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Serological lessons from the bovine lungworm Dictyocaulus viviparus: Antibody titre development is independent of the infection dose and reinfection shortens seropositivity
Veterinary Parasitology 242 (2017) 47–53
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Research paper
Serological lessons from the bovine lungworm Dictyocaulus viviparus: Antibody titre development is independent of the infection dose and reinfection shortens seropositivity
MARK
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Christina Strubea, , Andrea Springera, Anne-Marie Schunna, Andrew B. Forbesb a b
Institute for Parasitology, Centre for Infection Medicine, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany Scottish Centre for Production Animal Health and Food Safety, School of Veterinary Medicine, University of Glasgow, G61 1QH, Scotland, UK
A R T I C L E I N F O
A B S T R A C T
Keywords: Dictyocaulus Bovine lungworm Antibody titre Antibody level ELISA Milk ELISA BTM ELISA Infection dose Reinfection
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 MSPELISA 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 dosedependent. 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.
1. Introduction Infections with the bovine lungworm Dictyocaulus viviparus, the causative agent of parasitic bronchitis, are accompanied by substantial economic losses due to serious respiratory disease or even death of diseased cattle. In addition, subclinical lungworm infections negatively impact production parameters like milk yield, milk protein and fat content (Charlier et al., 2016; Dank et al., 2015). Despite the availability of a live attenuated vaccine and effective anthelminthics, dairy herd prevalence remains high (Bloemhoff et al., 2015; Höglund et al., 2010; Schunn et al., 2013). Because of its complex epidemiology, outbreaks of this pasture-borne disease can be difficult to predict (Eysker et al., 1994; Ploeger and Holzhauer, 2012; Wapenaar et al., 2007), resulting in the need for epidemiological surveillance. Clinical cases predominantly occur in youngstock, whereas adult dairy cows have traditionally played a role as silent carriers of the parasite,
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Corresponding author. E-mail address: [email protected] (C. Strube).
http://dx.doi.org/10.1016/j.vetpar.2017.05.023 Received 30 March 2017; Received in revised form 19 May 2017; Accepted 21 May 2017 0304-4017/ © 2017 Elsevier B.V. All rights reserved.
constituting the main source of pasture contamination (Saatkamp et al., 1994). However, during the last decades, disease outbreaks in adult cattle have become more frequent (David, 1997; van Dijk, 2004; Wapenaar et al., 2007). Dictyocaulus viviparus induces strong immune responses, resulting in protection against invading larvae as well as in reduced development and fecundity of pulmonary parasite stages during reinfections (Eysker et al., 2001; Jarrett et al., 1959; Michel, 1962), thus protecting most cattle from clinical disease. Protection is predominantly mediated by local immune responses, e.g. IgE directed against larval parasite stages (Kooyman et al., 2002) and local antibody production in the lungs (Scott et al., 1996). The degree of protection against reinfections is dependent on the initial level of infection (Eysker et al., 1993), and usually lasts only for a period of six to twelve months (Michel, 1962; Michel and Mackenzie, 1965). However, not all of the different manifestations of protection decline equally over time. Michel and Mackenzie (1965) found that protection against the establishment
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2008), serum samples were collected every two days p.i. until onset of seropositivity, and then approximately at weekly intervals. Animal experiments were approved by the ethics commission of the Institutional Animal Care and Use Committee (IACUC) of the Lower Saxony State Office for Consumer Protection and Food Safety (Niedersaechsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit) under reference number AZ 33.9-42502-0411/0371.
of challenge infection as well as the inhibition of early worm growth greatly declined during the first six months after first infection, whereas other phenomena like inhibition of development beyond the preadult stage persisted for more than a year. In the absence of regular boostering of immunity, outbreaks of parasitic bronchitis can occur. In addition, the so-called reinfection syndrome, a severe immunemediated inflammatory response, may cause disease in immune cattle upon exposure to large numbers of invading larvae, although these reinfections usually do not become patent (Breeze, 1985; Michel, 1957). To perform epidemiological studies and to assess herd infection status, for example at restocking of parasite-naïve animals, reliable and cost-efficient tools are needed. Routine examination of bulk-tank milk (BTM) may provide an inexpensive method to monitor herd health and thus potentially prevent disease and production losses (Charlier et al., 2016; Fiedor et al., 2009). An enzyme-linked immunosorbent assay (ELISA) based on recombinant major sperm protein (MSP), which is expressed in spermatocytes of adult male lungworms, has been developed for detection of lungworm-specific antibodies in serum and milk (Fiedor et al., 2009; Schunn et al., 2012; von Holtum et al., 2008), and has been used in several epidemiological studies (e.g. Bloemhoff et al., 2015; Charlier et al., 2016; Höglund et al., 2010; Schunn et al., 2013). However, accurately determining lungworm infections within a herd using BTM can be challenging, as within-herd seroprevalence may frequently be lower than the value of 20% which is required for the BTM ELISA result to reliably exceed the evaluated positivity threshold of an optical density ratio (ODR) of 0.410 (Schunn et al., 2012). In a recent field study including 15 dairy herds, the monthly within-herd seroprevalence of lungworm-positive herds − based on testing of approximately one third of cows in each herd − ranged between 0 and 29% of animals (Schunn et al., 2012). Several factors might be responsible for low within-herd seroprevalence despite potential occurrence of D. viviparus in many dairy herds. Because the level of protection against clinical disease is dependent on the initial infection dose, it is conceivable that low infection doses, as they may occur in the field, result in below-threshold levels of antibody production. Furthermore, as host immune responses affect invasion, development and fecundity of D. viviparus (Eysker et al., 2001; Jarrett et al., 1959; Michel, 1962), the antigenic stimulus during reinfections of partially protected animals might be insufficient to cause reliable antibody production against adult parasite stages, hampering serological diagnosis of reinfections. To test these hypotheses, a study was conducted to a) compare MSP antibody levels after initial infection with 12 different infection doses ranging from 10 to 3000 D. viviparus larvae, b) to evaluate the antibody response during reinfections at different time intervals after initial infection, and c) to assess patterns of MSP antibodies in adult dairy cows during naturally occurring reinfections.
2.2. D. viviparus reinfection experiments In a first reinfection experiment, two animals (animal A and B), which had been initially infected with 3000 L3, were re-infected with a dose of 3000 L3 eight months after initial infection (day 245 p. i.), and again about 3 months later (day 343 p. i.). The time span of eight months until reinfection was chosen to mimic either reinfection at turnout after the housing period or re-activation of hypobiotic larvae in spring, respectively. Reinfection another three months later was chosen to represent reinfection with the third generation of lungworms on the pasture, when herd prevalence as well as the level of pasture contamination usually shows a peak (Jørgensen, 1980; Pott et al., 1978). In a second reinfection experiment, two individuals (animal C and D) were initially infected with 2000 and 3000 L3, respectively, and then re-infected at day 90, day 150, and day 272 post initial infection with the same infection dose. During the study period, serum samples were collected every 2 days after infections until onset of seropositivity, followed by every 2–7 days. Faecal samples (2 × 10 g per animal) were analysed every 1–2 days as of day 20 after infections with the Baermann technique as described above to monitor larvae shedding. Animal experiments were approved by the ethics commission of the Institutional Animal Care and Use Committee (IACUC) of the Lower Saxony State Office for Consumer Protection and Food Safety under reference number AZ 33.9-42502-04-10/0078. 2.3. Field study on D. viviparus reinfections To assess patterns of MSP antibody levels during natural reinfections in dairy cows, individual milk samples were taken monthly from March 2009 to February 2010 at seven different dairy farms. Inclusion criteria for participating farms were as follows: dairy cows had to be kept on pasture during the grazing season and herd size had to be ≥20 dairy cows. Herd sizes of participating farms ranged from 20 to 86 cows. Lungworm-status of all cows included in the study was assessed in August the year before, when a minimum of 20 individual milk samples and one BTM sample per herd were tested for lungworm antibodies with the MSP-ELISA (Fiedor et al., 2009; Schunn et al., 2012). Additionally 2–5 cows per herd were randomly selected and tested for larvae shedding at this time. Based on these results, all seven farms were assigned a lungworm-positive status, whereas clinical signs of coughing were present on three farms only. Most of the participating dairy farms practised anthelmintic treatment of calves or heifers, whereas cows were not treated. For milk sample collection, tubes with lyophilised boric acid as preservative (Exactobac-L, nerbe plus GmbH, 21423 Winsen/Luhe, Germany) were used. Samples were stored at −20 °C until analysis by ELISA. Additionally, individual faecal samples were collected from each cow (n = 81) between June and September (and examined (2 × 40 g per cow)) for lungworm larvae via the Baermann technique.
2. Materials and methods 2.1. D. viviparus infections at different dose levels Twenty-four parasite-naïve, male 3 month old Holstein Friesian calves were used for infection experiments. Calves were reared at the Ruthe Research Farm of the University of Veterinary Medicine Hannover and were weaned before the start of the study. Two calves each were infected orally with the field isolates HannoverDv2000 or HannoverDv2010 (depending on the date of infection) at a dose of 10, 25, 50, 100, 250, 500, 1000, 1250, 1500, 2000, 2500 and 3000 thirdstage larvae (L3). The success of experimental infections was assessed from day 20 post infection (p.i.) by faecal examinations (2 × 2 g faeces per animal) using the Baermann technique. Specifically, faeces were placed on a sieve (150 μm mesh size) and larvae were allowed to migrate for approximately 18 h at room temperature. To monitor serum antibody development by use of the MSP-ELISA (von Holtum et al.,
2.4. ELISA Antibody response was evaluated using serum samples during dose and reinfection experiments, and using individual milk samples during the dairy cattle field study. MSP-ELISAs were carried out as previously described (Fiedor et al., 2009; Schunn et al., 2012; von Holtum et al., 48
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Fig. 1. Serum antibody levels against Dictyocaulus viviparus major sperm protein (MSP) of two calves each infected with 12 different infection doses from day 0 to day 66 post infection: (A) low, (B) medium and (C) high infection doses. Antibody levels are expressed as optical density ratios (ODR). The dashed line indicates the cut-off value of 0.500 ODR.
2008). All reactions were set up as duplicates. The arithmetic means of the ODs of the duplicates were converted into optical density ratios (ODR) using the following formula: ODR = (ODtest sample − Dblank)/ (ODpositive control − ODblank). To determine seropositivity, a cut-off value of 0.500 ODR was used for serum samples and a cut-off value of 0.573 ODR for milk samples as previously validated (Schunn et al., 2012; von Holtum et al., 2008). 3. Results 3.1. D. viviparus infections at different dose levels Infection of two calves with a single dose of 10 larvae resulted in one animal shedding 1–10 larvae/10 g faeces on three days during the examination period of 31 days, whereas no larval shedding was observed in the second animal. Neither of these two individuals became seropositive (Fig. 1A). All other infection doses resulted in patency as well as antibody production in both infected cattle (Fig. 1). Animals began shedding larvae between day 23 and day 29 p. i. (mean: 25.8, SD: 2.1) and became seropositive between day 28 and day 50 p. i. (mean: 34.5, SD: 5.7; Fig. 1). For those 22 animals which became seropositive, Spearman rank correlations were used to assess whether there was a relationship between infection dose and the onset of seropositivity or the maximum antibody level reached until day 66 p. i., respectively. As shown in Fig. 2, no significant correlation was found (N = 22, Spearman ρ = 0.11, P = 0.62, and Spearman ρ = −0.27, P = 0.233, respectively). Fig. 2. Scatterplots depicting (A) onset of seropositivity (day p.i.) and (B) maximum antibody level (expressed as optical density ratio [ODR]) against infection dose of 24 calves infected with 12 different doses of D. viviparus larvae.
3.2. Reinfection experiments Results of reinfection experiments are shown in Fig. 3. Reinfection of animals A and B eight months (245 days) after initial infection resulted in marked, but short periods of seropositivity relative to the period of seropositivity following primary infection (78 and 58 days versus 189 and 112 days, respectively, Fig. 3A). Reinfection of the same animals another 98 days later (day 343 post initial infection) resulted in another peak of seropositivity of even shorter duration (25 days) in animal A, whereas animal B remained seronegative. ODR values during both reinfections did not reach the same level as during initial infections. Furthermore, neither the first nor the second reinfections became patent. In the second experiment, reinfection 90 days after initial infection caused a rise in antibody levels, which started 46 days after reinfection (day 137 post initial infection) and lasted for approximately 10 days only until returning to previous levels. In contrast, seropositivity following initial infection had lasted for 50 days in animal D, whereas
animal C was still seropositive when reinfection occurred. However, via extrapolation the duration of seropositivity upon primary infection can be estimated at approximately 150 days without this second antigenic stimulus (Fig. 3B). Again, maximum ODR values caused by reinfection were lower compared to initial infection, and in animal D not sufficient to raise the ODR above the cut-off value of 0.500 (Fig. 3B), even though these reinfections became patent with low levels of larvae shedding in both individuals (between 1 and 7 larvae/2 × 10 g faeces for the duration of one week). The second reinfection of these animals another 60 days later (day 150 post initial infection) resulted in even lower patency (1 larva/2 × 10 g faeces at just one day) and caused only a barely noticeable increase of antibody levels 35 days after reinfection (day 185 post initial infection) in animal C, but no serological reaction or larvae shedding in animal D. However, when the animals were re49
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Fig. 3. Serum antibody levels directed against Dictyocaulus viviparus major sperm protein (MSP) over the course of two different reinfection experiments. In the first experiment (A) calves were reinfected on day 245 and day 343 post initial infection, in the second experiment (B) on day 90, day 150 and day 272 post initial infection. Antibody levels are expressed as optical density ratios (ODR). The dashed line indicates the cut-off value of 0.5.
infected a third time after MSP antibody levels had dropped to baseline levels in both individuals (day 272 post initial infection), animal C showed a strong seropositive antibody response 36 days after reinfection (day 308 post initial infection), which lasted – similar to initial infections – for 107 days. Furthermore, larval shedding was observed. By contrast, animal D remained seronegative and did not develop a patent infection.
Table 1 Number of animals included in the field study per dairy herd and percentage of animals showing a positive milk MSP antibody titre again during the course of the sampling period.
3.3. Field study on D. viviparus reinfections Overall, 52 dairy cows were included in the field reinfection study, corresponding to 3–11 cows per herd (mean 7.4), all of which displayed positive MSP titres at initial assessment. Over the course of the following year, a positive antibody level was detected in 11 of these cows (21.1%, 0–44.4% of sampled individuals per herd, Table 1). MSP50
Herd
Clinical signs at initial assessment
No. of animals sampled
Lungworm-positive (no./%)
1 2 3 4 5 6 7
No Yes Yes No Yes No No
3 11 8 9 5 7 9
0/0 3/27.3 1/12.5 1/11.1 1/20 1/14.3 4/44.4
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0.256 – 0.313 0.160 0.217 – – 0.380 0.145 – 0.364 0.262 0.286 – 0.329 0.250 0.195 – 0.618 0.338 0.384 – 0.426 0.353
0.218 – 0.293 0.174 0.252 – 0.937 0.315 0.248 – 0.366 0.350
Jan Dec
Feb
ELISA ODR results from monthly milk samples of those 11 cows are presented in Table 2. Short periods of seropositivity lasting only one to two months were observed in 10 of these individuals, whereas for one individual duration of seropositivity could not be conclusively determined because it became seropositive at the end of the study period. All faecal samples were negative for lungworm larvae, despite the fact that three individuals showed a positive antibody level at the time of faecal sampling.
0.246 0.417 0.223 0.312 0.151 – 0.503 0.273 0.376 – 0.451 0.328 0.407 (neg.) 0.230 (neg.) 0.455 (neg.) 0.290 0.919 – 0.349 0.404 – 0.390 0.530 0.442 0.465 0.173 0.408 0.509 – 0.457 0.264 – 0.416 0.350 0.513 0.395
– – – 0.712 (neg.) 0.322 0.350 (neg.) 0.332 (neg.) – (neg.) 0.704 (neg.) 0.489 (neg.) – 0.485
0.279 0.230 0.342 0.217 (neg.) 0.547 (neg.) – 0.327 0.584 0.402 0.339 0.709 (neg.) 0.398
0.245 0.386 0.242 0.342 0.121 – 0.348 0.325 0.360 – 0.473 0.316
Although dictyocaulosis is considered to be a herd problem, diagnosing subclinical lungworm infections can be challenging, particularly using bulk tank milk from dairy herds with low within-herd seroprevalence. In this study, potential causes of such low within-herd seroprevalences were investigated. The presented data demonstrate that the initial rise in antibody levels directed against the major sperm protein (MSP) of Dictyocaulus viviparus is independent of a primary infection at doses of ≥25 larvae, but that the magnitude and the duration of antibody response are reduced following reinfections. These results from experimental infections were confirmed by a field study, which revealed short periods of seropositivity as a major pattern in individual dairy cows exposed to natural D. viviparus reinfections. It was hypothesized that low infection doses in the field might lead to an attenuated production of antibodies against adult lungworms. However, when examining MSP antibody levels following infection with different doses ranging from 25 to 3000 larvae, no dose-dependency could be observed in this study on the onset of seropositivity and the magnitude of the response. Serum antibodies exceeded the cut-off value of 0.5 ODR on average 34.5 days after infection (range: 28–50), which supports the findings by Fiedor et al. (2009) (30-31 days, range: 26–41). Furthermore, the antibody curves showed a steep rise in the majority of animals, making it easy to distinguish seronegative from seropositive titres using the validated cut-off value of 0.500 (von Holtum et al., 2008). The absence of any dose-dependency was particularly noteworthy, as infection dose is positively associated with the level of protection against reinfections, with higher initial infection doses resulting in a higher level of protection (Ploeger and Eysker, 2002). Furthermore, it has been demonstrated that, independent of the infection dose, about 20–30% of larvae establish in the host (Ploeger and Eysker, 2002); a higher infection dose thus results in a higher parasite burden. Based on these data, it therefore has to be concluded that MSP antibody response during initial infection is independent of the number of adult parasites present in the host. Only the lowest infection dose of 10 larvae failed to result in a positive ELISA ODR, although a low level of patency was observed in one of the two individuals infected with this dose. It may be hypothesized that not enough or even no male lungworms reached adulthood in this animal and thus not enough or no sperm was produced to stimulate MSP antibody production. In conclusion, only very low levels of initial infection failed to evoke an antibody response, and low infection doses under natural conditions are unlikely to constitute a problem for serodiagnosis of primary infections. However, an effect of reinfection on MSP antibody levels was found, namely a reduction in magnitude and duration of the antibody response as compared to the initial response after primary infection. Adult lungworms are usually eliminated 60–90 days post infection and protection against challenge infection may then last for six to twelve months (Michel, 1962; Michel and Mackenzie, 1965). MSP antibodies have been shown to persist on average 95–112 days after initial infection (Fiedor et al., 2009). In our reinfection experiments, duration of seropositivity upon initial infections ranged on average 125 days (50–189 days). In contrast, the elevation in antibody levels observed after reinfections at eight months post initial infection lasted only 58 and 71 days (cf. Fig. 3A). As the MSP-ELISA only detects antibodies directed against proteins of adult male parasites, the short duration of
No milk sample available (dry period/animal sold or died). Information not available. b
a
2 2 2 3 4 5 6 7 7 7 7 35606 84462 86253 02381 78604 69227 1564 12140 43688 80565 97076 Mean ODR
2 2 3 4 4 2 n.a.b 6 5 4 3
0.723 0.882 0.872 0.896 0.850 0.646 0.993 0.737 0.989 0.743 0.673 0.819
0.303 0.146 0.555 0.149 0.307 0.286 0.26 – 0.272 0.235 0.305 0.282
0.663 0.186 0.667 0.210 – 0.724 0.309 – 0.343 0.308 0.349 0.418
–a 0.644 – 0.142 – 0.464 0.277 – 0.447 0.579 0.535 0.441
Aug Jun Initial assessment (Aug)
Herd
Lactation number
Individual milk ELISA ODR
Mar
Apr
May
Jul
Sep
Oct
Nov
4. Discussion
Animal ID
Table 2 ODR values of individual milk samples collected from adult dairy cows during the field study. Only animals are shown which became seropositive again during the year following initial assessment. Values exceeding the individual milk cut-off of 0.573 ODR are marked in grey. Results of faecal examinations using the Baermann technique are indicated in brackets.
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(cf. Fig. 3B, animal C). Similar patterns have already been observed by Höglund (2006). Despite serological evidence of reinfections, no evidence of larval shedding was found during the field study, even though three individuals displayed positive MSP antibody levels at the time of faecal sampling. This may indicate that reinfections did not lead to patency, as observed also in some of the reinfection experiments. However, animals were only sampled once or twice during the field study, not daily as during the infection experiments. Thus, it is possible that larvae shedding was missed due to limited sensitivity of the Baermann technique (Rode and Jørgensen, 1989), or because infections had already reached the postpatent period, whereas antibodies still persisted. Overall, the data collected from the infection experiments as well as the field study indicate that the attenuated antibody response and shortened seropositivity following reinfections may lead to low withinherd seroprevalence. In consequence, BTM antibody levels of immune herds may be too low to turn the BTM-ELISA, which shows 100% sensitivity and 97.32% specificity only as of an within-herd prevalence of ≥20% (Schunn et al., 2012), positive. However, it remains debatable whether this actually represents a disadvantage of the BTM-ELISA, as reinfections often do not become patent and seldom cause clinical symptoms, and are thus less important from a clinical perspective. In contrast, the BTM-ELISA is an excellent tool to investigate the incidence of lungworm infections in dairy herds, i.e. the number of newly infected herds in a certain time frame, as initial infections produce long periods of seropositivity. However, if a major aim of using the BTM-ELISA is to detect subclinical infections or reinfections in immune herds, the test should be carried out in August/September, when herd seroprevalence as well as antibody levels in BTM show a peak (Schunn et al., 2012).
seropositivity upon reinfections may reflect limited parasite development as a consequence of immune responses expressed in the lungs (Michel and MacKenzie, 1965), resulting in low adult worm burdens and reduced worm fecundity, leading to abbreviated seropositivity. In this context, immunization experiments with the commercially available attenuated live vaccine (Bovilis® Dictol) have shown that protected animals show 93–97% reduction in worm burden and 94% reduction in larvae shedding upon challenge infections as compared to control animals (Bain and Urquhart, 1988; Strube et al., 2015). When animals were reinfected at intervals of 90 and 150 days after initial infection, a low level of patency was observed. However, no pronounced secondary antibody response could be noted as the rise in antibody levels was less distinct and of shorter duration, or entirely absent. This may be due to protection against the establishment of challenge infection as well as the inhibition of early worm growth during the first months of acquired immunity (Michel and Mackenzie, 1965). A similar course of serum antibody levels despite lungworm reinfections during this time frame has already been reported from an early study using adult worm homogenate as antigen (Cornwell et al., 1960; Cornwell and Michel, 1960 Cornwell and Michel, 1960). By constrast, Scott et al. (1996), who also used adult worm homogenate as antigen, noted a further antibody increase following reinfection at day 65 post initial infection, whereas a second reinfection at day 112 post initial infection did not provoke any apparent antibody response. Two animals of the present study were subjected to a third reinfection 272 days after initial and 120 days after the preceding infection, when antibody levels in both animals had returned to baseline. Interestingly, this tertiary reinfection led to patency and a strong, rather long-lasting (107 days) antibody response in one of the two individuals (animal C, cf. Fig. 3B), whereas the other one remained seronegative and did not excrete larvae, indicating that it was still protected against challenge infection. Theoretically, differences in the viability of larvae between the two infection doses might have also been responsible for this discrepancy. However, during a total of 17 yearś experience by maintaining the lungworm field isolates used in the present study at the Institute for Parasitology, University of Veterinary Medicine Hannover, Germany, experimental infections of parasitenaïve cattle (doses of 1500–3000 L3) for parasite maintenance always resulted in patent lungworm infections (unpublished results). Furthermore, animals of each reinfection experiment were infected at the same time and from the same batch of larvae. Thus, we regard this possibility as extremely unlikely. A large variability between hosts in their response to infection is commonly known, possibly with a genetic basis (Ploeger, 2002). In summary, the reinfection experiments indicate that the antibody response is probably both influenced by the interval to the last infection and by individual host factors. The insights gained from these reinfection experiments have implications for the diagnosis of dictyocaulosis in the field. In the field study, only 21.1% of dairy cows which displayed a positive MSP milk antibody level upon initial assessment in August were detected seropositive again over the course of the following year. As the infection experiments have indicated, immune animals may not show a rise in antibody levels upon reinfection sufficient to result in a positive ELISA ODR, even though the reinfection may cause low levels of larval shedding. All – but one – dairy cows which turned seropositive again during the study period, displayed only short periods of positive MSP-ELISA results, lasting merely one or two months. This confirms the pattern of shortened seropositivity observed during the reinfection experiments. For one individual, the duration of seropositivity could not be conclusively determined; however, it appears that this individual displayed a rather long period of seropositivity lasting at least three months. This individual was seronegative for nine consecutive months before turning seropositive again. Thus, it is likely that a sufficient amount of time had elapsed for the animal to become susceptible again, resulting in a similiar strong antibody response upon reinfection as observed in one of the individuals in the second reinfection experiment
5. Conclusions During first infections with Dictyocaulus viviparus, MSP antibody levels are dose-independent as of an initial infection dose of 25 larvae. However, after reinfections, duration of seropositivity may be reduced or animals may not become seropositive at all, depending on the time interval until reinfection. Further experimental reinfection studies are needed to support the presented reinfection experiments and to elucidate the attenuated antibody response in more detail and regarding its underlying mechanisms. Short periods of seropositivity were detected as a major pattern in dairy cows subjected to natural D. viviparus reinfections, potentially resulting in low within-herd seroprevalence and low BTM antibody levels. The BTM MSP-ELISA may nevertheless serve as a useful tool in epidemiological studies as well as from a clinical perspective, as a negative result indicates absence of infection or protection of the herd due to immunity. Thus, a negative ELISA result in coughing herds indicates that dictyocaulosis is an unlikely cause for these symptoms. In such herds with acquired immunity, BTM testing in August/September can facilitate detection of antibodies. However, to confidently exclude lungworm infections it may ultimately be necessary to test individual milk (or serum) samples of a sufficient proportion of a herd, for example before restocking parasite-naïve individuals. Such efforts may focus on those animals which are likely to suffer from a primary infection and can thus be expected to have a high and long-lasting antibody level, such as recently introduced first-year grazing heifers.
Acknowledgements The authors wish to thank Sandra Buschbaum for excellent technical assistance. Parts of the study were funded by Merial. Study data interpretation is completely independent from the company’s opinion and there is no conflict with commercial interests.
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immunity resulting from experimental infection. Immunology 2, 252–261. Michel, J.F., Mackenzie, A., 1965. Duration of the acquired resistance of calves to infection with Dictyocaulus viviparus. Res. Vet. Sci. 6, 344–395. Michel, J., 1957. Husk in adult cattle. Agriculture 64, 224–228. Michel, J., 1962. Studies on resistance to Dictyocaulus infection: IV. The rate of acquisition of protective immunity in infection of D. viviparus. J. Comp. Pathol. 72, 281–285. Ploeger, H.W., Eysker, M., 2002. Protection against and establishment of Dictyocaulus viviparus following primary infection at different dose levels. Vet. Parasitol. 106, 213–223. Ploeger, H.W., Holzhauer, M., 2012. Failure to eradicate the lungworm Dictyocaulus viviparus on dairy farms by a single mass-treatment before turnout. Vet. Parasitol. 185, 335–338. Ploeger, H.W., 2002. Dictyocaulus viviparus: re-emerging or never been away? Trends Parasitol. 18, 329–332. Pott, J.M., Jones, R.M., Cornwell, R.L., 1978. Observations on parasitic gastroenteritis and bronchitis in grazing calves: untreated calves. Int. J. Parasitol. 8, 331–339. Rode, B., Jørgensen, R.J., 1989. Baermannization of Dictyocaulus spp. from faeces of cattle, sheep and donkeys. Vet. Parasitol. 30, 205–211. Saatkamp, H.W., Eysker, M., Verhoeff, J., 1994. Study on the causes of outbreaks of lungworm disease on commercial dairy farms in the Netherlands. Vet. Parasitol. 53, 253–261. Schunn, A.-M., Forbes, A., Schnieder, T., Strube, C., 2012. Validation of a Dictyocaulus viviparus MSP-ELISA and cut-off adjustment in a one-year longitudinal field study in dairy cattle herds. Vet. Parasitol. 189, 291–298. Schunn, A.-M., Conraths, F.J., Staubach, C., Fröhlich, A., Forbes, A., Schnieder, T., Strube, C., 2013. Lungworm infections in German dairy cattle herds—seroprevalence and GIS-supported risk factor analysis. PLOS One 8, e74429. Scott, C.A., McKeand, J.B., Devaney, E., 1996. A longitudinal study of local and peripheral isotype/subclass antibodies in Dictyocaulus viviparus-infected calves. Vet. Immunol. Immunopathol. 53, 235–247. Strube, C., Haake, C., Sager, H., Schorderet Weber, S., Kaminsky, R., Buschbaum, S., Joekel, D., Schicht, S., Kremmer, E., Korrell, J., Schnieder, T., von SamsonHimmelstjerna, G., 2015. Vaccination with recombinant paramyosin against the bovine lungworm Dictyocaulus viviparus considerably reduces worm burden and larvae shedding. Parasit. Vectors 8, 119. van Dijk, J., 2004. The epidemiology and control of dictyocaulosis in cattle. Cattle Pract. 12 (2), 133–145. von Holtum, C., Strube, C., Schnieder, T., von Samson-Himmelstjerna, G., 2008. Development and evaluation of a recombinant antigen-based ELISA for serodiagnosis of cattle lungworm. Vet. Parasitol. 151, 218–226. Wapenaar, W., Barkema, H.W., Eysker, M., O'Handley, R.M., 2007. An outbreak of dictyocaulosis in lactating cows on a dairy farm. J. Am. Vet. Med. Assoc. 231, 1715–1718.
References Bain, R.K., Urquhart, G.M., 1988. Parenteral vaccination of calves against the cattle lungworm Dictyocaulus viviparus. Res. Vet. Sci. 45, 270–271. Bloemhoff, Y., Forbes, A., Good, B., Morgan, E., Mulcahy, G., Strube, C., Sayers, R., 2015. Prevalence and seasonality of bulk milk antibodies against Dictyocaulus viviparus and Ostertagia ostertagi in Irish pasture-based dairy herds. Vet. Parasitol. 209, 108–116. Breeze, R., 1985. Parasitic bronchitis and pneumonia. Vet. Clin. North Am. Food Anim. Pract. 1, 277–287. Charlier, J., Ghebretinsae, A., Meyns, T., Czaplicki, G., Vercruysse, J., Claerebout, E., 2016. Antibodies against Dictyocaulus viviparus major sperm protein in bulk tank milkAssociation with clinical appearance, herd management and milk production. Vet. Parasitol. 232, 36–42. Cornwell, R.L., Michel, J.F., 1960. The complement fixing antibody response of calves to Dictyocaulus viviparus. I. Exposure to natural and experimental infection. J. Comp. Pathol. 70, 482–493. Dank, M., Holzhauer, M., Veldhuis, A., Frankena, K., 2015. Association between Dictyocaulus viviparus status and milk production parameters in Dutch dairy herds. J. Dairy Sci. 98, 7741–7747. David, G.P., 1997. An epidemiological study of husk in adult cows in 32 UK herds (Preliminary findings). Cattle Pract. 5, 295–297. Eysker, M., Saatkamp, H.W., Kloosterman, A., 1993. Infection build-up and development of immunity in calves following primary Dictyocaulus viviparus infections of different levels at the beginning or in the middle of the grazing season. Vet. Parasitol. 49, 243–254. Eysker, M., Claessens, E.W., Lam, T.J., Moons, M.J., Pijpers, A., 1994. The prevalence of patent lungworm infections in herds of dairy cows in the Netherlands. Vet. Parasitol. 53, 263–267. Eysker, M., Kooyman, F.N.J., Ploeger, H.W., 2001. Immunity in calves against Dictyocaulus viviparus following a low primary infection. Parasitology 123, 591–597. Fiedor, C., Strube, C., Forbes, A., Buschbaum, S., Klewer, A.-M., von SamsonHimmelstjerna, G., Schnieder, T., 2009. Evaluation of a milk ELISA for the serodiagnosis of Dictyocaulus viviparus in dairy cows. Vet. Parasitol. 166, 255–261. Höglund, J., Dahlström, F., Engström, A., Hessle, A., Jakubek, E.-B., Schnieder, T., Strube, C., Sollenberg, S., 2010. Antibodies to major pasture borne helminth infections in bulk-tank milk samples from organic and nearby conventional dairy herds in southcentral Sweden. Vet. Parasitol. 171, 293–299. Höglund, J., 2006. Targeted selective treatment of lungworm infection in an organic dairy herd in Sweden. Vet. Parasitol. 138, 318–327. Jørgensen, R.J., 1980. Epidemiology of bovine dictyocaulosis in Denmark. Vet. Parasitol. 7, 153–167. Jarrett, W.F.H., Jennings, F.W., Mcintyre, W.I.M., Mulligan, W., Thomas, B.A.C., Urquhart, G.M., 1959. Immunological studies on Dictyocaulus viviparus infection: the
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Research paper
Serological lessons from the bovine lungworm Dictyocaulus viviparus: Antibody titre development is independent of the infection dose and reinfection shortens seropositivity
MARK
⁎
Christina Strubea, , Andrea Springera, Anne-Marie Schunna, Andrew B. Forbesb a b
Institute for Parasitology, Centre for Infection Medicine, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany Scottish Centre for Production Animal Health and Food Safety, School of Veterinary Medicine, University of Glasgow, G61 1QH, Scotland, UK
A R T I C L E I N F O
A B S T R A C T
Keywords: Dictyocaulus Bovine lungworm Antibody titre Antibody level ELISA Milk ELISA BTM ELISA Infection dose Reinfection
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 MSPELISA 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 dosedependent. 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.
1. Introduction Infections with the bovine lungworm Dictyocaulus viviparus, the causative agent of parasitic bronchitis, are accompanied by substantial economic losses due to serious respiratory disease or even death of diseased cattle. In addition, subclinical lungworm infections negatively impact production parameters like milk yield, milk protein and fat content (Charlier et al., 2016; Dank et al., 2015). Despite the availability of a live attenuated vaccine and effective anthelminthics, dairy herd prevalence remains high (Bloemhoff et al., 2015; Höglund et al., 2010; Schunn et al., 2013). Because of its complex epidemiology, outbreaks of this pasture-borne disease can be difficult to predict (Eysker et al., 1994; Ploeger and Holzhauer, 2012; Wapenaar et al., 2007), resulting in the need for epidemiological surveillance. Clinical cases predominantly occur in youngstock, whereas adult dairy cows have traditionally played a role as silent carriers of the parasite,
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Corresponding author. E-mail address: [email protected] (C. Strube).
http://dx.doi.org/10.1016/j.vetpar.2017.05.023 Received 30 March 2017; Received in revised form 19 May 2017; Accepted 21 May 2017 0304-4017/ © 2017 Elsevier B.V. All rights reserved.
constituting the main source of pasture contamination (Saatkamp et al., 1994). However, during the last decades, disease outbreaks in adult cattle have become more frequent (David, 1997; van Dijk, 2004; Wapenaar et al., 2007). Dictyocaulus viviparus induces strong immune responses, resulting in protection against invading larvae as well as in reduced development and fecundity of pulmonary parasite stages during reinfections (Eysker et al., 2001; Jarrett et al., 1959; Michel, 1962), thus protecting most cattle from clinical disease. Protection is predominantly mediated by local immune responses, e.g. IgE directed against larval parasite stages (Kooyman et al., 2002) and local antibody production in the lungs (Scott et al., 1996). The degree of protection against reinfections is dependent on the initial level of infection (Eysker et al., 1993), and usually lasts only for a period of six to twelve months (Michel, 1962; Michel and Mackenzie, 1965). However, not all of the different manifestations of protection decline equally over time. Michel and Mackenzie (1965) found that protection against the establishment
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2008), serum samples were collected every two days p.i. until onset of seropositivity, and then approximately at weekly intervals. Animal experiments were approved by the ethics commission of the Institutional Animal Care and Use Committee (IACUC) of the Lower Saxony State Office for Consumer Protection and Food Safety (Niedersaechsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit) under reference number AZ 33.9-42502-0411/0371.
of challenge infection as well as the inhibition of early worm growth greatly declined during the first six months after first infection, whereas other phenomena like inhibition of development beyond the preadult stage persisted for more than a year. In the absence of regular boostering of immunity, outbreaks of parasitic bronchitis can occur. In addition, the so-called reinfection syndrome, a severe immunemediated inflammatory response, may cause disease in immune cattle upon exposure to large numbers of invading larvae, although these reinfections usually do not become patent (Breeze, 1985; Michel, 1957). To perform epidemiological studies and to assess herd infection status, for example at restocking of parasite-naïve animals, reliable and cost-efficient tools are needed. Routine examination of bulk-tank milk (BTM) may provide an inexpensive method to monitor herd health and thus potentially prevent disease and production losses (Charlier et al., 2016; Fiedor et al., 2009). An enzyme-linked immunosorbent assay (ELISA) based on recombinant major sperm protein (MSP), which is expressed in spermatocytes of adult male lungworms, has been developed for detection of lungworm-specific antibodies in serum and milk (Fiedor et al., 2009; Schunn et al., 2012; von Holtum et al., 2008), and has been used in several epidemiological studies (e.g. Bloemhoff et al., 2015; Charlier et al., 2016; Höglund et al., 2010; Schunn et al., 2013). However, accurately determining lungworm infections within a herd using BTM can be challenging, as within-herd seroprevalence may frequently be lower than the value of 20% which is required for the BTM ELISA result to reliably exceed the evaluated positivity threshold of an optical density ratio (ODR) of 0.410 (Schunn et al., 2012). In a recent field study including 15 dairy herds, the monthly within-herd seroprevalence of lungworm-positive herds − based on testing of approximately one third of cows in each herd − ranged between 0 and 29% of animals (Schunn et al., 2012). Several factors might be responsible for low within-herd seroprevalence despite potential occurrence of D. viviparus in many dairy herds. Because the level of protection against clinical disease is dependent on the initial infection dose, it is conceivable that low infection doses, as they may occur in the field, result in below-threshold levels of antibody production. Furthermore, as host immune responses affect invasion, development and fecundity of D. viviparus (Eysker et al., 2001; Jarrett et al., 1959; Michel, 1962), the antigenic stimulus during reinfections of partially protected animals might be insufficient to cause reliable antibody production against adult parasite stages, hampering serological diagnosis of reinfections. To test these hypotheses, a study was conducted to a) compare MSP antibody levels after initial infection with 12 different infection doses ranging from 10 to 3000 D. viviparus larvae, b) to evaluate the antibody response during reinfections at different time intervals after initial infection, and c) to assess patterns of MSP antibodies in adult dairy cows during naturally occurring reinfections.
2.2. D. viviparus reinfection experiments In a first reinfection experiment, two animals (animal A and B), which had been initially infected with 3000 L3, were re-infected with a dose of 3000 L3 eight months after initial infection (day 245 p. i.), and again about 3 months later (day 343 p. i.). The time span of eight months until reinfection was chosen to mimic either reinfection at turnout after the housing period or re-activation of hypobiotic larvae in spring, respectively. Reinfection another three months later was chosen to represent reinfection with the third generation of lungworms on the pasture, when herd prevalence as well as the level of pasture contamination usually shows a peak (Jørgensen, 1980; Pott et al., 1978). In a second reinfection experiment, two individuals (animal C and D) were initially infected with 2000 and 3000 L3, respectively, and then re-infected at day 90, day 150, and day 272 post initial infection with the same infection dose. During the study period, serum samples were collected every 2 days after infections until onset of seropositivity, followed by every 2–7 days. Faecal samples (2 × 10 g per animal) were analysed every 1–2 days as of day 20 after infections with the Baermann technique as described above to monitor larvae shedding. Animal experiments were approved by the ethics commission of the Institutional Animal Care and Use Committee (IACUC) of the Lower Saxony State Office for Consumer Protection and Food Safety under reference number AZ 33.9-42502-04-10/0078. 2.3. Field study on D. viviparus reinfections To assess patterns of MSP antibody levels during natural reinfections in dairy cows, individual milk samples were taken monthly from March 2009 to February 2010 at seven different dairy farms. Inclusion criteria for participating farms were as follows: dairy cows had to be kept on pasture during the grazing season and herd size had to be ≥20 dairy cows. Herd sizes of participating farms ranged from 20 to 86 cows. Lungworm-status of all cows included in the study was assessed in August the year before, when a minimum of 20 individual milk samples and one BTM sample per herd were tested for lungworm antibodies with the MSP-ELISA (Fiedor et al., 2009; Schunn et al., 2012). Additionally 2–5 cows per herd were randomly selected and tested for larvae shedding at this time. Based on these results, all seven farms were assigned a lungworm-positive status, whereas clinical signs of coughing were present on three farms only. Most of the participating dairy farms practised anthelmintic treatment of calves or heifers, whereas cows were not treated. For milk sample collection, tubes with lyophilised boric acid as preservative (Exactobac-L, nerbe plus GmbH, 21423 Winsen/Luhe, Germany) were used. Samples were stored at −20 °C until analysis by ELISA. Additionally, individual faecal samples were collected from each cow (n = 81) between June and September (and examined (2 × 40 g per cow)) for lungworm larvae via the Baermann technique.
2. Materials and methods 2.1. D. viviparus infections at different dose levels Twenty-four parasite-naïve, male 3 month old Holstein Friesian calves were used for infection experiments. Calves were reared at the Ruthe Research Farm of the University of Veterinary Medicine Hannover and were weaned before the start of the study. Two calves each were infected orally with the field isolates HannoverDv2000 or HannoverDv2010 (depending on the date of infection) at a dose of 10, 25, 50, 100, 250, 500, 1000, 1250, 1500, 2000, 2500 and 3000 thirdstage larvae (L3). The success of experimental infections was assessed from day 20 post infection (p.i.) by faecal examinations (2 × 2 g faeces per animal) using the Baermann technique. Specifically, faeces were placed on a sieve (150 μm mesh size) and larvae were allowed to migrate for approximately 18 h at room temperature. To monitor serum antibody development by use of the MSP-ELISA (von Holtum et al.,
2.4. ELISA Antibody response was evaluated using serum samples during dose and reinfection experiments, and using individual milk samples during the dairy cattle field study. MSP-ELISAs were carried out as previously described (Fiedor et al., 2009; Schunn et al., 2012; von Holtum et al., 48
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Fig. 1. Serum antibody levels against Dictyocaulus viviparus major sperm protein (MSP) of two calves each infected with 12 different infection doses from day 0 to day 66 post infection: (A) low, (B) medium and (C) high infection doses. Antibody levels are expressed as optical density ratios (ODR). The dashed line indicates the cut-off value of 0.500 ODR.
2008). All reactions were set up as duplicates. The arithmetic means of the ODs of the duplicates were converted into optical density ratios (ODR) using the following formula: ODR = (ODtest sample − Dblank)/ (ODpositive control − ODblank). To determine seropositivity, a cut-off value of 0.500 ODR was used for serum samples and a cut-off value of 0.573 ODR for milk samples as previously validated (Schunn et al., 2012; von Holtum et al., 2008). 3. Results 3.1. D. viviparus infections at different dose levels Infection of two calves with a single dose of 10 larvae resulted in one animal shedding 1–10 larvae/10 g faeces on three days during the examination period of 31 days, whereas no larval shedding was observed in the second animal. Neither of these two individuals became seropositive (Fig. 1A). All other infection doses resulted in patency as well as antibody production in both infected cattle (Fig. 1). Animals began shedding larvae between day 23 and day 29 p. i. (mean: 25.8, SD: 2.1) and became seropositive between day 28 and day 50 p. i. (mean: 34.5, SD: 5.7; Fig. 1). For those 22 animals which became seropositive, Spearman rank correlations were used to assess whether there was a relationship between infection dose and the onset of seropositivity or the maximum antibody level reached until day 66 p. i., respectively. As shown in Fig. 2, no significant correlation was found (N = 22, Spearman ρ = 0.11, P = 0.62, and Spearman ρ = −0.27, P = 0.233, respectively). Fig. 2. Scatterplots depicting (A) onset of seropositivity (day p.i.) and (B) maximum antibody level (expressed as optical density ratio [ODR]) against infection dose of 24 calves infected with 12 different doses of D. viviparus larvae.
3.2. Reinfection experiments Results of reinfection experiments are shown in Fig. 3. Reinfection of animals A and B eight months (245 days) after initial infection resulted in marked, but short periods of seropositivity relative to the period of seropositivity following primary infection (78 and 58 days versus 189 and 112 days, respectively, Fig. 3A). Reinfection of the same animals another 98 days later (day 343 post initial infection) resulted in another peak of seropositivity of even shorter duration (25 days) in animal A, whereas animal B remained seronegative. ODR values during both reinfections did not reach the same level as during initial infections. Furthermore, neither the first nor the second reinfections became patent. In the second experiment, reinfection 90 days after initial infection caused a rise in antibody levels, which started 46 days after reinfection (day 137 post initial infection) and lasted for approximately 10 days only until returning to previous levels. In contrast, seropositivity following initial infection had lasted for 50 days in animal D, whereas
animal C was still seropositive when reinfection occurred. However, via extrapolation the duration of seropositivity upon primary infection can be estimated at approximately 150 days without this second antigenic stimulus (Fig. 3B). Again, maximum ODR values caused by reinfection were lower compared to initial infection, and in animal D not sufficient to raise the ODR above the cut-off value of 0.500 (Fig. 3B), even though these reinfections became patent with low levels of larvae shedding in both individuals (between 1 and 7 larvae/2 × 10 g faeces for the duration of one week). The second reinfection of these animals another 60 days later (day 150 post initial infection) resulted in even lower patency (1 larva/2 × 10 g faeces at just one day) and caused only a barely noticeable increase of antibody levels 35 days after reinfection (day 185 post initial infection) in animal C, but no serological reaction or larvae shedding in animal D. However, when the animals were re49
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Fig. 3. Serum antibody levels directed against Dictyocaulus viviparus major sperm protein (MSP) over the course of two different reinfection experiments. In the first experiment (A) calves were reinfected on day 245 and day 343 post initial infection, in the second experiment (B) on day 90, day 150 and day 272 post initial infection. Antibody levels are expressed as optical density ratios (ODR). The dashed line indicates the cut-off value of 0.5.
infected a third time after MSP antibody levels had dropped to baseline levels in both individuals (day 272 post initial infection), animal C showed a strong seropositive antibody response 36 days after reinfection (day 308 post initial infection), which lasted – similar to initial infections – for 107 days. Furthermore, larval shedding was observed. By contrast, animal D remained seronegative and did not develop a patent infection.
Table 1 Number of animals included in the field study per dairy herd and percentage of animals showing a positive milk MSP antibody titre again during the course of the sampling period.
3.3. Field study on D. viviparus reinfections Overall, 52 dairy cows were included in the field reinfection study, corresponding to 3–11 cows per herd (mean 7.4), all of which displayed positive MSP titres at initial assessment. Over the course of the following year, a positive antibody level was detected in 11 of these cows (21.1%, 0–44.4% of sampled individuals per herd, Table 1). MSP50
Herd
Clinical signs at initial assessment
No. of animals sampled
Lungworm-positive (no./%)
1 2 3 4 5 6 7
No Yes Yes No Yes No No
3 11 8 9 5 7 9
0/0 3/27.3 1/12.5 1/11.1 1/20 1/14.3 4/44.4
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0.256 – 0.313 0.160 0.217 – – 0.380 0.145 – 0.364 0.262 0.286 – 0.329 0.250 0.195 – 0.618 0.338 0.384 – 0.426 0.353
0.218 – 0.293 0.174 0.252 – 0.937 0.315 0.248 – 0.366 0.350
Jan Dec
Feb
ELISA ODR results from monthly milk samples of those 11 cows are presented in Table 2. Short periods of seropositivity lasting only one to two months were observed in 10 of these individuals, whereas for one individual duration of seropositivity could not be conclusively determined because it became seropositive at the end of the study period. All faecal samples were negative for lungworm larvae, despite the fact that three individuals showed a positive antibody level at the time of faecal sampling.
0.246 0.417 0.223 0.312 0.151 – 0.503 0.273 0.376 – 0.451 0.328 0.407 (neg.) 0.230 (neg.) 0.455 (neg.) 0.290 0.919 – 0.349 0.404 – 0.390 0.530 0.442 0.465 0.173 0.408 0.509 – 0.457 0.264 – 0.416 0.350 0.513 0.395
– – – 0.712 (neg.) 0.322 0.350 (neg.) 0.332 (neg.) – (neg.) 0.704 (neg.) 0.489 (neg.) – 0.485
0.279 0.230 0.342 0.217 (neg.) 0.547 (neg.) – 0.327 0.584 0.402 0.339 0.709 (neg.) 0.398
0.245 0.386 0.242 0.342 0.121 – 0.348 0.325 0.360 – 0.473 0.316
Although dictyocaulosis is considered to be a herd problem, diagnosing subclinical lungworm infections can be challenging, particularly using bulk tank milk from dairy herds with low within-herd seroprevalence. In this study, potential causes of such low within-herd seroprevalences were investigated. The presented data demonstrate that the initial rise in antibody levels directed against the major sperm protein (MSP) of Dictyocaulus viviparus is independent of a primary infection at doses of ≥25 larvae, but that the magnitude and the duration of antibody response are reduced following reinfections. These results from experimental infections were confirmed by a field study, which revealed short periods of seropositivity as a major pattern in individual dairy cows exposed to natural D. viviparus reinfections. It was hypothesized that low infection doses in the field might lead to an attenuated production of antibodies against adult lungworms. However, when examining MSP antibody levels following infection with different doses ranging from 25 to 3000 larvae, no dose-dependency could be observed in this study on the onset of seropositivity and the magnitude of the response. Serum antibodies exceeded the cut-off value of 0.5 ODR on average 34.5 days after infection (range: 28–50), which supports the findings by Fiedor et al. (2009) (30-31 days, range: 26–41). Furthermore, the antibody curves showed a steep rise in the majority of animals, making it easy to distinguish seronegative from seropositive titres using the validated cut-off value of 0.500 (von Holtum et al., 2008). The absence of any dose-dependency was particularly noteworthy, as infection dose is positively associated with the level of protection against reinfections, with higher initial infection doses resulting in a higher level of protection (Ploeger and Eysker, 2002). Furthermore, it has been demonstrated that, independent of the infection dose, about 20–30% of larvae establish in the host (Ploeger and Eysker, 2002); a higher infection dose thus results in a higher parasite burden. Based on these data, it therefore has to be concluded that MSP antibody response during initial infection is independent of the number of adult parasites present in the host. Only the lowest infection dose of 10 larvae failed to result in a positive ELISA ODR, although a low level of patency was observed in one of the two individuals infected with this dose. It may be hypothesized that not enough or even no male lungworms reached adulthood in this animal and thus not enough or no sperm was produced to stimulate MSP antibody production. In conclusion, only very low levels of initial infection failed to evoke an antibody response, and low infection doses under natural conditions are unlikely to constitute a problem for serodiagnosis of primary infections. However, an effect of reinfection on MSP antibody levels was found, namely a reduction in magnitude and duration of the antibody response as compared to the initial response after primary infection. Adult lungworms are usually eliminated 60–90 days post infection and protection against challenge infection may then last for six to twelve months (Michel, 1962; Michel and Mackenzie, 1965). MSP antibodies have been shown to persist on average 95–112 days after initial infection (Fiedor et al., 2009). In our reinfection experiments, duration of seropositivity upon initial infections ranged on average 125 days (50–189 days). In contrast, the elevation in antibody levels observed after reinfections at eight months post initial infection lasted only 58 and 71 days (cf. Fig. 3A). As the MSP-ELISA only detects antibodies directed against proteins of adult male parasites, the short duration of
No milk sample available (dry period/animal sold or died). Information not available. b
a
2 2 2 3 4 5 6 7 7 7 7 35606 84462 86253 02381 78604 69227 1564 12140 43688 80565 97076 Mean ODR
2 2 3 4 4 2 n.a.b 6 5 4 3
0.723 0.882 0.872 0.896 0.850 0.646 0.993 0.737 0.989 0.743 0.673 0.819
0.303 0.146 0.555 0.149 0.307 0.286 0.26 – 0.272 0.235 0.305 0.282
0.663 0.186 0.667 0.210 – 0.724 0.309 – 0.343 0.308 0.349 0.418
–a 0.644 – 0.142 – 0.464 0.277 – 0.447 0.579 0.535 0.441
Aug Jun Initial assessment (Aug)
Herd
Lactation number
Individual milk ELISA ODR
Mar
Apr
May
Jul
Sep
Oct
Nov
4. Discussion
Animal ID
Table 2 ODR values of individual milk samples collected from adult dairy cows during the field study. Only animals are shown which became seropositive again during the year following initial assessment. Values exceeding the individual milk cut-off of 0.573 ODR are marked in grey. Results of faecal examinations using the Baermann technique are indicated in brackets.
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(cf. Fig. 3B, animal C). Similar patterns have already been observed by Höglund (2006). Despite serological evidence of reinfections, no evidence of larval shedding was found during the field study, even though three individuals displayed positive MSP antibody levels at the time of faecal sampling. This may indicate that reinfections did not lead to patency, as observed also in some of the reinfection experiments. However, animals were only sampled once or twice during the field study, not daily as during the infection experiments. Thus, it is possible that larvae shedding was missed due to limited sensitivity of the Baermann technique (Rode and Jørgensen, 1989), or because infections had already reached the postpatent period, whereas antibodies still persisted. Overall, the data collected from the infection experiments as well as the field study indicate that the attenuated antibody response and shortened seropositivity following reinfections may lead to low withinherd seroprevalence. In consequence, BTM antibody levels of immune herds may be too low to turn the BTM-ELISA, which shows 100% sensitivity and 97.32% specificity only as of an within-herd prevalence of ≥20% (Schunn et al., 2012), positive. However, it remains debatable whether this actually represents a disadvantage of the BTM-ELISA, as reinfections often do not become patent and seldom cause clinical symptoms, and are thus less important from a clinical perspective. In contrast, the BTM-ELISA is an excellent tool to investigate the incidence of lungworm infections in dairy herds, i.e. the number of newly infected herds in a certain time frame, as initial infections produce long periods of seropositivity. However, if a major aim of using the BTM-ELISA is to detect subclinical infections or reinfections in immune herds, the test should be carried out in August/September, when herd seroprevalence as well as antibody levels in BTM show a peak (Schunn et al., 2012).
seropositivity upon reinfections may reflect limited parasite development as a consequence of immune responses expressed in the lungs (Michel and MacKenzie, 1965), resulting in low adult worm burdens and reduced worm fecundity, leading to abbreviated seropositivity. In this context, immunization experiments with the commercially available attenuated live vaccine (Bovilis® Dictol) have shown that protected animals show 93–97% reduction in worm burden and 94% reduction in larvae shedding upon challenge infections as compared to control animals (Bain and Urquhart, 1988; Strube et al., 2015). When animals were reinfected at intervals of 90 and 150 days after initial infection, a low level of patency was observed. However, no pronounced secondary antibody response could be noted as the rise in antibody levels was less distinct and of shorter duration, or entirely absent. This may be due to protection against the establishment of challenge infection as well as the inhibition of early worm growth during the first months of acquired immunity (Michel and Mackenzie, 1965). A similar course of serum antibody levels despite lungworm reinfections during this time frame has already been reported from an early study using adult worm homogenate as antigen (Cornwell et al., 1960; Cornwell and Michel, 1960 Cornwell and Michel, 1960). By constrast, Scott et al. (1996), who also used adult worm homogenate as antigen, noted a further antibody increase following reinfection at day 65 post initial infection, whereas a second reinfection at day 112 post initial infection did not provoke any apparent antibody response. Two animals of the present study were subjected to a third reinfection 272 days after initial and 120 days after the preceding infection, when antibody levels in both animals had returned to baseline. Interestingly, this tertiary reinfection led to patency and a strong, rather long-lasting (107 days) antibody response in one of the two individuals (animal C, cf. Fig. 3B), whereas the other one remained seronegative and did not excrete larvae, indicating that it was still protected against challenge infection. Theoretically, differences in the viability of larvae between the two infection doses might have also been responsible for this discrepancy. However, during a total of 17 yearś experience by maintaining the lungworm field isolates used in the present study at the Institute for Parasitology, University of Veterinary Medicine Hannover, Germany, experimental infections of parasitenaïve cattle (doses of 1500–3000 L3) for parasite maintenance always resulted in patent lungworm infections (unpublished results). Furthermore, animals of each reinfection experiment were infected at the same time and from the same batch of larvae. Thus, we regard this possibility as extremely unlikely. A large variability between hosts in their response to infection is commonly known, possibly with a genetic basis (Ploeger, 2002). In summary, the reinfection experiments indicate that the antibody response is probably both influenced by the interval to the last infection and by individual host factors. The insights gained from these reinfection experiments have implications for the diagnosis of dictyocaulosis in the field. In the field study, only 21.1% of dairy cows which displayed a positive MSP milk antibody level upon initial assessment in August were detected seropositive again over the course of the following year. As the infection experiments have indicated, immune animals may not show a rise in antibody levels upon reinfection sufficient to result in a positive ELISA ODR, even though the reinfection may cause low levels of larval shedding. All – but one – dairy cows which turned seropositive again during the study period, displayed only short periods of positive MSP-ELISA results, lasting merely one or two months. This confirms the pattern of shortened seropositivity observed during the reinfection experiments. For one individual, the duration of seropositivity could not be conclusively determined; however, it appears that this individual displayed a rather long period of seropositivity lasting at least three months. This individual was seronegative for nine consecutive months before turning seropositive again. Thus, it is likely that a sufficient amount of time had elapsed for the animal to become susceptible again, resulting in a similiar strong antibody response upon reinfection as observed in one of the individuals in the second reinfection experiment
5. Conclusions During first infections with Dictyocaulus viviparus, MSP antibody levels are dose-independent as of an initial infection dose of 25 larvae. However, after reinfections, duration of seropositivity may be reduced or animals may not become seropositive at all, depending on the time interval until reinfection. Further experimental reinfection studies are needed to support the presented reinfection experiments and to elucidate the attenuated antibody response in more detail and regarding its underlying mechanisms. Short periods of seropositivity were detected as a major pattern in dairy cows subjected to natural D. viviparus reinfections, potentially resulting in low within-herd seroprevalence and low BTM antibody levels. The BTM MSP-ELISA may nevertheless serve as a useful tool in epidemiological studies as well as from a clinical perspective, as a negative result indicates absence of infection or protection of the herd due to immunity. Thus, a negative ELISA result in coughing herds indicates that dictyocaulosis is an unlikely cause for these symptoms. In such herds with acquired immunity, BTM testing in August/September can facilitate detection of antibodies. However, to confidently exclude lungworm infections it may ultimately be necessary to test individual milk (or serum) samples of a sufficient proportion of a herd, for example before restocking parasite-naïve individuals. Such efforts may focus on those animals which are likely to suffer from a primary infection and can thus be expected to have a high and long-lasting antibody level, such as recently introduced first-year grazing heifers.
Acknowledgements The authors wish to thank Sandra Buschbaum for excellent technical assistance. Parts of the study were funded by Merial. Study data interpretation is completely independent from the company’s opinion and there is no conflict with commercial interests.
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