Efficacy of spheroplastic and cell-wall competent vaccines for Mycobacterium avium subsp. paratuberculosis in experimentally-challenged baby goats

A Mycobacterium avium subspecies paratuberculosis (MAP) vaccine that reduced the incidence of clinical disease or reduced fecal shedding of MAP would aid control of Johne's disease (JD). The objective of the present study was to evaluate the efficacy of four MAP vaccine combinations, including cell-wall competent (CWC) alum adjuvant, CWC-QS21 adjuvant, cell-wall deficient (CWD) alum adjuvant and CWD-QS21 adjuvant vaccines. Eighty baby goats were vaccinated at 1 and 4 weeks of age with one of these vaccines or a sham control vaccine consisting of alum adjuvant. Kids were challenged orally with approximately 6.0 × 10⁹ organisms in four divided doses of 1.5 × 10⁹ organisms using a goat isolate of MAP. Vaccinated challenged and challenged control groups had 10 and 6 kids per group, respectively. Half of the kids within each group were necropsied at either 6 or 9 months post-challenge. Gross and microscopic lesions and relative number of acid-fast bacilli were evaluated and scored at necropsy. Results indicated all challenged kids had some lesions compatible with JD suggesting none of the vaccines prevented infection. Three vaccines (CWC-alum, CWC-QS21 and CWD-QS21) reduced lesion scores by 46–51% at 9 months. CWD-alum vaccine resulted in a more severe (+33.5%) lesion score than sham-vaccinated challenged control. Lesion scores were greater at 9 months than at 6 months post-challenge in the sham-vaccinated challenged group and CWD-alum vaccinated group, while lesion scores were generally stable with remaining vaccines. Mean fecal CFU/g were significantly different across time from challenge to 9-month necropsy (p = 0.043) and the CWC-QS21 vaccine group had a marked reduction in fecal CFU/g at all time points post-challenge. A reduction in MAP CFU/g was also detected in necropsy tissues from kids given the CWC-alum, CWC-QS21 and CWD-QS21 vaccines, and increased CFU/g were detected in tissues from kids given the CWD-alum vaccine. Immunological tests evaluated included, humoral response evaluation by AGID, ELISA and Western blot, and cell mediated response by comparative PPD skin testing (M. avium, Old Johnin, M. bovis and Lot 2 Johnin PPD's), and production of MAP induced γ-interferon. Vaccination also resulted in false-positive PPD skin test reactions for M. avium PPD, Old Johnin PPD and γ-interferon tests. When a 2-mm cutoff above normal skin thickness was used to define positive skin test reactions, false-positive reactions for M. bovis were detected in only 2 of 32 kids given a vaccine with QS21 adjuvant.     

Evaluation of serum and milk ELISAs for paratuberculosis in Danish dairy cattle

A milk and a serum ELISA for detection of antibodies against Mycobacterium avium ssp. paratuberculosis (MAP) were evaluated against the complement-fixation test (CFT) and culture of faecal samples from 580 cows collected between August 1996 and December 1996. Milk and serum were obtained concurrently from six dairy herds infected with MAP and from two dairy herds without history of infection with MAP.

A cut-off value of 7 OD% was used in the ELISAs. At this cut-off value, all six culture-positive herds were positive in the serum ELISA but one was negative in the milk ELISA. All six culture-positive herds were positive in the CFT. In the two culture-negative herds, the serum and the milk ELISA deemed all serum samples negative at this cut-off value, whereas four serum samples from one of these herds were positive in the CFT. The highest cut-off value enabling the milk ELISA to record all six culture-positive herds as positive was 4 OD%. The highest cut-off value enabling the serum ELISA to record all six culture-positive herds as positive was 17 OD%. Individual-sample relative sensitivities of the ELISAs ranged from 49 to 64% and relative specificities were 80–96% at the cut-off values of 4, 7 and 17 OD%.     

Limited phenotypic and functional maturation of bovine monocyte-derived dendritic cells following Mycobacterium avium subspecies paratuberculosis infection in vitro

After encountering antigen, dendritic cells (DC) must differentiate into a fully mature phenotype to induce a protective, lasting T cell immunity. Paratuberculosis is a disease caused by the intracellular pathogen Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) and is characterized by a transient cell mediated immune response, that when dissipates correlates to the onset of clinical disease. In order to study the mechanism of early cellular immunity associated with M. paratuberculosis infection, we tested the hypothesis that M. paratuberculosis infected bovine DC have impaired activation and maturation thus are defective in the initiation of a sustainable and protective Th1 immune response locally. Our results demonstrate that M. paratuberculosis infected DC showed decreased endocytosis of ovalbumin, indicating some functional maturation. Co-stimulatory molecules CD40 and CD80 mRNA expression from M. paratuberculosis infected DC was increased over untreated immature DC. M. paratuberculosis infection induced chemokine receptor CCR7 increase in DC, yet CCR5 remained high. MHC II surface expression remained low on M. paratuberculosis infected DC. M. paratuberculosis infection inhibited pro-inflammatory cytokine IL-12 production and promoted IL-10 secretion by bovine DC. Together, our findings showed evidence of phenotypic and functional maturation of DC. However, we did not see the expected antigen presentation via MHC II and cytokine responses as a fully mature DC. This may suggest semi-mature DC phenotype induced by M. paratuberculosis infection.     

Trends and advances in the diagnosis and control of paratuberculosis in domestic livestock

Paratuberculosis (pTB) is a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP) in a wide variety of domestic and wild animals. Control of pTB is difficult due to the lack of sensitive, efficacious and cost-effective diagnostics and marker vaccines. Microscopy, culture, and PCR have been used for the screening of MAP infection in animals for quite a long time. Besides, giving variable sensitivity and specificity, these tests have not been considered ideal for large-scale screening of domestic livestock. Serological tests like ELISA easily detects anti-MAP antibodies. However, it cannot differentiate between the vaccinated and infected animals. Nanotechnology-based diagnostic tests are underway to improve the sensitivity and specificity. Newer generation diagnostic tests based on recombinant MAP secretory proteins would open new paradigm for the differentiation between infected and vaccinated animals and for early detection of the infection. Due to higher seroreactivity of secretory proteins vis-à-vis cellular proteins, the secretory proteins may be used as marker vaccine, which may aid in the control of pTB infection in animals. Secretory proteins can be potentially used to develop future diagnostics, surveillance and monitoring of the disease progression in animals and the marker vaccine for the control and eradication of pTB.     

Sensitivity and specificity of two serological tests for the detection of ovine paratuberculosis

OBJECTIVE: To determine the sensitivity and specificity of an absorbed ELISA and an AGID test for the detection of clinical and subclinical paratuberculosis in sheep. DESIGN: By testing a panel of sera from 1257 Australian Merino and crossbred sheep greater than 1 year of age, of which 1137 sheep were not infected with Mycobacterium avium subsp paratuberculosis and 120 sheep had paratuberculosis. PROCEDURE: Sera were collected from 457 sheep in Victoria and 800 sheep in Western Australia. Presence of M a paratuberculosis infection in Victorian sheep was determined by histological examination of intestinal tissues, whereas sheep from Western Australia were presumed to be free of Johne's disease. The ability of an absorbed ELISA to discriminate between infected and uninfected sheep was described by test sensitivity and specificity, the distribution of ELISA OD, and the area under a receiver operating characteristic curve. RESULTS: The absorbed ELISA had a specificity of 98.2 to 99.5% (CI) and a sensitivity of 35 to 54% (CI). In sheep from infected flocks in Victoria, the AGID test had a specificity of 99 to 100% (CI) and a sensitivity of 38 to 56% (CI). The sensitivity of serological tests was higher in sheep with a body condition representative of the lower quintile of their flock of origin. CONCLUSION: The AGID test and absorbed ELISA are useful tests for the detection of ovine paratuberculosis. Although the tests had a similar accuracy, they detected different subpopulations of infected sheep with only moderate overlap. The AGID test had a higher specificity than the absorbed ELISA.     

Temporal patterns and quantification of excretion of Mycobacterium avium subsp paratuberculosis in sheep with Johne's disease

OBJECTIVES: To determine the frequency of excretion of Mycobacterium avium subsp paratuberculosis in Merino sheep with Johne's disease and to quantify excretion in a group of Merino sheep. DESIGN: A pen and laboratory experiment. PROCEDURE: Seven sheep selected from an affected flock on the basis of acid-fast bacilli in the sheep's faeces were housed and total daily faecal output was collected, weighed and subjected to culture for M avium subsp paratuberculosis. An end-point titration method was used to enumerate viable M avium subsp paratuberculosis in a 15 day pooled sample from five sheep that had acid-fast bacilli in their faeces while housed. RESULTS: Four sheep with subclinical multibacillary Johne's disease excreted M avium subsp paratuberculosis each day for 11 days of cultural observation. A further three sheep were intermittent excreters but lacked other evidence of infection with M avium subsp paratuberculosis. The average number of viable bacteria excreted was 1.09 x 10(8) per gram of faeces while total daily excretion was 8.36 x 10(10) viable M avium subsp paratuberculosis per sheep. Examination of faecal smears stained with Ziehl Neelsen was an unreliable means of assessing daily excretion in individual animals except in those with severe lesions. CONCLUSION: Excretion of M avium subsp paratuberculosis in Merino sheep with multibacillary Johne's disease occurred daily, proving that environmental contamination can be continuous on farms with endemic ovine Johne's disease. Faecal culture is a useful method for detecting infection as it does not appear to be affected by the timing of collection of a sample from sheep with multibacillary disease however, to maximise the sensitivity of disease surveillance using faecal culture, sampling rates should be adjusted to take account of the proportions of multibacillary and paucibacillary cases.     

Tissue predilection sites and effect of dose on Mycobacterium avium subs. paratuberculosis organism recovery in a short-term bovine experimental oral infection model

The objective of this study was to develop a short-term experimental infection model for Mycobacterium avium subsp. paratuberculosis (MAP) in cattle, using small oral doses of organisms. Specifically, the effect of dose size was evaluated, as well as specific tissue predilection sites for recovery of MAP. Oral doses as low as 1.5 x 10(6) CFU reliably produced infection that could be detected 3 weeks following infection. Detection of infection required culture of multiple intestinal samples (jejunum and ileum) for MAP. Histological examination did not permit detection at this early stage. Results from this study suggest intestinal mucosa, rather than tonsil, as the primary portal of entry for MAP. The experimental infection model described here is useful for studying the early effects of preventive and therapeutic interventions for paratuberculosis in cattle.     

Course of experimental infection of canine leishmaniosis: Follow-up and utility of noninvasive diagnostic techniques

This study compares the utility of a molecular diagnosis of experimental CanL on non-invasive samples (urine, conjunctival (CS), oral (OS) and vulvar (VS) swabs) with that of traditional invasive techniques during the course of infection. Eight dogs were experimentally infected with Leishmania infantum and followed monthly for 12 months to assess clinical, clinicopathological, immunological and parasitological variables. Active infection was produced in 100% of the dogs. The animals showed positive bone marrow (BM) cytologies and cultures, clinical signs, clinicopathological abnormalities and a high specific humoral immune response. The infection was detected at 90 days post-infection (p.i.) by real-time quantitative PCR (rtQ-PCR) on BM in all dogs and in blood in 2 dogs, while anti-L. infantum antibody seroconversion occurred between Days 120 and 180 days p.i. The tissue with the highest L. infantum kDNA load, as detected by rtQ-PCR, was BM (range 381.5–70,000 parasites/ml at the study end), this sample type showing greater sensitivity than peripheral blood (PB). The vulvar swabs used here for the first time to quantify parasite loads in dogs revealed a greater load than oral and conjunctival swabs at one year p.i. Urine samples showed the lowest concentrations of L. infantum DNA (maximum: 8.57 parasites/ml). Our results suggest that for the early detection of infection, adding to serology a test such as rtQ-PCR on OS or VS improves sensitivity and specificity.     

A long-term study in Angora goats experimentally infected with Mycobacterium avium subsp. paratuberculosis: clinical disease, faecal culture and immunological studies

Two longitudinal experiments involving Angora goats challenged with either bovine or ovine strains of Mycobacterium avium subspecies paratuberculosis (Map) have been conducted over a period of 54 and 35 months, respectively. Blood samples for the interferon-gamma (IFN-gamma) test and the absorbed ELISA and faecal samples for bacteriological culture were taken pre-challenge and monthly post-challenge. Persistent shedding, IFN-gamma production, seroconversion and clinical disease occurred earlier with the bovine Map gut mucosal tissue challenge inoculum than with cultured bacteria. The IFN-gamma responses of the gut mucosal tissue and bacterial challenge groups were substantially and consistently higher than those of the control group. The in vivo and cultured cattle strains were much more pathogenic for goats than the sheep strains with persistent faecal shedding, seroconversion and clinical disease occurring in the majority of bovine Map challenged goats. With the ovine Map, 3 goats developed persistent antibody responses but only one of these goats developed persistent faecal shedding and clinical disease. However, there was no significant difference between the IFN-gamma responses of the tissue challenged, bacterial challenged and control groups. Compared with sheep, the ELISA appeared to have higher sensitivity and the IFN-gamma test lower specificity.     

Cellular composition of granulomatous lesions in gut-associated lymphoid tissues of goats during the first year after experimental infection with Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) causes lesions in naturally and experimentally infected ruminants which greatly differ in severity, cellular composition and number of mycobacteria. Morphologically distinct lesions are already found during the clinically inapparent phase of infection. The complex local host response and number of MAP were characterized at the initial sites of lesions, organized gut-associated lymphoid tissue, in experimentally infected goats. Tissues were collected at 3, 6, 9 and 12 month post-inoculation (mpi) from goat kids that had orally received 10 times 10 mg of bacterial wet mass of MAP (JII-1961). The cellular composition of lesions in Peyer's patches in the jejunum and next to the ileocecal valve was evaluated in 21 MAP-inoculated goats, where lesions were compared with unaltered tissue of six control goats. CD68⁺, CD4⁺, CD8⁺, γδ T lymphocytes, B lymphocytes and plasma cells, MHC class II⁺ and CD25⁺ cells were demonstrated by immunohistochemistry in serial cryostat sections.At 3 mpi, extensive granulomatous infiltrates predominated, consisting of numerous epitheloid cells admixed with many CD4 and γδ T lymphocytes. Only single MAP were detected. This indicates a strong cellular immune reaction able to control MAP infection. γδ T lymphocytes were markedly increased in this type of lesion which may reflect their important role early in the pathogenesis of paratuberculosis. At 9 and 12 mpi, divergent lesions were observed which may reflect different outcomes of host–pathogen interactions. In five goats, minimal granulomatous lesions were surrounded by extensive lymphoplasmacytic infiltrates and no MAP were detected by immunohistochemistry. This was interpreted as effective host response that was able to eliminate MAP locally. In three goats, decreased numbers of lymphocytes, but extensive granulomatous infiltrates with numerous epitheloid cells containing increased numbers of mycobacteria were seen. This shift of the immune response resulted in uncontrolled mycobacterial multiplication. Focal and multifocal circumscribed granulomatous infiltrates of mainly epitheloid cells may represent sites of new infection, since they were observed in goats at all times after inoculation. Their presence in goats with minimal granulomatous lesions surrounded by extensive lymphoplasmacytic infiltrates may indicate that despite the local clearance, the infection may be perpetuated.The complex cellular immune reactions postulated for the pathogenesis of paratuberculosis were demonstrated at the local sites of infection. These early host–pathogen interactions are most likely essential for the eventual outcome of the MAP infection.     

Candidate gene and genome-wide association studies of Mycobacterium avium subsp. paratuberculosis infection in cattle and sheep: a review

Paratuberculosis (Johne's disease), caused by Mycobacterium avium subspecies paratuberculosis, is responsible for significant economic losses in livestock industries worldwide. This organism is also of public health concern due to an unconfirmed link to Crohn's disease. Susceptibility to paratuberculosis has been suggested to have a genetic component. In livestock, a number of candidate genes have been studied, selected on their association to susceptibility in other mycobacterial diseases, their known role in disease pathogenesis or links to susceptibility of humans to Crohn's disease. These genes include solute carrier family 11 member 1 (SLC11A1, formerly NRAMP1), toll-like receptors, caspase associated recruitment domain 15 (CARD15, formerly NOD2), major histocompatibility complex (MHC) and cytokines (interleukin-10 and interferon-gamma) and their receptors. Genome wide association studies have attempted to confirm associations found and identify new genes involved in pathogenesis and susceptibility. There are a number of limitations and difficulties in these approaches, some peculiar to paratuberculosis but others generally applicable to identification of genetic associations for complex traits. The technical approaches and available information for paratuberculosis have expanded rapidly, particularly relating to sheep and cattle. Here we review the current published evidence for a genetic association with paratuberculosis susceptibility, technological advances that have progressed the field and potential avenues for future research.     

Risk factors for methicillin-resistant Staphylococcus aureus (MRSA) infection in dogs and cats: a case-control study

Risk factors for methicillin-resistant Staphylococcus aureus (MRSA) infection in dogs and cats were investigated in an unmatched case-control study. A total of 197 animals from 150 veterinary practices across the United Kingdom was enrolled, including 105 MRSA cases and 92 controls with methicillin-susceptible S. aureus (MSSA) infection. The association of owners and veterinarian staff with the human healthcare sector (HCS) and animal-related characteristics such as signalment, antimicrobial and immunosuppressive therapy, and surgery were evaluated as putative risk factors using logistic regression. We found that significant risk factors for MRSA infection were the number of antimicrobial courses (p = 0.005), number of days admitted to veterinary clinics (p = 0.003) and having received surgical implants (p = 0.001). In addition, the odds of contact with humans which had been ill and admitted to hospital (p = 0.062) were higher in MRSA infected pets than in MSSA controls. The risk factors identified in this study highlight the need to increase vigilance towards identification of companion animal groups at risk and to advocate responsible and judicious use of antimicrobials in small animal practice.     

Laser acupuncture for acute inflammatory, visceral and neuropathic pain relief: An experimental study in the laboratory rat

Laser acupuncture is defined as the stimulation of traditional acupuncture points with low-intensity, non-thermal laser irradiation. We explored the clinical efficacy of a very low level diode laser wavelength 670 nm (Biolite LP020, RGM, Genoa, Italy), used to stimulate acupoints ST36 Zu San Li and TH5 Waiguan, on well-established experimental models of acute and persistent pain in the rat, e.g. acute inflammatory pain, muscle pain, visceral pain and neuropathic pain. We report the anti-edema and anti-hyperalgesia effects of laser acupuncture in models of acute inflammatory pain, e.g. CFA-induced inflammation and myofascial pain. We also indicate that spontaneous pain and thermal hyperalgesia are reduced in a neuropathic pain model, e.g. axotomy. On the contrary, no effects due to laser-acupuncture were observed on disconfort indices in a model of visceral pain, e.g. cystitis due to cyclophosphamide. We thus provide evidences that acupoints stimulation using a very low intensity laser irradiation can control pain and edema in specific experimental conditions.     

Maintenance of arytenoid abduction following carbon dioxide laser debridement of the articular cartilage and joint capsule of the cricoarytenoid joint combined with prosthetic laryngoplasty in horses: An in vivo and in vitro study

The objective was to evaluate CO₂ laser debridement of the cricoarytenoid joint (CAJ) combined with prosthetic laryngoplasty to prevent post-operative loss of arytenoid abduction in seven horses. Horses were assigned to either laser debridement of the left CAJ and laryngoplasty (laser treated, n = 5) or control laryngoplasty (sham, n = 2), and were evaluated with endoscopic examinations and measurement of right to left angle quotients (RLQ) to assess maintenance of arytenoid abduction. The animals were euthanased at intervals after surgery and larynges were harvested for post-mortem testing, including determination of translaryngeal flow, pressure, impedance and RLQ. Measurements were obtained under increasing vacuum-generated negative pressure with laryngoplasty sutures intact and with the knot/crimp of the laryngoplasty sutures removed. Following post-mortem testing the cricoarytenoid joints were examined histologically.Post-operative endoscopic examinations revealed no significant differences between RLQ measurements calculated for day 1 following surgery to the termination date of the study for the seven horses. Post-mortem RLQ at airflows of 10 and 60 L/s was significantly higher in sham than in laser treated horses both before and after knot/crimp removal. Translaryngeal impedance at 10 and 60 L/s was not statistically different between groups. Histopathology revealed necrosis and loss of articular cartilage in the laser treated horses. The lymphoid cell infiltration subsided but joint capsule and periarticular fibrosis increased over the course of the study. Post-operative loss of arytenoid abduction after laryngoplasty can be minimized with CO₂ laser debridement of the CAJ joint.     

Pathogens of zoonotic and biological importance in roe deer (Capreolus capreolus): Seroprevalence in an agro-system population in France

Antibody prevalence for several infectious and parasitic diseases in a population of roe deer (Capreolus capreolus) inhabiting a mixed agricultural landscape (south of France) has been analyzed. Serological analyses with ELISA in 245 animals captured from 2008 to 2012 has been performed. We found a high prevalence of Toxoplasma gondii (46.4%), Chlamydophila abortus (17.27%) and Coxiella burnetii (11.26%) compared to other studies in Europe. Seroprevalence varied strongly among years for T. gondii (27–91%), C. abortus (0–42%) and C. burnetii (0–27%). T. gondii prevalence was lower in juvenile females, compared to juvenile males and adults of both sexes. Other pathogens had low prevalences: Neospora caninum (1.56%), Bovine herpesvirus 1 (1.17%, 2008/09; 1.1%, 2010/11), Mycoplasma agalactiae (1.45%, 2009/10), Mycobacterium avium subsp. paratuberculosis (0.9%) and Slow viruses (CAEV–MVV) (0.15%, 2008/10; 0%, 2011/12). Antibodies to bluetongue virus and pestiviruses were not found in any individual.