Animals studied and collection of samples
Cattle in chronic phase of the disease
Fourteen adult cattle (11 females and three 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. 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, spleen, liver, heart, right kidney, diaphragm, subcutaneous connective tissue, skin muscle, nasal and tracheal mucosa) also were collected.
All samples were transported individually in an identified dry tube (Corning MCT-150-G) and stored at 4 °C before analysis the following day.
Asymptomatic cattle from slaughterhouses
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. Blood samples for western blot (WB) analyses were drawn from the jugular vein during bleeding and collected in BD Vacutainer® blood collection tubes without anticoagulants.
Asymptomatic cattle from herds showing high seroprevalence
Eight farms located in different regions of France (two in the center, six in the south) were included in the study (Table I). 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 ELISA-seroprevalence established just before the beginning of the survey was high (over 40%), and iii) the cattle producer volunteered to participate in the study. Overall, 518 seropositive individuals were tested by real-time PCR on skin biopsies. Detailed information is provided in Table I.
The skin sample for PCR was taken without anesthesia at the tail base using biopsy punches (8 mm diameter, Kruuse 273693). This area was previously cleaned with Betadine (Viatris 9451.8). The skin fragment was placed in a dry tube (Corning MCT-150-G), identified with the bovine individual ID and stored at + 4 °C before analysis. Aluminum spray (Aluspray® Vetoquinol) was sprayed on the biopsied area.
DNA Extraction And Quantitative Real-time PCR
In the ENVT 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 (ITS-1) 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 Ct value was inferior or equal to 36, the parasite DNA was considered detected and the animal was deemed to be a super-spreader, when Ct values ranged between 36 and 40, parasite DNA was at the limit of detection, and a Ct superior to 40 was considered as a negative real-time PCR result.
Sera were separated by centrifugation and tested for B. besnoiti antibodies using a commercial ELISA kit (ID Screen® Besnoitia Indirect 2.0, IDVET). 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.
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 . Three main antigenic reactivity areas are described : 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 .
Real-time PCR analyses were performed within a week 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 (Ct ≤ 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 ten months later on six animals considered to be super-spreaders on farm C, and seven animals with doubtful or negative PCR results were sampled three years later on farm A.
To assess the ability of this strategy to reduce the number of newly acquired B. besnoiti infections, serological analyses were performed on previously seronegative animals and young ones (over six 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.
In chronically infected cattle necropsied in the ENVT facilities, comparisons between mean Ct values from skin samples and mean Ct values from internal organs, and then between mean Ct 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. For cattle sampled at slaughterhouses, Ct values recorded in the neck, ear and tail base of the same individual were compared using the Friedman rank test. Individual variability of Ct values obtained in asymptomatic infected animals from the eight farms studied were presented according to classes of Ct (in steps of three Ct units between Ct ≤ 15 to NoCt). Distribution of individuals within these Ct classes was compared between farms using the Fisher exact chi-square test. The relationship between Ct values and ELISA sample to positive ratio (S/P value) was assessed on a subset of 147 animals (farms A, B, C and D) for which all serological analyses were performed in the same laboratory (LVD 09). The Spearman correlation was calculated, with individuals showing no DNA detection in their skin biopsies scored with a Ct value of 45. A binary logistic regression model was fitted to investigate factors related to the Ct value obtained in real-time PCR on the skin biopsies (positive for Ct value < 40 versus negative for Ct > 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%). Again, only the 147 individuals from farms A, B, C and D were used in this analysis. All statistical analyses were done using R software (version 3.5.2, R foundation).