Neglected tropical disease (NTD) diagnostics: current development and operations to advance control.

Neglected tropical diseases (NTDs) have become important public health threats that require multi-faceted control interventions. As late treatment and management of NTDs contribute significantly to the associated burdens, early diagnosis becomes an important component for surveillance and planning effective interventions. This review identifies common NTDs and highlights the progress in the development of diagnostics for these NTDs. Leveraging existing technologies to improve NTD diagnosis and improving current operational approaches for deployment of developed diagnostics are crucial to achieving the 2030 NTD elimination target. Point-of-care NTD (POC-NTD) diagnostic tools are recommended preferred diagnostic options in resource-constrained areas for mapping risk zones and monitoring treatment efficacy. However, few are currently available commercially. Technical training of remote health care workers on the use of POC-NTD diagnostics, and training of health workers on the psychosocial consequences of these diagnostics are critical in harnessing POC-NTD diagnostic potential. While the COVID-19 pandemic has challenged the possibility of achieving NTD elimination in 2030 due to the disruption of healthcare services and dwindling financial support for NTDs, the possible contribution of NTDs in exacerbating COVID-19 pandemic should motivate NTD health system strengthening.

Streamlining sporozoite isolation from mosquitoes by leveraging the dynamics of migration to the salivary glands.

BACKGROUND: Sporozoites isolated from the salivary glands of Plasmodium-infected mosquitoes are a prerequisite for several basic and pre-clinical applications. Although salivary glands are pooled to maximize sporozoite recovery, insufficient yields pose logistical and analytical hurdles; thus, predicting yields prior to isolation would be valuable. Preceding oocyst densities in the midgut is an obvious candidate. However, it is unclear whether current understanding of its relationship with sporozoite densities can be used to maximize yields, or whether it can capture the potential density-dependence in rates of sporozoite invasion of the salivary glands. METHODS: This study presents a retrospective analysis of Anopheles stephensi mosquitoes infected with two strains of the rodent-specific Plasmodium berghei. Mean oocyst densities were estimated in the midguts earlier in the infection (11-15 days post-blood meal), with sporozoites pooled from the salivary glands later in the infection (17-29 days). Generalized linear mixed effects models were used to determine if (1) mean oocyst densities can predict sporozoite yields from pooled salivary glands, (2) whether these densities can capture differences in rates of sporozoite invasion of salivary glands, and (3), if the interaction between oocyst densities and time could be leveraged to boost overall yields. RESULTS: The non-linear effect of mean oocyst densities confirmed the role of density-dependent constraints in limiting yields beyond certain oocyst densities. Irrespective of oocyst densities however, the continued invasion of salivary glands by the sporozoites boosted recoveries over time (17-29 days post-blood meal) for either parasite strain. CONCLUSIONS: Sporozoite invasion of the salivary glands over time can be leveraged to maximize yields for P. berghei. In general, however, invasion of the salivary glands over time is a critical fitness determinant for all Plasmodium species (extrinsic incubation period, EIP). Thus, delaying sporozoite collection could, in principle, substantially reduce dissection effort for any parasite within the genus, with the results also alluding to the potential for changes in sporozoites densities over time to modify infectivity for the next host.

Prevalence of Mutations in the Pfdhfr, Pfdhps, and Pfmdr1 Genes of Malarial Parasites Isolated from Symptomatic Patients in Dogondoutchi, Niger.

The effectiveness of artemisinin-based combination therapies (ACTs) depends not only on that of artemisinin but also on that of partner molecules. This study aims to evaluate the prevalence of mutations in the Pfdhfr, Pfdhps, and Pfmdr1 genes from isolates collected during a clinical study. Plasmodium genomic DNA samples extracted from symptomatic malaria patients from Dogondoutchi, Niger, were sequenced by the Sanger method to determine mutations in the Pfdhfr (codons 51, 59, 108, and 164), Pfdhps (codons 436, 437, 540, 581, and 613), and Pfmdr1 (codons 86, 184, 1034, and 1246) genes. One hundred fifty-five (155) pre-treatment samples were sequenced for the Pfdhfr, Pfdhps, and Pfmdr1 genes. A high prevalence of mutations in the Pfdhfr gene was observed at the level of the N51I (84.97%), C59R (92.62%), and S108N (97.39%) codons. The key K540E mutation in the Pfdhps gene was not observed. Only one isolate was found to harbor a mutation at codon I431V. The most common mutation on the Pfmdr1 gene was Y184F in 71.43% of the mutations found, followed by N86Y in 10.20%. The triple-mutant haplotype N51I/C59R/S108N (IRN) was detected in 97% of the samples. Single-mutant (ICS and NCN) and double-mutant (IRS, NRN, and ICN) haplotypes were prevalent at 97% and 95%, respectively. Double-mutant haplotypes of the Pfdhps (581 and 613) and Pfmdr (86 and 184) were found in 3% and 25.45% of the isolates studied, respectively. The study focused on the molecular analysis of the sequencing of the Pfdhfr, Pfdhps, and Pfmdr1 genes. Although a high prevalence of mutations in the Pfdhfr gene have been observed, there is a lack of sulfadoxine pyrimethamine resistance. There is a high prevalence of mutation in the Pfmdr184 codon associated with resistance to amodiaquine. These data will be used by Niger's National Malaria Control Program to better monitor the resistance of Plasmodium to partner molecules in artemisinin-based combination therapies.

Diagnosis of Schistosoma mansoni infections: what are the choices in Brazilian low-endemic areas?

The population of Brazil is currently characterised by many individuals harbouring low-intensity Schistosoma mansoni infections. The Kato-Katz technique is the diagnostic method recommended by the World Health Organization (WHO) to assess these infections, but this method is not sensitive enough in the context of low egg excretion. In this regard, potential alternatives are being employed to overcome the limits of the Kato-Katz technique. In the present review, we evaluated the performance of parasitological and immunological approaches adopted in Brazilian areas. Currently, the diagnostic choices involve a combination of strategies, including the utilisation of antibody methods to screen individuals and then subsequent confirmation of positive cases by intensive parasitological investigations.

Field Testing Integrated Interventions for Schistosomiasis Elimination in the People's Republic of China: Outcomes of a Multifactorial Cluster-Randomized Controlled Trial.

Despite significant progress, China faces the challenge of re-emerging schistosomiasis transmission in currently controlled areas due, in part, to the presence of a range of animal reservoirs, notably water buffalo and cattle, which can harbor Schistosoma japonicum infections. Environmental, ecological and social-demographic changes in China, shown to affect the distribution of oncomelanid snails, can also impact future schistosomiasis transmission. In light of their importance in the S. japonicum, lifecycle, vaccination has been proposed as a means to reduce the excretion of egg from cattle and buffalo, thereby interrupting transmission from these reservoir hosts to snails. A DNA-based vaccine (SjCTPI) our team developed showed encouraging efficacy against S. japonicum in Chinese water buffaloes. Here we report the results of a double-blind cluster randomized trial aimed at determining the impact of a combination of the SjCTPI bovine vaccine (given as a prime-boost regimen), human mass chemotherapy and snail control on the transmission of S. japonicum in 12 selected administrative villages around the Dongting Lake in Hunan province. The trial confirmed human praziquantel treatment is an effective intervention at the population level. Further, mollusciciding had an indirect ~50% efficacy in reducing human infection rates. Serology showed that the SjCTPI vaccine produced an effective antibody response in vaccinated bovines, resulting in a negative correlation with bovine egg counts observed at all post-vaccination time points. Despite these encouraging outcomes, the effect of the vaccine in preventing human infection was inconclusive. This was likely due to activities undertaken by the China National Schistosomiasis Control Program, notably the treatment, sacrifice or removal of bovines from trial villages, over which we had no control; as a result, the trial design was compromised, reducing power and contaminating outcome measures. This highlights the difficulties in undertaking field trials of this nature and magnitude, particularly over a long period, and emphasizes the importance of mathematical modeling in predicting the potential impact of control intervention measures. A transmission blocking vaccine targeting bovines for the prevention of S. japonicum with the required protective efficacy would be invaluable in tandem with other preventive intervention measures if the goal of eliminating schistosomiasis from China is to become a reality.

Prime-Boost Vaccine Regimen for SjTPI and SjC23 Schistosome Vaccines, Increases Efficacy in Water Buffalo in a Field Trial in China.

Schistosomiasis remains a serious zoonotic disease in China and the Philippines. Water buffalo and cattle account for the majority of transmission. Vaccination of water buffalo is considered a key strategy to reduce disease prevalence. Previously, we showed that vaccination of water buffalo with SjC23 or SjCTPI plasmid DNA vaccines, induced 50% efficacy to challenge infection. Here, we evaluated several parameters to determine if we can develop a two dose vaccine that maintains the efficacy of the three dose vaccine. We performed four trials evaluating: (1) lab produced vs. GLP grade vaccines, (2) varying the time between prime and boost, (3) the influence of an IL-12 adjuvant, and (4) a two dose heterologous (DNA-protein) prime-boost. We found the source of the DNA vaccines did not matter, nor did increasing the interval between prime and boost. Elimination of the IL-12 plasmid lowered homologous DNA-DNA vaccine efficacy. A major finding was that the heterologous prime boost improved vaccine efficacy, with the prime-boost regimen incorporating both antigens providing a 55% reduction in adult worms and 53% reduction in liver eggs. Vaccinated buffalo produced vaccine-specific antibody responses. These trials suggest that highly effective vaccination against schistosomes can be achieved using a two dose regimen. No adjuvants were used with the protein boost, and the potential that addition of adjuvant to the protein boost to further increase efficacy should be evaluated. These results suggest that use of these two schistosome vaccines can be part of an integrated control strategy to reduce transmission of schistosomiasis in Asia.

Diagnosis of Schistosoma mansoni infections: what are the choices in Brazilian low-endemic areas?

The population of Brazil is currently characterised by many individuals harbouring low-intensity Schistosoma mansoni infections. The Kato-Katz technique is the diagnostic method recommended by the World Health Organization (WHO) to assess these infections, but this method is not sensitive enough in the context of low egg excretion. In this regard, potential alternatives are being employed to overcome the limits of the Kato-Katz technique. In the present review, we evaluated the performance of parasitological and immunological approaches adopted in Brazilian areas. Currently, the diagnostic choices involve a combination of strategies, including the utilisation of antibody methods to screen individuals and then subsequent confirmation of positive cases by intensive parasitological investigations.

Vaccine self-assembling immune matrix is a new delivery platform that enhances immune responses to recombinant HBsAg in mice.

Vaccination remains the most effective public health tool to prevent infectious diseases. Many vaccines are marginally effective and need enhancement for immunocompromised, elderly, and very young populations. To enhance immunogenicity, we exploited the biphasic property of the (RADA)4 synthetic oligopeptide to create VacSIM (vaccine self-assembling immune matrix), a new delivery method. VacSIM solution can easily be mixed with antigens, organisms, and adjuvants for injection. Postinjection, the peptides self-assemble into hydrated nanofiber gel matrices, forming a depot with antigens and adjuvants in the aqueous phase. We believe the depot provides slow release of immunogens, leading to increased activation of antigen-presenting cells that then drive enhanced immunogenicity. Using recombinant hepatitis B virus surface antigen (rHBsAg) as a model immunogen, we compared VacSIM delivery to delivery in alum or complete Freund's adjuvant (CFA). Delivery of the rHBsAg antigen to mice via VacSIM without adjuvant elicited higher specific IgG responses than when rHBsAg was delivered in alum or CFA. Evaluating IgG subtypes showed a mixed Th1/Th2 type response following immunization with VacSIM, which was driven further toward Th1 with addition of CpG as the adjuvant. Increased specific IgG endpoint titers were observed in both C57BL/6 and BALB/c mice, representative of Th1 and Th2 environments, respectively. Restimulation of splenocytes suggests that VacSIM does not cause an immediate proinflammatory response in the host. Overall, these results suggest that VacSIM, as a new delivery method, has the potential to enhance immunogenicity and efficacy of numerous vaccines.

Schistosoma mansoni soluble egg antigens enhance T cell responses to a newly identified HIV-1 Gag H-2b epitope.

Schistosome infection induces significant T helper type 2 (Th2) and anti-inflammatory immune responses and has been shown to negatively impact vaccine efficacy. Our goal was to determine if the administration of schistosome soluble egg antigens (SEA) would negatively influence the induction of cytotoxic T lymphocyte (CTL) and Th1-type T cell responses to an HIV candidate vaccine in the Th1-biased C57BL/6 mouse strain. Initial experiments failed, as we were unable to detect any response to the defined class I epitope for HIV-1 IIIB Gag. Therefore, we initiated an epitope mapping study to identify C57BL/6 (H-2(b)) T cell epitopes in HIV-1 IIIB Gag in order to perform the experiments. This analysis defined two previously unreported minimal class I H-2(b) and class II I-A(b) epitopes for HIV-1 IIIB Gag. The newly defined HIV-1 IIIB Gag epitopes were used to evaluate the influence of SEA on the generation of CTL and Th1-type HIV-1 IIIB Gag responses. Surprisingly, in contrast to our hypothesis, we observed that the coadministration of SEA with a Listeria monocytogenes vector expressing HIV-1 IIIB Gag (Lm-Gag) led to a significantly increased frequency of gamma interferon (IFN-γ)-producing CD8(+) and CD4(+) T cells in C57BL/6 mice compared to mice immunized with Lm-Gag only. These observations suggest that SEA contains, in addition to Th2-type and immune-suppressive molecules, substances that can act with the Lm-Gag vaccine to increase CTL and Th1-type vaccine-specific immune responses.

Schistosoma mansoni soluble egg antigens enhance Listeria monocytogenes vector HIV-1 vaccine induction of cytotoxic T cells.

Vaccines are an important public health measure for prevention and treatment of diseases. In addition to the vaccine immunogen, many vaccines incorporate adjuvants to stimulate the recipient's immune system and enhance vaccine-specific responses. While vaccine development has advanced from attenuated organism to recombinant protein or use of plasmid DNA, the development of new adjuvants that safely increase immune responses has not kept pace. Previous studies have shown that the complex mixture of molecules that comprise saline soluble egg antigens (SEA) from Schistosoma mansoni eggs functions to promote CD4(+) T helper 2 (Th2) responses. Therefore, we hypothesized that coadministration of SEA with a Listeria vector HIV-1 Gag (Lm-Gag) vaccine would suppress host cytotoxic T lymphocyte (CTL) and T helper 1 (Th1) responses to HIV-1 Gag epitopes. Surprisingly, instead of driving HIV-1 Gag-specific responses toward Th2 type, we found that coadministration of SEA with Lm-Gag vaccine significantly increased the frequency of gamma interferon (IFN-γ)-producing Gag-specific Th1 and CTL responses over that seen in mice administered Lm-Gag only. Analysis of the functionality and durability of vaccine responses suggested that SEA not only enlarged different memory T cell compartments but induced functional and long-lasting vaccine-specific responses as well. These results suggest there are components in SEA that can synergize with potent inducers of strong and durable Th1-type responses such as those to Listeria. We hypothesize that SEA contains moieties that, if defined, can be used to expand type 1 proinflammatory responses for use in vaccines.

HIV-1 vaccine-specific responses induced by Listeria vector vaccines are maintained in mice subsequently infected with a model helminth parasite, Schistosoma mansoni.

In areas co-endemic for helminth parasites and HIV/AIDS, infants are often administered vaccines prior to infection with immune modulatory helminth parasites. Systemic Th2 biasing and immune suppression caused by helminth infection reduces cell-mediated responses to vaccines such as tetanus toxoid and BCG. Therefore, we asked if infection with helminthes post-vaccination, alters already established vaccine induced immune responses. In our model, mice are vaccinated against HIV-1 Gag using a Listeria vaccine vector (Lm-Gag) in a prime-boost manner, then infected with the human helminth parasite Schistosoma mansoni. This allows us to determine if established vaccine responses are maintained or altered after helminth infection. Our second objective asked if helminth infection post-vaccination alters the recipient's ability to respond to a second boost. Here we compared responses between uninfected mice, schistosome infected mice, and infected mice that were given an anthelminthic, which occurred coincident with the boost or four weeks prior, as well as comparing to un-boosted mice. We report that HIV-1 vaccine-specific responses generated by Listeria vector HIV-1 vaccines are maintained following subsequent chronic schistosome infection, providing further evidence that Listeria vector vaccines induce potent vaccine-specific responses that can withstand helminth infection. We also were able to demonstrate that administration of a second Listeria boost, which markedly enhanced the immune response, was minimally impacted by schistosome infection, or anthelminthic therapy. Surprisingly, we also observed enhanced antibody responses to HIV Gag in vaccinated mice subsequently infected with schistosomes.

Successful vaccination of immune suppressed recipients using Listeria vector HIV-1 vaccines in helminth infected mice.

Vaccines for HIV, malaria and TB remain high priorities, especially for sub-Saharan populations. The question is: will vaccines currently in development for these diseases function in populations that have a high prevalence of helminth infection? Infection with helminth parasites causes immune suppression and a CD4+ Th2 skewing of the immune system, thereby impairing Th1-type vaccine efficacy. In this study, we conduct HIV vaccine trials in mice with and without chronic helminth infection to mimic the human vaccine recipient populations in Sub-Saharan Africa and other helminth parasite endemic regions of the world, as there is large overlap in global prevalence for HIV and helminth infection. Here, we demonstrate that Listeria monocytogenes functions as a vaccine vector to drive robust and functional HIV-specific cellular immune responses, irrespective of chronic helminth infection. This observation represents a significant advance in the field of vaccine research and underscores the concept that vaccines in the developmental pipeline should be effective in the target populations.

Sine-wave current for efficient and safe in vivo gene transfer.

Recently, electro-gene transfer has become a powerful tool to enhance the efficiency of gene expression in different organs and particularly in muscle, which provides efficient secretion of therapeutic proteins into the circulation. However, its toxicity, owing to the high field strengths of conventional direct current (DC) square-waves, should be taken into account and should be minimized if electroporation is to be used routinely for the treatment of human diseases. In this study, we demonstrate that efficient in vivo gene transfer could be safely achieved using pulses of alternating current sine-waves (ACSWs) with a frequency of 60 Hz. Importantly, the field strength was decreased to as low as 20 V/cm and the efficiency of muscle gene transfer increased more than tenfold and showed less toxicity than with conventional DC square-wave pulses. Using ACSW pulses to transfer human clotting factor IX (hFIX) plasmid into muscle, we observed significant phenotypic correction in mice with hemophilia B.

Mechanism of naked DNA clearance after intravenous injection.

BACKGROUND: Injection of naked DNA has been viewed as a safer alternative to current gene delivery systems; however, the rate of clearance from the circulation has been a constant barrier in developing these methods. Naked DNA after intravenous (i.v.) injection will be taken up by the liver and depredated by serum nucleases. MATERIALS AND METHODS: Our study examines the mechanisms involved in clearance of naked DNA by each compartment, the blood and the liver, in an in vivo mouse model. Confocal microscopy and transmission electron microscopy were employed to identify the type of cells taking up DNA and the barrier to DNA access to hepatocytes, respectively. RESULTS: Our data showed the liver could take up over 50% of 5 microg perfused pDNA, with a maximum 25 microg of pDNA during a single pass, and a slower clearance rate compared to that of liver uptake. It was demonstrated that naked DNA is primarily taken up by the liver endothelial cells and this endothelial barrier to transfection could be overcome by manually massaging the liver, which enlarges the fenestrae. CONCLUSIONS: This study clarifies the mechanism by which naked DNA is eliminated from the circulation after i.v. injection, focusing on the role of both the liver and blood compartments in vivo (i.e. mouse). With this knowledge, we can more clearly understand the mechanism of naked DNA clearance and develop more efficient strategies for DNA transfer in vivo.

Novel nonviral vectors target cellular signaling pathways: regulated gene expression and reduced toxicity.

Advances in cell biology over the last several decades have allowed for a much greater understanding of the regulation of cellular processes. Many of these revelations have provided substantial details regarding the key players in cellular pathways and the role small-molecule ligands may play in controlling their function. Although much progress has been made in these areas, optimization of nonviral gene delivery vectors has not met with similar success. Many of the issues that have plagued the field, such as limited transgene activity, difficulty with specific cell targeting, inflammatory responses, and degradation of the vector, among others, continue to limit the efficacy of these delivery systems. In this study, we investigate several cellular pathways in an effort to develop more efficient nonviral vectors. To increase nuclear uptake of the transgene, we explored the use of nuclear localization sequences (NLS) incorporated into our plasmid. The results indicated that the NLS did significantly increase gene expression under several circumstances in the presence of small-molecule ligands, as indicated by both in vitro and in vivo studies. Furthermore, to decrease inflammatory response to the vectors, additional studies were performed to demonstrate that the incorporation of free anti-inflammatory ligands into liposome formulations does not affect transgene activity but is able to significantly decrease the inflammatory response. Overall, these examples provide hope that free ligand can be used to effectively mediate cellular processes to overcome some of the obstacles limiting the success of gene therapy.

Mechanism of in vivo DNA transport into cells by electroporation: electrophoresis across the plasma membrane may not be involved.

BACKGROUND: Recently, in vivo gene transfer with electroporation (electro-gene transfer) has emerged as a leading technology for developing nonviral gene therapies and nucleic acid vaccines. The widely hypothesized mechanism is that electroporation induces structural defects in the membrane and provides an electrophoretic force to facilitate DNA crossing the permeabilized membrane. In this study, we have designed a device and experiments to test the hypothesis. METHODS: In this study, we have designed a device that alternates the polarity of the applied electric field to elucidate the mechanism of in vivo electro-gene transfer. We also designed experiments to challenge the theory that the low-voltage (LV) pulses cannot permeabilize the membrane and are only involved in DNA electrophoresis, and answer the arguments that (1) the reversed polarity pulses can cause opposing sides of the cell membrane to become permeabilized and provide the electrophoresis for DNA entry; or (2) once DNA enters cytoplasmic/endosomal compartments after electroporation, it may bind to cellular entities and might not be reversibly extracted. Thus a gradual buildup of the DNA in the cell still seems quite possible even under the condition of the rapid reversal of polarity. RESULTS: Our results indicate that electrophoresis does not play an important role in in vivo electro-gene transfer. CONCLUSIONS: This study provides new insights into the mechanism of electro-gene transfer, and may allow the definition of newer and more efficient conditions for in vivo electroporation.

Non-immunostimulatory nonviral vectors.

The vectors for gene delivery are usually classified as viral and nonviral vectors. While the viral vectors are very efficient in transducing cells, safety concerns regarding their use in humans make nonviral vectors an attractive alternative. Among the nonviral vectors, the lipoplexes (complexes of cationic liposome/pDNA) are the most studied and represent the most promising approaches for human clinical trials. However, an inflammatory response is invariably associated with administration of the lipoplexes, which must be avoided in the clinical application. Here, we have successfully developed a nonimmunostimulatory vector for gene therapy. The vector possesses dual functions of: 1) efficiently delivering a gene to target cells and 2) codelivering DNA and inflammatory suppressors into the immune cells where the released suppressor can inhibit cytokine production. The inflammatory suppressors successfully delivered by the vector included glucocorticoids, a nonsteroidal anti-inflammatory drug (NSAID), an NF-kappaB inhibitor, and a natural compound from an herbal medicine. Intravenous injection of the vector dramatically suppressed the cytokine production induced by CpG motif pDNA, including TNF-alpha, IL-12 and IFN-gamma. This new gene vector has a great potential in clinical gene therapy. Another potential use of the vector is codelivery of an enhancer candidate, acting at the transcriptional and translational levels to improve the efficiency of gene transfer by the nonviral vector. Moreover, the unique feature of this vector is that it can be used as an easy and powerful tool for in vivo screening of anti-inflammatory drugs.

Enumeration, resuscitation, and infectivity of the sublethally injured erwinia cells induced by mild Acid treatment.

ABSTRACT Current knowledge about microbial injury was derived mostly from studies with bacteria associated with processed foods. Demonstration of injury and repair in phytopathogenic bacteria and its implication on pathogen detection and disease ecology have not been reported. The objective of this study was to investigate the lethal and sublethal effects of acetic acid (AA) on soft-rot Erwinia spp. and the limitation of using selective media for isolation of injured cells. Following exposure to 0.3% AA for 6 min, 90 to 99% of E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and E. chrysanthemi cells were killed. When AA-treated samples were plated on agar media, the number of bacteria recovered on nonselective media such as brain heart infusion agar was 3 log units higher than that recovered on selective media such as crystal violet pectate (CVP). Lethal and sublethal actions of AA on Erwinia spp. were affected greatly by acid concentration, exposure time, and bacterial strains tested. Injured Erwinia cells were able to repair and regain the ability to grow on CVP when placed in nutrient broth but not in selective broth containing crystal violet and sodium dodecyl sulfate. Injured cells also were able to resuscitate on cut surfaces of cucumber fruit and to induce soft rot on green bell pepper. Together, these results suggest that direct use of selective media for isolation of Erwinia spp. could limit the recovery of injured cells in samples that have been exposed to chemical or physical stresses. Enrichment of these samples in nutrient broth before plating on CVP is expected to improve the detection of injured Erwinia spp.