Knowledge gaps that hamper prevention and control of Mycobacterium avium subspecies paratuberculosis infection.

In the last decades, many regional and country-wide control programmes for Johne's disease (JD) were developed due to associated economic losses, or because of a possible association with Crohn's disease. These control programmes were often not successful, partly because management protocols were not followed, including the introduction of infected replacement cattle, because tests to identify infected animals were unreliable, and uptake by farmers was not high enough because of a perceived low return on investment. In the absence of a cure or effective commercial vaccines, control of JD is currently primarily based on herd management strategies to avoid infection of cattle and restrict within-farm and farm-to-farm transmission. Although JD control programmes have been implemented in most developed countries, lessons learned from JD prevention and control programmes are underreported. Also, JD control programmes are typically evaluated in a limited number of herds and the duration of the study is less than 5 year, making it difficult to adequately assess the efficacy of control programmes. In this manuscript, we identify the most important gaps in knowledge hampering JD prevention and control programmes, including vaccination and diagnostics. Secondly, we discuss directions that research should take to address those knowledge gaps.

SOLiD SAGE sequencing shows differential gene expression in jejunal lymph node samples of resistant and susceptible red deer (Cervus elaphus) challenged with Mycobacterium avium subsp. paratuberculosis.

This study compared in vivo lymph node gene expression levels between six young red deer that were either relatively resistant (R) or susceptible (S) to paratuberculosis following experimental challenge with Mycobacterium avium subsp. paratuberculosis. Intestinal lymph nodes were biopsied at 4, 12 and 50 weeks post challenge (pc) and parallel changes in histopathology, immunology and bacterial load monitored. SOLiD SAGE (serial analysis of gene expression) next generation sequencing of biopsied lymph node samples generated a total of 373 million transcript tags 26-28bp in length after filtering. A total of 36,632 unique transcripts were identified and 14,325 of these were able to be annotated. The copy number of each transcript was counted, averaged and compared for R and S animals (R-S). P values and False Discovery Rates (FDR) were calculated for each transcript. Genes differentially upregulated ≥2 fold (FDR<0.5) totalled 9, 40 and 32 in R animals (+ values) and 23, 164 and 47 in S animals (- values) at weeks 4, 12, and 50pc, respectively. Transcripts displaying greatest differential expression between R and S animals at each time point were IFIT2 (189 fold) and S100A8 (-32.7 fold) at week 4, LRR1 (52.7 fold), SERPINF2 (-214.6 fold) at week 12 and CEACAM8 (84.6 fold), and STK31 (-129.5 fold) at week 50, respectively. All 9 genes significantly upregulated at week 4 in R animals relate specifically to host defence and all involve Type I interferon stimulated genes. By contrast genes upregulated in S animals at week 4, relate predominantly to inflammation, but also involve adaptive immune responses, mitochondrial function and apoptosis regulation. At week 12, the genes differentially upregulated in R animals are linked predominantly to regulation of adaptive immunity and mucosal immunity, while many of the genes in S animals are associated with pro-inflammatory interleukins involved with innate and adaptive immunity. These correlated with greater lesion severity and higher MAP numbers in lymph nodes of S animals. By week 50 the number of upregulated genes declined in both groups. A number of genes upregulated in R animals appear to be associated with host resistance and regulation of adaptive immunity, especially CEACAM8. Genes upregulated in S animals involve antigen presentation (ENDOD1) and gut associated immune pathology (HSH2D). In conclusion, gene expression in jejunal lymph nodes of resistant and susceptible deer infer that the resistant phenotype is associated with pathways of adaptive immunity, while susceptibility is linked with upregulated non-protective pro-inflammatory responses, following experimental MAP infection.

Ovine rumen papillae biopsy via oral endoscopy; a rapid and repeatable method for serial sampling.

AIMS: To explore and validate the utility of rumen endoscopy for collection of rumen papillae for gene expression measurement. METHODS: Four adult Coopworth ewes were fasted for either 4 or 24 hours. Animals were sedated, placed in a dorsally recumbent position at 45 degrees with the head upright, and an endoscope inserted via a tube inserted into the mouth. Biopsies of rumen papillae were taken from the ventral surface of the rumen atrium under visual guidance. Two biopsies were collected from one of the animals that had been fasted for 4 hours, and three from one of the animals that had been fasted for 24 hours. Video of the rumen atrium and reticulum was also collected. The animals recovered uneventfully. Biopsies were subsequently used for extraction and sequencing of mRNA. RESULTS: The ventral surface of the rumen atrium was accessible after 4 hours off pasture, but a larger region was accessible after 24 hours of fasting. Sedation allowed access for endoscope use for around 5 to 10 minutes after which increased saliva flow was noted. Rumen papillae biopsies were easily collected, with samples from a variety of sites collected in the ∼10 minute time window. High quality RNA was obtained for stranded mRNA sequencing. Of the resulting reads, 69-70% mapped uniquely to version 3.1 of the ovine genome, and 48-49% to a known gene. The rumen mRNA profiles were consistent with a previously reported study. CONCLUSIONS: This method for obtaining rumenal tissue was found to be rapid and resulted in no apparent short or long term effects on the animal. High quality RNA was successfully extracted and amplified from the rumen papillae biopsies, indicating that this technique could be used for future gene expression studies. The use of rumen endoscopy could be extended to collection of a variety of rumen and reticulum anatomical measurements and deposition and retrieval of small sensors from the rumen. Rumen endoscopy offers an attractive and cost effective approach to repeated rumen biopsies compared with serial slaughter or use of cannulated animals.

Observations on the phenotypic relationships between anti-CarLA salivary IgA antibody response, nematode infection levels and growth rates in farmed red (Cervus elaphus) and wapiti hybrid deer (Cervus elaphus canadensis).

Nematode parasites are one of the most significant production limiting factors in farmed deer in New Zealand. One long term strategy to reduce reliance on anthelmintics is to select deer that develop resistance to parasites. It has been shown in sheep that secretory antibody (IgA) in the saliva against a Carbohydrate Larval Antigen (CarLA) on infective larvae (L3) of a wide range of gastro-intestinal nematodes protects against reinfection. This paper describes a longitudinal slaughter study undertaken to measure anti-CarLA IgA antibody (CarLA-IgA) levels in the saliva of 5-12 month old farmed red and wapiti-cross-red deer (wapx) grazed together and to attempt to relate these levels to parasite burdens and productivity. The study showed that salivary CarLA-IgA levels peaked in June (late autumn) and October (mid spring), but the levels in wapx deer were significantly lower than in red deer. Over the May-December period 55% of red deer had CarLA-IgA values ≥2 units compared with 26% of wapx deer and over this period red deer had consistently lower adult abomasal parasite burdens than wapx deer. The average number of adult abomasal nematodes was significantly lower at each slaughter from May to December for all deer with CarLA-IgA ≥2 units vs <2 units. There were no demonstrable correlations with liveweight gain in these small groups of deer.

Reduced efficacy of moxidectin and abamectin in young red deer (Cervus elaphus) after 20 years of moxidectin pour-on use on a New Zealand deer farm.

A study was undertaken on weaned 4-5 month old farmed red deer to test the efficacy of moxidectin and abamectin anthelmintics, given by three different routes of administration, compared with an untreated control. Faecal samples were collected on days 0, 7 and 14 for a faecal egg count reduction test (FECRT), blood samples were collected on days 0, 0.5, 1, 2, 3, 5, 7, 10 and 14 for pharmacokinetics, and the deer were killed on days 14 or 15 for total nematode count. The control group averaged 1264 adult Ostertagia-type nematode parasite species and treatment efficacy was 77.4% for moxidectin injection, 26% for oral moxidectin and 27.6% for pour-on moxidectin, while the treatment efficacy was 72.4% for abamectin injection, 70.1% for oral abamectin (Hi-Mineral) and 34.1% for pour-on abamectin. Both moxidectin and abamectin injections were significantly more efficacious than their equivalent pour-ons. There was a significant difference in efficacy between oral abamectin (Hi-Mineral) and oral moxidectin (P<0.01). The control group averaged 2956 adult lungworm (Dictyocaulus eckerti) and 50 Oesophagostomum venulosum in the large intestine and treatment efficacy against these nematodes was 100% for all treatments. There were negligible numbers of other gastro-intestinal nematodes. At slaughter, there was a significant correlation (P=0.02) between FEC and Ostertagia-type nematodes in the untreated controls. Relatively few eggs were found in faeces from treated animals at 7 and 14 days post-treatment despite significant worm burdens in all six treatment groups, suggesting egg-laying suppression in resistant nematodes, and all three different FECRT calculations tended to overestimate the efficacy of the treatments compared with actual nematode counts. Peak plasma concentrations (Cmax) for both actives were measured 12h after treatment for injection and oral and at 5 days for pour-on. Cmax (ng/ml) for moxidectin injection, oral and pour-on were 71.8, 8.3 and 0.4, respectively, and for abamectin injection, oral and pour-on were 62.1, 30.3 and 10.0, respectively. Area under the curve (AUC) estimates for moxidectin injection, oral and pour-on were 106.6, 12.9 and 6.1, respectively, and for abamectin injection, oral and pour-on were 162.7, 57.5 and 74.3, respectively. The results demonstrate that significant anthelmintic resistance to moxidectin and abamectin is present on this deer farm. However, the injection was the most effective route of administration in young deer for both anthelmintics, although <80% efficacious. We conclude that the FECRT is unreliable in deer when anthelmintic resistance is present.

A randomised controlled trial of Silirum vaccine for control of paratuberculosis in farmed red deer.

A randomised controlled trial to assess the efficacy of Silirum vaccine in control of paratuberculosis in young farmed deer was carried out in 2008-2009 in six New Zealand herds with a history of clinical disease. Vaccination with Silirum was carried out in four-month-old deer, and vaccinates (n=1671) and controls (n=1664) were weighed at vaccination and at 8 and 12 months old, when faecal samples were collected from 125 vaccinates and 123 controls on five farms. Deer were slaughtered between 11 and 20 months of age, and the incidence of gross visceral lymph node (VLN) pathology typical of paratuberculosis in deer, that is, enlarged and/or granulomatous VLN, was recorded. Clinical disease was confirmed in 18 controls and seven vaccinates, representing a vaccine efficacy estimate of 60 per cent (95% CI 3 per cent to 83 per cent, P=0.04). Forty-seven percent (95% CI 38 per cent to 56 per cent) of faecal samples from vaccinates and 55 per cent (95% CI 46 per cent to 64 per cent) from controls were Mycobacterium avium subspecies paratuberculosis positive (P=0.5). Average daily liveweight gain did not differ between the cohorts. At slaughter, 1.4 per cent of vaccinates and 4.5 per cent of controls had VLN pathology, RR=0.32 (95% CI 0.19 to 0.54, P<0.001). These data indicate that vaccination with Silirum may be useful as an aid to control losses associated with clinical paratuberculosis in young deer.

Gastrointestinal tract development in red deer (Cervus elaphus) calves from 1 to 12 months of age.

This study provides a detailed description of the development of the gastrointestinal tract (GIT) of farmed red deer (Cervus elaphus) calves over the first 12 months of age. GIT development was measured using a combination of computerised tomography (CT) scanning and traditional slaughter plus dissection techniques. Red deer calves of a known birth date were randomly assigned to two treatment groups. A group of five animals were repeatedly CT scanned at 31, 63, 92, 135, 207, 275 and 351 days of age to identify GIT organs and determine their volume. From a group of 20 animals, subsets of four individuals were also scanned at corresponding ages (except 135 days of age). They were immediately euthanised and dissected after CT scanning to compare CT-scanned results with actual anatomical measurements. Individual organ weights were compared with their respective organ volumes determined by CT scanning and were found to have a strong, positive relationship. The combined rumen and reticulum (RR) CT-scanned volume was compared with its volume determined by the water-displacement technique and this also showed good correlation between the two techniques (R = 0.92). The allometric growth rates of organs, relative to animal live weight gains, in descending order, were the rumen, omasum, reticulum, abomasum, caecum blind sac, kidneys, spleen and liver. The red deer GIT was continuing to grow and develop when the last measurement was taken at 351 days of age. The greatest growth of the RR, when expressed in terms of empty weight, was between 31 and 92 days of age. Compared with sheep and cattle, it appears that the red deer have a similar or greater rate of RR development up until approximately 60 to 90 days of age; however, the final increments of GIT maturity in deer may take longer to complete, with the empty weight of the RR gaining 7.5 g/day between 275 and 351 days of age. CT scanning was validated in this study as a viable technique to follow GIT development in the same animals over time, and it provided novel information on allometric organ growth. The success of CT scanning highlights the potential future use of diagnostic imaging for GIT development studies.

Prevalence of Mycobacterium avium subsp. paratuberculosis in farmed red deer (Cervus elaphus) with grossly normal mesenteric lymph nodes.

AIM: To estimate the prevalence of Mycobacterium avium subsp. paratuberculosis (Map) in farmed deer with no gross post-mortem evidence of Map infection slaughtered in New Zealand, and to assess predictors of infection. METHODS: Mesenteric lymph node (MLN) samples (n = 251) were collected from 60 lines of deer presented at two slaughterhouses in the North and two in the South Island of New Zealand between October 2008 and January 2009 and cultured for Map. Estimates of individual animal prevalence for each island were adjusted to account for the clustering of individual observations within herds. The national herd prevalence estimate was calculated as a weighted mean, with weights being the proportion of herds from which deer were slaughtered at North and South Island slaughterhouses among all herds slaughtering deer throughout New Zealand. Age, gender, and the presence of other carcasses with enlarged and/or granulomatous MLN in the same line (line status) were assessed as predictors of infection using multivariable logistic regression. RESULTS: A national cluster-adjusted individual animal prevalence of 45 (95% CI = 30-60)% was estimated, with North and South Island prevalences of 29 (95% CI = 16-45)% and 51 (95% CI = 36-66)%, respectively. Line status was a strong predictor of infection in young deer (OR 7.1, 95% CI = 2.4-21.5), but not in older deer. Herd-level prevalence was 44 (95% CI = 24-64)% in the North Island and 67 (95% CI = 49-85)% in the South Island. Weighted adjustment resulted in a national herd-level prevalence estimate of 59 (95% CI = 41-78)%. CONCLUSIONS: This study has provided a national baseline prevalence estimate for Map infection at the individual and herd-level, showing a contrast between the North and South Islands. More research to investigate the factors contributing to the difference in infection prevalence seen between the islands may help to identify suitable control measures for Map in deer herds.

Association between Mycobacterium avium subspecies paratuberculosis and lymph node size in New Zealand farmed deer (Cervus elaphus).

AIMS: To develop and validate criteria for identification of abnormal lymph nodes (LN) at commercial slaughter, for the purpose of national surveillance for Mycobacterium avium subspecies paratuberculosis (Map) in New Zealand farmed deer. This included estimation of the predictive value of abnormal LN for Map infection; a standard circumference cut-point for measurement of abnormal LN; and identification of risk factors associated with increasing LN circumference. METHODS: In Study 1, official assessors sampled 129 LN with macroscopically visible abnormalities (abnormal LN) from 76 deer herds between May and November 2007. LN samples were cultured for Map, with culture-negative LN further examined for typical histopathological changes. The predictive value of abnormal LN for Map infection was calculated and significance of herd location (North or South Island) assessed. In Study 2, the circumferences of 412 mesenteric LN (MLN) from 79 herds were measured between October 2007 and January 2009, with samples cultured for Map and examined for eight histopathological features. The minimum circumference of an abnormal MLN was defined, based on an arbitrary >95% specificity of a culture-positive Map diagnosis. Associations between the predictor variables Map culture status, carcase weight, animal age and gender, and histopathological features, and increasing MLN circumference were assessed using a linear mixed-effects model. RESULTS: Based solely on culture, the predictive value of abnormal LN for Map infection was 92.2 (95% CI: 86.2-96.2)% with no difference between the North and South Islands (p = 0.09). Inclusion of three culture-negative LN with histopathological changes typical of Map infection increased the predictive value estimate to 94.6 (95% CI: 89.2-97.3)%. The minimum circumference of an abnormal MLN was defined as 55 mm, with a sensitivity of Map detection at this cut-point of approximately 12%. Increasing MLN circumference was positively associated with the presence of moderate follicular hyperplasia (p < 0.01), focal granulomas (p < 0.01) and a synergistic interaction between focal granulomas and Map status (p = 0.03). CONCLUSIONS: Deer MLN with macroscopically visible abnormalities and/or a circumference of >55 mm have >95% likelihood of Map infection. However, sensitivity of Map diagnosis in MLN with circumference of >55 mm was 12%, indicating use of abnormal LN as a sole criterion in national surveillance for Map in slaughtered deer will underestimate animal-level prevalence.

Sensitivity, specificity and level of agreement of meat inspector detection of abnormal lymph nodes of farmed deer (Cervus elaphus) in New Zealand.

AIMS: To assess the sensitivity (Se), specificity (Sp) and level of agreement in the detection of abnormal deer mesenteric lymph nodes (MLN) by official assessors (meat inspectors) for the purpose of slaughter premise surveillance for Johne's disease in New Zealand farmed deer. METHODS: To determine Se and Sp, four meat inspectors in two commercial deer slaughter premises (DSP) each examined between 153 and 925 MLN under normal visual examination, palpation and/or incision inspection procedures. Abnormal MLN were independently defined as those with a circumference measurement of  ≥ 55 mm and/or grossly visible pathological changes such as caseation, necrosis and/or mineralisation, while those with a circumference of <55 mm and without gross lesions were defined as normal. The inspectors' diagnosis of normal or abnormal was retrieved from the national DSP-based surveillance database for Johne's disease in deer. To determine between-inspector level of agreement, 54 meat inspectors visually examined two photographic images each of 19 normal and 10 abnormal MLN, and recorded their diagnosis. Between-inspector agreement was calculated based on a modification of Cohen's kappa statistic for multiple raters. The influence of covariates such as inspector age and experience on the between-inspector level of agreement were assessed using the Breslow-Day statistic and test for equal kappa coefficients. RESULTS: The weighted average Se and Sp of inspector detection of abnormal MLN was 13.3 (minimum 4.8, maximum 41.2)% and 99.9 (minimum 99.5, maximum 100.0)%, respectively. The level of between-inspector agreement in the diagnosis of abnormal and normal MLN was fair (κ = 0.32). Employment location, inspector age, experience inspecting deer or other species, and the number of shifts inspecting deer within the previous four weeks had no influence on between-inspector agreement (p > 0.10). CONCLUSIONS: Inspectors diagnosed abnormal deer MLN with a high specificity, but low sensitivity. These data supported that visual assessment of MLN characteristics was suitable for national surveillance for paratuberculosis, while highlighting the need for further emphasis on training of inspectors in abnormal lymph node detection.

Effect of vaccination and natural infection with Mycobacterium avium subsp. paratuberculosis on specificity of diagnostic tests for bovine tuberculosis in farmed red deer (Cervus elaphus).

AIM: To assess the effect of vaccination against paratuberculosis (Johne's disease) on reactivity to diagnostic tests for bovine tuberculosis (Tb) in deer exposed to natural challenge with Mycobacterium avium subsp. paratuberculosis (Map), and to investigate Map infection as a factor in Tb test cross-reactivity at the herd level. METHODS: In Study 1, yearling deer (n=180 vaccinates and n=181 controls) were randomly selected from three commercial deer herds participating in a trial of a commercial vaccine against paratuberculosis. The deer were subjected to the comparative cervical skin test (CCT) for Tb at 44 weeks post-vaccination. Interpretation as a mid-cervical tuberculin skin test (MCT) was also recorded. Serum from deer positive to the CCT was collected 3-4 weeks after tuberculin injection and tested using the ELISA Tb test (ETB), with both standard and the modified-ETB interpretations. In Study 2, 102 herds were categorised as infected or uninfected with Map based on results of pooled faecal culture, and positive to MCT if one or more deer gave a positive reaction to the MCT following testing in 2005. Information on other potential risk factors for MCT reactivity was collected using a standardised questionnaire conducted on each farm. The data were analysed using a multivariable logistic regression model. RESULTS: In Study 1, 79/180 (44%) vaccinates and 42/181 (23%) controls were positive to the MCT (p<0.001). Two vaccinates (1.1%) that were CCT-positive were both positive to the standard ETB and negative to the modified ETB. One of three CCT-positive controls was negative to the standard ETB, and the other two were positive; both controls were modified ETB-positive. In Study 2, significantly more MCT-positive (41/58; 71%) than MCT-negative (18/44; 41%) herds were infected with Map (p=0.003). The OR for a positive MCT for herds infected with Map was 3.1 (95% CI=1.3-7.5), compared with uninfected herds. Herd size was positively associated with a positive MCT result (p=0.004). CONCLUSIONS: Infection with Map and vaccination increased the risk of non-specificity of the MCT in deer herds. The CCT and ancillary testing of CCT-positive animals using the modified ETB are effective tools to address the reduced specificity of the MCT. However, where use of these tests is not permitted, non-specificity related to infection and vaccination will be more difficult to resolve.

Immunological and pathological responses of red deer resistant or susceptible genotypes, to experimental challenge with Mycobacterium avium subsp. paratuberculosis.

This study aimed to monitor the clinical, immunological and pathological changes in red deer for 49 weeks after experimental oral challenge with Mycobacterium avium subsp. paratuberculosis (MAP) and to assess the heritability of resistance in the offspring of two red stags. Eighteen young deer, which were bred from unselected hinds and sired by two stags resistant (R) or susceptible (S) to paratuberculosis, were challenged with MAP and monitored for 49 weeks. Biopsy samples of the jejunal lymph node were collected at Weeks 4 and 13 and at necropsy after euthanasia of clinically affected animals or when electively killed at Week 49. Three animals (two S and one R) developed clinical disease and were euthanised. The nine S offspring had significantly more severe lesions than the nine R offspring (Mantel-Haenszel Chi-square P=0.017). The average Lesion Severity Score (LSS) of R offspring was 5.9 (mild), and 7/9 had no or very mild lesions. In contrast, the LSS of S offspring averaged 11.7 (severe), and 7/9 had severe lesions. Most of the resistant, but not the susceptible, animals showed evidence of resolving lesions and a reduction in the number of MAP between 13 and 49 weeks after challenge. One R offspring appeared to completely cure itself, and progressed from mild culture-positive paratuberculosis lesions at Week 13 to having no signs of disease or infection 36 weeks later. This study showed significant heritable resistance/susceptibility to paratuberculosis and key differences in immunological responses in the first 3 months after challenge, indicating different paths to relative success or failure to control MAP. In general, R deer had higher IFN-γ levels, low antibody titres and fewer MAP, while S deer had lower IFN-γ levels, higher antibody and more MAP.

Immunoregulatory cytokines are associated with protection from immunopathology following Mycobacterium avium subspecies paratuberculosis infection in red deer.

Although the causative agent of Johne's disease, Mycobacterium avium subsp. paratuberculosis, is well known, the etiology of disease and the immune responses generated in response to infection are still poorly understood. Knowledge of definitive markers of protective immunity, infection, and the establishment of chronic granulomatous Johne's disease is necessary to advance vaccine and diagnostic development. We sought to profile the immune responses occurring within jejunal lymph nodes of experimentally challenged red deer (Cervus elaphus). Quantitative PCR was utilized to measure a range of cytokines, signaling molecules, and transcription factors involved in Th1, Th2, Treg, and Th17 immune responses. Significant differences in gene expression were observed between control, minimally diseased, and severely diseased animals, with severely diseased animals showing elevated proinflammatory transcripts and reduced anti-inflammatory transcripts. We identified a proinflammatory cytokine milieu of gamma interferon, interleukin-1α (IL-1α), and IL-17, which may contribute to the immunopathology observed during clinical Johne's disease and suggest that Th2 and Treg immune responses may play an important role in controlling the development of immunopathology in infected animals.

Histopathology of grossly normal mesenteric lymph nodes of New Zealand farmed red deer (Cervus elaphus) including identification of lipopigment.

This article describes the histopathology of grossly normal mesenteric lymph nodes (MLNs) of New Zealand farmed red deer (Cervus elaphus). Eighty MLNs were sourced from 10 deer from 5 North Island herds and 5 South Island herds classified as low risk and high risk of Mycobacterium avium subspecies paratuberculosis (MAP) infection, respectively. Fixed sections were stained with hematoxylin and eosin; Ziehl-Neelsen; and, selectively, periodic acid-Schiff, Perl's, and Sudan black. Positive Ziehl-Neelsen stain, follicular hyperplasia, capsular eosinophil infiltration, focal granulomas, foci of macrophages containing lipopigment, parasitic granulomas, and calcified foci are described and severity graded where appropriate. Animal age, sex, and herd of origin are variably associated with the presence of one or more features. Trabecular fibrosis and dilated edema-filled sinusoids are described. These observations allow differentiation between likely nonpathologic histologic features in deer MLNs and features possibly attributable to infection with a pathogen such as MAP.

Regional seroprevalence of leptospirosis on deer farms in New Zealand.

AIM: To undertake a seroprevalence survey to estimate the distribution of infection by Leptospira spp. serovars Hardjobovis, Pomona and Copenhageni in farmed deer herds regionally throughout New Zealand. METHODS: Between March 2003 and February 2005, 111 red and red x wapiti deer herds not using a leptospiral vaccine were selected from nine regions from Northland to Southland. Eighty-three herds were selected for on-farm collection of 12-20 blood samples from hinds predominantly 9-30 months of age, by jugular venepuncture, while similar numbers, predominantly males 9-30 months of age, per farm were collected from 28 herds at sticking at deer slaughter premises (DSP). Serum was stored frozen until analysed using the microscopic agglutination test (MAT). Titre cut-points of 1:24 for serovar Hardjobovis and 1:48 for serovars Pomona and Copenhageni were considered positive at the individual animal level. Herds were considered seropositive if there were at least three animals per herd seropositive to any serovar. RESULTS: Eighty-one percent of herds were seropositive for Leptospira spp. Hardjobovis was the predominant serovar at the herd (78%) and individual animal (60.8%) levels. The seroprevalence of Pomona was 20% and 8.4% at the herd and animal levels, respectively. Dual seroreactivity to both Hardjobovis and Pomona was observed in 16% of herds and 6.6% of individuals; within-herd seroprevalence for both serovars ranged from 0 to 100%. No herds and 1.2% of animals were seropositive for serovar Copenhageni. There was a regional difference in the prevalence of herds seropositive to both Hardjobovis and Pomona (p=0.02), but no difference between regions in the prevalence of herds seropositive to either Hardjobovis or Pomona alone. CONCLUSION: The seroprevalence of Leptospira spp. serovars is high, and distributed in farmed deer herds throughout New Zealand. CLINICAL SIGNIFICANCE: Results confirm the risk of disease and potential loss of production in deer, and exposure of infection to humans and other livestock from deer is widespread, demonstrating that control of the disease needs to be considered at a national rather than regional level.

Johne's disease caused by Mycobacterium avium subsp. paratuberculosis infection in red deer (Cervus elaphus): an histopathological grading system, and comparison of paucibacillary and multibacillary disease.

AIM: To describe a grading system for evaluating lesions in the small intestine and mesenteric lymph nodes of red deer (Cervus elaphus) infected with Mycobacterium avium subsp. paratuberculosis (MAP), and report the distribution of granulomas and findings seen in paucibacillary and multibacillary forms of the disease. METHODS: Tissues were examined from red deer either experimentally (n=300) or naturally (n=131) infected with MAP. A disease severity score developed previously was expanded to help provide more sensitivity in assessing severity of disease. The distribution of granulomatous, paucibacillary and multibacillary lesions in sections of jejunum, ileocaecal valve and mesenteric lymph nodes was compared between sites and between animals with mild (severity score< or =7) and severe (severity score> or =8) forms of the disease. RESULTS: Based on the results of three published studies, the severity score related well with the clinical severity and gross lesions associated with the disease. Paucibacillary lesions tended to have smaller macrophages and increased numbers of Langhan's giant cells than multibacillary forms, but this was not a consistent finding. The multibacillary form of the disease had Langhan's giant cells, containing numerous acid-fast organisms (AFO), and in one form sheets of epithelioid-like macrophages with prominent vacuolated cytoplasm and few Langhan's giant cells. In deer experimentally infected with MAP, granulomatous lesions were more evident in mesenteric lymph nodes than in intestinal tissue, especially in the mild form of the disease. In mild cases, granulomas were significantly more evident in Peyer's patches than in the intestinal mucosa, but in severe cases, the difference was not significant. Paucibacillary forms of the disease were more evident in deer with the mild disease, and multibacillary forms were more evident in deer with the severe disease. CONCLUSIONS: The severity score provides an objective measure of the severity of Johne's disease, and is useful for comparing individuals and groups of deer in studies involving experimental or natural infection with MAP. In mild disease, lesions were more evident in mesenteric lymph nodes than in jejunum and ileocaecal tissue, and Langhan's giant cells were present in both paucibacillary and multibacillary forms of the disease. The posterior jejunum and ileocaecal-valve lymph nodes were the best sites for detecting mild lesions, while intestinal samples from the posterior jejunum and ileocaecal valve had a lesser but useful role.

Age susceptibility of red deer (Cervus elaphus) to paratuberculosis.

The aim of this study was to measure the relative susceptibility of three age classes of red deer (Cervus elaphus) to the various manifestations of infection and disease states due to Mycobacterium avium subsp. paratuberculosis (MAP) after experimental oral challenge. Three groups of seronegative female deer (30 three-month-old weaners, 20 fifteen-month-old yearlings and 20 adults) received four oral doses of approximately 10(9) colony forming units (cfu) of a bovine strain of MAP. They were monitored for 50 weeks by weighing, blood sampling for immunological assays, skin testing and faecal culturing. Clinically affected animals were promptly euthanised and the remaining deer were killed at the end of the study. Necropsies were carried out and samples of intestine and associated lymph nodes were taken for culture and histopathology from all deer. Ten weaners developed clinical paratuberculosis and were euthanased 20-28 weeks post-challenge (pc). No clinical cases occurred in the yearlings or adults. All 10 clinically affected weaners had severe gross and histopathological lesions typical of paratuberculosis (Johne's disease). At slaughter, gross lesions were seen in the jejunal lymph nodes of 8/17 weaners, 2/19 yearlings, and 0/20 adults. MAP was cultured from samples of the intestine and/or lymph nodes from all 10 clinical cases and from 16/17 weaners, 19/19 yearlings and 18/20 adult hinds at slaughter. Lesion Severity Scores of deer slaughtered 50 weeks pc averaged 4.9, 3.5 and 1.1 for the weaner, yearling and adult groups, respectively. At some time over the course of the trial, 24/28 weaners were antibody positive and immediately prior to slaughter, 13/17 weaners, 15/19 yearlings and 3/20 hinds were antibody positive. There is a strong age-related resistance against clinical disease and subclinical disease, but not to infection with MAP, after heavy oral challenge.

Efficacy, immune responses and side-effects of vaccines against Johne's disease in young red deer (Cervus elaphus) experimentally challenged with Mycobacterium avium subsp paratuberculosis.

AIMS: To test the efficacy of a commercially available and an experimental vaccine against Johne's disease in young red deer (Cervus elaphus), using experimental challenge with live virulent Mycobacterium avium subsp paratuberculosis (M. ptb), measure injection-site reactions, and assess the effects of vaccination and challenge on results of subsequent skin tests and ancillary blood tests for bovine tuberculosis (Tb). METHODS: Ninety 6-8-week-old red deer fawns were randomly allocated to three equal groups of 30, and received either a 1-ml S/C injection of either a commercially available whole-cell killed vaccine with a mineral-oil adjuvant (COM), or a live attenuated M. ptb experimental vaccine with a lipid adjuvant (EXP), or were unvaccinated controls. Ten weeks later (Week 10), all 90 fawns received an oral challenge with approximately 10(8) cfu of a bovine strain of M. ptb daily for 4 days. The fawns were regularly weighed and monitored for clinical signs of Johne's disease, and regularly blood-sampled and tested for antibodies to M. ptb, using the Paralisa test, an IgG1 ELISA, and for antibodies to Mycobacterium bovis, using a similar test. A mid-cervical tuberculin skin test (MCT) was administered at Week 23, and comparative cervical skin tests (CCTs) were administered at Weeks 37 and 57. All animals were electively killed at Week 59, injection sites inspected, gastrointestinal tracts examined for gross lesions, and samples taken for culture and histopathology. RESULTS: There were no clinical cases of Johne's disease but, at slaughter, more gross lesions in intestinal lymph nodes were observed in Control (20%) than COM animals (0%; p<0.05). This latter group also had less severe histopathological lesions in samples of intestines and lymph nodes compared with the Control group (p<0.05), but not deer in the EXP group. Over 89% of deer in all three groups were shown by culture to be infected with M. ptb, while only 21-33% of faecal samples were culture-positive. Time to positive culture was longer for COM vs EXP and Control groups (p<0.01), reflecting fewer M. ptb organisms in samples from the ileocaecal valve (ICV) in that group. Almost all (>or=90%) deer reacted to the MCT at Week 23, and there were no significant differences between groups. One or two deer in each group were classified as Tb reactors to the CCT at Week 37, and none were classified as Tb reactors to the CCT at Week 57. At the time of challenge, over 50% of deer in the COM group were classified as positive (9/28) or suspicious (7/28) for M. ptb antibodies in the Paralisa test, one animal in the EXP group was classified as suspicious, and all the Controls were negative. From Week 23 to the end of the trial, 25/28 (89%) deer in the COM group were Paralisa-positive or -suspicious. The proportion of animals in the EXP and Control groups that were Paralisa-positive peaked at Week 39 (60% and 55%, respectively). The majority of deer in the COM group had significant levels of antibody to M. bovis 10 weeks after vaccination, while the proportion of M. bovis-antibody positive Control deer rose gradually throughout the trial, reaching 23/30 (77%) at slaughter. Injection-site lesions in COM deer ranged from 10-38 mm in diameter 4 weeks after vaccination, and then resolved. Minimal injection-site lesions were observed in EXP deer. At slaughter, 14 months after vaccination, 19/28 deer in the COM group had 5-15-mm nodules that were easily trimmed from the carcass. CONCLUSIONS: The experimental challenge with M. ptb produced subclinical Johne's disease in the majority of deer, but did not cause any clinical disease. The number and severity of gross and microscopic lesions was significantly reduced in the COM compared with Control and EXP groups; vaccination of the EXP group did not appear to give significant protection. Deer vaccinated with the commercial vaccine are likely to give a false-positive reaction to the MCT but should have an avian reaction to the CCT, if it is carried out >12 months after vaccination. Most of the deer vaccinated with the commercial vaccine produced significant levels of antibodies against both M. ptb and M. bovis, which interfered with ancillary Tb tests. If this vaccine or similar oil-based vaccines are used on deer farms in the future, it may be advisable to only vaccinate animals destined for slaughter, that would not need to be Tb-tested, but would be 'works-monitored' for evidence of Tb instead.

Intra-uterine transmission of Mycobacterium avium subsp paratuberculosis in subclinically affected red deer (Cervus elaphus).

AIM: To determine the rate of transmission of Mycobacterium avium subsp paratuberculosis (M. ptb) from hind to fetus in utero, and the risk of transmission from dam to fawn via infected colostrum and milk in subclinically affected red deer hinds. METHODS: Hinds were sourced from farms in Otago or Southland and selected for the study if they were positive to the immunoglobulin G1 (IgG1) modified enzyme-linked immunosorbent assay (ELISA) (Paralisa) and exhibited no clinical signs of Johne's disease. The hinds (n=35) were sent to a deer slaughter premises (DSP; n=31) or were killed on-farm (n=4). All post-mortem samples were collected from the fetus first and then from the dam, taking care to avoid cross contamination between samples. Fresh samples (n=185) were collected for culture, and tissue samples (n=72) were collected from 24 hinds and their fetuses for histopathological examination. RESULTS: A total of 24/35 hinds selected were suitable for inclusion in the study. Eighteen of these pregnant hinds were culture-positive for M. ptb, and 14 of these had culture-positive fetuses, representing a transmission rate of 78% (95% confidence interval (CI) =0.58-0.98) from dam to fetus. Of the 16 mammary glands sampled, 11 (69%) were culture-positive for M. ptb while 12/15 (80%) mammary lymph nodes sampled were also culture-positive. CONCLUSIONS: This study demonstrated a high rate of transmission of M. ptb from dam to fetus in red deer, and a potential risk of transmission to fawns suckling from mothers that are subclinically affected with Johne's disease.