Recombinant Major Sperm Protein Research Articles

Risk factors for lungworm-associated milk yield losses in grazing dairy cattle

Dictyocaulus viviparus, the causative agent of bovine parasitic bronchitis, is an important parasite of dairy cattle. Infections can lead to substantial economic losses, due to mortality, reduced weight gain and milk production and treatment costs. There have been relatively few studies investigating herd management risk factors for infections with D. viviparus and lungworm-associated production losses. The aims of this study were (1) to assess the impact of (sub)clinical lungworm infections on productivity in dairy cows and, (2) to identify or confirm risk factors, related to herd management, for infections in grazing dairy cattle. Using a recombinant Major Sperm Protein (MSP)-based ELISA, the presence of D. viviparus antibodies in bulk tank milk (BTM) samples was evaluated on 717 and 634 farms at two-week intervals during two grazing seasons (2018 and 2019). Associations between milk antibody levels and production data (mean milk yield in kg/cow/day, percentage of fat and protein) were assessed, as well as associations with putative risk factors in the herd management, gathered through a questionnaire survey.In both years, there was a substantial, but non-significant, difference in the annual mean milk yield on farms with at least one BTM sample above the cut-off of 0.41 ODR, compared with the mean milk yield on farms that stayed under this threshold on each sampling day (-0.17 and -0.70 kg milk/cow/day in 2018 and 2019, respectively). In 2019, this association was stronger, and significant, when the cut-off was exceeded in at least two consecutive BTM samples (-1.74 kg milk/cow/day). BTM results were also significantly negatively associated with the closest milk production data during the two-weekly BTM sampling intervals in 2019.A single or two consecutive positive tests were used in the risk factor analysis as a proxy for lungworm-associated milk yield losses. Purchase of new animals (Odds Ratio (OR) = 2.68) and the proportion of the first grazing season covered by preventive anthelmintic treatment (OR up to 3.88, depending on proportion) were positively associated with lungworm-associated milk yield losses, while mowing at least 50 % of the pastures (OR = 0.57) was negatively associated with lungworm-associated milk yield losses.Our results suggest that the ELISA holds promise to identify herds with significant production losses due to lungworm infections, under the condition that BTM sampling is done repeatedly during the grazing season. Based on the confirmed risk factors, adjustments of the farm management could perhaps mitigate these losses.

Lungworm infections in harbour porpoises (Phocoena phocoena) in the German Wadden Sea between 2006 and 2018, and serodiagnostic tests.

Pseudaliid lungworm (Metastrongyloidea) infections and associated secondary bacterial infections may severely affect the health status of harbour porpoises (Phocoena phocoena) in German waters. The presented retrospective analysis including data from 259 harbour porpoises stranded between 2006 and 2018 on the German federal state of Schleswig-Holstein's North Sea coast showed that 118 (46%) of these stranded individuals harboured a lungworm infection. During this 13-year period, a significant difference in annual lungworm prevalence was only observed between the years 2006 and 2016. Lungworm coinfections of bronchi and pulmonary blood vessels were observed in 85.6% of positive cases. Mild infection levels were detected in 22.9% of infected animals and were most common in the age class of immature individuals (74.1%). Moderate and severe infections were present in 38.1% and 39.0% of the lungworm positive animals, respectively. Their distribution in immatures (51.1% and 54.3%) and adults (48.9% and 43.4%) did not show significant differences. In stranded animals, lungworm diagnosis can be easily obtained via necropsy, while reliable lungworm diagnosis in living porpoises requires invasive bronchoscopy or faecal examination, which is difficult to obtain in cetaceans. To overcome this issue, an enzyme-linked immunosorbent assay (ELISA) and immunoblot based on recombinant major sperm protein (MSP) of the cattle lungworm were evaluated as potential diagnostic tools in harbour porpoises. However, in contrast to hitherto other investigated host species, no reliable antibody response pattern was detectable in harbour porpoise serum/plasma or whole blood samples. Thus, MSP-based serological tests are considered unsuitable for lungworm diagnosis in harbour porpoises.

Association between Dictyocaulus viviparus bulk tank milk antibody levels and farmer-reported lungworm outbreaks

The lungworm Dictyocaulus viviparus can have a major impact on bovine health and productivity. Enzyme-Linked Immunosorbent Assays (ELISA), based on a recombinant Major Sperm Protein (MSP), have been developed to detect D. viviparus-specific antibodies in bulk tank milk (BTM). The objectives of this study are to assess the association between BTM optical density ratio’s (ODR) and farmer-reported lungworm outbreaks based on the clinical sign “coughing” throughout the grazing season and to compare the sensitivity and specificity of two ELISAs under field conditions. The Hannover MSP-ELISA and the prototype Svanova MSP-ELISA were used for the detection of D. viviparus antibodies in BTM samples on 717 dairy farms during the 2018 grazing season. Assuming all herds to be truly lungworm infected, the results show that the Svanova ELISA had a lower sensitivity (40-65%) and specificity (75-90%) for the detection of D. viviparus infections in BTM compared to the Hannover ELISA, which had a sensitivity of 42% and 74% and specificity of 100% and 98% at a cut-off of 0.41 ODR and 0.25 ODR, respectively. Therefore, analyses of the associations between milk antibody levels and farmer-reported outbreaks during the 2018 and 2019 grazing season were assessed using the Hannover ELISA, on 717 and 634 farms, respectively. A positive association was found between a farmer-reported outbreak and having at least two consecutive positive BTM ODR’s at a cut-off of 0.41 in 2018 (Odds Ratio (OR) = 5.5) and 2019 (OR = 2.8). Furthermore, there was a significant association between a farmer-reported outbreak and having a positive BTM ODR in August (OR 2018 = 4.4; OR 2019 = 2.8) and October (OR 2018 = 3.7; OR 2019 = 1.8). On the farms with a farmer-reported outbreak and positive BTM samples, over half (2018 = 77%; 2019 = 57%) of the positive ODR’s were situated before the outbreak and 47% (2018) and 42% (2019) within 12 weeks before the outbreak. In conclusion, there is a positive association between farmer-reported outbreaks and the occurrence of a positive BTM sample at the cut-off of 0.41 ODR using the Hannover ELISA.

Serological lessons from the bovine lungworm Dictyocaulus viviparus: Antibody titre development is independent of the infection dose and reinfection shortens seropositivity

Infections with the bovine lungworm Dictyocaulus viviparus, the causative agent of parasitic bronchitis, are accompanied by substantial economic losses due to impacts on production, clinical respiratory disease or even death of diseased cattle. To detect lungworm antibodies in cattle, an enzyme-linked immunosorbent assay (ELISA) based on recombinant major sperm protein (MSP) has been developed. However, it remained unknown whether the infection dose influences antibody levels, and how acquired immunity influences antibody level patterns during reinfections. The latter may lead to low within-herd seroprevalence and thus to negative MSP-ELISA results in examination of bulk tank milk (BTM). Thus, infection experiments with 12 different doses ranging from 10 to 3000 D. viviparus larvae were performed to assess whether the antibody response is dose-dependent. Second, the impact of reinfections on the antibody response was evaluated in infection experiments, and third, antibody patterns in dairy cows during naturally occurring reinfections were assessed in a longitudinal field study based on individual milk samples. Results of this study demonstrate that the rise in MSP antibodies during first infection is dose-independent at infection doses of 25 lungworm larvae and above. However, following reinfections the magnitude and duration of the MSP antibody response are reduced or lacking, depending on the interval to reinfection. The field study revealed short periods of seropositivity as a common pattern in dairy cows subjected to natural D. viviparus reinfections. Low within-herd seroprevalence in dairy herds can thus be a result of continuous reinfections. Low infection doses should not be a barrier to serodiagnosis of lungworm infection in first-time infected cattle.

Detection of specific antibodies in cats infected with the lung nematode Aelurostrongylus abstrusus

Feline aelurostrongylosis, caused by the metastrongylid nematode Aelurostrongylus abstrusus, is an underestimated respiratory parasitosis. Its diagnosis currently mainly relies on the isolation of first stage larvae from fresh faecal samples. The aim of our study was to develop a serological test for the detection of specific antibodies against A. abstrusus by ELISA. We used recombinant major sperm protein (MSP) of the bovine lungworm Dictyocaulus viviparus as detection antigen and evaluated two different ELISA plates (Maxisorp and Immobilizer™ Amino-plate, Nunc Roskilde, Denmark) with two different enzyme systems [alkaline phosphatase (AP) and horseradish peroxidase (HRP)]. Sera from cats experimentally (n=54) and naturally (n=17) infected with A. abstrusus and from randomly selected cats with different medical issues (n=160) were used to determine sensitivity and specificity. Furthermore, cross-reactions were evaluated using sera from cats naturally (n=71) and experimentally (n=8) infected with different nematodes. A sensitivity of 100% was obtained with sera from experimentally infected cats at 10 weeks post infection using MSP on the Immobilizer™ Amino-plate with HRP, while it ranged between 90.5 and 95.2% in the other ELISA set-ups. Using sera from naturally infected cats, a sensitivity of 88.2% (95% confidence interval: 63.6–98.5%) was achieved in all four set-ups. The specificity was 85.2–94.4% in sera from uninfected cats prior to experimental infection and 68.1–90% in randomly selected cats depending on the plate and enzyme system. The number of seropositive cats increased over time post infection. Serological follow-up showed a decrease of antibody levels within 30days after anthelmintic treatment. Seropositive reactions were observed with sera from stray cats naturally infected with Toxocara cati, Capillaria sp., hookworms and Taeniidae; however, coproscopic false negative A. abstrusus findings cannot be excluded. The serological detection of specific antibodies against A. abstrusus using ELISA requires a single serum sample and therefore represents a valid alternative for reliable individual diagnosis of A. abstrusus in cats and facilitates mass screening, overcoming the usually difficult collection of cat faeces.

Prevalence and seasonality of bulk milk antibodies against Dictyocaulus viviparus and Ostertagia ostertagi in Irish pasture-based dairy herds

Infections with Dictyocaulus viviparus and Ostertagia ostertagi nematode parasites are of importance to bovine health and production in temperate areas across the world. Losses due to these parasites in dairy herds can be considerable due to decreased milk productivity and fertility. However, information on current epidemiological patterns in Irish dairy herds is limited. Bulk milk samples were collected from a total of 319 dairy farms across the Republic of Ireland. The D. viviparus samples were tested with an ELISA based on recombinant major sperm protein, while the O. ostertagi samples were tested with an ELISA based on crude saline extract, whole worm O. ostertagi antigen. Management data were collected from the farms using a questionnaire. Logistic regression was used to find significant associations between the presence of antibodies against D. viviparus and O. ostertagi and management factors. The overall prevalence of D. viviparus infection was 62.8%, while over 98% of herds had antibodies to O. ostertagi at the specified cut-off. Both D. viviparus and O. ostertagi antibodies were highest in November, which could be explained by the accumulated uptake of larvae through the grazing season. In herds of farmers that dosed their in-calf heifers with anthelmintics were significantly more likely to be positive for antibodies against D. viviparus infection. This study highlights that both D. viviparus and O. ostertagi infections are widespread in dairy herds in Ireland throughout the grazing season.

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 28days 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 3–4weeks 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.

A survey on Dictyocaulus viviparus antibodies in bulk milk of dairy herds in Northern Germany

Parasitic bronchitis caused by the bovine lungworm, Dictyocaulus viviparus, occurs worldwide in temperate areas. The parasite is found predominantly in calves and heifers, but dairy cattle can suffer from lungworms when they become infected for the first time or if they have lost immunity due to lack of exposure to lungworm larvae during the grazing season. The present study was performed to determine the D. viviparus bulk milk antibody prevalence in dairy herds in the East Frisian region of northwestern Germany, Lower Saxony, by analysing bulk milk samples collected in January (860 samples), September (866 samples) and November (860 samples) 2008, thereby representing 906 dairy farms. These samples were tested for antibodies against D. viviparus by a milk ELISA. This test detects patent infections only since it is based on recombinant major sperm protein as antigen. While in January 12.8% of dairy farms were positive for D. viviparus antibodies, the bulk milk samples collected in September and November revealed 6.9% and 6.6% positive dairy herds. From the 906 dairy farms included in the study, 191 (21.1%) tested positive at least once for antibodies against lungworm. From 810 dairy farms from which bulk milk samples were obtained during all three samplings, 146 (18.0%) farms were positive at one sampling date, 27 (3.3%) at two, and 4 (0.5%) on all three sampling dates. The majority of the farms represented in the study belonged to four districts of East Frisia, which showed no significant difference in the proportion of positive dairy farms.

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.

Evaluation of a milk ELISA for the serodiagnosis of Dictyocaulus viviparus in dairy cows

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against the bovine lungworm Dictyocaulus viviparus in milk was established. This test is based on recombinant major sperm protein (MSP) as the antigen and ELISA results are expressed as optical density ratio (ODR) values. The cut-off value of the milk ELISA was determined as the arithmetic mean of negative milk samples plus three standard deviations (SD). Specificity and sensitivity were 100% and 97.5%, respectively, using either milk or serum samples as positive control to calculate the ODR. Therefore, the presented recombinant antigen-based ELISA is suitable for routine veterinary diagnosis of exposure to bovine lungworms using milk samples instead of sera. To assess the course of antibody titres following lungworm infection, milk and serum samples from experimentally infected dairy cows were collected over a period of 23–30 weeks in three trials. The milk and serum antibody titre curves showed strong Pearson correlation coefficients in all three trials (Trial 1 = 0.85; Trials 2 and 3 = 0.93). In milk D. viviparus-specific antibodies exceeded the cut-off value 30–32 days post-infection (dpi) and remained above this value until day 112–138 post-infection (pi) with an overall detection period of 79–107 days. Treatment with eprinomectin during the pre-patent period prevented larval shedding and the antibody response was eliminated; treatment during patency similarly caused a cessation of larval shedding, but had no effect on the pattern of antibody responses compared to the untreated, infected controls.

Development and evaluation of a recombinant antigen-based ELISA for serodiagnosis of cattle lungworm

An optimised enzyme-linked immunosorbent assay (ELISA) for the detection of Dictyocaulus viviparous-specific antibodies was developed and evaluated following the testing of various microtitration plates and anti-bovine Ig-conjugates. Based on recombinant major sperm protein (MSP) expressed as a glutathione- S-transferase (GST) fusion protein in Escherichia coli, sera collected from 112 cattle experimentally infected with D. viviparus, from 129 helminth-naïve calves, 8 calves experimentally infected with Ostertagia ostertagi, and 2 calves infected with Cooperia oncophora were tested. ELISA results showed a calculated specificity and sensitivity as well as positive and negative predictive values of >99%. No cross-reactions with sera from calves infected with O. ostertagi or C. oncophora were observed. Lungworm-specific immunoglobulins were first detected from 28 to 35 days post-infection onwards. To differentiate between antibody-binding to the MSP-part or the GST-part of the fusion protein, additional ELISAs were performed using pure recombinant MSP or GST. Optical densities obtained from the ELISAs with the MSP showed a similar pattern to optical densities measured in the ELISAs with the fusion protein, whereas GST gave only a low background. By testing serum samples from naturally infected calves, it was found that the MSP-ELISA is positive even for sera from calves showing very low faecal larval counts. Thus, we conclude that the ELISA using the recombinant MSP-fusion protein appears to be a suitable method for routine diagnosis and epidemiological studies of cattle lungworm.

Critical contact residues that mediate polymerization of nematode major sperm protein

The polymerization of protein filaments provides the motive force in a variety of cellular processes involving cell motility and intracellular transport. Regulated assembly and disassembly of the major sperm protein (MSP) underlies amoeboid movement in nematode sperm, and offers an attractive model system for characterizing the biomechanical properties of filament formation and force generation. To that end, structure-function studies of MSP from the nematode Caenorhabditis elegans have been performed. Recombinant MSP was purified from Escherichia coli using a novel affinity chromatography technique, and filament assembly was assessed by in vitro polymerization in the presence of polyethylene glycol. Prior molecular studies and structure from X-ray crystallography have implicated specific residues in protein-protein interactions necessary for filament assembly. Purified MSP containing substitutions in these residues fails to form filaments in vitro. Short peptides based on predicted sites of interaction also effectively disrupt MSP polymerization. These results confirm the structural determination of intermolecular contacts and demonstrate the importance of these residues in MSP assembly.

Swine Erysipelas

Infections with Dictyocaulus viviparus and Ostertagia ostertagi nematode parasites are of importance to bovine health and production in temperate areas across the world. Losses due to these parasites in dairy herds can be considerable due to decreased milk productivity and fertility. However, information on current epidemiological patterns in Irish dairy herds is limited. Bulk milk samples were collected from a total of 319 dairy farms across the Republic of Ireland. The D. viviparus samples were tested with an ELISA based on recombinant major sperm protein, while the O. ostertagi samples were tested with an ELISA based on crude saline extract, whole worm O. ostertagi antigen. Management data were collected from the farms using a questionnaire. Logistic regression was used to find significant associations between the presence of antibodies against D. viviparus and O. ostertagi and management factors. The overall prevalence of D. viviparus infection was 62.8%, while over 98% of herds had antibodies to O. ostertagi at the specified cut-off. Both D. viviparus and O. ostertagi antibodies were highest in November, which could be explained by the accumulated uptake of larvae through the grazing season. In herds of farmers that dosed their in-calf heifers with anthelmintics were significantly more likely to be positive for antibodies against D. viviparus infection. This study highlights that both D. viviparus and O. ostertagi infections are widespread in dairy herds in Ireland throughout the grazing season.