1 Michael J. Yabsley & Sarah Sapp [email protected] and [email protected]A One Health approach to the study of the raccoon roundworm Baylisascaris procyonis Michael J. Yabsley & Sarah Sapp and 1
2 Baylisascaris procyonisLarge intestinal ascarid nematode of raccoons (and sometimes dogs) Paratenic hosts can be used for transmission >150 sp. of birds and mammals, including humans 2
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4 Baylisascariasis Larva Migrans: Visceral (VLM); Neural (NLM); Ocular (OLM) NLM case fatality rate >40% Eosinophilic meningitis OLM – Diffuse unilateral subacute neuroretinitis Many with permanent vision defects In humans, ~50 confirmed cases Also mortality of numerous wildlife species, captive exotics, livestock Treatment complicated, expensive The clinical manifestations of baylisascaris infection are larva migrans syndromes that fall into these three categories …. Complicated because hard to get drug through blood brain barrier to kill migrating larvae, and also could cause harm killing larvae within brain (larval antigen release). Expensive— Wouldn’t think albendazole is expensive, but it fell into a similar fate as Darparim the Toxoplasmosis drug. 4
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9 Then a friend from Tallahassee calls...9
10 Then I’m on vacation...Ft. Lauderdale, 2010
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12 Kinkajou in a Tennessee pet storeMultiagency investigation TN DOH Local DVM USDA CDC FL DOH Various University partners Environment positive for eggs Established? Spreading? From raccoons? 12
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15 Baylisascaris diagnosticsRaccoons and dogs Fecal exam Distinguish eggs from Toxocara/Toxascaris in dogs Recovery of adult worms after treatment or at necropsy Paratenic hosts Recovery of larvae from tissues either by squash or digestion Histologic detection of larvae PCR testing of tissues 15
16 Baylisascaris diagnosticsHUMANS L3 within tissues, so serology invaluable as ante-mortem diagnostic tool Initial work on an ELISA using a recombinant E-S Antigen (BpRAG-1) (Dangoudoubiyam et al., 2011; Clin Vaccine Immunol) 20 Bp patients, 137 with other parasitic diseases, and 227 unknown/suspected parasite infections 85% Se and 86.9% Sp ~25% cross-reactivity with Toxocara spp. CDC-PDB and National Reference Centre for Parasitology (NRCP), QC, Canada have the rBpRAG-1 and utilize it in a western blot (Rascoe et. al 2013; Clin Vaccine Immunol) CDC: 15 Bp patients, 109 other diseases, and 150 healthy individuals NRCP: 7 Bp patients, 264 other diseases, and 130 healthy individuals 88% Se; 98% Sp (maybe higher?) Serum & CSF 16
17 Seroprevalence and risk factors for exposure to Baylisascaris procyonis in wildlife rehabilitators17
18 Epidemiology of baylisascariasisIncidence in people appears to be increasing Geographic expansion, increased raccoon density Cases generally restricted to children, mentally disabled Evidence for asymptomatic/subclinical infection in children & adults Dose related? Toxocara canis: closely related ascarid 14% seroprevalence nationally “Covert” infections: asthma, urticaria, cognitive delays, occult seizures May be due to inc. recognition, but also maybe other factorss 18
19 Baylisascaris seroprevalence and risk factors in wildlife rehabilitatorsHypothesis: B. procyonis infections in people may be occurring covertly, with some populations more at risk than others Objective: Determine seroprevalence of B.p. in wildlife rehabilitators and identify risk factors 19
20 Study Design 20 Cross-sectional serosurveyHealthy, adult wildlife rehabilitators 31-item QN assessing demographics, rehabilitation history, PPE use, raccoon- specific practices Antibody detection: Western Blot rBpRAG-1; recombinant excretory- secretory antigen (88% Se, 98% Sp) 20
21 Study Population 21 n=347 86% Female; 94% White Age: 18-89 (median 47)91% currently involved in wildlife rehabilitation 6% formerly; 4% other non-rehab wildlife involvement 21
22 Raccoon Rehab & ContactOf those who actively practice rehabilitation, over half rehabilitate raccoons. In a more general sense, the majority of participants reported some kind of contact with raccoons even if they did not actually rehab them. 22
23 Nature of Raccoon ContactOur participants reported a variety of types raccoon contact, with the majority reporting touching live animals or having contact with feces. Fewer reported bites, scratches, and necropsies. 23
24 Raccoon Contact: PPE UseLive animals Dead animals 24
25 Raccoon Feces Contact 25 PPE UseWith potential feces contact, fortunately most washed hands… over half consistently wore gloves as well. ¾ had daily contact with feces via enclosure cleaning. 25
26 Raccoon Husbandry: Parasite ControlDeworm raccoons on a regular basis? NO: 43% NO: 62% We assessed parasite control practices—over half had observed nematodes in feces; less had a confirmed dx YES: 57% YES: 38% Observed nematodes in feces Confirmed B.p. diagnosis 26
27 Seroprevalence of Baylisascaris in wildlife rehabilitatorsSeroprevalence: 7% (24/347) (Adjusted: 5.4%) 27
28 Univariate risk factor analysis: REGIONVariable n pos/n respondents cOR (95% CI) p Northeastern 4/106 (3.9%) 0.83 ( ) 1.000 Midwestern 8/74 (12.5%) 2.58 ( ) 0.141 Central 0/23 (0.0%) ND - Southern 5/110 (4.7%) Reference Western 7/34 (25.9%) 5.25 ( ) 0.009 28
29 29 Univariate risk factor analysis:STATE/PROV. LEVEL B.p. PREVALENCE IN RACCOONS Variable n pos/n respondents cOR (95% CI) p Hyperendemic (>50%) 14/79 (21.5%) 4.74 ( ) 0.006 High (25-50%) 5/127 (4.6%) 0.90 ( ) 1.000 Moderate (10-24%) 4/92 (4.3%) Reference - Low (<10%), Sporadic, or Unknown 1/49 (20.4%) 0.46 ( ) 0.658 29
30 Univariate risk factor analysis: PPE ComplianceVariable n pos/n respondents cOR (95% CI) p LIVE RACCOONS Always washes hands 12/214 (5.6%) Reference - Does not always wash hands 5/30 (16.7%) 3.37 ( ) 0.043 FECES CONTACT 10/198 (5.0%) 5/23 (21.7%) 5.22 ( ) 0.012 30
31 Serosurvey: Conclusions24/347 (7%) seropositive Significant risk factors included: Practicing rehabilitation in the Western region Practicing rehabilitation in B.p. hyperendemic areas Inconsistent hand-washing after handling live raccoons or after potential fecal contact Rehabilitators should always wash hands and wear gloves during contact with raccoons or their feces, deworm animals, clean enclosures with heat methods 31
32 In conclusion… Subclinical/covert baylisascariasis has been detected in the study population, indicating possibility for under- recognition. More work needed to understand B. procyonis epidemiology on larger scale Ongoing awareness/education efforts necessary 32
33 Future Directions 33 Knowledge/Attitudes/Practices Survey:Administered to wildlife rehabilitation community at large Analysis of risk factors and knowledge gaps within the (wildlife) community will guide intervention strategies and public health educational efforts. Serologic Assay Development: ELISA or Luminex format - higher throughput B.p. sero-epidemiologic studies in the general population 33
34 Baylisascariasis in rodentsParatenic hosts, ecological role in maintenance/transmission Has been reported from numerous species Most have been singleton cases or small ‘outbreaks’ Only two studies on wild healthy rodents Concern for some species such as Alleghany woodrat An experimental study showed that P. leucopus were less likely or took longer to develop disease compared with laboratory mice (50, 250, 500 eggs) Other experimental studies have showed that several other rodents developed disease but egg doses not provided 34
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36 Genus Peromyscus 36 “Deer Mice”Probably common intermediate hosts of B. procyonis Foraging/caching behavior Many species in North America Natural B. procyonis infections documented sporadically in a limited number of Peromyscus species Only P. leucopus studied in context of natural asympotomatic B. procyonis infections 36
37 37 P. maniculatus “Deer Mouse” P. californicus “California Mouse”TO investigate species level susceptibility, we used four species of Peromyscus in an experimental infection trial. First, the P.m. and P.l., the most widespread deer mouse species in North America; Then P. poilionotus… many species highly endangered. Finally, P. californicus, native to Southern CA and Baja Peninsula. These species represent a range of sizes (P.p. small – 12 g, Pc. Large – 50 g) come from a range of habitat types, and from variable areas of B. procyonis prevalence, which is very high in the upper midwest, northeast, and pacific nw, and a relatively recent introduction into the SE where prevalence is still relatievly low. P. leucopus “White-footed Mouse” P. polionotus “Oldfield Mouse” 37
38 brain examination for larvaeP. californicus P. maniculatus P. leucopus P. polionotus n=6 mice/dose group Euthanasia Upon CNS disease or 45 dpi 500, 50, or 10 eggs p.o. Necropsy & brain examination for larvae We acquired captive bred mice of each species, 6 per dose grop, and inoculated each group with 500, 50, or 10 embryonated B.p. eggs. Followed, euthanized upon severe neurologic disease or at 45 dpi if survived. These mice were necropsied, and the brains were examined microscopically for migrating larvae. Then, larvae were recovered from the rest of the carcass by artificial digestion with HCl/Pepsin, and counted seperately in muscle and visercal organs. Artificial digestion of skeletal muscle and viscera for larval enumeration 38
39 Survival 40 P. maniculatus P. leucopus P. californicus P. polionotusHigh dose- nearly uniformly euthanized b/w 10 and 22 days with Pl lasting the longest (Pc maybe a mistake) P. californicus P. polionotus 40
40 Survival Analysis 41 Weibull Regression Final Model:Others: 10 eggs P.l.: 10 eggs Survival Analysis Weibull Regression Final Model: Dose (p<0.0001) Species - P. leucopus vs . “others” (p=0.006) Others: 50 eggs P.l.: 50 eggs Weibull Regression – used to account for right censoring… Graph of fitted models - Survival estimates, 95% CI No significantly impact of weight or larval counts on survival Others: 500 eggs P.l.: 500 eggs 41
41 Larval Recovery 42 P. californicus P. leucopus P. polionotusAvg. Total Larvae (range) Avg. Larval Recovery (% of dose) P. californicus Low (10 eggs) 0.7 (0-3) 6.7% Medium (50 eggs) 3.2 (2-5) 6.3% High (500 eggs) 43.2 (16-59) 8.6% P. leucopus 1.0 (0-4) 10.0% 5.5 (2-9) 11.0% 39.0 (19-58) 7.8% P. maniculatus 0.8 (0-3) 8.3% 5.2 (2-9) 10.3% 39.2 (26-50) P. polionotus 0.3 (0-1) 3.3% 3.2 (0-7) 40.3 (30-53) 8.1% The total numebrs or larvae were surprisingly similar across species and dose groups, and the percent of larvae recovered was pretty constant over doses as well 42
42 Larval Recovery * * 43 L3 in Brain L3 in Skeletal MuscleL3 in Visceral Organs * 43
43 Discussion P. leucopus demonstrated superior tolerance towards severe baylisascariasis (delayed onset) Possibly better able to “wall off” more larvae in viscera and delay migration to CNS Did not detect survival differences among other species, despite size and habitat range differences 44
44 Discussion Tolerance: related to evolutionary history, behavioral attributes influencing selection pressure? P. maniculatus and P. leucopus broadly sympatric but demonstrate different microhabitat usage P.m. - arboreal, elevated refugia P.l. - terrestrial, low-lying refugia Page et al. - greater proportion of raccoon latrines in flat, less-elevated areas (stumps, tree roots, fallen logs). Differential contact rates/selection pressure even within same area? Of particular interest: differences between the sympatric species P.m. and P.l. native to areas of high Baylis endmicity The way this connects to B.p.,… page 45
45 Implications Differences should be taken into consideration for field studies on B. procyonis Only P.l. discussed in field studies— no reports of P.m. captures, even in sympatric areas? Slight difference in survival time could perhaps influence role of different species in B. procyonis ecology Possible conservation angle: Endangered P. polionotus subspecies and B. procyonis Alabama Beach Mouse P. polionotus ammobates 46
46 Serologic testing for animalsWestern Blot (BpRAG-1) adapted for Peromyscus, Rattus, Mus Differences noted in strength of responses so developed an ELISA to quantify ELISA (BpRAG-1) for Peromyscus 47
47 Both BpRAG-1 WB and ELISA showed significant differences in seroconversion between species and doseCurrently looking for an additional, somatic Ag that better represents a protective response (compared to ES) 48
48 Acknowledgements 49 All study participantsWildlife Rehabilitation Organizations: NWRA, IWRC, FWRA, Wildlife Center of VA Parasitic Diseases Branch, Centers for Disease Control and Prevention SCWDS/University of Georgia Clemson University University of South Carolina 49
49 [email protected] and [email protected]Thank you! and 50
50 Domestic dogs Natural and experimental infections show that dogs can be patent Limited number of puppies have also developed neurologic disease due to NLM circumstantial evidence of prenatal (transplacental) infection in dogs, similar to the situation with Toxocara canis Also lambs in Idaho born with infections 51
51 Canine Serology: What is role of dogs as hosts?Future Directions Canine Serology: What is role of dogs as hosts? Definitive and intermediate Bp host, but little known serum samples banked No effective positive control at the moment (blind testing?) Ideally would be conducted alongside experimental infections Could incorporate copro-antigen test from IDEXX along with blood ag capture test if developed (may distinguish between larval and adult infections) 52