Measurement of intestinal mucosal permeability in dogs with lymphocytic-plasmacytic enteritis

Lymphocytic-plasmacytic enteritis (LPE) is a type of canine inflammatory bowel disease (IBD). One of its most probable causes is a defect in the mucosal permeability barrier. In the present study, intestinal permeability in LPE dogs was examinated to evaluate its clinical value. Twenty-nine dogs with LPE diagnosed by clinical and histological examinations were included in this study. Intestinal permeability was evaluated by measuring the ratio of the concentrations of two sugars (lactulose (L) and rhamnose (R)) with different molecular weights in urine samples after oral administration of a solution containing them. Biopsy specimens of duodenum were evaluated according to histological criteria. The urinary L:R ratio in the 29 LPE dogs (1.68 +/- 1.17, mean +/- SD) was significantly higher than that in the 10 healthy control dogs (0.75 +/- 0.38, P<0.01). In the LPE dogs, a weak correlation was observed between the histopathological grading score of the duodenum and the urinary L:R ratio (r=0.408, P<0.05). The urinary L:R ratio in the 20 dogs showing hypoalbuminemia (< 2.5 g/dl) was significantly higher than that in the 9 dogs with normal serum albumin levels > 2.5 g/dl (P<0.01). In conclusion, permeability of the intestinal mucosa as determined by the urinary L:R ratio could be a useful laboratory parameter for evaluating intestinal damage in LPE dogs.     

Serum lipase activity in young dogs with acute enteritis or gastroenteritis

Blood serum lipase activity was determined in 48 young dogs with acute enteritis or gastroenteritis due to canine parvovirus (16 Cases) and presumably to other infectious agents (32 cases). Elevated serum lipase activity (> 500 U/L) was found in 13 dogs (27.1 %) with values ranging from 800 to 2,780 U/L. The hyperlipasemia of these cases may be attributed to acute pancreatitis secondary to acute gastroenteritis or to gastrointestinal upset.     

Dietary inclusion of arabinoxylo-oligosaccharides in response to broilers challenged with subclinical necrotic enteritis

1. This study investigated the prebiotic properties of arabinoxylo-oligosaccharides (AXOS) produced both in situ and in vitro for their activity against the onset of necrotic enteritis in broiler chickens.

2. A 2 × 3 factorial arrangement was applied, including necrotic enteritis challenge (challenged/unchallenged) and three dietary treatments from d 10 to 21. A wheat–soy commercial-type basal-grower diet was fed with 2% of the wheat proportion replaced by the same amount of either arabinoxylan (AX), AXOS produced from hydrolysing AX with 16 000 BXU (birch xylanase unit) xylanase in vitro or AX fed with 16 000 BXU xylanase (AX + E). Necrotic enteritis (NE) challenge was induced by orally infecting birds with a vaccine strain of Eimeria oocysts at d 9 of age followed by oral gavage of a freshly prepared Clostridium perfringens broth at d 14.

3. The challenge depressed growth performance, induced gross lesions and reduced ileal viscosity at d 10–21. Birds fed on the AXOS diet had numerically less severe gross lesions, improved feed conversion at d 0–16 and lower ileal viscosity at d 16 compared to birds fed on AX. Weight gain of the unchallenged birds ranked as follows in terms of the diets: AXOS > AX + E > AX. AX + E produced a lower ileal viscosity compared to the AX treatment but only led to marginal improvements in performance and intestinal lesion scores.

4. Caecal short-chain fatty acid (SCFA) concentration was higher in birds fed on AXOS and AX + E compared to those fed on AX and was higher in the challenged birds compared to the unchallenged birds. Gizzard pH was lower in birds fed on AX + E compared to those fed on AXOS at d 16. Challenged birds had lower ileum pH compared to the unchallenged birds at d 16 and 21.

5. Results of this study suggest that AXOS appeared to be efficacious prebiotics, as highlighted by improvements in feed conversion ratio and increased SCFA production. Future studies are warranted to elucidate the types of AXOS that are most active against NE and the mechanisms by which different levels of AXOS enhance bird performance.     

Characterization of mast cell numbers and subtypes in biopsies from the gastrointestinal tract of dogs with lymphocytic-plasmacytic or eosinophilic gastroenterocolitis

It has been suggested but not proven that hypersensitivity type I reactions are involved in the pathogenesis of canine inflammatory bowel disease (IBD). The main effector cells in type I hypersensitivity reactions are mast cells (MCs). Canine MCs, as human MCs, can be subdivided into three subtypes according to their content of mast cell-specific proteases: tryptase (MC_T), chymase (MC_C), or tryptase and chymase bearing MCs (MC_TC). In this study, numbers and subsets of mast cells were investigated in biopsies from the gastrointestinal tract of dogs with histopathologically confirmed lymphocytic-plasmacytic enteritis (LPE) (n = 4), lymphocytic-plasmacytic colitis (LPC) (n = 1) and eosinophilic gastroenterocolitis (EGE) (n = 11). Paraffin sections of formalin-fixed samples from the stomach, small intestine (duodenum, jejunum, ileum) and colon were stained by using a metachromatic staining method (kresylecht-violet; KEV) and a combined enzyme histochemical and immunohistochemical technique for chymase and tryptase. Additionally, immunohistochemistry with antibodies against T cells (CD3), macrophages (myeloid/histiocyte antigen) and IgA, IgG and IgM bearing cells was conducted. Quantitative evaluation of mast cells and semiquantitative scoring of immunohistochemically stained cells were performed. Between the two histopathologically defined groups clear differences concerning mast cell numbers were detected. In most affected intestinal tissue locations of dogs with LPE/LPC a decrease in metachromatically (kresylecht-violet) stained granule-containing MCs and immunohistochemically stained MC_T,C,TC was found. This reduction could be due to mast cell degranulation, a T helper cell 1 dominated reaction pattern or a “thinning out” due to increasing T cells, IgA and IgG bearing cells. Dogs with EGE displayed higher variability in mast cell numbers but most of the affected large and small intestinal locations had increased numbers of MCs. In these cases, T cells, IgA bearing cells and macrophages also increased. Increased numbers of MCs and eosinophils seen in the intestinal mucosa of dogs with EGE could indicate the presence of a type I hypersensitivity reaction (T helper cell 2 pattern) in response to dietary antigens. Changes in cell numbers occurred also in unaffected locations of dogs with LPE/LPC and EGE which showed reduced MC_T,C,TC, increased KEV positive cells and partially increased leucocytes and macrophages.     

Occurrence of canine parvovirus type 2c in the dogs with haemorrhagic enteritis in India

Canine parvovirus 2 (CPV-2) causes a highly contagious and often fatal disease in dogs. Since its sudden emergence in the early 1970s, CPV-2 has been evolving through the generation of novel genetic and antigenic variants (CPV-2a/b/c) that are unevenly distributed throughout the world. In the present study we have examined 36 clinical cases of dogs suspected of CPV collected during year 2006. A fragment of the VP2 gene of the virus was analyzed using polymerase chain reaction (PCR), restriction endonuclease (RE) and DNA sequence analysis. Out of the 36 samples analyzed, 16 were found positive for CPV-2a/2b by conventional PCR. DNA sequencing was done for 6 PCR positive samples, out of which three were characterized as CPV-2c, indicating that this CPV type 2c is currently circulating in India.     

Association between intestinal lymphangiectasia and expression of inducible nitric oxide synthase in dogs with lymphoplasmacytic enteritis

Intestinal lymphangiectasia (IL) is a common complication in dogs. Since nitric oxide (NO) is known to relax the lymphatic vessel, we evaluated inducible NO synthase (iNOS) expression using immunohistochemistry in 13 dogs with lymphoplasmacytic enteritis (LPE) with or without IL. The duodenal iNOS expressing cells were significantly increased in dogs with IL-negative or IL-positive LPE dogs (P=0.025, P=0.007) compared with control dogs. However, there was no significant difference in iNOS expression between IL-positive and IL-negative tissues. Based on these results, there is no clear evidence for the NO overproduction in the pathogenesis of IL in dogs with LPE. Factors other than NO could, thus, contribute to IL in dogs with LPE.     

Ehlers-Danlos-like syndrome in 2 dogs: clinical,histologic, and ultrastructural findings

Background: Ehlers-Danlos syndrome comprises a group of rare inherited connective tissue diseases characterized by skin hyperextensibility, joint laxity, skin and vessel fragility, and poor wound healing.
Objective: The purpose of this report was to describe the clinical, histologic, and ultrastructural findings in 2 dogs with collagenopathies consistent with Ehlers-Danlos syndrome.
Methods: Two dogs were examined clinically; skin extensibility index was calculated. Skin biopsies obtained from the dorsum were examined by light and electron microscopy.
Results: Both dogs had clinical signs of skin hyperextensibility and fragility, lower skin elasticity, vessel fragility, and poor wound healing. One dog had a hip dislocation, and the other had bilateral medial patellar luxation (grade II), subcutaneous hematomas produced by minimal trauma, and generalized periodontitis. Histologic and ultrastructural examination confirmed abnormalities in the structure and arrangement of collagen fibrils. Fibroblasts were characterized by variable dilatation of the rough endoplasmic reticulum, and anomalous elastic fibers (elaunin fibers) were present in the dermis.
Conclusion: Although the primary defects underlying collagenopathies in animals are still unknown, analysis of the ultrastructural changes in collagen fibrils and clinical findings could facilitate better characterization of these disorders in dogs.     

Evaluation of the circadian rhythm of anti-Leishmania IgG2 and IgA antibodies in serum and saliva of dogs with clinical leishmaniosis

In this study, the circadian rhythm of IgG2 and IgA specific antibodies in serum and saliva samples of 6 dogs experimentally infected with Leishmania infantum was assessed. Sampling was performed at 8.00, 12.00, 16.00, 20.00, and 00.00 h on two consecutive days. Anti-Leishmania antibody levels in serum were expressed without any correction, whereas in saliva were shown in different ways: without any correction, adjusted by protein concentration and corrected by the salivary flow rate. No significant differences in anti-Leishmania IgG2 antibody levels in serum and saliva samples with or without correction were found. Significant differences were found when anti-Leishmania IgA levels were corrected by the salivary flow rate. In addition, a greater intra-individual variation of antibody levels was observed in saliva than in serum. However, this variation did not modify the serological status of the dogs. Therefore, it could be concluded that there is no circadian rhythm in serum and saliva samples and sampling can be performed at any time of the day.     

Serum leptin and ghrelin levels in response to methylprednisolone injection in healthy dogs

The aim of this study was to investigate the effects of methylprednisolone treatment on serum leptin and ghrelin levels in healthy dogs (n=40). After 14 h of fasting, the dogs were injected intramuscularly with saline (control group) or methylprednisolone (1, 5 or 10mg/kg). Blood samples were collected prior to (baseline) and 2, 3, 4, 8, 12 and 24h subsequent to the treatments. Serum leptin and ghrelin were measured by radioimmunoassay. The mean baseline serum leptin and ghrelin were 2.5+/-0.1 ng/mL (n=40) and 35.0+/-2.1 pg/mL (n=40), respectively. In the control dogs, serum leptin, but not ghrelin levels showed a significant fluctuation during the 24h observation period. Serum leptin increased significantly (p<0.05-0.01) between 2 and 12h after 1mg/kg of methylprednisolone. Serum leptin levels showed biphasic response to 5mg/kg of methylprednisolone: its level decreased to 1.9+/-0.1 ng/mL (p<0.01) at 2h and increased at 12h (2.6+/-0.1 ng/mL) (p<0.01). In response to 10mg/kg of methylprednisolone, serum leptin levels decreased significantly (p<0.01) for 24h. Serum ghrelin levels decreased to 19+/-5 pg/mL at 2-3h (p<0.01) or increased to 87+/-18 pg/mL at 3-8h (p<0.05-0.01) after 1mg/kg of methylprednisolone or 10mg/kg of methylprednisolone, respectively. Serum ghrelin levels did not change at any time point during 24h observation period after 5mg/kg of methylprednisolone. There was a significant (p<0.001) inverse correlation (r=-0.635) between serum leptin and ghrelin levels. In conclusion, we found that methylprednisolone increases or decreases serum leptin and ghrelin levels depending upon its dose and there is a negative correlation between serum leptin and ghrelin levels after methylprednisolone administration.     

Evaluation of serum cystatin-C in dogs with visceral leishmaniasis

Serum Cystatin C (sCys-C) is one of the most important serum markers of renal function assessment in dogs. The purpose of this study was to determine the sCys-C concentration in dogs with visceral leishmaniasis (VL). In the study, 16 dogs with VL and 10 clinical healty dogs (control) were used. Mean sCys-C concentration of the infected dogs was significantly higher than that of the control group (p < 0.05). Mean serum creatinine concentration was lower and mean blood urea nitrogen, albumin and globulin concentrations were higher in dogs with VL; however, these changes were not statistically significant. Mean total protein and phosphorus concentrations were found to be higher in dogs with VL than healthy dogs (p < 0.05). No significant correlation had been determined between sCys-C and other variables. Visceral leishmaniasis in dogs has increased sCys-C concentration indicating a possible renal impairment; however, further studies are needed to be performed together with renal biopsies in the investigation sCys-C in dogs with VL.     

Plasma and urinary trypsinogen activation peptide in healthy dogs, dogs with pancreatitis and dogs with other systemic diseases

OBJECTIVE: To determine the specificity and sensitivity of plasma and urinary trypsinogen activation peptide (TAP) concentrations in diagnosing pancreatitis in dogs. DESIGN: Retrospective analysis of clinical cases. PROCEDURE: Dogs were classified into three groups: healthy animals, dogs with confirmed pancreatitis and dogs with nonpancreatic disease, which clinically or biochemically resembled pancreatitis. This last group was further subdivided into dogs with renal and those with nonrenal disease. The plasma and urinary TAP concentration was determined by a competitive enzyme immunoassay. Clinical cases additionally had serum trypsin-like immunoreactivity concentration measured, as well as radiography and ultrasound of the abdomen and further diagnostic procedures. Nonparametric analysis of variance (Kruskal-Wallis test) was performed using Statistix 4.0 program. RESULTS: There was a wide range of urinary TAP concentration in healthy dogs (mean 52.30 nmol/L, standard deviation 55.25) that made interpretation of urinary TAP concentrations difficult in the other groups. There was a narrow reference range for plasma TAP (mean 2.67 nmol/L, standard deviation 0.93). Plasma and urinary TAP concentrations, as well as urinary TAP to creatinine ratio, were all increased in dogs that died with necrotising pancreatitis. Values were not increased in mild, interstitial pancreatitis. Increased plasma TAP concentrations were also present in dogs with severe renal disease. CONCLUSION: Plasma TAP concentration is a good prognostic indicator in naturally occurring pancreatitis in dogs. The failure of TAP to increase in mild pancreatitis, and the increase present in severe renal disease, suggests its measurement has limited application as a sole diagnostic tool for canine pancreatitis. Further investigations are required in order to explain the large variability of urinary TAP concentration and the presence of circulating TAP in healthy dogs.     

Evaluation of kidney injury in dogs with pyometra based on proteinuria, renal histomorphology, and urinary biomarkers

Background: Proteinuria is a feature of pyometra‐associated renal dysfunction, but its prevalence and clinical relevance are not well characterized. Objectives: To define which subset of dogs with pyometra has clinically relevant kidney injury by quantification of proteinuria; light, immunofluorescence, and electron microscopic examination of kidney biopsy specimens; and measurement of urinary biomarkers. Animals: Forty‐seven dogs with pyometra. Ten clinically healthy intact bitches of comparable age. Methods: Prospective study. Routine clinicopathological variables including urinary protein to creatinine ratio (UPC) were analyzed. Validated assays were used to quantify urinary biomarkers for glomerular (urinary albumin, urinary immunoglobulin G, urinary C‐reactive protein, urinary thromboxane B₂) and tubular function (urinary retinol‐binding protein, urinary N‐acetyl‐β‐d ‐glucosaminidase). Kidney biopsy specimens from 10 dogs with pyometra and dipstick urine protein concentrations of 2+ or 3+ were collected during ovariohysterectomy. Urinalysis was repeated within 3 weeks after surgery in 9 of the 10 dogs. Results: UPC (median, range) was significantly higher in dogs with pyometra (0.48, 0.05–8.69) compared with healthy bitches (0.08, 0.02–0.16) (P < .01). Twenty‐two of 47 dogs with pyometra had UPC>0.5, 12 had UPC>1.0, and 7 had UPC>2.0. Glomerulosclerosis and tubulointerstitial nephritis were common kidney biopsy findings in proteinuric dogs with pyometra. Dogs with glomerulosclerosis (5/10), either global or focal and segmental, had UPC>1.0 at ovariohysterectomy and afterward. Dogs with structural glomerular and tubular changes mostly had urinary biomarker to creatinine ratios above the 75th percentile. Conclusion: Dogs with pyometra and UPC>1.0 or high ratios of urinary biomarkers appear likely to have clinically relevant renal histologic lesions and require monitoring after ovariohysterectomy. Future studies should evaluate the role of pyometra‐associated pathogenic mechanisms in causing or exacerbating focal and segmental glomerulosclerosis in dogs.     

中药肠炎康治疗猪大肠杆菌病的临床试验

[摘 要] 为了验证中药肠炎康对猪大肠杆菌的临床效果,对猪人工感染大肠杆菌进行治疗性试验。利用大肠杆菌O301对试验组猪进行人工感染后,分为肠炎康低、中、高,庆大霉素治疗试验。结果表明拌入饲料肠炎康（拌料10g/kg ,连用3d）治疗猪大肠杆菌病疗效可靠,效果明显。对人工感染引起的猪大肠杆菌病治愈为86.7%,有效率100%,并能显著降低猪死亡率。     

Characterisation of the inflammatory reaction in equine idiopathic focal eosinophilic enteritis and diffuse eosinophilic enteritis

REASONS FOR PERFORMING STUDY: Idiopathic focal eosinophilic enteritis (IFEE) and diffuse eosinophilic enteritis (DEE) are primary eosinophilic intestinal conditions without a known cause that are associated with an increasing number of surgical colic cases. Histology may be helpful in defining disease aetiology and pathogenesis. OBJECTIVES: To characterise further the inflammatory infiltrate in equine IFEE and to compare the condition with DEE. METHODS: Twenty-three IFEE cases and 5 DEE cases were examined by light microscopy including immunohistology to identify infiltrating leucocytes. Inflammatory infiltrates in mucosa and submucosa were characterised in IFEE lesions (Group 1), the intestine distant from the lesions in IFEE (Group 2) and DEE (Group 3). RESULTS AND CONCLUSIONS: IFEE lesions represented an accumulation of leucocytes in submucosa and muscularis, with dominance of eosinophils and macrophages and smaller numbers of lymphocytes, plasma cells and neutrophils. T cells represented the dominant lymphocytes. The mucosa overlying the lesion and both mucosa and submucosa in IFEE nonlesion sites and in DEE exhibited a similar composition, with different prevalence of various cell types. Macrophages were significantly more prevalent in the mucosal and submucosal infiltrates in IFEE nonlesion sites than in DEE, and lymphocytes significantly more prevalent in the mucosa in DEE than in IFEE nonlesion sites. The findings confirm IFEE as a primary eosinophilic intestinal disorder and indicate that IFEE represents a focally exacerbated inflammatory reaction in horses with DEE, possibly due to functional changes in the macrophage and T cell components, with subsequent excessive recruitment of both eosinophils and macrophages.     

Dynamics of Leishmania-specific immunoglobulin isotypes in dogs with clinical leishmaniasis before and after treatment

Concentrations of Leishmania-specific immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin A (IgA) isotypes were analyzed by enzyme-linked immunosorbent assay (ELISA) in 23 dogs naturally infected with Leishmania infantum before and 1 year after initiating drug therapy. Results showed a high expression and prevalence of Leishmania-specific IgG (176.4 +/- 89 ELISA units [EU]), IgM (105.3 +/- 95.5 EU), and IgA (153.6 +/- 98 EU) in dogs before treatment (median +/- interquartile range EU). One year after treatment was started, dogs were classified as responsive dogs (RDs; n = 13) or unresponsive dogs (UDs; n = 10) based on clinicopathologic findings. Both groups of dogs experienced a statistically significant decrease (P < .05) in Leishmania-specific IgG (RDs = 27%, UDs = 41%), IgM (RDs = 42%, UDs = 29%), and IgA (RDs = 56%, UDs = 46%). Concentrations of specific IgG and IgM were not different at diagnosis or after treatment between the 2 groups. However, the median value for Leishmania-specific IgA 1 year after treatment was significantly lower (P < .05) in RDs (60.8 +/- 67 EU) than in UDs (117 +/- 54 EU). Examination of our data indicates that both the IgA isotype, which is mostly produced by mucosal plasma cells, and the IgM isotype are increased in infected symptomatic dogs, as previously reported for IgG. These 3 isotypes decreased significantly 1 year after initiation of medical treatment.     

Parvoviral enteritis and salmonellosis in raccoons with sudden death

Eight of 9 juvenile raccoons at a rehabilitation center died without obvious prior clinical signs. Gross changes were unremarkable except for mildly distended intestines. Microscopically, crypt loss, distension, necrosis, and regeneration with intranuclear viral inclusions were observed in the small intestine, with marked lymphoid depletion and necrosis in Peyer patches and mesenteric lymph nodes. Immunohistochemistry with a canine parvovirus antibody showed intensive signals of parvoviral antigens in the crypts and lymphoid germinal centers. Metagenomic sequencing allowed assembly of a complete parvoviral genome with >99% identity to canine parvovirus 2a, as well as Salmonella enterica subsp. enterica. Also, S. enterica subsp. enterica serovar Thompson with multiple antimicrobial resistance was isolated from the intestinal contents. Concurrent infection with parvovirus and Salmonella should be included as a differential diagnosis in raccoons with sudden death.     

Ketoconazole in the treatment of dermatomycosis in cats and dogs

Ketoconazole (Nizoral^®, Janssen Pharmaceutica) a new systemic antimycotic was tested in dermatomycosis in cats and dogs. The daily dose (10 mg/kg body weight) was administered for 10 or 20 days without any other measures being taken. After the 20‐day treatments new hair growth was observed in 96.7 per cent of the cats and 89.9 percent of the dogs. Clinical cure was complete in 96.8 per cent of the cats and 90.5 per cent of the dogs.

Particularly the good tolerance in the cat was appreciated. There were practically no side‐effects in dogs (except vomiting in two pups) or in cats.     

Serum cobalamin concentrations in dogs with leishmaniosis before and during treatment

Hypocobalaminemia in dogs is most commonly associated with gastrointestinal disorders leading to impaired absorption and utilization of cobalamin. The objectives of this study were to compare serum cobalamin concentrations between dogs with leishmaniosis and clinically healthy dogs, and to assess possible alterations of serum cobalamin concentrations in dogs with leishmaniosis at different timepoints during treatment. Fifty-five dogs with leishmaniosis and 129 clinically healthy dogs were prospectively enrolled. Diagnosis of leishmaniosis was based on clinical presentation, positive serology and microscopic detection of Leishmania amastigotes in lymph node aspiration smears. Twenty of the dogs with leishmaniosis were treated with a combination of meglumine antimonate and allopurinol for 28 days and serum cobalamin concentrations were measured in blood samples that were collected before initiation of treatment (timepoint 0) and on days 14 and 28. In order to estimate alterations of serum cobalamin concentrations during treatment, cobalamin concentrations were measured in blood samples from 20 out of 55 dogs with leishmaniosis at all timepoints. Serum cobalamin concentrations were significantly lower in dogs with leishmaniosis before treatment (median: 362 ng/L; IQR: 277−477 ng/L) compared to clinically healthy dogs (median: 470 ng/L; IQR: 367−632 ng/L; P = 0.0035). Serum cobalamin concentrations increased significantly in dogs with leishmaniosis on day 14 of treatment compared to timepoint 0 (P = 0.02).In the present study, serum cobalamin concentrations were significantly lower in dogs with leishmaniosis compared to clinically healthy dogs. In addition, there was an increase in serum cobalamin concentrations during treatment. The clinical significance of hypocobalaminemia in dogs with leishmaniosis remains to be determined.     

Clinical signs,diagnosis and treatment of three dogs with angiostrongylosis in Ireland

: Infection with Angiostrongylus vasorum was diagnosed at necropsy on a dog that died from acute pulmonary haemorrhage, and on recovery of L1 larvae by Baermann examination of faeces from two dogs, one of which had abdominal pain and retroperitoneal haemorrhage, while the other had right-sided heart failure due to cor pulmonale. The presenting signs included syncope (one dog), exercise intolerance (two dogs), cough (two dogs), abdominal pain (one dog) and depression (one dog). One-stage prothrombin time and activated partial thromboplastin time were prolonged in two dogs, buccal mucosal bleeding time was prolonged in one dog and globulin was elevated in all three dogs. Two dogs were treated with fenbendazole and recovered.