Effectiveness of Lonomia antivenom in recovery from the coagulopathy induced by Lonomia orientoandensis and Lonomia casanarensis caterpillars in rats

Abstract

In South America, accidental contact with Lepidoptera larvae can produce a diversity of reactions that vary from dermatological problems to severe hemorrhagic syndromes, such as those caused by contact with caterpillars of the genus Lonomia (Saturniidae). Lonomia venom can alter the hemostatic system and lead to renal failure, internal and brain bleeding, and in severe cases, death. The only specific treatment available for these envenomations is the Lonomia Antivenom (LAV) produced by the Butantan Institute, in Brazil, using an extract of Lonomia obliqua scoli as the antigen. LAV has been used to treat exposure to other Lonomia species across South America. However, no experimental studies have been performed to test the efficacy of LAV in neutralizing the venom of species other than L. obliqua found in Southern Brazil. In this study, we tested the effectiveness of LAV in reversing the hemostatic disturbances induced by injecting Lonomia casanarensis (Lca) and Lonomia orientoandensis (Lor) scolus extracts into rats and compared the effects to the case of L. obliqua (Lob) scolus extract-induced envenomation. Lca and Lor caterpillars were collected in Colombia, and some of them were reared to adults for identification. The Minimum Defibrinating Doses (MDD) of Lca and Lor were estimated. Rats were injected (i.d.) with a dose of 3 MDD per rat of each scolus extract and treated (i.v.) with 1.5 mL of LAV or 1.5 mL of saline. Twenty-four hours after the treatment, the fibrinogen levels and platelet counts had recovered to the hemostatic levels in the groups treated with LAV. The groups treated with the saline solution had fibrinogen levels and platelet counts at non-hemostatic levels. Thromboelastometric analyses confirmed these results. In conclusion, the results showed that LAV is effective at neutralizing the envenomation induced by Lca and Lor spine extracts in rats and restoring hemostasis.

Fig 1. Geographic location of caterpillar’s collection sites.

Map of Colombia highlighting the Casanare department and showing the localities where Lonomia casanarensis (triangle) and Lonomia orientoandensis (circle) caterpillars were collected. This figure was built using ArcGis 10.5 software, licensed to Universidad de Los Andes; the layers are available for free: elevation from the Hydro 1K available at https://www.usgs.gov/ and political division by the National administrative department of statistics (Dane by its acronym in Spanish) at https://geoportal.dane.gov.co. This figure doesn't include any satellite image from a copyrighted source.

Fig 2. Recovery of fibrinogen levels in rats envenomed with Lonomia scoli extracts and treated with LAV, in comparison to envenomed but non-treated animals.

The animals were injected i.d. with Lob (150 µg), Lor (450 µg), or Lca (300 µg), which corresponded to 3 MDD of each extract. One hour (Lob), or two hours (Lor and Lca) later, the animals were treated with the LAV or sterile saline (1.5 mL, i.v.), and blood was collected 24 h after the treatment. Fibrinogen was measured in the citrated plasma as described in the Material and Methods section. The results are expressed as the means±SEM, n = 6 rats/group. Significant differences are indicated as follows (p<0.05): (*) different from the control non-envenomed group; (#) different from the respective envenomed and saline-treated groups, and (§) different from the other groups.

Fig 3. Recovery of platelet counts in rats envenomed with the Lonomia scoli extracts and treated with LAV, in comparison to envenomed but non-treated animals.

Animals were injected i.d. with 3 MDD of each extract: Lob (150 µg), Lor (450 µg), or Lca (300 µg). One hour (Lob), or two hours (Lor and Lca) later, animals were treated with the LAV or sterile saline (1.5 mL, i.v.), and blood was collected 24 h after the treatment. The platelets were counted in EDTA-anticoagulated blood using an automated cell counter as described in the Material and Methods section. The results are expressed as the means±SEM, n = 6 rats/group. Significant differences are indicated as (p<0.05): (*) different from the control non-envenomed group; (#) different from the respective envenomed and saline-treated groups; and (§) different from the other groups.

Fig 4. Thromboelastometry of rat blood samples collected 24 hours after the i.v. treatment with LAV or saline in rats envenomed (i.d.) with scoli extracts of L. obliqua (150 µg/rat), L. orientoandensis (450 µg/rat), L. casanarensis (300 µg/rat) caterpillars.

Graphical representations of the assay in citrated whole blood with addition of 0.2 M calcium chloride and saline (NATEM) and specific reagents for the analysis of extrinsic pathway (EXTEM), intrinsic pathway (INTEM), or fibrinogen (FIBTEM). The control was a non-envenomed rat that was treated with Lonomia antivenom. The data were captured by the ROTEM software in the time interval of 40–60 min. Each set of graphs represents one animal in each experimental group.

Fig 5. Quantitative values of the Clotting Time—CT (A, C, E) and Clot Formation Time–CFT (B, D, F) obtained from the thromboelastometry of NATEM (A, B), INTEM (C, D), and EXTEM (E, F) of rats envenomed with the Lonomia scoli extracts and treated with LAV, in comparison to envenomed but non-treated animals.

The animals were injected i.d. with scoli extract of L. obliqua (Lob-150 µg), L. orientandensis (Lor-450 µg), or L. casanarensis (Lca–300 µg), which corresponded to 3 MDD (Minimum Defibrinating Dose) of each extract. One hour (Lob), or two hours (Lor and Lca) later, the animals were treated with LAV (1.5 mL, i.v.) or sterile saline, and blood was collected 24 h after the treatment. Thromboelastography was performed on citrated whole blood with the addition of 0.2 M calcium chloride (NATEM) or specific reagents for the analysis of the extrinsic pathway (EXTEM), or intrinsic pathway (INTEM). The control group consisted of non-envenomed rats that were treated with the Lonomia antivenom. The data were captured by the ROTEM software in the time interval of 40–60 min. n = 6 rats/ group. Significant differences are indicated as follows (p<0.05): (*) = different from the non-envenomed control group and different from the respective group treated with the antivenom; (#) = different from the respective group treated with the antivenom, but not different from the non-envenomed control group; (§) = different from other groups; and (n.c.) = no clot.

Fig 6. Quantitative values of the Maximum Clot Firmness—MCF (A, C, E) and Alpha (α) Angle (B, D, F) determined from the thromboelastometry of NATEM (A, B), INTEM (C, D), and EXTEM (E, F) of rats envenomed with the Lonomia scoli extracts and treated with LAV, in comparison to envenomed but non-treated animals.

The animals were injected i.d. with scoli extract of L. obliqua (Lob-150 µg), L. orientoandensis (Lor-450 µg), or L. casanarensis (Lca–300 µg), which corresponded to 3 MDD (Minimum Defibrinating Doses) of each extract. One hour (Lob), or two hours (Lor and Lca) later, the animals were treated with the LAV or sterile saline (1.5 mL, i.v.), and blood was collected 24 h after the treatment. Thromboelastography was performed in citrated whole blood with the addition of 0.2 M calcium chloride (NATEM) or specific reagents for the analysis of the extrinsic pathway (EXTEM), or intrinsic pathway (INTEM). The control group consisted of non-envenomed rats treated with LAV. The data were captured by the ROTEM software in the time interval of 40–60 min, n = 6 rats/ group. Significant differences are indicated as follows (p<0.05): (*) = different from the non-envenomed control group and different from the respective group treated with the antivenom; (#) = different from the respective group treated with antivenom but not different from the non-envenomed control group; (§) = different from other groups; (&) = different from the non-envenomed control group, but not different from the respective antivenom-treated group; and (n.c.) = no clot.