Study 1: Distribution of parasite DNA in the severe chronic stage of the disease
Fourteen adult (from 2 to 12 years-old) cattle (11 females and 3 males) of various breeds, including Salers, Limousine, Charolaise, Gasconne des Pyrénées and Blonde d’Aquitaine, showing advanced clinical signs of the chronic phase of the disease were sent to the National Veterinary School of Toulouse by cattle breeders located within a 200 km radius around Toulouse. Before euthanasia, to confirm the diagnosis of besnoitiosis, blood samples for western blot (WB) analyses were collected in BD Vacutainer® (Dutscher, Brumath, France) blood collection tubes without anticoagulants. These animals were humanly euthanized by a veterinary surgeon using a lethal intra-venous injection of embutramide (T61, Intervet) and immediately necropsied.
For each animal, skin samples (1 cm2) were collected immediately after death from several sites: right foreleg; right hind leg; udder for females; right inner thigh; backline; right flank; right shoulder; right eyelid; dewlap; umbilicus area; tail base; and ocular sclera for external zones. Tissue samples (1 cm2 or 1 cm3) from internal organs, lung (lower respiratory tract), spleen, liver, heart, right kidney, diaphragm, subcutaneous connective tissue, cutaneous muscle collected from right flank, nasal and tracheal mucosa (upper respiratory tract)) also were collected. All samples were transported individually in an identified dry tube (Corning MCT-150-G; Fisher Scientific, Illkirch, France) and stored at 4 °C before DNA extraction and PCR analysis carried out the following day.
DNA extraction and quantitative real-time PCR
In the ENVT (Ecole Nationale Vétérinaire Toulouse) laboratory, DNA was extracted from tissue biopsies using a commercial kit (QIAmp® DNA Mini Kit; Qiagen, Courtaboeuf, France). Following the manufacturer’s recommendations, 50 mg aliquots of tissue biopsies were processed after an over-night incubation with proteinase K. Besnoitia spp. internal transcribed spacer 1 (ITS1) amplification was performed with the commercial PCR kit AdiaVetTM Besnoitia (AES Chemunex, Bruz, France). The quantitative PCR was performed with the Stratagene MX3005P thermal cycler (Agilent Technologies, La Jolla, CA, USA). Positive and negative controls were provided by the manufacturer. Results were analyzed using the MxPro QPCR version 4.10 software (Agilent Technologies). When the Cq value was inferior or equal to 36, the parasite DNA was considered detected and the animal was deemed to be a super-spreader, when Cq values ranged between 36 and 40, parasite DNA was at the limit of detection, and a Cq superior to 40 was considered as a negative real-time PCR result.
Western blot analyses
WB analyses were performed on animals necropsied in the ENVT facilities, on animals collected in the slaughterhouses, and when doubtful ELISA results were recorded on farms A to H. For tachyzoite-based WB analysis, the coated membranes and the immunoblots were performed as previously described [18]. Three main antigenic reactivity areas are described [19]: area I, 12–20 kDa; area II, 23–38 kDa; and area III, 60–90 kDa. The minimal criterion for serological positivity was the recognition of at least four bands in at least two domains. This test is considered to be highly specific [20].
Study 2: Comparison of the DNA concentration between three skin locations in asymptomatic infected cattle
One hundred and sixty adult cattle were sampled in three slaughterhouses located in endemic areas: Ariège (n = 105), Hautes-Alpes (n = 33) and Alpes de Haute-Provence (n = 22). These animals did not show any clinical sign of bovine besnoitiosis and their status (infected or not) was unknown at the time of sampling. Skin biopsies for PCR were collected from the ear, neck and tail base, transported individually in an identified dry tube (Corning MCT-150-G) and stored at 4 °C until PCR analysis carried out the following day as described in Study 1. Blood samples for WB analyses were drawn from the jugular vein during bleeding and collected in BD Vacutainer® blood collection tubes without anticoagulants. WB analyses were processed as described in Study 1.
Study 3: Evaluation of the DNA concentrations in skin samples from herds with high seroprevalence
Eight farms located in different regions of France (2 in the center, 6 in the south) were included in the study (Table 1). Both dairy (n = 4) and beef cattle herds (n = 4) were selected. Farms A, B and C were recruited in the spring of 2017, farms D, E and F in the spring of 2018 and farms G and H in the spring of 2019. These farms were chosen because (i) all had experienced at least one clinical case of bovine besnoitiosis before the beginning of the survey, (ii) the enzyme-linked immunosorbent assay (ELISA)-seroprevalence established in the two weeks before the beginning of the survey was high (over 40%), and (iii) the cattle producer volunteered to participate in the study. All seropositive individuals (n = 518) of these 8 farms were tested by real-time PCR on skin biopsies. Detailed information is provided in Table 1.
According to the results of Study 2, the skin sample for PCR was taken without anesthesia at the tail base using biopsy punches (8 mm diameter; Kruuse, Langeskov, Denmark). This area was previously cleaned with Betadine® (Centravet, Castelnaudary, France). The skin fragment was placed in a dry tube (Corning MCT-150-G), identified with the bovine individual ID, stored at +4 °C and analyzed within two days as described in Study 1. Aluminum spray (Aluspray® Vetoquinol; Centravet, Castelnaudary, France) was sprayed on the biopsied area.
ELISA analyses
Sera were separated by centrifugation and tested for B. besnoiti antibodies using a commercial ELISA kit (ID Screen® Besnoitia Indirect 2.0; IDVET, Grabels, France). Serological analyses were performed by the Departmental Veterinary Laboratory of Ariège (LVD 09) for farms A, B, C and D, LVD 26 (Drôme) for farms G and H, LVD 18 (Cher) for farm E, and LVD 36 (Indre) for farm F.
Study 4: Efficacy of the culling of super-spreaders in the control of bovine besnoitiosis
Real-time PCR analyses were performed within two days after the skin biopsy samples were collected. The results were immediately communicated to farmers and veterinarian practitioners in charge of the farm so that animals deemed to be super-spreaders (Cq ≤ 36) could be culled as soon as possible. In this way, super-spreaders were discarded from the herd before the high activity period of hematophagous flies began. However, this culling strategy was only applied on farms A, D and H. To assess whether the status of individuals persisted over time, PCR samples were collected again 10 months later on 6 animals considered to be super-spreaders on farm C, and 7 animals with doubtful or negative PCR results were sampled three years later on farm A. All real-time analyses were performed as described in Study 1.
To assess the ability of this strategy to reduce the number of newly acquired B. besnoiti infections, serological ELISA analyses were performed on previously seronegative animals and young ones (over 6 months-old) that were not present on the farm at the previous sampling date. The time intervals between the initial and subsequent analyses differed per farm as follows: 12, 24 and 36 months for farm A; 12 and 24 months for farm D; 12 months only for farms B, C and H. ELISA analyses were performed as described in Study 3.
Statistical analyses
In chronically infected cattle necropsied in the ENVT facilities, comparisons between mean Cq values from skin samples and mean Cq values from internal organs, and then between mean Cq values from skin of the upper side of the body and from skin of the lower side of the body, were performed using Mann-Whitney-Wilcoxon (MWW) rank tests. Individual variability of Cq values obtained in asymptomatic infected animals from the 8 farms studied were presented according to classes of Cq (in steps of 3 Cq units between Cq ≤ 15 to no Cq). Distribution of individuals within these Cq classes was compared between farms using the Fisherʼs exact Chi-square test. A binary logistic regression model was fitted to investigate factors (farm, age and ELISA S/P value) related to the Cq value obtained in real-time PCR on the skin biopsies (positive for Cq value < 40 versus negative for Cq > or equal to 40). These factors included farm (A, B, C or D), age (below or above 24 months-old) and ELISA S/P value (below and above 110%). Only the 147 individuals from farms A, B, C and D were used in this analysis because these analyses were performed in the same laboratory (Laboratoire Vétérinaire Départemental de l’Ariège). All statistical analyses were done using R software (version 3.5.2, R foundation; www.r-project.org).