Paratuberculosis Research Articles

Genomic epidemiology of Mycobacterium avium subsp. paratuberculosis isolates from Canadian dairy herds provides evidence for multiple infection events.

Mycobacterium avium subsp. paratuberculosis (MAP) is the pathogen responsible for paratuberculosis or Johne's Disease (JD) in ruminants, which is responsible for substantial economic losses worldwide. MAP transmission primarily occurs through the fecal-oral route, and the introduction of an MAP infected animal into a herd is an important transmission route. In the current study, we characterized MAP isolates from 67 cows identified in 20 herds from the provinces of Quebec and Ontario, Canada. Whole genome sequencing (WGS) was performed and an average genome coverage (relative to K-10) of ∼14.9 fold was achieved. The total number of SNPs present in each isolate varied from 51 to 132 and differed significantly between herds. Isolates with the highest genetic variability were generally present in herds from Quebec. The isolates were broadly separated into two main clades and this distinction was not influenced by the province from which they originated. Analysis of 8 MIRU-VNTR loci and 11 SSR loci was performed on the 67 isolates from the 20 dairy herds and publicly available references, notably major genetic lineages and six isolates from the province of Newfoundland and Labrador. All 67 field isolates were phylogenetically classified as Type II (C-type) and according to MIRU-VNTR, the predominant type was INMV 2 (76.1%) among four distinct patterns. Multilocus SSR typing identified 49 distinct INMV SSR patterns. The discriminatory index of the multilocus SSR typing was 0.9846, which was much higher than MIRU-VNTR typing (0.3740). Although multilocus SSR analysis provides good discriminatory power, the resolution was not informative enough to determine inter-herd transmission. In select cases, SNP-based analysis was the only approach able to document disease transmission between herds, further validated by animal movement data. The presence of SNPs in several virulence genes, notably for PE, PPE, mce and mmpL, is expected to explain differential antigenic or pathogenetic host responses. SNP-based studies will provide insight into how MAP genetic variation may impact host-pathogen interactions. Our study highlights the informative power of WGS which is now recommended for epidemiological studies and to document mixed genotypes infections.

Improved DNA Amplification of the Hallmark IS900 Element in Mycobacterium avium subsp. paratuberculosis: a Reexamination Based on Whole-Genome Sequence Analysis.

Amplification of the IS900 multicopy element is a hallmark nucleic acid-based diagnostic test for Mycobacterium avium subsp. paratuberculosis, which causes Johne's disease in ruminants. This assay is frequently used to determine the presence of the bacterium in feces of infected cattle and sheep. Two IS900 primer sets developed in the 1990s were widely used for decades, and their use has continued in current studies. However, these primers were developed prior to the availability of complete genome sequences. Recent sequence analysis of the binding locations for one primer pair (P90/P91) identified errors and binding inefficiencies that can be easily corrected to further increase detection sensitivity. The P90 primer is missing two nucleotides that should be present near the 3' end, and it does not bind all copies of IS900 due to 5' deletions at some IS900 loci. These IS900 primer pairs, along with newly developed primers, were tested by real-time PCR on purified genomic DNA to determine which primer set performed the best and how primer design errors affect amplification efficiencies. The newly designed PCR primer set (JB5) showed increased sensitivity by two to three quantification cycles using purified genomic DNA and was similar in efficiency to 150C/921. These tests were extended using DNA from feces and tissues of infected cows, which showed similar results. Finally, a 167-bp partial duplication of IS900 was found in type I strains. Although P90 and P91 primers successfully amplify M. avium subsp. paratuberculosis DNA, their use should be discontinued in favor of more efficient primer pairs in future studies. IMPORTANCE This study is an example of how applied genomic analysis can aid diagnostic test improvements. Detection of Mycobacterium avium subsp. paratuberculosis infection of livestock prior to the appearance of clinical disease signs is very difficult but essential for identifying animals shedding the bacterium to prevent transmission of Johne's disease. Total M. avium subsp. paratuberculosis quantity in the feces as determined by real-time PCR (qPCR) using the IS900 target indicates bacterial shedding status and potential for transmission of the pathogen. However, legacy primers designed prior to the availability of complete genome sequences that are used in these tests to detect M. avium subsp. paratuberculosis were based on data from only a single copy of IS900 and not considering all copies collectively as a group. This approach resulted in primer design errors which can be easily corrected to improve test sensitivities. We tested original primers that contain these errors and their corrected versions by qPCR and showed improved sensitivity on purified genomic DNA as well as fecal and tissue samples. These findings may help detect the organism from environmental samples on farms where sensitivity is currently lacking.

Reproductive and seminal characteristics of Pelibuey rams infected with Mycobacterium avium subsp. paratuberculosis in the subclinical stage

Objective: To evaluate the reproductive and seminal characteristics of Pelibuey rams infected with Mycobacterium avium subsp. paratuberculosis in the subclinical phase.
 Design/methodology/approach: In order to determine whether paratuberculosis (PTB) affects the reproductive variables and seminal quality in Pelibuey rams naturally infected with Mycobacterium avium subsp. paratuberculosis (MAP) in the subclinical phase, weekly evaluations were conducted, over a period of two months, of the variables live weight, scrotal circumference, ejaculation latency and seminal characteristics: volume, pH, masal motility and progressive individual motility, concentration, live spermatozoids and dead spermatozoids, in five rams infected by MAP and a further five uninfected rams, of average weight and age 53.58 kg (± 3.26) and 2.91 years (± 0.59), respectively.
 Results: PTB was not found to affect the reproductive and seminal variables evaluated.
 Limitations of the study/implications: It would have been convenient to carry out the evaluation of the seminal characteristics for a longer time, however, the sampling was stopped in order to ensure the animal welfare of the rams as far as possible, since they began to show signs of PTB.
 Findings/Conclusions: The Pelibuey rams diagnosed with PTB in subclinical phase did not present any effect on the reproductive and seminal characteristics. Meanwhile, reproductive management can be carried out with PTB-infected Pelibuey lambs in a subclinical phase.

Characterization of early immune responses elicited by live and inactivated vaccines against Johne's disease in goats.

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease, a chronic debilitating condition affecting ruminants causing significant economic losses to the dairy industry. Available inactivated vaccines are not effective in controlling the disease and vaccinated animals can continue to infect newly born calves. Recently, we have shown that a live-attenuated vaccine candidate (pgsN) is protective in goats and calves following challenge with virulent strains of M. paratuberculosis. To decipher the dynamics of the immune responses elicited by both live-attenuated and inactivated vaccines, we analyzed key immunological parameters of goats immunized through different routes when a marker-less pgsN vaccine was used. Within a few weeks, the inactivated vaccine triggered the formation of granulomas both at the site of inoculation and in regional lymph nodes, that increased in size over time and persisted until the end of the experiment. In contrast, granulomas induced by the pgsN vaccine were small and subsided during the study. Interestingly, in this vaccine group, histology demonstrated an initial abundance of intra-histiocytic mycobacterial bacilli at the site of inoculation, with recruitment of very minimal T lymphocytes to poorly organized granulomas. Over time, granulomas became more organized, with recruitment of greater numbers of T and B lymphocytes, which coincided with a lack of mycobacteria. For the inactivated vaccine group, mycobacterial bacilli were identified extracellularly within the center of caseating granulomas, with relatively equal proportions of B- and T-lymphocytes maintained across both early and late times. Despite the differences in granuloma-specific lymphocyte recruitment, markers for cell-mediated immunity (e.g., IFN-γ release) were robust in both injected pgsN and inactivated vaccine groups. In contrast, the intranasal live-attenuated vaccine did not elicit any reaction at site of inoculation, nor cell-mediated immune responses. Finally, 80% of animals in the inactivated vaccine group significantly reacted to purified protein derivatives from M. bovis, while reactivity was detected in only 20% of animals receiving pgsN vaccine, suggesting a higher level of cross reactivity for bovine tuberculosis when inactivated vaccine is used. Overall, these results depict the cellular recruitment strategies driving immune responses elicited by both live-attenuated and inactivated vaccines that target Johne's disease.

Assessment of Paratuberculosis international official reporting in Europe using the information supplied to the WOAH by National Veterinary Services.

The present study characterizes the epidemiological situation of Paratuberculosis (PTB) in Europe during the last 24 years, using the information officially reported to the World Organisation for Animal Health (WOAH) by veterinary services of the European countries. The prevalence of PTB at country level was described during the study period. A Cox proportional hazards (PH) regression analysis was implemented to evaluate the notification behaviours. Results from this work indicate that the most affected countries are in Southern and Western Europe, whereas PTB presence appears lower in Northern and Eastern Europe. PTB was routinely declared as a notifiable disease in 65% of the countries. Less than 50% of the countries routinely implemented passive surveillance, and only 19%, reported active surveillance for disease detection. Results from the Cox PH regression indicate that the Gross National Income (GNI) per capita and the application of active surveillance significantly influence the recurrence of PTB reporting. In countries with lower and upper middle income, the hazard of recurrence is 0.13 and 0.18 times lower than in countries with high income. The hazard of recurrence in countries that infrequently and moderately applied active surveillance is 1.99 and 1.65 times higher than in countries that routinely applied active surveillance. Findings from this work highlight an important variation in reporting behaviours, disease status and surveillance across Europe.

First report of paratuberculosis (Johne's disease) in livestock farms of river buffaloes (Bubalus Bubalis) in Nineveh, Iraq.

The present study was designed to investigate Mycobacterium avium subsp. paratuberculosis (MAP) in dairy buffalo herds from six different geographical areas in Nineveh, Iraq. A total of 87 individual faecal samples from river buffaloes, representing 12 dairy herds, were investigated for detection of MAP using cultural, Ziehl‑Neelsen and MAP‑specific PCR‑based methods. Overall, MAP was detected at a higher frequency at herd‑level (4/12; 33%) compared to the total individual faecal samples (14/87; 16%) with a cell density ranging from 101 to 103 CFU g‑1. A significantly (p < 0.05) higher frequency (9/17; 53%) of MAP was observed in faecal samples collected from clinically diseased as compared to healthy (5/70; 7%) buffaloes selected for the study. However, no statistically significant difference (p ≥ 0.05) was observed in the frequency of MAP occurrence between clinical (9; 64%) and apparently healthy (5; 36%) cases. This report, which is the first MAP study based on data from Iraqi dairy buffalo herds suggests that MAP transmission is a significant health risk for grazing livestock. In conclusion, this study would help farm owners and regulatory authorities to realise the importance of developing and applying best farm management practices in order to prevent transmission of MAP to healthy animals and the environment. In addition, effective diagnostic tests should be taken into account when carrying out the screening tests.

Management practices associated with Johne's bulk tank milk ELISA positivity

Johne's disease (JD) control is often based on the culling of positive animals and the adoption of management practices that minimize exposure of young stock to the pathogen (Mycobacterium avium ssp. paratuberculosis). Throughout 2010 to 2013, the province of Ontario, Canada, instituted a voluntary Johne's control program consisting of whole-herd testing and risk assessment. The JD risk assessment evaluated 5 management areas to characterize herd JD risk. Using a modified milk ELISA technique with an optical density cut-off of 0.089, province-wide bulk tank milk (BTM) testing was used to assess the prevalence of JD high-risk herds at the end of the control program and again 4 yr after its completion. Approximately 71% of Ontario bulk tanks were classified as positive in 2017 compared with roughly 46% in 2013. In 2019, the same JD risk assessment used in the original program was readministered on 180 Ontario dairy farms. Using this cross-sectional approach, logistic regression models were built using data from the original program risk assessment and follow-up risk assessment as well as the BTM ELISA results to determine management factors associated with the control of JD. We demonstrated that management of the maternity area is an important factor in the control of Johne's disease. Although it is believed that the highest risk group for JD infection is calves under 6 mo, the cleanliness scores of older heifers and their exposure to mature cow manure was significantly associated with JD control; farms with highly contaminated weaned and bred heifers and those that had exposure to mature cow manure were more likely to be unsuccessful in their JD control efforts. Careful management of young calves appears to be important for JD control, and this management should continue even after calves have left the maternity area.

Evaluation of total sialic acid, paraoxonase activity and malondialdehyde in cows with subclinical paratuberculosis

In this study, it was aimed to evaluate total sialic acid (TSA), paraoxonase activity (PON1), malondialdehyde (MDA) and some serum biochemical parameters in cows with subclinical paratuberculosis. Paratuberculosis (PT) test was performed on 500 cows aged 2-6 years by ELISA method. Twenty-four cows diagnosed with paratuberculosis constituted the PT group and 12 healthy cows constituted the control group. According to the results, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and UREA levels were found to be statistically higher (p &lt;0.05) in the group with paratuberculosis compared to the control group. Iron (Fe) levels were found to be lower than the control group (p&gt; 0.05). Compared to the control group, there was a decrease in PON1 activity and a significant increase in TSA and MDA levels in the PT group (p &lt;0.05). In conclusion, in subclinical paratuberculosis infection, the long incubation period and the absence of clinical findings until advanced stages constitute a handicap. However, our findings show that the determination of lipid peroxidation (MDA), PON1 activity and TSA concentration caused by the inflammatory and oxidative stress state caused by the infection can be used as new biomarkers for this infection.

Molecular and serological survey of paratuberculosis in cattle in selected districts of Western Uganda

Knowledge of Mycobacterium avium subsp. paratuberculosis (MAP) herd infection status is important to plan appropriate control and prevention strategies for Paratuberculosis (PTB); however, in Uganda MAP infection status of most herds is unknown. This study aimed at determining the MAP infection status of cattle herds and the associated risk factors for MAP infection in six western districts of Uganda. The survey covered a total of 93 herds where faecal and blood samples were collected from 1814 cattle. A Recombinase Polymerase Amplification (RPA) and an antibody-based (ELISA) assays were used to test for the presence of MAP DNA in faeces and MAP antibodies in serum, respectively. The apparent cow-level prevalence of MAP infection was 3.2 and 2.7% using ELISA and RPA respectively and the true cow-level prevalence using ELISA and RPA was 4.9 and 3% respectively. A herd-level prevalence of 43% (ELISA) and 40.8% (RPA) and a within-herd prevalence of 3.8 ± 2.1% based on ELISA were obtained. Among the risk factors investigated, long dry spells were significantly associated with high MAP infection (p < 0.05). These results indicate that MAP is actively present in most areas where surveillance was carried out. This poses a serious threat to the livestock industry and potentially to public health as MAP is highly suspected to play a role in the pathogenesis of several diseases in humans. Other areas of the country are to be surveyed as well in order to establish full data on MAP infection status to enable interventions for the control and prevention of the disease.

Culture of Mycobacterium avium subsp. paratuberculosis: challenges, limitations and future prospects.

Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis (Johne's disease) in ruminants and is suspected to be involved in the development of Crohn's disease and several autoimmune disorders. As such, sensitive and specific MAP detection methods are required to confirm infection in animals and identify potential sources of animal and human exposure. Despite recent developments in immunological and nucleic acid-based detection methods, culture-based detection of MAP remains the 'gold standard' against which the sensitivity and specificity of other detection methods are measured. However, not all culture-based approaches are equivalent in terms of detection capability, which can lead to errors in the evaluation of other detection methods. This review will provide an overview of the chronological development of culture methods for MAP, and will consider the unique growth requirements of MAP, the merits of solid versus liquid culture media, the relative performance of the commonly used MAP culture media, and sample preparation/decontamination protocols for different sample types. The limitations of current MAP culture methods and prospects for improvements are discussed.

Genetic Features of Mycobacterium avium subsp. paratuberculosis Strains Circulating in the West of France Deciphered by Whole-Genome Sequencing.

Paratuberculosis is a chronic infection of the intestine, mainly the ileum, caused by Mycobacterium avium subsp. paratuberculosis in cattle and other ruminants. This enzootic disease is present worldwide and has a negative impact on the dairy cattle industry. For this subspecies, the current genotyping tools do not provide the needed resolution to investigate the genetic diversity of closely related strains. These limitations can be overcome by the application of whole-genome sequencing (WGS), particularly for clonal populations such as M. avium subsp. paratuberculosis. The purpose of the present study was to undertake a WGS analysis with a panel of 200 animal field M. avium subsp. paratuberculosis strains selected based on a previous large-scale longitudinal study of Prim'Holstein and Normande dairy breeds naturally infected with M. avium subsp. paratuberculosis in the West of France. The pangenome analysis revealed that M. avium subsp. paratuberculosis has a closed pangenome. The phylogeny, based on alignment of 2,786 nonhomoplasic single nucleotide polymorphisms (SNPs), showed that the strain population is structured into three clades independently of the cattle breed or geographic distribution. The increased resolution of phylogeny obtained by WGS confirmed the homoplasic nature of the markers variable-number tandem repeat (VNTR) and short sequence repeat (SSR) used for M. avium subsp. paratuberculosis genotyping. These phylogenetic data also revealed independent introductions of the different genotypes in two main waves since at least 2003. WGS applied to this sampling demonstrated the presence of mixed infections in herds and at the individual animal level. Collectively, the phylogeny results inferred with French isolates compared to M. avium subsp. paratuberculosis isolates from around the world suggest introductions of M. avium subsp. paratuberculosis genotypes through the animal trade. Relationships between genetic traits and epidemiological data can now be investigated to better understand transmission dynamics of the disease. IMPORTANCE Mycobacterium avium subsp. paratuberculosis causes Johne's disease in ruminants, which is present worldwide and has significant negative impacts on the dairy cattle industry and animal welfare. Prevention and control of M. avium subsp. paratuberculosis infection are hampered by knowledge gaps in strain virulence, genotype distribution, and transmission dynamics. This work has revealed new insights into M. avium subsp. paratuberculosis strains currently circulating in western France and how they are related to strains circulating globally. We applied whole-genome sequencing (WGS) to obtain comprehensive information on genome evolution and discrimination of closely related strains. This approach revealed the history of M. avium subsp. paratuberculosis infection in France, refined the pangenomic characteristics of M. avium subsp. paratuberculosis, and demonstrated the existence of mixed infection in animals. Finally, this study identified predominant genotypes, which allow a better understanding of disease transmission dynamics. This information will facilitate tracking of this pathogen on farms and across agricultural regions, thus informing transmission pathways and disease control points.

Prevalence of Mycobacterium avium Subsp. paratuberculosis in Feral Pigeons (Columba livia) Associated with Difficulties Controlling Paratuberculosis in a Bovine Herd (Fighting Bull Breed).

A bovine herd with a high prevalence of paratuberculosis (PTB) cohabiting with a population of pigeons was studied (2011−2020). After finding the disease in 2011, annual monitoring was performed in 2012−2014 by obtaining blood samples for ELISA and intradermal tuberculinization (IT) tests for Mycobacterium avium subsp. paratuberculosis (MAP). Positive animals were eliminated. PTB prevalence dropped from 10% to 0% but returned to similar values (9.5%) after 6 years without tests. In all animals, Ac values according to the optical density (OD) determined by ELISA increased each year and could be used to isolate herds close to the cutoff point to improve PTB control. Possible reservoirs were considered after evaluating the little success of the PTB control program, and the population of feral pigeons was studied. Specifically, 10% of the pigeon population (n = 13) was necropsied. Samples of intestine, feces, and foot skin for PCR study for MAP and samples of terminal intestine for histopathological analysis were taken. Eleven pigeons were PCR-positive against MAP, in the intestine (10/11), foot skin (3/11), and feces (1/11). The presence of MAP in pigeon feet could demonstrate its role as a mechanical disseminator of PTB, while the presence in pigeon intestine and feces could also suggest its role as a reservoir.

First Report on Ovine Paratuberculosis in the Sudan: Diagnosis Using Different Techniques.

Paratuberculosis (PTB) has been reported in the Sudan in cattle and goats for more than 50 years but has never been reported in sheep. However, suspicion of the disease in a breeding flock of sheep in Khartoum North locality was made due to a history of unknown cause of loss of weight. Blood and faecal samples were collected from all animals (N = 59): harvested sera were tested for anti-Mycobacterium avium subsp. paratuberculosis (MAP) antibodies by Enzyme Linked Immunosorbent Assay (ELISA); faeces were screened for acid-fast bacilli by Ziehl-Neelsen staining, tested for MAP DNA by recombinase polymerase amplification (RPA) and some faecal samples were cultured for MAP isolation. Typical MAP acid-fast bacilli were seen in 10.2% (6/59) of the faecal smears, 37.5% of the tested faecal samples (12/32) were positive for MAP DNA and only 3 (5.1%) animals were seropositive for MAP. MAP positive cultures were obtained from 2 out the 6 samples showing typical MAP acid-fast bacilli; the isolates were confirmed by real-time PCR and sequencing. As sheep are animals of utmost economic importance as the main export animals for the country, this first report of ovine PTB warrants special considerations and more investigations for planning control programmes of the disease.

Investigating effective testing strategies for the control of Johne's disease in western Canadian cow-calf herds using an agent-based simulation model.

Johne's disease is an insidious infectious disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Johne's disease can have important implications for animal welfare and risks causing economic losses in affected herds due to reduced productivity, premature culling and replacement, and veterinary costs. Despite the limited accuracy of diagnostic tools, testing and culling is the primary option for controlling Johne's disease in beef herds. However, evidence to inform specific test and cull strategies is lacking. In this study, a stochastic, continuous-time agent-based model was developed to investigate Johne's disease and potential control options in a typical western Canadian cow-calf herd. The objective of this study was to compare different testing and culling scenarios that included varying the testing method and frequency as well as the number and risk profile of animals targeted for testing using the model. The relative effectiveness of each testing scenario was determined by the simulated prevalence of cattle shedding MAP after a 10-year testing period. A second objective was to compare the direct testing costs of each scenario to identify least-cost options that are the most effective at reducing within-herd disease prevalence. Whole herd testing with individual PCR at frequencies of 6 or 12 months were the most effective options for reducing disease prevalence. Scenarios that were also effective at reducing prevalence but with the lowest total testing costs included testing the whole herd with individual PCR every 24 months and testing the whole herd with pooled PCR every 12 months. The most effective method with the lowest annual testing cost per unit of prevalence reduction was individual PCR on the whole herd every 24 months. Individual PCR testing only cows that had not already been tested 4 times also ranked well when considering both final estimated prevalence at 10 years and cost per unit of gain. A more in-depth economic analysis is needed to compare the cost of testing to the cost of disease, taking into account costs of culling, replacements and impacts on calf crops, and to determine if testing is an economically attractive option for commercial cow-calf operations.

Exploration of Solanum xanthocarpum Schrad. & Wendl. against Mycobacterium avium Subspecies paratuberculosis and Assessment of Its Immunomodulatory and Anti-Inflammatory Potential.

Mycobacterium avium subspecies paratuberculosis (MAP), being a dairy-borne pathogen, resistant of pasteurization and other sterilization techniques, is a major cause for development of inflammatory bowel disorders such as Johne's disease (JD) in dairy animals and Crohn's Disease (CD) in humans, for which no therapy is available to date. In the absence of effective therapy or a vaccine, management of CD has been accomplished by removal of the affected intestines. However, usually, even after removal of 2/3 of the intestine, CD reoccurs. Hence, there exists a need to develop an alternative therapy for such infection. The potential of herbals remains unexplored against MAP and related infections. Therefore, the conducted study is a novel initiative for the evaluation of anti-mycobacterial activity of bioactive extracts of Solanum xanthocarpum Schrad. & Wendl. against MAP infection. The said plant was authenticated according to the Ayurvedic Pharmacopoeia of India. Qualitative and quantitative evaluation of the extracts were done using chromatographic and spectroscopic techniques. Preliminary in vitro pharmacological assessments revealed the immunomodulatory and anti-inflammatory potential of the extracts. REMA assay was conducted to determine their anti-MAP activity along with determination of the best active extract. The hydro-alcoholic extract showed the best inhibition of MAP, providing a potential ray of hope against this emerging major pathogen of animals, and associated with Crohn's disease and other autoimmune disorders in human beings.

Genome-wide association study for antibody response to Mycobacterium avium ssp. paratubeculosis in goats.

Mycobacterium avium ssp. paratuberculosis (MAP), causes Johne's disease (JD), or paratuberculosis, a chronic enteritis of ruminants, which in goats is characterized by ileal lesions. The work described here is a case-control association study using the Illumina Caprine SNP50 BeadChip to unravel the genes involved in susceptibility of goats to JD. Goats in herds with a high occurrence of Johne's disease were classified as healthy or infected based on the level of serum antibodies against MAP, and 331 animals were selected for the association study. Goats belonged to the Jonica (157) and Siriana breeds (174). Whole-genome association analysis identified one region suggestive of significance associated with an antibody response to MAP on chromosome 7 (p-value=1.23 × 10-5 ). These results provide evidence for genetic loci involved in the antibody response to MAP in goats.

Cattle Review

Introduction: In this Cattle Review we consider open access papers on Johne's disease and the management of surplus dairy calves.

Descriptive analysis of the changes in Johne's disease management practices on Ontario dairy farms through repeat risk assessment.

Johne's disease (JD) control is often based on the culling of positive animals and the adoption of management practices that minimize exposure of calves to the pathogen Mycobacterium avium ssp. paratuberculosis (MAP). From 2010 to 2013, Ontario, Canada, instituted a voluntary Johne's control program consisting of whole-herd testing and a Risk Assessment and Management Plan (RAMP). The RAMP consisted of 38 questions that evaluated 5 different management areas to characterize herd risk for MAP introduction and within-herd spread. The RAMP produced a numerical score for each area, with higher scores associated with higher risk. The RAMP focused on animal purchases, calving management, calf management, and heifer and cow cleanliness and management. In the summer of 2019, the RAMP was repeated on 180 farms that had participated in the JD program of 2010 to 2013 and had bulk tank milk ELISA results from 2013 and 2017. This cross-sectional study demonstrated that many producers changed management practices over the 4- to 7-year period. Producers changed their cattle buying practices, with a reduction in purchasing from multiple sources and more herds refraining from buying in animals. However, overall scores were higher in 2019 than in 2013. The 2019 RAMP indicated that fewer farms were utilizing individual calving pens in 2019 than in 2013 (13% vs. 26%), yet more farms had policies in place to deal with sick or suspect JD cows entering the maternity area (92% vs. 74%). Management changes occurred over time, some of which represent increased risk (crowded maternity pens) and others decreased risk (closed herd, protocols in place for JD-positive cows) for MAP introduction and transmission. These results highlight the importance of frequent risk assessments and the documentation of changes to management practices on-farm as a means to assess herd disease risk more accurately.

Double immunofluorescence staining of whole-mount small intestinal mucosa samples as a tool for characterization of three-dimensional paratuberculosis granulomas

Bovine paratuberculosis (PTB) is a chronic granulomatous enteritis, caused by Mycobacterium avium subsp. paratuberculosis (Map). The progression of PTB from subclinical to the clinical stage of the disease is determined locally at the level of the granuloma, a host defence hallmark against mycobacterial infection. Therefore, in-depth characterization of distinct cell populations controlling granuloma formation is critical to understanding PTB progression. Confocal laser scanning microscopy (CLSM) has been extensively used to visualize two or more proteins of interest concomitantly within a variety of cellular structures. As such, it is an invaluable tool for the correct identification and characterization of different cell populations. In this study, a novel approach, CLSM of whole-mount small intestinal mucosa samples, is used to characterize three-dimensional (3-D) paratuberculosis granulomas and epithelioid macrophages. Detailed optimized procedures to perform CLSM in whole mount small intestinal mucosa samples and also in formalin fixed paraffin embedded (FFPE) intestinal tissue sections of Holstein Friesian cows presenting different types of PTB-associated histological lesions are described.

Identification of essential genes in Mycobacterium avium subsp. paratuberculosis genome for persistence in dairy calves.

To cause disease Mycobacterium avium subsp. paratuberculosis needs to enter mammalian cells, arrest phagosomal maturation and manipulate the host immune system. The genetic basis of the bacterial capacity to achieve these outcomes remains largely unknown. Identifying these genes would allow us to gain a deeper understanding of MAP's pathogenesis and potentially develop a live attenuated Johne's disease vaccine by knocking out these genes. MAP genes demonstrated to be essential for colonization in the natural host, ruminants, are unknown. Genome-wide transposon mutagenesis and high-throughput sequencing were combined to evaluate the essentiality of each coding region in the bacterial genome to survive in dairy calves. A saturated library of 3,852 MAP Tn mutants, with insertions in 56% of TA sites, interrupting 88% of genes, was created using a MycoMarT7 phagemid containing a mariner transposon. Six calves were inoculated with a high dose of a library of MAP mutants, 1011 CFUs, (input) at 2 weeks of age. Following 2 months of incubation, MAP cells were isolated from the ileum, jejunum, and their associated lymph nodes of calves, resulting in approximately 100,000 colonies grown on solid media across 6 animals (output). Targeted next-generation sequencing was used to identify the disrupted genes in all the mutants in the input pool and the output pool recovered from the tissues to identify in vivo essential genes. Statistical analysis for the determination of essential genes was performed by a Hidden Markov Model (HMM), categorizing genes into essential genes that are devoid of insertions and growth-defect genes whose disruption impairs the growth of the organism. Sequence analysis identified 430 in vivo essential and 260 in vivo growth-defect genes. Gene ontology enrichment analysis of the in vivo essential and growth-defect genes with the highest reduction in the tissues revealed a high representation of genes involved in metabolism and respiration, cell wall and cell processing, virulence, and information pathway processes. This study has systematically identified essential genes for the growth and persistence of MAP in the natural host body.