Evaluation of Point-of-Care Tests for Identification of Pathogens to Inform Clinical Mastitis Treatment Decisions in Pasture- and Confinement-Managed Dairy Cows in Australia.

To support antimicrobial stewardship in livestock production, there is a growing array of point of care diagnostics to guide antimicrobial treatment. The primary objective of this observational study was to evaluate the diagnostic performance of 5 point of care tests currently available in Australia for guiding lactational treatment of non-severe clinical mastitis. A secondary objective was to describe the pathogen profiles of mastitis-causing organisms in cows managed in barns ("intensive") and on pasture ("non-intensive"). Foremilk samples (n = 641) were collected by farm staff in dairy herds in Australia (n = 30) and tested at a university laboratory using a reference test and 5 index tests. The reference test was aerobic culture on Trypticase Soy Agar with 5% sheep blood followed by MALDI-TOF for identification of isolates. The following point of care tests were evaluated as index tests: Accumast®, biplate, Check-Up, Mastatest®, and 3M Petrifilm. We found that 23% of samples were contaminated, with the median herd contamination prevalence being 22%. After excluding contaminated samples, the most common diagnoses (according to the reference test) in intensive herds were no growth (31.7%), Klebsiella spp. (28.1%), E. coli (15.0%), and Strep. uberis (8.4%). The most common diagnoses in non-contaminated samples from cows in non-intensive herds were Strep. uberis (35.0%), no growth (26.9%), and E. coli (13.3%). After 24 h of incubation, all index tests demonstrated limited diagnostic sensitivity for identification of pathogens of interest (range: 0.06 to 0.63). Diagnostic performance was better at the group-level, with sensitivity and specificity for identification of non-contaminated gram-positive growths (i.e., cases that are widely considered to be candidates for antimicrobial treatment) being 0.84 and 0.75 (biplate), 0.76 and 0.90 (Accumast), 0.89 and 0.79 (Check-Up), 0.67 and 0.83 (Petrifilm), and 0.55 and 0.81 (Mastatest). In intensive herds, 22.7 to 40% of cases were classified as antimicrobial treatment candidates by index tests, which was less than for cows in non-intensive herds (41.3 to 61.0%). Despite limited diagnostic reliability at genus and species level, and the need to ensure samples are collected aseptically, our findings indicate that implementation of selective treatment protocols using the tests evaluated in this study would likely reduce antimicrobial usage in Australian herds.

The Effect of Month and District on 100-Day In-Calf Rate in Year-Round Calving Dairy Herds.

Monitoring 100-day in-calf rate (100DICR) is an integral part of the assessment of reproductive performance in year-round calving dairy herds. The objective of this study was to investigate the effect of month on 100DICR in year-round calving herds in New South Wales (NSW), Australia and determine whether a fluctuating 100DICR target is an appropriate alternative to a constant 100DICR target. The 100DICR is defined as the percentage of all current lactating cows over 100 days in milk (DIM) that conceive on or before 100 DIM. As dairy cows are typically dried off 7 months after conception, 100DICR was an approximate 7-month rolling average. Mean monthly 100DICRs were calculated with a generalised linear model for six NSW north coast herds located 15-140 km from the coast and four NSW south coast herds located less than 10 km from the coast, over a two-year period. The mean 100DICR was lowest in May at 28.62% (95%CI 28.31-28.93) and increased during winter and spring, peaking in December at 34.74% (95%CI 34.32-35.15). The observed trend was similar for north and south coast herds, although north coast herds experienced a greater change in 100DICR from the peak to a nadir of 27.58% (95%CI 27.18-27.98), a 7.15-point difference, compared to south coast herds with a nadir of 30.18% (95%CI 29.69-30.67), a 4.67-point difference between the peak and nadir. In conclusion, 100DICR is affected by month with the lowest 100DICRs observed in late autumn and the highest 100DICRs observed in late spring and early summer. Therefore, a fluctuating target 100DICR is an appropriate alternative to a constant target when assessing reproductive performance in year-round calving herds. While the district does not affect mean 100DICR per se, the district does affect the difference between peak and nadir 100DICR.

Antimicrobial susceptibility testing to evaluate minimum inhibitory concentration values of clinically relevant antibiotics.

Antimicrobial susceptibility testing is used to determine the minimum inhibitory concentration (MIC), the standard measurement of antibiotic activity. Here, we present a protocol for evaluating MIC values of clinically relevant antibiotics against bacterial isolates cultured in standard bacteriologic medium and in mammalian cell culture medium. We describe steps for pathogen identification, culturing bacteria, preparing MIC plates, MIC assay incubation, and determining MIC. This protocol can potentially optimize the use of existing antibiotics while enhancing efforts to discover new ones. For complete details on the use and execution of this protocol, please refer to Heithoff et al.1.

Re-evaluation of FDA-approved antibiotics with increased diagnostic accuracy for assessment of antimicrobial resistance.

Accurate assessment of antibiotic susceptibility is critical for treatment of antimicrobial resistant (AMR) infections. Here, we examine whether antimicrobial susceptibility testing in media more physiologically representative of in vivo conditions improves prediction of clinical outcome relative to standard bacteriologic medium. This analysis reveals that ∼15% of minimum inhibitory concentration (MIC) values obtained in physiologic media predicted a change in susceptibility that crossed a clinical breakpoint used to categorize patient isolates as susceptible or resistant. The activities of antibiotics having discrepant results in different media were evaluated in murine sepsis models. Testing in cell culture medium improves the accuracy by which MIC assays predict in vivo efficacy. This analysis identifies several antibiotics for treatment of AMR infections that standard testing failed to identify and those that are ineffective despite indicated use by standard testing. Methods with increased diagnostic accuracy mitigate the AMR crisis via utilizing existing agents and optimizing drug discovery.

A broad-spectrum synthetic antibiotic that does not evoke bacterial resistance.

BACKGROUND: Antimicrobial resistance (AMR) poses a critical threat to public health and disproportionately affects the health and well-being of persons in low-income and middle-income countries. Our aim was to identify synthetic antimicrobials termed conjugated oligoelectrolytes (COEs) that effectively treated AMR infections and whose structures could be readily modified to address current and anticipated patient needs. METHODS: Fifteen chemical variants were synthesized that contain specific alterations to the COE modular structure, and each variant was evaluated for broad-spectrum antibacterial activity and for in vitro cytotoxicity in cultured mammalian cells. Antibiotic efficacy was analyzed in murine models of sepsis; in vivo toxicity was evaluated via a blinded study of mouse clinical signs as an outcome of drug treatment. FINDINGS: We identified a compound, COE2-2hexyl, that displayed broad-spectrum antibacterial activity. This compound cured mice infected with clinical bacterial isolates derived from patients with refractory bacteremia and did not evoke bacterial resistance. COE2-2hexyl has specific effects on multiple membrane-associated functions (e.g., septation, motility, ATP synthesis, respiration, membrane permeability to small molecules) that may act together to negate bacterial cell viability and the evolution of drug-resistance. Disruption of these bacterial properties may occur through alteration of critical protein-protein or protein-lipid membrane interfaces-a mechanism of action distinct from many membrane disrupting antimicrobials or detergents that destabilize membranes to induce bacterial cell lysis. INTERPRETATION: The ease of molecular design, synthesis and modular nature of COEs offer many advantages over conventional antimicrobials, making synthesis simple, scalable and affordable. These COE features enable the construction of a spectrum of compounds with the potential for development as a new versatile therapy for an imminent global health crisis. FUNDING: U.S. Army Research Office, National Institute of Allergy and Infectious Diseases, and National Heart, Lung, and Blood Institute.

Coagulation factor protein abundance in the pre-septic state predicts coagulopathic activities that arise during late-stage murine sepsis.

BACKGROUND: Although sepsis accounts for 1 in 5 deaths globally, few molecular therapies exist for this condition. The development of effective biomarkers and treatments for sepsis requires a more complete understanding of host responses and pathogenic mechanisms at early stages of disease to minimize host-driven pathology. METHODS: An alternative to the current symptom-based approach used to diagnose sepsis is a precise assessment of blood proteomic changes during the onset and progression of Salmonella Typhimurium (ST) murine sepsis. FINDINGS: A distinct pattern of coagulation factor protein abundance was identified in the pre-septic state- prior to overt disease symptoms or bacteremia- that was predictive of the dysregulation of fibrinolytic and anti-coagulant activities and resultant consumptive coagulopathy during ST murine sepsis. Moreover, the changes in protein abundance observed generally have the same directionality (increased or decreased abundance) reported for human sepsis. Significant overlap of ST coagulopathic activities was observed in Gram-negative Escherichia coli- but not in Gram-positive staphylococcal or pneumococcal murine sepsis models. Treatment with matrix metalloprotease inhibitors prevented aberrant inflammatory and coagulopathic activities post-ST infection and increased survival. Antibiotic treatment regimens initiated after specific changes arise in the plasma proteome post-ST infection were predictive of an increase in disease relapse and death after cessation of antibiotic treatment. INTERPRETATION: Altered blood proteomics provides a platform to develop rapid and easy-to-perform tests to predict sepsis for early intervention via biomarker incorporation into existing blood tests prompted by patient presentation with general malaise, and to stratify Gram-negative and Gram-positive infections for appropriate treatment. Antibiotics are less effective in microbial clearance when initiated after the onset of altered blood proteomics as evidenced by increased disease relapse and death after termination of antibiotic therapy. Treatment failure is potentially due to altered bacterial / host-responses and associated increased host-driven pathology, providing insight into why delays in antibiotic administration in human sepsis are associated with increased risk for death. Delayed treatment may thus require prolonged therapy for microbial clearance despite the prevailing notion of antibiotic de-escalation and shortened courses of antibiotics to improve drug stewardship. FUNDING: National Institutes of Health, U.S. Army.

Development of a qPCR for the detection and quantification of Salmonella spp. in sheep feces and tissues.

Salmonella spp. are common causes of disease in intensive livestock production systems, and contamination of foodstuffs is of significant concern for public health. Therefore, the identification and quantification of Salmonella spp. is important for monitoring the level of fecal shedding or tissue colonization in infected animals and animal products. We developed and evaluated a quantitative PCR (qPCR) method on spiked sheep tissue and fecal samples for the detection and quantification of Salmonella spp. Without the use of a pre-enrichment step, the qPCR limit of detection (LOD) results for sheep fecal (4 × 104-6 × 103 cfu/g) and tissue (4 × 105-4 × 103 cfu/g) samples were not adequate for detection purposes. With the inclusion of a 6-h pre-enrichment step in buffered peptone water (BPW), the LOD was 9 cfu/g (2.57 × 101 copies/g) in sheep feces, and 5.4 cfu/g (3.22 copies/g) sheep tissue. Comparison of the 6-h BPW qPCR method with a 24-h mannitol-selenite-cystine broth enrichment culture method using spiked samples revealed a sensitivity of 91% and 92%, respectively, and a specificity of 100% for both methods. The correlation was significant between the quantity (copies/mL) of Salmonella spp. in BPW at 6 h and at 0 h, allowing semiquantitative analysis. Our results demonstrate that, following inclusion of a 6-h pre-enrichment step in BPW, qPCR is semiquantitative with improved LODs of Salmonella spp. in sheep fecal and tissue samples.

A review of mycoplasma diagnostics in cattle.

Mycoplasma species have a global distribution causing serious diseases in cattle worldwide including mastitis, arthritis, pneumonia, otitis media and reproductive disorders. Mycoplasma species are typically highly contagious, are capable of causing severe disease, and are difficult infections to resolve requiring rapid and accurate diagnosis to prevent and control disease outbreaks. This review discusses the development and use of different diagnostic methods to identify Mycoplasma species relevant to cattle, with a particular focus on Mycoplasma bovis. Traditionally, the identification and diagnosis of mycoplasma has been performed via microbial culture. More recently, the use of polymerase chain reaction to detect Mycoplasma species from various bovine samples has increased. Polymerase chain reaction has a higher efficiency, specificity, and sensitivity for laboratory diagnosis when compared with conventional culture-based methods. Several tools are now available for typing Mycoplasma spp. isolates, allowing for genetic characterization in disease outbreak investigations. Serological diagnosis through the use of indirect ELISA allows the detection of antimycoplasma antibodies in sera and milk, with their use demonstrated on individual animal samples as well as BTM samples. While each testing method has strengths and limitations, their combined use provides complementary information, which when interpreted in conjunction with clinical signs and herd history, facilitates pathogen detection, and characterization of the disease status of cattle populations.

Correcting a Fundamental Flaw in the Paradigm for Antimicrobial Susceptibility Testing.

The emergence and prevalence of antibiotic-resistant bacteria are an increasing cause of death worldwide, resulting in a global 'call to action' to avoid receding into an era lacking effective antibiotics. Despite the urgency, the healthcare industry still relies on a single in vitro bioassay to determine antibiotic efficacy. This assay fails to incorporate environmental factors normally present during host-pathogen interactions in vivo that significantly impact antibiotic efficacy. Here we report that standard antimicrobial susceptibility testing (AST) failed to detect antibiotics that are in fact effective in vivo; and frequently identified antibiotics that were instead ineffective as further confirmed in mouse models of infection and sepsis. Notably, AST performed in media mimicking host environments succeeded in identifying specific antibiotics that were effective in bacterial clearance and host survival, even though these same antibiotics failed in results using standard test media. Similarly, our revised media further identified antibiotics that were ineffective in vivo despite passing the AST standard for clinical use. Supplementation of AST medium with sodium bicarbonate, an abundant in vivo molecule that stimulates global changes in bacterial structure and gene expression, was found to be an important factor improving the predictive value of AST in the assignment of appropriate therapy. These findings have the potential to improve the means by which antibiotics are developed, tested, and prescribed.

Comparison of culture and a multiplex probe PCR for identifying Mycoplasma species in bovine milk, semen and swab samples.

Mycoplasma spp. are a major cause of mastitis, arthritis and pneumonia in cattle, and have been associated with reproductive disorders in cows. While culture is the traditional method of identification the use of PCR has become more common. Several investigators have developed PCR protocols to detect M. bovis in milk, yet few studies have evaluated other sample types or other important Mycoplasma species. Therefore the objective of this study was to develop a multiplex PCR assay to detect M. bovis, M. californicum and M. bovigenitalium, and evaluate its analytical performance against traditional culture of bovine milk, semen and swab samples. The PCR specificity was determined and the limit of detection evaluated in spiked milk, semen and swabs. The PCR was then compared to culture on 474 field samples from individual milk, bulk tank milk (BTM), semen and swab (vaginal, preputial, nose and eye) samples. Specificity analysis produced appropriate amplification for all M. bovis, M. californicum and M. bovigenitalium isolates. Amplification was not seen for any of the other Mollicutes or eubacterial isolates. The limit of detection of the PCR was best in milk, followed by semen and swabs. When all three Mycoplasma species were present in a sample, the limit of detection increased. When comparing culture and PCR, overall there was no significant difference in the proportion of culture and PCR positive samples. Culture could detect significantly more positive swab samples. No significant differences were identified for semen, individual milk or BTM samples. PCR identified five samples with two species present. Culture followed by 16S-23S rRNA sequencing did not enable identification of more than one species. Therefore, the superior method for identification of M. bovis, M. californicum and M. bovigenitalium may be dependent on the sample type being analysed, and whether the identification of multiple target species is required.

Genetic characterization of Australian Mycoplasma bovis isolates through whole genome sequencing analysis.

Mycoplasma bovis is a major pathogen in cattle causing mastitis, arthritis and pneumonia. First isolated in Australian cattle in 1970, M. bovis has persisted causing serious disease in infected herds. To date, genetic analysis of Australian M. bovis isolates has not been performed. With whole genome sequencing (WGS) becoming a common tool for genetic characterization, this method was utilized to determine the degree of genetic diversity among Australian M. bovis isolates collected over a nine year period (2006-2015) from various geographical locations, anatomical sites, and from clinically affected and non-clinical carrier animals. Eighty-two M. bovis isolates underwent WGS from which single nucleotide polymorphism (SNP) analysis, comparative genomics and analysis of virulence genes was completed. SNP analysis identified a single M. bovis strain circulating throughout Australia with marked genomic similarity. Comparative genomics suggested minimal variation in gene content between isolates from clinical and carrier animals, and between isolates recovered from different anatomical sites. A total of 50 virulence genes from the virulence factors database (VFDB) were identified as highly similar in the Australian isolates, while the presence of variable surface lipoprotein (vsp) genes was greatly reduced compared to reference strain M. bovis PG45. These results highlight that, while the introduction of multiple M. bovis strains has been prevented, elimination of the current strain has not been successful. The persistence of this strain may be due to the significant role that carrier animals play in harboring the pathogen. The similarity of clinical and non-clinical isolates suggests host and environmental factors play a significant role in determining host pathogen outcomes.

Development of a loop-mediated isothermal amplification assay for the detection of Streptococcus agalactiae in bovine milk.

Streptococcus agalactiae is a well-characterized bovine mastitis pathogen that is known to be highly contagious and capable of spreading rapidly in affected dairy herds. Loop-mediated isothermal amplification (LAMP) is a novel molecular diagnostic method that has the capability to provide rapid, cost-effective screening for pathogens to support on-farm disease control and eradication programs. In the current study, a LAMP test was developed to detect S. agalactiae in milk. The assay was validated on a bank of existing clinical mastitis milk samples that had previously been identified as S. agalactiae positive via traditional microbiological culture techniques and PCR. The LAMP assay was conducted on bacterial colonies and DNA extracted from milk in tube- and plate-based formats using multiple detection platforms. The 1-h assay conducted at 64 °C exhibited repeatability (coefficient of variation) of 2.07% (tube) and 8.3% (plate), sensitivity to ~20 pg of extracted DNA/reaction, and specificity against a panel of known bacterial mastitis pathogens. Of the 109 known S. agalactiae isolates assessed by LAMP directly from bacterial cells in culture, 108 were identified as positive, in accordance with PCR analysis. The LAMP analysis from the corresponding milk samples indicated that 104 of these milks exhibited a positive amplification curve. Although exhibiting some limitations, this assay provides an opportunity for rapid screening of milk samples to facilitate on-farm management of this pathogen.

Host-dependent Induction of Transient Antibiotic Resistance: A Prelude to Treatment Failure.

Current antibiotic testing does not include the potential influence of host cell environment on microbial susceptibility and antibiotic resistance, hindering appropriate therapeutic intervention. We devised a strategy to identify the presence of host-pathogen interactions that alter antibiotic efficacy in vivo. Our findings revealed a bacterial mechanism that promotes antibiotic resistance in vivo at concentrations of drug that far exceed dosages determined by standardized antimicrobial testing. This mechanism has escaped prior detection because it is reversible and operates within a subset of host tissues and cells. Bacterial pathogens are thereby protected while their survival promotes the emergence of permanent drug resistance. This host-dependent mechanism of transient antibiotic resistance is applicable to multiple pathogens and has implications for the development of more effective antimicrobial therapies.

Development of a Salmonella cross-protective vaccine for food animal production systems.

Intensive livestock production is associated with increased Salmonella exposure, transmission, animal disease, and contamination of food and water supplies. Modified live Salmonella enterica vaccines that lack a functional DNA adenine methylase (Dam) confer cross-protection to a diversity of salmonellae in experimental models of murine, avian, ovine, and bovine models of salmonellosis. However, the commercial success of any vaccine is dependent upon the therapeutic index, the ratio of safety/efficacy. Herein, secondary virulence-attenuating mutations targeted to genes involved in intracellular and/or systemic survival were introduced into Salmonella dam vaccines to screen for vaccine candidates that were safe in the animal and the environment, while maintaining the capacity to confer cross-protective immunity to pathogenic salmonellae serotypes. Salmonella dam mgtC, dam sifA, and dam spvB vaccine strains exhibited significantly improved vaccine safety as evidenced by the failure to give rise to virulent revertants during the infective process, contrary to the parental Salmonella dam vaccine. Further, these vaccines exhibited a low grade persistence in host tissues that was associated with reduced vaccine shedding, reduced environmental persistence, and induction of cross-protective immunity to pathogenic serotypes derived from infected livestock. These data indicate that Salmonella dam double mutant vaccines are suitable for commercial applications against salmonellosis in livestock production systems. Reducing pre-harvest salmonellae load through vaccination will promote the health and productivity of livestock and reduce contamination of livestock-derived food products, while enhancing overall food safety.

Intraspecies variation in the emergence of hyperinfectious bacterial strains in nature.

Salmonella is a principal health concern because of its endemic prevalence in food and water supplies, the rise in incidence of multi-drug resistant strains, and the emergence of new strains associated with increased disease severity. Insights into pathogen emergence have come from animal-passage studies wherein virulence is often increased during infection. However, these studies did not address the prospect that a select subset of strains undergo a pronounced increase in virulence during the infective process- a prospect that has significant implications for human and animal health. Our findings indicate that the capacity to become hypervirulent (100-fold decreased LD(50)) was much more evident in certain S. enterica strains than others. Hyperinfectious salmonellae were among the most virulent of this species; restricted to certain serotypes; and more capable of killing vaccinated animals. Such strains exhibited rapid (and rapidly reversible) switching to a less-virulent state accompanied by more competitive growth ex vivo that may contribute to maintenance in nature. The hypervirulent phenotype was associated with increased microbial pathogenicity (colonization; cytotoxin production; cytocidal activity), coupled with an altered innate immune cytokine response within infected cells (IFN-ß; IL-1ß; IL-6; IL-10). Gene expression analysis revealed that hyperinfectious strains display altered transcription of genes within the PhoP/PhoQ, PhoR/PhoB and ArgR regulons, conferring changes in the expression of classical virulence functions (e.g., SPI-1; SPI-2 effectors) and those involved in cellular physiology/metabolism (nutrient/acid stress). As hyperinfectious strains pose a potential risk to human and animal health, efforts toward mitigation of these potential food-borne contaminants may avert negative public health impacts and industry-associated losses.

Salmonella in calves.

Salmonellae are endemic on most large intensive farms and salmonellosis is a common cause of neonatal morbidity and mortality. Disease and mortality usually reflect a variety of management events and environmental stressors that contribute to compromised host immunity and increased pathogen exposure. The diversity of salmonella serovars present on farms, and the potential for different serovars to possess different virulence factors, require the implementation of broad prophylactic strategies that are efficacious for all salmonellae. This article discusses strategies to promote host immunity and minimize pathogen exposure at the farm level. The benefits of control include a reduction in disease incidence and mortality, reduced drug and labor costs, and improved growth rates.

Human Salmonella clinical isolates distinct from those of animal origin.

The global trend toward intensive livestock production has led to significant public health risks and industry-associated losses due to an increased incidence of disease and contamination of livestock-derived food products. A potential factor contributing to these health concerns is the prospect that selective pressure within a particular host may give rise to bacterial strain variants that exhibit enhanced fitness in the present host relative to that in the parental host from which the strain was derived. Here, we assessed 184 Salmonella enterica human and animal clinical isolates for their virulence capacities in mice and for the presence of the Salmonella virulence plasmid encoding the SpvB actin cytotoxin required for systemic survival and Pef fimbriae, implicated in adherence to the murine intestinal epithelium. All (21 of 21) serovar Typhimurium clinical isolates derived from animals were virulent in mice, whereas many (16 of 41) serovar Typhimurium isolates derived from human salmonellosis patients lacked this capacity. Additionally, many (10 of 29) serovar Typhimurium isolates derived from gastroenteritis patients did not possess the Salmonella virulence plasmid, in contrast to all animal and human bacteremia isolates tested. Lastly, among serovar Typhimurium isolates that harbored the Salmonella virulence plasmid, 6 of 31 derived from human salmonellosis patients were avirulent in mice, which is in contrast to the virulent phenotype exhibited by all the animal isolates examined. These studies suggest that Salmonella isolates derived from human salmonellosis patients are distinct from those of animal origin. The characterization of these bacterial strain variants may provide insight into their relative pathogenicities as well as into the development of treatment and prophylactic strategies for salmonellosis.

Cross-protective immunity in calves conferred by a DNA adenine methylase deficient Salmonellaenterica serovar Typhimurium vaccine.

The global trend towards intensive livestock production is associated with increased fecal oral pathogen transmission resulting in a high prevalence of Salmonella. Since many pathogenic Salmonella serovars are often endemic to livestock production systems, it is desirable to develop a vaccine that is capable of eliciting immunity to more than one serovar. Here we examined whether immunization with a modified live Salmonella enterica serovar Typhimurium vaccine strain lacking the DNA adenine methylase (Dam) conferred protection in calves against a heterologous S. enterica Dublin challenge. Vaccinated animals challenged with a virulent Dublin strain exhibited a significant attenuation of clinical disease (improved attitude scores and reduced fever and diarrhea) and a concomitant reduction in Dublin fecal shedding and colonization of mesenteric lymph nodes (MLN) compared to non-vaccinated control animals. These data suggest that vaccination with a dam(-) Typhimurium vaccine strain conferred significant cross-protection against clinical disease in cattle attributable to heterologous challenge with Dublin.