Lack of teratogenicity of microcystin-LR in the mouse and toad.

Microcystin-LR (MC-LR) is a cyanobacterial toxin generated by the organism Microcystis aeruginosa. Although the hepatotoxicity of this chemical has been characterized, the potential developmental toxicity in vertebrates has not been well studied. The purpose of this study was to elucidate the effects of this toxin on the in vivo and in vitro development of mammals and the development of an Anuran (toad). Initial acute toxicity experiments with female CD-1 mice were accomplished with MC-LR administered i.p. in saline. Lethality occurred at 128 and 160 microg kg (-1) and histopathology revealed massive hepatic necrosis with diffuse hemorrhage. Developmental toxicity studies were done with MC-LR administered i.p. for 2-day periods: gestation days 7-8, 9-10 or 11-12. Doses used ranged from 2 to 128 microg kg(-1). On gestation day 17, fetuses were weighed and analyzed for gross morphological and skeletal defects. No treatment-related differences were seen in litter size, viability, weight or the incidence of anomalies. Groups of dams dosed with 32-128 microg kg(-1) on gestation days 7-8, 9-10 or 11-12 were allowed to give birth and the growth and development of their pups were followed postnatally. There were no significant effects noted in the offspring of the treated dams. Neurulation-staged CD-1 mouse conceptuses were exposed to 50-1000 nM MC-LR in whole embryo culture for 24 h. No significant increase in abnormalities or developmental delays was observed. Finally, exposure of the developing toad. Bufo arenarum was done from stage 17 (tail bud) for 10 days at concentrations of 1-20 mg l(-1). No effect on morphological development or survival was noted in any exposed groups. These data indicate that microcystin does not appear to affect development adversely in the mouse (in vivo or in vitro) or the toad at the doses and exposure parameters used.

Placental transfer and pharmacokinetics of a single oral dose of [14C]p-nitrophenol in rats.

The pharmacokinetics and placental transfer of a single oral dose of 100 mg/kg (10 microCi/kg, 16% of acute oral LD50) of uniformly phenyl-labeled [14C]p-nitrophenol were investigated in pregnant Sprague-Dawley rats at 14-18 days of gestation. Three animals were killed on gestation day 18, at 0.5, 1, 2, 4, 12, 24, and 48 h after dosing. Radioactivity was rapidly absorbed and distributed throughout the maternal and fetal tissues. The gastrointestinal tract contents retained 20% and 2% of the dose at 0.5 h and 4 h after dosing. The peak maternal plasma concentration of radioactivity (microg p-nitrophenol equivalent/ml) was 7.17 compared with 0.37 for fetal plasma at 0.5 h. Maximum concentration of radioactivity (microg p-nitrophenol equivalent/g fresh tissue) was detected in most tissues 0.5 h after dosing and was in descending order: kidney 23.27, liver 12.37, placenta 3.56, fetus 2.17, and brain 1.99. Radioactivity was eliminated from plasma and all tissues beiexponentially. The half-lives of elimination of 14C were 34.65 h and 69.30 h for maternal and fetal plasma, respectively. p-Nitrophenol, detected by HPLC, was the major compound identified in plasma and tissues. While p-nitrophenol disappeared biphasically from maternal plasma and kidney, it was eliminated monophasically from brain, placenta, and liver. p-Nitrocatechol and p-aminophenol were detected in the liver with peak concentrations at 0.5 h of 1.13 and 1.00 microg/g fresh tissue, respectively. While the change in the concentration of p-nitrocatechol with time was monophasic, that of p-aminophenol showed a biphasic pattern with elimination half-lives of 1.93 h and 4.95 h, respectively. Radioactivity was rapidly excreted in the urine mostly as polar metabolites, while only 3% of the dose was recovered in the feces. Radioactive materials excreted in the urine comprised: glucuronides 4%, sulfates 8%, hot-acid hydrolysates 11%, nonconjugated compounds 16%, and water-soluble metabolites 61%. This study demonstrated that although orally administered p-nitrophenol is a rapidly absorbed and excreted compound, it is transported to the maternal brain and the fetus and may pose a health risk following exposure to toxic doses during pregnancy.

Perceptions of veterinarians regarding privatization of veterinary services delivery in Ghana and Jamaica.

There are on-going reforms in the delivery of veterinary services in many developing countries, with privatization of certain veterinary activities as one of the approaches. In Jamaica, with the support of veterinarians, clinical aspects of veterinary services were privatized in 1992. In contrast, Ghanaian veterinarians are generally wary of the government's on-going privatization process. The objective of this study was to find out if perceptions of the veterinarians from these two countries on certain issues of privatization were sufficiently different to explain the willingness or reluctance to go into private practice. The response proportions for predominantly self-administered questionnaires were 83% (121/145) and 92% (35/38) for Ghana and Jamaica, respectively. There was a very good (92%) agreement in the perceptions of veterinarians in Ghana and Jamaica on a battery of 24 responses pertaining to privatization of veterinary services. Generally, the perceptions of the veterinarians in Ghana and Jamaica were similar even though the predominant delivery systems for animal health services were different. Therefore, reasons other than those examined in this study may explain the differences in willingness to go into private practice. The need to account for these other reasons is discussed.

Financing the delivery of animal health services in developing countries: a case study of Ghana.

Inadequate financing for the delivery of animal health services in many developing countries has been blamed for lack of efficiency and effectiveness of veterinary services. There are no reports of how the delivery of veterinary services in Ghana is financed. The aim of this paper is to provide information on the funding of veterinary services in Ghana to help in decision making on resource allocation. Various indicators and measures were used in assessing the adequacy of financing and resource allocation from 1990 to 1995. These measures were the veterinary budget as proportions of the national budget, GDP and AGDP; the proportions of the veterinary budget allocated to salaries; the ratios of salaries to non-staff expenditure and of non-staff expenditure to veterinary livestock units and technical staff; coefficient of efficacy; and R-ratio. These generally declined or worsened over the period, deviating from recommended norms where such norms exist. This confirmed the paucity of financing and resource allocation for the delivery of veterinary services. Revenue generation from cost recovery over the 1993-95 period was a potential source of funding, exceeding 100% of non-staff expenditure for 1993 and 1994. However, the revenue generated was not channelled back to veterinary services but went to the national coffers. This served as a disincentive. There is an urgent need to review how veterinary services are financed in Ghana, if the delivery of services is to improve in efficiency and effectiveness.

Perceived constraints to privatization of delivery of veterinary services in Ghana.

Ghana is on the verge of privatizing selected activities in the delivery of animal health services. However, various constraints are being encountered. The aim of this paper is to identify these constraints so as to help find and solutions to them. Questionnaires were administered to veterinarians in Ghana to elicit their responses on various issues concerning privatization. A significant proportion (61%) of government veterinarians, who formed 94% of the respondents, were unwilling to go into private practice. Among the reasons given were that private practice was too risky, that farmers were unwilling or unable to pay for services, that capital to start practices was lacking and that the societal value for animals was low. Also, low livestock densities in many areas and the absence of commercial livestock farming were perceived as deterrents to the sustainability of private practice. Furthermore, the poor macroeconomic environment of high inflation, high interest rates and unstable currency discouraged investment. If privatization of veterinary services is to succeed in Ghana, these perceptions have to be addressed and solutions found, since veterinarians are the targets of the privatization process.

Perceptions of veterinarians regarding privatization of veterinary services delivery in Ghana and Jamaica

There are on-going reforms in the delivery of veterinary services in many developing countries, with privatization of certain veterinary activities as one of the approaches. In Jamaica, with the support of veterinarians, clinical aspects of veterinary services were privatized in 1992. In contrast, Ghanian veterinarians are generally wary of the government's on-going privatization process. The objective of this study is to find out if perceptions of the veterinarians from these two countries on certain issues of privatization were sufficiently different to explain the willingness or reluctance to go into private practice. The response proportions for predominantly self-administered questionnaires were 83 percent (121/145) and 92 percent (35/38) for Ghana and Jamaica, respectively. There was a very good (92 percent) agreement in the perceptions of veterinarians in Ghana and Jamaica on a battery of 24 responses pertaining to privatization of veterinary services. Generally, the perceptions of the veterinarians in Ghana and Jamaica were similar even though the predominant delivery systems for animal health services were different. Therefore, reasons other than those examined in this study may explain the differences in willingness.(Au)

A preliminary survey of antibiotic residues and viable bacteria in milk from three Caribbean Basin countries.

There is widespread concern about the presence of antimicrobial drugs in milk. The presence of drug residues in milk may have public health implications. Milk samples (n = 25 to 65/country) were collected from bulk tanks and commercial vendors in Barbados, Costa Rica, and Jamaica between February 1996 and August 1997. Bulk tank samples were collected from high milk-producing regions of Jamaica and Costa Rica and from 26 dairy farms in Barbados. Milk pH, bacterial growth (total CFU/ml and the presence of Streptococcus agalactiae and Staphylococcus aureus), and the presence of antimicrobials were determined. Milk samples were tested by a microbial inhibition test (Delvotest-P, Gist-Brocades Food Ingredients, Inc.) to screen for antimicrobial drugs. All positives were retested for the presence of beta-lactam antibiotics after incubating with penicillinase and some positives were identified by high-pressure liquid chromatography-UV. Mean pH values ranged from 6.5 to 6.7. S. aureus was identified in bulk tank samples from Costa Rica (52%), Barbados (44%), and Jamaica (46%). S. agalactiae was identified in bulk tank samples from Costa Rica (28%), Barbados (8 and 16%), and Jamaica (18%). Antimicrobial residues were detected in some bulk tank samples from Barbados (8%) and Jamaica (10%) but not in samples from Costa Rica. All positives in milk from Jamaica and Barbados were determined to be beta-lactams. No residues were detected in pasteurized milk samples from Barbados or ultrahigh-temperature milk from Jamaica. The presence of beta-lactam residues in some of these samples suggests the appropriateness of testing milk prior to processing for consumption.

A preliminary survey of antibiotic residues and viable bacteria in milk from three Caribbean Basin countries

There is widespread concern about the presence of antimicrobial drugs in milk. The presence of drug residues in milk may have public health implications. Milk samples (n = 25 to 65/country) were collected from bulk tanks and commercial vendors in Barbados, Costa Rica, and Jamaica between February 1996 and August 1997. Bulk tank samples were collected from high milk-producing regions of Jamaica and Costa Rica and from 26 dairy farms in Barbados. Milk pH, bacterial growth (total CFU/ml and the presence of Streptococcus agalactiae and Staphylococcus aureus), and the presence of antimicrobials were determined. Milk samples were tested by a microbial inhibition test (Delvotest-P, Gist-Brocades Food Ingredients, Inc.) to screen for antimicrobial drugs. All positives were retested for the presence of beta-lactam antibiotics after incubating with penicillinase and some positives were identified by high-pressure liquid chromatography-UV. Mean pH values ranged from 6.5 to 6.7. S. aureus was identified in bulk tank samples from Costa Rica (52 percent), Barbados (44 percent), and Jamaica (46 percent). S. agalactiae was identified in bulk tank samples from Barbados (8 percent) and Jamaica (10 percent) but not in samples from Costa Rica. All positives in milk from Jamaica and Barbados were determined to be beta-lactams. No residues were detected in pasteurized milk samples from Barbados or ultrahigh-temperature milk from Jamaica. The presence of beta-lactam residues in some of these samples suggests the appropriateness of testing milk prior to processing for consumption.(Au)

Financing the delivery of public-sector animal health services in Jamaica: pre- and post-privatization

Lack of adequate financing was a major reason for the privatization of veterinary services in Jamaica in 1992. The belief was that, with privatization, funding of animal health services delivery would improve, since staff numbers and clinical activities undertaken by the Veterinary Division were reduced. However, analyses of data revealed that, in most cases, privatization neither improved nor stemmed the declines, that had started before privatization, in the measures or indicators used. It was concluded that privatization of veterinary services did not result in any appreciable improvement in the financing of the delivery of public-sector animal health services in Jamaica in the short term (Au)

Differentially expressed genes associated with 5-Aza-2'-deoxycytidine-induced hindlimb defects in the Swiss Webster mouse.

5-Aza-2'-deoxycytidine (d-AZA) inhibits methylation of DNA, a process that serves as an epigenetic regulator of gene expression. We have shown that d-AZA causes temporally related defects in mice. Gestational day (GD) 10 treatment induced severe long-bone defects of the hindlimb but not the forelimb. Exposure of younger embryos (GD 8 or 9) does not induce similar defects in forelimbs. This limb-dependent response suggests that methylation alterations in genes specific for fore- or hindlimbs may contribute to the observed pattern of defects. Subtraction hybridization (SH) studies were conducted to identify differential expression of DNA subsequent to the administration of d-AZA to mice on GD 10. Hindlimb buds collected from both treated and untreated embryos at 4, 12, and 24 hours post-treatment were used. A clone isolated from the untreated sample (down-regulation in treated tissue) was identified as a member of the murine B1 family of repetitive sequences. The two other clones isolated from the treated tissue (up-regulation) were homologous to avian myogenic regulatory protein mRNA and activin receptor type II gene. Both species are active during embryogenesis. These findings suggest that the isolated clones may have roles in abnormal embryonic development when inappropriately expressed.

Financing the delivery of public-sector animal health services in Jamaica: pre- and post-privatization.

Lack of adequate financing was a major reason for the privatization of veterinary services in Jamaica in 1992. The belief was that, with privatization, funding of animal health services delivery would improve, since staff numbers and clinical activities undertaken by the Veterinary Division were reduced. However, analyses of data revealed that, in most cases, privatization neither improved nor stemmed the declines, that had started before privatization, in the measures or indicators used. It was concluded that privatization of veterinary services did not result in any appreciable improvement in the financing of the delivery of public-sector animal health services in Jamaica in the short term.

Critical period of carbon tetrachloride-induced pregnancy loss in Fischer-344 rats, with insights into the detection of resorption sites by ammonium sulfide staining.

Several low-molecular weight halocarbons have been shown to cause full-litter resorption (FLR), i.e., pregnancy loss, in Fischer-344 rats treated during organogenesis. To determine periods of gestation sensitive to acute exposure, a single dose of 150 mg carbon tetrachloride (CCl4)/kg was administered on gestation day (GD) 6, 7, 8, 10, or 12. Fetuses were delivered by cesarean section on GD 20. Non-gravid uteri were examined for resorption sites, placed in 10% ammonium sulfide, and re-examined for stained resorption sites approximately 1 and 4.5 hr later. FLR was seen in 4% (1/27) of control dams and 36% (4/11), 54% (7/13), 72% (18/25), 54% (7/13), and 0% (0/12) of dams treated on GD 6, 7, 8, 10, and 12, respectively. Ammonium sulfide staining clearly yielded a more accurate account of the incidence of FLR. The technique was most effective when the staining period was extended to 4.5 hr, as two cases of FLR were revealed that had been undetected after 1 hr of staining. For dams with FLR, staining was required to detect resorption sites in all dams treated on GD 6 or 7, most dams treated on GD 8, and one dam treated on GD 10. Fewer implantation sites were detected in the dams treated on GD 6, and the size of the stained resorption sites increased as the day of treatment was delayed. These findings demonstrate a relationship between the time of toxicant exposure and the size and detectability of resorption sites near term, suggesting that the size of the resorption site may reliably reflect the time of embryonic death. Treatment on GD 8 caused the highest incidence of FLR and will be used in subsequent mechanistic research.

Teratogenic effects of the demethylating agent 5-aza-2'-deoxycytidine in the Swiss Webster mouse.

5-Aza-2'-deoxycytidine (d-AZA) replaces cytidine in DNA thereby altering gene expression by passively removing methyl groups. This study determined the temporal patterns of morphological defects induced by d-AZA in mice. The dosages (0, 0.3, or 1.0 mg/kg) were administered by a single i.p. injection on gestational days (GD) 8, 9, 10, or 11. Mice were killed on GD 17 and fetal skeletons examined. The 1.0 mg/kg dose elicited characteristic defects for each treatment day: GD 8, supernumerary ribs, (significantly above background), fused vertebrae and ribs; GD 9, cleft palate and vertebral variations; GD 10, hind limb defects (especially phocomelia); GD 11, digital defects of fore and hindlimbs. The known demethylating ability of d-AZA coupled with the induction of longbone defects only in the hindlimbs suggests that d-AZA may act by disrupting specific hindlimb gene function through DNA hypomethylation.

Supernumerary lumbar rib: manifestation of basic alteration in embryonic development of ribs.

Supernumerary ribs (SNR) are a common variant in some strains of mice used in standard teratology bioassays. We have previously demonstrated that increased incidence of SNR may be induced by a wide variety of xenobiotics and/or general maternal stress. The significance of this defect in cross-species extrapolations has been problematic and recent studies, including this one, have shown that this anomaly is more complex than previously thought. The SNR in mice have a bimodal distribution composed of 'rudimentary ribs' (RR) with a mode of 0.3-0.4 mm and 'extra ribs' (ER) with a mode of 0.9-1.1 mm. The studies reported here examine the relationship between the presence of SNR and the 13th rib length and the gross morphological development of the anomaly. Supernumerary ribs were induced in CD-1 mice by surgical stress (subcutaneous micropump implanted on gestational day (GD), restraint stress (GD8), food and water deprivation (GD8) or maternal administration of the pesticide dinoseb (50 mg kg-1 on GD7 and GD8). Fetuses from untreated litters were also examined. Dinoseb-treated mice were killed on GD14, 15, 16 or 17. All other groups were killed on GD17. The lengths of the 13th and 14th ribs were measured and other anomalies were recorded. Femur length was used as an indicator of fetal size. The SNR frequency was higher in all treatment groups compared to controls. We found that ER and RR were morphologically distinct. The ER were flat ended and distally joined by a cartilaginous portion, while RR were usually rounded distally and were without cartilaginous extensions. The 13th ribs were significantly longer in fetuses having SNR than in those not having SNR, whether treated or untreated. This relationship was present in all fetal ages examined and with both ER and RR groups. These results suggest that SNR are indicative of basic alterations in the development of the axial skeleton.

The use of ascorbic acid in the treatment of 2 cases of red maple (Acer rubrum)-poisoned horses.

Two horses with red maple (Acer rubrum) toxicity responded to treatment with high doses of vitamin C (ascorbic acid), in addition to blood transfusions, and intravenous fluid therapy. The clinical course included Heinz body anemia, marked methemoglobinemia, depression, and evidence of severe tissue anoxia. Clinical recovery was dramatic with stabilization achieved 36 hours following the initiation of ascorbic acid therapy.

Evaluation of an indirect enzyme-linked immunosorbent assay for screening antibody against aflatoxins.

Enzyme-linked immunosorbent assay screening of antibody produced against aflatoxin was accomplished by a new and simple procedure. To demonstrate the new indirect ELISA technique used, antibody against aflatoxin M1 was produced in female BALB/CJ mice by immunization with an aflatoxin M1-bovine serum albumin conjugate. Instead of coating test-plate wells with purified antibody (direct ELISA) or synthesizing a second protein-aflatoxin conjugate (aflatoxin M1-poly-L-lysine) to coat test-plate wells, wells were coated with the readily available aflatoxin M1-bovine serum albumin and aflatoxin B1-bovine serum albumin. This method, applicable for any aflatoxin conjugated by the common cyclopentano-carboxymethoxyl-oxime technique, eliminates the more time-consuming and technically difficult portions of earlier direct and indirect ELISA. The new technique can be valuable in continued efforts toward development of new and improved immunoassays against aflatoxin metabolites.

Bilateral renal agenesis in two litters of Shetland sheepdogs.

Two cases of bilateral renal agenesis (1 pup from each of 2 related bitches) were observed in a purebred Shetland sheepdog breeding colony. Gross postmortem and histopathologic examinations confirmed a diagnosis of bilateral renal agenesis as the cause of death. Limited available data strongly suggests an inherited etiology for bilateral renal agenesis. (Editor's Note: This report is published as an example of a situation where circumstantial evidence suggested a cause-and-effect relationship between the change in heartworm medication and the anomalies. Careful scientific data collection and review avoided falling into that trap and identified the genetic basis for the observed defects.

Preliminary study on serum enzyme changes in Long Evans rats given parenteral ochratoxin A, aflatoxin B1 and their combination.

Long Evans rats of both sexes were each administered a total of 250 micrograms aflatoxin B1 (AFB1), ochratoxin-A (OCH-A) or 500 micrograms AFB1 + OCH-A (1:1 ratio) in corn oil over 5 equal daily intraperitoneal (IP) injections of 50 micrograms/rat/day. Control rats were given a total of 1.25 ml corn oil over 5 equal daily IP injections of 0.25 ml/rat. All rats were observed daily for clinical signs of toxicity. Twenty-four hr following the last injection all rats were weighed, killed, examined for gross pathologic lesions and blood samples collected for routine hematologic and serum chemistry evaluation. All rats gained weight over the treatment period. Though not significantly different among the treatment groups, weight gain was significantly greater for males (54.0 g) than females (33.8 g). Routine hematology showed no difference among treatment groups. Serum enzyme aspartate aminotransferase (AST = SGOT) activities were indicative of hepatoxicity. Lactate dehydrogenase (LDH) activity was significantly increased in the AFB1 and AFB1 + OCH-A treatment groups, signifying a possible interaction between these 2 mycotoxins. LDH isoenzyme fractionation studies would be helpful in delineating the organ system(s) involved and the possible diagnostic value of this interaction.

An epidemiological investigation associating aflatoxin M1 with milk production in dairy cattle.

An attempt was made to utilize the analysis for aflatoxin M2 in milk, instead of aflatoxin B1 in feed for a pilot epidemiological study addressing the association of M1 levels and various production parameters. Four dairy farms, representing above average to below average management practices, were identified with 10 animals being randomly selected from each farm for this study. Milk samples and production data were evaluated from these animals in order to gain insight into the possible effects of low level mycotoxin ingestion on production and to test whether or not this approach offered potential for future epidemiological studies. Feed samples were basically negative for the presence of aflatoxin B1, yet milk analysis revealed the presence of M1 in a large percentage of the animals. Further, the M1 levels appeared to be associated with a decrease in daily milk production. This study indicates that employing M1 analysis in milk for epidemiological investigations of low level mycotoxin ingestion effects, instead of B1 feed analysis, is feasible and offers a potential for more definitive studies in this area.