Paratuberculosis vaccination response in cattle, sheep and goats. Geijo MV, Garrido JM, Aduriz G, Sevilla I, Berriatua E and Juste RA

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1 Paratuberculosis vaccination response in cattle, sheep and goats Geijo MV, Garrido JM, Aduriz G, Sevilla I, Berriatua E and Juste RA Instituto Vasco de Investigación y Desarrollo Agrario (NEIKER), Berreaga,. 486 Derio, Bizkaia, Spain. Corresponding author: Geijo MV. Phone Fax mgeijo@neiker.net Keywords: paratuberculosis, immune response, vaccination, ELISA, Interferon-γ, PCR. SUMMARY Diagnostic and control criteria for paratuberculosis (PTB) are based on the assumption that all ruminant species have a similar response to the infection. However, there is some evidence that infections in each species have quite different features, like the strains that infect cattle and sheep or the pathological picture in each species. We designed a study involving cattle, sheep and goats in order to compare the immune response against PTB in natural conditions and to measure the effects of vaccination according to age of immunisation and PTB status of the herd. Three age cohorts (5 days, 3 to 6 months, and.5 to 2 years old) for each species were selected in three farms with recorded cases of PTB and in three farms free of PTB. Animals were tested by PPA3-ELISA, IFN-γ-ELISA, and PCR every 6 months for.5 years. For each age group and species there were 5-7 vaccinated and non-vaccinated animals. Individual antibody and IFN-γ ELISA results at the first control were subtracted from results at subsequent controls in order to have comparable data for all species. Overall, cell mediated response to vaccination was found to be stronger in all PTB-free herds / flocks than in clinically affected ones. By PCR, Mycobacterium avium subsp. paratuberculosis (Map) was at least as frequently detected in PTB-free as in PTB-affected herds, both before and after vaccination. These results indicate that Map distribution might not be related to a previous clinical history of PTB, and that there are different patterns of PTB immune responses according to species, age, and clinical history. INTRODUCTION After many years of increasing efforts towards improving diagnosis and control of paratuberculosis (PTB), this infection remains a challenge that is still far from a definitive solution and imposes a heavy burden to ruminant production. In addition to this, the suspected potential for causing disease in humans makes even more relevant to improve the understanding of the dynamics of infection and the mechanisms by which exposure to the causative agent, Mycobacterium avium subsp. paratuberculosis (Map) leads to clinical disease and further spread in the environment. Although substantial progress has been made in the characterisation of strains that cause PTB in sheep and cattle, the differences in the immune response against Map exhibited by the different ruminant species are less understood. This is an aspect of great importance given the fact that it is well known that exposure to the agent does not necessarily lead to infection and disease. Furthermore, advanced cases of PTB show a variety of immunopathological pictures (2,9) (4,2). The knowledge of these forms is essentially limited to the final stages of the disease, and very little is known about the factors that influence the progress in one or another way. Since individual immune responses vary greatly in the face of a bacterium of low virulence, we have hypothesised that sensitisation in early life with Map might be one of the most important factors in determining the course of the infection (). In order to investigate this aspect we examined the immunological and the PCR status of sheep, cattle and goats of different ages from PTB clinically and non-clinically affected flocks and herds. To this end, we designed cohort studies for each species where we addressed two main points: a) pattern of PTB immune response by measuring IFN-γ and antibodies. b) differences in the immune response to vaccine sensitisation according to PTB status of the herd and age of vaccination. MATERIALS AND METHODS Animals in study. Two dairy cattle herds, two sheep flocks and two goat herds were selected. One herd / flock of each species had a clinical history of PTB, while the other one was clinically free of PTB for the last years. The PTB free cattle herds and sheep flock were property of the Basque Government, whereas the herds / flocks with a clinical history of PTB belonged to private farmers who initially 65

2 requested a laboratory confirmation of PTB at NEIKER. These flocks and herds were well managed and animals were easy to trace. In contrast the goat herds were both privately owned, extensively managed and animals were more difficult to trace. The flock with a history of clinical PTB had had a serious PTB outbreak in the 99s. The PTB-free herd was from a neighbouring farm and had no recorded evidence of PTB. Three age cohorts (< 5 days, 3 6 months and.5 2 years old) of -4 animals of each species were followed in this study. Vaccination. In each age group half the animals were vaccinated, and other half were left as unvaccinated controls. One ml of the Spanish commercial vaccine (Gudair ) was administered subcutaneously in the brisket area. This vaccine contains 2.5 mg/ml of dried heat inactivated Map 36F strain. Follow-up. Samples were taken beginning on the vaccination day (month or pre-vaccination sampling), and then at 6, 2 and 8 months postvaccination (mpv). On each sampling, blood was drawn from the jugular vein of sheep and goats and from the coccygeal vein in cattle into a ml EDTA coated vacuum tube (for ELISA and PCR) and into a similar heparin coated tube (for IFN-γ). Diagnostic Tests. Humoral immune response (HIR) detection. An indirect ELISA with PPA3 as antigen and protein G as conjugate according to Garrido (5) was used. The EDTA tube was centrifuged and the plasma transferred to a 2 ml eppendorf tube where samples were frozen at 2 ºC and stored until use. Briefly, polystyrene microtiter plates (Greiner Microlon) were coated with µl/well of.4 mg/ml solution of PPA3. Plates were washed 3 times with PBS-T and frozen at 2 C until use. The sera were adsorbed (:) with a saline suspension of M. phlei (5 gr/l). After an overnight incubation at 4 C, the supernatants were diluted to /6 for goat and cattle, and to /2 for sheep, and µl were added into duplicate wells of an antigen-coated plate. Positive and negative controls were included in every plate. After a two hours incubation at room temperature in a wet chamber, plates were washed three times with PBS-T. 5 l of Protein G horseradish peroxidase conjugate (,25 µg/ml) was added and incubation was repeated in the same conditions. After three washings with PBS-T, µl of substrate were added and plates incubated for 2 min in the dark. The reaction was stopped with 5 µl/well fluorhidric acid (2 %) and optical density (OD) was measured in an spectrophotometer at double wave length (45/ 45 nm). The average serum OD value was divided by the average positive control OD, and this was taken as the final serum reading. Since the study goal was to compare responses according to other variables these quantitative results were used for all analysis and no attempt was made to score sera as positive or negative. Cell-mediated immune response (CMR) detection. Within 6 h of collection,.5 ml heparinised whole blood were incubated with l of PBS (NA: no antigen), avian (PPDa) or bovine (PPDb) purified protein derivative (.3 mg/ml) for 24 h in 2 ml eppendorf tubes at 37 ºC. The tubes were centrifuged (5 x g for min at room temperature) and about 5 µl of the supernatant collected and kept frozen at -2 ºC until used. The IFN-γ testing was carried out according to the Bovigam TM Interferon-γ ELISA assay (CSL Veterinary, Melbourne, Australia). The level of IFN-x was taken as a measure of the CMR (5) and the final sample value calculated as follows: (PPDa OD - NA OD) / (Positive control OD mean - Negative control OD mean) Map detection. Five millilitres of peripheral blood in EDTA where transferred to a 5 ml tube. After two washes with ml.83 % ammonium chloride with centrifugation at 2 x g for min at room temperature, the supernatant was discarded and the pellet was resuspended in ml PBS. After a new centrifugation, the pellet was resuspended in 3 ml PBS and split into two 2 ml eppendorf tubes. After a further centrifugation for min at 5 x g, the supernatant was discarded and the tubes were frozen at -2 ºC until used. Bacterial cells were broken by a freezingboiling method (6) consisting of three cycles of a 5 min incubation of tubes in liquid nitrogen at 96 ºC, a 5 min boiling in a dry bath at ºC and a chloroform : octanol (24:) DNA extraction in 2 ml tubes. The octanol fraction was centrifuged at 6 x g during 5 min, and the pellet washed twice in absolute ethanol and once in 7 % ethanol. After a centrifugation at 6 x g during min, the ethanol was discarded and the tubes allowed to dry in inverted position on a sheet of filter paper. Finally the DNA was resuspended in l nuclease-free distilled water. A PCR reaction in a 25 l volume was carried out using primers P9 (2) and P9 according to the protocol reported by Innis et al. (8). The thermal profiling consisted of: 96 C for 2 min, 4 cycles at 95 C for 3 s, 55 C for 3 s and 72 C for min, and a final extension at 72 C for min. The reaction products were separated on a 2 % agarose gel with.5 % Ethidium bromide at 5 V for 2 min. A reaction was considered positive when the specific 389 bp product was visible and positive and negative controls validated the gel. Analysis of results. In order to have the same scale for all species, the ELISA and IFN-γ values were transformed into the difference between the value of the actual test and the value at month 66

3 zero of the study (pre-vaccination). ELISA and IFN-γ optical density values were logarithmically transformed to normalise their distribution and Analysis of Variance was used to compare mean ELISA and IFN-γ values according to the independent variables, including, species, age, PTB status and time. RESULTS Each ruminant species showed a different pattern of immune response. The cattle ELISA results of the PTB-free and affected cattle herds are shown in Figure. It is interesting to emphasise a fall in the antibodies level of the < 5 days vaccinated calves group, which was specially noticeable in the PTB-affected herd, and that was followed by an increase in the last sampling, at 8 months old. With regard to 3-6 months and.5-2 year old groups, antibody levels in vaccinated animals were numerically higher than in non-vaccinated animals, but the difference was smaller in the PTB-affected herd compared to the PTB-free herd. The evolution of the bovine CMR is shown in Figure 2. A strong and progressive increase of the CMR was observed in the PTB-free herd in the < 5 days vaccinated calves, while in the clinically affected herd these calves suffered an initial rise followed by a fall. However the 3-6 months old and.5-2 years old vaccinated animals in the PTBaffected herd had a remarkable positive response. The ELISA results of goats are shown in Figure 3. A fall of the HIR of the < 5 days vaccinated calves is observed in kids too, specially in the clinically affected herd. With regard to CMR, noticeable differences were observed between the free herd and the affected one (Figure 4). Whereas the free herd showed a good response to vaccine, which was numerically higher in vaccinated compared to control animals, in the affected herd the levels of the CMR of the vaccinated goats were similar to initial pre-vaccination values and to those in the controls. Finally, at the last sampling CMR fell to initial levels in all age groups. The difference of the immune response between clinically free flocks and affected flocks is more evident in sheep. In the PTB free flock (Figure 5) a positive HIR can be observed in all vaccinated groups. Response to vaccination was first observed in < 5 days vaccinated lambs and latter with numerically lower values in the older sheep. However, in the PTB-affected flock the humoral response to the vaccine was less strong than in the PTB-free flock, differences between groups were not evident and only slightly larger in vaccinated animals compared to nonvaccinated animals. The results of the IFN-γ test in sheep showed a similar trend to the HIR results; On the one hand there was a notable response to the vaccinal challenge in the free flock and only a limited response in the affected flock (Figure 6). BOVINE ELISA,6, Age <5 days Age 3-6 months Age,5-2 years Figure. Cattle ELISA results. of transformed ELISA index for each sampling, treatment group and herd status (PTB-free and PTB-affected herds). Serum samples were drawn at, 6, 2 and 8 months post vaccination. BOVINE IFN-γ 2,4 2,2 2,8,6, Age <5 days Age 3-6 months Age,5-2 years Figure 2. Cattle IFN-γ results. of transformed γ-ifn index for each sampling, treatment group and herd status (PTB-free and PTB-affected herds). Serum samples were drawn at, 6, 2 and 8 months post vaccination. 67

4 CAPRINE ELISA 2,5, Age <5 days Age 3-6 months Age,5-2 years Figure 3. Goat ELISA results. of transformed ELISA index for each sampling, treatment group and herd status (PTB-free and PTB-affected herds). Serum samples were drawn at, 6, 2 and 8 months post vaccination. C APRINE IFN-γ,3,,9 N O 2 2 N O 2 2 PTB-FR EE PTB -AFF ECTE D Age <5 days Age 3-6 m onths Age,5-2 years Figure 4. Goat IFN-γ results. of transformed γ-ifn index for each sampling, treatment group and herd status (PTB-free and PTB-affected herds). Serum samples were drawn at, 6, 2 and 8 months post vaccination. OVINE ELISA,3,, Age <5 days Age 3-6 months Age,5-2 years Figure 5. Sheep ELISA results. of transformed ELISA index for each sampling, treatment group and flock status (PTB-free and PTB-affected flocks). Serum samples were drawn at, 6, 2 and 8 months post vaccination. OVINE IFN- γ,3, γ, Age <5 days Age 3-6 months Age,5-2 years Figure 6. Sheep IFN-γ results. of transformed IFN-γ index for each sampling, treatment group and flock status (PTB-free and PTB-affected flocks). Serum samples were drawn at, 6, 2 and 8 months post vaccination. 2 The overall, average post-vaccination 68

5 The overall, average post-vaccination ELISA and IFN-γ for each age group are represented in Tables and 2. In cattle the immune response to the vaccine was positive in the three ages of vaccination in the free herd, with a significantly higher increase of the CMR in < 5 days vaccinated calves (p <.). In contrast, in the youngest animals in the affected herd both ELISA and IFN-γ post-vaccination levels were lower than pre-vaccination levels. The PCR results are shown in Figure 7. There were PCR positive animals in all herds / flocks. The proportion of PCR positives ranged between % in the goat herds and in the PTB-affected cattle herd, and % in the PTB affected cattle herd and in the sheep flocks. The proportion of PCR positive animals was similar within goat herds and within sheep flocks but was significantly higher in the PTB-free herd than in the PTB-affected herd (p <.5). Table. Average post vaccination ELISA results. Six, 2 and 8 months mean post-vaccination transformed ELISA index according to age at vaccination and herd status for each species and treatment group. In parentheses p value for the difference to pre-vaccination baseline ELISA index (equal to ). PTB FREE PTB AFFECTED CATTLE GOAT SHEEP CINATED CINATED CINATED <5 days 3-6 months,5-2 years <5 days 3-6 months,5-2 years,66,9,594 7, (p=,693) (p=,4574) (p=,6662) (p=36) (p=,9666) (p=,56),827,52 968,9764,23,65 (p=,56) (p=,9687) (p=,42) (p=654) (p=528) (p=,27), ,68,584,534,482 (p=,6789) (p=,78) (p=,8) (p=<,) (p=,6) (p=,2652) 4,49,387,9386,494,224 (p=,275) (p=,7438) (p=6) (p=,665) (p=,778) (p=<,) 272,335,726,392,563,233 (p=,737) (p=,36) (p=,757) (p=,2233) (p=,965) (p=,2886),84,275,98,555,3,244 (p=742) (p=52) (p=,9328) (p=,6575) (p=,766) (p=39) Table 2. Average post vaccination IFN-γ results. Six, 2 and 8 months mean post-vaccination transformed IFN-γ index according to age at vaccination and herd status for each species and treatment group. In parentheses p value for the difference to pre-vaccination baseline IFN-γ index (equal to ). PTB FREE PTB AFFECTED CATTLE GOAT SHEEP CINATED CINATED CINATED <5 days 3-6 months,5-2 years <5 days 3-6 months,5-2 years,74,22 727,924,63,3624 (p=<,) (p=,46) (p=,63) (p=,5332) (p=,743) (p=,96) 428,96,928, ,9239 (p=,9) (p=,75) (p=,5335) (p=,54) (p=,77) (p=,4994),69,392,34,246,29,9926 (p=,382) (p=,799) (p=,9273) (p=64) (p=,984) (p=,958),9,978,9599,66 983,4 (p=,939) (p=,5453) (p=,7784) (p=,6732) (p=,575) (p=,992),67,74,278,97,3,66 (p=,393) (p=,54) (p=6) (p=,7772) (p=,7853) (p=76),248, , ,997 (p=262) (p=,699) (p=,3278) (p=,558) (p=,394) (p=,9398) In the goat herds similar observations were made. Whereas in the free herd a positive immune response was observed in the three vaccination ages, in the affected herd a fall of the immune response was observed in < 5 days vaccinated kids, which was specially significant in the HIR. Finally, in the sheep flocks, in contrast to the observations in cattle and goats, the < 5 days vaccinated lambs of the PTB free flock had a strong immune response, even higher than the response observed in older vaccinated sheep. In contrast, in the < 5 days vaccinated lambs in the affected flock the post-vaccination HIR increased and the CMR suffered a reduction, but differences were not statistically significant (p >.5). %Blood PCR Peripheral Blood PCR CATTLE GOAT SHEEP PTB-free 53,4 55,56 24,83 PTB-affected 27,4 56,4 27,2 Figure 7. Percentage of peripheral blood PCR positive results according to species and PTB status of the herd / flock. In the PTB-free cattle herd the number of positive animals was significantly higher than in the affected herd. 69

6 DISCUSSION No clear pattern across species has been observed in this study and therefore no general immune response model could be postulated. Instead, each species appeared to behave in slightly different ways. By species, the failure of the < 5 days vaccinated calves and kids to develop an ELISA response to vaccination can be compared to results obtained by Spangler et al. (4) who explained it either as a prenatal exposure-induced immunological tolerance or as a result of interference with colostral immunoglobulins. Therefore it could be taken as a general behaviour in these two species. The strong response of sheep, both in PTB-free and in PTB-affected flocks, may indicate a better capability of this species to counter Map infections. This, however, contrasts the results obtained by Corpa et al. (3) that found some decrease in the HIR of young lambs which they attributed to a lack of full maturation of the immune system. The rise of antibody levels in the youngest cattle at the last sampling could be explained as a response to changes in the management, as at this age, cattle leave the heifer herd and suffer a change in the diet and by joining the adult herd for the first time in their lives they possibly become exposed to a more heavily contaminated environment. This rise may have not been observed in sheep and goats because animals remain with the adult flock all the time or until weaning at several months of age and are hence, exposed to infection from an early age. At other ages, there were differences related to vaccination by 6 mpv, which tended to disappear after 6-2 months. This group yielded the strongest HIR in PTB-free flock / herds. Regarding the PTB status, overall, the immune response seemed to be better in PTB-free herds than in PTB-affected herds where individual responses seemed to result in a larger variability, maybe because a compromised immune system of animals in affected farms favours clinical presentation of PTB. With regard to the CMR, the PTB-free groups of all three species reacted to vaccination by producing large amounts of IFN-γ, while the PTB-affected groups seemed to react less. This suggests a certain impairment of CMR associated to infection or alternatively, a certain predisposition of lower reactors to be present in PTB-affected flocks. In any case, the lack of CMR in PTB-affected and the variable HIR observed might be the result of a compromised immune response of some individuals. These observations are in concordance with Gwozdz et al. (7) which found that the mean IFN-γ response in blood of experimentally infected sheep with clinical signs of PTB was diminished in comparison with experimentally infected but clinically normal animals. On the other hand, Pérez et al. (2) found a close association between CMR and lesion type in naturally infected sheep. Positive results in the IFN-γ assay were found in animals with focal (tuberculoid) and diffuse lesions (borderline tuberculoid), and had scant or no acid fast bacilli in the intestine. These findings agree with the paradigm of a relation between a strong CMR and the animal s ability to control infection avoiding Map proliferation and restricting lesions to tuberculoid or borderline tuberculoid forms. The return of vaccinated goat and sheep IFN-γ results to initial values at the last sampling (8 months post-vaccination) is consistent with the results obtained by Leslie et al. () in a longitudinal vaccination trial where a loss of the CMR of vaccinated goats was observed after a year or more. The presence of Map in peripheral blood was similar in the different species, ages and independent of the PTB status. These observations support an epidemiological model where Map is widespread (confirmed by isolation from environment in some PTB-free farms) and young animals may develop clinical PTB only in herds or flocks with high rate of infection and poor management as a result of heavy exposure to Map. Otherwise, the prevalence of PTB in infected herds or flocks is expected to be very low and losses due to clinical disease may not occur or remain unnoticed (6). This hypothesis implies that the presence of Map is necessary but not sufficient for developing clinical disease, but other factors as genetic predisposition, immune constitution or subtle unbalances of nutrition may be necessary. This is consistent with observations of experimental inoculations with large amounts of organism which were not % effective in inducing persistent infection () and open a suggestive way for comparison with Crohn s disease, since Map also appears in healthy individuals (3). Regarding the PTB status, overall, the immune response seemed to be better in PTBfree herds than in PTB-affected herds where individual responses seemed to result in a larger variability. Good HIR and CMR were observed at all ages in PTB-free herds, and therefore it could be assumed that it is not necessary to vaccinate new-born animals as often has been recommended. However, this conclusion becomes somewhat weaker according to the results in PTB affected herds because of the wide variability in their capability to mount an effective immune response. This could be attributed to Map environmental sensitisation, but since Map was found in all herds, it is even more likely that it could indicate the existence of a pre-existing altered immune response that would favour the development of infections into 7

7 clinical cases. In this sense, the protective effects of vaccination in these herds (the only ones where vaccination should be considered as a control measure) cannot be evaluated solely on the light of the variables considered here, and a study specifically addressing these points should be designed. CONCLUSIONS Humoral response to vaccination in sheep was found to be stronger than response to natural infection. In cattle and goat this response was more variable including a failure of < 5 days vaccinated calves and kids to develop an ELISA response to vaccination. With regard to CMR, the response to vaccination was stronger in all PTB-free herds / flocks, while in clinically affected ones showed a larger variability. On the other hand the presence of Map in peripheral blood did not appear positively related to clinical history of the herds. These observations support the hypothesis that the animal s capability to control Map infection and the development of clinical signs can be related with its CMR strength. ACKNOWLEDGEMENTS This work was supported by the Comisión Internministerial de Ciencia y Tecnología (CICYT) Project AGF C3-2 Mycobacteriosis of ruminants: Development of host response related to factors modifying it. REFERENCES. Adams JL, Collins MT and Czuprynski CJ Polymerase chain reaction analysis of TNFalpha and IL-6 mrna levels in whole blood from cattle naturally or experimentally infected with Mycobaterium paratuberculosis. Can J Vet Res, 6: Clarke CJ The pathology and pathogenesis of paratuberculosis in ruminants and other species. J Comp Path, 6: Corpa JM, Pérez V and García Marín JF Differences in the Immune Responses in lambs and kids vaccinated against paratuberculosis, according to the age at vaccination. Proc Int Coll PTBC VI, Corpa JM, Garrido JM, García Marín JF and Pérez V. 2. Classification of lesions observed in natural cases of paratuberculosis in goats. J Comp Pathol, 22: Garrido JM. 2. Puesta a punto de las técnicas de PCR en heces y de ELISA para el diagnóstico de la paratuberculosis. Estudio de prevalencia en ganado bovino. Thesis/Dissertation. Universidad de Zaragoza. 6. Garrido JM, Cortabarria N, Oguiza JA, Aduriz G and Juste RA. 2. Use of a PCR method on fecal samples for diagnosis of sheep paratuberculosis. Vet Microbiol, 77: Gwözdz JM, Thompson KG, Manktelow BW, Murray A and West DM. 2. Vaccination against paratuberculosis of lambs already infected experimentally with Mycobacterium avium subspecies paratuberculosis. Aust Vet J, 78: Innis MA and Gelfand DH. 99. Optimization of PCRs. In: Innis MA, Gelfand DH, Sninsky JJ and White TJ (Eds.), PCR protocols. A guide to methods and applications. Academic Press. San Diego, : Juste RA Johne's disease: a review of current knowledge. Int Congr Sheep Vet, IV. University of New England. Armidale, Australia, pp Leslie P, Riemann HP, West G and Moe A Vaccination field trial for Mycobacterium paratuberculosis (Johne's disease) in the caprine species. In: Thorel MF and Merkal RS (Eds.), Proc Int Coll PTBC, II. Laboratoire Central de Recherches Veterinaires. Maisons-Alfort, France, pp Manning EJ and Collins MT. 2. Mycobacterium avium subsp. paratuberculosis: pathogen, pathogenesis and diagnosis. Rev Sci Tech. Off Int Epiz, 2: Moss MT, Sanderson JD, Tizard MLV, Hermon-Taylor J, El-Zaatari FAK, Markesich DC and Graham DY Polymerase chain reaction detection of Mycobacterium paratuberculosis and Mycobacterium avium subsp. silvaticum in long term cultures from Crohn's disease and control tissues. Gut, 33: Pérez V, Tellechea J, Corpa JM, Gutiérrez M and García-Marín JF Relation between pathologic findings and cellular immune responses in sheep with naturally acquired paratuberculosis. Am J Vet Res, 6: Schwartz D, Shafran I, Romero C and Naser SA. 2. Rapid isolation of Mycobacterium avium subsp. paratuberculosis form tissue specimens of Crohn's disease patients. Am Soc Microbiol Meet. Los Angeles, CA, p U Spangler E, Heider LE, Bech-Nielsen S and Dorn CR. 99. Serologic enzyme-linked immunosorbent assay responses of calves vaccinated with a killed Mycobacterium paratuberculosis vaccine. Am J Vet Res, 52: Stabel JR Production of interferon-γ by peripheral blood mononuclear cells: an important diagnostic tool for detection of subclinical paratuberculosis. J Vet Diagn Invest, 8: Whittington RJ and Sergeant ES. 2. Progress towards understanding the spread, detection and control of Mycobacterium avium subsp. paratuberculosis in animal populations. Aust Vet J, 79: