Quantitative trait loci exploration and characterization of gestation length in Holstein cattle.

Gestation length (GL) is a moderately heritable trait in cattle with economic and management implications. This study aimed to characterize the gestation length of an Argentinian Holstein cattle population, understand contributing factors, and explore the GL effect on production performance. Further objectives were to estimate direct and maternal heritabilities for this trait and to identify genomic regions affecting it. Data consisted of GL records from 45,738 births corresponding to 17,004 Holstein cows and heifers. The effects of age and calving season over GL were analyzed using a Student's t-test for homoscedastic samples. The effects of the GL category (GL shorter than 1.5 SD, within ±1.5 SD, and longer than 1.5 SD from the mean) on production performance were studied by analysis of variance. A single-step genome-wide association study was performed using the BLUPF90 suite of programs with genotypes from 654 Holstein animals on 40,339 SNP. The results showed that the younger the age at calving, the shorter the GL. Moreover, gestations ending in warmer seasons were, in general, statistically shorter than those ending in colder seasons for both heifers and cows. Regarding the effect of GL on production performance, cows with gestation periods within ±1.5 SD from the population mean exhibited the highest 305-day cumulative milk, fat, and protein productions. Direct and maternal heritabilities for GL were 0.42 and 0.03, respectively. We detected a SNP suggestively associated with direct gestation length at 57.7 Mb on Bos taurus autosome 18, a locus included in a region described in the literature as associated with the trait. The information obtained on the environmental and genetic factors affecting GL in Argentinian Holstein cows contributes to characterizing the population in pursuit of improving the performance of national dairy cattle breeding systems.

Association of TNF rs668920841 and INRA111 polymorphisms with caprine brucellosis: A case-control study of candidate genes involved in innate immunity.

Caprine brucellosis is an infectious, contagious zoonotic disease caused by Brucella melitensis. Multiple factors, including host genetics, can influence the outcome of the exposure to Brucella; and it is expected that genetic variants that affect the host innate immune response could have a key role in Brucella infection and pathogenesis. In this study, we evaluated if polymorphisms in innate immunity-related genes are associated with results of Brucella infection in goats. Nine polymorphisms within interferon gamma (IFNG), tumor necrosis factor (TNF), MyD88 innate immune signal transduction adaptor (MYD88), interleukin 10 (IL10) and IL-10 receptor subunit alpha (IL10RA) genes and two molecular markers (BMS2753 and INRA111) were resolved by PCR-capillary electrophoresis in samples from 81 seronegative and 61 seropositive goats for brucellosis. A heterozygous genotype at INRA111, a microsatellite near the VRK serine/threonine kinase 2 (VRK2) gene, was associated with absence of Brucella-specific antibodies in goats naturally exposed to the pathogen (P = .004). Conversely, variants in the TNF gene (rs668920841) and near the IFN gamma receptor 1 (IFNGR1) gene (microsatellite BMS2753) were significantly associated with presence of Brucella-specific antibodies at allelic (P = .042 and P = .046) and genotypic level (P = .012 and P = .041, respectively). Moreover, an in silico analysis predicted a functional role of the insertion-deletion polymorphism rs668920841 on the transcriptional regulation of the caprine TNF gene. Altogether, these results contribute to the identification of genetic factors that have a putative effect on the resistance / susceptibility phenotype of goats to Brucella infection.

BOLA-DRB3 gene polymorphisms influence bovine leukaemia virus infection levels in Holstein and Holstein × Jersey crossbreed dairy cattle.

Bovine leukemia virus (BLV) infections, causing persistent lymphocytosis and lethal lymphosarcoma in cattle, have reached high endemicity on dairy farms. We observed extensive inter-individual variation in the level of infection (LI) by assessing differences in proviral load in peripheral blood. This phenotypic variation appears to be determined by host genetics variants, especially those located in the BoLA-DRB3 MHCII molecule. We performed an association study using sequencing-based typed BOLA-DRB3 alleles from over 800 Holstein and Holstein × Jersey cows considering LI in vivo and accounting for filial relationships. The DBR3*0902 allele was associated with a low level of infection (LLI) (<1% of circulating infected B-cells), whereas the DRB3*1001 and DRB3*1201 alleles were related to a high level of infection (HLI). We found evidence that 13 polymorphic positions located in the pockets of the peptide-binding cleft of the BOLA-DRB3 alleles were associated with LI. DRB3*0902 had unique haplotypes for each of the pockets: Ser13 -Glu70 -Arg71 -Glu74 (pocket 4), Ser11 -Ser30 (pocket 6), Glu28 -Trp61 -Arg71 (pocket 7) and Asn37 -Asp57 (pocket 9), and all of them were significantly associated with LLI. Conversely, Lys13 -Arg70 -Ala71 -Ala74 and Ser13 -Arg70 -Ala71 -Ala74 , corresponding to the DRB3*1001 and *1201 alleles respectively, were associated with HLI. We showed that the specific amino acid pattern in the DRB3*0902 peptide-binding cleft may be related to the set point of a very low proviral load level in adult cows. Moreover, we identified two BOLA-DRB3 alleles associated with a HLI, which is compatible with a highly contagious profile.

Genetic analysis of the 3' untranslated region of the bovine SLC11A1 gene reveals novel polymorphisms.

Polymorphisms in microsatellites at the 3' untranslated region (3'UTR) of the SLC11A1 (solute carrier family 11 member A1) gene have been associated with natural resistance to Brucella abortus and Mycobacterium bovis infection in livestock species. Here, we carried out an individual genetic analysis of the two microsatellites present at the 3'UTR SLC11A1 gene in 254 Bos taurus purebred, 125 B. indicus purebred and 54 B. taurus × B. indicus crossbred cattle. The genotyping by capillary electrophoresis showed the presence of four alleles (157, 159, 161 and 163) for the first microsatellite (MS1) and six alleles (175, 177, 179, 181, 183 and 185) for the second microsatellite (MS2). The alleles 159 and 175 were the most frequent in all breeds analyzed. B. taurus showed the most homogeneous haplotype and genotype for both microsatellites, whereas B. indicus showed the most heterogeneous haplotype and genotype. Two novel variants (alleles 161 and 163) within the MS1 are reported as well as novel variants in MS2 in Holstein breed. The knowledge of the polymorphisms distribution in both microsatellites at the 3'UTR of the SLC11A1 gene in cattle breeds is useful for future experimental design to evaluate the association between reported genotypes and natural resistance to pathogens infection.

CAPN1 markers in three Argentinean cattle breeds: report of a new InDel polymorphism within intron 17.

In this study, the genotype distribution and allelic frequencies of CAPN1 (Calcium activated neutral protease) single nucleotide polymorphisms (SNPs) were analyzed taking advantage of the different genetic backgrounds provided by Hereford, Brahman and Braford cattle. We report a new insertion/deletion (InDel) polymorphism, consisting of a change of seven nucleotides for only one nucleotide (TCTGGGT → C) within intron 17 of the CAPN1 gene. The segregation pattern of this polymorphism was analyzed together with the markers CAPN316, CAPN530 and CAPN4751 already described. The allele distribution of CAPN1 markers in the Braford crossbreed (3/8 Brahman 5/8 Hereford) is described for the first time. Four assays of allelic discrimination were designed: the tetra primer ARMS-PCR technique for genotyping the new InDel and the CAPN4751 marker, and a PCR-RFLP method for genotyping the markers CAPN316 and CAPN530. The genotypic and minor allele frequencies (MAFs) obtained showed that the InDel polymorphism does not provide redundant information to that already provided by the other CAPN1 markers and segregates differently between breeds, being a common SNP (MAF ≥ 0.05) in the herds with a high percentage of Bos indicus background. The high percentage of heterozygous individuals found in the Braford crossbreed for the markers assessed reveals enough genetic variation that could help to solve the tenderness problem of tropical-adapted cattle.

Merino sheep: a further look at quantitative trait loci for wool production.

A quantitative trait loci (QTL) analysis of wool traits from experimental half-sib data of Merino sheep is presented. A total of 617 animals distributed in 10 families were genotyped for 36 microsatellite markers on four ovine chromosomes OAR1, OAR3, OAR4 and OAR11. The markers covering OAR3 and OAR11 were densely spaced, at an average distance of 2.8 and 1.2 cM, respectively. Body weight and wool traits were measured at first and second shearing. Analyses were conducted under three hypotheses: (i) a single QTL controlling a single trait (for multimarker regression models); (ii) two linked QTLs controlling a single trait (using maximum likelihood techniques) and (iii) a single QTL controlling more than one trait (also using maximum likelihood techniques). One QTL was identified for several wool traits on OAR1 (average curvature of fibre at first and second shearing, and clean wool yield measured at second shearing) and on OAR11 (weight and staple strength at first shearing, and coefficient of variation of fibre diameter at second shearing). In addition, one QTL was detected on OAR4 affecting weight measured at second shearing. The results of the single trait method and the two-QTL hypotheses showed an additional QTL segregating on OAR11 (for greasy fleece weight at first shearing and clean wool yield trait at second shearing). Pleiotropic QTLs (controlling more than one trait) were found on OAR1 (clean wool yield, average curvature of fibre, clean and greasy fleece weightand staple length, all measured at second shearing).

QTL detection for milk production traits in goats using a longitudinal model.

Eight paternal half-sib families were used to identify chromosomal regions associated with variation in the lactation curves of dairy goats. DNA samples from 162 animals were amplified by PCR for 37 microsatellite markers, from Capra hircus autosomes CHI3, CHI6, CHI14 and CHI20. Milk samples were collected during 6 years, and there were 897 records for milk yield (MY) and 814 for fat (FP) and protein percentage (PP). The analysis was conducted in two stages. First, a random regression model with several fixed effects was fitted to describe the lactation function, using a scale (alpha) plus four shape parameters: beta and gamma, both associated with a decrease in the slope of the curve, and delta and phi that are related to the increase in slope. Predictions of alpha, beta, gamma, delta and phi were regressed using an interval mapping model, and F-tests were used to test for quantitative trait loci (QTL) effects. Significant (p < 0.05) QTLs were found for: (i) MY: CHI6 at 70-80 cM for all parameters; CHI14 at 14 cM for delta and phi; (ii) FP: CHI14, at 63 cM was associated with beta; CHI20, at 72 cM, showed association with alpha; (iii) PP: chromosomal regions associated with beta were found at 59 cM in CHI3 and at 55 cM in CHI20 with alpha and gamma. Analyses using more families and more animals will be useful to confirm or to reject these findings.

African-derived mitochondria in South American native cattle breeds (Bos taurus): evidence of a new taurine mitochondrial lineage.

This article reports the nucleotide diversity within the control region of 42 mitochondrial chromosomes belonging to five South American native cattle breeds (Bos taurus). Analysis of these data in conjunction with B. taurus and B. indicus sequences from Africa, Europe, the Near East, India, and Japan allowed the recognition of eight new mitochondrial haplotypes and their relative positions in a phylogenetic network. The structure of genetic variation among different hypothetical groupings was tested through the molecular variance decomposition, which was best explained by haplotype group components. Haplotypes surveyed were classified as European-related and African-related. Unexpectedly, two haplotypes within the African cluster were more divergent from the African consensus than the latter from the European consensus. A neighbor-joining tree shows the position of two haplotypes compared to European/African mitochondrial lineage splitting. This different and putatively ancestral mitochondrial lineage (AA) is supported by the calibration of sequence divergence based on the Bos-Bison separation. The European/African mitochondria divergence might be subsequent (67,100 years before present) to that between AA and Africans (84,700 years before present), also preceding domestication times. These genetic data could reflect the haplotype distribution of Iberian cattle five centuries ago.

A phase II study of sequential 5-fluorouracil, epirubicin and cyclophosphamide (FEC) and paclitaxel in advanced breast cancer (Protocol PV BC 97/01).

Sequential administration of the association of 5-fluorouracil, epirubicin and cyclophosphamide (FEC) and paclitaxel could be better tolerated than the association of an anthracycline and paclitaxel while having a similar antitumour effect. 69 patients with advanced breast cancer previously untreated with anthracyclines or paclitaxel entered a phase II multicentre study in which FEC was followed by paclitaxel. Both regimens were administered 4 times every 21 days. The median follow-up is 20 months and 38/69 patients have died. Grade III-IV toxicity was acceptable. Leukopenia occurred in 26% of patients, thrombocytopenia in 2% and anaemia in 4%. One patient had reversible heart failure during FEC therapy. Peripheral neuropathy and arthralgia-myalgia occurred in 9% and 4% of patients, respectively and one patient had respiratory hypersensitivity during paclitaxel treatment. 9 patients did not complete therapy because of: treatment refusal (n = 1), cardiac toxicity (n = 1), early death during FEC chemotherapy (n = 1), major protocol violations (n = 4), hypersensitivity reaction (n = 1) and early death during paclitaxel chemotherapy (n = 1). The overall response rate was 65% (95% CI = 53-76), and 7% of patients had stable disease. Therapy was defined as having failed in 28% of patients because they were not evaluable (13%) or had progressive disease (15%). The median time to progression and survival are 13.2 and 23.5 months, respectively. Sequential FEC-paclitaxel is a suitable strategy for patients with metastatic breast cancer who have not been previously treated with anthracyclines and/or taxanes. In fact, it avoids major haematologic toxicity and has a good antitumour effect.

Evidence for PSP in mussels in Trinidad

Herein we present the first evidence for the presence of Paralytic Shellfish Poison (PSP) in Trinidadian waters. The toxin was found in a meat extract of the mussel, Perna viridis. PSP has not previously been demonstrated in the shellfish of Caribbean islands. The presence of PSP in Trinidad is therefore significant in that it presents an opportunity to better understand the dynamics of PSP and algal blooms in both a region and island environment not normally associated with PSP.P. viridis is not native to Trinidad, but rather originates from eastern Asia. It presented itself only recently in Trinidadian waters. Interestingly, shellfish consumption and algal blooms have had a long history of coexistence in Trinidad without any record of human intoxications. In this context, potential Public Health implications of finding PSP in a non-native shellfish species are briefly discussed.

Evidence for PSP in mussels in Trinidad.

Herein we present the first evidence for the presence of Paralytic Shellfish Poison (PSP) in Trinidadian waters. The toxin was found in a meat extract of the mussel, Perna viridis. PSP has not previously been demonstrated in the shellfish of Caribbean islands. The presence of PSP in Trinidad is therefore significant in that it presents an opportunity to better understand the dynamics of PSP and algal blooms in both a region and island environment not normally associated with PSP.P. viridis is not native to Trinidad, but rather originates from eastern Asia. It presented itself only recently in Trinidadian waters. Interestingly, shellfish consumption and algal blooms have had a long history of coexistence in Trinidad without any record of human intoxications. In this context, potential Public Health implications of finding PSP in a non-native shellfish species are briefly discussed.

Neurotoxic shellfish poisoning and brevetoxin metabolites: a case study from Florida.

In June of 1996, three family members were diagnosed as suffering from neurotoxic shellfish poisoning (NSP) as a result of eating shellfish harvested from Sarasota Bay, Florida. Urine from two of these patients and extracts of shellfish collected from the same location were analyzed by radioimmunoassay (RIA) and by receptor binding assay. Activity consistent with brevetoxins was present in both urine and shellfish extracts. High performance liquid chromatographic (HPLC) analysis of shellfish extracts demonstrated multiple fractions recognized by specific anti-brevetoxin antibodies, suggesting metabolic conversion of parent brevetoxins. Affinity-purification of these extracts yielded four major peaks of activity. One peak was identified by HPLC-mass spectroscopy (HPLC-MS) to be PbTx-3, which was likely produced metabolically from the dominant parent toxin PbTx-2. No PbTx-2, however, was detected. Other peaks of activity were determined to consist of compounds of apparent masses of [M + H]+ of 1018, 1034, and 1005. These higher masses are suggestive of conjugated metabolites, but their structures have yet to be determined. The material associated with these latter three peaks were recognized by both RIA and receptor binding assay, but they quantitated differently. This finding suggests that these metabolites react differently in the two assays, and this result may have important implications for seafood safety and regulation. We suggest these metabolites to be the true cause of NSP, and they should be taken into account during regulatory testing.

Development of sensitive colorimetric capture elisas for Clostridium botulinum neurotoxin serotypes A and B.

Sensitive and specific enzyme-linked immunosorbent assays were developed to detect Clostridium botulinum neurotoxin serotypes A (BoNT A) and B (BoNT B) in assay buffer and human serum. The assay is based upon affinity-purified horse polyclonal antibodies directed against the approximately 50 kDa C-fragments of each toxin. Standard curves were linear over the range of 0.1-10 ng mL. Detection was possible at 0.2 ng mL (20 pg/well) and accurate quantitation at 0.5 ng/mL (50 pg well) in assay buffer and 10% human serum. Variations between triplicates was typically 5-10%. Less than 1% cross reactivity occurred between other serotypes when each assay was performed against serotypes A, B and E. When tested against toxins complexed to their associated nontoxic proteins, interference was absent (BoNT B) or < 25% (BoNT A). These assays demonstrate sensitivity close to that of the mouse bioassay without the use of animals and in a much simpler format than other reported assays of similar sensitivity.

Hypertension and identification of toxin in human urine and serum following a cluster of mussel-associated paralytic shellfish poisoning outbreaks.

Following four outbreaks of paralytic shellfish poisoning on Kodiak Island, Alaska, during 1994, medical records of ill persons were reviewed and interviews were conducted. Urine and serum specimens were analyzed at three independent laboratories using four different saxitoxin binding assays. High-performance liquid chromatography was used to determine the presence of specific toxin congeners. Among 11 ill persons, three required mechanical ventilation and one died. Mean peak systolic and diastolic blood pressure measurements were 172 (range 128-247) and 102 (range 78-165) mmHg, respectively, and blood pressure measurements corresponded with ingested toxin dose. All four different laboratory methodologies detected toxin in serum at 2.8-47 nM during acute illness and toxin in urine at 65-372 nM after acute symptom resolution. The composition of specific paralytic shellfish poisons differed between mussels and human biological specimens, suggesting that human metabolism of toxins had occurred. The results of this study indicate that saxitoxin analogues may cause severe hypertension. In addition, we demonstrate that saxitoxins can be detected in human biological specimens, that nanomolar serum toxin levels may cause serious illness and that human metabolism of toxin may occur. Clearance of paralytic shellfish poisons from serum was evident within 24 hr and urine was identified as a major route of toxin excretion in humans.

Identification of Caribbean ciguatoxins as the cause of an outbreak of fish poisoning among U.S. soldiers in Haiti.

On 24 February 1995, six U.S. soldiers serving with the Multinational Force in Haiti became ill after eating a locally caught fish identified as the greater amberjack Seriola dumerili. The victims presented with nausea, vomiting, watery diarrhea and abdominal cramps 5-8 hr after consumption. Also present in some victims were numbness in the extremities or perioral region, bradycardia and scalp paresthesia. Patients were treated with i.v. hydration therapy and antiemetics. All recovered without sequelae over the course of 1-3 months. A portion of the cooked fish was obtained for analysis. A semipurified lipid extract was prepared according to standard methods and analyzed for the presence of Na+ channel site 5 binding activity using a brevetoxin receptor binding assay. By this assay, the fish sample contained the equivalent of approximately 20 ng Caribbean ciguatoxin/g flesh. The presence of the major Caribbean ciguatoxin (C-CTX-1) was confirmed by liquid chromatography-mass spectrometry. Using the receptor binding assay to monitor activity in TSK and PRP-1 column fractions, two minor toxins were detected in addition to C-CTX-1. One of these minor toxins was more polar, and the other less polar, than C-CTX-1. These data provide firm evidence that a family of C-CTX-1 is responsible for ciguatera in the Caribbean.

Comparison of the pulmonary distribution and efficacy of antibodies given to mice by intratracheal instillation or aerosol inhalation.

The respiratory tract is the portal of entry and target organ of many aerosolized toxins and infective agents, and there is increasing need for testing the efficacy of potential therapeutic agents delivered directly into the lungs. Intratracheal instillation and aerosol inhalation are the two methods most often used to introduce drugs, toxins, or infective agents into the respiratory tract of experimental animals. In this study we compared the distribution and efficacy of antibodies given to mice by aerosol inhalation or intratracheal instillation. We determined the pulmonary distribution of these antibodies by immunohistochemistry and observed the distribution and severity of pulmonary lesions that developed after exposure to aerosolized ricin. Although antibodies administered by either method prevented death, we found that instilled antibodies tended to concentrate around terminal airways and often failed to reach peripheral lung fields. Sometimes entire lung lobes were missed by the instillation route. Acute and chronic pulmonary lesions developed in the unprotected areas of instillation-treated lungs. In contrast, aerosolized antibodies covered all pulmonary surfaces and effectively prevented ricin-induced lesions throughout the lungs. Our findings suggest that the aerosol inhalation method may be preferable for evaluating the efficacy of therapeutic agents in the respiratory tract because of the failure of instilled agents to reach and protect peripheral alveoli.

Aerosolized specific antibody protects mice from lung injury associated with aerosolized ricin exposure.

Parenteral vaccination with ricin toxoid, although protective against death after a lethal aerosol ricin challenge, only partially protects against lung lesions. Therefore, we tested whether passive protection with aerosolized specific anti-ricin IgG (goat polyclonal, affinity-purified) could protect against both lethality and lung lesions in unvaccinated mice. Healthy CD-l mice were administered antibody (Ab) by small particle aerosol. Group 1 received non-specific control Ab (2160 mg/min/m3), and groups 2 and 3 received anti-ricin IgG (960 and 3280 mg/min/m3, respectively). Each group was challenged with a lethal dose of aerosolized ricin 1 hr after Ab exposure. All group 1 (control Ab) mice developed diffuse airway epithelial necrosis, with severe interstitial edema and inflammation involving all lung lobes, and died 48-96 hr post-challenge (PC). In contrast, in groups 2 and 3 at 24 hr PC, lung lesions were absent to very mild although there was rare epithelial necrosis in the upper airways in both groups. By 48 hr PC, necrosis of the tracheal epithelium and peritracheal inflammation were noted in some group 3 mice only. By 4 days PC, lungs and airways did not differ from cage controls in most group 2 and 3 mice. Weight gain in group 2 and 3 mice paralleled that of control mice. At 14 days PC, lungs were no different in controls than in group 3 mice. However, two non-survivors in group 3 had obstructions due to proximal airway epithelial damage. All group 2 mice survived, although a mild lymphoplasmacytic perivasculitis was present at 14 days PC which was not noted in the group 3 mice.

PCR screening for carriers of bovine leukocyte adhesion deficiency (BLAD) and uridine monophosphate synthase (DUMPS) in Argentine Holstein cattle.

BLAD (Bovine Leukocyte Adhesion Deficiency) and DUMPS (Deficiency of Uridine Monophosphate Synthase) are monogenic autosomal, recessive inherited diseases of Holstein cattle. Single nucleotide changes (point mutations) responsible for the genetic disorders were detected by polymerase chain reaction coupled with restriction fragment length polymorphism assays (PCR-RFLP). Using oligonucleotide primers, DNA fragments of predicted sizes were amplified, and the products' specificity was assessed by nucleotide sequencing. Mutations were detected in DNA samples from bovine blood and semen by the presence or absence of restriction sites within the PCR amplification products (Taq I, Hae III for BLAD, Ava I for DUMPS). The test included 104 bulls and 950 cows of Argentinean Holstein breed. Defective alleles frequencies were as follows: 2.88% BLAD in bulls used in artificial insemination, 1.79% in cows; 0.96% DUMPS in bulls and 0.11% in cows.

Prophylaxis and treatment with a monoclonal antibody of tetrodotoxin poisoning in mice.

The ability of a tetrodotoxin (TTX)-specific monoclonal antibody to confer passive protection against lethal TTX challenge was investigated. The monoclonal antibody, T20G10, has an estimated affinity for TTX of approximately 10(-9) M and is about 50-fold less reactive with anhydrotetrodotoxin and unreactive with tetrodonic acid by competitive immunoassay. T20G10 specifically inhibited TTX binding in an in vitro radioligand receptor binding assay, but had no effect on the binding of saxitoxin to the sodium channel on rat brain membranes. In prophylaxis studies, mice were administered T20G10 via the tail vein 30 min prior to i.p. TTX challenge (10 micrograms/kg). Under these conditions, 100 micrograms T20G10 protected 6/6 mice, whereas 3/6 mice were protected with 50 micrograms T20G10. Non-specific control monoclonal antibody did not protect against lethality. Therapy studies simulating oral intoxication were performed with mice given a lethal dose of TTX by gavage in a suspension of non-fat dry milk in phosphate-buffered saline. Death occurred within 25-35 min in 6/6 mice not treated with T20G10. However, 500 micrograms T20G10 administered via the tail vein 10-15 min after oral TTX exposure prevented death in 6/6 mice. Lower doses of mAb conferred less protection.

Radioimmunoassay for PbTx-2-type brevetoxins: epitope specificity of two anti-PbTx sera.

Antiserum against PbTx-2-type brevetoxins was produced by immunizing rabbits with a PbTx-2-bovine serum albumin (BSA) conjugate. This serum had a higher affinity, but lower titer, than our current goat serum. Using 4 natural brevetoxins and 6 synthetic derivatives as competitors in our brevetoxin radioimmunoassay, we determined the epitope specificity of both sera. Modification of the backbone structure at C-42 on the K-ring had little or no effect on the antigen-binding capability of either serum. Reduction of the double bond between C-2 and C-3 on the A-ring by reduction of the lactone decreased binding 500 to 750-fold. Epoxidation of the double bond between C-27 and C-28 on the H-ring did not affect binding, which suggested that the goat serum is specific for the A-ring region of the brevetoxin backbone. In contrast, modifying the A-ring had no effect on rabbit serum binding. However, epoxidation of the H-ring decreased binding 5 to 20-fold, which suggested that the rabbit antiserum is specific for the H-ring region of the molecule. These results suggest that assays utilizing only one antibody may not adequately detect toxin metabolites if molecules are altered in the critical region of antibody recognition.