An in-depth investigation of the microbiota and its virulence factors associated with severe udder cleft dermatitis lesions.

Udder cleft dermatitis (UCD) is a skin condition affecting the anterior parts of the udder in dairy cattle. In the present study, we aimed to shed light on the microbiota in severe UCD lesions versus healthy udder skin by putting forward a taxonomic and functional profile based on a virulence factor analysis. Through shotgun metagenomic sequencing, we found a high proportion of bacteria in addition to a low abundance of archaea. A distinct clustering of healthy udder skin versus UCD lesion samples was shown by applying principal component analysis and (sparse) partial least squares analysis on the metagenomic data. Proteobacteria, Bacillota, and Actinomycetota were among the most abundant phyla in healthy udder skin samples. In UCD samples, Bacteroidota was the most abundant phylum. At genus level, Bifidobacterium spp. had the highest relative abundance in healthy skin samples, whereas Porphyromonas spp. and Corynebacterium spp. had the highest relative abundance in UCD samples. In the differential abundance analysis, Porphyromonas spp. and Bacteroides spp. were significantly differentially abundant in UCD samples, whereas Bifidobacterium spp., Staphylococcus sp. AntiMn-1, and Staphylococcus equorum were more commonly found in healthy samples. Moreover, the abundance of several treponeme phylotypes was significantly higher in lesion samples. The streptococcal cysteine protease speB was among the most abundant virulence factors present in severe UCD lesions, while a plethora of virulence factors such as the antitoxin relB were downregulated, possibly contributing to creating the ideal wound climate for the dysbiotic community. Network analysis showed healthy lesion samples had a large network ofpositive, correlations between the abundances of beneficial species such as Aerococcus urinaeequi and Bifidobacterium angulatum, indicating that the healthy skin microbiome forms an active protective bacterial network, which is disrupted in case of UCD. In UCD samples, a smaller microbial network mainly consisting of positive correlations between the abundances of Bacteroides fragilis and anaerobic Bacteroidota was exposed. Moreover, a high correlation between the taxonomic data and virulence factors was revealed, concurrently with 2 separate networks of microbes and virulence factors. One network, matching with the taxonomic findings in the healthy udder skin samples, showcased a community of harmless or beneficial bacteria, such as Bifidobacterium spp. and Butyrivibrio proteoclasticus, associated with hcnB, hcnC, relB, glyoxalase, and cupin 2. The other network, corresponding to UCD samples, consisted of pathogenic or facultative pathogenic and mainly anaerobic bacteria such as Treponema spp., Mycoplasmopsis spp., and bovine gammaherpesvirus 4, that correlated with virulence factors SpvB, fhaB, and haemagglutination activity domain-associated factor. Our results point toward a dysbiotic community with a notable decrease in diversity and evenness, with a loss of normal skin inhabitants and innocuous or useful species making way for predominantly anaerobic, facultative pathogens. The shift in the abundance of virulence factors such as fhaB and SpvB could play a role in the manifestation of a local micro-environment favorable to the microbiome associated with udder skin lesions. Lastly, the presence of specific networks between microbial species, and between microbes and virulence factors was shown.

Severe udder cleft dermatitis lesion transcriptomics points to an impaired skin barrier, defective wound repair and a dysregulated inflammatory response as key elements in the pathogenesis.

This study is the first to investigate the transcriptomic changes occurring in severe udder cleft dermatitis lesions (UCD) in Holstein-Friesian cows. An examination of the gene expression levels in natural UCD lesions and healthy udder skin through RNA Seq-Technology provided a deeper insight into the inflammatory pathways associated with this disease. A clear distinction between the gene expression patterns of UCD lesions and healthy skin was shown in the principal component analysis. Genes coding for inflammatory molecules were upregulated such as the chemokines C-X-C motif ligand 2 (CXCL2), 5 (CXCL5) and 8 (CXCL8), and C-C motif ligand 11 (CCL11). Moreover, the genes coding for the multifunctional molecules ADAM12 and SLPI were amongst the highest upregulated ones, whereas the most downregulated genes included the ones coding for keratins and keratin-associated molecules. Predominantly inflammatory pathways such as the chemokine signaling, cytokine receptor interaction and IL-17 signaling pathway were significantly upregulated in the pathway analysis. These results point towards a fulminant, dysregulated inflammatory response concomitant with a disruption of the skin barrier integrity and a hampered wound repair mechanism in severe UCD lesions.

Evaluation of a Cooperia oncophora double-domain ASP-based vaccine against Cooperia spp. infections in cattle and sheep.

A double-domain activation-associated secreted protein (dd-Co-ASP) isolated from the bovine small intestinal parasite Cooperia oncophora was previously shown to be an effective vaccine candidate to protect calves against a homologous challenge infection. The aim of this study was to investigate whether the dd-Co-ASP protein, purified from a Belgian C. oncophora isolate, would offer protection against a C. oncophora isolate from the southern hemisphere as well as other Cooperia species such as C. punctata in cattle and C. curticei in sheep. Two vaccination studies were performed, i.e. one in cattle and one in sheep, in which the protective effects of dd-Co-ASP, supplemented with Quil A as an adjuvant, were compared with an adjuvant control. Whereas our results showed a 75 % reduction in Cooperia spp. cumulative faecal egg counts, the results obtained in sheep demonstrated that dd-Co-ASP was ineffective in raising a protective immune response against a C. curticei challenge infection. Even though sequence analysis of the dd-Co-ASP gene revealed restricted sequence heterogeneity in the double domain ASP within and between bovine Cooperia species, the results of the vaccine study suggest that there is sufficient conservation at the protein level to yield cross-protection, holding promise for the development of a general Cooperia vaccine for use in cattle.

Regulatory role of the intestinal microbiota in the immune response against Giardia.

Giardia duodenalis is one of the most commonly found intestinal parasites in mammalian hosts. Infections can generally be cleared by mounting an adequate protective immune response that is orchestrated through IL-17A. This study was aimed to investigate if and how the intestinal microbiome affects the protective Th17 response against Giardia by analysing and comparing the immune response following a G. muris and G. duodenalis infection in antibiotic treated and untreated mice. Depletion of the intestinal flora by antibiotic treatment had a severe effect on the infection dynamics of both Giardia species. Not only duration of infection was affected, but also the parasite burden increased significantly. Markers associated with a protective immune response, such as IL-17A and mannose binding lectin 2 were still significantly upregulated following infection in the antibiotic-treated mice, despite the lack of protection. On the other hand, the antibiotic treatment significantly decreased the level of IgA in the intestinal lumen by affecting its transporter and by reducing the number of IgA+ B-cells at the Peyer's patches. Furthermore, the depletion of the gut microbiota by antibiotics also significantly lowered the intestinal motility. The combination of these factors likely results in a decreased clearance of the parasite from the intestinal tract.

Mind the gaps in research on the control of gastrointestinal nematodes of farmed ruminants and pigs.

Gastrointestinal (GI) nematode control has an important role to play in increasing livestock production from a limited natural resource base and to improve animal health and welfare. In this synthetic review, we identify key research priorities for GI nematode control in farmed ruminants and pigs, to support the development of roadmaps and strategic research agendas by governments, industry and policymakers. These priorities were derived from the DISCONTOOLS gap analysis for nematodes and follow-up discussions within the recently formed Livestock Helminth Research Alliance (LiHRA). In the face of ongoing spread of anthelmintic resistance (AR), we are increasingly faced with a failure of existing control methods against GI nematodes. Effective vaccines against GI nematodes are generally not available, and anthelmintic treatment will therefore remain a cornerstone for their effective control. At the same time, consumers and producers are increasingly concerned with environmental issues associated with chemical parasite control. To address current challenges in GI nematode control, it is crucial to deepen our insights into diverse aspects of epidemiology, AR, host immune mechanisms and the socio-psychological aspects of nematode control. This will enhance the development, and subsequent uptake, of the new diagnostics, vaccines, pharma-/nutraceuticals, control methods and decision support tools required to respond to the spread of AR and the shifting epidemiology of GI nematodes in response to climatic, land-use and farm husbandry changes. More emphasis needs to be placed on the upfront evaluation of the economic value of these innovations as well as the socio-psychological aspects to prioritize research and facilitate uptake of innovations in practice. Finally, targeted regulatory guidance is needed to create an innovation-supportive environment for industries and to accelerate the access to market of new control tools.

Serological detection of Ascaris suum at fattening pig farms is linked with performance and management indices.

The aim of the present study was to determine the association between the presence of Ascaris suum at fattening pig farms, using different serological methods and the percentage of affected livers at slaughter, with performance and management indices. In total, 21 fattening pig farms from the North of Spain were included in the study. Serum samples were collected from pigs at slaughter and analysed for the presence of anti-Ascaris antibodies. For this, two different ELISAs were used. The first was based on the antibody recognition of the A. suum haemoglobin (As-Hb) molecule whereas the second test used the total extract of A. suum lung stage L3. The serological results were subsequently correlated with the percentage of condemned livers at slaughter, management practices and technical performance parameters including average daily gain (ADG) and feed conversion ratio (FCR). According to the data from the slaughterhouse, 12 out of the 21 farms had livers condemned due to liver white spots. A total of 10 farms (48%) had an average optical density ratio (ODr) exceeding the test cutoff when the As-Hb ELISA was used. This number increased to 18 farms (81%) when using the As-Lung-L3 ELISA. The average ODr of the farms on both ELISAs correlated positively with the percentage of affected livers (P<0.01). Only the average ODr values obtained with the As-Lung-L3 ELISA were positively correlated with the FCR (P<0.01). No correlation was found between percentage of affected livers or serology and the ADG. In relation to management practices, farms with greater than or equal to 50% slatted flooring and that applied the 'all-in/all-out' flow system showed a lower percentage of liver condemnations (P<0.01), lower average ODr results on the As-Lung-L3 ELISA (P<0.05) and lower FCR (P<0.01) compared with those with less than 50% slatted flooring. This study emphasizes that serology is a promising diagnostic tool for diagnosing ascariasis at fattening pig farms. It also supports earlier findings that the presence of A. suum can have a significant negative impact on farm productivity and that stable infrastructure or management practices can have a considerable impact on the control of this parasite.

Progress in the development of subunit vaccines for gastrointestinal nematodes of ruminants.

The global increase in anthelmintic resistant nematodes of ruminants, together with consumer concerns about chemicals in food, necessitates the development of alternative methods of control for these pathogens. Subunit recombinant vaccines are ideally placed to fill this gap. Indeed, they are probably the only valid option for the long-term control of ruminant parasitic nematodes given the increasing ubiquity of multidrug resistance in a range of worm species across the world. The development of a subunit multicellular parasite vaccine to the point of practical application would be a groundbreaking step in the control of these important endemic infections of livestock. This review summarizes the current status of subunit vaccine development for a number of important gastrointestinal nematodes of cattle and sheep, with a focus on the limitations and problems encountered thus far, and suggestions as to how these hurdles might be overcome.

Host Th1/Th2 immune response to Taenia solium cyst antigens in relation to cyst burden of neurocysticercosis.

Neurocysticercosis (NCC), Taenia solium larval infection of the brain, is an important cause of acquired seizures in endemic countries, which relate to number, location and degenerating cysts in the brain. Multicyst infections are common in endemic countries although single-cyst infection prevails in India. Single-cyst infections in an endemic country suggest a role for host immunity limiting the infection. This study examined ex vivo CD4(+) T cells and in vitro Th1 and Th2 cytokine responses to T. solium cyst antigens of peripheral blood mononuclear cells of healthy subjects from endemic and nonendemic regions and of single- and multicyst-infected patients for association with cyst burden of NCC. T. solium cyst antigens elicited a Th1 cytokine response in healthy subjects of T. solium-endemic and T. solium-non-endemic regions and those with single-cyst infections and a Th2 cytokine response from subjects with multicyst neurocysticercosis. Multicyst neurocysticercosis subjects also exhibited low levels of effector memory CD4(+) T cells. Th1 cytokine response of T. solium exposure and low infectious loads may aid in limiting cyst number. Th2 cytokines and low effector T cells may enable multiple-cyst infections to establish and persist.

Analysis of the protective immune response following intramuscular vaccination of calves against the intestinal parasite Cooperia oncophora.

Recently we reported the successful vaccination of calves against Cooperia oncophora with a double domain activation-associated secreted protein, purified from the excretory-secretory material of adult stage parasites. In an attempt to elucidate the immune mechanisms involved in protection, the humoral and cell-mediated immune responses following vaccination and infection were compared with non-vaccinated control animals. Antigen-specific IgG1, IgG2 and IgA levels were significantly increased in sera of vaccinated animals post vaccination, whereas no effect was observed for IgM. Antigen-specific intestinal IgG1 levels were significantly increased in the vaccinated animals, whereas no differences were observed for antigen-specific IgA, IgM and IgG2 levels. Upon re-stimulation in vitro with the vaccine antigen, a significant proliferation of both αß- and γδ-T cells, and B cells, collected from mesenteric lymph nodes, was only observed in vaccinated animals. RNA-seq analysis of intestinal tissue yielded a list of 67 genes that were differentially expressed in vaccinated animals following challenge infection, amongst which were several cell adhesion molecules, lectins and glycosyl transferases. A correlation analysis between all immunological and parasitological parameters indicated that intestinal anti-double domain activation-associated secreted protein IgG1 levels correlated negatively with cumulative faecal egg counts and positively with the proportion of L4s and L5s. The proportion of immature stages was also positively correlated with the proliferation of αß T cells. Worm length was negatively correlated with the transcript levels of several lectins and cell adhesion molecules. Overall, the results indicate that intramuscular administration of the vaccine resulted in an immune memory response particularly characterised by increased antigen-specific IgG1 levels in the intestinal mucosa.

Evaluation of cellular and humoral systemic immune response against Giardia duodenalis infection in cattle.

Giardia duodenalis causes diarrhoea in humans and a wide range of mammals, including cattle. In cattle, the infection often has a chronic character. Infected calves may excrete cysts for several months, suggesting that Giardia is able to suppress and evade the immune response. In this study six calves were infected with G. duodenalis assemblage A and E and housed in an environment that allowed reinfection. Cyst excretion was monitored twice a week and blood was collected every 2 weeks, until decreasing cyst counts indicated the development of protective immunity. The kinetics of the circulating memory cells and serum antibodies were followed up throughout this period. Cyst excretion started 1 week post-infection and remained high until week 14. Low cyst counts from week 15 p.i. onwards indicated that the calves had developed immunity. From week 5 p.i. significant proliferation of CD4(+) αß T-cells was observed after in vitro stimulation with G. duodenalis antigen. Characterisation of the proliferating CD4(+) T-cells using real time qPCR showed that at the peak of antigen driven PBMC proliferation the majority of cells were CD4(+) T-cells expressing IL-17 and to a lesser extent FoxP3. The cell proliferation was strongly reduced after plastic adhesion of the PBMC, suggesting a role for antigen-presenting cells. Failure to restore proliferation of depleted PBMC with Giardia-stimulated monocyte-derived dendritic cells (MoDC) and unchanged proliferation after depletion of CD21(+) B-cells showed that other antigen-presenting cells than MoDC and B-cells were important for T-cell proliferation. Analysis of the antibody response indicated that serum IgG1 and IgA levels against G. duodenalis assemblage A and E increased from week 11 post-infection. From the start of the antibody response, all trophozoites stained positive in an immunofluorescence assay with serum antibodies, indicating that a broad repertoire of antibodies was produced against all variant-specific surface proteins. Further research is necessary to determine which effector T-cell subset produces IL-17 and which cells play a role in antigen presentation.

Analysis of the mucosal immune responses induced by single and trickle infections with the bovine abomasal nematode Ostertagia ostertagi.

The purpose of this study was to provide more information on the kinetics of the immunological changes occurring in the abomasal mucosa after single and trickle infections with the bovine parasite Ostertagia ostertagi. The time course analysis of gene expression revealed that the major changes coincided with the emergence of adult worms from the gastric glands. These changes consisted of a simultaneous upregulation of Th1- and Th2-type cytokines. In addition, a single O. ostertagi infection elicited an upregulation of the epithelial-derived cytokine IL33, while TSLP expression levels were not impacted. Apart from the massive increase in inflammatory cytokines IL6, IL17 and IL21, O. ostertagi infection also elicited an upregulation of the immunosuppressors TGFB, IL10 and ARG1, as well as NK and γδ-T cell markers. Furthermore, the cytotoxic factors granulysin, perforin and granzyme B were upregulated following an O. ostertagi infection. Analysis of cytokine transcript levels in animals receiving trickle infections for 60 days showed a similar trend as observed following a single infection except for IL33, IL6, GATA-3, TBX21 and NCR1, which were no longer upregulated after trickle infections. Finally, the long trickle infections were associated with mucosal eosinophilia and mastocytosis.

Giardia duodenalis stimulates partial maturation of bovine dendritic cells associated with altered cytokine secretion and induction of T-cell proliferation.

Giardia duodenalis is an important intestinal parasite in animals and humans. The role of dendritic cells (DC) in the initiation of the immune response against G. duodenalis is poorly documented. The aim of this study was to test the hypothesis that G. duodenalis interferes with bovine DC function. Therefore, the effect of trophozoites and excretion/secretion products on bovine monocyte-derived dendritic cells (MoDC) was investigated. We assessed MoDC maturation and cytokine production of G. duodenalis-stimulated MoDC and the ability of these MoDC to take up antigen and induce lymphocyte proliferation. Little or no upregulation of maturation markers CD40 and CD80 was measured, but MHCII expression was increased after stimulation with low parasite concentrations. A dose-dependent decrease in ovalbumin uptake was observed in G. duodenalis-stimulated MoDC. In addition, stimulated MoDC induced proliferation of CD3(-) , γδ-T-cells and TCRαß(+) CD4(+) and CD8(+) T-cells. Increased transcription of TGF-ß was shown in CD4(+) T cells, and increased TNF-α, TGF-ß, IL-10 and IL-4 were seen in γδ-T-cells. We found no evidence that G. duodenalis has a regulatory or inhibitory effect on bovine MoDC. MoDC stimulated with G. duodenalis are functionally active and able to induce proliferation of T cells that produce both pro- and anti-inflammatory cytokines.

Gene expression analysis of ABC transporters in a resistant Cooperia oncophora isolate following in vivo and in vitro exposure to macrocyclic lactones.

Members of the ATP-binding cassette (ABC) transporter family (P-glycoproteins, Half-transporters and Multidrug Resistant Proteins) potentially play a role in the development of anthelmintic resistance. The aim of this study was to investigate the possible involvement of ABC transporters in anthelmintic resistance in the bovine parasite, Cooperia oncophora. Partial sequences of 15 members of the ABC transporter protein family were identified, by mining a transcriptome dataset combined with a degenerate PCR approach. Reverse transcriptase PCR showed that most of the ABC transporters identified were constitutively transcribed throughout the life cycle of C. oncophora. Constitutive differences in gene transcript levels between a susceptible and resistant isolate were only observed for Con-haf-9 and Con-mrp-1 in eggs of the resistant isolate, while no differences were observed in L3 or the adult life stage. Analysis of resistant adult worms, collected from calves 14 days after treatment with either ivermectin or moxidectin, showed a significant 3- to 5-fold increase in the transcript levels of Con-pgp-11 compared to non-exposed worms. Interestingly, a 4-fold transcriptional up-regulation of Con-pgp-11 was also observed in L3 of the resistant isolate, after in vitro exposure to different concentrations of ivermectin, whereas this effect was not observed in exposed L3 of the susceptible isolate. The results suggest that the worms of this particular resistant isolate have acquired the ability to up-regulate Con-pgp-11 upon exposure to macrocyclic lactones. Further work is needed to understand the genetic basis underpinning this process and the functional role of PGP-11.

Assessing resistance against macrocyclic lactones in gastro-intestinal nematodes in cattle using the faecal egg count reduction test and the controlled efficacy test.

The main objective of the present study was to evaluate the accuracy of the faecal egg count reduction test (FECRT) to assess the resistance status of ivermectin (IVM)-resistant isolates of the cattle nematodes Ostertagia ostertagi and Cooperia oncophora, using the controlled efficacy test (worm counts) as a reference. The second objective was to investigate whether both IVM-resistant isolates showed side-resistance against moxidectin (MOX) under controlled conditions. Thirty male Holstein calves were experimentally infected with 25,000 L3 of an IVM-resistant O. ostertagi isolate and 25,000 L3 of an IVM-resistant C. oncophora isolate. Twenty-eight days later the calves were randomly divided into 2 treatment groups and 1 untreated control group. Animals in groups 1 and 2 received MOX (Cydectin(®) 1%, Pfizer) and IVM (Ivomec(®) 1%, Merial) respectively, by subcutaneous injection at a dose rate of 0.2mg/kg bodyweight. Faecal samples were collected 7 and 14 days after treatment and animals were necropsied 14/15 days post-treatment. Both the FECRT and the controlled efficacy test demonstrated that the O. ostertagi and C. oncophora isolates were resistant against IVM, with efficacies below 90%. The IVM-resistant O. ostertagia isolate was still susceptible to MOX treatment, as shown by over 99% reduction in egg counts and worm burden. The FECRT suggested borderline resistance against MOX in the IVM-resistant C. oncophora isolate, with egg count reductions between 97% (95% CI: 76; 100) at day 7 and 86% (95% CI: 49; 96) at day 14. However, the controlled efficacy test clearly showed MOX-resistance, with a decrease of only 31% (95% CI: -12; 57) in C. oncophora worm numbers. After MOX treatment, a significantly lower number of eggs per female C. oncophora worms was counted compared to the control group (43% reduction). Due to this reduced fecundity, the FECRT may fail to detect MOX-resistance.

Intramuscular inoculation of cattle with Sarcocystis antigen results in focal eosinophilic myositis.

Bovine eosinophilic myositis (BEM) is a subclinical myopathy characterized by multifocal white to grey-green discolorations in skeletal muscles, heart, tongue and oesophagus. These lesions are found at slaughter or during meat cutting and result in considerable economic losses. The etiology and pathogenesis are unclear, although it has been suggested, that Sarcocystis species are involved. To elucidate their role, two calves were repeatedly injected intramuscularly with adjuvanted Sarcocystis antigen. The morphological changes at the injection sites in these calves were histologically and immunohistochemically compared to spontaneous lesions from 44 BEM condemned carcasses sampled in slaughterhouses. Experimental intramuscular injection of Sarcocystis antigen resulted in lesions at the injection sites that were similar to the lesions of natural cases of BEM. They were characterized by massive infiltration of eosinophilic granulocytes, reactive macrophages (MAC387(+) cells), T-cells (CD3(+)) and B-cells (CD20(+)). Both in the experimental and in the natural cases, COX-2 expression was present in endothelial cells adjacent to lesional areas. MHC class II(+) staining was found amongst others in muscle cells surrounding the lesion. These results show that Sarcocystis antigens can induce an inflammatory response in bovine muscle having the characteristics of natural BEM.

Immunologically based control strategies for ostertagiosis in cattle: where do we stand?

Ostertagia ostertagi is one of the most important gastrointestinal parasites infecting millions of cattle in temperate climate areas. Because infection leads to significant losses in productivity, farmers are pushed, because of the on-going intensification of the livestock production systems, to frequently administer anthelmintics to minimize the exposure of their animals to this parasite. However, whether such approach is sustainable in the long term, especially in an era of emerging drug resistance and global climatic changes, is still a matter of debate. Immunological control of worm infections through vaccination is often put forward as the most rational and cost-effective alternative for anthelmintics to control helminth infections. However, the development of an immunologically based control strategy requires a thorough knowledge of the host-parasite interactions, the immune responses involved and the biology of the parasite itself. The aim of this review is to consolidate information available in these areas, specifically for O. ostertagi, and identify some critical gaps in our current knowledge.

Doramectin resistance in Haemonchus contortus on an alpaca farm in Belgium.

Parasitism by gastrointestinal nematodes is a health concern in New World Camelids (NWC) worldwide, and anthelmintic treatment is often needed for parasite control. Although anthelmintic resistance has been reported in ruminants worldwide, data in NWC are only scarce. In the present study, a case of suspected doramectin resistance in alpacas was examined. A field efficacy study was conducted for the evaluation of two different dosages of doramectin using a faecal egg count reduction test. A group of 8 alpacas was treated with a subcutaneous injection of doramectin at 0.2mg/kg bodyweight. Individual faecal samples were collected before treatment and 7 days after treatment. The faecal egg counts indicated a treatment efficacy of only 68%. To determine whether the treatment failure was caused by true anthelmintic resistance or suboptimal dosage in this animal species, a group of 4 alpacas was subsequently treated at 0.3mg/kg bodyweight. Faecal egg counts 7 days post treatment were reduced by only 41%, indicating that the treatment failure was more likely to be caused by the presence of resistant parasites on this farm. Coprocultures of faecal samples collected after treatment indicated the presence of 98.5% Haemonchus contortus and a small percentage of Cooperia oncophora (<1.5%). A controlled efficacy trial in sheep, for which the optimal dosage of doramectin is known, was conducted to ensure that this truly was a case of resistant parasites. Infective larvae collected from the faeces of these alpacas were used to infect eight nematode-free lambs. These lambs were assigned to one of two groups based on faecal egg counts post infection. One group was treated with doramectin injectable at 0.2mg/kg bodyweight, the other group served as a non treated control group. Pharmacokinetics indicated that the doramectin treatment was adequate, yet an efficacy of only 16% was determined on day 7 after treatment. Identification of the larvae after treatment revealed 100% H. contortus. On day 7 after treatment, H. contortus worm counts were only reduced by 8% in the treated lambs. The results of the present study report for the first time a case of doramectin resistance in alpacas, mainly in H. contortus.

Galectin-11 induction in the gastrointestinal tract of cattle following nematode and protozoan infections.

Galectin-11 (LGALS11) has been suggested to play an important role in protective immunity against gastrointestinal nematodes in ruminants. However, in cattle, this molecule has not been characterized in detail. In the current study, it was shown that transcription of LGALS11 was highly inducible in the bovine abomasal mucosa after an Ostertagia ostertagi infection. LGALS11 protein expression was also increased in the abomasal mucosa following O. ostertagi infection and localized to the nucleus and cytoplasm of epithelial cells and the mucus. Using in vitro abomasal epithelial cell cultures, it was shown that LGALS11 induction was associated with the proliferative and dedifferentiated status of cells. However, LGALS11 was not induced following stimulation with O. ostertagi excretory-secretory products. These results suggest that LGALS11 induction in vivo may be an indirect rather than a direct effect of the parasite on the epithelium. In addition, LGALS11 transcript was also detected in the abomasal lymph nodes where it was shown to be transcribed in MHCII+ cells; however, transcription levels in the lymph nodes were not altered after O. ostertagi infection. In addition, LGALS11 was also induced in the small intestine by different types of parasites, including the nematode Cooperia oncophora and the protozoan parasite Giardia duodenalis.

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 parasite's 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.

Immunizing pigs with Ascaris suum haemoglobin increases the inflammatory response in the liver but fails to induce a protective immunity.

To determine whether purified Ascaris suum haemoglobin (AsHb) is a suitable vaccine candidate for the control of Ascaris infections, pigs were vaccinated with AsHb in combination with QuilA adjuvant and challenged with A. suum eggs. The number of liver lesions and worms in the intestine was assessed on day 14, 28 and 56 post-infection (p.i.). No significant differences were found in the number of worms recovered between vaccinated and control pigs on any of these days. However, significantly more white spots were counted on the livers of vaccinated pigs on day 14 (+86%) and day 28 (+118%) p.i. compared with nonvaccinated controls. To investigate whether the increased immunoreactivity against the liver stage L3s in vaccinated pigs was triggered by and directed against AsHb, the transcription and expression of AsHb in this larval life stage was analysed by RT-PCR and immunoblotting. The results showed that neither the AsHb transcript nor protein was detectable in freshly hatched L3. However, the immunoblot analysis showed that vaccination with AsHb resulted in the production of antibodies binding to several other antigens of the L3, suggesting that these might be involved in the increased white spot development.