Estimation of the sensitivity and specificity of four serum ELISA and one fecal PCR for diagnosis of paratuberculosis in adult dairy cattle in New Zealand using Bayesian latent class analysis.

In New Zealand, a new diagnostic approach for the control of paratuberculosis in mixed aged milking cows has been developed using a combination of ELISA and quantitative fecal PCR (f-qPCR). Our analysis was designed to evaluate performance of these individual tests in infected or infectious mixed aged cows across the prevalence of infection typically encountered on NZ dairy farms and calculate test accuracy when used as a screening test of serological ELISAs for four separate antigens read in parallel followed by a confirmatory quantitative f-qPCR test. Data from a cross-sectional study of 20 moderate prevalence herds was combined with existing data from 2 low and 20 high prevalence herds forming a dataset of 3845 paired serum and fecal samples. Incidence of clinical Johne's disease (JD) was used to classify herds into three prevalence categories. High (≥ 3% annual clinical JD for the last three years), moderate (<3 - 1%) and low (<1% incidence for at least the last five years). Positive tests were declared if> 50 ELISA units and f-qPCR at two cut-points (≥1 × 104 genomes/mL or >1 × 103 genomes/mL). Fixed Bayesian latent class models at both f-qPCR cut-points, accounted for conditional independence and paired conditional dependence. Mixed models at both f-qPCR cut-points, using a different mechanism to account for conditional dependencies between tests were also implemented. Models (24 in number) were constructed using OpenBUGS. The aim was to identify Mycobacterium avium subsp. paratuberculosis (MAP) infected cows that met at least one of two criteria: shedding sufficient MAP in feces to be detected by f-qPCR or mounting a detectable MAP antibody response. The best fit to the data was obtained by modelling pairwise dependencies between tests in a fixed model or by accounting for dependencies in a mixed model at a fecal cut-off of ≥1 × 104 genomes/mL. Test performance differed with prevalence, but models were robust to prior assumptions. For the fixed model, at a prevalence of 0.29 (95 % probability interval (PI) = 0.25-0.33), as a screening plus confirmatory f-qPCR, post-test probability for disease in a positive animal was 0.84 (95 %PI = 0.80-0.88) and 0.16 (95 %PI = 0.15-0.18) for disease in a test negative animal. In low prevalence herds (0.01(95 %PI = 0.00-0.04)) the equivalent figures were 0.84 (95 %PI = 0.08-0.92) and 0.00 (95 %PI = 0.00-0.02). These results suggest this is a useful tool to control JD on dairy farms, particularly in herds with higher levels of infection, where the sampling and testing cost per animal is defrayed across more detected animals.

Control of Mycobacterium avium subsp. paratuberculosis infection on a New Zealand pastoral dairy farm.

BACKGROUND: Johne's disease is a major production limiting disease of dairy cows caused by infection with Mycobacterium avium subsp. paratuberculosis in calf-hood. The disease is chronic, progressive, contagious and widespread with no treatment and no cure. Economic losses arise from decreased productivity through reduced growth, milk yield, fertility and also capital losses due to premature culling or death. Control chiefly centers upon removing those animals which actively shed bacteria and protecting calves from infection. A prolonged pre-clinical shedding phase, lack of test sensitivity, organism persistence and abundance in the environment as well as management systems that expose susceptible calves to infection make control challenging, particularly in pastoral, seasonal dairy systems. Combining a novel testing strategy to remove infected cows along with limited measures to protect vulnerable calves at pasture, this study reports the successful reduction over a four-year period of seroprevalence of cows testing positive for MAP infection in a New Zealand pastoral dairy herd. RESULTS: For all age groups considered the apparent seroprevalence of cows testing positive decreased from 297 / 1,122 (26%) in 2013-2014, to 24 / 1,030 (2.3%) in 2016-2017. Over the same period, the apparent seroprevalence in primiparous cows decreased from 39 / 260 (15%) to 7 / 275 (2.5%) and in multiparous cows from 258 / 862 (29.9%) to 17 / 755 (2.3%). The reported proportion of calved cows culled annually from suspected clinical Johne's disease fell from 55 / 1,201 (5%) in the year preceding the control program to 5 / 1,283 (0.4%) in the final year of the study. CONCLUSIONS: On this farm, reduction in the prevalence of infection was achieved by reducing the infectious pressure through targeted culling of heavily shedding animals together with limited measures to protect calves at pasture from exposure to Mycobacterium avium subsp. paratuberculosis. Whilst greater protection of young animals through separation from infected cows and their colostrum and milk would have reduced the risk of neonatal infection, this study demonstrates, in this case, that these management measures while prudent were not essential for effective reduction in the prevalence of MAP infection.

Control of paratuberculosis: who, why and how. A review of 48 countries.

Paratuberculosis, a chronic disease affecting ruminant livestock, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It has direct and indirect economic costs, impacts animal welfare and arouses public health concerns. In a survey of 48 countries we found paratuberculosis to be very common in livestock. In about half the countries more than 20% of herds and flocks were infected with MAP. Most countries had large ruminant populations (millions), several types of farmed ruminants, multiple husbandry systems and tens of thousands of individual farms, creating challenges for disease control. In addition, numerous species of free-living wildlife were infected. Paratuberculosis was notifiable in most countries, but formal control programs were present in only 22 countries. Generally, these were the more highly developed countries with advanced veterinary services. Of the countries without a formal control program for paratuberculosis, 76% were in South and Central America, Asia and Africa while 20% were in Europe. Control programs were justified most commonly on animal health grounds, but protecting market access and public health were other factors. Prevalence reduction was the major objective in most countries, but Norway and Sweden aimed to eradicate the disease, so surveillance and response were their major objectives. Government funding was involved in about two thirds of countries, but operations tended to be funded by farmers and their organizations and not by government alone. The majority of countries (60%) had voluntary control programs. Generally, programs were supported by incentives for joining, financial compensation and/or penalties for non-participation. Performance indicators, structure, leadership, practices and tools used in control programs are also presented. Securing funding for long-term control activities was a widespread problem. Control programs were reported to be successful in 16 (73%) of the 22 countries. Recommendations are made for future control programs, including a primary goal of establishing an international code for paratuberculosis, leading to universal acknowledgment of the principles and methods of control in relation to endemic and transboundary disease. An holistic approach across all ruminant livestock industries and long-term commitment is required for control of paratuberculosis.

The effect of sub-clinical infection with Mycobacterium avium subsp. paratuberculosis on milk production in a New Zealand dairy herd.

BACKGROUND: Johne's disease is a major production limiting disease of dairy cows. The disease is chronic, progressive, contagious and widespread; there is no treatment and there is no cure. Economic losses arise from decreased productivity through reduced growth, milk yield and fertility and capital losses due to premature culling or death. This study attempts to address the effect of subclinical JD on milk production under New Zealand pastoral dairy farming conditions using a new testing approach. Blood samples were taken from all lactating animals from a single seasonally calving New Zealand dairy herd in the autumn of 2013 and 2014. Samples were subject to serological assay for antibodies to Mycobacterium avium subsp. paratuberculosis using a combination of four ELISA tests in parallel followed by selective quantitative fecal PCR to confirm the fecal shedding characteristics of ELISA positive cows. ELISA status was classified as Not-Detected, Low, Moderate or High and fecal PCR status as Not-Detected, Moderate or High. RESULTS: A mixed generalized regression model indicated that, compared to cows where MAP was not detected, daily milk solids production was 4% less for high ELISA positive cows (p = 0.004), 6% less for moderate fPCR cows (p = 0.036) and 12% less for high fPCR cows (p < 0.001). CONCLUSIONS: This study confirms that sub-clinical JD can have a significant impact on milk production and that the testing methodology used stratified the animals in this herd on their likely impact on production and disease spread. This allowed the farmer to prioritize removal of heavily shedding, less-productive animals and so reduce the risk of infection of young stock. This is the first longitudinal study based in New Zealand looking at the effect of Johne's infection status on daily milk production allowing for intermediary and confounding factors.

Bovine milk RNases modulate pro-inflammatory responses induced by nucleic acids in cultured immune and epithelial cells.

Activation of innate immune receptors by exogenous substances is crucial for the detection of microbial pathogens and a subsequent inflammatory response. The inflammatory response to microbial lipopolysaccharide via Toll-like receptor 4 (TLR4) is facilitated by soluble accessory proteins, but the role of such proteins in the activation of other pathogen recognition receptors for microbial nucleic acid is not well understood. Here we demonstrate that RNase4 and RNase5 purified from bovine milk bind to Salmonella typhimurium DNA and stimulate pro-inflammatory responses induced by nucleic acid mimetics and S. typhimurium DNA in an established mouse macrophage cell culture model, RAW264.7, as well as in primary bovine mammary epithelial cells. RNase4 and 5 also modulated pro-inflammatory signalling in response to nucleic acids in bovine peripheral blood mononuclear cells, although producing a distinct response. These results support a role for RNase4 and RNase5 in mediating inflammatory signals in both immune and epithelial cells, involving mechanisms that are cell-type specific.

Farmed deer: A veterinary model for chronic mycobacterial diseases that is accessible, appropriate and cost-effective.

Although most studies in immunology have used inbred mice as the experimental model to study fundamental immune mechanisms they have been proven to be limited in their ability to chart complex functional immune pathways, such as are seen in outbred populations of humans or animals. Translation of the findings from inbred mouse studies into practical solutions in therapeutics or the clinic has been remarkably unproductive compared with many other areas of clinical practice in human and veterinary medicine. Access to an unlimited array of mouse strains and an increasing number of genetically modified strains continues to sustain their paramount position in immunology research. Since the mouse studies have provided little more than the dictionary and glossary of immunology, another approach will be required to write the classic exposition of functional immunity. Domestic animals such as ruminants and swine present worthwhile alternatives as models for immunological research into infectious diseases, which may be more informative and cost effective. The original constraint on large animal research through a lack of reagents has been superseded by new molecular technologies and robotics that allow research to progress from gene discovery to systems biology, seamlessly. The current review attempts to highlight how exotic animals such as deer can leverage off the knowledge of ruminant genomics to provide cost-effective models for research into complex, chronic infections. The unique opportunity they provide relates to their diversity and polymorphic genotypes and the integrity of their phenotype for a range of infectious diseases.

Composite testing for ante-mortem diagnosis of Johne's disease in farmed New Zealand deer: correlations between bacteriological culture, histopathology, serological reactivity and faecal shedding as determined by quantitative PCR.

BACKGROUND: In the absence of overt clinical signs of Johne's Disease (JD), laboratory based tests have largely been limited to organism detection via faecal culture or PCR and serological tests for antibody reactivity. In this study we describe the application of quantitative faecal PCR for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in New Zealand farmed deer to quantify the bacterial load in cervine faecal samples as an adjunct to an existing serodiagnostic test (Paralisa™) tailored for JD diagnosis in deer. As ELISA has potential as a cheap, high throughput screening test for JD, an attempt was made to assess the sensitivity, specificity and positive/negative predictive (PPV/NPV) values of Paralisa™ for estimating levels of faecal shedding of MAP as a basis for JD management in deer. RESULTS: Correlations were made between diagnostic tests (ELISA, qPCR, culture and histopathology) to establish the precision and predictive values of individual tests. The findings from this study suggest there is strong correlation between bacterial shedding, as determined by faecal qPCR, with both culture (r = 0.9325) and histopathological lesion severity scoring (r = 0.7345). Correlation between faecal shedding and ELISA reactivity in deer was weaker with values of r = 0.4325 and r = 0.4006 for Johnin and Protoplasmic antigens, respectively. At an ELISA Unit (EU) cutoff of >50 (Johnin antigen) the PPV of Paralisa™ for significant faecal shedding in deer (>104 organisms/g) was moderate (0.55) while the NPV was higher (0.89). At an EU cutoff of ≥ 150, the PPV for shedding >105 organisms/g rose to 0.88, with a corresponding NPV of 0.85. CONCLUSIONS: The evidence available from this study suggests that Paralisa™ used at a cutoff of 50EU could be used to screen deer herds for MAP infection with sequential qPCR testing used to cull all Paralisa™ positive animals that exhibit significant MAP faecal shedding.

The mammalian secreted RNases: mechanisms of action in host defence.

The mammalian ribonucleaseA family comprises a large group of structurally similar proteins which are secreted by a range of tissues and immune cells. Their physiological role is unclear. It has been suggested that some of these RNases contribute to host defence, notably eosinophil-derived neurotoxin, eosinophil cationic protein, eosinophil-associated RNases, RNase4, angiogenin (RNase5), RNase7, RNase8 and bovine seminal RNase. This review summarises data supporting the involvement of these proteins in host defence, focusing on their antimicrobial, cytotoxic and immunomodulatory activities. The extent to which the data support possible mechanisms of action for these proteins is discussed. This compilation of findings and current hypotheses on the physiological role of these RNases will provide a stimulus for further research and development of ideas on the contribution of the RNases to host defence.

Longitudinal Pathogenesis Study of Young Red Deer (Cervus elaphus) after Experimental Challenge with Mycobacterium avium subsp. paratuberculosis (MAP).

Paratuberculosis progresses more quickly in young red deer than in sheep or cattle. This study describes the clinical, immunological and pathological changes over a 50-week period in fourteen 4-month-old red deer that received heavy oral challenge with Mycobacterium avium subsp. paratuberculosis (MAP). At 4 and 12 weeks post challenge they were anaesthetized and a section of jejunal lymph node was surgically removed for culture, histopathology, and genetic studies. All 14 deer became infected, none were clinically affected, and they had varying degrees of subclinical disease when killed at week 50. Week 4 biopsies showed no paratuberculosis lesions, but MAP was cultured from all animals. At weeks 12 and 50 histopathological lesions ranged from mild to severe with corresponding low-to-high antibody titres, which peaked at 12-24 weeks. IFN-γ responses peaked at 8-15 weeks and were higher in mildly affected animals than in those with severe lesions.

Differential immune responses of red Deer (Cervus elaphus) following experimental challenge with Mycobacterium avium subsp. paratuberculosis.

Immune responses of red deer (Cervus elaphus) that presented with different levels of paucibacillary pathology were profiled to detail immune changes during the progression of Johne's disease. Immune responses were monitored using an immunoglobulin G1 (IgG1) antibody enzyme-linked immunosorbent assay (ELISA), a gamma interferon (IFN-gamma) ELISA, and flow cytometry. Animals in the study were divided into outcome groups postmortem according to disease severity. All animals mounted IgG1 antibody and IFN-gamma responses to both the vaccination and experimental challenges. The Mycobacterium avium subsp. paratuberculosis-specific IgG1 antibody responses in the challenged group showed marked differences between infected and severely diseased animals. Slightly higher IFN-gamma responses were seen in infected animals compared with severely diseased animals. No significant changes were seen in the phenotype of lymphocyte populations investigated. Vaccination with killed M. avium subsp. paratuberculosis in mineral oil adjuvant reduced the level of severe disease; however, it obscured immunological differences between the infected and severely diseased groups. This suggests protection is not exclusively mediated via the presence of a type 1 response and, furthermore, the presence of a type 2 response is compatible with protection. These profiles provide information on the different immune processes in Johne's disease progression.