Paratuberculosis progresses more quickly in young red deer than in sheep or cattle. This study describes the clinical, immunological and pathological changes over a 50-week period in fourteen 4-month-old red deer that received heavy oral challenge with
Paratuberculosis (Johne’s disease), caused by
Although early immune responses have been studied in cattle [
An experimental paratuberculosis challenge model for red deer has been developed to reproduce a typical range of disease outcomes from mild subclinical infection to clinical disease [
The two main objectives of this study were to monitor the clinical, immunological, and pathological changes over the course of a year and to collect biopsy samples of jejunal lymph node at three time points for future study of gene expression related to resistance/susceptibility MAP.
Seventeen red deer calves were born to 22 randomly chosen mixed-age seronegative hinds that had been inseminated with frozen semen from two unrelated red stags (7 to sire 1, 10 to sire 2) on a farm which had no previous history of clinical paratuberculosis and has been tested free of bovine tuberculosis for 30 years. These calves were weaned in late February when they were ~3 months of age. They were treated in early March with pour-on moxidectin (Cydectin; Fort Dodge Animal Health Ltd., Auckland, NZ), dosed with copper oxide wire particles (4 g Copper Capsule; Bayer NZ Ltd., Auckland, NZ), and vaccinated with Yersiniavax (AgVax Developments Ltd., Upper Hutt, NZ). This study was approved by the AgResearch Invermay Ethics Committee (AEC 11425).
In late March 2008, the 17 deer, which were all Paralisa test negative, were challenged with MAP using a standard infection model described previously [
The deer were grazed together at pasture throughout the 50-week study and their diet was supplemented with hay and barley over the winter. Their physical condition was monitored daily and they were weighed monthly in April, May, and June, two weekly in July and weekly from August to early March. The protocol required that any deer that developed early clinical signs of paratuberculosis (loss of 5–10% body weight over a two week period, loss of muscle mass, ± diarrhoea) would be euthanised.
At 4 and 12 weeks post challenge (pc) each deer was anaesthetized using intravenous fentanyl citrate, xylazine, and azaperone (Fentazin 5; Bomac Laboratories Ltd., Manukau), intubated and maintained on halothane (Halothane-Vet; Merial Ancare, Manukau) and oxygen. The animal was placed in dorsal recumbency and the abdomen was clipped, prepped, and draped. A 15 cm long midline laparotomy was performed and the gross appearance of the intestines, mesenteric lymph nodes, and mesenteries noted. A 40–50 mm piece of posterior JJLN was excised and cut into five pieces; three pieces were placed in cryotubes and immediately frozen in liquid nitrogen for future gene expression studies, one piece was fixed in 10% buffered formalin for histopathological examination, and one piece was cultured for MAP. The incision was closed with Maxon sutures (Davis & Geck, USA) in the linea alba and Vicryl sutures (Ethicon, USA) in the skin. The animals were injected with long-acting penicillin (Norocillin LA; Norbrook NZ Ltd., Rangiora) to prevent postoperative infection and with meloxicam (Metacam 20; Boeringer Ingelheim NZ Ltd., Auckland) for postoperative pain relief. The anaesthetic was reversed with yohimbine and naloxone (Contran H; Bomac Laboratories Ltd., Manukau). The deer were monitored closely for two weeks following each biopsy session.
All surviving deer were euthanised 50 weeks pc and necropsied and samples were taken from the anterior, mid, and posterior jejunum (JJ), ileocaecal valve (ICV), and associated lymph nodes and processed as for the biopsies. At biopsy and necropsy any gross lesions of paratuberculosis in the JJ, ICV, JJLN, ileocaecal lymph node (ICLN), and mesenteries were described and graded according to the following gross lesion scores: 0, no visible lesions (NVLs); 1, slightly enlarged JJLN/ICLN; 2, moderately enlarged JJLN/ICLN; 3, very enlarged JJLN/ICLN; 4, enlargement plus a single caseogranulomatous JJLN or ICLN lesion; and 5, multiple JJLN and ICLN lesions.
Blood samples were taken at regular intervals during the trial and tested with the Paralisa test and gamma interferon (IFN-
All tissue and faecal samples (5 g) were cultured for MAP using BACTEC 12B liquid culture medium containing egg yolk and mycobactin [
The JJLN biopsy samples taken at week 4 and week 12 pc were chilled and cultured within 48 hours of collection. At week 50 a pool of four intestinal samples (anterior, mid and posterior JJ and ICV) and a pool of four corresponding lymph node samples (JJLN and ICLN) from each animal were collected immediately after death, chilled overnight, and cultured the next day. Faecal samples collected from the rectum at week 50 pc were chilled overnight, processed using the double incubation method [
Sections of fixed samples from each animal were stained with H&E and Ziehl-Neelsen (ZN) and the histopathological lesion severity scores (LSSs) were assessed using a modified 7-point scale for both the mesenteric lymph nodes (MLN) and the enteric lesions. The week 50 MLN and enteric scores were added together for a total LSS score on a 14-point scale [
There were insufficient animals in this study for meaningful statistical analysis.
No animals showed signs of clinical paratuberculosis and the deer all gained weight throughout the study, with hinds averaging 35 kg and stags 56 kg weight gain over the 50 week study, and there was no significant difference in weight gain between Sire 1 and Sire 2 offspring. Three deer died of misadventure unrelated to paratuberculosis.
There were no gross lesions apparent in the JJLN at week 4, but by week 12 half the deer had slightly or moderately enlarged JJLN (Table
Gross lesion score (Weeks 12 and 50), histopathological lesion severity scores (LSSs) for mesenteric lymph node (MLN), enteric (ENT) and total, and description of acid fast organisms (AFO) in the lesions as none, paucibacillary (PB), or multibacillary (MB) for samples biopsied at weeks 4 and 12 and taken at week 50 necropsy of the offspring of two unrelated sires.
Sire | Tag | Week 4 | Week 12 | Week 50 | |||||||
Sex | MLN LSS | Gross | MLN LSS | AFO | Gross | MLN LSS | ENT LSS | Total LSS | AFO | ||
1 | 506 | F | 2 | 0 | 5 | PB | 0 | 2 | 0 | 2 | None |
1 | 511 | F | 0 | 0 | 4 | PB | 0 | 2 | 2 | 4 | PB |
1 | 513 | M | 0 | 2 | 7 | MB | 0 | 4 | 4 | 8 | PB |
1 | 508 | F | 0 | 2 | 7 | MB | 4 | 6 | 6 | 12 | PB |
1 | 507 | M | 0 | 2 | 7 | MB | 5 | 7 | 5 | 12 | MB |
2 | 514 | M | 0 | 2 | 7 | PB | 4 | 4 | 2 | 6 | PB |
2 | 504 | F | 0 | 1 | 7 | PB | 0 | 3 | 4 | 7 | PB |
2 | 509 | M | 0 | 0 | 5 | PB | 0 | 4 | 4 | 8 | PB |
2 | 502 | M | 0 | 2 | 7 | PB | 3 | 5 | 4 | 9 | PB |
2 | 501 | M | 0 | 0 | 7 | MB | 0 | 5 | 5 | 10 | PB |
2 | 500 | F | 0 | 0 | 5 | MB | 0 | 4 | 6 | 10 | PB |
2 | 512 | M | 0 | 0 | 5 | MB | 1 | 5 | 6 | 11 | PB |
2 | 510 | M | 2 | 2 | 7 | MB | 1 | 5 | 6 | 11 | PB |
2 | 517 | F | 0 | 0 | 5 | MB | 0 | 6 | 6 | 12 | PB |
Gross lesion scores: 0, no visible lesions (NVLs); 1, slightly enlarged jejunal lymph nodes (JJLNs) and/or ileocaecal lymph nodes (ICLNs); 2, moderately enlarged JJLN/ICLN; 3, very enlarged JJLN/ICLN; 4, enlargement plus a single caseogranulomatous JJLN or ICLN lesion; 5, multiple JJLN and ICLN lesions.
MLN and ENT LSSs of 1 and 2 were regarded as very mild nonspecific, 3 as suggestive of very mild paratuberculosis, 4 as mild, 5 as moderate, 6 as moderately severe and 7 as severe paratuberculosis. Lesions were also described as no AFO, paucibacillary (PB), or multibacillary (MB).
There were no specific lesions seen at Week 4, but by Week 12 MLN LSSs ranged from mild to severe (4–7) (Table
Over the period from Week 12 to Week 50 the following changes in MLN LSS occurred; 11 of 14 reduced, one increased and two remained the same. Of those that became less severe, one went from severe to very mild, two from severe to mild, three from severe to moderate, one from severe to moderately severe, one from moderate to mild/nonspecific, two from moderate to mild, and one mild went to nonspecific. The one that increased in severity went from mild to moderate. One severe and one moderately severe remained the same. At Week 50, MLN and enteric LSS generally had similar scores.
Interestingly, an increased concentration of AFO in calcified tissue present in two of the animals at Week 50 was associated with PB, as per criteria described by Clark et al. in 2010 [
There was no apparent difference in LSS, the changes in LSS with time or PB/MB ratio between offspring of the two sires throughout the study.
MAP was isolated from all JJLN biopsies at weeks 4 and 12 and from pooled LNs at Week 50 of all 14 deer (Table
Summary of BACTEC culture results in terms of the number of days to positive (dtp) for jejunal lymph node (JJLN) samples taken at Weeks 4 and 12, and for a pool of three samples of JJLN plus ileocaecal lymph node (ICLN) and a pool of three jejunum and ileo-caecal valve samples (JJ + ICV) and faecal samples (FSs) at week 50, compared with MLN lesion severity scores (LSSs) at each time point, with the animals sorted by total LSS at week 50 and designated as least or most affected.
Tag | Sire | Week 4 | Week 12 | Week 50 | ||||||
MLN LSS | JJLN | MLN LSS | JJLN | MLN LSS | JJLN + ICLN | JJ + ICV | FS | Affected | ||
506 | 1 | 2 | 36 | 5 | 21 | 2 | 34 | 34 | Neg | Least |
511 | 1 | 0 | 36 | 4 | 21 | 2 | 34 | 34 | Neg | Least |
509 | 2 | 0 | 36 | 5 | 15 | 4 | 26 | 26 | Neg | Least |
Mean | 5 | |||||||||
514 | 2 | 0 | 28 | 7 | 15 | 4 | 34 | 26 | Neg | |
504 | 2 | 0 | 32 | 7 | 15 | 3 | 26 | 21 | Neg | |
513 | 1 | 0 | 32 | 7 | 15 | 4 | 18 | 18 | 31 | |
502 | 2 | 0 | 36 | 7 | 12 | 5 | 18 | 26 | Neg | |
501 | 2 | 0 | 42 | 7 | 12 | 5 | 18 | 34 | 37 | |
500 | 2 | 0 | 35 | 5 | 12 | 4 | 21 | 18 | Neg | |
510 | 2 | 2 | 36 | 7 | 12 | 5 | 18 | 26 | Neg | |
512 | 2 | 0 | 36 | 5 | 12 | 5 | 18 | 34 | 31 | |
Mean | ||||||||||
517 | 2 | 0 | 36 | 5 | 12 | 6 | 18 | 26 | Neg | Most |
508 | 1 | 0 | 32 | 7 | 15 | 6 | 18 | 26 | Neg | Most |
507 | 1 | 0 | 36 | 7 | 12 | 7 | 18 | 18 | 37 | Most |
Mean | 7 |
Over the course of the 50 week study, four of the animals (506, 509, 511, and 512) had low titres to Johnin throughout and all peaked at <60 EU (Figure
(a) Paralisa titres (PPA antigen) in ELISA units (EU) over the 50 week study for the four deer (506, 509, 511, 512) that had the lowest serological response to the Paralisa Johnin antigen (black lines), and the four deer (507, 508, 510, 513) that had the highest and most sustained serological response to Johnin (gray lines). (b) Paralisa titres (PPA antigen) in ELISA units (EU) over the 50 week study for the six deer that had an intermediate serological response to the Paralisa Johnin antigen.
The IFN
Mean gamma interferon (IFN-
The increase in skin thickness at the avian and bovine sites averaged 9.7 mm (1.8–13.4) and 3.1 mm (0.6–8.4), respectively, with
The red deer MAP infection model allowed a number of variables to be controlled so that their immune response to challenge could be studied efficiently. The offspring were all the same age, having been bred on the same day by artificial insemination of synchronised hinds, which calved within a few days of each other. The animals were all run together in one mob from birth, treated regularly with anthelmintics, vaccinated against yersiniosis, treated with mineral supplements, grazed at pasture, and given the same infective dose of MAP on the same days. The only common variable that was not controlled was gender, but there was no obvious gender bias in the results. In previous studies a proportion of young red deer challenged by MAP have developed clinical disease [
The use of the infection model together with the sampling of jejunal lymph nodes at 4, 12, and 50 weeks pc and periodic serology and IFN-
There have been a small number of studies in sheep and cattle that have used surgical biopsies of small intestine and mesenteric lymph nodes to diagnose paratuberculosis [
An interesting outcome of this study was the spectrum of changes in disease severity that took place over the course of a year. At Week 4 MAP was cultured from the JJLN of all animals but there were no paratuberculosis-specific lesions seen on histology. Between Weeks 4 and 12 all the deer showed an increase in MAP numbers and they developed JJLN lesions ranging from mild to severe. However, MAP numbers increased more in the animals that went on to become more severely affected, suggesting that differences in relative resistance/susceptibility were influencing the development of lesions and rate of MAP multiplication. Between Week 12 and Week 50, some animals remained mildly affected, some remained severely affected, while some improved and others got worse. About half the animals managed to suppress the multiplication of MAP and limit the extent of disease, such that the LSS and the number of MAP in the JJLN declined, as indicated by an increase in dtp. In contrast, some of the animals appeared to be unable to suppress MAP multiplication, their LSS worsened, and number of MAP present increased or stayed the same over this 9-month period. Two animals that were MB at Week 12 were found to be PB at Week 50 but had a few foci of high numbers of AFO in mineralized tissue. This type of lesion has been observed previously in naturally infected animals [
The most severely affected animals also had the highest antibody titres, while the two least affected animals remained seronegative throughout the study and had higher earlier IFN-
Similar observations have recently been reported in naturally infected sheep that were monitored over a 36-month period using lymph node and intestine biopsies [
The longitudinal changes in lesion severity and number of MAP in this study are similar although not as extreme as those seen in a subsequent study undertaken in 2009, which also showed significant heritable resistance/susceptibility to experimental paratuberculosis challenge in the offspring of two sires that had displayed differences in resistance/susceptibility to paratuberculosis [
The longitudinal monitoring of histopathology and culture of the jejunal lymph nodes, as well as serology and IFN-
The authors wish to acknowledge the assistance of staff at AgResearch Invermay, the Disease Research Laboratory, Otago University, and the Wallaceville Tb Laboratory for field work and laboratory work in carrying out this study. The study was funded by the Johne’s Disease Research Consortium.