Calves

Thirty-two newborn Holstein–Friesian bull calves were purchased from 13 Alberta (Canada) dairy farms selected based on annual testing as part of the Alberta Johne’s Disease Initiative [27] and participation in the JD herd health status program in Alberta. All farms had tested negative for at least 4 years using 6 environmental samples and 1 of the following: bacteriological culture of 60 individual fecal samples tested as pooled samples into groups of 5, individual milk ELISA of the whole milking herd, or serum ELISA of the entire herd.

Nutrition, health and husbandry

All calves were collected immediately after birth (to prevent contamination from fecal material on farm or ingesting colostrum), and transported to the research facility. Nutrition was similar to that described by Mortier et al. [16]. In short, calves were fed 6 L (in 2-L portions) of high-quality colostrum within the first 8 h after birth. Colostrum was collected from 4 of the 13 farms that had tested negative consistently for ≥ 4 years. Starting the 2^nd day of their life, calves were fed milk replacer, followed by calf starter (without antimicrobials) and high-quality hay. Calves were gradually weaned by 8 week of age, and had ad libitum access to water and hay (supplemented with concentrates).

Calves were housed in a biosecurity Level 2 facility. The facility included 15 custom-built housing units with waterproof liners to contain all bedding and fecal material. Group-housing pens were 10 × 10 feet and 6 feet tall (3.05 × 3.05 × 1.82 m). Each housing unit consisted of a marked-off area containing the pen, 2 pairs of boots, 2 pairs of coveralls and gloves dedicated specifically for use in the pen within the unit. All personnel were trained to monitor health daily, and to observe strict biosafety and isolation protocols to prevent transmission of MAP between pens by any vectors, e.g. buckets, scoops for feed, personnel, etc. All protocols and the experimental design were approved by the University of Calgary Veterinary Sciences Animal Care Committee (protocol AC14-0168).

Study design

Calves were assigned to pens based on time of birth and entry into the research facility. The first 14 calves were designated to be inoculated animals (IN), with 2 calves in each of the 7 experimental pens. The next 14 calves to enter the barn were assigned as contact-exposed (CE) and individually housed temporarily in separate pens from the IN calves. The last 4 calves to enter the barn were designated as the control group, and placed together in the control pen. At 2 weeks of age, the IN calves in each pen were inoculated over 2 consecutive days. After 2 weeks (to allow the inoculum to pass through the calves), pens were relined with new liners and bedded with fresh shavings and straw. Calves designated as CE had to reach a minimum of 1 week of age with no health complications to ensure that they could drink from a bucket without assistance, and that only healthy calves were added to the study. When both CE calves entering the same pen reached a minimum 1 week of age, they were placed into the clean, re-lined experimental pen with the IN calves. Four calves (2 IN and 2 CE) were then group-housed for 3 months following the first day of group housing. The IN calves were euthanized and necropsied after 3 months of group housing. The CE calves were then individually housed in relined and clean pens for an additional 3 months. All 4 control calves were group housed (1 pen) for the entirety of the study.

Inoculum

The inoculum was a virulent MAP cattle type strain from a clinical JD case in Alberta (Cow 69) [16]. In short, a culture was prepared in 7H9/mycobactin/OADC liquid broth, from a first passage frozen stock and quantified using a combination of optical density (OD) at 600 nm, the wet weight method, and qPCR, as described [28]. Once culture grew to a concentration of 5 × 10⁸ CFU/mL, 1 mL aliquots were frozen at −80 °C until 1 week prior to inoculation. Before each inoculation, 1 tube was thawed and suspended in 50 mL 7H9 broth for 1 week, during which time inoculum was tested for contamination. 2.5 × 10⁸ CFU’s was quantified using the wet weight method, diluted in 20 mL of broth, placed in a 20-mL syringe and transported to the research facility. Calves were allowed to suckle the syringe containing the inoculum and it was expelled at the root of the tongue (on 2 consecutive days).

Fecal sampling and culture

Fecal samples were collected daily for 14 days following inoculation of IN calves to ensure viability of the inoculum, and monitor passive shedding. As of 14 days after inoculation, shedding was attributed to active MAP infection. For the remainder of the trial, fecal samples from each calf were collected three times/week during group housing for all calves. Following group housing, when calves were housed individually, fecal samples were collected weekly from CE calves for the remainder of the trial. Samples were stored at 4 °C until processing, which occurred within 7 days after collection.

All samples were processed using a modified TREK ESP II culture media (TREK para-JEM^®; TREK Diagnostic Systems, Cleveland, OH, USA) with subsequent F57-specific qPCR, as described [15]. Briefly, 2 g of fecal sample was thoroughly mixed with 30 mL of distilled water and left to settle for 30 min. Then, 5 mL of supernatant was transferred to 25 mL of a 0.9% hexadecylpyridinium chloride (HPC) half-strength brain heart infusion (BHI) solution for decontamination. Samples were then incubated for 24 h at 37 °C, followed by centrifugation at 3000 × g for 20 min, and the pellet re-suspended in a mixture of antibiotic solution (AS; para-Jem^®), water, and full strength BHI. Tubes were incubated again for 24 h at 37 °C and then 1 mL was added to liquid culture medium in TREK para-JEM^® culture bottles (TREK Diagnostic Systems, Cleveland, OH, USA) and incubated at 37 °C for 49 days.

Environmental sampling and culture

Environmental samples were collected once per week from each pen for the duration of the trial. Samples were collected from 5 locations within the pen, and mixed together, resulting in 1 composite sample from each pen. Samples were collected from the surface of the bed pack (individual piles of feces were avoided). Samples were stored at 4 °C until processing, and were subjected to the same protocol (described above) as fecal samples.

Necropsies and tissue cultures

The IN calves were euthanized after 3 months of group housing at 4 months of age by intravenous injection of barbiturate (Euthanyl Forte^®, DIN 00241326, Bimeda-MTC Animal Health Inc., Cambridge, ON, Canada), whereas CE were euthanized at 6 months of age, after an additional 3 months of individual housing. Control calves were euthanized last, after all other animals had exited the trial. Necropsies were performed immediately after euthanasia. No other ruminants were examined in the pathology room during necropsies, and the pathology room and tables were thoroughly cleaned and disinfected before and after each necropsy. Thirteen tissue samples were collected from each calf, including two sections of the duodenum, the ileum (including ileal-cecal valve), three sections of jejunum, and spleen. All associated lymph nodes with each gastrointestinal tract section were also collected, as well as the inguinal lymph nodes. Sample locations were marked and isolated with zip ties prior to collection (to prevent movement of intestinal contents). A new set of disinfected instruments and a new pair of gloves was used for collection of each new sample to prevent cross contamination, and PBS was used to rinse fecal content from intestinal tissues.

Samples were transported to the laboratory, and processed immediately on the same day using a modified version of a previous protocol [16]. Briefly, 2.5 g of tissue was dissociated using gentleMACS M tubes (Miltenyi Biotech Inc, Auburn, CA, USA) in 10 mL 0.5% triton x-100 PBS solution. Samples were then transferred to a falcon tube and centrifuged at 4700 × g for 15 min and the pellet re-suspended in 25 mL of 0.75% HPC, ½ strength BHI, 4-mm sterile glass beads and vortexed vigorously for 1–2 min. Samples were then incubated at 37 °C for 3 h, before centrifugation at 4700 × g for 15 min. The pellet was then re-suspended in 3 mL of antibiotic brew (paraJEM^®) and incubated overnight, and 1 mL added to paraJEM^® culture bottles and incubated at 37 °C for 49 days.

qPCR procedure

Following liquid culture of fecal and tissue samples, DNA was extracted as described [29]. A duplex qPCR targeting the MAP-specific F57 region and an internal amplification control (IAC) was performed, with primers, probes, and IAC sequences identical to those described [30]. Amplification conditions for qPCR were as follows: 50 °C for 2 min, 95 °C for 20 s to allow for initial denaturation, then 42 cycles of 95 °C for 30 s and 59 °C for 30 s. Samples were considered positive when the cycle threshold (CT) value was < 40.

Blood sampling, IFN-γ release assay and ELISA

Serum was collected for antibody testing following centrifugation and stored at −20 °C until antibody ELISA testing was performed, based on manufacturer’s directions (IDEXX Laboratories Inc.), with analysis as described [33]. Briefly, sample results were expressed as a proportion of the positive control corrected for the negative control (S/P ratio), and a ratio ≥ 60 was considered positive.

Data and statistical analyses

All statistical analyses were performed using STATA 11.2 (StataCorp LP, College Station, TX, USA). For all analyses, P < 0.05 was considered significant.

To define shedding events, isolated fecal culture-positive samples (sample collected week prior and subsequent week were negative), and groups of positive samples in which a positive sample was immediately followed by a subsequent positive fecal sample(s), were categorized as a single shedding event. Difference in mean number of fecal positive samples and shedding events, and length of shedding period between IN and CE calves was evaluated using a Student’s t test. The average length of events for IN and CE calves was calculated separately. Calves were also separated into fecal shedding categories based on the number of positive samples during group housing, where: 1 = calves with 0–4 positive fecal samples; 2 = calves with 5–9 positive fecal samples; 3 = calves 10–14 positive fecal samples; and 4 = calves with ≥ 15 positive fecal samples of all 38 samples collected during group housing.

The INF-γ results were dichotomized using a cutoff of 100% IFN-γ by calculating the average of presumed negative calves (control calves) + 1.96 the standard deviation [34]. All samples with a value of  % IFN-γ exceeding 100, immediately followed by a sample below 100% IFN-γ, were considered false-positive spikes and removed from analysis (28 samples were excluded).

Differences in fecal shedding category, tissue culture and IFN-γ results between IN and CE calves, as well as the association between having at least 1 positive IFN-γ sample and having at least 1 tissue-positive sample, were evaluated using a Fisher’s Exact test.

Tissue culture

All IN calves had at least 3 MAP-positive tissue cultures (range 3–11), whereas 7 (50%) of the CE calves had at least 1 MAP-positive tissue culture, but no more than 2 positive tissue samples (Table 1; Figure 1; p < 0.001). None of the control calves had positive tissue cultures.

Calf status	Fecal culturea				Tissue cultureb
	1	2	3	4	0	1	2	3
Inoculated (n = 14)	0	1	3	10	0	1	4	9
Contact-exposed (n = 14)	3	10	1	0	7	7	0	0
Control (n = 4)	4	0	0	0	4	0	0	0

^a1, calves with 0–4 fecal culture positive samples; 2, calves with 5–9 fecal culture-positive samples; 3, calves with 10–14 culture-positive samples; and 4, calves with ≥ 15 fecal culture-positive samples.

^b0, calves with 0 tissue culture-positive samples; 1, calves with 1–3 tissue culture-positive samples; 2, calves with 4–6 tissue culture-positive samples; and 3, calves with > 6 tissue culture-positive samples.

All tissue locations were positive in at least 2 IN calves. No location was MAP culture-positive in all calves; however, lymph nodes associated with the jejunum were most frequently MAP-positive, especially among tissue-positive CE calves (Figure 1).

Immune responses

For all calves, all samples were antibody ELISA-negative, except for 2 pre-infection samples of Calves 15 and 16 that tested positive on 1 occasion before testing negative for the remainder of the study, and this may be due to the transfer of maternal antibodies absorbed from colostrum intake.

All IN calves had at least 5 positive IFN-γ responses, whereas 5 (36%) CE calves had at least 1 positive IFN-γ response (Figure 2). Additionally, 1 control calf (C29) had 2 positive INF-γ samples at 2 consecutive time points (35 and 42 days after beginning of group housing) during the experimental trial.

The IN calves started to have positive INF-γ samples on average 55 days after inoculation (41 days after the start of group housing), with the earliest and latest being 33 and 73 days after inoculation, respectively. Eight (64%) of the IN calves had their first INF-γ response on or after 56 days following inoculation and the average interval after the start of group housing for the CE calves to have an INF-γ response was 45 days, with the earliest being the day of exposure, and the latest day 89 of group housing (p = 0.32); however, 3 (60%) of these CE calves had positive INF-γ response on or before 33 days after the start of group housing.

Among CE calves, the tissue sample outcome was not associated with the INF-γ result (p = 0.58).

Fecal shedding of MAP

No calves fecally shed MAP prior to exposure (inoculation for IN, and group housing for CE). Positive fecal samples were detected consistently in all IN animals for at least 7 days after inoculation (14 days before group housing), and as many as 10 days. The first positive fecal sample collected from a CE calf occurred 5 days after the start of group housing, whereas the latest first shedding event was detected 31 days after the start of group housing (Figure 2). All IN and CE calves had at least 2 positive fecal samples during the 3 months of group housing (Figure 2); however, all fecal samples from CE calves were negative after group housing ended and they were housed individually (Figure 2). Fecal samples from control calves were culture-negative for all time points, except for day 21 after the start of group housing at which time all calves had a positive sample (Figure 2), whereas Calf 29 also had 1 additional positive sample on day 56.

Mean number of shedding events was 5.6 (95% CI 4.6–6.7) and 4.1 (95% CI 3.4–4.9) in IN and CE calves, respectively (p = 0.02). Mean number of positive samples was 17.6 (95% CI 14.4–20.9) and 5.1 (95% CI 3.9–6.2) in IN and CE calves, respectively (p < 0.001). Thirteen (93%) of the IN calves were categorized into Groups 3 and 4, whereas 13 (93%) CE calves were categorized into Groups 1 and 2 (Table 1). Average length of a shedding event among the IN calves was 7.5 days, ranging from 2 to 54 days, whereas an average shedding event of CE calves lasted 2.9 days, ranging from 2 to 9 days (p = 0.001; Figure 2).

Environmental culture