Type C botulism in dogs from rural properties located in Goiânia, Brazil

Botulism is a disease usually fatal, caused by the ingestion of neurotoxins produced by Clostridium botulinum. In dogs, intoxication is caused by the ingestion of botulinum toxin type C, and animals often recover spontaneously. The present study describes the occurrence of type C botulism in two dogs domiciled on neighboring rural properties in the municipality of Goiânia, state of Goiás, Brazil, probably associated with ingestion of decomposing bovine carcass. Upon clinical evaluation, the dogs were alert in the lateral decubitus position with ascending flaccid paralysis, absence of eyelid reflexes, and reduced muscle tone. Due to their worsening clinical symptoms, the animals died within 12 h and 3 days after supportive treatment. Botulinum toxin type C was identified, in the serum and feces of both dogs, by seroneutralization in mice with homologous monovalent antitoxin. The results of the high-throughput gene sequencing showed that the abundance of C. botulinum in the fecal microbiota of one of the affected dogs was low (0.53%). In this way, the present study highlights the need of sanitary practices related to the appropriate collection and disposal of bovine carcasses in rural areas since they represent a risk factor for the occurrence of botulism in dogs domiciled on rural properties.

Surto de botulismo tipo C em aves de subsistência em Santa Catarina, Brasil / Outbreak of type C botulism in backyard poultry in Santa Catarina, Brazil

Background: Botulism is a disease caused by the ingestion of neurotoxin produced by Clostridium botulinum, characterizedby flaccid paralysis, which can lead to high mortality. They have seven types of neurotoxins (A, B, C, D, E, F, and G) and,in birds, most cases are attributed to type C. They are considered sources of botulinum toxins where the decomposition oforganic matter occurs, like stagnant water and rotting food. The main feature of the disease in birds is ascending symmetricflaccid paralysis. The present study aims to describe an outbreak of type C botulism in backyard poultry in the state ofSanta Catarina, Southern Brazil.Case: A visit was made to the property with 160 backyard poultry with a history of high mortality in the municipality ofAgrolândia, Santa Catarina. Clinical signs were characterized by paralysis of the pelvic limbs, neck and pendular wings,which progressed to death within 48 h. There was a mortality rate of 37.5% (60/160) between March and May 2019. Thesebirds were kept in an overcrowded environment, with different species (chickens, ducks, teals, and turkeys) fed irregularly.The water supplied was provided from kitchen exhaust, accumulating in puddles on the floor that contained organic matterresidues such as animal feces, food waste and bone fragments. The disposal of the carcasses of birds that died was in thesame enclosure, buried superficially, facilitating the access of other birds to dig them up and consume them. Necropsywas performed on 2 chickens and one duck, no macroscopic or histopathological lesions were observed. Blood, liver, andgastrointestinal content samples were sent for research and identification of botulinum toxin through the serum neutralization test in mice. The presence of type C botulinum toxin was confirmed in the liver chicken of one sampled animals.Discussion: The identification of type C botulism toxin enabled the characterization of the outbreak, which is...

Surto de botulismo tipo C em aves de subsistência em Santa Catarina, Brasil / Outbreak of type C botulism in backyard poultry in Santa Catarina, Brazil

Background: Botulism is a disease caused by the ingestion of neurotoxin produced by Clostridium botulinum, characterizedby flaccid paralysis, which can lead to high mortality. They have seven types of neurotoxins (A, B, C, D, E, F, and G) and,in birds, most cases are attributed to type C. They are considered sources of botulinum toxins where the decomposition oforganic matter occurs, like stagnant water and rotting food. The main feature of the disease in birds is ascending symmetricflaccid paralysis. The present study aims to describe an outbreak of type C botulism in backyard poultry in the state ofSanta Catarina, Southern Brazil.Case: A visit was made to the property with 160 backyard poultry with a history of high mortality in the municipality ofAgrolândia, Santa Catarina. Clinical signs were characterized by paralysis of the pelvic limbs, neck and pendular wings,which progressed to death within 48 h. There was a mortality rate of 37.5% (60/160) between March and May 2019. Thesebirds were kept in an overcrowded environment, with different species (chickens, ducks, teals, and turkeys) fed irregularly.The water supplied was provided from kitchen exhaust, accumulating in puddles on the floor that contained organic matterresidues such as animal feces, food waste and bone fragments. The disposal of the carcasses of birds that died was in thesame enclosure, buried superficially, facilitating the access of other birds to dig them up and consume them. Necropsywas performed on 2 chickens and one duck, no macroscopic or histopathological lesions were observed. Blood, liver, andgastrointestinal content samples were sent for research and identification of botulinum toxin through the serum neutralization test in mice. The presence of type C botulinum toxin was confirmed in the liver chicken of one sampled animals.Discussion: The identification of type C botulism toxin enabled the characterization of the outbreak, which is...(AU)

Type C botulism outbreak in free-ranging waterfowl in Goiás / Surto de botulismo tipo C em aves aquáticas de vida livre em Goiás

Botulism is generally a fatal disease caused by ingestion of neurotoxins produced by Clostridium botulinum. The present study describes the epidemiological, clinical, and laboratory aspects of a type C botulism outbreak in free-living aquatic birds residing in an urban park in Quirinópolis, Goiás, Brazil. Among a population of approximately 80 waterfowl, a total of 30 birds, including ducks (Cairina moschata), teals (Anas platyrhynchos), and geese (Anser cygnoides), died within 10 days. Of these, six birds showed signs of flaccid paralysis of the pelvic limbs, eyelids, neck, and wings. To confirm the suspicion of botulism, four lake water samples, two samples of the feed consumed by the birds, and samples of serum, intestinal content, stomach content, and liver tissue from two teals that died after presenting clinical signs were analyzed. Using bioassay and neutralization with homologous antitoxin in mice, it was possible to detect the presence of botulinum toxin type C in a water sample and in the intestinal content of one of the necropsied teals. Additionally, the presence of C. botulinum type C was identified in the lake water using polymerase chain reaction. Based on the clinical signs and laboratory results, a diagnosis of botulism caused by botulinum toxin type C was confirmed with probable transmission by lake water.

O botulismo é uma doença geralmente fatal, causada pela ingestão de neurotoxinas produzidas pelo Clostridium botulinum. O presente estudo descreve os aspectos epidemiológicos, clínicos e laboratoriais de um surto de botulismo tipo C em aves aquáticas de vida livre habitantes de parque urbano em Quirinópolis, Goiás. De uma população de cerca de 80 aves aquáticas, um total de 30 aves, entre patos (Cairina moschata), marrecos (Anas platyrhynchos) e gansos (Anser cygnoides), morreram no intervalo de 10 dias. Destes, seis aves apresentaram sinais de paralisia flácida de membros pélvicos, pálpebras, pescoço e asas. Para confirmar a suspeita de botulismo, foram analisadas quatro amostras da água do lago, duas amostras da ração consumida pelas aves e amostras de soro, conteúdo intestinal, conteúdo estomacal e fígado de dois marrecos que morreram após apresentarem os sinais clínicos. Pelo bioensaio e neutralização com antitoxina homóloga em camundongos foi possível detectar a presença de toxina botulínica tipo C em uma amostra de água e no conteúdo intestinal de um dos marrecos necropsiados. Adicionalmente, pela reação em cadeia da polimerase identificou-se a presença de C. botulinum tipo C na água do lago. Com base nos sinais clínicos e resultados laboratoriais estabeleceu-se o diagnóstico de botulismo causado pela toxina botulínica tipo C e veiculada provavelmente pela água do lago.

Venom composition of Trimeresurus albolabris, T. insularis, T. puniceus and T. purpureomaculatus from Indonesia

Background: Several studies have been published on the characterization of Trimeresurus venoms. However, there is still limited information concerning the venom composition of Trimeresurus species distributed throughout Indonesia, which contributes to significant snakebite envenomation cases. The present study describes a comparative on the composition of T. albolabris, T. insularis, T. puniceus, and T. purpureomaculatus venoms originated from Indonesia. Methods: Protein content in the venom of four Trimeresurus species was determined using Bradford assay, and the venom proteome was elucidated using one-dimension SDS PAGE nano-ESI- LCMS/MS shotgun proteomics. Results: The venom of T. albolabris contained the highest protein content of 11.1 mg/mL, followed by T. puniceus, T. insularis and T. purpureomaculatus venom with 10.7 mg/mL, 8.9 mg/mL and 5.54 mg/mL protein, respectively. In total, our venomic analysis identified 65 proteins belonging to 16 protein families in T. purpureomaculatus; 64 proteins belonging to 18 protein families in T. albolabris; 58 different proteins belonging to 14 protein families in T. puniceus; and 48 different proteins belonging to 14 protein familiesin T. insularis. Four major proteins identified in all venoms belonged to snake venom metalloproteinase, C-type lectin, snake venom serine protease, and phospholipase A2. There were 11 common proteins in all venoms, and T. puniceus venom has the highest number of unique proteins compared to the other three venoms. Cluster analysis of the proteins and venoms showed that T. puniceus venom has the most distinct venom composition. Conclusions: Overall, the results highlighted venom compositional variation of four Trimeresurus spp. from Indonesia. The venoms appear to be highly similar, comprising at least four protein families that correlate with venom's toxin properties and function. This study adds more information on venom variability among Trimeresurus species within the close geographic origin and may contribute to the development of optimum heterologous antivenom.(AU)

Exploring the five-paced viper (Deinagkistrodon acutus) venom proteome by integrating a combinatorial peptide ligand library approach with shotgun LC-MS/MS

Abstract Background Snake venoms are complex mixtures of toxic proteins or peptides encoded by various gene families that function synergistically to incapacitate prey. In the present study, in order to unravel the proteomic repertoire of Deinagkistrodon acutus venom, some trace abundance components were analyzed. Methods Shotgun proteomic approach combined with shotgun nano-LC-ESI-MS/MS were employed to characterize the medically important D. acutus venom, after collected samples were enriched with the combinatorial peptide ligand library (CPLL). Results This avenue helped us find some trace components, undetected before, in D. acutus venom. The results indicated that D. acutus venom comprised 84 distinct proteins from 10 toxin families and 12 other proteins. These results are more than twice the number of venom components obtained from previous studies, which were only 29 distinct proteins obtained through RP-HPLC for the venom of the same species. The present results indicated that in D. acutus venom, the most abundant components (66.9%) included metalloproteinases, serine proteinases, and C-type lectin proteins; the medium abundant components (13%) comprised phospholipases A2 (PLA2) and 5-nucleotidases and nucleases; whereas least abundant components (6%) were aminopeptidases, L-amino acid oxidases (LAAO), neurotoxins and disintegrins; and the trace components. The last were undetected before the use of conventional shotgun proteomics combined with shotgun nano-LC-ESI-MS/MS, such as cysteine-rich secretory proteins Da-CRPa, phospholipases B-like 1, phospholipases B (PLB), nerve growth factors (NGF), glutaminyl-peptide cyclortransferases (QC), and vascular non-inflammatory molecules 2 (VNN2). Conclusion These findings demonstrated that the CPLL enrichment method worked well in finding the trace toxin proteins in D. acutus venom, in contrast with the previous venomic characterization of D. acutus by conventional LC-MS/MS. In conclusion, this approach combined with the CPLL enrichment was effective for allowing us to explore the hidden D. acutus venomic profile and extended the list of potential venom toxins.

De novo venom gland transcriptomics of Calliophis bivirgata flaviceps: uncovering the complexity of toxins from the Malayan blue coral snake

The Malayan blue coral snake, Calliophis bivirgata flaviceps, is a medically important venomous snake in Southeast Asia. However, the complexity and diversity of its venom genes remain little explored. Methods: To address this, we applied high-throughput next-generation sequencing to profile the venom gland cDNA libraries of C. bivirgata flaviceps. The transcriptome was de novo assembled, followed by gene annotation, multiple sequence alignment and analyses of the transcripts. Results: A total of 74 non-redundant toxin-encoding genes from 16 protein families were identified, with 31 full-length toxin transcripts. Three-finger toxins (3FTx), primarily delta-neurotoxins and cardiotoxin-like/cytotoxin-like proteins, were the most diverse and abundantly expressed. The major 3FTx (Cb_FTX01 and Cb_FTX02) are highly similar to calliotoxin, a delta-neurotoxin previously reported in the venom of C. bivirgata. This study also revealed a conserved tyrosine residue at position 4 of the cardiotoxin-like/cytotoxin-like protein genes in the species. These variants, proposed as Y-type CTX-like proteins, are similar to the H-type CTX from cobras. The substitution is conservative though, preserving a less toxic form of elapid CTX-like protein, as indicated by the lack of venom cytotoxicity in previous laboratory and clinical findings. The ecological role of these toxins, however, remains unclear. The study also uncovered unique transcripts that belong to phospholipase A2 of Groups IA and IB, and snake venom metalloproteinases of PIII subclass, which show sequence variations from those of Asiatic elapids. Conclusion: The venom gland transcriptome of C. bivirgata flaviceps from Malaysia was de novo assembled and annotated. The diversity and expression profile of toxin genes provide insights into the biological and medical importance of the species.(AU)

De novo venom gland transcriptomics of Calliophis bivirgata flaviceps: uncovering the complexity of toxins from the Malayan blue coral snake

The Malayan blue coral snake, Calliophis bivirgata flaviceps, is a medically important venomous snake in Southeast Asia. However, the complexity and diversity of its venom genes remain little explored. Methods: To address this, we applied high-throughput next-generation sequencing to profile the venom gland cDNA libraries of C. bivirgata flaviceps. The transcriptome was de novo assembled, followed by gene annotation, multiple sequence alignment and analyses of the transcripts. Results: A total of 74 non-redundant toxin-encoding genes from 16 protein families were identified, with 31 full-length toxin transcripts. Three-finger toxins (3FTx), primarily delta-neurotoxins and cardiotoxin-like/cytotoxin-like proteins, were the most diverse and abundantly expressed. The major 3FTx (Cb_FTX01 and Cb_FTX02) are highly similar to calliotoxin, a delta-neurotoxin previously reported in the venom of C. bivirgata. This study also revealed a conserved tyrosine residue at position 4 of the cardiotoxin-like/cytotoxin-like protein genes in the species. These variants, proposed as Y-type CTX-like proteins, are similar to the H-type CTX from cobras. The substitution is conservative though, preserving a less toxic form of elapid CTX-like protein, as indicated by the lack of venom cytotoxicity in previous laboratory and clinical findings. The ecological role of these toxins, however, remains unclear. The study also uncovered unique transcripts that belong to phospholipase A2 of Groups IA and IB, and snake venom metalloproteinases of PIII subclass, which show sequence variations from those of Asiatic elapids. Conclusion: The venom gland transcriptome of C. bivirgata flaviceps from Malaysia was de novo assembled and annotated. The diversity and expression profile of toxin genes provide insights into the biological and medical importance of the species.(AU)

Exploring the five-paced viper (Deinagkistrodon acutus) venom proteome by integrating a combinatorial peptide ligand library approach with shotgun LC-MS/MS

Snake venoms are complex mixtures of toxic proteins or peptides encoded by various gene families that function synergistically to incapacitate prey. In the present study, in order to unravel the proteomic repertoire of Deinagkistrodon acutus venom, some trace abundance components were analyzed. Methods Shotgun proteomic approach combined with shotgun nano-LC-ESI-MS/MS were employed to characterize the medically important D. acutus venom, after collected samples were enriched with the combinatorial peptide ligand library (CPLL). Results This avenue helped us find some trace components, undetected before, in D. acutus venom. The results indicated that D. acutus venom comprised 84 distinct proteins from 10 toxin families and 12 other proteins. These results are more than twice the number of venom components obtained from previous studies, which were only 29 distinct proteins obtained through RP-HPLC for the venom of the same species. The present results indicated that in D. acutus venom, the most abundant components (66.9%) included metalloproteinases, serine proteinases, and C-type lectin proteins; the medium abundant components (13%) comprised phospholipases A2 (PLA2) and 5'-nucleotidases and nucleases; whereas least abundant components (6%) were aminopeptidases, L-amino acid oxidases (LAAO), neurotoxins and disintegrins; and the trace components. The last were undetected before the use of conventional shotgun proteomics combined with shotgun nano-LC-ESI-MS/MS, such as cysteine-rich secretory proteins Da-CRPa, phospholipases B-like 1, phospholipases B (PLB), nerve growth factors (NGF), glutaminyl-peptide cyclortransferases (QC), and vascular non-inflammatory molecules 2 (VNN2). Conclusion These findings demonstrated that the CPLL enrichment method worked well in finding the trace toxin proteins in D. acutus venom, in contrast with the previous venomic characterization of D. acutus by conventional LC-MS/MS. In conclusion, this approach combined with the CPLL enrichment was effective for allowing us to explore the hidden D. acutus venomic profile and extended the list of potential venom toxins.(AU)

Exploring the five-paced viper (Deinagkistrodon acutus) venom proteome by integrating a combinatorial peptide ligand library approach with shotgun LC-MS/MS

Snake venoms are complex mixtures of toxic proteins or peptides encoded by various gene families that function synergistically to incapacitate prey. In the present study, in order to unravel the proteomic repertoire of Deinagkistrodon acutus venom, some trace abundance components were analyzed. Methods Shotgun proteomic approach combined with shotgun nano-LC-ESI-MS/MS were employed to characterize the medically important D. acutus venom, after collected samples were enriched with the combinatorial peptide ligand library (CPLL). Results This avenue helped us find some trace components, undetected before, in D. acutus venom. The results indicated that D. acutus venom comprised 84 distinct proteins from 10 toxin families and 12 other proteins. These results are more than twice the number of venom components obtained from previous studies, which were only 29 distinct proteins obtained through RP-HPLC for the venom of the same species. The present results indicated that in D. acutus venom, the most abundant components (66.9%) included metalloproteinases, serine proteinases, and C-type lectin proteins; the medium abundant components (13%) comprised phospholipases A2 (PLA2) and 5'-nucleotidases and nucleases; whereas least abundant components (6%) were aminopeptidases, L-amino acid oxidases (LAAO), neurotoxins and disintegrins; and the trace components. The last were undetected before the use of conventional shotgun proteomics combined with shotgun nano-LC-ESI-MS/MS, such as cysteine-rich secretory proteins Da-CRPa, phospholipases B-like 1, phospholipases B (PLB), nerve growth factors (NGF), glutaminyl-peptide cyclortransferases (QC), and vascular non-inflammatory molecules 2 (VNN2). Conclusion These findings demonstrated that the CPLL enrichment method worked well in finding the trace toxin proteins in D. acutus venom, in contrast with the previous venomic characterization of D. acutus by conventional LC-MS/MS. In conclusion, this approach combined with the CPLL enrichment was effective for allowing us to explore the hidden D. acutus venomic profile and extended the list of potential venom toxins.(AU)

Fecal microbiota transplantation via colonoscopy in a dog with Clostridioides (Clostridium) difficile infection / Transplante de microbiota fecal via colonoscopia em um cão com infecção por Clostridioides (Clostridium) difficile

In dogs, antimicrobial therapy for Clostridioides (Clostridium) difficile infection (CDI) is based solely on metronidazole, leaving limited treatment options in case of recurrent disease. Fecal microbiota transplantation (FMT) has been successfully used in humans with recurrent CDI, whereas the usefulness of this approach is largely unknown in dogs. In the present study, a dog with a chronic-recurring diarrhea was treated with FMT via colonoscopy. CDI was confirmed by A/B toxin detection and isolation of toxigenic C. difficile from ribotype 106, a strain also commonly associated with nosocomial infection in humans. The dog recovered well after the procedure and C. difficile was no longer isolated from its stool sample. The present research suggested that FMT could be a useful tool to treat recurrent CDI in dogs, corroborating the actual protocol in humans.(AU)

Em cães, a terapia antimicrobiana para infecções por Clostridioides (Clostridium) difficile é baseada apenas no uso de metronidazol, limitando as opções de tratamento nos casos de recorrência. O transplante de microbiota fecal (FMT) tem sido utilizado com sucesso em seres humanos com infecções recorrentes por C. difficile, porém a utilidade desse método é ainda amplamente desconhecida em cães. O presente trabalho relata a utilização de FMT para o tratamento de um cão com diarreia crônica-recorrente por C. difficile. A infecção foi confirmada por detecção das toxinas A/B e isolamento de uma estirpe toxigênica do ribotipo 106, linhagem comumente associada a infecção em seres humanos. Após o transplante via colonoscopia, o animal se recuperou do quadro e C. difficile não mais foi encontrado em novas amostras fecais. O presente trabalho sugere que o FMT possa ser útil para o tratamento de quadros de C. difficile em cães, corroborando protocolo atual de tratamento em seres humanos.(AU)

Jingzhaotoxin-X, a gating modifier of Kv4.2 and Kv4. 3 potassium channels purified from the venom of the Chinese tarantula Chilobrachys jingzhao

The tarantula Chilobrachys jingzhao is one of the largest venomous spiders in China. In previous studies, we purified and characterized at least eight peptides from C. jingzhao venom. In this report, we describe the purification and characterization of Jingzhaotoxin-X (JZTX-X), which selectively blocks Kv4.2 and Kv4.3 potassium channels. Methods: JZTX-X was purified using a combination of cation-exchange HPLC and reverse-phase HPLC. The amino-acid sequence was determined by automated Edman degradation and confirmed by mass spectrometry (MS). Voltage-gated ion channel currents were recorded in HEK293t cells transiently transfected with a variety of ion channel constructs. In addition, the hyperalgesic activity of JZTX-X and the toxin´s effect on motor function were assessed in mice. Results: JZTX-X contained 31 amino acids, with six cysteine residues that formed three disulfide bonds within an inhibitory cysteine knot (ICK) topology. In whole-cell voltage-clamp experiments, JZTX-X inhibited Kv4.2 and Kv4.3 potassium channels in a concentration- and voltage-dependent manner, without affecting other ion channels (Kv1.1, 1.2, 1.3, 2.1, delayed rectifier potassium channels, high- and low-voltage-activated Ca2+ channels, and voltage-gated sodium channels Nav1.5 and 1.7). JZTX-X also shifted the voltage-dependent channel activation to more depolarized potentials, whereas extreme depolarization caused reversible toxin binding to Kv4.2 channels. JZTX-X shifted the Kv4.2 and Kv4.3 activities towards a resting state, since at the resting potential the toxin completely inhibited the channels, even in the absence of an applied physical stimulus. Intrathecal or intraplantar injection of JZTX-X caused a long-lasting decrease in the mechanical nociceptive threshold (hyperalgesia) but had no effect on motor function as assessed in the rotarod test. Conclusions: JZTX-X selectively suppresses Kv4.2 and Kv4.3 potassium channel activity in a concentration- and voltage-dependent manner and causes long-lasting mechanical hyperalgesia.(AU)

Geographic variation of individual venom profile of Crotalus durissus snakes

South American rattlesnakes are represented in Brazil by a single species, Crotalus durissus, which has public health importance due to the severity of its envenomation and to its wide geographical distribution. The species is subdivided into several subspecies, but the current classification is controversial. In Brazil, the venoms of C. d. terrificus and C. d. collilineatus are used for hyperimmunization of horses for antivenom production, even though the distinction of these two subspecies are mostly by their geographical distribution. In this context, we described a comparative compositional and functional characterization of individual C. d. collilineatus and C. d. terrificus venoms from three Brazilian states. Methods: We compared the compositional patterns of C. d. terrificus and C. d. collilineatus individual venoms by 1-DE and RP-HPLC. For functional analyzes, the enzymatic activities of PLA2, LAAO, and coagulant activity were evaluated. Finally, the immunorecognition of venom toxins by the crotalic antivenom produced at Butantan Institute was evaluated using Western blotting. Results: The protein profile of individual venoms from C. d. collilineatus and C. d. terrificus showed a comparable overall composition, despite some intraspecific variation, especially regarding crotamine and LAAO. Interestingly, HPLC analysis showed a geographic pattern concerning PLA2. In addition, a remarkable intraspecific variation was also observed in PLA2, LAAO and coagulant activities. The immunorecognition pattern of individual venoms from C. d. collilineatus and C. d. terrificus by crotalic antivenom produced at Butantan Institute was similar. Conclusions: The results highlighted the individual variability among the venoms of C. durissus ssp. specimens. Importantly, our data point to a geographical variation of C. durissus ssp. venom profile, regardless of the subspecies, as evidenced by PLA2 isoforms complexity, which may explain the increase in venom neurotoxicity from Northeastern through Southern Brazil reported for the species.(AU)

Analgesic and side effects of intravenous recombinant Ph alfa 1 beta

Background: Intrathecal injection of voltage-sensitive calcium channel blocker peptide toxins exerts analgesic effect in several animal models of pain. Upon intrathecal administration, recombinant Phα1β exerts the same analgesic effects as the those of the native toxin. However, from a clinical perspective, the intrathecal administration limits the use of anesthetic drugs in patients. Therefore, this study aimed to investigate the possible antinociceptive effect of intravenous recombinant Phα1β in rat models of neuropathic pain, as well as its side effects on motor, cardiac (heart rate and blood pressure), and biochemical parameters. Methods: Male Wistar rats and male Balb-C mice were used in this study. Giotto Biotech® synthesized the recombinant version of Phα1β using Escherichia coli expression. In rats, neuropathic pain was induced by chronic constriction of the sciatic nerve and paclitaxel-induced acute and chronic pain. Mechanical sensitivity was evaluated using von Frey filaments. A radiotelemeter transmitter (TA11PA-C10; Data Sciences, St. Paul, MN, USA) was placed on the left carotid of mice for investigation of cardiovascular side effects. Locomotor activity data were evaluated using the open-field paradigm, and serum CKMB, TGO, TGP, LDH, lactate, creatinine, and urea levels were examined. Results: Intravenous administration of recombinant Phα1β toxin induced analgesia for up to 4 h, with ED50 of 0.02 (0.01-0.03) mg/kg, and reached the maximal effect (Emax = 100% antinociception) at a dose of 0.2 mg/kg. No significant changes were observed in any of the evaluated motor, cardiac or biochemical parameters. Conclusion: Our data suggest that intravenous administration of recombinant Phα1β may be feasible for drug-induced analgesia, without causing any severe side effects.(AU)

Analgesic and side effects of intravenous recombinant Ph alfa 1 beta

Background: Intrathecal injection of voltage-sensitive calcium channel blocker peptide toxins exerts analgesic effect in several animal models of pain. Upon intrathecal administration, recombinant Phα1β exerts the same analgesic effects as the those of the native toxin. However, from a clinical perspective, the intrathecal administration limits the use of anesthetic drugs in patients. Therefore, this study aimed to investigate the possible antinociceptive effect of intravenous recombinant Phα1β in rat models of neuropathic pain, as well as its side effects on motor, cardiac (heart rate and blood pressure), and biochemical parameters. Methods: Male Wistar rats and male Balb-C mice were used in this study. Giotto Biotech® synthesized the recombinant version of Phα1β using Escherichia coli expression. In rats, neuropathic pain was induced by chronic constriction of the sciatic nerve and paclitaxel-induced acute and chronic pain. Mechanical sensitivity was evaluated using von Frey filaments. A radiotelemeter transmitter (TA11PA-C10; Data Sciences, St. Paul, MN, USA) was placed on the left carotid of mice for investigation of cardiovascular side effects. Locomotor activity data were evaluated using the open-field paradigm, and serum CKMB, TGO, TGP, LDH, lactate, creatinine, and urea levels were examined. Results: Intravenous administration of recombinant Phα1β toxin induced analgesia for up to 4 h, with ED50 of 0.02 (0.01-0.03) mg/kg, and reached the maximal effect (Emax = 100% antinociception) at a dose of 0.2 mg/kg. No significant changes were observed in any of the evaluated motor, cardiac or biochemical parameters. Conclusion: Our data suggest that intravenous administration of recombinant Phα1β may be feasible for drug-induced analgesia, without causing any severe side effects.

Jingzhaotoxin-X, a gating modifier of Kv4.2 and Kv4.3 potassium channels purified from the venom of the Chinese tarantula Chilobrachys jingzhao

Background:The tarantula Chilobrachys jingzhao is one of the largest venomous spiders in China. In previous studies, we purified and characterized at least eight peptides from C. jingzhao venom. In this report, we describe the purification and characterization of Jingzhaotoxin-X (JZTX-X), which selectively blocks Kv4.2 and Kv4.3 potassium channels.Methods:JZTX-X was purified using a combination of cation-exchange HPLC and reverse-phase HPLC. The amino-acid sequence was determined by automated Edman degradation and confirmed by mass spectrometry (MS). Voltage-gated ion channel currents were recorded in HEK293t cells transiently transfected with a variety of ion channel constructs. In addition, the hyperalgesic activity of JZTX-X and the toxin´s effect on motor function were assessed in mice.Results:JZTX-X contained 31 amino acids, with six cysteine residues that formed three disulfide bonds within an inhibitory cysteine knot (ICK) topology. In whole-cell voltage-clamp experiments, JZTX-X inhibited Kv4.2 and Kv4.3 potassium channels in a concentration- and voltage-dependent manner, without affecting other ion channels (Kv1.1, 1.2, 1.3, 2.1, delayed rectifier potassium channels, high- and low-voltage-activated Ca2+ channels, and voltage-gated sodium channels Nav1.5 and 1.7). JZTX-X also shifted the voltage-dependent channel activation to more depolarized potentials, whereas extreme depolarization caused reversible toxin binding to Kv4.2 channels. JZTX-X shifted the Kv4.2 and Kv4.3 activities towards a resting state, since at the resting potential the toxin...(AU)

Venomics and antivenomics of the poorly studied Brazil's lancehead, Bothrops brazili (Hoge, 1954), from the Brazilian State of Pará

The Brazil's lancehead, Bothrops brazili, is a poorly studied pit viper distributed in lowlands of the equatorial rainforests of southern Colombia, northeastern Peru, eastern Ecuador, southern and southeastern Venezuela, Guyana, Suriname, French Guiana, Brazil, and northern Bolivia. Few studies have been reported on toxins isolated from venom of Ecuadorian and Brazilian B. brazili. The aim of the present study was to elucidate the qualitative and quantitative protein composition of B. brazili venom from Pará (Brazil), and to carry out a comparative antivenomics assessment of the immunoreactivity of the Brazilian antibothropic pentavalent antivenom [soro antibotrópico (SAB) in Portuguese] against the venoms of B. brazili and reference species, B. jararaca. Methods: We have applied a quantitative snake venomics approach, including reverse-phase and two-dimensional electrophoretic decomplexation of the venom toxin arsenal, LC-ESI-MS mass profiling and peptide-centric MS/MS proteomic analysis, to unveil the overall protein composition of B. brazili venom from Pará (Brazil). Using third-generation antivenomics, the specific and paraspecific immunoreactivity of the Brazilian SAB against homologous (B. jararaca) and heterologous (B. brazili) venoms was investigated. Results: The venom proteome of the Brazil's lancehead (Pará) is predominantly composed of two major and three minor acidic (19%) and two major and five minor basic (14%) phospholipase A2 molecules; 7-11 snake venom metalloproteinases of classes PI (21%) and PIII (6%); 10-12 serine proteinases (14%), and 1-2 L-amino acid oxidases (6%). Other toxins, including two cysteine-rich secretory proteins, one C-type lectin-like molecule, one nerve growth factor, one 5'-nucleotidase, one phosphodiesterase, one phospholipase B, and one glutaminyl cyclase molecule, represent together less than 2.7% of the venom proteome. Third generation antivenomics profile of the Brazilian pentabothropic antivenom showed paraspecific immunoreactivity against all the toxin classes of B. brazili venom, with maximal binding capacity of 132.2 mg venom/g antivenom. This figure indicates that 19% of antivenom's F(ab')2 antibodies bind B. brazili venom toxins. Conclusion: The proteomics outcome contribute to a deeper insight into the spectrum of toxins present in the venom of the Brazil's lancehead, and rationalize the pathophysiology underlying this snake bite envenomings. The comparative qualitative and quantitative immunorecognition profile of the Brazilian pentabothropic antivenom toward the venom toxins of B. brazili and B. jararaca (the reference venom for assessing the bothropic antivenom's potency in Brazil), provides clues about the proper use of the Brazilian antibothropic polyvalent antivenom in the treatment of bites by the Brazil's lancehead.(AU)

Epidemiological and clinical aspects of snakebites in the upper Juruá River region, western Brazilian Amazonia / Aspectos clínicos e epidemiológicos de acidentes ofídicos na região do Alto Juruá, oeste da Amazônia brasileira

This study addresses the clinical and epidemiological aspects of envenoming cases resulting from snakebites treated at a hospital in Cruzeiro do Sul, in the upper Juruá River region, western Brazilian Amazonia. The specific identity of snakes that caused the envenomings was inferred (a) from the diagnosis of patient symptoms and signs upon hospital admission, (b) by enzyme immunoassay for detection of Bothrops atrox and Lachesis muta venom from serum samples taken from patients before antivenom therapy, or (c) by direct identification of the snake, when it was brought along to the hospital or photographed. There were 133 snakebites (76.2 cases per 100,000 inhabitants) registered during one year (July 2017 to June 2018). Most snakebites (88.7%) were caused by Bothrops spp., and the rest by non-venomous snakes or dry bites. Snakebites tended to occur more often during the rainy season, coinciding with the period of greater reproductive activity of the snakes and greater availability of their prey. In addition, the increase in the water level of rivers and lakes during the rainy season tends to concentrate snakes in dry places and, thus, to increase encounters with humans. Information campaigns on prevention and first aid, specially among the most vulnerable groups (indigenous people, farmers, and children and teenagers in rural areas), and the importance of using protective equipment (boots, leggings, leather gloves) in certain high risk activities (e.g. agriculture and extractivism in forests) are fundamental for the reduction of snakebite morbidity.(AU)

Este estudo aborda os aspectos clínicos e epidemiológicos dos envenenamentos ofídicos tratados em um hospital em Cruzeiro do Sul, Acre, resultantes de acidentes que ocorreram na região do Alto Juruá, no oeste da Amazônia brasileira. A identidade específica das serpentes que causaram os envenenamentos foi inferida (a) pelos sinais e sintomas apresentados pelo paciente na admissão hospitalar, (b) por imunoensaio enzimático para detecção de veneno de Bothrops atrox e Lachesis muta em amostras de soro retiradas de pacientes antes da soroterapia, e (c) pela identificação direta da serpente, quando esta foi levada para o hospital ou fotografada. Houve 133 casos (76,2 casos por 100.000 habitantes) de acidentes ofídicos registrados durante um ano (julho 2017 a junho 2018). A maioria das picadas de serpentes (88,7%) foi causada por Bothrops spp., e o restante por espécies não peçonhentas ou picadas secas. Os acidentes ofídicos tenderam a ocorrer com maior frequência durante a estação chuvosa, coincidindo com o período de maior atividade reprodutiva das serpentes e maior disponibilidade de suas presas. Além disso, o aumento dos níveis de rios e lagos pode fazer com que esses animais procurem locais mais secos, aumentando a frequência de encontro com seres humanos. Campanhas educativas sobre prevenção e primeiros socorros, principalmente entre os grupos mais vulneráveis (indígenas, agricultores, crianças e adolescentes em áreas rurais), e sobre a importância da utilização de equipamentos de proteção (botas, perneiras, luvas de couro) em determinadas atividades de maior risco (e.g., agricultura e extrativismo em florestas), são fundamentais para reduzir a morbidade de picadas de serpentes.(AU)

Clostridium perfringens α and ß recombinant toxoids in equine immunization / Toxóides recombinantes α e ß de Clostridium perfringens na imunização de equinos

Clostridium perfringens is considered one of the main causative agents of superacute enterocolitis, usually fatal in the equine species, due to the action of the ß toxin, and is responsible for causing severe myonecrosis, by the action of the α toxin. The great importance of this agent in the equine economy is due to high mortality and lack of vaccines, which are the main form of prevention, which guarantee the immunization of this animal species. The aim of this study was to evaluate three different concentrations (100, 200 and 400µg) of C. perfringens α and ß recombinant toxoids in equine immunization and to compare with a group vaccinated with a commercial toxoid. The commercial vaccine was not able to stimulate an immune response and the recombinant vaccine was able to induce satisfactory humoral immune response in vaccinated horses, proving to be an alternative prophylactic for C. perfringens infection.(AU)

Clostridium perfringens é considerado um dos principais agentes causadores de enterocolites superagudas, geralmente fatais na espécie equina, devido à ação da toxina ß, além de ser responsável por causar quadros graves de mionecrose, pela ação da toxina α. A grande importância desses agentes na equinocultura, deve-se a elevada mortalidade e a inexistência de vacinas, principal forma de prevenção, que garantam a imunização dessa espécie animal. O objetivo deste trabalho foi avaliar três diferentes concentrações (100, 200 e 400µg) dos toxóides recombinantes α e ß de C. perfringens na imunização de equinos, bem como comparar com um grupo vacinado com um toxóide comercial. A vacina comercial não se mostrou capaz de estimular uma resposta imune e a vacina recombinante foi capaz de induzir resposta imune humoral satisfatória em equinos vacinados, provando ser uma alternativa profilática para infecção por C. Perfringens.(AU)

Clostridium perfringens α and ß recombinant toxoids in equine immunization / Toxóides recombinantes α e ß de Clostridium perfringens na imunização de equinos

Clostridium perfringens is considered one of the main causative agents of superacute enterocolitis, usually fatal in the equine species, due to the action of the ß toxin, and is responsible for causing severe myonecrosis, by the action of the α toxin. The great importance of this agent in the equine economy is due to high mortality and lack of vaccines, which are the main form of prevention, which guarantee the immunization of this animal species. The aim of this study was to evaluate three different concentrations (100, 200 and 400µg) of C. perfringens α and ß recombinant toxoids in equine immunization and to compare with a group vaccinated with a commercial toxoid. The commercial vaccine was not able to stimulate an immune response and the recombinant vaccine was able to induce satisfactory humoral immune response in vaccinated horses, proving to be an alternative prophylactic for C. perfringens infection.(AU)

Clostridium perfringens é considerado um dos principais agentes causadores de enterocolites superagudas, geralmente fatais na espécie equina, devido à ação da toxina ß, além de ser responsável por causar quadros graves de mionecrose, pela ação da toxina α. A grande importância desses agentes na equinocultura, deve-se a elevada mortalidade e a inexistência de vacinas, principal forma de prevenção, que garantam a imunização dessa espécie animal. O objetivo deste trabalho foi avaliar três diferentes concentrações (100, 200 e 400µg) dos toxóides recombinantes α e ß de C. perfringens na imunização de equinos, bem como comparar com um grupo vacinado com um toxóide comercial. A vacina comercial não se mostrou capaz de estimular uma resposta imune e a vacina recombinante foi capaz de induzir resposta imune humoral satisfatória em equinos vacinados, provando ser uma alternativa profilática para infecção por C. Perfringens.(AU)