrot was experimentally induced and then controlled through vaccination with an homologous rDNA pilus vaccine. The four main probe/enzyme combinations detected extensive polymorphism; in total 74 bands were detected of which only 2 were present in all sheep. A similar level of polymorphism was also seen with the EcoRI/DRβ-signe probe/enzyme combination, which was greater than previously published for sheep, cattle or goats. After accounting for cross-hybridising bands, the sheep DRβ region appeared to be more polymorphic than the DQα region. Similarly the TaqI restriction enzyme revealed more polymorphism than the PvuI enzyme (40 and 34 bands respectively). For all 74 bands, 7 main clusters were identified both within and across probe/enzyme combinations. After removing bands/clusters which were of extreme frequency (< 0.10 and > 0.90), a total of 53 bands/clusters were analysed for their potential association with footrot and antibody responses. One band was significantly associated with susceptibility to footrot over the 27-week period during which footrot was measured. In addition, two single bands and one cluster were significantly (P > 0.001) associated with residual footrot infection after vaccination. Nine bands/clusters were associated with antibody titre during infection, and one cluster with antibody titre after vaccination. These results suggest a possible involvement for genetic polymorphism within the ovine Class II region in variation in response to footrot. ZUSAMMENFASSUNG: Krankheitsresistenz bei Merinos, II. RFLP's in Klasse II MHC und ihre Verbindung mit der Moderhinkeresistenz Restriktionsfragmentl?ngenpolymorphismus (RFLP) innerhalb des Haupthistokompatibilit?tskomplexes (MHC) Klasse II Region wurde bei 83 Schafen untersucht. Zwei Restriktionsenzyme (TaqI und PvuII) und zwei humane cDNA-Sonden (DQα-folke und DRβ-malta) wurden für Southern blots bei allen Schafen verwendet. Zus?tzlich wurde eine zweite humane DRβ-Sonde (signe) und das Restriktionsenzym EcoRI bei 15 Schafen zum Vergleich mit früher publizierten Polymorphismus mit den selben Enzym/Sondenkombinationen durchgeführt. Alle Schafe waren Teil eines Versuches, in welchem Moderhinke experimentell induziert und dann durch Vaccination mit homologer rDNA-pilus vaccine bek?mpft worden war. Die 4 Enzym/Sondenkombinationen entdeckten extensiven Polymorphismus. Insgesamt 74 Banden wurden entdeckt, wovon nur zwei in allen Schafen vorhanden waren. Ein ?hnliches Ausma? von Polymorphismus wurde auch mit der EcoRI/DRβ-signe Enzym/Sondenkombination entdeckt, das gr??er war als bisher für Schafe, Rinder oder Ziegen publiziert. Nach Berücksichtigung der Kreuz-Hybridisierungsbande scheint die Schaf-DRβ-Region st?rker polymorph als die DQα-Region zu sein. ?hnlicherweise zeigte das TaqI Restriktionsenzym mehr Polymorphismus als das PvuII Enzym (40 bzw. 34 Bande). Bei allen 74 Banden wurden sieben Hauptkluster identifiziert, sowohl innerhalb als auch über Enzym/Sondenkombinationen. Nach Entfernung von Banden/Kluster mit extremen H?ufigkeiten (< 0,10 bzw. > 0.9) wurden insgesamt 53 Bande/Kluster hinsichtlich ihrer potentiellen Verbindung mit Moderhinke und Antik?rperreaktion analysiert. Ein Band war signifikant mit Moderhinkeempfindlichkeit w?hrend der 27-Wochenperiode assoziiert. Zus?tzlich waren zwei einzelne Bande und ein Kluster signifikant (p > 0,001) assoziiert mit Moderhinkeinfektion nach der Impfung. 9 Band/Kluster waren mit Antik?rperspiegel w?hrend der Infektion verbunden und ein Kluster mit Antik?rperspiegel nach Impfung. Die Ergebnisse deuten auf Zusammenhang des genetischen Polymorphismus innerhalb der ovinen Klasse II-Region mit Variabilit?t im Moderhinkeverlauf.     

MHC class II expression by mast cells in the genital tract of cows

This work examined the presence of MHC class II molecules expressing mast cells in oviduct, uterus and vaginal tissues in cows. The tissue samples of five cows were collected from a local slaughterhouse. Toluidine blue pH 0.5 (TB) and avidin-biotin peroxidase complex (ABC) staining procedures applied to adjacent sections from tissue samples. It was determined that some TB + cells were also gave positive reaction with strept ABC staining for MHC II molecules. To our knowledge this is the first evidence indicating the presence of MHC class II molecules expressing mast cells in the cow.     

Genomic tools to improve parasite resistance.

The natural genetic variability of the ruminant immune system provides a feasible means to control gastrointestinal (GI) parasite infection without anthelmintics. However, the paradigm of traditional selection has not been effectively applied to the moderately heritable traits of parasite resistance (h approximately equal to 0.3) due to the difficulty and expense of gathering accurate phenotypes in a commercial production setting. These characteristics make host traits related to GI nematode infection ideal candidates for genomics-based research. To initiate explanation of important allelic differences, economic trait loci (ETL) are being identified and mapped using a resource population of Angus cattle segregating for GI nematode resistance and susceptibility to the two most common nematode parasites of US cattle, Ostertagia ostertagi and Cooperia oncophora. The population is composed of five generations of half-sib progeny with complete phenotypic records produced from controlled infections. To detect the genomic locations of the three distinct phenotypic traits being expressed (innately immune, acquired immune, and immunologically non-responsive), genotypes have been generated for DNA markers (N=199) spaced at regular intervals (approximately 20cm intervals) throughout the entire genome (3000cm). Although initial ETL detection may be limited by half-sib family size, the unique structure of this population provides additional statistical power for refining map position of potential ETL. After allele frequency and contribution to phenotype are determined in this population, marker tests associated with ETL most beneficial for controlling parasite infection can be accurately used for selection. Comparative map and functional genomic information from humans and other species of biomedical importance will be utilized in further investigations to elucidate the genes underlying ETL.     

Multigeneric resistance to benzimidazole anthelmintics in sheep

Benzimidazole resistance involving several gastrointestinal nematode genera on a single sheep farm in New Zealand is reported for the first time. A controlled slaughter trial showed that at the recommended dose rate of 12.5 mg/kg, mebendazole had efficacies of 0, 60, 66, 90, 54 and 38% against Haemonchus contortus, Ostertagia spp., Nematodirus spp., intestinal Trichostrongylus spp., Strongyloides spp. and Oesophagostomum venulosum, respectively. The relevance of such multigeneric resistance to possible future options for controlling anthelmintic resistant sheep trichostrongylids is briefly discussed.