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.

Spirocerca lupi induced oesophageal neoplasia: Predictors of surgical outcome.

Canine spirocercosis is caused by the nematode Spirocerca lupi. Migration results in oesophageal fibro-inflammatory nodules that may undergo neoplastic transformation. No studies have assessed pre- or post-surgical prognostic indicators in dogs that undergo intervention for S. lupi induced oesophageal neoplasia. This observational, multi-center study aimed to assess the outcome of dogs with Spirocerca induced sarcoma undergoing endoscopic-guided ablation (n = 12) or surgery (n = 18), and identify prognostic indicators. Parameters evaluated included: age, weight, gender, presenting complaints, duration of clinical signs, complete blood count, serum biochemistry, neoplasia size, placement of percutaneous endoscopically-placed gastrostomy tube, histopathological mitotic indices, days to discharge and chemotherapy administration. Kaplan-Meier survival curves showed no difference in survival between ablation and surgery {(median: 73.5 days (range: 0-1511) vs. 108 days (range: 0-1550), respectively (p = 0.982)}. Reduced survival was documented in patients presenting with weight loss (P = 0.027), hypochromasia (MCHC <33 g/dL, P = 0.023) or leucocytosis (>15 × 109/L, P = 0.017) with a hazard ratio of 2.51 (CI95% = 1.071-6.018, P = 0.034), 2.71 (CI95% = 1.10-6.65, P = 0.03) and 4.39 (CI95%: 1.21-15.97, P = 0.025) respectively. In the dogs surviving more than 21 days, Ht <36% and leucocytosis >15.0 × 109/L at presentation were associated with reduced survival (p = 0.016, p = 0.021 respectively) and hazard ratio of 3.29 (CI95% = 1.18-9.2, P = 0.023) and 3.81 (CI95% = 1.15-12.55, P = 0.028) respectively. Intra-intervention-group survival analysis identified increased survival time in dogs receiving chemotherapy, but only within the surgical group (P = 0.02).The hospitalisation time of dogs undergoing ablation (median: 0 days, range: 0-4) was significantly shorter than dogs undergoing surgery (9 days, 1-21) (P < 0.001). In this study, no clear benefit was identified for surgery, thus when ablation is technically possible it should be considered advantageous, as hospitalisation time is significantly shorter. Weight loss, hypochromasia and leucocytosis were identified as long-term prognostic indicators at presentation.

Serratospiculosis in Captive Peregrine Falcons (Falco peregrinus) in Switzerland.

Infection with Serratospiculum species was identified in a captive peregrine falcon (Falco peregrinus) in Switzerland. Pathologic and parasitologic examination results revealed generalized severe granulomatous airsacculitis, with intralesional adults, larvae, and eggs of Serratospiculum species. Subsequently, an individual coprological analysis of the remaining 15 falcons (peregrine falcons and gyrfalcons [Falco rusticolus]) from the same owner was performed. Eggs of Serratospiculum species (4 birds) and Capillaria species (11 birds), and oocysts of Caryospora species (1 bird) were detected. Treatment with ivermection (2 mg/kg SC) was effective, as none of the falcons excreted Serratospiculum species eggs 10 days after one dose. To our knowledge, this is the first report of infection with Serratospiculum species in captive falcons in Europe.

Long-term outcome of transendoscopic oesophageal mass ablation in dogs with Spirocerca lupi-associated oesophageal sarcoma.

Oesophageal sarcoma is a potential sequel of Spirocerca lupi infection. Oesophageal mass excision can be performed by open chest surgery. The objectives of this observational study were to evaluate the feasibility, short-term morbidity and long-term outcome of transendoscopic oesophageal mass ablation in dogs with spirocercosis-associated oesophageal neoplasia. A 9 mm video-endoscope and laser or electrocauterisation were used to debulk the oesophageal mass. Long-term follow-up was done by telephonic interviews. Fifteen dogs were included. The median tumour size was 5 cm (range 3.5-9). The median procedure time was 75 minutes (range 35-165) and was deemed successful in 12/15 dogs (80 per cent). Recovery was uneventful in all dogs. Immediate complications included oesophageal damage (two dogs) oesophageal perforation (one dog) and a focal thermal damage (one dog). The median hospitalisation time of all dogs was less than one day, with all but two discharged on the procedure day. The median survival time, available in nine dogs that were followed, was 202 days (range 51-691). Four of these dogs (44 per cent) survived more than six months, of which three survived more than one year. In conclusion, transendoscopic oesophageal mass ablation might be considered an alternative, palliative procedure for open-chest oesophageal surgery. It has comparable long-term survival, lower morbidity, short hospitalisation time and relatively low cost.

Serum acute phase protein concentrations in dogs with spirocercosis and their association with esophageal neoplasia - a prospective cohort study.

Spirocerca lupi, the dog esophageal worm, typically induces formation of esophageal nodules, which may transform to sarcoma. Ante mortem discrimination between benign and malignant esophageal masses is challenging. Serum acute phase proteins (APPs) are utilized in diagnosis and prognosis of various canine diseases as markers of inflammation. This study characterized serum APPs concentrations in dogs with benign and malignant esophageal spirocercosis and evaluated their accuracy in differentiating benign from malignant lesions. Seventy-eight client-owned dogs with esophageal spirocercosis were included. Serum C-reactive protein (CRP), haptoglobin, serum-amyloid A (SAA) and albumin concentrations were measured upon diagnosis and follow-up visits, and compared with healthy dogs, and between malignant and benign cases. Haptoglobin, CRP and SAA concentrations were higher, and albumin concentration was lower (P<0.001 for all) in infected dogs compared to healthy controls. Dogs with suspected neoplasia had significantly higher CRP (P=0.011), haptoglobin (P=0.008) and SAA (P=0.05), and lower albumin (P=0.012) concentrations compared to dogs with benign esophageal nodules. APPs moderately discriminated between suspected malignant and benign esophageal disease. None of the dogs with suspected neoplasia had concurrent normal concentrations of all APPs. The present results indicate that canine spirocercosis is characterized by an acute phase reaction, both at presentation and during treatment. When concentrations of all four APPs are within reference range, esophageal malignancy is highly unlikely. Although concentrations of all positive APPs were significantly higher in suspected neoplastic cases compared to benign ones, moderate discriminatory power limits their clinical use. Neither APP was useful to monitor response to treatment.

Hypovitaminosis D in dogs with spirocercosis.

BACKGROUND: Spirocercosis in dogs is characterized by esophageal nodules that can undergo neoplastic transformation. Hypovitaminosis D has been associated with neoplasia formation. We hypothesized hypovitaminosis D in neoplastic spirocercosis and that it could be a risk factor for neoplastic transformation. OBJECTIVE: To measure and compare vitamin D status, assessed by serum 25-hydroxyvitamin D [25(OH)D] concentrations in non-neoplastic (n = 25) and neoplastic (n = 26) spirocercosis client-owned dogs and healthy dogs (n = 24). ANIMALS: Twenty-five non-neoplastic dogs, 26 neoplastic dogs, and 24 healthy dogs. METHODS: Fifty-one dogs were randomly selected from 119 dogs diagnosed with spirocercosis presenting to our hospital, and further divided into non-neoplastic or neoplastic groups. Exclusion criteria included dogs less than 1 year old, with concurrent diseases, received corticosteroids, or treated prophylactically for spirocercosis. Serum 25(OH)D concentration was measured by high-performance liquid chromatography. Spirocercosis dogs' appetites were graded and compared. RESULTS: Serum 25(OH)D concentrations were significantly different among all groups (P < .001). 25-Hydroxyvitamin D concentrations were significantly lower in neoplastic group (median 30.7 nmol/L [range 14.7-62.2]) compared to non-neoplastic (median 52.7 nmol/L [range 19.1-129.7, P < .05]) and healthy groups (median 74.6 nmol/L [range 37.4-130.5, P < .005]). 25-hydroxyvitamin D concentrations were significantly lower in non-neoplastic spirocercosis dogs compared to healthy ones (P < .05). Neoplastic and non-neoplastic spirocercosis dogs had similar appetite scores (P = 1.0). 25-Hydroxyvitamin D concentrations were not significantly different between dogs with normal (P = .087) and abnormal (P = .125) appetites within neoplastic and non-neoplastic spirocercosis groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Further studies are warranted to determine potential use of vitamin D treatment in spirocercosis and explore role of hypovitaminosis D in pathogenesis of malignant transformation.

Canine spirocercosis-associated extraskeletal osteosarcoma with central nervous system metastasis.

A five-year-old male Boerboel presented for examination, collapsed for an unknown period of time. On clinical examination, multifocal subcutaneous masses and enlarged prescapular lymph nodes as well as neurological deficits that suggested a multifocal neurological syndrome were found. Fine needle aspirates of the prescapular lymph nodes revealed cells suggestive of osteosarcoma. Radiographs showed foci of mineralisation within the soft tissue masses as well as diffuse pulmonary metastasis and a caudodorsal mediastinal mass believed to be a Spirocerca lupi nodule. Computed tomography imaging, necropsy and histopathology confirmed S. lupi oesophageal neoplastic transformation (extraskeletal osteosarcoma), believed to be the primary lesion, and the majority of secondary metastasis to the brain, spine, heart, multiple muscular groups and abdominal organs. This is the first known report of extraskeletal osteosarcoma metastasis to the brain and spinal cord in a dog.

Successful transendoscopic oesophageal mass ablation in two dogs with Spirocerca lupi associated oesophageal sarcoma.

This report describes two cases of oesophageal tumours managed by transendoscopic neodymium:yttrium-aluminum-garnet laser ablation and polypectomy snare electrocautery. In each dog oesophagoscopy revealed caudal oesophageal masses, suspected to be Spirocerca lupi-induced oesophageal neoplasia. To resect the masses, transendoscopic neodymium:yttrium-aluminum-garnet laser ablation was used in the first case and polypectomy snare electrocautery in the second. Recovery was uneventful. Histopathology was consistent with oesophageal fibrosarcoma and osteosarcoma in each case, respectively. Follow-up oesophagoscopy revealed apparently healthy oesophageal tissue except for focal scar tissue in the first case. Transendoscopic laser ablation and polypectomy snare electrocautery is a potential non-invasive, cost effective alternative for surgical oesophageal mass resection.

Computed tomographic and radiographic characteristics of aortic lesions in 42 dogs with spirocercosis.

Spirocerca lupi is a common cause of vomiting, regurgitation, and sudden death in dogs that live in tropical or subtropical regions. Sudden death due to aortic rupture may occur with no preceding clinical signs. The purpose of this prospective study was to compare radiographic and computed tomographic (CT) characteristics of aortic lesions in a cohort of 42 dogs with endoscopically confirmed spirocercosis. Dorsoventral and right lateral recumbent thoracic radiographic findings were compared with pre- and postcontrast thoracic CT findings. Aortic mineralization was detected using CT in 18/42 dogs (43%). Three dogs had faint diffuse aortic wall mineralization. Using CT as the reference standard, radiographs had a sensitivity and specificity of 6% and 96%, respectively, for detecting aortic mineralization. A total of 20 aortic aneurysms were detected using CT in 15/42 dogs (36%). Using CT as the reference standard, radiographs had a sensitivity and specificity of 86% and 56%, respectively, for detecting aortic aneurysms. Respiratory motion, aortic displacement by esophageal masses and Spirocerca nodules adjacent to the aorta mimicked aneurysm formation on radiographs. Aortic thrombi were seen in two dogs in postcontrast CT images. Findings from this study indicated that aortic mineralization and aneurysm formation are common in dogs with spirocercosis. Findings also supported the use of pre- and postcontrast CT as effective methods for detecting and characterizing these lesions.

Salivary gland enlargement and sialorrhoea in dogs with spirocercosis: a retrospective and prospective study of 298 cases.

This longitudinal cross-sectional clinical study investigated the incidence of sialorrhoea in dogs with spirocercosis and determined whether breed, body weight and the extent of the oesophageal involvement was associated with this presentation. A retrospective analysis was performed on the medical records of 233 dogs and information pertaining to 65 dogs was collected as part of a prospective study. All the animals were client-owned. Patients from the retrospective study underwent thoracic radiography or oesophageal endoscopy to diagnose and characterise the infection and were placed on therapy with a macrocyclic lactone, whereas the patients in the prospective study had both radiography and endoscopy routinely performed and biopsies of the oesophageal nodules collected where possible. Tru-cut biopsies of affected salivary glands were taken in 10 of 13 patients demonstrating clinical signs of sialorrhoea and salivary gland enlargement. The entire salivary gland was sectioned in an additional three dogs with spirocercosis and no sialorrhoea that were presented for post mortem examination. Sialorrhoea was present in 33/298 cases (11%). Fox terrier breeds were over-represented in the patients with sialorrhoea, comprising 36% of cases, whereas they only comprised 1.5% of the patients without sialorrhoea (p < 0.001, chi squared test) and 5% of the combined group. Dogs weighing 12 kg or less were significantly over-represented in the sialorrhoea group, 69% versus 19.5% (p < 0.001, chi square test). Age was not significantly different between the two groups (p < 0.08, Mann-Whitney test). The number of oesophageal nodules per case was significantly higher in the non-sialorrhoea cases (p = 0.048, Mann-Whitney test). The prevalence of distal oesophageal and lower oesophageal sphincter involvement, and neoplastic transformation of the nodules were not statistically different between the two groups. None of the fox terriers in either group showed neoplastic transformation of the parasitic nodules even though they were over-represented as a breed. Mandibular salivary glands were affected in 86% of cases showing sialorrhoea. Histopathology revealed acinar hyperplasia in all cases with concurrent necrosis detected in only two cases.Sialorrhoea and salivary gland enlargement has an incidence of 11% (33/298 cases) in canine spirocercosis. Small breeds (≤ 12 kg) and particularly fox terrier breeds are over-represented in the group demonstrating sialorrhoea and this appeared to be the only risk factor.The conclusion was that sialorrhoea secondary to canine spirocercosis occurs frequently and its presence should prompt further investigation for oesophageal and gastro-intestinal disease. Severely affected patients can be managed with phenobarbitone to control the dysphagia in addition to the routine macrocyclic lactones treatment.

Imaging diagnosis--spinal cord chondrosarcoma associated with spirocercosis in a dog.

A 7-year-old neutered female Boerboel cross was examined for progressive left pelvic limb lameness. There was no left patellar reflex but the remaining pelvic limb reflexes were hyperreflexic. Radiographically, there was a poorly mineralized opacity occupying the intervertebral foramen at LA-L5. On computed tomography images there was a hyperattenuating intramedullary lesion at LA-L5 that continued caudally, lateralized to the left and became extramedullary, terminating at L5-L6. In addition, well marginated, hyperattenuating lesions were noted at two muscular sites. The dog underwent euthanasia and a caudal esophageal mass was found at post mortem examination. The tumors in the spinal cord, the esophagus, and the skeletal muscles were diagnosed histologically as low-grade chondrosarcoma undergoing endochondral ossification. Spirocerca lupi-induced esophageal chondrosarcoma was believed to be the primary site from which the other, presumably metastatic, lesions originated.

Fatal aortic aneurysm and rupture in a neotropical bush dog (Speothos venaticus) caused by Spirocerca lupi.

This report details the post-mortem discovery of a larva of Spirocerca lupi in the caudal thoracic aorta of a 2-year, male bush dog (Speothos venaticus). This individual presented no clinical symptoms of the parasite's presence prior to its sudden death. The cause of death was determined to be acute bleeding following the rupture of an aneurysm in the caudal thoracic aorta as a result of the parasite located there. This is the first report of S. lupi in a bush dog.

Eosinophilic meningitis due to Angiostrongylus and Gnathostoma species.

Eosinophilic meningitis can be the result of noninfectious causes and infectious agents. Among the infectious agents, Angiostrongylus cantonensis and Gnathostoma spinigerum are the most common. Although angiostrongyliasis and gnathostomiasis are not common in the United States, international travel and immigration make these diseases clinically relevant. Both A. cantonensis and G. spinigerum infection can present as severe CNS compromise. Diagnoses of both infections can be challenging and are often clinical because of a paucity of serological assays readily available in the United States. Furthermore, there are conflicting recommendations about treatment for angiostrongyliasis and gnathostomiasis. To further explore the emerging nature of these helminthic infections, a case description and review of A. cantonensis and G. spinigerum infections are presented. The clinical severity of eosinophilic meningitis and diagnosis of these infections are highlighted.

Hypereosinophilia and abdominopulmonary gnathostomiasis.

A 16-year-old Thai male presented with sudden onset severe epigastric and right upper quadrant pain, fever (39 degrees C), chills and malaise. He gave no history of underlying disease, migratory swelling or urticarial skin rash. He had a history of frequently eating raw pork. Physical examination revealed a soft abdomen with markedly tender hepatomegaly. His blood count showed extreme leukocytosis with hypereosinophilia. After admission he developed a non-productive cough with left sided chest pain, a chest x-ray showed a left pleural effusion. Serological findings were positive for Gnathostoma larval antigen but not Fasciola antigen. The patient recovered completely after albendazole treatment. His clinical presentation is compatible with abdominopulmonary hypereosinophilic syndrome or visceral larva gnathostomiasis. The presented case is interesting not only for physicians who work in endemic areas of gnathostomiasis but also for clinicians who work in travel medicine clinics in developed countries, to consider abdominopulmonary gnathostomiasis when patients present with the signs and symptoms of visceral larva migrans.

Clinical differentiation between dogs with benign and malignant spirocercosis.

Spirocerca lupi is a nematode infesting the canine oesophagus, where it induces the formation of a nodule that may transform into a malignant sarcoma. The current, retrospective study compared the clinical presentation, haematology, serum albumin and globulin and radiology of benign cases (n=31) and malignant cases (n=31) of spirocercosis. Dogs with spirocercosis-induced sarcoma were significantly older (6.4+/-1.91 years) than benign cases (4.93+/-2.87). In the malignant cases there were significantly (p=0.03) more sterilized females (10/31) and fewer intact males (4/31) compared to 2/31 and 13/31, respectively, in the benign cases. Hypertrophic osteopathy was observed in 38.7% of malignant cases and in none of the benign cases (p=0.0002). Common clinical signs included weight loss, regurgitation, anorexia, pyrexia (T>or=39.5 degrees ), respiratory complications and salivation but did not differ in prevalence between groups. On haematology, the malignant group had significantly (p<0.05) lower haematocrit (0.34+/-0.08 vs. 0.41+/-0.07) and higher white cell count (31.6+/-27.83 vs. 17.71+/-13.18 x 10(3)microl(-1)), mature neutrophil count (26.06+/-26.08 vs. 12.23+/-9.96 x 10(3)microl(-1)) and thrombocyte count (493.15+/-151.61 vs. 313.27+/-128.54 x 10(9)microl(-1)). There were no differences in the mean corpuscular volume and immature neutrophil count. On radiology, the mass length was not significantly different, but the height and the width of the malignant masses were significantly larger (62.59+/-15.15 mm and 73.93+/-20.94 mm) compared to the benign group (46.43+/-23.62 and 49.29+/-25.56, respectively). Spondylitis was more prevalent in the malignant group (67.86% vs. 38.46%, p=0.03). Examining secondary pulmonary changes revealed significantly higher prevalence of bronchial displacement in the malignant group (52% vs. 17%, p=0.008). Hypertrophic osteopathy appeared to be a very specific but relatively rare (poor sensitivity) marker of malignancy. Female gender, anaemia, leukocytosis, thrombocytosis, spondylitis and bronchial displacement are significantly more common in malignant cases, but appear in benign cases as well. However, if found together in a specific case, they should increase the index of suspicion for malignancy in a diagnosed spirocercosis case.

Imaging diagnosis--aortic thromboembolism associated with spirocercosis in a dog.

An 8-year-old neutered female Rhodesian Ridgeback developed acute, nonneurological right pelvic limb lameness. Femoral pulsation was poor, and oscillometric blood pressure measurements between the two pelvic limbs differed markedly. A caudal aortic right external iliac embolus was detected sonographically. Radiographically, there was a caudal esophageal mass and thoracic vertebral spondylitis typical of spirocercosis. Using CT-angiography, a caudal thoracic aortic aneurysm with a mural thrombus was detected. The dog recovered following heparin and aspirin therapy but signs recurred 7 months later. Subsequently, the patient improved on treatment and remains asymptomatic. This report illustrates the value of CT-angiography in detecting aortic thrombosis in dogs with spirocercosis.

Spirocercosis-associated pyothorax in dogs.

The records of five dogs diagnosed with spirocercosis-associated pyothorax were retrospectively reviewed. On admission, the most common clinical findings were weakness, anorexia, depression, dyspnoea and fever. All dogs had typical oesophageal Spirocerca lupi granulomas. Contrast thoracic radiography revealed contrast material leakage from the oesophagus into the pleural cavity in two dogs only. All dogs were medically treated with broad-spectrum antibiotics, repeated chest tube drainage and thoracic lavage, and four of the dogs were treated with doramectin. One dog was euthanased due to a concurrent non-related disease at the owners request while the remaining four survived. S. lupi infection should be included in the differential diagnoses of canine pyothorax in endemic areas. Conservative medical management of pyothorax with antihelmintic medication was shown to be effective in the treatment of S. lupi-associated pyothorax.

Orbital apex syndrome from gnathostomiasis.

A 16-year-old Thai girl presented with acute unilateral visual loss, proptosis, and ophthalmoparesis. CT demonstrated thickening and enhancement of orbital tissues including the orbital apex. A history of consumption of raw fish, together with the findings of cutaneous migratory swelling and eosinophilia, made the diagnosis of gnathostomiasis likely. Her serum was positive for Gnathostoma spinigerum using an immunoblotting technique. Parasites removed from the skin lesions revealed the typical head bulbs with 4 circumferential rows of hooklets and fine cuticular spines on their surface. Treatment with an antihelminthic and systemic corticosteroids led to resolution of orbital inflammation but left a persistent optic neuropathy marked by nerve fiber bundle visual field loss with normal visual acuity. Gnathostomiasis should be suspected in patients with an orbital apex syndrome who live or travel in an endemic area, have eaten raw fish, and develop a migratory skin rash.

Outward migration of Gnathostoma spinigerum in interferon alpha treated hepatitis C patient.

After the first dose injection of pegylated interferon alpha-2b (Peg-IFN alpha-2b) to a HCV infected Thai woman, she developed cyclic painful swelling nodules on right upper quadrant of abdomen and right anterior lower chest wall. The nodules subsided spontaneously within 1-2 days but were recurrent after every Peg-IFN alpha-2b injection. She also experienced acute urticaria. After nine months of therapy, an immature male of G. spinigerum migrated out from the skin nodule shortly after a Peg-IFN alpha-2b injection as scheduled. The worm showed a head-bulb bearing 8 transverse rows of spines which indicated immature stage. It had well defined four pairs of caudal papillae on posterior body part which were used to identify male gender. Painful migratory swelling and urticaria disappeared after the parasite was removed. She was continually treated and had sustained both virological and biochemical responses to HCV treatment. This case demonstrates that the outward migration of G. spinigerum may be stimulated by the injection of Peg-IFN alpha-2b.