Disseminated microsporidiosis due to Encephalitozoon hellem in an Egyptian fruit bat (Rousettus aegyptiacus).

Disseminated microsporidiosis was diagnosed in an adult female Egyptian fruit bat that died unexpectedly in a zoo. Gross findings, which were minimal, included poor body condition, bilateral renomegaly, and mottling of the liver. Histopathological lesions, which were particularly pronounced in the urogenital tract and liver, consisted primarily of inflammation associated with intracytoplasmic microsporidian spores. Polymerase chain reaction -based methods were used to establish the identity of the microsporidian as Encephalitozoon hellem. E. hellem is an emerging cause of human and avian disease, manifested mainly as opportunistic infection in immunosuppressed patients. This report describes the first documented case of E. hellem in a non-human mammalian species.

Field investigation of Mycoplasma gallisepticum infections in house finch (Carpodacus mexicanus) eggs and nestlings.

We conducted a field study to investigate the occurrence of Mycoplasma gallisepticum (MG) in eggs and nestlings from nests of house finches (Carpodacus mexicanus). Forty-three nests were located between the months of April and August 1998 and were followed with one to three sampling efforts. Vitelline membrane of fresh eggs, whole embryos, or swabs from the choanal cleft or conjunctiva of nestlings were inoculated into mycoplasma broth for MG isolation and polymerase chain reaction (PCR) testing. No isolation of MG was made from 39 eggs or 110 nestlings sampled during the study. Pooled choanal and conjunctival swab samples from two broods of nestlings, however, tested positive for MG by PCR. None of the nestlings examined showed clinical signs of conjunctivitis, and no nestling mortality could be linked to MG infection. Serologic tests from 37 older nestlings were negative for antibodies to MG. The results suggest transmission of MG is occurring between breeding adults and their dependent offspring (pseudovertical transmission). Evidence supporting transovarian transmission of MG was not found in these house finches.

Bacteremia and vegetative endocarditis associated with a heart murmur in a blue-and-gold macaw.

A 6-year-old male blue-and-gold macaw (Ara ararauna) was presented with severe weakness, anorexia, and weight loss of 2 weeks duration. Cardiac auscultation revealed a soft systolic murmur. Blood cultures collected both antemortem and postmortem yielded pure isolates of Enterobacter cloacae. At necropsy, vegetative endocarditis was found involving the left atrioventricular valve. Microscopically, the lesion on the valve was characterized by a mixture of necrotic material, colonies of gram-negative bacteria, fibrosis, and inflammatory infiltrate consisting primarily of heterophils.

Subcutaneous filariasis (Pelecitus sp.) in a yellow-collared macaw (Ara auricollis).

Clinical manifestations of subcutaneous filariasis in a yellow-collared macaw (Ara auricollis) included lameness induced by subcutaneous nodule formation, which was attributed to the presence of the filarial parasite Pelecitus sp. Following anthelmintic and corticosteroid therapeutic failure, the problem was resolved by surgically removing the adult worms.

Comparative cardiopulmonary effects of intramuscularly administered etorphine and carfentanil in goats.

OBJECTIVE: To determine comparative cardiopulmonary effects of IM administered etorphine and carfentanil in goats. ANIMALS: Seven clinically normal adult female goats. DESIGN: Each goat received at least 9 drug treatments (etorphine HCl, 5 [twice], 10, 20, and 40 and carfentanil citrate, 5, 10, 20 and 40 micrograms/kg of body weight), with a minimal 2-day interval between trials. Although drug dosages were randomized, etorphine and carfentanil treatments were alternated. To assess for drug tolerance, the first and last treatments always were etorphine (5 micrograms/kg). PROCEDURE: All goats were instrumented for long-term cardiopulmonary variable data collection. RESULTS: Both drugs induced rapid catatonic immobilization, characterized by limb and neck hyperextension, with occasional vocalization and bruxation. Etorphine elicited transient violent struggling and vocalization immediately. Time to immobilization appeared dose-dependent, and was more rapid with carfentanil (< or = 5 minutes) than etorphine (5 to 10 minutes) at all dosages. Recovery to standing occurred earlier for etorphine (1 to 2 hours) than carfentanil (> 2 hours) at all dosages. Both drugs at all dosages significantly (P < or = 0.05) increased systemic and left ventricular (LV) end-diastolic pressures, LV peak negative dP/dt, total peripheral resistance (TPR), hemoglobin concentration, and left atrial (LA) and pulmonary O2 contents. They also significantly decreased heart and respiration rates, and TPR. A significant increase was observed at some dosages for LV stroke volume and index, LV peak positive dP/dt, mean pulmonary artery pressure, PaO2, pulmonary artery oxygen partial pressure, PaCO2, pulmonary mixed venous carbon dioxide partial pressure, LA hemoglobin saturation, LA transport index, and body temperature. Pulmonary and systemic mixed venous carbon dioxide and oxygen contents were significantly decreased at some dosages. CONCLUSIONS: Intramuscularly administered etorphine and carfentanil induce hypertension, bradycardia, and bradypnea in goats. The hypertension appears attributable to an increase in TPR. CLINICAL RELEVANCE: Although the cardiopulmonary effects of carfentanil occurred more rapidly, these effects were similar in magnitude for etorphine and carfentanil over the evaluated dosage range.

Development of monoclonal antibodies for measurement of immunoglobulin G antibody responses in blue and gold macaws (Ara ararauna).

OBJECTIVES: To produce monoclonal antibodies (MAB) with specificity for the heavy chain of macaw IgG; to incorporate these MAB into an ELISA to measure IgG responses of macaws inoculated with bovine serum albumin (BSA); and to evaluate the antigenicity of BSA in Blue and Gold Macaws. ANIMALS: Four 1-year-old Blue and Gold Macaws, 2 males and 2 females. PROCEDURE: 1 male and 1 female 1 were randomly assigned to each of 2 study groups. Group-1 birds were inoculated with 200 micrograms of BSA on days 0, 14, 28, and 42. Group-2 birds were inoculated with 200 micrograms of BSA on days 0 and 28. Blood was collected weekly for measurement of anti-BSA titer. Hybridomas were prepared from mice immunized with Scarlet Macaw (Ara macao) IgG purified by salt precipitation and gel chromatography. Specificity for IgG of Scarlet Macaw and other macaw species was confirmed by ELISA and western blot analysis. Hybridoma HL544 was cloned and the antibody purified. Following biotinylation, MAB HL544 was incorporated into an ELISA that measured IgG responses of macaws inoculated with BSA. RESULTS: Adult Blue and Gold Macaws developed strong primary and secondary anti-BSA antibody titers 14 days after inoculation with 200 micrograms of BSA. An inoculation interval of 28 days resulted in stronger secondary responses than an interval of only 14 days. CONCLUSIONS: MAB specific for macaw immunoglobulins can be used in ELISA to evaluate the humoral immune responses of macaws. 1-year-old Blue and Gold Macaws developed strong anti-BSA titer when inoculated with 200 micrograms of BSA. An inoculation interval of 28 days resulted in stronger secondary responses than did an interval of only 14 days. CLINICAL RELEVANCE: These MAB, the first reported to have specificity for a psittacine antibody class, will be useful in the evaluation of psittacine antibody responses and in the development of psittacine diagnostic assays.

Efficacy of acyclovir against herpesvirus infection in Quaker parakeets.

We evaluated the efficacy of acyclovir against experimentally induced herpesvirus infection (Pacheco's parrot disease) in Quaker parakeets. Thirty-two of 40 birds were challenge-exposed with 0.1 ml of a suspension of herpesvirus (10(4) median cell culture infective doses [CCID50]) given IM. Treatment with acyclovir was started 24 hours later and was continued for 7 days. The birds were allotted to 5 groups of 8 birds each. There was a considerable difference in mortality between groups 1-5. Of 8 bird in each group, 6 died in group 1 (control), 1 died in group 2 (gavage), 3 died in group 3 (low dose, IM), 4 died in group 4 (high dose, IM), and none died in group 5 (contact controls). There was a significant (P = 0.023) difference in mortality between groups 1 and 2, thus the oral form of acyclovir administered by gavage was the most efficacious therapeutic regimen. Clinical signs and death occurred after discontinuation of acyclovir in groups 2 and 3, whereas the mean time of death for the control group was 6 days after challenge exposure. Herpesvirus was recovered by inoculation of chick embryo cell culture with pooled tissue suspensions from all birds that died. Histologic evidence of herpesvirus infection was found in most birds that died, with the control group having the most severe lesions. Surviving Quaker parakeets were transferred to cages with seronegative Quaker parakeets with no known exposure to herpesvirus. There have been no deaths attributable to herpesvirus infection in a period exceeding 2 years.

Maternal immunoglobulin G antibody transfer and development of immunoglobulin G antibody responses in blue and gold macaw (Ara ararauna) chicks.

OBJECTIVE: To document the maternal transfer of IgG antibodies from Blue and Gold Macaw hens to chicks via the egg; to measure serum IgG half-life in macaw chicks; and to measure the ability of 2- to 10-week-old macaw chicks to generate primary and secondary IgG responses. PROCEDURE: 4 adult Blue and Gold Macaw hens were inoculated with 200 micrograms of bovine serum albumin (BSA) every 21 days throughout the breeding season. Eggs laid by these hens were incubator hatched to eliminate the possibility of antibody transfer through crop secretions during feeding. Anti-BSA titer was measured just prior to each inoculation in hens and in chicks from 14 to 42 days of age. 1 chick from each of 5 macaw clutches hatched to nonimmunized hens was assigned to 1 of 2 experimental groups. Group-1 chicks were inoculated with 200 micrograms of BSA at 2 and 6 weeks of age. Group-2 chicks were inoculated with 200 micrograms of BSA at 6 and 10 weeks of age. Anti-BSA titer was measured weekly for 8 weeks after primary inoculation. Blood samples were centrifuged, and serum was harvested and frozen at -85 C until analyzed. Anti-BSA IgG titer were measured by ELISA. In the maternal transfer experiment, an exponential decay model was used to calculate the half-life of BSA antibodies in chicks. In the BSA antibody response experiment, comparison of primary and secondary anti-BSA responses of 2- and 6-week-old chicks was performed, using a two-way repeated measures ANOVA, with significance set at P < 0.05. RESULTS: Hens maintained high anti-BSA titer throughout the breeding season. Maternal transfer of anti-BSA IgG antibodies was documented in all 7 chicks. Anti-BSA titer in chicks decreased in exponential fashion with an average serum IgG half-life of 3.85 days. By 42 days of age, antibodies to BSA were virtually undetectable in all chicks. The primary antibody response of 6-week-old chicks was significantly higher than that of 2-week-old chicks (P = 0.016). No significant difference was observed in the magnitude of the secondary antibody responses between these age groups. Peak anti-BSA IgG antibody responses were reached by 14 days after primary and secondary immunization. Chicks of both age groups generated lower anti-BSA IgG titer than did adult Blue and Gold Macaws. CONCLUSIONS: Blue and Gold Macaw hens transfer IgG antibodies to their chicks through the egg. The half-life of IgG in newly hatched chicks is approximately 3.85 days. 6-week-old chicks develop higher anti-BSA titers than do 2-week-old chicks, but significantly lower titers than do adult macaws. CLINICAL RELEVANCE: Information on the nondomestic avian immune system will be useful in the development of vaccination and other preventive health programs for psittacine birds.

Mycoplasmal conjunctivitis in songbirds from New York.

A field study was conducted to determine the prevalence of conjunctivitis and Mycoplasma gallisepticum (MG) infections in house finches (Carpodacus mexicanus) and other songbirds common to bird feeders in Tompkins County (New York, USA). Eight hundred two individuals of 23 species and nine families of birds were captured and given physical examinations during the 14 mo study beginning in February 1998. Clinical conjunctivitis (eyelid or conjunctival swelling, erythema, and discharge) was observed in 10% (19/196) of house finches examined, and only in the winter months from November to March. Unilateral conjunctivitis was observed in 79% (15/19) of affected house finches; one case developed bilateral disease between 8 and 18 days following initial examination. Conjunctivitis was observed in a similar proportion of males and females sampled, and body condition scores and wing chord lengths were not significantly different between diseased and non-diseased house finches. Mycoplasma gallisepticum was isolated from 76% (13/17) of finches with conjunctivitis and 2% (3/168) of clinically normal house finches sampled during the study. DNA fingerprints of 11 MG isolates using random amplification of polymorphic DNA (RAPD) techniques showed no apparent differences in banding patterns over the course of the study, suggesting persistence of a single MG strain in the study population. The prevalence of conjunctivitis and MG infections declined in house finches between February/March 1998 and February/March 1999 (23% to 6%, and 20% to 5%, respectively), but only the former was significant (P < 0.05). Conjunctivitis was also observed in four American goldfinches (Carduelis tristis) and one purple finch (Carpodacus purpureus). Mycoplasma gallisepticum infection was confirmed in the purple finch, the first documented case of MG-associated conjunctivitis in this species. The purple finch isolate was similar to house finch isolates from the study site by RAPD analysis. Positive plate agglutination (PA) tests were recorded in one other goldfinch and two purple finches, suggesting exposure of these individuals to MG. Positive PA tests were also obtained from two brown-headed cowbirds (Molothrus ater) and four tufted titmice (Parus bicolor), but MG infection could not be confirmed in these cases due to lack of samples. Based on these findings, the prevalence of MG infections in hosts other than house finches appear to be low in the population sampled. There is growing evidence, however, that songbird species other than house finches are susceptible to MG infection and disease.

An outbreak of toxoplasmosis in wallaroos (Macropus robustus) in a California zoo.

Four wallaroos (Macropus robustus) from a central California zoo colony of 22 died within a three week period. Histopathologic findings in all four cases were suggestive of toxoplasmosis. Brain, lung and heart were the most frequently involved organs. Within 4 months of the last death sera from nine colony members were tested for antibodies against Toxoplasma gondii using the indirect hemagglutination test. All nine had serological titers ranging from 1:64 to 1:131,072.

Exertional myopathy in translocated river otters from New York.

Lesions consistent with exertional myopathy (EM) were documented postmortem in four North American river otters (Lutra canadensis) during translocation for a population restoration project. Clinical signs in these otters included depression, anorexia and shock. Gross lesions in one otter included locally extensive linear, pale areas within the subscapularis, rectus abdominis, quadriceps, and dorsal laryngeal muscles. Microscopic lesions were characterized by acute to subacute myofiber necrosis of varying severity, and occurred in a variety of skeletal muscles as well as cardiac muscle in one otter. Based on these observations, we conducted a retrospective review of records of otters which experienced similar capture, transfer, and holding protocols between 1995 and 1997, but with a successful outcome (n = 69). Significant elevations in serum aspartate aminotransferase (AST) and creatine kinase (CK) were observed in 19 (28%) of the otters, but may have been higher due to delayed sample collection from some otters. However, none of the otters with elevated enzymes exhibited clinical signs suggestive of EM. These findings indicate that river otters may develop EM when translocated, but many cases may be mild or clinically inapparent.

Risk factors associated with mycoplasmal conjunctivitis in house finches.

Observations from a citizen-based survey were used to identify potential risk factors associated with mycoplasmal conjunctivitis (Mycoplasma gallisepticum) in eastern house finches (Carpodacus mexicanus). Between November 1994 and October 1996, 778 volunteers provided 7,224 monthly observations at residential bird feeding sites across an eight state region in the eastern USA. Information collected by questionnaires included health status of house finches and four sympatric passerine species, types and number of bird feeders maintained, neighborhood housing locale and altitude of the observation site. Bivariate analyses revealed that house finches were 14 to 72 times as likely to be observed with conjunctivitis than the sympatric species studied. Year of the study, season, and the presence of platform, hopper, and tube type feeders were significantly associated with conjunctivitis in house finches. Multivariate analysis using a logistic regression model suggests that increased risk of conjunctivitis in house finches was associated with the second year of the study (the third year of the outbreak), the cooler non-breeding periods from September through March, and the presence of tube style feeders. In addition, the presence of raised platform type feeders may have been protective against conjunctivitis in house finches. Prevention of spread of this disease may include modifying bird feeding activities based on season and type of feeder.

Host range and dynamics of mycoplasmal conjunctivitis among birds in North America.

An epidemic of conjunctivitis among house finches (Carpodacus mexicanus) caused by Mycoplasma gallisepticum (MG) bacterial infections was first described in 1994. The disease exhibits high primary host specificity, but has been isolated from a limited number of secondary avian hosts at various times and locations. We used records from the House Finch Disease Survey, a continent-wide, volunteer monitoring project, to document the host range of conjunctivitis in birds at feeding stations and to investigate how disease in house finches might influence the spread of conjunctivitis to other hosts. Between 1994 and 1998, participants recorded 675 cases of conjunctivitis in 31 species other than house finches in eastern North America. Seventy five % of these cases were observed among three species: American goldfinches (Carduelis tristis), purple finches (Carpodacus purpureus) and house sparrows (Passer domesticus). The proportion of sites with diseased wintering populations of the three species increased over the 4 yr study and coincided with range expansion of conjunctivitis in house finches. Sites with diseased house finches present were significantly more likely to report conjunctivitis in each of the three species during the same month. These observations are most consistent with transmission of an infectious agent (presumably MG) from house finches to these secondary hosts via spillover of localized epidemics, rather than sustained interspecific transmission.

Seroprevalence of infectious disease agents in free-ranging Florida panthers (Felis concolor coryi).

Serum samples obtained from 38 free-ranging Florida panthers (Felis concolor coryi) in southern Florida, March 1978 through February 1991, were tested for antibodies against eight bacterial, parasitic, and viral disease agents. Sera were positive for antibodies against feline panleukopenia virus (FPV) (78%), feline calicivirus (56%), feline immunodeficiency virus/puma lentivirus (37%), feline enteric coronavirus/feline infectious peritonitis virus (19%), and Toxoplasma gondii (9%). All samples were seronegative for Brucella spp., feline rhinotracheitis virus, and pseudorabies virus. In addition, all the animals tested were negative for feline leukemia virus p27 antigen as determined by enzyme-linked immunosorbent assay. Feline panleukopenia virus was considered to be a potentially significant disease agent; FPV antibodies occurred in the highest prevalences in older age classes (P = 0.027) and in panthers living in the dense mixed hardwood swamps in the western portion of their range compared to the open cypress and sawgrass prairies to the east (P = 0.096). Because < 50 animals remain in this relict population and the probable resultant depression of genetic diversity and lowered disease resistance, FPV or other disease agents could contribute to the extinction of this endangered subspecies.

Evaluation of comparative cervical tuberculin skin testing in cervids naturally exposed to mycobacteria.

A comparative cervical tuberculin skin test was evaluated in cervids with naturally acquired mycobacterial infections. The test exhibited a high degree of sensitivity (84%) and specificity (80%) in detecting infected animals but a low level of accuracy (57%) in differentiating Mycobacterium bovis infections from infections caused by M avium and other Runyon groups III and IV mycobacteria. This phenomenon was attributed to a low recovery rate of more than 1 species of mycobacteria from individuals with mixed infections.

Effects of yohimbine on combined xylazine-ketamine-induced sedation and immobilization in juvenile African elephants.

Twenty-two juvenile African elephants were given a combination of xylazine (mean +/- SD = 0.14 +/- 0.03 mg/kg of body weight) and ketamine (1.14 +/- 0.21 mg/kg) as a single IM injection; one elephant was immobilized twice, 77 days apart. After injection, 14 elephants were immobilized, 4 were sedated deeply, 2 were sedated moderately, and 2 were sedated minimally. Immobilized elephants had a mean immobilization time of 11.6 +/- 6.9 minutes. At the conclusion of a variety of clinical procedures, 12 of the 14 elephants immobilized with a single dose combination of xylazine and ketamine were given yohimbine (0.13 +/- 0.03 mg/kg) IV, and the remaining 2 elephants were allowed to recover spontaneously; the elephants given yohimbine had a mean standing time of 2.4 +/- 1.1 minutes. Of the 8 sedated elephants, 5 were given an additional dose of combined xylazine (0.08 +/- 0.03 mg/kg), and ketamine (0.61 +/- 0.19 mg/kg) IM, and 1 elephant was given ketamine (0.47 mg/kg) IV. After injection, 4 of the 8 elephants were recumbent laterally within 17 minutes and 2 remained standing, under deep sedation. Seven of the 8 elephants were given yohimbine (0.13 +/- 0.03 mg/kg) IV; all were ambulatory in 2 minutes. Results indicated that yohimbine may be useful in controlling duration of xylazine-ketamine sedation and immobilization in juvenile African elephants.

Use of halothane to maintain anesthesia induced with etorphine in juvenile African elephants.

Sixteen 3- to 5-year-old African elephants were anesthetized one or more times for a total of 27 diagnostic and surgical procedures. Xylazine (0.1 +/- 0.04 mg/kg of body weight, mean +/- SD) and ketamine (0.6 +/- 0.13 mg/kg) administered IM induced good chemical restraint in standing juvenile elephants during a 45-minute transport period before administration of general anesthesia. After IM or IV administration of etorphine (1.9 +/- 0.56 micrograms/kg), the mean time to lateral recumbency was 20 +/- 6.6 and 3 +/- 0.0 minutes, respectively. The mean heart rate, systolic blood pressure, and respiration rate during all procedures was 50 +/- 12 beats/min, 106 +/- 19 mm of Hg, and 10 +/- 3 breaths/min, respectively. Cardiac arrhythmias were detected during 2 procedures. One elephant with hypotension responded to a decrease in the concentration of halothane and IV infusion of dobutamine HCl. Alterations in systolic blood pressure, ear flapping, and trunk muscle tone were useful for monitoring depth of anesthesia. Results indicated that halothane in oxygen was effective for maintenance of surgical anesthesia in juvenile African elephants after induction with etorphine.

Cutaneous papillomas associated with a herpesvirus-like infection in a herd of captive African elephants.

Proliferative cutaneous lesions developed in a herd of captive African elephants (33 from an animal importer in Texas [group 1], and 63 young elephants collected in Zimbabwe [group 2]). Group-1 elephants were purchased 8 months before the arrival of the group-2 elephants. On arrival, 7 group-1 elephants had raised nodular fibrous growths, located predominantly on their trunks. Lesions were not observed in the group-2 elephants until approximately 3 months after they were acquired. Lesions on group-2 elephants began as small focal proliferative growths that regressed or that progressed into large nodular fibrous growths that were similar in appearance to those seen in the group-1 elephants. Lesions at various stages of development were biopsied and examined. Histologically, early lesions were inverted papillomas, with hyperplastic and hypertrophic epithelial cells containing amphoteric intranuclear inclusions in the lesion center. Older, large, nodular fibrous growths were ulcerated and were composed predominantly of a thickened dermis containing fibroblasts, collagen, and a mixed inflammatory cell infiltrate; inclusions were not observed in adjacent epidermal cells. Using a peroxidase-antiperoxidase technique, we did not detect group-specific papillomavirus antigens. Southern blot hybridization analysis of DNA from lesion specimens did not indicate papillomavirus-specific genomes. Electron-microscopically, inclusions consisted of aggregates of virus particles. The particles had electron-dense and electron-lucent cores and were 95 to 103 nm in diameter. Virions developed envelopes from nuclear membranes. Mature particles were seen within the cytoplasm and filled the intercellular spaces. On the basis of size, location, conformation, and envelopment, the particles most closely resembled those of herpesviruses.

Medical and surgical management of benign prostatic hyperplasia, diskospondylitis, and penile paresis in an ocelot.

Benign prostatic hyperplasia, diskospondylitis at the T13-L1 intervertebral disk space, and penile paresis were diagnosed in a 16-year-old male ocelot. Treatment consisted of castration for the benign prostatic hyperplasia and long-term administration of antibiotics for the diskospondylitis. On physical examination 8 weeks after initial referral, the prostate gland was no longer palpable and could not be visualized radiographically or ultrasonographically. The diskospondylitis lesion was found to be healing, on the basis of radiographic results.

Needle catheter duodenostomy: a technique for duodenal alimentation of birds.

A technique for duodenal alimentation (needle catheter duodenostomy) of birds was developed, using the domestic pigeon (Columba livia) as the experimental model. A needle catheter was inserted into the descending duodenum of 5 pigeons and was secured to the body wall and dorsum of each bird. A liquid diet was administered daily (in equal amounts of 0, 4, 8, 12, and 16 hours) for 14 days without adverse effects. On day 15, the catheters were removed, and the birds immediately resumed normal consumption of a pigeon ration and water diet. Although 4 of the 5 birds had minor weight loss, dietary alterations probably could be used on an individual basis to alleviate this problem. After oral alimentation was resumed, the 5 birds exceeded their initial body weight within 7 days. Four weeks after catheter removal, positive-contrast radiographic evaluations indicated that the duodenum of each pigeon appeared normal. Needle catheter duodenostomy was a viable method of alimentation in the domestic pigeon. This technique should be applicable for other avian species requiring bypass of the upper gastrointestinal tract proximal to the region of catheter insertion in the duodenum.