Larvoscopic study on Dictyocaulus sp. in the faeces of beef cattle in northeastern Brazil.

The lungworm Dictyocaulus viviparus has an important role in cattle health and productivity worldwide, since infections can lead to substantial economic losses. Despite its importance, few studies investigating the epidemiological aspects of infection by this parasite have been conducted. The aim of this study was to report the occurrence of lungworm infection in beef cattle herds reared in an area of livestock production in the northeastern region of Brazil. From September 2020 to August 2021, monthly fecal samples (n = 493) were collected from 46 beef cattle. Among all the animals assessed, lungworm larvae were detected in 8.7% (4/46). None of them presented any clinical sign suggestive of infection by lungworm parasites. Twenty larvae were retrieved, with the minimum number (n = 1) detected in October and December, and the maximum number (n = 13) in November. These presented a mean length of 363 µm (± 28.65 µm) and mean width of 19 µm (± 1.03 µm), and were morphologically similar to Dictyocaulus sp.. This study reports the occurrence of this parasite in this livestock production area. Lastly, local veterinarians need to be aware of inclusion of this parasite in the differential diagnosis of other respiratory infections in beef cattle.

Larvoscopic study on Dictyocaulus sp. in the faeces of beef cattle in northeastern Brazil / Estudo larvoscópico de Dictyocaulus sp. em fezes de bovinos de corte do nordeste do Brasil

Abstract The lungworm Dictyocaulus viviparus has an important role in cattle health and productivity worldwide, since infections can lead to substantial economic losses. Despite its importance, few studies investigating the epidemiological aspects of infection by this parasite have been conducted. The aim of this study was to report the occurrence of lungworm infection in beef cattle herds reared in an area of livestock production in the northeastern region of Brazil. From September 2020 to August 2021, monthly fecal samples (n = 493) were collected from 46 beef cattle. Among all the animals assessed, lungworm larvae were detected in 8.7% (4/46). None of them presented any clinical sign suggestive of infection by lungworm parasites. Twenty larvae were retrieved, with the minimum number (n = 1) detected in October and December, and the maximum number (n = 13) in November. These presented a mean length of 363 μm (± 28.65 μm) and mean width of 19 μm (± 1.03 μm), and were morphologically similar to Dictyocaulus sp.. This study reports the occurrence of this parasite in this livestock production area. Lastly, local veterinarians need to be aware of inclusion of this parasite in the differential diagnosis of other respiratory infections in beef cattle.

Resumo O parasito pulmonar Dictyocaulus viviparus tem um papel importante na saúde e produtividade bovina em todo o mundo, uma vez que infecções podem levar a perdas econômicas. Além de sua importância, poucos estudos que investigam aspectos epidemiológicos das infecções por esse parasito têm sido realizados. Objetivou-se, nesta pesquisa, relatar a ocorrência de infecção por nematódeos pulmonares em rebanhos bovinos de corte, criados em uma importante área de produção pecuária, na região Nordeste do Brasil. De setembro de 2020 a agosto de 2021, foram coletadas, mensalmente, amostras fecais (n = 493) de 46 bovinos de corte. De todos os animais avaliados, larvas de nematódeos pulmonares foram detectadas em 8,7% (4/46). Os animais não apresentaram nenhum sinal clínico sugestivo de infecção por parasitos pulmonares. Vinte larvas foram recuperadas, com o número mínimo (n = 1) detectado em outubro e dezembro, e o número máximo (n = 13) em novembro. Apresentavam comprimento médio de 363 μm (± 28,65 μm), largura média de 19 μm (±1,03 μm) e eram morfologicamente semelhantes a Dictyocaulus sp.. Este estudo relata a ocorrência desse parasito nesta área. Logo, os veterinários locais devem estar atentos à inclusão desse parasito no diagnóstico diferencial de outras infecções respiratórias em bovinos de corte.

An assessment of the use of cox1 and cox3 mitochondrial genetic markers for the identification of Dictyocaulus spp. (Nematoda: Trichostrongyloidea) in wild ruminants.

Lungworms of the genus Dictyocaulus Railliet and Henry, 1907 (Nematoda: Trichostrongyloidea) are the causative agents of parasitic bronchitis (dictyocaulosis, husk) of various ungulate hosts, including domestic and wild ruminants. Correct diagnosis of lungworm species and a better understanding of the transmission patterns of Dictyocaulus spp. are crucial in minimising the risk of its cross transmission between wildlife and livestock, and for the control of dictyocaulosis. The study was conducted on large lungworms collected from European bison, roe deer and red deer. The study resulted in 14 sequences of the partial cox1 region of Dictyocaulus spp. and 10 novel DNA sequences of partial cox3 region, including the first available mt cox3 sequence, of the roe deer lungworm (D. capreolus). The European bison was infected with bison genotype of D. viviparus, whereas red deer and roe deer were infected with D. cervi and D. capreolus respectively. The current study revealed that the cox3 nucleotide sequences of D. capreolus and D. viviparus were 100% homologous to each other. Our findings indicate that the mt cox3 gene does not serve as an efficient mt marker for systematic, population genetic or molecular epidemiological studies of Dictyocaulus lungworms.

Diagnosis of bovine dictyocaulosis by bronchoalveolar lavage technique: A comparative study using a Bayesian approach.

Bovine dictyocaulosis is a pulmonary parasitic disease present in temperate countries, with potential important clinical and economic impacts. The Baermann technique is routinely used despite its low sensitivity in adult cows. Recently developed serological tests seem to offer better sensitivity, but validations of these tests in field conditions are few. We aimed to study two non-previously evaluated diagnosis methods of dictyocaulosis based on bronchoalveolar lavage sampling (BAL), which allows finding lungworm stages in the lungs as well as determination of eosinophilia. We compared them to the Baermann technique and serological tests. As no gold standard was available, we performed a Bayesian analysis by the simultaneous use of latent class and mixture models. The study was carried out during the 2015 pasture season on 60 adult cows originating from 11 herds with clinical signs of dictyocaulosis, and 10 apparently healthy cows originating from the teaching herd of VetAgro Sup, in France. Prevalence of infection was highly variable among herds with clinical signs (10-90%). Despite a maximal specificity (100%), the sensitivity of parasitological methods was low (7.4% for the Baermann sedimentation and 24.7% for the examination of BAL fluids). Better results were observed with serology (Se = 74.9%, Sp = 85.5%) with an optimal cut-off value estimated at 0.397 for the optical density ratio. Even better results were obtained with the count of eosinophil in BAL (Se = 89.4%, Sp = 85.2%) with an optimal cut-off value estimated at 4.77% for the eosinophil proportion. The BAL is a relevant diagnostic method of dictyocaulosis for practitioners due to the opportunity to perform two analyses (direct parasitic research and the eosinophil count) and to its good sensitivity and specificity.

Antibodies against Dictyocaulus viviparus major sperm protein in bulk tank milk: Association with clinical appearance, herd management and milk production.

The objective of this study was to conduct a comprehensive field survey using a Dictyocaulus viviparus major sperm protein ELISA on bulk tank milk samples from Belgian dairy herds to gain insights in: (1) the sensitivity (Se) and specificity (Sp) of the test under field conditions; (2) the value of the test to predict a future clinical lungworm outbreak; (3) its associations with milk production parameters and (4) its associations with herd management factors. A total of 1248 herds were sampled, with samplings occurring in the middle ("August") and towards the end ("October") of the grazing season. A completed questionnaire on potential risk factors and potentially lungworm-induced clinical signs was obtained from 587 farms and milk production records could be obtained from 343 herds. The median (25th-75th percentile) D. viviparus antibody level (ODR) was 0.25 (0.19-0.31) in "August" and 0.24 (0.19-0.32) in "October". At a threshold of 0.41 ODR, the Se and Sp were estimated using mixture models at 50 and 99%, respectively. At the same threshold, the positive and negative predictive value of the ELISA applied in "August" on the occurrence of farmer-reported lungworm symptoms in the period August-November were 65% and 69%, respectively. D. viviparus antibody levels were significantly higher in the north vs. the south of the country, in large herds and in herds that did not mow pastures or that frequently purchased new animals. An increase in the ELISA result of "August" over the interquartile range was associated with a drop in the annual average milk yield, milk protein% and milk fat% of -0.50kgcow-1day-1, 0.02 and 0.02, respectively. The relationships between the ELISA results in "October" and milk production parameters were also negative, but lower and non- or only marginally significant. We conclude that the bulk tank milk ELISA has a low value to predict lungworm disease on an individual farm based on a fixed sampling date in the middle of the grazing season. On the other hand, the test has been potential to detect subclinical production impacts and study risk factors through epidemiological surveys.

Cattle respiratory disease in late summer and autumn.

Increased diagnoses of husk in cattle are seen as summer turns into autumn. In this article, Arthur Otter of the APHA Cattle Expert Group discusses how the disease is diagnosed, and other respiratory diseases that it might be confused with.

Association between Dictyocaulus viviparus status and milk production parameters in Dutch dairy herds.

The objective of this study was to estimate the association between Dictyocaulus viviparus bulk tank milk (BTM) test results and milk production and milk composition parameters in adult Dutch dairy cattle herds. Bulk tank milk samples were collected in August and November 2013, and ELISA tests were performed. Two hundred BTM positive (BTM+) and 200 BTM negative (BTM-) herds were selected based on their BTM test result of November 2013, obtained from a list of farms that participated in the Dutch GD Animal Health voluntary monitoring program for controlling nematode infections. The relationship between D. viviparus BTM status and 3 production parameters (milk production, milk fat %, and milk protein %) in summer (June to August 2013) and autumn (September and October 2013) was investigated using generalized linear mixed models. Production data were available for 126 BTM- herds and 109 BTM+ herds. Results showed that a positive D. viviparus status was associated with decreased milk production (June: -1.01, July: -1.19, August: -1.68, September and October: -1.33kg/cow per d). Milk fat percentage was 0.14% and 0.08% lower during summer and autumn, respectively, in BTM+ herds. No significant association was demonstrated between a positive BTM test result and milk protein percentage. Because a strong correlation was present between the BTM status for D. viviparus and that for Ostertagia ostertagi, these losses cannot be attributed to one of the two parasites. However, it is clear that these parasite infections have a considerable effect on production.

Development of a multiplex fluorescence immunological assay for the simultaneous detection of antibodies against Cooperia oncophora, Dictyocaulus viviparus and Fasciola hepatica in cattle.

BACKGROUND: A major constraint for the effective control and management of helminth parasites is the lack of rapid, high-throughput, routine diagnostic tests to assess the health status of individual animals and herds and to identify the parasite species responsible for these helminthoses. The capability of a multiplex platform for the simultaneous detection of three pasture associated parasite species was evaluated and compared to existing ELISAs. METHODS: The recombinant antigens 14.2 kDa ES protein for Cooperia oncophora, major sperm protein for Dictyocaulus viviparus and Cathepsin L1 for Fasciola hepatica were recombinantly expressed either in Escherichia coli or Pichia pastoris. Antigens were covalently coupled onto magnetic beads. Optimal concentrations for coupling were determined following the examination of serum samples collected from experimentally mono-infected animals, before and after their infection with the target species. Absence of cross-reactivity was further determined with sera from calves mono-infected with Haemonchus contortus, Ostertagia ostertagi and Trichostrongylus colubriformis. Examination of negative serum samples was characterised by low median fluorescence intensity (MFI). RESULTS: Establishment of the optimal serum dilution of 1:200 was achieved for all three bead sets. Receiver Operating Characteristic analyses were performed to obtain cut-off MFI values for each parasite separately. Sensitivity and specificity at the chosen cut-off values were close to, or 100% for all bead sets. Examination of serum samples collected on different days post infection from different animals showed a high reproducibility of the assays. Serum samples were additionally examined with two already established ELISAs, an in-house ELISA using the recombinant MSP as an antigen and a DRG ELISA using Cathepsin L1 for liver fluke. The results between the assays were compared and kappa tests revealed an overall good agreement. CONCLUSIONS: A versatile bead-based assay using fluorescence detection (xMAP technology) was developed to simultaneously detect antibodies against C. oncophora, D. viviparus and F. hepatica in cattle serum samples. This platform provides rapid, high-throughput results and is highly sensitive and specific in comparison to existing serological as well as coproscopical diagnostic techniques.

Comparison of two serum and bulk-tank milk ELISAs for diagnosing natural (sub)clinical Dictyocaulus viviparus infection in dairy cows.

Lungworm antibody ELISAs developed in Germany (DE) and The Netherlands (NL) were compared using four sets of serum (S) and bulk-tank milk (BTM) samples from adult dairy cows. The samples originated from 37 farms with or without a suspected clinical lungworm infection during August-October 2010 (Dataset 1), from cows excreting lungworm larvae or not during August-October 2010 (n=59) or May-June 2011 (n=164) (Dataset 2), from 305 farms in a national survey during October 2010 (Dataset 3), and 14 zero-grazing farms during February-April 2011 (Dataset 4). During August-October 2010, covering the second half of the grazing season, the NL-S and NL-BTM ELISA outperformed the DE-S and DE-BTM ELISAs in terms of sensitivity. For at least the NL-S and DE-S ELISA the opposite was found during May-June 2011, covering the end of the winter housing period and the early grazing season. Of the 305 farms in the survey 62.6% were found positive with the NL-BTM ELISA, whereas 2.6% was found positive with the DE-BTM ELISA. ODR values for the zero-grazing farms indicated that a cut-off value of 30% for the DE-BTM ELISA might be more appropriate than the currently used 41%. Results suggest that the NL ELISAs also respond to lungworm antigens other than Major Sperm Protein as used in the DE ELISAs. It is concluded that the generally higher sensitivity of the NL-BTM ELISA makes it better suited for large-scale prevalence studies and herd health monitoring programmes than the DE-BTM ELISA, positivity of which is more associated with the presence of clinical lungworm infection. Reducing the cut-off value of the DE-BTM ELISA from the original 49.3% to the current 41% or the possibly more appropriate 30% increased its sensitivity for detecting lungworm infections, but did not lead to similar sensitivity estimates as found for the NL-BTM ELISA.

Validation of a Dictyocaulus viviparus MSP-ELISA and cut-off adjustment in a one-year longitudinal field study in dairy cattle herds.

A one-year field study analysing lungworm seropositivity by use of the MSP-ELISA was performed (1) to investigate the antibody dynamics in individual milk samples following field (re-)infections of dairy cows with the bovine lungworm Dictyocaulus viviparus, (2) to investigate the correlation between individual and bulk tank milk (BTM) antibody titres and (3) to review the current individual as well as BTM cut-off value, which was extrapolated from dilution experiments (Fiedor et al., 2009). Over a one-year period individual and BTM samples were collected monthly on 15 dairy farms. Following a critical review of previous cut-off values, individual and BTM samples were subjected to different cut-off thresholds. Following Receiver-Operating-Characteristics (ROC) analysis, individual milk samples were assessed with the cut-off value 0.573, previously shown to be associated with each 100% sensitivity and specificity. In addition, the present study enabled BTM cut-off adjustment based on field data. To ensure reliable detection of herds with an in-herd prevalence of ≥20% the BTM cut-off was lowered from 0.493 to 0.410, corresponding to 100% sensitivity and 97.32% specificity. Regression analysis showed that the percentage of seropositive animals related to the corresponding BTM ODR correlated moderately (r=0.581, P<0.001), whereas a strong correlation (r=0.764, P<0.001) was found between mean individual and BTM ODR per herd and sampling month. Seasonal antibody pattern became obvious in a single-peaked antibody curve in late summer/early autumn for individual milk whilst BTM showed a two-peaked distribution with an additional spring peak besides the late summer/early autumn peak. This leads to the conclusion that the BTM-ELISA could be a useful tool to detect and control pasture contamination in the spring, following sexual maturation of hypobiotic lungworm larvae harboured by clinically asymptomatic carrier animals. In addition to the knowledge gained on antibody patterns in dairy herds and the relationship of individual and BTM, the present study enabled sensitivity and specificity calculations for the obsolete BTM cut-off value 0.493 to be performed.

The value of a bulk-tank milk ELISA and individual serological and faecal examination for diagnosing (sub)clinical Dictyocaulus viviparus infection in dairy cows.

To test the value of a recently developed bulk-tank milk (BTM) ELISA for diagnosing (sub)clinical Dictyocaulus viviparus infection in lactating dairy herds under field conditions, bulk milk samples were collected from farms with or without clinical symptoms suspected to be caused by lungworm infection. Results of the BTM ELISA were compared against individual examinations for lungworm larvae in faeces and lungworm antibodies in serum from up to 20 heifers (parity 1) and up to 20 cows (parity ≥ 2) on the same farms. This also allowed, for the first time, to examine the value of individual faecal and serological examinations in the diagnosis of (sub)clinical lungworm infections. In total, 33 farms participated. Of these, 16 reported clinical symptoms possibly related to lungworm infection (defined as a suspected positive clinical status or CS(+)) and 17 reported having no such symptoms (CS(-)). In total, 503 heifers and 649 cows were sampled. Of all faeces samples positive for lungworm larvae, 94 were from heifers (18.9% of all heifers) and 75 from cows (11.7% of all cows) (P<0.001). Of all sera positive for lungworm antibodies, 130 were from heifers (26.1% of all heifers) and 113 from cows (17.5% of all cows) (P<0.001). Of the CS(-) farms 41% had at least one heifer or cow shedding larvae and 71% had at least one seropositive heifer or cow. Of the CS(+) farms this was 81% and 94%, respectively. There were only 4 farms, all CS(-), where none of the animals were found shedding larvae and all animals tested seronegative. This implies that on 76% of the CS(-) farms lungworm infection circulated unnoticed. On all CS(+) farms the suspicion that lungworm caused the respiratory symptoms was confirmed by the individual faecal and serological examinations, whereas the BTM ELISA confirmed presence of lungworm on half of the CS(+) farms. The latter in particular occurred on farms with the more severe outbreaks. Overall, of 32 available BTM samples 10 tested positive (8 of 15 CS(+) and 2 of 17 CS(-) farms). For diagnosing suspected lungworm disease it was concluded that testing a BTM sample might suffice in case of moderate to severe outbreaks. However, in case of a mild outbreak with just a few animals coughing, examining individual animals has to be preferred over testing a BTM sample. The likelihood to detect lungworm infection is higher if heifers are sampled compared to cows. Sensitivity of the BTM ELISA was 35.7% if the presence of at least one seropositive and/or one larvae shedding animal in the herd was used to define lungworm positive farms. On average, at least 30% of the herd had to be seropositive before the BTM ELISA was found positive for lungworm antibodies. Results indicate that the BTM ELISA in its current form does not appear to be suitable for surveys on the prevalence of lungworm presence on farms. However, this BTM ELISA might be used in large-scale surveys to detect, for instance, annual changes in percentage positive farms, as long as it is recognized that positivity is more closely related to incidence of lungworm disease than to prevalence of lungworm infection.

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.

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.

Lungworms of ruminants.

Several species of nematodes parasitize the respiratory tract of ruminants. Disease due to infection with these parasites occurs less frequently in North America than in other parts of the world but can be locally and regionally important. The life cycles, epidemiology, pathology, diagnosis and treatment of Dictyocaulus viviparus, Dictyocaulus filaria, and Muellerius capillaris are summarized in this article.