Social media as a tool for detecting underdiagnosed parasitic infections: the case of spirocercosis.

Spirocercosis is a parasitic disease caused by the nematode Spirocerca lupi that affects several canids around the world. In Costa Rica, few spirocercosis cases have been reported; however, it is suspected that the infection is currently underdiagnosed. As seen during the COVID-19 pandemic, social media has sped up the spread of scientific knowledge, raising practioner's awareness of the epidemiology and signs and manifestations of infections. The aim of this work was to use social media as a tool for increasing awareness of spirocercosis among veterinarians and investigate the frequency of cases occurring in Costa Rica. Infographic material about the infection was disseminated by Whatsapp, Facebook, and Instagram from January 2021 to July 2022 using a public account of the Laboratory of Helminthology of the University of Costa Rica. In addition, Google Forms questionnaires were completed by veterinarians to report the number of spirocercosis cases in their clinics, associated manifestations, diagnosis, treatment, and outcome of the disease. Remarkably, since 2020, a six-fold increase in the number of cases per year was noted. Clinical manifestations matched those reported elsewhere, and one of every three dogs infected with S. lupi died spontaneously or required euthanasia because of the infection. This work suggests that S. lupi infection in Costa Rica may have been underdiagnosed in the last decade, and the knowledge and awareness of clinical personnel play a major role in the detection of this and other infectious diseases.

Spirocerca vulpis sp. nov. (Spiruridae: Spirocercidae): description of a new nematode species of the red fox, Vulpes vulpes (Carnivora: Canidae).

Previous studies have reported nematodes of the Spirocercidae family in the stomach nodules of red foxes (Vulpes vulpes) described as Spirocerca sp. or Spirocerca lupi (Rudolphi, 1819). We characterized spirurid worms collected from red foxes and compared them to S. lupi from domestic dogs by morphometric and phylogenetic analyses. Nematodes from red foxes differed from S. lupi by the presence of six triangular teeth-like buccal capsule structures, which are absent in the latter. Additionally, in female worms from red foxes, the distance of the vulva opening to the anterior end and the ratio of the glandular-to-muscular oesophagus lengths were larger than those of S. lupi (P < 0.006). In males, the lengths of the whole oesophagus and glandular part, the ratio of the glandular-to-muscular oesophagus and the comparison of the oesophagus to the total body length were smaller in S. lupi (all P < 0.044). Phylogenetic analyses revealed that S. lupi and the red foxes spirurid represent monophyletic sister groups with pairwise nucleotide distances of 9.2 and 0.2% in the cytochrome oxidase 1 and 18S genes, respectively. Based on these comparisons, the nematodes from red foxes were considered to belong to a separate species, for which the name Spirocerca vulpis sp. nov. is proposed.

The use of primary murine fibroblasts to ascertain if Spirocerca lupi secretory/excretory protein products are mitogenic ex vivo.

BACKGROUND: Spirocerca lupi is a nematode that parasitizes vertebrates in particular canids, by forming nodules in the thoracic cavity specifically in the oesophagus. In 25% of Spirocerca infections of the domestic dog, nodules progress from inflammatory to pre-neoplastic to sarcomatous neoplasia. With the mechanism of neoplastic transformation being incompletely understood, this study investigates if S. lupi parasite proteinaceous secretory/excretory products (ESPs) play a role in the neoplastic transformation. METHODS: To facilitate collection of ESPs, we maintained naturally harvested adult parasites in the laboratory under artificial conditions. Media in which the parasites were grown was subsequently evaluated for the presence of proteinaceous compounds using a mass spectroscopy library as well as for their ability to be mitogenic in primary murine fibroblastic cells. RESULTS: Chromatrography of the ethyl acetate extracted incubation media showed the presence of 9 protein compounds, of which three were identified as non-specific proteins isolated from Nematostella vectensis, Caenorhabditis brenneri and Sus scrofa, with the rest being unknown. Acetone, methanol, hexane and ethylacetate extracted culture media were unable to induce a mitogenic change in primary murine fibroblasts in comparison to the controls. CONCLUSION: While no mitogenic effect was evident, further studies are required to understand the role of worm excretory/secretory products on clastogenesis under chronic exposure. In addition, while not of primary importance for this study, the observed duration of parasite survival indicates that ex vivo studies on S. lupi are possible. For the latter we believe that the worm culture method can be further optimized if longer survival times are required.

Genotyping and comparative pathology of Spirocerca in black-backed jackals (Canis mesomelas) in South Africa.

BACKGROUND: The pathology of spirocercosis, a disease caused by the infestation of carnivores with the nematode Spirocerca lupi, has been extensively described in domestic dogs and coyotes. However, it has not been described in wild carnivores in South Africa. The aim of this study was to evaluate whether black-backed jackals are a host for Spirocerca species and to provide a detailed description of the associated pathology. Jackals were also stratified according to age and the Spirocerca species recovered were characterized using molecular techniques. METHODS: Standard necropsies were performed on routinely culled jackals from three of the nine provinces of South Africa during the period June 2012 to February 2013. Jackals were screened for the presence of pathognomonic Spirocerca-induced lesions and for evidence of aberrant migration. Relevant samples were submitted for histopathology and collected larvae were genotyped at nine microsatellite loci. RESULTS: Spirocerca lupi-associated aortic lesions were found in 16 of 93 (17%) black-backed jackals. Of these, four (25%) were associated with S. lupi larvae. Genotyping of the larvae revealed amplification of all nine loci that amplified dog-derived S. lupi, with the same level of polymorphism in the allele size ranges. Only 1 of 93 jackals had an esophageal nodule with concurrent S. lupi-induced aortic aneurysms. The single esophageal nodule found did not contain adult nematodes, nor did it communicate with the esophageal lumen. None of the jackals that were examined had macroscopically evident spondylitis, which is frequently reported in the dog. Histopathology of the S. lupi-induced aortic lesions in the jackal revealed replacement of elastic and smooth muscle fibers by fibrous connective tissue. In cases where inflammation was present, the inflammatory infiltrate consisted predominantly of eosinophils. The single esophageal nodule histologically resembled the early inflammatory nodule described in dogs and consisted of fibrous connective tissue, multifocal accumulation of lymphocytes, plasma cells and rare hemosiderin-laden macrophages. CONCLUSIONS: These lesions suggest that the life cycle of S. lupi may not or only rarely be completed in jackals. A possible explanation might be that jackals are relatively resistant to developing significant pathology associated with S. lupi-infection. However, before any conclusions can be drawn, many more jackals, including those that die naturally will have to be investigated for evidence of S. lupi infection.

GASTROINTESTINAL PARASITES OF CAPTIVE AND FREE-LIVING LEMURS AND DOMESTIC CARNIVORES IN EASTERN MADAGASCAR.

Fecal samples from captive and free-living lemurs at Ivoloina Zoological Park (IZP) and domestic carnivores from six villages surrounding IZP were evaluated between July and August 2012. Free-living lemurs from Betampona Natural Reserve (BNR), a relatively pristine rainforest fragment 40 km away, were also evaluated in November 2013. All 33 dogs sampled (100%) and 16 of 22 cats sampled (72.7%) were parasitized, predominantly with nematodes (strongyles, ascarids, and spirurids) as well as cestodes and protozoans. Similar types of parasites were identified in the lemur populations. Identification of spirurid nematodes and protozoans in the lemur fecal samples were of concern due to previously documented morbidity and mortality in lemurs from these parasitic agents. Twelve of 13 free-living (93%) and 31 of 49 captive (63%) lemurs sampled at IZP had a higher parasite prevalence than lemurs at BNR, with 13 of 24 (54%) being parasitized. The lemurs in BNR are likely at risk of increased exposure to these parasites and, therefore, increased morbidity and mortality, as humans and their domestic animals are encroaching on this natural area.

Triple phase dynamic computed tomographic perfusion characteristics of spirocercosis induced esophageal nodules in non-neoplastic versus neoplastic canine cases.

Neoplastic transformation of Spirocerca lupi induced esophageal nodules carries a poor prognosis. Clinical, clinicopathological, endoscopic, and radiographic characteristics may be indicative of neoplastic transformation but variable sensitivity and specificity of these parameters makes their use questionable. We hypothesized that CT would be a better diagnostic modality to discriminate between non-neoplastic and neoplastic nodules. In this prospective study of 38 dogs, the appearance and perfusion characteristics of confirmed spirocercosis-induced neoplastic and non-neoplastic esophageal nodules were described using survey CT and triple phase dynamic CT angiography (CTA). Pre- and post-contrast early arterial, late arterial, and venous CTA images were evaluated. Non-neoplastic nodules were smooth and nonmineralized with a higher proportion of hypoattenuating necropurulent cavities compared to neoplastic nodules that had a more irregular surface, with 93% having mineralized foci and rarely any hypoattenuating pockets. Non-neoplastic nodules were significantly more perfused than neoplastic nodules with the difference being up to 23 Hounsfield units. The difference was most marked in the early and late arterial phases (P = 0.0005 and 0.00005, respectively). Ratios of the normal esophagus adjacent to the neoplastic and non-neoplastic nodules did not differ significantly from each other. Perfusion findings demonstrated relative hypoperfusion of the esophageal sarcomas. Findings from the current study indicated that CT characteristics of relative postcontrast hypoperfusion, combined with nodule irregularity and mineralization warrant a high level of concern for neoplastic transformation in canine spirocercosis-induced esophageal nodules.

Spirocerca lupi draft genome, vaccine and anthelmintic targets.

Spirocerca lupi is a parasitic nematode affecting predominantly domestic dogs. It causes spirocercosis, a disease that is often fatal. The assembled draft genome of S. lupi consists of 13,627 predicted protein-coding genes and is approximately 150 Mb in length. Several known anthelmintic gene targets such as for ß-Tubulin, glutamate, and GABA receptors as well as known vaccine gene targets such as cysteine protease inhibitor and cytokines were identified in S. lupi by comparing orthologs of C. elegans anthelmintic gene targets as well as orthologs to known vaccine candidates. New anthelmintic targets were predicted through an inclusion-exclusion strategy and new vaccine targets were predicted through an immunoinformatics approach. New anthelminthic targets include DNA-directed RNA polymerases, chitin synthase, polymerases, and other enzymes. New vaccine targets include cuticle collagens. These gene targets provide a starting platform for new drug identification and vaccine design.

Detection of Spirocerca lupi and an unknown Trichinella-like nematode in raccoon (Procyon lotor).

The raccoon Procyon lotor (Carnivora: Procyonidae) is an invasive species of growing importance for the introduction of alien pathogens or as additional hosts for autochthonous pathogens in Europe, including zoonotic parasites. As the population is steadily increasing and outcompeting the red fox (Vulpes vulpes) in Germany, the consumption of raccoon meat raises concerns about pathogens they may transmit. Therefore the presence of Trichinella larvae was here investigated in muscle samples (n = 904) of raccoons from northern Germany. No Trichinella larvae were found, thus confirming the general low occurrence of this parasite in Germany. However, Spirocerca lupi (n = 12) and an unidentified Trichinella-like nematode (n = 1) were accidently detected in the examined samples. The first is not a zoonotic parasite but has a high veterinary relevance as it can cause severe diseases in dogs. It is the first documented autochthonous infection of this nematode in Germany. The larvae of an unidentified Trichinella-like nematode were found in high abundance in all examined muscles of one raccoon, though they could not be identified to species level. Histological investigation revealed intramuscular cystic structures. This is the largest study investigating muscular parasites of raccoons in Europe so far, which suggests that this invasive animal species is infected by S. lupi and by a yet unknown Trichinella -like parasite.

Spirocercosis and Parvovirus in an Imported Dog: Implications for Biosecurity and Foreign Infectious Disease Risk.

Increased international travel and importation of animals brings with it the potential for spread of infectious diseases. This report details a case in which an 8-month-old male mixed breed dog died shortly after arrival to the United States from complications secondary to spirocercosis, despite having been deemed healthy and approved for international travel. Four weeks following arrival, the dog developed mild tachypnea and diarrhea. Physical examination revealed moderate pyrexia with generalized cerebellar ataxia, moderate anemia, a mature neutrophilia, and severe panhypoproteinemia. Packed red blood cells were administered. The dog was diagnosed with pyothorax and decompensated with septic shock shortly thereafter. Necropsy revealed severe transmural esophagitis with intralesional Spirocerca lupi. Evaluation of the brain revealed cerebellar hypoplasia, and polymerase chain reaction of brain tissue was positive for canine parvovirus. Despite receiving a clean bill of health in its country of origin, this patient ultimately had evidence of 2 different infectious processes that pre-dated its arrival into the United States. While neither of these diseases posed a significant public health risk, this case highlights the role veterinarians play before and after international travel as important barriers against the spread of exotic diseases, and emphasizes that maintaining vigilance is paramount to that task.

Subcutaneous spirocercosis in a dog from Costa Rica and first molecular confirmation of Spirocerca lupi in Central America.

Spirocerca lupi is the etiological agent of spirocercosis in canids from tropics and subtropics in the world. This parasite can lead to life threatening complications, including the formation of fibro and osteosarcomas. A 5-year-old crossbreed Maltese dog from Costa Rica was presented to a veterinary clinic with two nodules in the subcutaneous tissues of the cervical region. One of the nodules resolved with intramuscular antibiotic and anti-inflammatory treatment. However, the other nodule persisted and became purulent. The content of the nodule was excised releasing two coiled 3-cm reddish worms. Identity of the specimens was confirmed by amplifying an 850 bp fragment of the 18S rDNA. The obtained sequence showed 99.96% similarities with S. lupi from Hungary. The present study highlights the need of veterinary clinicians' awareness towards nematode aberrant migrations, and the require of molecular studies to gain insight into the parasite's evolutionary history.

Morphological and molecular characterization of Spirocerca lupi (Nematoda: Spirocercidae) from domestic dogs in Vietnam with reference to Spirocerca vulpis.

Spirocerca lupi is a common parasitic nematode associated with esophageal cancer of canids. Recent surveys have revealed an increasing number of canids infected with Spirocerca spp. in Africa, the Americas, Europe and Western Asia, and described a new species, Spirocerca vulpis, from red foxes (Vulpes vulpes). However, in Southeast Asia, research on Spirocerca spp. is scarce. Therefore, the aim of this study is to explore Spirocerca infection in domestic dogs in Vietnam and to identify the Spirocerca species by analyzing their morphometric and molecular data. We found Spirocerca spp. specimens in 51 (17.7%) out of 287 dogs examined with the intensity of infection ranging from one to 29 nematodes per dog. These nematodes were morphologically and molecularly identified as S. lupi. For morphology, the presence/absence of teeth, the ratio of glandular to muscular esophagus lengths, and the position of the vulva opening of S. lupi and S. vulpis varied between reports, suggesting caution in identification of Spirocerca species based exclusively on morphological characteristics. The molecular analysis based on a partial cox1 sequence revealed that S. lupi from Vietnam is genetically close to those from India and China, but far different from those of Israel, South Africa, Peru and Hungary. Given high genetic and morphological variations, more extensive surveys on Spirocerca spp. from various mammalian hosts at a greater scale are necessary to elucidate the divergence of this nematode.

Balloon expandable aortic stent-graft for the treatment of aortic aneurysm and rupture in a dog.

OBJECTIVE: To describe the successful placement of an aortic stent graft in a dog suffering from aortic aneurysm and rupture. CASE SUMMARY: A 4-year-old neutered female mixed breed dog weighing 25 kg was presented with a chief complaint of acute onset of weakness and respiratory distress. On presentation, the dog was lateral and unresponsive, hypothermic, tachycardic, and tachypneic. Pleural effusion was identified in the right hemi-thorax on thoracic-focused sonographic scan for trauma. Thoracic radiographs showed a fluid/soft tissue opacity in the cranial mediastinum and pleural effusion on the right side. Thoracocentesis yielded noncoagulating blood. Hematology revealed normal PCV and total plasma protein that decreased within 24 hours to 20% and 50 g/L (20%/5.0 g/dL), respectively. The dog was treated with IV fluids, tranexamic acid, and fresh frozen plasma. An aortic aneurysm with irregular mineralization of the right ventrolateral aortic wall was identified on computed tomography examination, which also demonstrated a peri-aortic hematoma. A decision was made to attempt treatment with a stent graft. Under general anesthesia, a marker catheter was inserted into the esophagus. A 10-Fr sheath was inserted into the right femoral artery, and an angiography catheter was advanced over a wire. After angiography, a stent graft (12-mm × 50-mm) was placed over the guide wire and deployed under fluoroscopic guidance. The dog recovered uneventfully, with no evidence of recurrent bleeding, and was discharged 2 days after the procedure with antimicrobials, anthelmintic, and antithrombotic treatment. At 90-day follow-up, the dog was doing well, and stent position was radiographically confirmed. NEW OR UNIQUE INFORMATION PROVIDED: This report describes successful management of aortic aneurysm and dissection, an uncommon and challenging pathology in the veterinary practice with few available treatment options. Stent graft placement is a minimally invasive procedure in which a covered stent is placed over the lesion to prevent rupture.

Comparative mitogenomics of Spirocerca lupi from South Africa and China: Variation and possible heteroplasmy.

The complete mitochondrial genome of Spirocerca lupi isolated from a dog in South Africa was sequenced using next generation sequencing (NGS) technology and the 12 protein coding genes along with the two rRNA genes were compared to 18 other nematode species as well as S. lupi from China. The mitochondrial genome of S. lupi South Africa had a mean genetic diversity of 6.1 % compared to S. lupi China with some variation in nucleotide composition, gene positioning and size. Pairwise distance results indicated slightly higher variation when compared to the pairwise distances of other closely related species, however, this variation was not high enough for it to be considered a cryptic species. Phylogenetic analysis indicated that S. lupi from the two continents are very similar. In addition, single nucleotide polymorphisms were detected in the nad2 gene with ten sequence variants identified from 10 clones from a single nematode, suggesting possible heteroplasmy. The origin of the heteroplasmy is currently unknown but it is speculated to have arisen from accumulated mutations in the mitochondria during somatic replication.

Esophageal spirocercosis with pulmonary egg deposition and secondary hypertrophic osteopathy in a dog from Costa Rica.

Spirocerca lupi is the etiologic agent of spirocercosis in canids from the tropical and subtropical regions of the world. A 1-year-old dog was presented with weight loss and diarrhea. Upon examination, thickened limbs, a mass in the lung's right middle lobe and an esophageal mass were found. The pulmonary mass aspirate revealed S. lupi eggs surrounded by a purulent infiltrate. Limb bone biopsies showed irregular mineralization and cartilaginous metaplasia. Treatment with doramectin resolved the initial clinical signs. However, regurgitation was reported 5 months later. Therefore, the dog received additional doramectin and later vomited a worm morphologically consistent with a S. lupi adult. Herein we report the esophageal migration of S. lupi with egg deposition to the lung parenchyma, accompanied by secondary hypertrophic osteopathy. This report highlights the presence of S. lupi in Costa Rica and the importance of including this parasite as a differential diagnosis when compatible signs are identified.

First report of Spirocerca lupi larva in dung beetles (Scarabaeus armeniacus) in the central region of Iran: A morphological and molecular identification.

Dung beetles as detritivores insects, naturally use feces of vertebrates as foods and reproduction beds. This leads to frequent contacts between dung beetles and parasitic helminths. The current study was carried out to assess infections of dung beetles with larval stages of helminths in rural areas of Taleqan County, Alborz Province, Iran. A total number of 200 dung beetles were collected randomly in June 2017 from the highlands of Taleqan County. Beetles were dissected in normal saline and carefully studied using a stereomicroscope. Morphological characteristics of the recovered larvae were drawn using a camera lucida equipped microscope at 400× magnification. Then, genomic DNAs of the recovered larvae extracted and PCR amplifications of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were carried out and the amplicons were sequenced. All collected dung beetles identified as Scarabaeus armeniacus from the Scarabaeidae family (55.5 % were male and 44.5 % female). Three females of the beetles have been found to be naturally infected with the third stage larvae of Spirocerca lupi. The average length and width of the larvae were 2.95 (2.81-3.15; CI 95 %) and 0.12 (0.1-0.15; CI 95 %) mm, respectively. The phylogenetic analysis showed that S. lupi belonged to a clade within the Spirocercidae family. In the current study, S. armeniacus introduced as a potential biological vector for transmission of S. lupi to vertebrates in the region. To the best of the authors' knowledge, this is the first report on the larval stages of S. lupi in S. armeniacus.

Spirocerca lupi Proteomics and Its Role in Cancer Development: An Overview of Spirocercosis-Induced Sarcomas and Revision of Helminth-Induced Carcinomas.

Spirocerca lupi is a parasitic nematode of canids that induces a myriad of clinical manifestations in its host and, in 25% of infections, leads to the formation of sarcomas. The description of the protein composition of the excretory and secretory products (Sl-ESP) of S. lupi has shed light on its possible interactions with the host environment, including migration within the host and mechanisms of immunomodulation. Despite this, the process by which S. lupi induces cancer in the dog remains poorly understood, and some hypotheses have arisen regarding these possible mechanisms. In this review, we discuss the role of specific ESP from the carcinogenic helminths Clonorchis sinensis, Opisthorchis viverrini and Schistosoma haematobium in inducing chronic inflammation and cancer in their host's tissues. The parasitic worms Taenia solium, Echinococcus granulosus, Heterakis gallinarum, Trichuris muris and Strongyloides stercoralis, which have less-characterized mechanisms of cancer induction, are also analyzed. Based on the pathological findings in spirocercosis and the mechanisms by which other parasitic helminths induce cancer, we propose that the sustained inflammatory response in the dog´s tissues produced in response to the release of Sl-ESP homologous to those of other carcinogenic worms may lead to the malignant process in infected dogs.

Insights on Spirocerca lupi, the Carcinogenic Dog Nematode.

Spirocerca lupi is a nematode transmitted by dung beetles that infects domestic and wild canids in tropical and subtropical regions and is associated with neoplasia. It produces a distinctive pathology with the formation of esophageal nodules classified as inflammatory, preneoplastic, or neoplastic with metastasis to distant organs. Aberrant central nervous system migration of this nematode is also responsible for severe neurological manifestations. Reports of spirocercosis have increased over the last two decades showing spread of this canine helminth in five continents. S. lupi from different geographical locations is genetically distinct with two genotypes, genotype I from Africa, Asia, and Australia, and genotype II from Europe, and recently separated from Spirocerca vulpis, a new species described in red foxes from Europe.

Esophageal Infection of Spirocerca lupi with Vulpes rueppellii in Yazd Province, Iran: Case Report.

Spirocerca lupi is an international nematode of Canine and wild carnivores. The generic clinical symptoms are regurgitation, vomiting, and pursiness. The parasite usually procreates nodular masses in the esophagus and pectoral aorta. On July 2014, a four-month male sandy fox was found accidentally killed on the road in Bafgh City, Southeast of Yazd, Yazd Province, Iran during necropsy, referring to esophagus, parasites were collected from esophagus and transmissive up to 70% of alcohol. Parasites were recognized as S. lupi. After clarification of lactophenol and staining, the nematodes were recognized as S. lupi according to key guidelines of Taylor. Besides, the tissue samples were isolated from esophagus wall - the appendix site of nematode for histopathological investigation - so they were transmissive up to 10% of buffered formalin and stained with haematoxylin and eosin. Microscopic diagnoses in esophagus were included epithelial destruction, wall thickening, inflammatory cells infiltration, necrosis and vascular thrombosis. With the author's knowledge, this is the first report and histopathological investigation of S. lupi in sandy fox.

Detection of Intraspinal Spirocerca lupi in Canine Cerebrospinal Fluid by Polymerase Chain Reaction.

Aberrant migration of Spirocerca lupi into the spinal cord is an important cause of severe progressive neurological dysfunction in dogs. Although early diagnosis is essential to prevent deterioration, ante-mortem diagnosis of this condition remains challenging. The aim of this study was to evaluate the detection of the 18S ribosomal DNA (rDNA) S. lupi gene in the cerebrospinal fluid (CSF) of presumptively-affected dogs using polymerase chain reaction (PCR). Dogs with a non-compressive spinal cord lesion, pleocytosis with presence of eosinophils in the CSF and a characteristic clinical presentation were included. CSF samples from eight dogs were available for the study, of which seven were definitively diagnosed with intraspinal spirocercosis by PCR of either the CSF samples (6/7) or tissue samples obtained at necropsy examination (3/7), or both (2/7). Of these seven positive cases, only one dog had a negative CSF PCR, indicating a sensitivity of 86% for detecting nematode DNA in the CSF of infected dogs using this PCR protocol. The nematode DNA sequences obtained from the CSF of six dogs and the spinal cord tissue of three dogs were 98-100% identical to the publicly available sequences of S. lupi, confirming the diagnosis. These findings indicate that PCR targeting the 18S rDNA of S. lupi in CSF is useful for the ante-mortem diagnosis of canine intraspinal spirocercosis.

Secretome of the carcinogenic helminth Spirocerca lupi reveals specific parasite proteins associated with its different life stages.

Spirocerca lupi is a parasitic and carcinogenic nematode of canids distributed in tropical and subtropical regions around the world. The excretion-secretion proteins (PES) of S. lupi have been suggested to play a role in the pathogenesis of its infection. We aimed to identify the PES of different stages of S. lupi and search for proteins that would be useful for diagnostic, therapeutic and vaccination purposes as well as understand their functions. A nano-UPLC mass spectrometry de novo analysis was performed on proteins collected from cultures of S. lupi L3 larvae, L4 females, adult females and adult males from naturally infected hosts. A total of 211 proteins were identified in all cultures. Accordingly, 117, 130, 99 and 116 proteins were detected in L3 larva, L4 females, adult females and adult males, respectively, with a strong correlation in the biological replicates (Pearson coefficients > 0.73). Fourty-four proteins were detected in all developmental stages, 64 were stage-specific and 49 were exclusively identified in L4 females. Cell compartment enrichment analysis revealed that proteins common to all stages were cytoplasmatic (p < 9.x10-6), whereas L4 unique proteins were in collagen trimers, and macromolecular complexes (p < 0.00001). Functional enrichment analysis of proteins showed significant enrichment in lipid metabolism in L3-unique proteins (p<0.00005), in mannose metabolism and protein de-glycosylation for L4-unique proteins (p < 0.00004), and in phosphorus metabolism in proteins shared by all stages (p <  2.1 x10-9). Interestingly, annexin 6, associated with cancer in humans, was detected in all life stages, but in a larger abundance in L4 females and adults. These findings indicate that S. lupi establishes complex interactions with its hosts by an arsenal of proteins expressed in different patterns in each life stage which influence the pathogenesis and oncogenesis of S. lupi and may be used as potential targets for diagnostic assays, drug targets or vaccine candidates.