A. Van Zeveren

Monitoring the efficacy of ivermectin and albendazole against gastro intestinal nematodes of cattle in Northern Europe

Faecal egg count reduction tests (FECRT) using ivermectin (IVM) and benzimidazole (BZ) were conducted to investigate the prevalence of anthelmintic resistance in gastro-intestinal nematodes on cattle farms in Germany, Belgium and Sweden in 2006 and 2007. Based on sufficient numbers of eggs prior to the study, between 3 and 10 farms per country were selected. 10-15 animals were randomly selected per farm and subcutaneously treated with 0.2 mg IVM/kg bodyweight (Ivomec, Merial). Faecal samples were collected individually from every animal on day 0 (treatment), day 7 (Belgium & Sweden) or 14 (Germany), and day 21 (Germany, Belgium and Sweden). Faecal egg counts (FEC) were performed at each sampling occasion to estimate the eggs per gram of faeces (EPG) and the reduction of eggs after treatment. The FECRT using IVM in 2006 revealed mean reduction of egg counts between 69-100% on day 7/14 (95% confidence interval (CI) 19-102) and 35-96% (95% CI 0-102) on day 21. Farms with a suggested problem of anthelmintic resistance have been re-visited in 2007 and except for one case all results obtained in 2006 were confirmed in 2007. Larvae obtained from faecal cultures were identified using microscopic identification keys or genus-specific real time PCR. Cooperia oncophora was the predominant species detected after treatment, but Ostertagia ostertagi was found in samples on 3 farms in Germany and 3 farms in Sweden post-treatment. In 2007 additionally a FECRT using benzimidazoles was conducted in Germany and Sweden. In Germany oral Valbazen (albendazole, 10%, Pfizer) was used at a concentration of 7.5 mg albendazole/kg bodyweight; in Sweden Valbazen Vet (albendazole, 10%, Orion Pharma) at a dose of 8 mg/kg was used. For benzimidazoles an efficacy of 100% was obtained on all tested farms in both countries. This is the first report of a multinational anthelmintic efficacy investigation in cattle in Europe. The results suggest that testing of anthelmintic efficacy should be performed more intensively due to possible insufficient efficacy of current drugs.

Influence of oxygen tension on apoptosis and hatching in bovine embryos cultured in vitro.

Various oxygen tensions are employed for in vitro embryo production. Since it is known that oxygen tension can influence the efficiency of embryo production and embryo quality, the aim of our study was to define an optimal oxygen concentration for bovine embryo production in vitro in synthetic oviduct fluid (SOF). Embryo quality criteria were hatching ability and the degree of apoptosis as assessed by TUNEL staining and Bax gene expression. In Experiment 1, the effects of 2, 5 and 20% O(2) tensions on embryo development were compared. The highest rate of eight-cell embryos (47%) at 72 hpi was obtained under 20% O(2). However, it seemed that 2 and 5% O(2) were also suitable as assessed by embryo survival rates at 144 hpi (29 and 30% at morula stage), 168 hpi (21 and 19% at blastocyst stage) and 216 hpi (14 and 17% at hatched blastocyst stage). In Experiment 2, comparisons were made between effects of 5, 20% and alternating O(2) (20% O(2) to 72 hpi and then changed to 5% O(2) up to 216 hpi) on embryo development. Alternating the O(2) tension significantly reduced the number of hatching blastocysts to 7%. Staining with TUNEL revealed that apoptosis occurred in all tested hatched blastocysts, but a significantly lower apoptotic cell ratio was found in embryos cultured under 5% O(2) (P<0.05). Total cell number of embryos cultured under 5% and alternating oxygen was significantly higher than that of other groups (P<0.05). Bax gene expression was detected by means of RT-PCR in only 2 of 66 hatched blastocysts. It can be concluded that 5% oxygen is optimal for bovine embryo culture in cell free media. Moreover, it is very likely that the apoptosis detected by TUNEL staining in this study is Bax-independent.

A detailed physical map of the porcine major histocompatibility complex (MHC) class III region: comparison with human and mouse MHC class III regions.

A detailed physical map of the porcine MHC class III region on Chr 7 was constructed with a panel of probes in a series of hybridizations on genomic pulsed field gel electrophoresis (PFGE) Southern blots. A precise organization of the 700-kb segment of DNA between G18 and BAT1 can now be proposed, with more than 30 genes mapped to it. Comparison of this region with homologous regions in human and mouse showed only minor differences. The biggest difference was observed in the CYP21/C4 locus with only one CYP21 gene and one C4 gene found, whereas in human and mouse these genes are duplicated. These results show the class III region is very well conserved between pig, human, and mouse, in contrast with the class I and class II regions, which seem more prone to rearrangements.

Integration of porcine chromosome 13 maps

In order to expand the comparative map between human chromosome 3 (HSA3) and porcine chromosome 13 (SSC13), seven genes from HSA3 were mapped on SSC13 by fluorescence in situ hybridisation (FISH), viz. ACAA1, ACPP, B4GALT4, LTF, MYLK, PDHB and RARB. With a view to integrating this expanded comparative map with the existing SSC13 linkage map, we used the INRA-University of Minnesota porcine Radiation Hybrid panel (IMpRH) to localize more precisely and to order 15 genes on the SSC13 map, viz. ACPP, ADCY5, APOD, BCHE, CD86, DRD3, GAP43, PCCB, RAF1, RHO, SI, TF, TFRC, TOP2B and ZNF148. In this way, we were able to create an integrated map, containing 38 type I and 81 type II markers, by correlating the linkage, radiation hybrid (RH) and cytogenetic maps of SSC13. This integrated map will give us the opportunity to take maximal advantage of the comparative mapping strategy for positional candidate cloning of genes responsible for economically important traits.

Porcine PPARGC1A (peroxisome proliferative activated receptor gamma coactivator 1A): coding sequence, genomic organization, polymorphisms and mapping

We report here the characterisation of porcine PPARGC1A. Primers based on human PPARGC1A were used to isolate two porcine BAC clones. Porcine coding sequences of PPARGC1A were sequenced together with the splice site regions and the 5′ and 3′ regions. Using direct sequencing nine SNPs were found. Allele frequencies were determined in unrelated animals of five different pig breeds. In the MARC Meishan-White Composite resource population, the polymorphism in exon 9 was significantly associated with leaf fat weight. PPARGC1A has been mapped by FISH to SSC8p21. A (CA)n microsatellite (SGU0001) has been localised near marker SWR1101 on chromosome 8 by RH mapping and at the same position as marker KS195 (32.5 cM) by linkage mapping. The AseI (nt857, Asn/Asn489) polymorphism in exon 8 was used to perform linkage analysis in the Hohenheim pedigrees and located the gene in the same genomic region. Transcription of the gene was detected in adipose, muscle, kidney, liver, brain, heart and adrenal gland tissues, which is in agreement with the function of PPARGC1A in adaptive thermogenesis.

Altered avr-14B gene transcription patterns in ivermectin-resistant isolates of the cattle parasites, Cooperia oncophora and Ostertagia ostertagi☆

Ivermectin (IVM) resistance is an emerging problem for the control of gastrointestinal nematodes of cattle such as Cooperia oncophora and Ostertagia ostertagi. Although there is still a poor understanding of the molecular basis of macrocyclic lactone (ML)-resistance, it is clear that IVM exerts its activity by binding to glutamate-gated chloride (GluCl) channels within the parasites neuromuscular system. One of the GluCl genes (avr-14) encodes, via alternative splicing, two subunits, AVR-14A and AVR-14B; the latter is suggested to be the main target for IVM. The genomic DNA (gDNA) sequence of avr-14 in C. oncophora contains 21 exons separated by 20 introns and spans approximately 10 kb of gDNA. Intron 13 contains a sequence with high homology to a mammalian mariner transposase. The L256F polymorphism in the avr-14 gene, which was shown to be associated with IVM resistance in a UK isolate of C. oncophora, was not found in the IVM-resistant C. oncophora and O. ostertagi isolates investigated in this study. However, genetic analyses on C. oncophora indicated a loss in allelic diversity of the avr-14 gene in the resistant isolates compared with the susceptible isolate. This suggests that the avr-14 gene, or another genetically linked locus, is under selection in these Belgian C. oncophora isolates. Comparison of the full-length avr-14B coding sequence in the susceptible and resistant C. oncophora isolates did not show any polymorphisms specifically linked to IVM resistance, although a decrease in the number of avr-14B isoforms was observed in the resistant isolates compared with the susceptible one. Measuring the transcription levels of avr-14B in adult male and female C. oncophora and O. ostertagi worms showed significantly lower levels in resistant worms compared with susceptible ones. Whether the down-regulation of this IVM target actually contributes to the resistance mechanism in these worms remains unclear.

Protein disulphide isomerase of Ostertagia ostertagi: an excretory-secretory product of L4 and adult worms?

A pepstatin A-agarose column was used in an attempt to purify a previously described antibody-degrading aspartyl proteinase from excretory-secretory material from the L4 and the adult stages of the bovine abomasal nematode Ostertagia ostertagi. However, no aspartyl proteinase activity was detected in the eluted fractions (L4Pepst and AdPepst). Screening of cDNA libraries with polyclonal antibodies raised against L4Pepst and AdPepst showed that a protein disulphide isomerase (Ost-PDI2) was present in both antigen fractions. This multifunctional enzyme was detected in extracts of L3, L4 and adult parasites and, interestingly, also in excretory-secretory material of L4 and adult O. ostertagi. By immunohistochemistry, the Ost-PDI2 enzyme was localised in some parts of the hypodermis of L4 and adult worms and in the intestinal cells of all three parasitic life stages. Two-dimensional Western blot analysis indicated that Ost-PDI2 is recognised by calves during a natural O. ostertagi infection, which suggests that Ost-PDI2 could be used for immunological control of ostertagiosis.

Comparative mapping between human chromosome 3 and porcine chromosome 13

Zoo-FISH and somatic cell hybrid panels have earlier demonstrated extended synteny conservation between human chromosome 3 (HSA3) and pig chromosome 13 (SSC13). In the present study, eight human genes viz., ADCY5, CASR, COL7A1, COL8A1, ITIH1, RHO, SIAT1 and XPC, spread along the length of HSA3, were chosen for expanding the comparative map between the two chromosomes. Using human and rat cDNAs, or human- and porcine-specific PCR products as probes, 8 porcine lambda clones were isolated. After subcloning and partial sequence determination, identity of the clones with regards to the specific genes was established. The eight type 1 markers thus obtained were biotin labeled and FISH mapped to pig metaphase spreads. All lambda clones localized to SSC13. In combination with the hitherto published mapping data of coding sequences on SSC13, a preliminary comparative status depicting the relative organization of this chromosome with respect to HSA3 was developed. The comparative map thus obtained bears significance in searching for candidate genes of economically important traits mapped to SSC13.

Effect of stress susceptibility on some reproductive traits in Belgian Landrace pigs

Abstract The investigation included 1413 Belgian Landrace litters (305 first, 278 second and 832 later farrowings) issued from matings between 190 halothane-positive (HP) and 304 halothane-negative (HN) sows and 139 HP and 49 HN boars on seven commercial farms of West Flanders. Four groups of matings were investigated: HP × HP; HP × HN; HN × HP and HN × HN. All HN sows and boars were heterozygotes for halothane sensitivity. The results were statistically analysed by a linear model according to Harvey (1977), including the following factors: mean, farm, year, sensitivity group, interaction year × sensitivity group. This model explained between 10 and 24% of the total variance; within this an important portion is caused by the sensitivity × farm interaction factor. Abstracting this interaction there are indications that HN sows are more prolific, but only from the second farrowings, the number of piglets born alive in the second litter being on average 9.2 in HP vs 9.6 in HN sows and in the following litters on average 9.3 vs 10.3. In all litters of HN sows an additional gain of 0.2 piglets at weaning was found. No significant conclusions could be drawn for the effect of halothane sensitivity on the age at first farrowing and on the farrowing interval.

An Abortion of Monozygotic Twins in a Warmblood Mare

Naturally occurring monozygotic twins are extremely rare in the horse. This paper describes an abortion in a mare after 260 days of pregnancy with monozygotic twins, one a fresh foal and the other a mummified foal.