The Health Status of Horses Used for at Least Six Complete Cycles of Loxoscelic Antivenom Production.

Antivenom production against Loxosceles venom relies on horses being immunized and bled for plasma harvest. One horse can partake in several cycles of antivenom production, which will require years of constant venom and adjuvant inoculation and bleeding. The actual impact on the health of horses that participate in several antivenom-producing cycles is unknown. Therefore, this study aimed to evaluate the general health status of horses that underwent at least six cycles of loxoscelic antivenom production. Seven crossbred horses that had partaken in six to eight complete antivenom-producing cycles were used and established as the immunized group (IG). Under the same handling and general management, eleven horses were established as the control group (CG). The horses were evaluated regarding their general clinical status and had their blood sampled, and an ECG recorded. The IG presented lower RBC and PCV, despite keeping values within inferior limits for the species. Renal function was not impaired, and liver-related enzymes were higher than those in the CG, probably due to liver exertion from immunoglobulin synthesis. ECG showed some abnormalities in the IG, such as atrioventricular block and a wandering atrial pacemaker, corroborated by an increase in CK-MB. The cardiovascular abnormalities were mainly found in the horses that participated in several antivenom-producing cycles. The overall results indicate that these horses had some impairment of their general health status. Once available, some alternative, less toxic antigens should replace the venom for immunization of horses used for antivenom production.

An effective strategy for the humanization of antibody fragments under an accelerated timeline.

The use of monoclonal antibodies (mAbs) in therapy is gradually advancing and discussions entail its safety, rentability and effectiveness. To this date, around a hundred mAbs have been approved by the FDA for the treatment of various diseases. Aiming for their large-scale production, recombinant DNA technology is mainly employed, and antibodies can be expressed in various eukaryotic and prokaryotic systems. Moreover, considering their heterologous origin and potential immunogenicity, various strategies have been developed for mAb humanization, considering that around 50 % of commercial mAbs are humanized. Hence, we introduce LimAb7, a mouse mAb capable of binding and neutralizing brown spider's Loxosceles intermedia dermonecrotic toxins in vivo/in vitro. This antibody has been produced in mouse and humanized scFv and diabody formats, however results indicated losses in antigen-binding affinity, stability, and neutralizing ability. Intending to develop evolved, stable, and neutralizing antibody fragments, we report for the first time the design of humanized antibody V-domains produced as Fab fragments, against spider venom toxins. Improvements in constructs were observed regarding their physicochemical stability, target binding and binding pattern maintenance. As their neutralizing features remain to be characterized, we believe this data sheds new light on antibody humanization by producing a parental molecule in different recombinant formats.

Validation of a colorimetric LAMP to detect Loxosceles experimental envenomation.

Diagnostic tests for brown spider accidents are unavailable and impact treatment decisions, increasing costs and patient risks. In this work, we used for the first time a fast, simple, and visual method based on the loop-mediated isothermal amplification assay (LAMP) to detect Loxosceles envenomation. Using the DNA from L. similis legs, we observed a high sensitivity using this test since as low as 0.32 pg of DNA could be detected. This pH-dependent colorimetric assay was 64 times more sensitive than PCR to detect spider DNA. The test was specific for Loxosceles once no cross-reaction was observed when testing DNA from different agents that cause similar dermonecrotic injuries. The test allowed the detection of Loxosceles intermedia DNA from hair, serum, and exudate samples obtained from experimentally-envenomed rabbit within 72 h. The method sensitivity varied according to the sample and the collection time, reaching 100% sensitivity in serum and hair, respectively, 1 h and 24 h after the experimental envenomation. Due to its ease of execution, speed, sensitivity, and specificity, LAMP presents an excellent potential for identifying Loxosceles spp. Envenomation. This can reduce the burden on the Health System and the morbidity for the patient by implementing the appropriate therapy immediately.In addition, this work opens up the perspective to other venomous animal accident identification using LAMP.

Protective Effectiveness of an Immunization Protocol Against the Toxic Effects of Loxosceles intermedia Venom in Rabbits.

Loxosceles spp. (brown spiders) bites are responsible for the development of a syndrome consisting mainly of dermonecrotic lesions, and also systemic effects. Rabbits are one of the main experimental models used for better understanding the systemic and local effects of Loxosceles venom. The aim of this study is to evaluate the toxic and protective effects of rabbits immunized with Loxosceles spp. venom. Male New Zealand rabbits were allocated as a control group (CG; n = 5) that received adjuvant (Montanide) and phosphate-buffer saline (PBS), or as venom group (VG; n = 5) that received 21 µg of Loxosceles venom using Montanide as adjuvant. After five immunization cycles, a trial with 7 µg of Loxosceles intermedia (L. intermedia) venom was performed, and dermonecrotic lesions were measured. The rabbits were then euthanized, and their organs were collected for histopathology analysis. Rabbits that had undergone Loxosceles venom immunization protocol showed minor clinical disturbances during the experimental period. The used immunization protocol protected the rabbits against the toxic effect of the Loxosceles venom because they showed minor clinical disturbances during the experimental period.

Clinical Effects of the Immunization Protocol Using Loxosceles Venom in Naïve Horses.

Bites of brown spiders (Loxosceles spp.) are responsible for dermonecrotic lesions and potentially systemic envenoming that can lead to death. The only effective therapy is the use of the antivenom, usually produced in horses. However, little is known about the consequences of the systematic use of the Loxosceles venom and adjuvants and of the bleedings on antivenom-producing horses. Thus, the aim of this study was to evaluate the clinical changes in horses in their first immunization protocol for Loxosceles antivenom production. Eleven healthy horses, never immunized, were evaluated in three different periods: T0 (before immunization); T1 (after their first venom immunization); and T2 (after their first bleeding). Horses were clinically evaluated, sampled for blood, and underwent electrocardiographic (ECG) recordings. Several suppurated subcutaneous abscesses occurred due to the use of Freund's adjuvants and thrombophlebitis due to systematic venipunctures for the bleeding procedures. ECG showed arrhythmias in few horses in T2, such as an increase in T and R waves. In summary, the immunization protocol impacted on horses' health, especially after bleeding for antivenom procurement.

Production and Functional Evaluation of Anti-Loxosceles Sera Raised by Immunizations of Rabbits with Mutated Recombinant Phospholipases-D.

Loxoscelism is the clinical condition triggered after the bite of spiders of the genus Loxosceles. The main species involved in accidents in South America are L. intermedia, L. laeta, and L. gaucho. The only specific treatment is the anti-Loxosceles serum produced with crude venoms. As phospholipases D (PLDs) trigger most of the effects observed in accidents, we developed and evaluated second-generation sera using mutated PLDs as antigens. Three isoforms of PLDs with site-directed mutations without biological activities were used for rabbit immunizations: D32A-E34A (L. gaucho), W230A (L. intermedia), and H12A-H47A (L. laeta). Sera were produced using crude venoms of three species of Loxosceles enriched with mutated recombinant PLDs (MIX) or using only mutated PLDs (REC). Immunizations stimulated the immune system from the second immunization with higher antibody production in the REC group. In vivo neutralization assays demonstrated that both sera reduced edema and dermonecrosis caused by Loxosceles intermedia crude venom. Follow-up of animals during the immunization protocols and in the neutralization assays demonstrated that the mutated proteins and the sera are safe. Results demonstrate the potential of using mutated recombinant PLDs in total or partial replacement of Loxosceles venoms in animal immunizations to produce anti-Loxosceles sera for treatments of Loxoscelism.

A protective vaccine against the toxic activities following Brown spider accidents based on recombinant mutated phospholipases D as antigens.

Accidents involving Brown spiders are reported throughout the world. In the venom, the major toxins involved in the deleterious effects are phospholipases D (PLDs). In this work, recombinant mutated phospholipases D from three endemic species medically relevant in South America (Loxosceles intermedia, L. laeta and L. gaucho) were tested as antigens in a vaccination protocol. In such isoforms, key amino acid residues involved in catalysis, magnesium-ion coordination, and binding to substrates were replaced by Alanine (H12A-H47A, E32A-D34A and W230A). These mutations eliminated the phospholipase activity and reduced the generation of skin necrosis and edema to residual levels. Molecular modeling of mutated isoforms indicated that the three-dimensional structures, topologies, and surface charges did not undergo significant changes. Mutated isoforms were recognized by sera against the crude venoms. Vaccination protocols in rabbits using mutated isoforms generated a serum that recognized the native PLDs of crude venoms and neutralized dermonecrosis and edema induced by L. intermedia venom. Vaccination of mice prevented the lethal effects of L. intermedia crude venom. Furthermore, vaccination of rabbits prevented the cutaneous lesion triggered by the three venoms. These results indicate a great potential for mutated recombinant PLDs to be employed as antigens in developing protective vaccines for Loxoscelism.

Prospective Use of Brown Spider Venom Toxins as Therapeutic and Biotechnological Inputs.

Brown spider (genus Loxosceles) venoms are mainly composed of protein toxins used for predation and defense. Bites of these spiders most commonly produce a local dermonecrotic lesion with gravitational spread, edema and hemorrhage, which together are defined as cutaneous loxoscelism. Systemic loxoscelism, such as hematological abnormalities and renal injury, are less frequent but more lethal. Some Loxosceles venom toxins have already been isolated and extensively studied, such as phospholipases D (PLDs), which have been recombinantly expressed and were proven to reproduce toxic activities associated to the whole venom. PLDs have a notable potential to be engineered and converted in non-toxic antigens to produce a new generation of antivenoms or vaccines. PLDs also can serve as tools to discover inhibitors to be used as therapeutic agents. Other Loxosceles toxins have been identified and functionally characterized, such as hyaluronidases, allergen factor, serpin, TCTP and knottins (ICK peptides). All these toxins were produced as recombinant molecules and are biologically active molecules that can be used as tools for the potential development of chemical candidates to tackle many medical and biological threats, acting, for instance, as antitumoral, insecticides, analgesic, antigens for allergy tests and biochemical reagents for cell studies. In addition, these recombinant toxins may be useful to develop a rational therapy for loxoscelism. This review summarizes the main candidates for the development of drugs and biotechnological inputs that have been described in Brown spider venoms.

Description of a serpin toxin in Loxosceles (Brown spider) venoms: Cloning, expression in baculovirus-infected insect cells and functional characterization.

Several classes of toxins are present in the venom of Brown spiders (Loxosceles genus), some of them are highly expressed and others are less expressed. In this work, we aimed to clone the sequence of a little expressed novel toxin from Loxosceles venom identified as a serine protease inhibitor (serpin), as well as to express and characterize its biochemical and biological properties. It was named LSPILT, derived from Loxoscelesserine protease inhibitor-like toxin. Multiple alignment analysis revealed high identity between LSPILT and other serpin molecules from spiders and crab. LSPILT was produced in baculovirus-infected insect cells, resulting in a 46-kDa protein fused to a His-tag. Immunological assays showed epitopes in LSPILT that resemble native venom toxins of Loxosceles spiders. The inhibitory activity of LSPILT on trypsin was found both by reverse zymography and fluorescent gelatin-degradation assay. Additionally, LSPILT inhibited the complement-dependent lysis of Trypanosoma cruzi epimastigotes, reduced thrombin-dependent clotting and suppressed B16-F10 melanoma cells migration. Results described herein prove the existence of conserved serpin-like toxins in Loxosceles venoms. The availability of a recombinant serpin enabled the determination of its biological and biochemical properties and indicates potential applications in future studies regarding the pathophysiology of the envenoming or for biotechnological purposes.

Production of a novel recombinant brown spider hyaluronidase in baculovirus-infected insect cells.

Hyaluronidases are low expressed toxins of brown spider venoms, but, as highly active molecules, they present an important role as spreading factors. By degrading extracellular matrix components, these enzymes favor the diffusion of toxins in the affected tissue and at systemic level. Here, a novel isoform of hyaluronidase of Loxosceles intermedia Mello-Leitão (1934) venom was cloned, expressed in a baculovirus-insect cell expression system and fully active purified. This recombinant enzyme, named LiHyal2 (Loxosceles intermedia Hyaluronidase isoform 2), shares high identity with hyaluronidases of other spiders and scorpions. The catalytic and sugar binding amino acid residues are conserved in LiHyal2, human, and honeybee venom hyaluronidases and the molecular model of LiHyal2 shares major similarities with their crystal structures, including the active site. LiHyal2 was expressed as a 45 kDa protein and degraded hyaluronic acid (HA) and chondroitin sulphate as demonstrated by HA zymography and agarose gel electrophoresis. Lectin blot analysis revealed that LiHyal2 is post-translationally modified by the addition of high mannose N-linked carbohydrates. In vivo experiments showed that LiHyal2 potentialize dermonecrosis and edema induced by a recombinant phospholipase-D (PLD) of L. intermedia venom, as well as enhance the increase in capillary permeability triggered by this PLD, indicating that these toxins act synergistically during envenomation. Altogether, these results introduce a novel approach to express spider recombinant toxins, contribute to the elucidation of brown spider venom mechanisms and add to the development of a more specific treatment of envenomation victims.

Brown Spiders' Phospholipases-D with Potential Therapeutic Applications: Functional Assessment of Mutant Isoforms.

Phospholipases-D (PLDs) found in Loxosceles spiders' venoms are responsible for the dermonecrosis triggered by envenomation. PLDs can also induce other local and systemic effects, such as massive inflammatory response, edema, and hemolysis. Recombinant PLDs reproduce all of the deleterious effects induced by Loxosceles whole venoms. Herein, wild type and mutant PLDs of two species involved in accidents-L. gaucho and L. laeta-were recombinantly expressed and characterized. The mutations are related to amino acid residues relevant for catalysis (H12-H47), magnesium ion coordination (E32-D34) and binding to phospholipid substrates (Y228 and Y228-Y229-W230). Circular dichroism and structural data demonstrated that the mutant isoforms did not undergo significant structural changes. Immunoassays showed that mutant PLDs exhibit conserved epitopes and kept their antigenic properties despite the mutations. Both in vitro (sphingomyelinase activity and hemolysis) and in vivo (capillary permeability, dermonecrotic activity, and histopathological analysis) assays showed that the PLDs with mutations H12-H47, E32-D34, and Y228-Y229-W230 displayed only residual activities. Results indicate that these mutant toxins are suitable for use as antigens to obtain neutralizing antisera with enhanced properties since they will be based on the most deleterious toxins in the venom and without causing severe harmful effects to the animals in which these sera are produced.

LALLT (Loxosceles Allergen-Like Toxin) from the venom of Loxosceles intermedia: Recombinant expression in insect cells and characterization as a molecule with allergenic properties.

Bites evoked by Brown spiders (Loxosceles genus) are associated with skin injuries (cutaneous rash, itching, swelling, erythema and dermonecrosis) and systemic manifestations. Transcriptome analyses of Loxosceles venom glands showed that the venom has a complex composition containing toxins such as phospholipases-D, metalloproteases and hyaluronidases. Here, by screening the RNA from L. intermedia venom glands, we cloned a novel allergen toxin, and named LALLT (LoxoscelesAllergen-Like Toxin). Sequence analysis showed that LALLT is closely related to allergens from other spiders and RNA screening indicated the presence of LALLT orthologues in the venom of other Loxosceles spiders. Recombinant LALLT was expressed (~45 kDa) in baculovirus-infected insect cells and purified by affinity chromatography. Antibodies against different Loxosceles venoms cross-reacted with LALLT and antibodies against LALLT recognized three Loxosceles venoms, revealing epitopes identity. LALLT triggered paw edema in mice and erythema, edema and leukocyte infiltration into the dermis of rabbit skin. Also, LALLT induced vascular permeability in mice, degranulation of rat mesentery mast cells, as well as prompted degranulation and increased calcium influx in RBL-2H3 cells. Data reported suggest for the first time the existence of allergens in Loxosceles venoms and make LALLT available for clinical studies about allergenic events arisen by Loxosceles envenoming.

Forty Years of the Description of Brown Spider Venom Phospholipases-D.

Spiders of the genus Loxosceles, popularly known as Brown spiders, are considered a serious public health issue, especially in regions of hot or temperate climates, such as parts of North and South America. Although the venoms of these arachnids are complex in molecular composition, often containing proteins with distinct biochemical characteristics, the literature has primarily described a family of toxins, the Phospholipases-D (PLDs), which are highly conserved in all Loxosceles species. PLDs trigger most of the major clinical symptoms of loxoscelism i.e., dermonecrosis, thrombocytopenia, hemolysis, and acute renal failure. The key role played by PLDs in the symptomatology of loxoscelism was first described 40 years ago, when researches purified a hemolytic toxin that cleaved sphingomyelin and generated choline, and was referred to as a Sphingomyelinase-D, which was subsequently changed to Phospholipase-D when it was demonstrated that the enzyme also cleaved other cellular phospholipids. In this review, we present the information gleaned over the last 40 years about PLDs from Loxosceles venoms especially with regard to the production and characterization of recombinant isoforms. The history of obtaining these toxins is discussed, as well as their molecular organization and mechanisms of interaction with their substrates. We will address cellular biology aspects of these toxins and how they can be used in the development of drugs to address inflammatory processes and loxoscelism. Present and future aspects of loxoscelism diagnosis will be discussed, as well as their biotechnological applications and actions expected for the future in this field.

In silico and in vitro Evaluation of Mimetic Peptides as Potential Antigen Candidates for Prophylaxis of Leishmaniosis.

Antigen formulation is the main feature for the success of leishmaniosis diagnosis and vaccination, since the disease is caused by different parasite species that display particularities which determine their pathogenicity and virulence. It is desirable that the antigens are recognized by different antibodies and are immunogenic for almost all Leishmania species. To overcome this problem, we selected six potentially immunogenic peptides derived from Leishmania histones and parasite membrane molecules obtained by phage display or spot synthesis and entrapped in liposome structures. We used these peptides to immunize New Zealand rabbits and determine the immunogenic capacity of the chimeric antigen. The peptides induced the production of antibodies as a humoral immune response against L. braziliensis or L. infantum. Next, to evaluate the innate response to induce cellular activation, macrophages from the peptide mix-immunized rabbits were infected in vitro with L. braziliensis or L. infantum. The peptide mix generated the IFN-γ, IL-12, IL-4 and TGF-ß that led to Th1 and Th2 cellular immune responses. Interestingly, this mix of peptides also induced high expression of iNOS. These results suggest that the mix of peptides derived from histone and parasites membrane molecules was able to mimic parasites proteins and induce cytokines important to CD4+ T cell Th1 and Th2 differentiation and effector molecule to control the parasite infection. Finally, this peptide induced an immune balance that is important to prevent immunopathological disorders, inflammatory reactions, and control the parasite infection.

Biological evaluation of mimetic peptides as active molecules for a new and simple skin test in an animal model.

A skin test is a widely used tool in diagnostic evaluations to investigate cutaneous leishmaniases (CL). The actual antigen (Montenegro skin test [MST] antigen) presents some difficulties that pertain to its manufacturing and validation. To contribute to overcoming this problem, we propose the application of new-generation molecules that are based on skin antigen tests. These antigens were obtained through biotechnology pathways by manufacturing synthetic mimetic peptides. Three peptides, which were selected by phage display, were tested as skin test antigens in an animal model (Cavia porcellus) that was immunized with Leishmania amazonensis or Leishmania braziliensis. The peptide antigens, individually (PA1, PA2, PA3) or in a mix (PAMix), promoted induration reactions at 48 and 72 h after the test was performed. The indurations varied from 0.5 to 0.7 cm. In the animals immunized with L. amazonensis, the PA3 antigen showed better results than the standard MST antigen. In animals immunized with L. braziliensis, two peptide antigens (PA2 and PAMix) promoted induration reactions for a longer period of time than the standard MST antigen. These results validate our hypothesis that peptides could be used as antigens in skin tests and may replace the current antigen for CL diagnosis.

Next-generation sequencing reveals new insights about gene usage and CDR-H3 composition in the horse antibody repertoire.

Horse serum antibodies have been used for greater than a century for the treatment and prophylaxis of infectious diseases and envenomations. Little is known, however, about the immunogenetic diversity that produces horse serum antibodies. Here, we employed next-generation sequencing for a first-in-kind comprehensive analysis of the equine B-cell repertoire. Nearly 45,000 and 30,000 clonotypes were obtained for the heavy-chain (IGH) and lambda light-chain (IGL) loci, respectively. We observed skewed use of the common subgroups IGHV2 (92.49%) and IGLV8 (82.50%), consistent with previous reports, but also novel use of the rare genes IGHV6S1 and IGLV4S2. CDR-H3 amino acid composition revealed different amino acid patterns at positions 106 and 116 compared to human, rabbit, and mouse, suggesting that an extended conformation predominates among horse CDR-H3 loops. Our analysis provides new insights regarding the mechanisms employed to generate antibody diversity in the horse, and could be applicable to the optimized design of synthetic antibodies intended for future therapeutic use.

Immunoprotection elicited in rabbit by a chimeric protein containing B-cell epitopes of Sphingomyelinases D from Loxosceles spp. spiders.

Accidents with venomous animals pose a health issue in Brazil, and those involving brown spiders (Loxosceles sp.) figure between the most frequent ones. The accidental envenomation by brown spiders causes a strong local dermonecrotic effect, which can be followed by systemic manifestations that in some cases lead to death. The production of antivenoms for the treatments of such accidents relies on a variety of animal experiments, from the spider venom extraction to the production of antivenom in horses. In the present work, there is an attempt to reduce and optimize animal experiments with the construction and production of a chimeric protein, named Lil, containing immunodominant epitopes previously mapped from the main proteins of the Loxosceles venom, the Sphingomyelinases D. The Lil protein contains epitopes from Sphinomyelinases D of the three-main species found in Brazil and this chimeric protein was found capable of inducing antibodies with the potential to partially neutralize the toxic effects of Loxosceles intermedia venom in an animal model. Therefore, in order to reduce spider usage and to improve the lifespan of the horses used for immunization we suggest the Lil protein as a potential candidate to replace the venom usage in the antivenom production protocols.

New strategy to improve quality control of Montenegro skin test at the production level

Abstract INTRODUCTION: The production of the Montenegro antigen for skin test poses difficulties regarding quality control. Here, we propose that certain animal models reproducing a similar immune response to humans may be used in the quality control of Montenegro antigen production. METHODS: Fifteen Cavia porcellus (guinea pigs) were immunized with Leishmania amazonensis or Leishmania braziliensis , and, after 30 days, they were skin tested with standard Montenegro antigen. To validate C. porcellus as an animal model for skin tests, eighteen Mesocricetus auratus (hamsters) were infected with L. amazonensis or L. braziliensis , and, after 45 days, they were skin tested with standard Montenegro antigen. RESULTS: Cavia porcellus immunized with L. amazonensis or L. braziliensis , and hamsters infected with the same species presented induration reactions when skin tested with standard Montenegro antigen 48-72h after the test. CONCLUSIONS: The comparison between immunization methods and immune response from the two animal species validated C. porcellus as a good model for Montenegro skin test, and the model showed strong potential as an in vivo model in the quality control of the production of Montenegro antigen.

Anti-hMC2RL1 Functionalized Gold Nanoparticles for Adrenocortical Tumor Cells Targeting and Imaging.

The low rate of cure of adrenocortical carcinomas (ACC) in children and adults is related to germ line TP53 mutation, late diagnosis, incomplete surgical resection, and lack of an efficient adjunctive therapy. To provide a new approach for the improvement of ACC diagnosis and therapy, the present study aimed to explicitly target ACC cells using gold nanoparticle (AuNP) probes bound to specific antibodies. Immunohistochemistry of ACC and positive and negative control tissue micro-sections under light microscopy was used to test a purified polyclonal antibody raised against the 80­93, outer loop 1 position of the human melanocortin receptor 2 (hMC2R). Both this and a control commercial antibody were found to specifically target cells known to express hMC2R. These were bound to FITC-labeled AuNPs and tested via direct immunofluorescence using the H295R ACC cell line. Both probes recognized only cells expressing hMC2R and exhibited very low background. Further studies are required to ascertain the potential of AuNPs bound to ACC cells for tumor diagnostics via imaging analysis or as a delivery device for targeted therapy.

New strategy to improve quality control of Montenegro skin test at the production level.

INTRODUCTION: The production of the Montenegro antigen for skin test poses difficulties regarding quality control. Here, we propose that certain animal models reproducing a similar immune response to humans may be used in the quality control of Montenegro antigen production. METHODS: Fifteen Cavia porcellus (guinea pigs) were immunized with Leishmania amazonensis or Leishmania braziliensis , and, after 30 days, they were skin tested with standard Montenegro antigen. To validate C. porcellus as an animal model for skin tests, eighteen Mesocricetus auratus (hamsters) were infected with L. amazonensis or L. braziliensis , and, after 45 days, they were skin tested with standard Montenegro antigen. RESULTS: Cavia porcellus immunized with L. amazonensis or L. braziliensis , and hamsters infected with the same species presented induration reactions when skin tested with standard Montenegro antigen 48-72h after the test. CONCLUSIONS: The comparison between immunization methods and immune response from the two animal species validated C. porcellus as a good model for Montenegro skin test, and the model showed strong potential as an in vivo model in the quality control of the production of Montenegro antigen.