# Unexpected decrease in milk production after fenbendazole treatment of dairy Cows during early grazing season

Nadine Ravinet, Christophe Chartier, Nathalie Bareille, Anne Lehébel, Adeline Ponnau, Nadine Brisseau, Alain Chauvin

► To cite this version:

Nadine Ravinet, Christophe Chartier, Nathalie Bareille, Anne Lehébel, Adeline Ponnau, et al.. Unexpected decrease in milk production after fenbendazole treatment of dairy Cows during early grazing season. PLoS ONE, 2016, 11 (1), pp.e0147835. 10.1371/journal.pone.0147835. hal-02637379

HAL Id: hal-02637379

https://hal.inrae.fr/hal-02637379

Submitted on 27 May 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

RESEARCH ARTICLE

# Unexpected Decrease in Milk Production after Fenbendazole Treatment of Dairy Cows during Early Grazing Season

Nadine Ravinet¹,²,³*, Christophe Chartier²,³, Nathalie Bareille²,³, Anne Lehebel³,², Adeline Ponnau², Nadine Brisseau²,³, Alain Chauvin²,³

1 IDELE, French Livestock Institute, UMT Cattle Herd Health Management, Nantes, France, 2 LUNAM Université, Oniris, Nantes-Atlantic College of Veterinary Medicine and Food sciences and Engineering, UMR BioEpAR, Nantes, France, 3 INRA, UMR1300 Biology, Epidemiology and Risk Analysis in animal health, Nantes, France

* ravinet.nadine@wanadoo.fr

## Abstract

Gastrointestinal nematodes (GIN) infection can impair milk production (MP) in dairy cows. To investigate whether MP would be optimized by spring targeted-selective anthelmintic treatment in grazing cows, we assessed (1) the effect on MP of an anthelmintic treatment applied 1.5 to 2 months after turn-out, and (2) herd and individual indicators associated with the post-treatment MP response. A randomized controlled clinical trial was conducted in 13 dairy farms (578 cows) in western France in spring 2012. In each herd, lactating cows of the treatment group received fenbendazole orally, control cows remained untreated. Daily cow MP was recorded from 2 weeks before until 15 weeks after treatment. Individual serum pep-sinogen and anti-Ostertagia antibody levels (expressed as ODR), faecal egg count and bulk tank milk (BTM) Ostertagia ODR were measured at treatment time. Anthelmintic treatment applied during the previous housing period was recorded for each cow. In each herd, information regarding heifers' grazing and anthelmintic treatment history was collected to assess the Time of Effective Contact (TEC, in months) with GIN infective larvae before the first calving. The effect of treatment on weekly MP averages and its relationships with herd and individual indicators were studied using linear mixed models with two nested random effects (cow within herd). Unexpectedly, spring treatment had a significant detrimental effect on MP (-0.92 kg/cow/day on average). This negative MP response was particularly marked in high producing cows, in cows not treated during the previous housing period or with high pepsinogen levels, and in cows from herds with a high TEC or a high BTM ODR. This post-treatment decrease in MP may be associated with immuno-inflammatory mechanisms. Until further studies can assess whether this unexpected result can be generalized, non-persistent treatment of immunized adult dairy cows against GIN should not be recom-mended in early grazing season.

<figure><img src="image_3.png" /></figure>

OPEN ACCESS

**Citation:** Ravinet N, Chartier C, Bareille N, Lehebel A, Ponnau A, Brisseau N, et al. (2016) Unexpected Decrease in Milk Production after Fenbendazole Treatment of Dairy Cows during Early Grazing Season. PLoS ONE 11(1): e0147835. doi:10.1371/journal.pone.0147835

**Editor:** Graciela Andrei, Rega Institute for Medical Research, BELGIUM

**Received:** July 2, 2015

**Accepted:** November 27, 2015

**Published:** January 25, 2016

**Copyright:** © 2016 Ravinet et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability Statement:** Data are only available upon request from the authors. Indeed, the milk production data used and analysed in this study belong to farmers who accepted to work with us (milk production data were directly recovered and extracted from the farm computer). Farmers agreed to participate in this study on the condition that their data would only be used for this study. This is the reason why data will only be available upon request, and only after obtaining the agreement of farmers. Moreover, a description of the data is provided within the paper.
**Funding:** The study was funded by the CASDAR project N°1127 (Ministère de l'Agriculture, de l'Agroalimentaire et de la Forêt). CASDAR: Compte d'Affectation Spécial Développement Agricole et Rural. Authors who received the funding: NR, AC, CC, N. Bareille. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Competing Interests:** The authors have declared that no competing interests exist.

Introduction

Gastrointestinal nematode (GIN) infections are highly prevalent in young and adult grazing cattle [1-4]. The most frequent and pathogenic GIN in cattle is *Ostertagia ostertagi*, an abomasal worm responsible for clinical disease and growth retardation in young stock [5-9], and subclinical decrease in milk production (MP) in adult dairy cows [10-12].

Control measures are essential and depend heavily on blanket anthelmintic treatments, applied to whole groups or herds, to remove or prevent infection with GIN. Nevertheless, the regular use of anthelmintics exerts a heavy selection pressure on parasite populations leading to possible emergence of anthelmintic resistance. This risk is no longer negligible in cattle [13-19], threatening the long term efficacy of usual control programs aiming at securing growth of young stocks and MP in adult dairy cows. Changes in treatment practices are therefore needed, towards a more rational and sustainable use of anthelmintics.

In adult grazing dairy cows, looking at the large variability of the effect of anthelmintic treatment on MP among studies, between herds and between cows [10,11,20-26], there appears both a need and an opportunity to use anthelmintics in a more targeted and selective way. In targeted treatment (TT) and targeted-selective treatment (TST) strategies, treatments are restricted to herds, and cows within herds, that will most benefit from treatment (TST), drug being administered at the most appropriate time(s) (TT), bearing in mind the need to maintain susceptible parasites in *refugia* (parasites unexposed to drug, i.e. free-living stages on pasture, and parasitic stages within untreated hosts) [27-30]. The implementation of such treatment strategies against GIN should enable: (i) MP optimization, while (ii) limiting the use of anthelmintics, and (iii) lowering the risk of development of anthelmintic resistance thanks to the preservation of a susceptible parasite population in *refugia*.

TST administered during the winter housing period allows the preservation of a *refugia* population made up mainly of parasites within untreated hosts. Indeed, housed cows are no longer in contact with infective stages at that time, and the population of free-living stages on pasture decline in a substantial level during winter in cold temperate climate areas [5, 31]. Conversely, with TST during grazing season, the size of the *refugia* population could be theoretically greater, because it could be increased by the free-living stages on pasture resulting from spring parasitic cycles. To optimize MP in adult dairy cows while maintaining a large reservoir of susceptibility to anthelmintics in the GIN population, TST during grazing season, if it is associated with a significant increase in MP, could thus be optimal.

The effect of an anthelmintic treatment applied in autumn-winter (housing period) on MP has been broadly studied: it differs from one study to another, but even if it is sometimes non-significant or slight, a post-treatment increase in MP has often been observed [20-22, 25, 32-37]. In contrast, the MP response of anthelmintic treatment applied during the grazing season is less documented. Only one study reported a positive effect of a single treatment applied 1.5 months after turn out [38], but this study was conducted with a small sample size (40 cows in one herd). In a few other studies, several treatments were applied repeatedly on lactating cows during the grazing season, with a positive or a non-significant effect on MP [23, 39, 40]. But this whole herd repeated-treatment strategy is of course questionable if we keep in mind the need to preserve a large population of parasites in *refugia*. In other studies, a single treatment was applied, not in a given season and to the entire herd at the same time, but all over the year, at a given physiological stage, namely at calving or drying off [24, 26, 41-47]. Unfortunately, the majority of these studies did not provide a comparison of the effects on MP of treatments applied during housing period (autumn-winter) versus grazing season (spring, summer, and early autumn). Indeed, either the effect of season on the MP response after anthelmintic treatment was not investigated [24, 43, 45-47], or failed to be detected because of a possible lack of
statistical power, as pointed out by the authors [44], or because seasons were not sufficiently distinguishable to be compared [41, 42]. Only one recent study reported that the treatment effects were similar during housing *versus* pasture period [26].

Consequently, the MP response after a single anthelmintic treatment during grazing season would deserve more in-depth examination, to determine whether such treatment strategies could offer a good compromise between the optimization of MP and the preservation of a large population of parasites in *refugia*.

The objectives of this study were (i) to assess the effect on MP of a single anthelmintic treatment applied at early grazing season, 1.5 to 2 months after turn out, (ii) to evaluate whether TST treatment could be possible at that time by analyzing the relationships between treatment response and several parasitological or production-based indicators, at both herd- and individual-levels.

# Materials and Methods

## Farms and animals

Thirteen dairy herds in the North-West of France were visited twice during spring 2012. The first visit took place around the date of turn out, and the second 1.5 to 2 months after turn out. Eleven of these herds were already included in a previous randomized controlled clinical field trial conducted in autumn 2011 [25]. The major herd recruitment criteria were the breed (Holstein), an access to pasture (with at least 1/3 of grazed grass in the cows' diet), no intention of anthelmintic treatment on adult dairy cows in the upcoming grazing season, and a daily recording of MP (automatic milking system, or milk parlor with milk meters). In each herd, the majority of lactating cows were included provided that they were planned to be milked for at least 5 weeks after the second visit and in apparent good health.

This study was carried out in commercial dairy farms, so on "private lands". On the basis of an accurate description of the objectives of the study and its design, the owners of the lands (the farmers) gave permission to conduct the study on their farm, accepted to follow the protocol and to participate in the handling of cows. Except this informed consent of each farmer and his permission to conduct the study on his farm and on his animals, no other specific permission was required.

This field study did not involve endangered or protected species. No cows were sacrificed for the purposes of the study.

## Anthelmintic treatment

On the first visit, the history of anthelmintic treatment applied during autumn-winter 2011–2012 was recorded for each cow. In fact, some cows had actually been treated during the previous housing period either by the farmer, or by researchers in the context of the autumn clinical trial [25]. On the second visit, in each herd, cows were stratified according to three criteria: treated during the previous housing period (yes *versus* no), parity (first, second, third and greater), and days in milk (DIM) classes (less than 35 DIM, 35 to 100 DIM, 100 to 200 DIM, and more than 200 DIM). Then, in each stratum, cows were ranked and paired by ascending expected production level (last test-day milk yield before the second visit). Finally, cows of each pair were randomly assigned to a treatment group or a control group using a random number table. Cows belonging to the treatment group received orally on the day of the second visit a single 60 mL dose of fenbendazole (Panacur® 10%), which is the dose for 800 Kg body weight (7.5 mg/Kg). Cows from the control group remained untreated.
## Samples and laboratory analysis

On the first visit, individual blood samples were taken from all cows (5mL blood sample at the coccygeal vein). On the day of treatment (second visit), individual blood and faecal samples (around 100g taken directly into the rectum, using gloves and lubricating gel) were taken from all treated and control cows, and a bulk tank milk (BTM) sample was collected in each herd.

Anti-*Ostertagia* antibody levels were determined with the ELISA SVANOVIR kit (Svanova Biotech, Uppsala, Sweden) on the individual blood samples and on the BTM samples. Results were expressed as an optical density ratio (ODR) [25]. Individual serum pepsinogen levels were also determined on blood samples, following the simplified method described by Ker-boeuf et al. (2002) [48]. Results were expressed in milli-Units of Tyrosine (mUTyr). Individual faecal egg counts (FEC) per 5g of feces were determined using the modified Wisconsin Sugar Centrifugal flotation method [49].

This study was conducted by a veterinarian researcher who holds a licence to experiment on live animals (diploma issued by the National Veterinary School of Nantes (Oniris) which relies on the Ministère de l'Agriculture, de l'Agroalimentaire et de la Forêt). The protocol was pain-less for cows, all efforts were made to minimize stress and holding time, and every samples and handling were performed by a veterinarian, helped by the farmer when needed.

## Daily milk production data

Daily individual cow MP data were recorded and extracted from the farm computer from 14 days before treatment until 105 days after treatment. The average daily MP over the period of 14 days before treatment was calculated (one reference point for the pre-treatment period). Then, for the post-treatment period (15 weeks), daily MPs were averaged by week.

## Indicators characterizing cows and herds

Each cow was characterized by its daily MP averaged by week, and by 3 production-based indi-cators: parity, days in milk at the time of treatment (DIMt) and pre-treatment production level. To estimate this latter indicator, additional daily MP data, up to 49 days before the day of treat-ment, were recovered. The pre-treatment production level was then evaluated for each cow cal-culating the average daily MP over this period (maximum 49 days, and minimum 3 days according to DIMt).

Each cow was also characterized by 6 individual parasitological indicators: history of treat-ment applied during the previous housing period, serum pepsinogen levels and individual serum anti-*Ostertagia* antibody levels at the time of the first visit and on the day of treatment (second visit), and FEC on the day of treatment.

Each herd was characterized by its BTM anti-*Ostertagia* antibody level measured on the day of treatment. Moreover, on the basis of information regarding heifers' grazing and anthelmin-tic treatment history, the Time of Effective Contact (TEC, in months) with GIN infective larvae before the first calving was calculated in each herd according to Ravinet et al. (2014) [25]: each herd was characterized by a minimal TEC (TECmin) and a maximal TEC (TECmax) according to its pattern of dates of birth and age at first calving.

## Statistical analysis

**Coding and classification of variables.** Raw data were entered into an Access database (Microsoft Corp., Redmond, WA). They were then transferred into SAS 9.2 (SAS Institute Inc., Cary, NC) to build the variables of interest and carry out statistical analyses.
The treatment was coded as described by Ravinet et al. (2014) [25]: a combined variable between treatment and time was created ('week-trt') and divided into 17 categories: for a treated cow, 'week-trt' took the values -1 (reference point before treatment), 0, 1, 2, 3... up to 14 (after treatment period, week 0 being the week of treatment); and for a control cow, 'week-trt' only took the value 99 whatever the week.

The 3 production-based indicators were categorized. Three classes were constructed for parity (1, 2, 3 and more), and days in milk at the time of treatment (DIMt≤100, 100<DIMt≤200, DIMt>200 days). The pre-treatment production level was corrected for parity and days in milk and categorized in 3 classes (low, moderate, high) according to terciles of the distribution of the average MP calculated on the 3 to 49 days period before treatment (see above).

The 6 individual parasitological indicators were also categorized. Two classes were defined for FEC at the time of treatment (positive versus negative) and for the history of treatment applied during the previous housing period (yes versus no, whatever the drug used). For individual serum pepsinogen and anti-Ostertagia antibody levels measured on the first and on the second visit, three classes were defined according the terciles of their respective distribution.

At the herd-level, the BTM anti-Ostertagia antibody level was categorized in two classes, according to the median of its distribution. Two classes were also defined for TEC: high-TEC when TECmin ≥ 8 months (every heifers in the herd experienced a long TEC with GIN infective larvae before the first calving), and low-TEC otherwise (TECmax < 8 months, or TECmax ≥ 8 months but TECmin < 8 months).

To ensure that the number of treated cows and control cows was evenly distributed among the different categories of each indicator, the proportion of treated cows and control cows was compared using Chi-square tests (level of significance set at $p<0.05$).

**Comparison of individual parasitological indicators between cows treated or not treated during the previous housing period.** Cows treated and not treated during the previous housing period were first compared on the basis of their individual serum pepsinogen levels and anti-Ostertagia antibody levels measured on the first visit around the date of turn out (parametric or non-parametric mean comparison tests according to the normality of the distribution of quantitative variables, level of significance set at $p<0.05$).

These comparisons were also performed for the 3 parasitological indicators measured on the second visit (1.5 to 2 months after turn out) (chi-square tests for categorized variables, and parametric or non-parametric mean comparison tests according to the normality of the distribution of quantitative variables, level of significance set at $p<0.05$).

**Assessment of the overall treatment effect over time on milk production.** The overall effect, over time, of spring treatment on daily MP averaged by week was studied using a first linear mixed model (model 0) with two nested random effects (cow within herd). The outcome variable was the daily MP averaged by week, expressed in Kg per day, and normally distributed (8393 observations, mean = 28.6 Kg/day, sd = 8.2 Kg/day). This model 0 included the following independent variables: week-trt (variable of interest, reference = 99), parity (1, 2, 3 or greater), days in milk (DIM) (expressed as week in milk: 1 to 52, and 52 or greater), pre-treatment production level corrected for parity and DIM (low, moderate, high), month of milk production (April to September), and a two way interaction between parity and DIM.

This model 0 was of the following form:

$$ (\text{Daily MP average by week})_{wij} = \mu + \sum \beta_{wij}X_{wij} + \sum \beta_{ij}X_{ij} + v_j + \omega_{ij} + \varepsilon_{wij} $$
with

$$ v_j \sim N(0, \sigma_v^2), \omega_{ij} \sim N(0, \sigma_{\omega}^2) \\ \varepsilon_{wij} = [\varepsilon_{1ij}, \varepsilon_{2ij}, \dots, \varepsilon_{14ij}] \sim N(0, \sigma_{\varepsilon}^2) $$
where (Daily MP average by week)_{wij} = MP for cow i in farm j, on week w, μ = average MP after adjusting for covariates, β_{wij} = coefficients for X_{wij}, X_{wij} = variables varying between daily MP averaged by week (DIM, week-trt, month of milk production, and DIM* parity), β_{ij} = coefficients for X_{ij}, X_{ij} = variables varying between cows (parity, pre-treatment production level), v_j = farm random effect, ω_{ij} = cow random effect nested into herd, ε_{wij} = residual at week w. The random effects v_j, ω_{ij} and the residual ε_{wij} were assumed to be normally distributed with mean 0 and variance σ_v², σ_ω² and σ_ε² respectively.

Residuals and predicted values were plotted to evaluate their heteroscedasticity and their normality.

**Assessment of the variations of the treatment effect according to indicators characterizing herds and cows on the second visit.** The relationships between the treatment response and (i) the 3 categorical individual production-based indicators, (ii) the 3 categorical individual parasitological indicators measured on the second visit, plus the history of treatment applied during the previous housing period, and (iii) the 2 categorical herd-level indicators were evaluated using one linear mixed model per indicator. Each of these models included the same independent variables as the model 0, plus the indicator of interest in interaction with week-trt. For each of these models, residuals and predicted values were plotted to evaluate their heteroscedasticity and their normality.

**Estimation method to assess the milk production gain week after week.** The statistical models estimated each week the average daily MP of treated cows versus control cows, for the whole population of our study sample (model 0), or within each category of the investigated indicators (models including the interaction terms between week-trt and indicators). On the basis of these estimations, the treated cows' MP gain was calculated each week as described in Ravinet et al. (2014) [25]. Finally, with Gᵢ being the estimated treated cows' MP gain in weekᵢ, the average treated cows' MP gain for the whole period of follow up after treatment (from week₀ to week₁₄) was the arithmetic mean of the Gᵢs.

To test each week if Gᵢs were significantly different from zero, we used student tests with adjusted p-values for multiple testing.

# Results

## Description of the study sample

625 cows in 13 herds were initially included in the study. 11 herds were equipped with an automatic milking system, and 2 with a milking parlor with milk meters (herd n°1 and 9). General information regarding spring grazing practices of dairy cows in our study sample is provided in Table 1. Cows were turned out between 2012-02-28 and 2012-03-28. The proportion of grazed grass in the cows' spring diet increased gradually from turn out onwards (from 20% to 75%) (Table 1), and was not lower in herds with automatic milking systems. The first visits took place between 2012-03-07 and 2012-03-28. In five herds it was on the day of turn out, or 2 to 13 days before, whereas in 7 herds it was 6 to 14 days after. The time lag between turn out and this first visit was longer (25 days) only for one herd (herd n°13) (Table 1). Anthelmintic treatment was administered between 2012-04-16 and 2012-05-29, that is to say 41 to 68 days after turn out. In two herds (n°4 and 10), the actual time spent on pasture between turn out and treatment was shorter (37 and 34 days, respectively) because cows returned to the barn for 4 and 1 weeks respectively, because of adverse weather conditions.

47 cows were excluded from the dataset for the following reasons: insufficient number of MP data recovered before treatment to calculate the pre-treatment production level (10 cows), days in milk at the time of treatment exceeding 516 days (11 cows), daily MP data not correctly
<table><caption>Table 1. Dates of visits and description of the spring grazing management practices of dairy cows in the 13 herds included in the study carried out on spring 2012.</caption><thead><tr><th>N° herd</th><th>Date of turn out</th><th>Date of first visit</th><th>Time lag between turn out and first visit (days)</th><th>Date of treatment (second visit)</th><th>Time lag between turn out and treatment (days)</th><th>% of grazed grass in the spring cows' diet</th><th>Grazing time per day (hours)*</th><th>Number of plots grazed by dairy cows in spring (size)</th><th>Time spent on each plot (days)</th><th>Average number of lactating cows</th></tr></thead><tbody><tr><td>1</td><td>03-28</td><td>03-28</td><td>0</td><td>05-16</td><td>49</td><td>33 to 66%</td><td>6</td><td>4 (1.5 to 4 ha)</td><td>8</td><td>65</td></tr><tr><td>2</td><td>03-23</td><td>03-19</td><td>-4</td><td>05-24</td><td>62</td><td>50 to 66%</td><td>19*</td><td>3 (4.5 ha)</td><td>5 to 6</td><td>60</td></tr><tr><td>3</td><td>03-02</td><td>03-16</td><td>14</td><td>05-03</td><td>62</td><td>50 to 70%</td><td>4 à 6</td><td>7 (3 to 4 ha)</td><td>5</td><td>70</td></tr><tr><td>4</td><td>03-26</td><td>03-13</td><td>-13</td><td>05-29</td><td>64<sup>1</sup></td><td>20 to 50%</td><td>13*</td><td>2 (4 ha)</td><td>2</td><td>40</td></tr><tr><td>5</td><td>03-15</td><td>03-13</td><td>-2</td><td>05-22</td><td>68</td><td>30 to 40%</td><td>24*</td><td>5 (1.2 ha)</td><td>5</td><td>50</td></tr><tr><td>6</td><td>03-01</td><td>03-07</td><td>6</td><td>04-24</td><td>54</td><td>30 to 60%</td><td>10*</td><td>3 (8 to 10 ha)</td><td>15</td><td>70</td></tr><tr><td>7</td><td>03-13</td><td>03-23</td><td>10</td><td>05-09</td><td>57</td><td>30 to 75%</td><td>24*</td><td>9 (0.5 to 2 ha)</td><td>2 to 7</td><td>47</td></tr><tr><td>8</td><td>03-02</td><td>03-08</td><td>6</td><td>04-26</td><td>55</td><td>30%</td><td>9*</td><td>5 (2 ha)</td><td>3</td><td>47</td></tr><tr><td>9</td><td>03-12</td><td>03-22</td><td>10</td><td>05-14</td><td>63</td><td>30%</td><td>4</td><td>3 (2.5 to 4 ha)</td><td>1</td><td>120</td></tr><tr><td>10</td><td>03-22</td><td>03-12</td><td>-10</td><td>05-02</td><td>41<sup>2</sup></td><td>30 to 50%</td><td>12*</td><td>5 (0.7 to 1.6 ha)</td><td>3 to 4</td><td>40</td></tr><tr><td>11</td><td>03-14</td><td>03-21</td><td>7</td><td>05-07</td><td>54</td><td>30 to 50%</td><td>17*</td><td>6 (1 to 3 ha)</td><td>6 to 10</td><td>50</td></tr><tr><td>12</td><td>03-01</td><td>03-14</td><td>13</td><td>04-18</td><td>48</td><td>15 to 65%</td><td>NA<sup>3</sup></td><td>14 (1 ha)</td><td>2 to 4</td><td>70</td></tr><tr><td>13</td><td>02-28</td><td>03-24</td><td>25</td><td>04-16</td><td>48</td><td>25 to 70%</td><td>9 à 24*</td><td>5 (2 to 3 ha)</td><td>7 to 15</td><td>50</td></tr></tbody></table>

*In herds where the barn is always open (access to the automatic milking system, or access to feed in the trough), cows freely move from the barn to the pasture. The information given here is thus the duration of free access to pasture per day

¹In this herd, cows returned to the barn between 04–18 and 05–15, the actual time spent on pasture between turn out and treatment was thus 37 days.

²In this herd, cows returned to the barn between 04–25 and 05–02, the actual time spent on pasture between turn out and treatment was thus 34 days.

³NA = Not available.

doi:10.1371/journal.pone.0147835.t001

recorded on farm (1 cow), duration of the post-treatment follow-up shorter than 4 weeks or health issue indicated by the farmer (25 cows).

Finally, 578 cows were included to carry out our statistical analysis: 295 treated cows and 283 control cows, 31 to 72 cows per herd. The final dataset is described in Tables 2–4. Among these 578 cows, 556 were present on the two visits, and 22 were sampled only on the second visit. Thus, serum pepsinogen levels and anti-Ostertagia antibody levels measured on the first visit were not available for these 22 cows.

Several cows were dried off or sold during the period of post-treatment follow-up. The number of cows therefore decreased gradually from one week to another, similarly in treated cows and control cows. On week₁₄, there were still 428 cows in the dataset (226 treated cows and 202 control cows).

For each indicator, there was no difference in the proportion of treated cows between categories (*p*>0.05) (Tables 2–4).

## Comparison of individual parasitological indicators between cows treated or not treated during the previous housing period

**At the time of the first visit.** On the first visit, on average, serum pepsinogen levels tended to be slightly higher in cows not treated during the previous housing period compared to cows treated during the previous housing period (1708 versus 1632 mUTyr, *p* = 0.07). In the five herds sampled before or on the day of turn-out, this difference was not significant (1575 versus 1600 mUtyr, *p* = 0.7). In contrast, in the 8 herds sampled 6 to 25 days after turn-out, this
<table><caption>Table 2. Description of the dataset (578 cows in 13 herds, 295 treated cows, 283 control cows): distribution and classification of individual production-based indicators characterizing cows, number of treated cows and control cows per classes for each categorized indicator, and distribution of the daily milk production average by week.</caption><thead><tr><th rowspan="2">Indicator</th><th rowspan="2">Minimum</th><th rowspan="2">Q1</th><th rowspan="2">Median</th><th rowspan="2">Q3</th><th rowspan="2">Maximum</th><th rowspan="2">Mean (std)</th><th colspan="4">Classes threshold and number of cows per classes</th><th rowspan="2">Total</th><th rowspan="2">Chi-square test</th></tr><tr><th>Control cows</th><th>Treated cows</th></tr></thead><tbody><tr><td rowspan="3">Parity</td><td rowspan="3">1</td><td rowspan="3">1</td><td rowspan="3">2</td><td rowspan="3">3</td><td rowspan="3">8</td><td rowspan="3">2.1 (1.2)</td><td>1</td><td>111</td><td>110</td><td>196</td><td rowspan="3">221</td><td rowspan="3">p = 0.89</td></tr><tr><td>2</td><td>94</td><td>102</td><td>161</td></tr><tr><td>3 and greater</td><td>78</td><td>83</td><td>161</td></tr><tr><td>Days in milk at the time</td><td>4</td><td>89</td><td>193</td><td>260</td><td>516</td><td>187 (114)</td><td>DIMt ≤ 100</td><td>79</td><td>83</td><td>162</td><td rowspan="3">p = 0.91</td></tr><tr><td>of treatment (days)</td><td></td><td></td><td></td><td></td><td></td><td></td><td>100 &lt; DIMt ≤ 200</td><td>68</td><td>75</td><td>143</td></tr><tr><td>(DIMt)</td><td></td><td></td><td></td><td></td><td></td><td></td><td>DIMt &gt; 200</td><td>136</td><td>137</td><td>273</td></tr><tr><td rowspan="3">Production level* (kg/day)</td><td rowspan="3">13.5</td><td rowspan="3">26.8</td><td rowspan="3">32.3</td><td rowspan="3">38.6</td><td rowspan="3">62.4</td><td rowspan="3">32.8 (8.1)</td><td>Low</td><td>87</td><td>92</td><td>179</td><td rowspan="3">p = 0.98</td></tr><tr><td>Moderate</td><td>94</td><td>96</td><td>190</td></tr><tr><td>High</td><td>102</td><td>107</td><td>209</td></tr><tr><td>Daily milk production average by week</td><td>5.2</td><td>22.8</td><td>27.9</td><td>33.9</td><td>61.6</td><td>28.6 (8.2)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

doi:10.1371/journal.pone.0147835.t002

difference was significant: cows not treated during the previous housing period had a higher serum pepsinogen level than cows treated during this autumn-winter period (1770 versus 1645 mUTyr, respectively, *p* = 0.02).

On average, anti-*Ostertagia* antibody levels were not different between cows treated or not during the previous housing period (average ODR = 0.53 and 0.54 respectively, *p* = 0.32), whatever the date of first visit compared to the date of turn-out.

**At the time of the second visit.** Table 5 describes the individual parasitological indicators measured 1.5 to 2 months after turn-out according to the history of anthelmintic treatment applied during the previous housing period. Anti-*Ostertagia* antibody levels and pepsinogen levels were on average significantly higher in cows not treated *versus* cows treated during the previous housing period (*p* < 0.0001). Moreover, those previously untreated cows had more often a positive FEC (39% *versus* 28%, *p* = 0.007).

## Overall milk production response after spring anthelmintic treatment over time

In model 0, all the usual factors associated with the variations of MP were significant (parity, DIM, interaction between parity and DIM, pre-treatment production level, and month of milk production) (*p*<0.0001). The combined variable of interest ‘week-trt’ was significant (*p* < 0.0001), and the effect of spring treatment on MP was negative: in comparison with the 283 control cows, the 295 treated cows’ daily MP averaged by week decreased significantly (Gis were negative). The evolution of the treated cows’ MP over time is displayed in Fig 1. The drop in MP after treatment was sharp: -0.92 Kg/cow/day on average for the whole period of follow up after treatment, with a maximum of -1.8 Kg/cow/day on week9 after treatment (Fig 1).

<footnote>*Average daily MP calculated on the 3 to 49 days period before treatment and corrected for parity and days in milk.</footnote>
<table><caption>Table 3. Description of the dataset (578 cows in 13 herds, 295 treated cows, 283 control cows): distribution and classification of individual parasitological indicators characterizing cows, number of treated cows and control cows per classes for each categorized indicator.</caption><thead><tr><th rowspan="2">Indicator</th><th rowspan="2">Minimum</th><th rowspan="2">Q1</th><th rowspan="2">Median</th><th rowspan="2">Q3</th><th rowspan="2">Maximum</th><th rowspan="2">Mean (std)</th><th colspan="3">Classes threshold and number of cows per classes</th><th rowspan="2">Total</th><th rowspan="2">Chi-square test</th></tr><tr><th>Control cows</th><th>Treated cows</th><th>Total</th></tr></thead><tbody><tr><td>Serum pepsinogen level on the first visit<sup>1</sup></td><td>243</td><td>1270</td><td>1622</td><td>2042</td><td>4391</td><td>1677 (592)</td><td>Pepsi<sub>1</sub> ≤ 1401<sup>2</sup></td><td>88</td><td>100</td><td>188</td><td>p = 0.31</td></tr><tr><td>(Pepsi<sub>1</sub>)(mUTyr)</td><td></td><td></td><td></td><td></td><td></td><td></td><td>1401&lt; Pepsi<sub>1</sub> ≤ 1815</td><td>95</td><td>82</td><td>177</td><td></td></tr><tr><td>Serum pepsinogen level on the second visit<sup>3</sup></td><td>350</td><td>1258</td><td>1762</td><td>2265</td><td>5509</td><td>1878 (847)</td><td>Pepsi<sub>1</sub> &gt; 1815<sup>2</sup></td><td>89</td><td>102</td><td>191</td><td>p = 0.61</td></tr><tr><td>(Pepsi<sub>2</sub>) (mUTyr)</td><td></td><td></td><td></td><td></td><td></td><td></td><td>1419 &lt; Pepsi<sub>2</sub> ≤ 2025</td><td>93</td><td>90</td><td>183</td><td>p = 0.61</td></tr><tr><td>Anti-Ostertagia antibody level on the first visit<sup>1</sup></td><td>-0.10</td><td>0.37</td><td>0.56</td><td>0.73</td><td>1.26</td><td>0.54 (0.27)</td><td>Pepsi<sub>2</sub> &gt; 2025<sup>2</sup></td><td>91</td><td>106</td><td>197</td><td>p = 0.32</td></tr><tr><td>(ODR<sub>1</sub>)</td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.452 &lt; ODR<sub>1</sub> ≤ 0.670</td><td>89</td><td>87</td><td>176</td><td></td></tr><tr><td>Anti-Ostertagia antibody level on the second visit<sup>3</sup></td><td>-0.081</td><td>0.22</td><td>0.48</td><td>0.64</td><td>1.10</td><td>0.43 (0.28)</td><td>ODR<sub>1</sub> &gt; 0.670<sup>2</sup></td><td>96</td><td>89</td><td>185</td><td>p = 0.45</td></tr><tr><td>(ODR<sub>2</sub>)</td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.309 &lt; ODR<sub>2</sub> ≤ 0.578</td><td>89</td><td>93</td><td>182</td><td>p = 0.45</td></tr><tr><td>FEC on the second visit</td><td>0</td><td>0</td><td>0</td><td>1</td><td>66</td><td>1.5 (4.8)</td><td>ODR<sub>2</sub> &gt; 0.578<sup>2</sup></td><td>105</td><td>96</td><td>201</td><td>p = 0.55</td></tr><tr><td>(eggs per 5g of feces)<sup>4</sup></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Positive FEC</td><td>99</td><td>98</td><td>197</td><td>p = 0.55</td></tr><tr><td>Treatment during the previous housing period</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>Negative FEC</td><td>179</td><td>197</td><td>376</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>Yes</td><td>106</td><td>122</td><td>228</td><td>p = 0.34</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>No</td><td>177</td><td>173</td><td>350</td><td></td></tr></tbody></table>

doi:10.1371/journal.pone.0147835.t003

Variations of the milk production response after spring anthelmintic treatment according to the indicators characterizing cows and herds

**Variations of the treatment response according to individual production-based indicators.** The interaction between week-trt and pre-treatment production level was significant (*p* < 0.0001). The high-producing treated cows' daily MP averaged by week decreased significantly and markedly compared to the one of high-producing control cows (-2.3 Kg/cow/day on average, until -3.7 Kg/cow/day on week<sub>8</sub> after treatment), whereas moderate and low-producing treated cows did not experience significant losses in milk production after treatment (Fig 2).

Interactions between parity or days in milk at the time of treatment and week-trt were not significant (*p* = 0.44 and *p* = 0.41, respectively).

<footnote n="1">¹ 22 missing data</footnote>

<footnote n="2">² Terciles of the global distribution of the quantitative variable</footnote>

<footnote n="3">³ 1 missing data</footnote>

<footnote n="4">⁴ 5 missing data.</footnote>
<table><caption>Table 4. Description of the dataset (578 cows in 13 herds, 295 treated cows, 283 control cows): distribution and classification of indicators characterizing herds, number of treated cows and control cows per classes for each categorized indicator.</caption><thead><tr><th rowspan="2">Indicators</th><th rowspan="2">Minimum</th><th rowspan="2">Q1</th><th rowspan="2">Median</th><th rowspan="2">Q3</th><th rowspan="2">Maximum</th><th rowspan="2">Mean (std)</th><th colspan="2">Classes threshold and number of cows (and herds) per classes</th><th rowspan="2">Total</th><th rowspan="2">Chi-square test</th></tr><tr><th>Control cows</th><th>Treated cows</th></tr></thead><tbody><tr><td>TEC<sup>5</sup> with GIN larvae before first calving (months)</td><td>3</td><td>6.5</td><td>8.5</td><td>10.4</td><td>15</td><td>8.5 (3)</td><td>High-TEC (TEC<sub>min</sub> ≥ 8)</td><td>118</td><td>127</td><td>245 (5 herds)</td><td>p = 0.74</td></tr><tr><td>Bulk tank milk anti-Ostertagia antibody level</td><td>0.546</td><td>0.652</td><td>0.717</td><td>0.863</td><td>0.994</td><td>0.766 (0.151)</td><td>Low-TEC (otherwise)</td><td>165</td><td>168</td><td>333 (8 herds)</td><td>p = 0.72</td></tr><tr><td>on the second visit (BTM ODR)</td><td></td><td></td><td></td><td></td><td></td><td></td><td>BTM ODR &lt; 0.717<sup>6</sup></td><td>152</td><td>150</td><td>302 (7 herds)</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>BTM ODR ≥ 0.717</td><td>133</td><td>143</td><td>276 (6 herds)</td><td></td></tr></tbody></table>

doi:10.1371/journal.pone.0147835.t004

Variations of the treatment response according to individual parasitological indicators. The evolution of MP after spring treatment differed significantly between cows which had been treated during the previous housing period and cows which had not been treated during this autumn-winter period (interaction between week-trt and history of anthelmintic treatment: *p* < 0.0001). The decrease in MP after spring treatment was marked and significant in cows not previously treated during the housing period (-1.2 Kg/cow/day on average, until -2.4 Kg/cow/day on week<sub>9</sub> after spring treatment), whereas this decrease remained non-significant in cows previously treated during the housing period (Fig 3).

Serum pepsinogen level measured on the second visit was significantly associated with the evolution of MP after treatment (interaction between week-trt and pepsinogen level 1.5 to 2 months after turn-out: *p* < 0.0001). Moderate and high-pepsinogen level cows (between 1409 and 2025 mUTyr, and > 2025 mUTyr, respectively) experienced higher MP losses after treatment than low-pepsinogen level cows: -1.1 Kg/cow/day on average for moderate and high-

<table><caption>Table 5. Comparison of individual parasitological indicators measured 1.5 to 2 months after turn-out (second visit, spring 2012) according to the history of anthelmintic treatment applied during the previous housing period (autumn-winter 2011–2012).</caption><thead><tr><th>Treatment during the previous housing period</th><th>NO</th><th>YES</th><th>Mean comparison test</th><th>Chi-square test</th></tr></thead><tbody><tr><td>Anti-Ostertagia antibody level (ODR)</td><td>n = 350 cows<br>mean = 0.48 (std = 0.26)<br>Q1 = 0.30; Q2 = 0.51; Q3 = 0.66</td><td>n = 227 cows<br>mean = 0.36 (std = 0.29)<br>Q1 = 0.12; Q2 = 0.40; Q3 = 0.59</td><td>p < 0.0001</td><td>-</td></tr><tr><td>Serum pepsinogen level (mUtyr)</td><td>n = 350 cows<br>mean = 1997 mUtyr (std = 858)<br>Q1 = 1390; Q2 = 1855; Q3 = 2493 mUtyr</td><td>n = 227 cows<br>mean = 1694 mUtyr (std = 797)<br>Q1 = 1107; Q2 = 1587; Q3 = 2025 mUtyr</td><td>p < 0.0001</td><td>-</td></tr><tr><td>FEC (positive versus negative)</td><td>n = 346 cows<br>Positive: 134 cows (39%)<br>Negative: 212 cows (61%)</td><td>n = 227 cows<br>Positive: 63 cows (28%)<br>Negative: 164 cows (72%)</td><td>-</td><td>p = 0.007</td></tr></tbody></table>

doi:10.1371/journal.pone.0147835.t005

<footnote n="5">⁵TEC = Time of Effective Contact</footnote>

<footnote n="6">⁶Median of the between-herd distribution.</footnote>
<figure><img src="image_4.png" /><figcaption>**Fig 1. Evolution of the treated cows' milk production over time in comparison with control cows (G<sub>i</sub>s < 0) (model 0, n = 578 cows, 295 treated cows, 283 control cows).** Week<sub>0</sub> = week of treatment, the first day of week<sub>0</sub> is the day of treatment. Black dots on the curve indicate that the corresponding negative G<sub>i</sub> are significantly different from zero, adjusted p-value<0.05: the drop in milk production for treated cows is significant during these weeks).</figcaption></figure>

doi:10.1371/journal.pone.0147835.g001

pepsinogen level cows, whereas the post-treatment decrease in MP was not significant in low-pepsinogen level cows (Fig 4).

FEC and anti-Ostertagia antibody level measured at the time of treatment were not associated with the evolution of MP after treatment: interactions between these two variables and the combined variable week-trt were not significant (*p* = 0.21 and *p* = 0.81, respectively).

**Variations of the treatment response according to herd-level indicators.** Fig 5 displays the evolution of the treated cows' MP in comparison with control cows' MP (G<sub>i</sub>s) according to the two herd-level indicators. The interaction between week-trt and TEC was significant (*p* = 0.001): after treatment, the MP of cows from high-TEC herds decreased somewhat more markedly than the MP of cows from low-TEC herds (-1.2 *versus* -0.7 Kg/cow/day, on average) (Fig 5a). The interaction between week-trt and BTM ODR was also significant (*p* < 0.0001): cows from high-BTM ODR herds (BTM ODR ≥ 0.717) experienced significant MP losses after treatment (-1.7 Kg/cow/day on average, down to -3.1 Kg/cow/day on week<sub>0</sub> after treatment),

<figure><img src="image_5.png" /><figcaption>**Fig 2. Evolution of the treated cows' milk production over time in comparison with control cows (G<sub>i</sub>s) according to the pre-treatment production level (corrected for parity and days in milk).** Week<sub>0</sub> = week of treatment, the first day of week<sub>0</sub> is the day of treatment. Black dots on the curve indicate that the corresponding negative G<sub>i</sub> are significantly different from zero, adjusted p-value<0.05: the drop in milk production for treated cows is significant during these weeks). See Table 2 for the number of treated cows and control cows per category.</figcaption></figure>

doi:10.1371/journal.pone.0147835.g002
<figure><img src="image_6.png" /><figcaption>**Fig 3.** Evolution of the treated cows' milk production over time in comparison with control cows (G<sub>i</sub>s) according to the history of anthelmintic treatment (treated versus not treated during the previous housing period). Week<sub>0</sub> = week of treatment, the first day of week<sub>0</sub> is the day of treatment. Black dots on the curve indicate that the corresponding negative G<sub>i</sub> are significantly different from zero, adjusted p-value<0.05: the drop in milk production for treated cows is significant during these weeks).—See Table 3 for the number of treated cows and control cows per category.</figcaption></figure>

doi:10.1371/journal.pone.0147835.g003

whereas the decrease in MP for treated cows from low-BTM ODR herds (BTM ODR < 0.717) remained much lower (-0.33 Kg/cow/day, down to -1.3 Kg/cow/day on week<sub>8</sub> after treatment) (Fig 5b).

Discussion

This is the first study dealing with the effect on milk production (MP) of a single non-persistent
anthelmintic treatment (fenbendazole) applied on lactating cows in spring, in the first part of
the grazing season, and taking into account the history of treatment administered during the
previous housing period. By treating 1.5 to 2 months after turn-out, one could expect (i) either
a positive effect on MP due the removal of parasites from hosts, with in addition the

<figure><img src="image_7.png" /><figcaption>**Fig 4.** Evolution of the treated cows' milk production over time in comparison with control cows (G<sub>i</sub>s) according to serum pepsinogen level at the time of treatment (second visit, 1.5 to 2 months after turn-out). Week<sub>0</sub> = week of treatment, the first day of week<sub>0</sub> is the day of treatment. Black dots on the curve indicate that the corresponding negative G<sub>i</sub> are significantly different from zero, adjusted p-value<0.05: the drop in milk production for treated cows is significant during these weeks). See Table 3 for the number of treated cows and control cows per category.</figcaption></figure>

doi:10.1371/journal.pone.0147835.g004
<figure><img src="image_8.png" /><figcaption>**Fig 5.** Evolution of the treated cows' milk production over time in comparison with control cows (Gᵢs) according to the 2 herd-level indicators (n = 578 cows): 5a) Time of Effective Contact (TEC) with GIN infective larvae before the first calving, 5b) Anti-Ostertagia antibody level in the bulk tank milk (BTM ODR). Week₀ = week of treatment, the first day of week₀ is the day of treatment. Black dots on the curve indicate that the corresponding negative Gᵢ are significantly different from zero, adjusted p-value<0.05: the drop in milk production for treated cows is significant during these weeks). See Table 4 for the number of treated cows and control cows per category.</figcaption></figure>

doi:10.1371/journal.pone.0147835.g005

preservation of a parasite population in *refugia* (free-living stages on pastures resulting from the spring development of eggs excreted since turn-out), (ii) or an absence of effect on MP due the possible rapid re-infection after this non-persistent anthelmintic treatment (persistent exposure to parasites as cows are grazing). Therefore, this prolonged negative marked effect of spring treatment on MP is quite surprising and contrary to what was initially expected. This negative effect made it impossible to identify the categories of cows that could be treated selectively in spring (TST strategy) on the basis of the demonstration of their improved milk production after treatment. However, a possible explanation of this negative effect of the treatment may be found by looking at the factors associated with the treatment response. The decrease in MP was particularly marked in cows (i) with a high pre-treatment production level, (ii) not treated with anthelmintics during the previous housing period (autumn-winter 2011–2012), (iii) with moderate or high pepsinogen level at the time of spring treatment (pepsinogen level being associated with the history of anthelmintic treatment), (iv) from herds with a high bulk tank milk anti-*Ostertagia* antibody level (BTM ODR > 0.717, median of our sample), and (v) from high-TEC herds.

Studies investigating the effect of a benzimidazole treatment on MP sometimes reported negative values, but the decrease in MP always remained non-significant [10, 11], except in one Norwegian study, conducted in 22 dairy herds, where fenbendazole treatment was applied at
calving from September to the end of January, while cows were housed [50]. In this previous Norwegian study, the significant reduction in milk yield of the fenbendazole-treated cows was -151 kg in the subsequent lactation. The authors did not put forward any hypothesis to explain this decrease in MP. Jara et al. (1984) [51] showed that albendazole, fenbendazole and oxfenda-zole could cause a decrease in volatile fatty acid concentration and digestibility of cellulose in the rumen of sheep. However, according to Tharaldsen and Helle (1989) [50], those effects of fenbendazole treatment are unlikely to influence significantly MP for several consecutive weeks, in light of its rapid elimination from the body.

Forbes et al. (2004) [38] reported a significant positive MP response on weeks 2 and 3 after a single anthelmintic treatment applied, as in our study, 1.5 month after turn-out. However, in this previous study, cows were treated with eprinomectin, a persistent-activity product which prevents from re-infection with *Ostertagia* for 28 days [52]; and MP was followed for only 4 weeks after treatment, a period over which cows could therefore not be re-infected. The same was true in studies where several treatments were applied repeatedly on lactating cows during the grazing season [23, 39, 40]: positive effects of these repeated treatment strategies on MP were mainly reported but with highly limited post-treatment reinfections during the period of follow-up. On the contrary, in our study, treated cows could be re-infected during the 105 days period of follow up after the non-persistent fenbendazole treatment, because they kept grazing together with control cows on pastures where parasitic cycles had been re-initiated for 1.5 to 2 months. As a result, it may be assumed that the prolonged negative marked effect of spring treatment on MP was associated with post-treatment re-infection. This hypothesis is supported by the more severe and significant post-treatment decrease in MP observed in cows from high-BTM ODR herds at the time of treatment, i.e. from herds with a higher exposure to and therefore more prone to re-infection, due to a higher pasture infectivity [53].

Moreover, our observations suggest that immunological and inflammatory mechanisms may be associated with this negative effect of spring treatment on MP. Indeed, the negative post-treatment MP response was more marked in herds where the higher exposure to GIN could have induced a heavier antigenic stimulation (high BTM ODR herds). The negative post-treatment MP response was also somewhat more marked in cows from high-TEC herds, i.e. in herds where the acquired immune response controlling the establishment of ingested L3 could be stronger [25, 54], causing a stronger reaction when new contact with L3 occurred. This phenomenon is known in resistant sheep: a new larval challenge can induce a strong inflammatory reaction, with clinical signs of diarrhea, even though worm burdens are low [55-59]. As it leads to the elimination of worms, this phenomenon is commonly described as “self-cure”. It has been demonstrated that it is based on mechanisms similar to type I hypersensitivity [60], explaining the deleterious consequences for hosts. In cattle, high pepsinogen levels observed in second-grazing-season cattle exposed to an initial low level of infection [5, 31] and clinical forms of oedematous ostertagiosis [61] are also considered as deleterious effects of these particular immuno-inflammatory processes in immune animals.

This hypothetical association between inflammatory processes and negative MP response is also supported by the fact that the post-treatment decrease in MP was particularly marked in cows not treated during the previous housing period. In such cows, which contributed the most to the overall negative spring treatment response, the inflammatory state of the abomasal mucosa could have been more pronounced. Indeed, their worm burden is expectedly higher than the one of cows treated during the previous housing period, resulting in the resumption of development of a larger number of inhibited larvae, with higher damage of the abomasal mucosa (as evidenced by higher pepsinogen levels found in these cows). In addition, serum pepsinogen levels of these cows were higher when they were measured after turn-out, particularly on the second visit 1.5 to 2 months after turn-out, demonstrating higher abomasal
mucosal damages potentially also caused by the response to larval challenge between turn-out and the date of anthelmintic treatment.

Those inflammatory processes may have been exacerbated by the persistence of dead *Ostertagia* larvae in the abomasal mucosa following fenbendazole treatment. Two studies come in support of this hypothesis. (i) In yearling beef cattle naturally infected with *O. ostertagi*, treated with fenbendazole while a large number of early fourth stage inhibited larvae had been established, and then slaughtered 7 to 12 days after treatment, Snider III et al. (1985) [62] observed an aggravation of the abomasal inflammatory lesions compared to those found in untreated calves. This aggravation was related to the persistence of dead and degenerate *O. ostertagi* larvae in the abomasal mucosa of fenbendazole treated calves. Indeed, these degenerate larvae were not observed in the tissues of untreated calves nor in calves treated with ivermectin, and were thus attributed to the fenbendazole treatment. Histologically, the presence of a marked eosinophil and lymphocyte infiltration and of an unidentified material coating the degenerate larvae suggested an inflammatory and/or immunological response initiated or exacerbated by the presence of such larvae [62]. (ii) Similar results were found in horses naturally infected with cyathostomins and treated either with fenbendazole or moxidectin [63]: in contrast to moxidectin effects, the killing of larvae due to fenbendazole treatment was associated with severe tissue damage. These observations argue in favor of the involvement of fenbendazole treatment in the maintenance of inflammatory reaction of the abomasal mucosa. However, considering the persistence of the negative effect of treatment on MP, one may wonder what the duration of the pro-inflammatory effect of dead larvae is. Snider III et al. (1985) [62] indicated that these degenerate *Ostertagia* larvae were found up to 20 days after treatment, but in smaller proportion than between 7 to 12 days after treatment, and that the time needed for dead nematode clearance is unknown for *O. ostertagi* and other GIN. Moreover, other observations are in favour of the pro-inflammatory effect of larvae killed by the treatment. In the study conducted by Ravinet et al. (2014) [25], where adult dairy cows were treated with fenbendazole at housing and where the pattern/kinetics of the treatment response is described, the global effect of treatment on MP was positive but was not immediate, and treated cows tended to produce less milk than control cows during the first two weeks following the date of treatment. This could suggest a slight deleterious effect of treatment, before tissue repair and increased feed intake following the removal of parasites could allow a moderate increase in MP [25].

Further studies are needed to assess whether this unexpected negative effect of spring anthelmintic treatment on MP can be generalized, and to identify and understand the biological mechanisms underlying this decrease in MP. A study investigating more closely the inflammatory and immune responses would be necessary to assess the potential adverse effects of (i) the larval challenge at turn-out, and of (ii) the spring anthelmintic treatment under different exposures to GIN, and with different immunological status (TEC). Moreover, the approach outlined in this study should be replicated with another anthelmintic (eprinomectin), to assess whether our observed negative effect of treatment is related, at least in part, to the use of fenbendazole. Finally, a longer post-treatment follow-up of MP would enable to assess if this deleterious effect of treatment on MP is reversible and how long it lasts.

## Conclusion

The idea of optimizing milk production while preserving a large GIN population in *refugia* by spring TST in grazing dairy cows ran up against an unexpected result: the anthelmintic treatment applied 1.5 to 2 months after turn-out had a significant negative effect on milk production.
Our result suggests that treatment for GIN of dairy cows cannot be generally recommended and must be associated with seasonal considerations. Until further studies can assess whether this unexpected result can be generalized or is tied to specific conditions, anthelmintic treatment of adult dairy cows immunized against GIN, specifically non-persistent treatment, should be discouraged at the beginning of the grazing season, and should rather be targeted in autumn at housing.

## Acknowledgments

The authors thank all the farmers for their participation, their help and co-operation. They are very grateful for all the assistance provided on the field and at the laboratory by Jean-Yves Audiart, Marie-Astrid Malard, Françoise Leray, Emmanuelle Blandin, Didier Billon, Manon Joly and Melissa Girard (ONIRIS). The authors would also like to thank all the veterinary practitioners who provided their support to the recruitment and on the field. The study was conducted within the UMT Cattle Herd Health Management.

## Author Contributions

Conceived and designed the experiments: NR CC N. Bareille AC. Performed the experiments: NR CC N. Brisseau AP AC. Analyzed the data: NR AL N. Brisseau. Wrote the paper: NR AC CC N. Bareille.

## References

1. Agneessens J, Claerebout., Dorny P, Borgsteede FHM, Vercruysse J. Nematode parasitism in adult dairy cows in Belgium. Vet Parasitol 2000; 90: 83-92. PMID: 10828514

2. Borgsteede FHM, Tibben J, Cornelissen JB, Agneessens J, Gaasenbeek CP. Nematode parasites of adult dairy cattle in the Netherlands. Vet Parasitol 2000; 89: 287-296. PMID: 10799842

3. Chartier C, Le Corre A, Ravinet N, Chauvin A. Nematode parasites from the abomasa of adult dairy cows in France: an abattoir survey. In: Proceeding WAAVP, Perth, Australia, 25-29 August 2013, p. 540.

4. Slocombe JOD. Abomasal nematodes in cattle in Ontario. Can J Comp Med 1974; 38: 18-21. PMID: 4272952

5. Armour J, Bairden K, Duncan JL, Jennings FW, Parkins JJ. Observations on ostertagiasis in young cattle over two grazing seasons with special references to plasma pepsinogen levels. Vet Rec 1979; 105: 500-503. PMID: 524707

6. Anderson N, Armour J, Jarrett WFH, Jennings FW, Ritchie JSD, Urquhart GM. A field study of parasitic gastritis in cattle. Vet Rec 1965; 77: 1196-1204. PMID: 5890703

7. Anderson N. Aspects of the biology of *Ostertagia ostertagi* in relation to the genesis of ostertagiasis. Vet Parasitol 1988; 27: 13-21. PMID: 3284162

8. Shaw DJ, Vercruysse J, Claerebout E, Dorny P. Gastrointestinal nematode infections of first-grazing season calves in Western Europe: general patterns and the effect of chemoprophylaxis. Vet Parasitol 1998; 75: 115-131. PMID: 9637215

9. Shaw DJ, Vercruysse J, Claerebout E, Dorny P. Gastrointestinal nematode infections of first-grazing season calves in Western Europe: associations between parasitological, physiological and physical factors. Vet Parasitol 1998; 75: 133-151. PMID: 9637216

10. Gross SJ, Ryan WG, Ploeger HW. Anthelminthic treatment of dairy cows and its effect on milk production. Vet Rec 1999; 144: 581-587. PMID: 10378289

11. Sanchez J, Dohoo I, Carrier J, DesCôteaux L. A meta-analysis of the milk-production response after anthelminthic treatment in naturally infected adult dairy cows. Prev Vet Med 2004; 63: 237-256. PMID: 15158573

12. Charlier J, Höglund J, Samson-Himmelstjerna G, Dorny P, Vercruysse J. Gastrointestinal nematode infections in adult dairy cattle: Impact on production, diagnosis and control. Vet Parasitol 2009; 164: 70-79. doi: 10.1016/j.vetpar.2009.04.012 PMID: 19414223

13. Sutherland IA, Leathwick DM. Anthelmintic resistance in nematode parasites of cattle: a global issue? Trends Parasitol 2011; 27: 176-180. doi: 10.1016/j.pt.2010.11.008 PMID: 21168366
14. Demeler J, Van Zeveren AMJ, Kleinschmidt N, Vercruysse J, Höglund J, Koopmann R, et al. Monitoring the efficacy of ivermectin and albendazole against gastrointestinal nematodes of cattle in Northern Europe. Vet Parasitol 2009; 160: 109–115. doi: 10.1016/j.vetpar.2008.10.030 PMID: 19062191

15. Gasbarre L.C., 2014. Anthelmintic resistance in cattle nematodes in the US. Vet Parasitol 2014; 204: 3–11. doi: 10.1016/j.vetpar.2014.03.017 PMID: 24798800

16. Geurden T, Chartier C, Frangipane di Regalbono A, Traversa D, von Samson-Himmelstjerna G, Otranto D, et al. Anthelmintic resistance of cattle gastro-intestinal nematodes in 4 European countries. In: Proceeding WAAVP, Perth, Australia, 25–29 August 2013, p. 167.

17. Waghorn TS, Leathwick DM, Rhodes AP, Jackson R, Pomroy WE, West DM, et al. Prevalence of anthelmintic resistance on 62 beef cattle farms in the North Island of New Zealand. N Z Vet J 2006; 54: 278–282. PMID: 17151725

18. O'Shaughnessy J, Earley B, Mee JF, Doherty ML, Crosson P, Barrett D, et al. Detection of anthelmintic resistance on two Irish beef research farms. Vet Rec 2014; 175: 120. doi: 10.1136/vr.102556

19. Rose H, Rinaldi L, Bosco A, Mavrot F, de Waal T, Skuce P, et al. Widespread anthelmintic resistance in European farmed ruminants: a systematic review. Vet Rec 2015 doi: 10.1136/vr.102982

20. O'Farrell KJ, Downey NE, Sherington J. The effect of anthelmintic treatment at calving on the subsequent milk production characteristics of dairy cows. Ir Vet J 1986; 40: 116–123.

21. Ploeger HW, Schoenmaker GJW, Kloosterman A, Borgsteede FHM. Effect of anthelminthic treatment of dairy cattle on milk production related to some parameters estimating nematode infection. Vet Parasitol 1989; 34: 239–253. PMID: 2617828

22. Ploeger HW, Kloosterman A, Bargeman G, Wuijckhuise L, Van Der Brink R. Milk yield increase after anthelminthic treatment of dairy cattle related to some parameters estimating helminth infection. Vet Parasitol 1990; 35: 103–116. PMID: 2343521

23. Mason WA, Pomroy WE, Lawrence KE, Scott I. The effect of a repeated, four-weekly eprinomectin treatment on milk production in pasture-based, seasonally-calving dairy cattle. Vet Parasitol 2012; 189: 250–259. doi: 10.1016/j.vetpar.2012.05.003 PMID: 22664339

24. Vanderstichel R, Dohoo I, Sanchez J, Sithole F, Keefe G, Stryhn H. Predicting the effect of anthelmintic treatment on milk production of dairy cattle in Canada using an *Ostertagia ostertagi* ELISA from individual milk samples. Prev Vet Med 2013; 111: 63–75. doi: 10.1016/j.prevetmed.2013.04.001 PMID: 23647705

25. Ravinet N, Bareille N, Lehebel A, Ponnau A, Chartier C, Chauvin A. Change in milk production after treatment against gastrointestinal nematodes according to grazing history, parasitological and production-based indicators in adult dairy cows. Vet Parasitol 2014; 201: 95–109. doi: 10.1016/j.vetpar.2013.12.031 PMID: 24468428

26. Verschave SH, Vercruysse J, Forbes A, Opsomer G, Hostens M, Duchateau L, et al. Non invasive indicators associated with the milk yield response after anthelmintic treatment at calving in dairy cows. BMC Vet Res 2014; 10:264. doi: 10.1186/s12917-014-0264-x PMID: 25394846

27. van Wyk JA. Refugia—overlooked as perhaps the most important factor concerning the development of anthelmintic resistance. Onderstepoort J Vet Res 2001; 68: 55–67. PMID: 11403431

28. Kenyon F, Greer AW, Coles GC, Cringoli G, Papadopoulos E, Cabaret J, et al. The role of targeted selective treatments in the development of refugia-based approaches to the control of gastrointestinal nematodes of small ruminants. Vet Parasitol 2009; 164: 3–11. doi: 10.1016/j.vetpar.2009.04.015 PMID: 19450930

29. Kenyon F, Jackson F, 2012. Targeted flock/herd and individual ruminant treatment approaches. Vet Parasitol 2012; 186: 10–17. doi: 10.1016/j.vetpar.2011.11.041 PMID: 22188928

30. Charlier J, Morgan ER, Rinaldi L, van Dijk J, Demeler J, Höglund J, et al. Practices to optimize gastrointestinal nematode control on sheep, goat and cattle farms in Europe using targeted (selective) treatments. Vet Rec 2014; 175: 250–255. doi: 10.1136/vr.102512 PMID: 25217603

31. Armour J. The epidemiology of helminth disease in farm animals. Vet. Parasitol. 1980; 6: 7–46.

32. McBeath DG, Dean SP, Preston NK. The effect of a preparturient fenbendazole treatment on lactation yield in dairy cows. Vet Rec 1979; 105: 507–509. PMID: 524709

33. Fréchette JL, Lamothe P. Milk production effect of a morantel tartrate treatment at calving in dairy cows with subclinical parasitism. Can Vet J 1981; 22: 252–254. PMID: 7340928

34. Bisset SA, Marshall ED, Morrison L. Economics of a dry-cow anthelminthic drenching programme for dairy cows in New Zealand. Part 1. Overall response in 47 herds. Vet Parasitol 1987; 26: 107–118. PMID: 3439001

35. Reist M, Medjitna TDE, Braun U, Pfister K. Effect of a treatment with eprinomectin or thrichlorfon on the yield and quality of milk produced by multiparous dairy cows. Vet Rec 2002; 151: 377–380. PMID: 12403517
36. Charlier J, Duchateau L, Claerebout E, Vercruysse. Predicting milk-production responses after an autumn treatment of pastured dairy herds with eprinomectin. Vet Parasitol 2007; 143: 322–328. PMID: 16989949

37. Charlier J, Vercruysse J, Smith J, Vanderstichel R, Stryhn H, Claerebout E, et al. Evaluation of anti-Ostertagia ostertagi antibodies in individual milk samples as decision parameter for selective anthelmintic treatment in dairy cows. Vet Parasitol 2010; 93: 147–152.

38. Forbes AB, Huckle CA, Gibb MJ. Impact of eprinomectin on grazing behaviour and performance in dairy cattle with sub-clinical gastro-intestinal nematode infections under continuous stocking management. Vet Parasitol 2004; 125: 353–364. PMID: 15482891

39. Gibb MJ, Huckle CA, Forbes AB. Effects of sequential treatments with eprinomectin on performance and grazing behaviour in dairy cattle under daily-paddock stocking management. Vet Parasitol 2005; 133: 79–90. PMID: 16129562

40. McQueen IPM, Cottier K, Hewitt SR, Wright DF. Effects of anthelmintics on dairy cow yields. New Zeal J Exp Agr 1977; 5: 115–119.

41. Michel JF, Richards M, Altman JFB, Mulholland JR, Gould CM, Armour J. Effect of anthelmintic treatment on the milk yield of dairy cows in England, Scotland and Wales. Vet Rec 1982; 111: 546–550. PMID: 7164331

42. Kloosterman A, Ploeger HW, Pieke EJ, Lam TJGM, Verhoeff J. The value of bulk milk ELISA Ostertagia antibody titres as indicators of milk production response to anthelmintic treatment in the dry period. Vet Parasitol 1996; 64: 197–205. PMID: 8888553

43. McPherson WB, Gogolewski RP, Slacek B, Familton AS, Gross SJ, Maciel AE, et al. Effect of a peri-parturient eprinomectin treatment of dairy cows on milk production. N Z Vet J 2001; 49: 106–110. PMID: 16032172

44. Nødtvedt A, Dohoo I, Sanchez J, Conboy G, DesCôteaux L, Keefe G. Increase in milk yield following eprinomectin treatment at calving in pastured dairy cattle. Vet Parasitol 2002; 105: 191–206. PMID: 11934459

45. Sanchez J, Dohoo I, Nødtvedt A, Keefe G, Markham F, Leslie K, et al. A longitudinal study of gastrointestinal parasites in Canadian dairy farms. The value of an indirect Ostertagia ostertagi ELISA as a monitoring tool. Vet Parasitol 2002; 107: 209–226. PMID: 12127251

46. Sanchez J, Dohoo I, Leslie K, Keefe G, Markham F, Sithole F. The use of an indirect Ostertagia oster tagi ELISA to predict milk production response after anthelmintic treatment in confined and semi-con fined dairy herds. Vet Parasitol 2005; 130: 115–124. PMID: 15893078

47. Sithole F, Dohoo I, Leslie K, DesCôteaux L, Godden S, Campbell J, et al. Effect of eprinomectin treatment at calving on milk production in dairy herds with limited outdoor exposure. J Dairy Sci 2005; 88: 929–937. PMID: 15738227

48. Kerboeuf D, Koch C, Le Dréan E, Lacourt A. Méthode simplifiée de mesure de la concentration en pepsinogène dans le sérum. Revue Méd. Vét. 2002; 153: 707–712.

49. Bliss DH, Kvasnicka WG. The Fecal Examination: a Missing Link in Food Animal Practice. The Compendium 1997; 4: 104–109.

50. Tharaldsen J, Helle O. The effect of periparturient treatment with fenbendazole on the milk production of cows. Acta Vet Scand 1989; 30: 247–252. PMID: 2629501

51. Jara W, Sumano H, Ocampo L. Effects of three benzimidazole anthelmintics on the ruminal fermentation ability of sheep. Vet Rec 1984; 115: 565–566. PMID: 6523680

52. Cramer LG, Pitt SR, Rehbein S, Gogolewski RP, Kunkle BN, Langhoff WK, et al. Persistent efficacy of topical eprinomectin against nematode parasites in cattle. Parasitol Res 2000; 86: 944–946. PMID: 11097305

53. Eysker M, van Aarle D, Kooyman FNJ, Nijzink AM, Orsel K, Ploeger HW. Exposure of dairy cows to nematode infections at the end of the grazing season in the Netherlands. Vet Parasitol 2002; 110: 93–100. PMID: 12446093

54. Vercruysse J, Claerebout E. Immunity development against Ostertagia ostertagi and other gastrointestinal nematodes in cattle. Vet. Parasitol. 1997; 72: 309–326. PMID: 9460204

55. Miller HRP, Jackson F, Newlands G, Appleyard WT. Immune exclusion, a mechanism of protection against the ovine nematode Haemonchus contortus. Res Vet Sci 1983; 35: 357–363. PMID: 6665320

56. Balic A, Bowles VM, Meeusen ENT. The immunobiology of gastrointestinal nematodes infections in ruminants. Adv Parasit 2000; 45: 181–241.

57. Larsen JW, Anderson N, Vizard AL, Anderson GA, Hoste H. Diarrhoea in Merino ewes during winter: association with trichostrongylid larvae. Aust Vet J 1994; 71: 365–372. PMID: 7726760
58. Larsen JW, Anderson N, Vizard AL. The pathogenesis and control of diarrhea and breech soiling in adult Merino sheep. Int J Parasitol 1999; 29: 893-902. PMID: 10480726

59. Larsen JW, Anderson N. The relationship between the rate of intake of trichostrongylid larvae and the occurrence of diarrhoea and breech soiling in adult merino sheep. Aust Vet J 2000; 78: 112-116. PMID: 10736672

60. Meeusen ENT. Immunology of helminth infections, with special reference to immunopathology. Vet Parasitol 1999; 84: 259-273. PMID: 10456418

61. Raynaud JP, Bouchet A. Bovine ostertagiosis, a review. Analysis of types and syndromes found in France by post mortem examinations and total worm counts. Ann rech vétér 1976; 7: 253-280.

62. Snider III TG, Williams JC, Knox JW, Roberts ED, Romaine TL. Persistence of dead *Ostertagia ostertagi* in the abomasal mucosa following anthelminthic treatment. Vet Rec 1985; 116: 69-72. PMID: 3838393

63. Steinback T, Bauer C, Sasse H, Baumgärtner W, Ray-Moreno C, Hermosilla C, et al. Small strongyle infection: consequences of larvicide treatment of horses with fenbendazole and moxidectine. Vet Parasitol 2006; 139: 115-131. PMID: 16675126