Technologies for Detecting Botulinum Neurotoxins in Biological and Environmental Matrices

Biomonitoring of food and environmental matrices is critical for the rapid and sensitive diagnosis, treatment, and prevention of diseases caused by toxins. The U.S. Centers for Disease Control and Prevention (CDC) has noted that toxins from bacteria, fungi, algae, and plants present an ongoing public health threat, especially since some of these toxins could compromise security of the food supply. Botulinum neurotoxins (BoNTs), produced by Clostridium spp., are among those bacterial toxins that pose life-threatening danger to humans. BoNTs inhibit the release of acetylcholine at peripheral cholinergic nerve terminals and cause flaccid paralysis. BoNTs are grouped in seven serotypes and many subtypes within these groups. Rapid and accurate identification of these toxins in contaminated food as well as in environmental matrices can help direct treatment. Herein, we discuss current methods to detect BoNTs with a focus on how these technologies have been used to identify toxins in various food and environmental matrices. We also discuss the emergence of new serotypes and subtypes of BoNTs and the increasing number of cases of botulism in wildlife. Finally, we consider how environmental changes impact food safety for humans and present new challenges for detection technology

Horizontal transmission of S. choleraesuis in weaned pigs exposed to different challenge levels

Salmonella choleraesuis, the serotype most often isolated from swine in the United States, is the agent responsible for swine paratyphoid. As a host adapted pathogen, S. choleraesuis is rarely isolated from sources other than swine and thus is spread primarily via horizontal transmission from diseased or asymptomatic carrier animals (Schwartz, 1990). Carriers persistently colonized by S. choleraesuis have been shown to shed the pathogen in their feces for up to 12 weeks post inoculation (Gray et al., 1996b) and that Salmonella-free pigs can be infected following exposure to infected pigs shedding Hi\u27 CFU S. Choleraesuis of feces (Gray et al., 1996a). Recurrent shedding by carriers can be stimulated by events such as recent transport, parturition, or weaning (Gray and Fedorka-Cray, 1996). In many production settings, weaned pigs from different litters are commingled and thus shedding by even one pig can result in transmission to many

Modelling S. choleraesuis infection by oral route of inoculation; titration of dose strength

Salmonella infections cost the United States swine industry more than 100 million dollars annually (Schwartz, 1990). In the United States, salmonellosis caused by Salmonella choleraesuis, the etiologic agent of swine parathyphoid, occurs most frequently. While pigs can be exposed to broad host range salmonellae, such as Salmonella typhimurium, from a variety of sources, S. choleraesuis is a host adapted pathogen rarely isolated from non-swine sources (Schwartz, 1990). Thus, natural infection of new animals by S. choleraesuis occurs primarily via horizontal transmission from infected animals which shed the pathogen from their gastrointestinal tract

Effect of competitive exclusion on salmonella shedding in swine

Despite the efforts of researchers and public health agencies, the incidence of human salmonellosis has continued to increase over the past 20 years. Salmonellosis is now the most common cause of food-borne gastroenteritis. The number of reported cases of human Salmonella infection exceeds 40,000 per year. The Communicable Disease Center estimates that the true annual incidence of human salmonellosis in the United States may be as high as 4 million cases

Evaluation of two commercially available ELISA test kits for detection of Salmonellae in swine lymph nodes and cecal and fecal contents

We conducted a survey to determine the prevalence of salmonellae in pigs in an integrated swine operation. Isolation of salmonellae from lymph nodes, cecal contents, and fecal samples was by routine microbiological culture techniques. Simultaneously, and using the same samples, we evaluated two commercially available ELISA-based Salmonella test systems (Neogen Reveal and EiaFosslM) for detection of salmonellae and compared these results to our culture results. The advantages of the Neogen test were that it was simple to use and had up to an 87% agreement with culture. The disadvantages of the Neogen test were that it was expensive, it was not suitable for cecal or fecal samples (cross-reacted with Citrobacter spp.), it had a high rate of false negatives, and positive endpoints were subjective and difficult to read. The advantages of the Foss test were that it had up to a 93% agreement with culture results, it had low rates of false negatives and false positives, it was applicable for cecal and fecal samples, and it reduced time and labor compared to culture procedures. The disadvantages of the Foss test were that it required purchase of an expensive auto analyzer and not all of the 3 protocols recommended by the manufacturer were equally effective in recovery of salmonellae. We concluded that under our study conditions, the Neogen test was not acceptable, but that the Foss system was suitable for detection of salmonellae from field samples

Competitive exclusion of Salmonella serovar Typhimurium from the gut of early weaned pigs

Strategies to curb infection of pigs by Salmonella are sought throughout the fann to table continuum. Competitive exclusion (CE), which involves colonizing the gastrointestinal tract of young animals with a healthy gut microflora, has been shown to be an attractive strategy for protecting poultry from salmonellae infections (4-7, 14, 16-17, 19-20, 23). More recent evidence suggests that CE may enhance resistance of young pigs against infections by Salmonella serovar Choleraesuis (2, 9, 15, 21), the serotype responsible for the vast majority of swine salmonellosis cases in the United States (22). Salmonella serovar Typhimurium can also infect and cause disease in swine and may be considered a greater food safety concern than Salmonella Choleraesuis but until now, reports regarding the effect of CE treatment on colonization resistance of early weaned pigs to this serotype have been few if not nonexistent

Long term shedding of Salmonella choleraesuis following experimental infection of very young piglets

In the United States, more than 90% of diagnosed swine salmonellosis cases can be attributed to infections by Salmonella Choleraesuis, the etiologic agent of swine paratyphoid (9). Salmonellosis caused by this serotype is primarily manifested as a post weaning septicentia or enterocolitis and often occurs on operations that comntingle pigs of different ages (9). Salmonella Choleraesuis is rarely isolated from sources other than swine which suggests an important role of carrier swine in the spread of this host adapted pathogen (4-6, 9). Since Salmonella Choleraesuis infections can recur in pigs previously infected, an important role for latent carriers is further implicated (9). The development of a Salmonella Choleraesuis carrier state has been investigated experimentally with weaned piglets (1, 4- 6) but not much is known regarding the potential of neonatal piglets to become long term carriers. This may be because neonatal piglets, while capable of beconting infected, rarely exhibit clinical salmonellosis (9). The objective of the present study was to experimentally infect suckling piglets with Salmonella Choleraesuis and then examine their daily shedding pattern and pattern of tissue colonization well beyond the initial infection period

Comparison of two enrichment schemes for qualitative recovery of Salmonella serovar Choleraesuis from rectal swabs collected from neonatal and early weaned pigs

Traditional bacteriological cultivation of samples for salmonellae typically have involved a preenrichment in buffered peptone water, particularly for samples suspected of harboring low numbers of salmonellae. Many researchers; however, have since dispensed with this initial step (4-6, 13, 17-20), opting instead for direct enrichment in tetrathionite broth, especially for samples suspected of containing nonhost adapted serotypes. However, direct enrichment in tetrathionite may be too harsh for optimal recovery of Salmonella serovar Choleraesuis (3, 16). Thus, GN-Hajna broth has been recommended as a preenrichment broth for samples suspected of containing this host adapted serotype (7) and has been used in several recent studies (I, 4, 8-10). Direct comparative evidence, however, substantiating this recommendation are lacking. Presently, we report findings supporting the use of GN-Hajna as a preenrichment medium for recovery of Salmonella Choleraesuis

Preliminary experiments involving Salmonella-immune lymphokine in vivo effects on neutrophil function in weaned pigs and Salmonella choloraesuis organ invasion in neonatal pigs

Using knowledge gained from the administration of Salmonella enteritidis-immune lymphokines (ILK) derived from the t cells of S. enteritidis-immune chickens to neonatal poultry, the idea ofimmunopotentiation of the immune response of immune-compromised animals was applied to swine. AS. enteritidis-immune lymphokine (PILK) was isolated from the splenic t cells of S. enteritidis-immune pigs. PILK was then administered orally to weaned pigs, considered to possess deficient immune responses, which were subsequently challenged with both lethal and non-lethal doses of the swine pathogenS. choleraesuis (SC). PILKtreated pigs were shown to have a 50-60% decrease in organ invasion and a similar reduction in cecal colonization by SC. PILK also enhanced growth performance in both SC challenged and non challenged pigs, with PILK-treated pigs gaining an average of 5 pounds more than both nonchallenged controls and SC challenged controls. PILK also significantly reduced morbity and mortality as compared to control pigs. Neutrophils isolated from the peripheral blood of PILK-treated pigs exhibited increased functional capabilities when compared to control pigs. Significant increases in the oxidative burst, adherence to nylon wool and bovine serum albumin-coated slides, and increased movement towards chemotactic stimuli were shown by neutrophils from PILK-treated pigs when compared to neutrophils from control pigs. We have shown that PILK protects pigs from SC organ invasion, cecal colonization and enhances growth performance and neutrophil functions in weaned pigs

Epidemiological survey of antimicrobial-resistant of Salmonella in market-age swine

We conducted a survey of antibiotic resistance in Salmonella isolaled from market-age swine at five different farms. These farms, which were sampled between October 1997 and June 1998, were part of a completely integrated Texas swine operation. Four of the farms sampled were farrow-to-finish farms and were sampled three times each. The fifth farm provided replacement gilts for the other farms in the operation and was sampled once. Samples were taken from the lymph nodes and cecal contents at the time of slaughter. Of the 559 Salmonella isolates recovered, 420 were sent to the National Veterinary Services Laboratory for serotyping. Resistance patterns were determined by disk diffusion using thitteen antibiotics, and 320 isolates were analyzed. Resistance was observed to ampicillin, chloramphenicol, chlortetracycline, nitrofurantoin, penicillin G, streptomycin and sulfisoxazole. Only 21 isolates (6.9%) demonstrated no resistance to any of the antibiotics tested. Multidrug resistance (resistance to two or more antibiotics) was observed in 183 isolates (57.2%) with 34 of these isolates 18.6%) resistant to three antibiotics and 26 (8.1 %) resistant to four antibiotics. The most common three drug resistance pattern consisted of chlortetracycline, penicillin G and streptomycin (37 .3% ). All four drug resistance patterns consisted of chlortetracycline, penicillin G, streptomycin and sulfisoxazole ( 100% ). A significant difference was observed between serotypes and between somatic serogroups in their antibiotic resistance patterns. Variation also was observed between farms. As a step in understanding the connection between antibiotic use in agriculture and medicine and emergence of antibiotic resistant bacteria, programs that monitor the levels of antibiotic resistance must be continued. In animal production, where subtherapeutic administration of antibiotics can be common, continued surveillance is especially important