Avium Subsp Research Articles

Expression Profiles of Different Cytokine Genes in Peripheral Blood Mononuclear Cells of Goats Infected Experimentally with Native Strain of Mycobacterium Avium Subsp. Paratuberculosis

Paratuberculosis (ParaTB), caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a chronic enteritis of ruminants and may contribute to Crohn's disease in humans. Key features of host immunity to MAP infection include an early pro-inflammatory (Th1-like) response that eventually gives way to a predominant anti-inflammatory (Th2-like) response. Many studies have been conducted to understand the underlying mechanism of misdirected host immune response, however, these studies mainly focused on cattle. The present study is the first attempt to test the hypothesis of shift in Th1 to Th2 like responses during the progression of ParaTB in caprine species (small ruminant). Ten healthy male kids (<6 months old) of the same breed were selected for this study. Of the 10 kids, 6 were experimentally infected with native strain (S5) of MAP (“Indian Bison Type”) and the remaining 4 kids were control. Kids were monitored for a period of 12 months post infection (MPI) and were tested for establishment of infection. Expression levels of IFNG, IL2, IL12, IL4, and IL10 genes were estimated before infection and at 4, 8, and 12 MPI in stimulated peripheral blood mononuclear cells (PBMCs) of infected and control kids. The study demonstrated the expression of IFNG and IL2 as classic Th1-like pro-inflammatory signatures; whereas, IL10 exhibited itself as classical Th2-like signature. The study also reports unexpected lowered expression of the IL12 gene simultaneously with increased expression of IFNG, lowered expression of the IL2 gene (compared to IFNG), and suppressed expression of the IL4.

Sensitivity ofMycobacterium aviumsubspparatuberculosis,Escherichia coliandSalmonella entericaserotype Typhimurium to low pH, high organic acids and ensiling

To evaluate the persistence of Mycobacterium avium subsp paratuberculosis (Myco. paratuberculosis), Salmonella enterica serotype Typhimurium (Salm.Typhimurium) and a commensal Escherichia coli (E. coli) isolate under the low pH and high organic acid (OA) conditions of ensiling of forages. Decay rates and the time required to obtain a 90% reduction in cell concentration were calculated following (i) exposure to buffered OA (pH 4.0, 5.0, 6.0 or 7.0) (ii) exposure to silage exudates and (iii) survival through ensiling of forage materials. Salm. Typhimurium had higher decay rates in silage exudates (-0.5601 day(-1) ) than did E. coli (-0.1265 day(-1) ), but both exhibited lower decay rates in silage than in OA or silage exudates. Myco. paratuberculosis showed no decrease in silage and decay rates in silage exudates were significantly lower (2-12 times) than for the other two organisms. Escherichia coli, Salm. Typhimurium and Myco. paratuberculosis exhibit marked differences in response to acidity. All three organisms show acid resistance, but Myco. paratuberculosis in particular, if present in manure and applied to forage grasses, may survive the low pH and high OA of the ensilaging process; silage may therefore be a potential route of infection if ingested by a susceptible animal. This information contributes to the understanding of potential risks associated with silage preservation and contamination of livestock feed with manure-borne pathogens.

Is an individual calving pen better than a group calving pen for preventing transmission of Mycobacterium avium subsp paratuberculosis in calves? Results from a field trial

The objective of this study was to quantify the efficacy of using individual calving pens (ICP) from which manure was removed between successive calving compared with group calving pens (GCP) for limiting transmission of Mycobacterium avium subsp paratuberculosis (MAP) in Holstein calves. Every other pregnant cow in three Minnesota MAP endemic herds was assigned to calve in either the ICP or the GCP within 48–72h prior to expected calving. Heifer calves born in the ICP were assigned to the intervention group (n=238) while heifer calves born in the GCP were considered controls (n=211). Calves were separated from their dams as soon as was possible once the calf was found. The intervention within the ICP relative to the GCP was the removal of fecal material in the ICP immediately after each birth. Upon enrollment in 2005, calves were monitored into adulthood. Of the original animals enrolled, 318 were tested for MAP at least once in 2007, 2009, or 2010 using serum ELISA (ICP, n=165; GCP, n=141) and bacterial culture of feces (ICP, n=173; GCP, n=145) tests. Cox regression analysis was performed to evaluate the time until MAP test positivity. Cows born in the ICP had a hazard ratio of 0.37 (95% CI=0.34–0.4) for testing MAP serum ELISA positive, compared with cows born in GCP. Similarly, cows born in the ICP had a hazard ratio of 0.09 (95% CI=0.06–0.14) for testing MAP fecal culture positive, compared with cows born in GCP. The Cox proportional-hazard assumption was violated in both models such that differences observed in the instantaneous hazards of MAP positive outcomes between groups (ICP vs. GCP) subsequently diminished overtime. These findings indicate that using ICP for calving delays exposure to MAP in calves and provides an effective strategy for reducing peripartum MAP transmission risks in herds attempting to limit the impact of paratuberculosis.

In vitro expression of the SLC11A1 gene in goat monocyte-derived macrophages challenged with Mycobacterium avium subsp paratuberculosis

Johne’s disease or paratuberculosis is a chronic, progressive intestinal disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). One of the genes that have been targeted with regard to resistance or sensitivity to paratuberculosis is the SLC11A1 (solute carrier family 11 member A1). Here we extend our previous work to the sequence and structure analysis of the caprine SLC11A1 gene and we assess the functional impact of the most frequent polymorphisms of the 3′ UTR region of the SLC11A1 gene to its expression in goat macrophages exposed in vitro to MAP. The role of these polymorphisms in primary immune response is also investigated with connection to gene expression of two interleukins (IL), one of which pro (IL-1a), and the other anti-inflammatory (IL-10). In order to assess gene response, quantitative detection of the SLC11A1, IL-10 and IL1a mRNA was performed by real time PCR before, and at 1, 3 and 24h after exposure of primary cultures of peripheral blood monocyte-derived macrophages to MAP, collected from 54 goats of the Greek native goat breed. Sequence analysis of the 3′ UTR end of the caprine SLC11A1 gene determined its full length to be 522 bases. Structure analysis confirmed the presence of two microsatellites consisted of a variable number of guanine-thymine repeats (regions A and B). The homozygous B7 genotype [B(GTn)7/7] was associated at a statistically significant level with increased expression of the SLC11A1 and IL-1α genes indicating increased in vitro responsiveness and therefore resistance of mononuclear derived macrophages to MAP infection.

Assessing the Inactivation of Mycobacterium avium subsp. paratuberculosis during Composting of Livestock Carcasses

Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants, with substantial economic impacts on the cattle industry. Johne's disease is known for its long latency period, and difficulties in diagnosis are due to insensitivities of current detection methods. Eradication is challenging as M. avium subsp. paratuberculosis can survive for extended periods within the environment, resulting in new infections in naïve animals (W. Xu et al., J. Environ. Qual. 38:437-450, 2009). This study explored the use of a biosecure, static composting structure to inactivate M. avium subsp. paratuberculosis. Mycobacterium smegmatis was also assessed as a surrogate for M. avium subsp. paratuberculosis. Two structures were constructed to hold three cattle carcasses each. Naturally infected tissues and ground beef inoculated with laboratory-cultured M. avium subsp. paratuberculosis and M. smegmatis were placed in nylon and plastic bags to determine effects of temperature and compost environment on viability over 250 days. After removal, samples were cultured and growth of both organisms was assessed after 12 weeks. After 250 days, M. avium subsp. paratuberculosis was still detectable by PCR, while M. smegmatis was not detected after 67 days of composting. Furthermore, M. avium subsp. paratuberculosis remained viable in both implanted nylon and plastic bags over the composting period. As the compost never reached a homogenous thermophilic (55 to 65°C) state throughout each structure, an in vitro experiment was conducted to examine viability of M. avium subsp. paratuberculosis after exposure to 80°C for 90 days. Naturally infected lymph tissues were mixed with and without compost. After 90 days, M. avium subsp. paratuberculosis remained viable despite exposure to temperatures typically higher than that achieved in compost. In conclusion, it is unlikely composting can be used as a means of inactivating M. avium subsp. paratuberculosis associated with cattle mortalities.

Coexistence of Granulomatous Enteric Inflammation and Neoplasia in an Adult Sheep

A 7-year-old dairy sheep suffering from chronic loss of weight without diarrhea or anorexia was euthanized after failing to respond to any treatment (antibiotic and antiparasitic). The main findings at the necropsy of this animal were multifocal miliary nodules in several organs, mainly in the Peyer's patches of the small intestine, and a segmental thickening of the jejunal wall. Histologic examination of the samples taken at the necropsy showed a multifocal chronic granulomatous inflammation, with mineralization and caseous necrosis at the core of the larger granulomas and scarce intrahistiocytic acid-fast bacilli consistent with a disseminated digestive tuberculosis. Polymerase chain reaction and bacteriological culture from these samples confirmed Mycobacterium avium subsp avium to be the etiologic agent of this infection. Histologically, the cause of the segmental thickening of the jejunal wall was found to be a small intestine adenocarcinoma, which in some areas coexisted with the granulomatous lesion.

Evaluation of the effects of a killed whole-cell vaccine against Mycobacterium avium subsp paratuberculosis in 3 herds of dairy cattle with natural exposure to the organism

To evaluate effects of vaccination with a killed whole-cell vaccine against Mycobacterium avium subsp paratuberculosis (MAP) on fecal shedding of the organism, development of clinical paratuberculosis (Johne's disease [JD]), milk production, measures of reproduction, and within-herd longevity of dairy cattle naturally exposed to MAP. Controlled clinical trial. 200 vaccinated and 195 unvaccinated (control) dairy cows from 3 herds in Wisconsin. Every other heifer calf born in each herd received the MAP vaccine; 162 vaccinates and 145 controls that had ≥ 1 lactation were included in analyses. Bacteriologic culture of fecal samples for MAP was performed annually for 7 years; results were confirmed via histologic methods and PCR assay. Production records and culture results were evaluated to determine effects of vaccination on variables of interest in study cows. Annual whole-herd prevalence of MAP shedding in feces was also determined. Vaccinates had a significantly lower hazard of testing positive for MAP via culture of fecal samples than did controls over time (hazard ratio, 0.57; 95% confidence interval, 0.34 to 0.97). Fewer vaccinates developed clinical JD than did controls (n = 6 and 12, respectively), but these differences were nonsignificant. Overall within-herd longevity, total milk production, and calving-to-conception intervals were similar between vaccinates and controls. In all herds, prevalence of MAP shedding in feces decreased over time. Vaccination with a killed whole-cell MAP vaccine appeared to be an effective tool as part of a program to control the spread of JD in dairy cattle.

Pathological Findings in Young and Adult Sheep Following Experimental Infection With 2 Different Doses of Mycobacterium avium Subspecies paratuberculosis

Mycobacterium avium subsp paratuberculosis (Map) is assumed to infect young ruminants; however, little is known concerning the possibility of adult animals becoming infected. An experimental infection was conducted to establish the effect of age and doses of Map on susceptibility to paratuberculosis in sheep. Sixteen of twenty-four 1.5-month-old Churra lambs and 23 of 30 adult ewes (from 2-11 years old) were orally challenged with an ovine field strain of Map. Thirteen ewes and 8 lambs were infected with a high dose (HD) and 10 adult sheep and 8 lambs with a low dose (LD) of Map. The remaining animals were unchallenged controls. Animals were euthanized at 110 to 120 and 210 to 220 days postinfection. Histological, bacteriological, and nested polymerase chain reaction (PCR) studies were conducted in samples of intestine and related lymphoid tissue (Peyer patches, lymph nodes). Animals were classified according to their lesions. The number of granulomas was counted in 3 tissue sections from each sample. Only the HD groups showed lesions associated with paratuberculosis (92.3% of ewes and 100% of lambs). Adults had lesions characterized by few small demarcated focal granulomas restricted to the lymphoid tissue, whereas granulomas were more numerous and larger, appearing in the lamina propria unrelated to lymphoid tissue, in the lambs. Only HD-infected lambs were positive to culture, whereas nested PCR also detected positive HD ewes and some LD animals. These results suggest that adult sheep can become infected by Map, as seen by the development of lesions, but they are focal and restricted to the lymphoid tissue.

Histopathological study of avian tuberculosis in naturally infected domestic pigeons with Mycobacterium avium subsp. avium.

The aim of this study was to investigate the histopathology of avian tuberculosis in naturally infected domestic pigeons (Columba livia var. domestica) with Mycobacterium aviumsubsp.avium. Avian tuberculosis is one of the most important diseases that affect all species of birds, and is most often caused by mycobacterium avium and mycobacterium genavense. Eighty out of more than 600 pigeons were selected based on their clinical signs and poor healthconditions and under standard conditions were euthanized, necropsied, and followed by bacterial culture on specific media for Mycobacterium avium subsp.avium. Fifty Mycobacterium avium subsp. Avium was isolated from pigeons. All acid fast bacilli isolates were tested by the PCR assays targeting the 16S rRNA, IS1245 and IS901 genes. After definitive identification of Mycobacterium aviumsubsp. avium by culturing and PCR assay, 45 fixed samples including liver, gizzard, proventriculus, intestines, kidneys and lungs from positive pigeons were subjected for histopathology studies. Tissues sections were prepared as usual and stained by hematoxylin and eosin, Ziehl-Neelsen and Congo red. Based on gross findings, liver and intestines were the most affected organs. Histologically, caseative uncalcified granulomatous inflammation was noted in the affected organs. Also histopathology examinations showed that most of the granulomatous lesions in the lungs were in microscopic size and it seems that lungs were affected more than it was expected. In Ziehl-Neelsen’s staining, a large number of acid-fast bacilli were observed within multinucleated giant cells and in necrotic areas. Also in Congo red staining, deposition of amyloid in liver and kidneys sections were observed. In conclusion, histopathology findings were typical of avian tuberculosis, including acid fast bacilli and uncalcified caseous necrosis centers which were surrounded by multinucleated giant cells, macrophages and lymphocytes.

Relationship between Paratuberculosis and the microelements Copper, Zinc, Iron, Selenium and Molybdenum in Beef Cattle

To study the deficiency of minerals and its relationship with Paratuberculosis, blood, serum, and fecal samples were obtained from 75 adult bovines without clinical symptoms of the disease and from two bovines with clinical symptoms of the disease, from two beef herds with a previous history of Paratuberculosis in the Province of Buenos Aires, Argentina. Serum samples were processed by ELISA and feces were cultured in Herrolds medium. Copper, zinc and iron in serum were quantified by spectrophotometry and selenium was measured by the activity of glutathione peroxidase. We also determined copper, zinc, iron and molybdenum concentrations in pastures and the concentration of sulfate in water. Mycobacterium avium subsp paratuberculosis (Map) was isolated from 17.3% of fecal samples of asymptomatic animals and from the fecal samples from the two animals with clinical symptoms. All the Map-positive animals were also ELISA-positive or suspect, and among them, 84.6% presented low or marginal values of selenium and 69.2% presented low or marginal values of copper. The two animals with clinical symptoms, and isolation of Map from feces and organs were selenium-deficient and had the lowest activity of glutathione peroxidase of all the animals from both herds. All the animals negative to Map in feces and negative to ELISA had normal values of Se, while 13.8% of animals with positive ELISA or suspect and culture negative presented low levels of Se. Half of the animals that were negative both for ELISA and culture in feces were deficient in copper but none of them presented low values of selenium. The content of molybdenum and iron in pasture was high, 2.5 ppm and 1.13 ppm in one herd and 2.5 ppm and 2.02 ppm in the other, respectively, whereas the copper:molybdenum ratio was 1.5 and 5.2, respectively. These results do not confirm an interaction between imbalances of the micronutrients and clinical Paratuberculosis, but show evidence of the relationship between selenium deficiencies in animals with Map infection and ELISA positive results.

Evaluation of environmental culture to determine herd status for Johne's disease on Atlantic Canadian dairy farms

Environmental culture (EC) is a non-invasive and relatively inexpensive method to detect the presence of Mycobacterium avium subsp paratuberculosis (MAP) in a herd. Research into the test characteristics of EC has generally focused on large, MAP-positive herds. This study was designed to evaluate the test characteristics of EC within typical Atlantic Canadian dairy herds. These herds tend to be smaller in size than herds that have been used in previous EC studies. Furthermore, many of these herds were suspected to be test-negative or have low MAP prevalence.

Isolation of Mycobacterium avium ssp paratuberculosis from the saliva of cattle

Johne's disease can be an economically devastating disease on dairy farms. Current risk assessment (RA)-based control programs use best management practices to prevent the transmission of the causative pathogen, Mycobacterium avium subsp paratuberculosis (MAP), between animals. Most effort is focused on the fecal-oral route of transmission between infectious dams and calves. Because MAP has been isolated from water troughs, the objective of this pilot study was to determine whether MAP can be detected in the saliva (a previously overlooked route of transmission) of infected cattle.

Effect of season and lactation stage on the diagnostic sensitivity of direct real-time PCR assay for detection of fecal shedding of Mycobacterium avium subsp paratuberculosis

Mycobacterium avium subsp paratuberculosis (MAP) is the causative organism of Johne's disease. Dairy Farmers of Canada lists this production-limiting disease as one of the top two animal health priorities for the Canadian dairy industry. Infection of newborn calves from ingestion of MAP-infected feces, colostrum, or milk is considered the main route of transmission and a major concern in a herd. Current diagnostics rely on identification of the bacterium in feces via culture and molecular tests or identification of MAP antibodies in milk or serum via enzyme-linked immunosorbent assays (ELISA). However, these tests are inadequate to meet industry needs for herd biosecurity and environmental-transmission control of MAP, especially when MAP-infected cows are in the subclinical stages of the disease. Milk ELISAs have poor predictive values due to imperfect sensitivity and specificity, especially when used in herds with a low prevalence of MAP-infected cows. Although culture of fecal samples for MAP is currently the gold standard diagnostic test for identification of MAP-infected cows, the long incubation times, costs, and intermittent shedding of MAP in feces hinder its use as an efficient screening tool. The goal of this study was to assess how shedding patterns of MAP in feces vary with lactation stage and season, as determined via direct real-time polymerase chain reaction (PCR) assay.

Evaluation of the milk ELISA to determine herd status for Johne's Disease

Results from individual cow milk ELISAs can be used to determine herd status for Mycobacterium avium subsp paratuberculosis (MAP). Milk samples for the MAP ELISA are less invasive to collect than fecal samples for MAP culture. Moreover, MAP results from the milk ELISA can be obtained quicker and at less expense than MAP results from the culture of feces. The objective of this study was to evaluate the test characteristics of the milk ELISA for determination of herd MAP status in Atlantic Canadian dairy herds. It was expected that many of the MAP-positive herds in this region had low prevalence, and it was of particular interest to evaluate the performance of the milk ELISA within these low-prevalence MAP herds.

First isolation of Mycobacterium avium subsp paratuberculosis from commercial pasteurized milk in Argentina

Mycobacterium avium subsp paratuberculosis was isolated from two out of seventy samples (2.86 %) of pasteurized and ultra-pasteurized milk. The isolates were positives to IS900 PCR and showed a C17 RFLP pattern, the most prevalent in Argentina. The present study is the first report of Mycobacterium avium subsp paratuberculosis culture from pasteurized milk in Argentina.

Proteomic analysis of plasma from Holstein cows testing positive for mycobacterium avium subsp. Paratuberculosis (MAP)

Johne's disease (JD) is a widespread and economically important chronic inflammatory disease of the small intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Although there are several techniques available for diagnosis of JD, their sensitivity is questionable. New proteome profiling methods, such as serum/plasma protein fingerprinting by 2-Dimensional Fluorescence Difference Gel Electrophoresis (2D-DIGE), may therefore be useful for identifying novel protein biomarkers of MAP infection. In this study, plasma samples were collected from 380 Holstein cows and screened for the presence of MAP infection using the M.pt. Johne's antibody Kit (IDEXX). Five negative (MAP-), and 5 strongly positive (MAP+) cows were selected for proteomic analysis. Highly abundant proteins were depleted from the plasma samples using the ProteoMiner technology (Bio-Rad) to enhance the resolution of low abundance proteins. Plasma samples from MAP-, MAP+, and a pooled internal control were labelled with different fluorescent dyes and separated based on their isoelectrical point (IP) and then their molecular weight. Gel images of the fluorescent plasma protein maps were acquired using a Typhoon scanner and analyzed using the DeCyder software. Proteins that were differentially expressed were excised from the gels, trypsin digested, and subjected to MS/MS analysis for identification. Six proteins were identified as being up-regulated at least 2-fold in MAP+ cows including: transferrin, gelsolin isoforms α & β (actin binding protein - ABP), complement subcomponent C1r, complement component C3, amine oxidase - copper containing 3 (AOC3), and coagulation factor II (thrombin) (p<0.05). Two proteins that were down-regulated approximately 2-fold in the MAP+ cows included coagulation factor XIII -B polypeptide (COAFXIII), and fibrinogen γ chain (FGG) and its precursor.

Immunogenicity evaluation of in silico identified Mycobacterium avium subsp paratuberculosis recombinant proteins that were upregulated under stress conditions (160.3)

Abstract Mycobacterium avium subsp paratuberculosis (MAP) is the causative agent of Johne’s disease (JD) in ruminants. It is known to enter a dormant phase outside the host typically on soil and survive inside the host macrophage. In vitro experiments have reported differential regulation of certain MAP proteins when exposed to stressors similar to dormancy. It is believed that in vivo regulation of dormancy genes and associated proteins by MAP may play an important role in evading the host defence mechanisms and the host may also mount an immune response against these dormancy related proteins. Evaluation of such proteins may provide insight to host-pathogen interaction during the course of MAP infection and may be of diagnostic interest. A group of dormancy genes upregulated under stress conditions were examined using in silico analysis to identify B and T-cell epitopes. Five potential candidate genes based on epitope prediction results were selected and cloned. Recombinant proteins were produced, purified and evaluated for their immunogenicity using a panel of sera from sheep with a spectrum of JD and sheep free of MAP infection by detection of serum antibodies. Individually, the five proteins were found to have the ability to partially discriminate between sera from sheep unexposed and exposed to MAP infections. This study showed that the MAP proteins upregulated under in vitro stress conditions were recognised by host immune system.

Effect of lipoarabinomannan from Mycobacterium avium subsp avium in Freund’s incomplete adjuvant on the immune response of cattle

The aim of the present study was to determine whether lipoarabinomannan (LAM), in combination with Freund's incomplete adjuvant (FIA), was able to improve cell-mediated and antibody-mediated immune responses against ovalbumin (OVA) in cattle. Twenty-three calves were assigned to four treatment groups, which were subcutaneously immunized with either OVA plus FIA, OVA plus FIA and LAM from Mycobacterium avium subsp avium, FIA plus LAM, or FIA alone. Lymphoproliferation, IFN-γ production and cell subpopulations on peripheral blood mononuclear cells before and 15 days after treatment were evaluated. Delayed hypersensitivity was evaluated on day 57. Specific humoral immune response was measured by ELISA. Inoculation with LAM induced higher levels of lymphoproliferation and IFN-γ production in response to ConA and OVA (P < 0.05). Specific antibody titers were similar in both OVA-immunized groups. Interestingly, our results showed that the use of LAM in vaccine preparations improved specific cell immune response evaluated by lymphoproliferation and IFN-γ production by at least 50 and 25%, respectively, in cattle without interfering with tuberculosis and paratuberculosis diagnosis.

Evaluation of an alternative method of herd classification for infection with paratuberculosis in cattle herds in the United States

To develop a better system for classification of herd infection status for paratuberculosis (Johne's disease [JD]) in US cattle herds on the basis of the risk of potential transmission of Mycobacterium avium subsp paratubeculosis. Simulated data for herd size and within-herd prevalence; sensitivity and specificity for test methods obtained from consensus-based estimates. Interrelationships among variables influencing interpretation and classification of herd infection status for JD were evaluated by use of simulated data for various herd sizes, true within-herd prevalences, and sampling and testing methods. The probability of finding ≥ 1 infected animal in herds was estimated for various testing methods and sample sizes by use of hypergeometric random sampling. 2 main components were required for the new herd JD classification system: the probability of detection of infection determined on the basis of test results from a sample of animals and the maximum detected number of animals with positive test results. Tables were constructed of the estimated probability of detection of infection, and the maximum number of cattle with positive test results or fecal pools with positive culture results with 95% confidence for classification of herd JD infection status were plotted. Herd risk for JD was categorized on the basis of 95% confidence that the true within-herd prevalence was ≤ 15%, ≤ 10%, ≤ 5%, or ≤ 2%. Analysis of the findings indicated that a scientifically rigorous and transparent herd classification system for JD in cattle is feasible.

Identification and Characterization of a Spore-Like Morphotype in Chronically Starved Mycobacterium avium Subsp. Paratuberculosis Cultures

Mycobacteria are able to enter into a state of non-replication or dormancy, which may result in their chronic persistence in soil, aquatic environments, and permissive hosts. Stresses such as nutrient deprivation and hypoxia provide environmental cues to enter a persistent state; however, a clear definition of the mechanism that mycobacteria employ to achieve this remains elusive. While the concept of sporulation in mycobacteria is not novel, it continues to spark controversy and challenges our perceptions of a non-replication. We investigated the potential role of sporulation in one-year old broth cultures of Mycobacterium subsp. paratuberculosis (MAP).We show that dormant cultures of MAP contain a mix of vegetative cells and a previously unknown morphotype resembling a spore. These spore-like structures can be enriched for using sporulating media. Furthermore, purified MAP spore forms survive exposure to heat, lysozyme and proteinase K. Heat-treated spores are positive for MAP 16SrRNA and IS900. MAP spores display enhanced infectivity as well as maintain acid-fast characteristics upon germination in a well-established bovine macrophage model. This is the first study to demonstrate a new MAP morphotype possessing spore-like qualities. Data suggest that sporulation may be a viable mechanism by which MAP accomplishes persistence in the host and/or environment. Thus, our current understanding of mycobacterial persistence, pathogenesis, epidemiology and rational drug and vaccine design may need to be reevaluated.