Annie Engström

Antibodies to major pasture borne helminth infections in bulk-tank milk samples from organic and nearby conventional dairy herds in south-central Sweden

The objective of this randomised pairwise survey was to compare the regional distribution of antibody levels against the three most important helminth infections in organic and conventional dairy herds in Sweden. Bulk-tank milk from 105 organic farms and 105 neighbouring conventional dairy farms with access to pasture in south-central Sweden were collected in September 2008. Samples were also collected from 8 organic and 8 conventional herds located in a much more restricted area, on the same as well as 3 additional occasions during the grazing season, to reveal evidence for seasonal patterns against cattle stomach worm (Ostertagia ostertagi). Antibody levels to the stomach worm (O. ostertagi), liver fluke (Fasciola hepatica) and lungworm (Dictyocaulus viviparus) were then determined by detection of specific antibodies using three different enzyme-linked immunosorbent assays (ELISAs). According to the Svanovir Ostertagia ELISA, the mean optical density ratio (ODR) was significantly higher in the milk from organic compared to conventional herds, i.e. 0.82 (95% CL=0.78-0.86) versus 0.66 (0.61-0.71). However, no significant differences were observed in the samples collected at different time points from the same 16 herds (F(3,39)=1.18, P=0.32). Antibodies to D. viviparus infection were diagnosed with an ELISA based on recombinant major sperm protein (MSP), and seropositivity was found in 21 (18%) of the 113 organic herds and 11 (9%) of the 113 conventional herds. The seroprevalence of D. viviparus was somewhat higher in the organic herds (Chi-square=3.65, P=0.056), but with the positive conventional herds were located in the vicinity of infected organic herds. Of the 16 herds that were sampled on repeated occasions, as many as 10 (63%), were seropositive on at least one sampling occasion. Many of these turned positive towards the end of the grazing season. Only one herd was positive in all 4 samples and 3 were positive only at turn-out. Considering F. hepatica there was no difference in seroprevalence between organic and conventional herds according to the Institute Pourquier ELISA. In general, liver fluke infection was low and it was only diagnosed in 8 (7%) organic and 7 (6%) conventional herds.

Population genetics of the bovine/cattle lungworm ( Dictyocaulus viviparus ) based on mtDNA and AFLP marker techniques

Mitochondrial DNA (mtDNA) sequence data and amplified fragment length polymorphism (AFLP) patterns were compared for the lungworm Dictyocaulus viviparus, a nematode parasite of cattle. Eight individual D. viviparus samples from each of 8 herds in Sweden and 1 laboratory isolate were analysed, with the aim of describing the diversity and genetic structure in populations using different genetic markers on exactly the same DNA samples. There was qualitative agreement between the whole-genome AFLP data and the mtDNA sequence data, both indicating relatively strong genetic differentiation among the Swedish farms. However, the AFLP data detected much more genetic variation than did the mtDNA data, even after allowing for the different inheritance patterns of the markers, and indicated that there was much less differentiation among the populations. The mtDNA data therefore seemed to be more informative about the most recent history of the parasite populations, as the general patterns were less obscured by detailed inter-relationships among individual worms. The 4 mtDNA genes sequenced (1542 bp) showed consistent patterns, although there was more genetic variation in the protein-coding genes than in the structural RNA genes. Furthermore, there appeared to be at least 3 distinct genetic groups of D. viviparus infecting Swedish cattle, 1 of which was predominant and showed considerable differentiation between farms, but not necessarily within farms. Second, the 2 smaller genetic groups occurred on farms where the predominant group also occurred, suggesting that these farms have had multiple introductions of D. viviparus.

Real-time PCR detection for quantification of infection levels with Ostertagia ostertagi and Cooperia oncophora in cattle faeces

A quantitative real-time polymerase chain reaction (qPCR) based on hydrolysis (TaqMan) probes is described for robust and sensitive detection of the infection levels with eggs and third stage larvae (L3) of Cooperia oncophora and Ostertagia ostertagi isolated from cattle faeces. The current microscopic method for identification of strongyle nematodes in cattle faeces is labour-intensive where reliable species determination also requires trained expertise, which is increasingly lacking. The goal of this study was to develop a sustainable non-labour intensive diagnostic qPCR assay to detect and determine the levels of infection with the two most common gastro-intestinal nematodes (GIN) in cattle faeces targeting the second internal transcribed spacer of nuclear ribosomal DNA (ITS2) region (rDNA). According to our results, this procedure allows to reliably detect the relative proportions of eggs and L3 for each of the two species. This assay produced consistent results when mixtures with known numbers of L3 of both species were tested, although it was also demonstrated that the calculated copy numbers of ITS2 between single L3 sometimes varied very much. In addition, a positive correlation (r(2)=0.23) between the proportion of eggs and L3 in different paired samples collect in the field was observed for both species. Thus, for the first time a qPCR assay is reported, which can discriminate between the two most important cattle nematode parasites in temperate regions. This is of major importance to the livestock sector as it can be used with great precision to demonstrate strategic treatment efficacy that is important for the detection of anthelmintic resistance (AR).

Optimization of in-house ELISA based on recombinant major sperm protein (rMSP) of Dictyocaulus viviparus for the detection of lungworm infection in cattle.

The aim of this study was to optimize an in-house ELISA based on a recombinant version of the major sperm protein (MSP) of Dictyocaulus viviparus for routine diagnosis of lungworm infection in cattle. A recombinant MSP (rMSP) was cloned into pGEX-6P-1 vector and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21 (DE3) chemically competent cells. The product was then employed as capture antigen in an ELISA, and validated against 304 samples of known status (216 negative and 88 positive) in which the antibody levels in sera had also been measured earlier with a commercial ELISA kit (Ceditest® lungworm ELISA). The receiver operating characteristic (ROC) curve analysis of the ELISA results estimated the optimized diagnostic sensitivity and specificity as 97.7% (95% confidence interval [CI]: 91.9-99.7%) and 98.1% (CI: 95.3-99.5%), respectively. The results from the in-house rMSP-based ELISA were compared with results obtained on both fecal examination and the Ceditest® lungworm ELISA. Rising antibody levels in sera of experimentally infected calves were observed between 21 and 28 days post infection, when patency was also confirmed by the presence of larvae in feces. Notably, using the in-house rMSP-based ELISA infection was confirmed in calves shedding larvae approximately 3-4 weeks post inoculation, while using the Ceditest® lungworm ELISA those animals remained negative. Additionally, 251 sera samples from calves naturally exposed to the parasites on pasture were used to evaluate the test. In in-house rMSP-based ELISA no cross-reactions were observed with sera from calves infected with the gastrointestinal nematodes (Ostertagia ostertagi and Cooperia oncophora), even though the presence of eggs in the feces was confirmed. Overall, the in-house rMSP-based ELISA optimized in this study showed excellent diagnostic performance for detection of lungworm infection in cattle.

Population genetic structure of Ascaridia galli re-emerging in non-caged laying hens

BackgroundThe poultry roundworm Ascaridia galli has reappeared in hens kept for egg production in Sweden after having been almost absent a decade ago. Today this is a frequent intestinal nematode parasite in non-caged laying hens. The aim of this study was to investigate the genetic diversity (Fst) in A. galli collected from different poultry production sites in southern Sweden, to identify possible common routes of colonization.MethodsAdult parasites (n = 153) from 10 farms, including both broiler breeder parents and laying hens, were investigated by amplified restriction fragment length polymorphism analysis (AFLP). Worms from a Danish laying hen farm were also included for comparison. Most of the farms were represented by worms from a single host, but on two farms multiple samples from different hosts were assessed in order to study flock variation.ResultsA total of 97 fragments (loci) were amplified among which 81% were variable alleles. The average genetic diversity was 0.13 (range = 0.09-0.38), which is comparable to other AFLP studies on nematodes of human and veterinary importance. Within-farm variation showed that worms harboured by a single hen in a flock covered most of the A. galli genetic variation within the same flock (Fst = 0.01 and 0.03 for two farms). Between-farm analysis showed a moderate population genetic structure (Fst = 0.13), along with a low mutational rate but high gene flow between different farms, and absence of strong genetic selection. Network analysis showed repeated genetic patterns among the farms, with most worms on each farm clustering together as supported by high re-allocation rates.ConclusionsThe investigated A. galli populations were not strongly differentiated, indicating that they have undergone a genetic bottlenecking and subsequent drift. This supports the view that the investigated farms have been recently colonized, and that new flocks are reinfected upon arrival with a stationary infection.

Population genetics of Parascaris equorum based on DNA fingerprinting

The large roundworm of horses, Parascaris equorum is considered ubiquitous in breeding operations, and is regarded as a most important helminth pathogen of foals. Over the past decade, this parasite has been reported increasingly resistant to anthelmintic drugs worldwide. This paper reports analysis of the population genetic structure of P. equorum. Adult parasites (n=194) collected from Sweden, Norway, Iceland, Germany, Brazil and the USA were investigated by amplified restriction fragment length polymorphism (AFLP) analysis. The genetic variation was low (Hj=0.12-0.4), for the global population of worms. This was accompanied by a weak degree of population structure (Fst=0.2), low gene flow (Nm=1.0) and low mutation rate (4 Nμ=0.07). Thus, the low genetic diversity is probably a result of a low mutation rate in DNA, although the gene flow (due to global movement of horses) is large enough to allow the spread of novel mutations. Surprisingly, isolates from Icelandic horses were not found to be different from other isolates, in spite of the fact that these have been isolated for thousands of years. The study indicates that the global P. equorum population is essentially homogenous, and continents do not appear to be strong barriers for the population structure of this species. Consequently, the potential spread of rare anthelmintic resistance genes may be rapid in a homogenous population.

Limited sequence variation in the major sperm protein 1 (MSP) gene within populations and species of the genus Dictyocaulus (Nematoda)

Populations of the bovine lungworm, Dictyocaulus viviparus, are genetically structured based on variation in mtDNA and AFLP data. Our aim was to investigate if this genetic variability also is reflected in a protein recognized by the host immune system. We focused on the major sperm protein (MSP), a small and abundant protein used in diagnostic immunoassays, which has been shown to be variable in some nematodes but not others. MSP was sequenced using worm DNA from eight adult worms from each of nine populations whose genetic structure previously had been quantified. For comparison, we also analyzed MSP sequences of the closely related Dictyocaulus eckerti and Dictyocaulus capreolus and from nematodes with sequences deposited in GenBank. In contrast to previous results, this study shows that the MSP of D. viviparus is similar to that of other nematodes. Almost no sequence variation, and thus no antigenic diversity, was detected in MSP between worms from different sub-populations or in the other Dictyocaulus species investigated. A functional test of a recombinant variant of the MSP showed that the expressed protein was recognized by antibodies in sera from infected cattle. This has practical implications for the development of species-specific markers, recombinant vaccines, and immunodiagnostics.

Lymnaea fuscus (Pfeiffer, 1821) as a potential intermediate host of Fascioloides magna in Europe

Experimental infections of two different populations of Lymnaea fuscus in France and Sweden, with a Czech isolate of Fascioloides magna were carried out to determine if this lymnaeid species enables parasite larval development. Species identification of both snail populations was performed using the morphology of the copulatory organ, and also confirmed by sequencing of the internal transcribed spacer 2 (ITS2) region of the snail genomic rDNA. Only juvenile snails measuring less than 3mm (1-3 weeks of age) were successfully infected (the viable cercariae were recorded) and infection prevalence decreased with age, as documented by increased shell height. In both French and Swedish L. fuscus populations, prevalence ranged between 1.1% and 58.8%. The mean number of metacercariae obtained from cercariae-shedding snails was 13.7 (±11.4), while the total cercarial production noted in snails dissected at day 85 post-exposure was 147.5 (±56.6). Compared to uninfected control snails, we observed reduced growth of infected snails. Despite age-related resistance of snail to the parasite, and limited cercarial production in these experimentally infected snails, F. magna was still able to complete larval development in L. fuscus.

Sequencing of the β-tubulin genes in the ascarid nematodes Parascaris equorum and Ascaridia galli

Benzimidazoles (BZ) are used to control infections of the equine roundworm Parascaris equorum and the poultry roundworm Ascaridia galli. There are still no reports of anthelmintic resistance (AR) to BZ in these two nematodes, although AR to BZ is widespread in several other veterinary parasites. Several single nucleotide polymorphisms (SNP) in the β-tubulin genes have been associated with BZ-resistance. In the present study we have sequenced β-tubulin genes: isotype 1 and isotype 2 of P. equorum and isotype 1 of A. galli. Phylogenetic analysis of all currently known isotypes showed that the Nematoda has more diversity among the β-tubulin genes than the Vertebrata. In addition, this diversity is arranged in a more complex pattern of isotypes. Phylogenetically, the A. galli sequence and one of the P. equorum sequences clustered with the known Ascaridoidea isotype 1 sequences, while the other P. equorum sequence did not cluster with any other β-tubulin sequences. We therefore conclude that this is a previously unreported isotype 2. The β-tubulin gene sequences were used to develop a PCR for genotyping SNP in codons 167, 198 and 200. No SNP was observed despite sequencing 95 and 100 individual adult worms of P. equorum and A. galli, respectively. Given the diversity of isotype patterns among nematodes, it is likely that associations of genetic data with BZ-resistance cannot be generalised from one taxonomic group to another.