Botulinum neurotoxin (BoNT) serotype E is one of three serotypes that cause the preponderance of human botulism cases and is a Tier 1 Select Agent. BoNT/E is unusual among BoNT serotypes for its rapid onset and short duration of intoxication. Here we report two large panels of unique, unrelated camelid single-domain antibodies (VHHs) that were selected for their ability to bind to BoNT/E holotoxin and/or to the BoNT/E light chain protease domain (LC/E). The 19 VHHs which bind to BoNT/E were characterized for their subunit specificity and 8 VHHs displayed the ability to neutralize BoNT/E intoxication of neurons. Heterodimer antitoxins consisting of two BoNT/E-neutralizing VHHs, including one heterodimer designed using structural information for simultaneous binding, were shown to protect mice against co-administered toxin challenges of up to 500 MIPLD₅₀. The 22 unique VHHs which bind to LC/E were characterized for their binding properties and 9 displayed the ability to inhibit LC/E protease activity. Surprisingly, VHHs selected on plastic-coated LC/E were virtually unable to recognize soluble or captured LC/E while VHHs selected on captured LC/E were poorly able to recognize LC/E coated to a plastic surface. This panel of anti-LC/E VHHs offer insight into BoNT/E function, and some may have value as components of therapeutic antidotes that reverse paralysis following BoNT/E exposures.

Leptospirosis is a zoonotic disease with worldwide distribution caused by pathogenic Leptospira spp. Pathogenic Leptospira spp. are shed in urine of infected hosts and transmitted via ingestion of contaminated food or water, inoculation, inhalation of aerosolized urine, and absorption through mucous membranes. Leptospirosis is of particular concern in tropical and subtropical regions such as Barranquilla, Colombia. Recent reports indicate that in Barranquilla, rodents, dogs, and humans have a high leptospiral seroprevalence; and amongst zoo mammals, nonhuman primates have a high prevalence of Leptospira spp. infection. We therefore sought to determine whether primates in captivity at the Barranquilla Zoo were exposed to Leptospira spp. and whether there was a probable causal transmission link between the primates and peridomestic rodents. Samples were collected from 29 captive nonhuman primates, 15 free-ranging rats (Rattus rattus), and 10 free-ranging squirrels (Sciurus granatensis). Serum samples from primates, rats, and squirrels were evaluated via microagglutination test (MAT) vs 24 reference Leptospira serovars. Blood and urine from the primates and kidney tissue from the rats and squirrels were cultured in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium and polymerase chain reaction (PCR) of lipL32 was performed to determine whether active infection was present. Leptospiral seroprevalence was found to be 66.7% (10/15) in rats, 60% (6/10) in squirrels, and 6.9% (2/29) in neotropical primates. Ateles hybridus and Ateles fusciceps had positive titers to serogroups Cynopteri and Ictohaemorrhagiae, respectively. Of the rodents that had antibodies against Leptospira spp., 90% of the rats and 66.7% of the squirrels corresponded to the serovar australis. Interestingly, all animals were culture and PCR negative, indicating Leptospira spp. exposure in the absence of current infection. While their status as maintenance hosts needs to be investigated further, this is the first study to show leptospiral seropositivity in red-tailed squirrels (S. granatensis).