The contraceptive efficacy of a self-assembling intra-uterine device in domestic mares.

OBJECTIVE: The primary aim of this study was to investigate the contraceptive efficacy of a self-assembling uterine device (iUPOD™) in the mare. In addition, the effects of iUPODs on oestrous cyclicity, uterine health and circulating concentrations of cortisol were evaluated. METHODS: Domestic mares underwent oestrous monitoring and artificial insemination. After subsequent ovulation, mares underwent either placement (n = 7) or sham placement (n = 7; controls) of an iUPOD device. Devices were left in place for at least 3 months. Pregnancy diagnoses were carried out 14 days post-ovulation, with any pregnancies terminated at 28 days post-ovulation. All mares underwent weekly blood sampling with or without reproductive examinations throughout the study. Towards the end of the study, multiple serum samples collected over three consecutive days were analysed for concentrations of cortisol. Endometrial biopsies were collected before artificial insemination and during the subsequent breeding season. Endometrial cytology and bacterial cultures were performed before device removal (iUPOD mares) or at the end of the study (control mares). RESULTS: Pregnancies were diagnosed in 0 of 7 iUPOD mares versus 7 of 7 control mares. Placement of iUPODs was associated with extended luteal phases and variable accumulations of intra-uterine fluid. Bacterial culture results suggested that the mild endometritis associated with iUPODs was sterile in six of seven mares. Short-term placement of iUPODs had no detrimental effects on endometrial architecture. Mean serum cortisol concentrations were significantly lower in iUPOD mares than control mares. CONCLUSION: iUPODs represent a promising means of fertility control in the mare.

Evaluation of PCR assays for the detection of Campylobacter fetus in bovine preputial scrapings and the identification of subspecies in South African field isolates.

As a result of the high lability and slow growth of Campylobacter fetus subspecies, the laboratory diagnosis of bovine genital campylobacteriosis has always been difficult. This is especially true under South African conditions, where farms are far apart, laboratories are only present in major centres and there are high ambient temperatures. In order to overcome the shortcomings associated with traditional diagnostic methods, the implementation of a molecular assay was sought. This work describes how a previously published PCR assay (MG3F/ MG4R primers) was adapted, optimised and applied in the diagnostic laboratory to test preputial samples directly for the presence of Campylobacter fetus. Field evaluation of the assay revealed an analytical sensitivity and specificity of 85.7% and 99%, respectively. Subsequent genotyping and phenotyping of a diverse collection of South African field isolates revealed that South Africa has an unexpected and previously unreported high incidence of Campylobacter fetus subsp. venerealis biovar intermedius strains. These strains were not identified correctly by the subspecies-specific primer set evaluated. Until such time that cost- effective genotyping methods are available to diagnostic laboratories in South Africa, and other countries with these atypical Campylobacter fetus subsp. venerealis strains, the need for bacterial culture will persist. Identification to subspecies level of isolates at present remains dependent upon a single phenotypic criterion, namely tolerance to 1% glycine.

A serological survey of antibodies to Leptospira species in dogs in South Africa.

Leptospirosis, a disease more common in the tropics, can cause a life-threatening multisystemic syndrome in humans and animals. Immunity, whether natural or vaccine-induced, is serogroup-specific with the infecting serovars varying according to geographical locality. In South Africa, in spite of the fact that the bacterin vaccine for some Leptospira serovars is often used, there is no recent information on the incidence of canine leptospirosis as well as the infecting serovar/s. The aim of this study, which was undertaken on sera collected in 2008 and 2009 from both strays and owned dogs predominantly in the coastal regions of South Africa, was to determine the presence of leptospiral antibodies to 15 serovars known to infect dogs. Of the 530 samples tested, 25 tested positive to 7 different serovars with the microscopic agglutination test (MAT). Nine of the 25 samples tested positive to more than one serovar. The 2 serovars most frequently represented were Canicola, which reacted to 17 sera, and Pyrogenes, which reacted to 10 sera. Currently the only vaccines available in South Africa in different combinations contain serovars Canicola, Icterohaemorrhagiae, Pomona and Grippotyphosa. The results showed that the use of vaccines containing serovar Canicola is still justifiable in certain regions of the country. However, the presence of antibodies to serovar Pyrogenes in several dogs, pending a broader investigation, indicates that this serovar should also be included in the range of Leptospira vaccines for use in South Africa.

Antimicrobial susceptibility in thermophilic Campylobacter species isolated from pigs and chickens in South Africa.

Campylobacter jejuni is one of the leading causes of sporadic food-borne bacterial disease in humans. In intensive poultry and pig rearing systems the use of oral antibiotics is essential to maintain health. Consequently, there is a high risk for the thermophilic Campylobacter jejuni and C. coli resident in the intestinal tract of food animals to develop resistance to commonly used antibiotics. Contamination of meat or eggs with pathogenic strains of resistant Campylobacter could, therefore, result in a form of campylobacteriosis in humans that is difficult to treat. The aim of this investigation was to determine the antimicrobial susceptibility of thermophilic Campylobacter spp. isolated from pigs and poultry by the broth microdilution minimum inhibitory concentration (MIC) test. A total of 482 samples from the Western Cape and Gauteng provinces was collected and analysed. Thirty-eight Campylobacter isolates were obtained. Analysis of data revealed that C. jejuni strains mainly of poultry origin were more resistant to the fluoroquinolones, macrolides and tetracyclines and the C. coli strains were more resistant to the macrolides and lincosamides. Multi-resistance was also detected in 4 Campylobacter strains from the Western Cape. With the exception of tetracyclines, strains from high health Gauteng broiler farms were susceptible to antibiotics used to treat Campylobacter infections.

Case-control study to determine whether river water can spread tetracycline resistance to unexposed impala (Aepyceros melampus) in Kruger National Park (South Africa).

A case-control study was performed in the Kruger National Park (KNP), South Africa, to find out whether impala (Aepyceros melampus) were more likely to harbor tetracycline-resistant Escherichia coli (TREC) in their feces when they drank from rivers that contained these bacteria than when they drank from rivers that were uncontaminated with TREC. The following five perennial rivers were selected: the Crocodile, the Letaba, the Olifants, the Sabie, and the Sand. Samples of river water (n = 33) and feces (n = 209), collected at 11 different sites, were cultured for E. coli. The resulting colonies were screened for tetracycline resistance by use of the Lederberg replica plating method (breakpoint, 4 mg/liter). A resistant and/or a susceptible isolate was then selected from each sample and subjected to the CLSI MIC broth microdilution test for tetracyclines. Among the 21 water specimens contaminated by E. coli, 19.05% (n = 4) were found to be resistant by the MIC method (breakpoint, >/=8 mg/liter). This led to the Crocodile, Olifants, and Letaba rivers being classified as TREC positive. Among the 209 impala feces sampled, 191 were positive for the presence of E. coli (91.38%). Within these (n = 191), 9.95% (n = 19) of the isolates were shown to be TREC by the MIC method. It was found that 1.11% (n = 1) of the E. coli isolates cultured from the feces of the control group (n = 90) were TREC, in comparison with 17.82% (n = 18) of those in feces from the exposed group (n = 101). The calculation of the odds ratio showed that impala drinking from TREC-contaminated rivers were 19.3 (2.63 to 141.69) times more likely to be infected with TREC than were unexposed impala. This is a significant finding, indicating that surface water could be a possible source of antimicrobial resistance in naïve animal populations and that impala could act as sentinels for antimicrobial resistance.

Antimicrobial drug resistance of Escherichia coli isolated from poultry abattoir workers at risk and broilers on antimicrobials.

Antimicrobial usage in food animals increases the prevalence of antimicrobial drug resistance among their enteric bacteria. It has been suggested that this resistance can in turn be transferred to people working with such animals, e.g., abattoir workers. Antimicrobial drug resistance was investigated for Escherichia coli from broilers raised on feed supplemented with antimicrobials, and the people who carry out evisceration, washing and packing of intestines in a high-throughput poultry abattoir in Gauteng, South Africa. Broiler carcasses were sampled from 6 farms, on each of which broilers are produced in a separate 'grow-out cycle'. Per farm, 100 caeca were randomly collected 5 minutes after slaughter and the contents of each were selectively cultured for E. coli. The minimum inhibitory concentration (MIC) of each isolate was determined for the following antimicrobials: doxycycline, trimethoprim, sulphamethoxazole, ampicillin, enrofloxacin, fosfomycin, ceftriaxone and nalidixic acid. The same was determined for the faeces of 29 abattoir workers and 28 persons used as controls. The majority of isolates from broilers were resistant, especially to antimicrobials that were used on the farms in the study. Overall median MICs and the number of resistant isolates from abattoir workers (packers plus eviscerators) tended to be higher than for the control group. However, no statistically significant differences were observed when the median MICs of antimicrobials used regularly in poultry and percentage resistance were compared, nor could an association between resistance among the enteric E. coli from packers and those from broilers be demonstrated.

Prevalence of virulent Rhodococcus equi in isolates from soil collected from two horse farms in South Africa and restriction fragment length polymorphisms of virulence plasmids in the isolates from infected foals, a dog and a monkey.

The prevalence of virulent Rhodococcus equi in soil isolates from two horse farms in South Africa and nine clinical isolates from six foals, a foal foetus, a dog, and a monkey was investigated. The isolates were tested for the presence of virulence plasmid DNA and 15- to 17-kDa antigens by immunoblotting. Rhodococcus equi was isolated from almost all of the soil samples obtained from the two farms with 5.0 x 10(1) to 3.3 x 10(4) colony forming units per gram of soil. Virulent R. equi was isolated from three soil samples from one of the farms and appeared in 3.8% (three of 80 isolates), but not in any of the 182 isolates from the other farm. Of the three virulent R. equi isolates, one contained an 85-kb type I plasmid and two an 87-kb type I plasmid. Of nine clinical isolates from the foals, foal foetus, dog and monkey, five from the foals were virulent R. equi which expressed the virulence-associated antigens and contained a virulence plasmid 85-kb type I, and were all isolated from cases of pneumonia typical of that induced by R. equi in young foals living in widely separated areas in South Africa. The isolates from the other four foals, the dog and the monkey were avirulent R. equi.

Temporary remission of disseminated paecilomycosis in a German shepherd dog treated with ketoconazole.

Disseminated mycosis caused by Paecilomyces varioti in a female German shepherd dog presented with chronic forelimb lameness is described. Radiographs of the swollen carpal joint revealed geographic lysis of the radial epiphysis. Diagnosis was based on cytological demonstration of fungal hyphae and chlamydiospores, as well as fungal culture of fluid obtained by arthrocentesis. Temporary remission was characterised by markedly improved clinical signs and laboratory parameters, following treatment with ketoconazole. The dog was euthanased 9 months after the initial diagnosis, following the diagnosis of multifocal discospondylitis. This appears to be the longest described period of temporary remission obtained with treatment in dogs with paecilomycosis. Clinical, clinicopathological and necropsy findings of this disease in another German shepherd dog are briefly described.

Structure-activity relationships and pharmacokinetic analysis for a series of potent, systemically available biphenylsulfonamide matrix metalloproteinase inhibitors.

A series of biphenylsulfonamide derivatives of (S)-2-(biphenyl-4-sulfonylamino)-3-methylbutyric acid (5) were prepared and evaluated for their ability to inhibit matrix metalloproteinases (MMPs). For this series of compounds, our objective was to systematically replace substituents appended to the biphenyl and alpha-position of 5 with structurally diverse functionalities to assess the effects these changes have on biological and pharmacokinetic activity. The ensuing structure-activity relationship (SAR) studies showed that biphenylsulfonamides substituted with bromine in the 4'-position (11c) significantly improved in vitro activity and exhibited superior pharmacokinetics (C(max), t(1/2), AUCs), relative to compound 5. Varying the lipophilicity of the alpha-position by replacing the isopropyl group of 11c with a variety of substituents, in general, maintained potency versus MMP-2, -3, and -13 but decreased the oral systemic availability. Subsequent evaluation of its enantiomer, 11c', showed that both compounds were equally effective MMP inhibitors. In contrast, the corresponding hydroxamic acid enantiomeric pair, 16a (S-isomer) and 16a' (R-isomer), stereoselectivity inhibited MMPs. For the first time in this series, 16a' provided nanomolar potency against MMP-1, -7, and -9 (IC(50)'s = 110, 140, and 18 nM, respectively), whereas 16a was less potent against these MMPs (IC(50)'s = 24, 78, and 84 microM, respectively). However, unlike 11c, compound 16a' afforded very low plasma concentrations following a single 5 mg/kg oral dose in rat. Subsequent X-ray crystal structures of the catalytic domain of stromelysin (MMP-3CD) complexed with inhibitors from closely related series established the differences in the binding mode of carboxylic acid-based inhibitors (11c,c') relative to the corresponding hydroxamic acids (16a,a').

Cryptococcosis in captive cheetah (Acinonyx jubatus): two cases.

Cryptococcus neoformans is a yeast-like organism associated with pulmonary, meningoencephalitic, or systemic disease. This case report documents 2 cases of cryptococcosis with central nervous system involvement in captive cheetah (Acinonyx jubatus). In both cases the predominant post mortal lesions were pulmonary cryptococcomas and extensive meningoencephalomyelitis. Both cheetahs tested negative for feline immunodeficiency virus and feline leukaemia virus. The organism isolated in Case 2 was classified as Cryptococcus neoformans var. gattii, which is mainly associated with disease in immunocompetent hosts.

alpha-Substituted malonester amides: tools to define the relationship between ACAT inhibition and adrenal toxicity.

We prepared a series of alpha-substituted malonester amides that were evaluated for their ability to inhibit acyl-CoA:cholesterol O-acyl transferase activity in vitro and to lower plasma total cholesterol levels in a variety of cholesterol-fed animal models. Compounds of this series were also useful in examining the relationship between adrenal toxicity and ACAT inhibition. One compound from this series, 9f, was a potent inhibitor of ACAT in both the microsomal and cellular assays. It was also bioavailable as determined by both a bioassay and a HPLC-UV assay. This compound was evaluated in both guinea pig and dog models of adrenal toxicity and compared to tetrazole amide 15. In the most sensitive species, the dog, both of these compounds achieved good plasma levels; however, compound 9f caused adrenal necrosis, whereas compound 15 had no effect on the adrenal gland. This adds to the growing body of evidence that the adrenal toxicity observed with ACAT inhibitors may not be mechanism related.

Inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT) as hypocholesterolemic agents: synthesis and structure-activity relationships of novel series of sulfonamides, acylphosphonamides and acylphosphoramidates.

Sulfoacetic acid, phosphoramidate, and phosphoramide analogs of the ACAT inhibitors, CI-999 and CI-1011 were synthesized. The structure-activity relationships of these compounds as ACAT inhibitors are described.

Heterocyclic amides: inhibitors of acyl-CoA:cholesterol O-acyl transferase with hypocholesterolemic activity in several species and antiatherosclerotic activity in the rabbit.

A series of heterocyclic amides were synthesized and evaluated as inhibitors of acyl-CoA: cholesterol O-acyltransferase (ACAT) in vitro and for cholesterol lowering in cholesterol-fed rats. Compounds were evaluated for cell-based macrophage ACAT inhibition, bioactivity, and adrenal toxicity. Candidates were selected for evaluation in cholesterol-fed dogs and, ultimately, the injured cholesterol-fed rabbit model of atherosclerosis. The heterocyclic amides potently inhibited rabbit liver ACAT (IC50's = 0.014-0.11 microM), and the majority of compounds significantly lowered plasma cholesterol (42-68%) in an acute cholesterol-fed rat model at 3 mg/kg. The most efficacious compounds in the rat were evaluated for bioactivity in vivo and arterial ACAT inhibition in a cell-based macrophage ACAT assay. Two highly bioactive analogs, (+/-)-2-(3-dodecylisoxazol-5-yl)-2-phenyl-N-(2,4,6-trimethoxypheny l) acetamide (13a) and (+/-)-2-(5-dodecylisoxazol-3-yl)-2-phenyl-N-(2,4,6-trimethoxypheny l) acetamide (16a), were selected for further study and were found to be nontoxic in a guinea pig model of adrenal toxicity. Compounds 13a and 16a lowered total cholesterol in the cholesterol-fed rat, rabbit, and dog models of pre-established hypercholesterolemia. Compound 13a in the injured cholesterol-fed rabbit model of atherosclerosis was effective in slowing the development of cholesteryl ester-rich thoracic aortic lesions, reducing lesion coverage by 53% at a dose of 1 mg/kg.

Inhibitors of acyl-CoA:cholesterol O-acyltransferase. synthesis and pharmacological activity of (+/-)-2-dodecyl-alpha-phenyl-N-(2,4,6-trimethoxyphenyl)-2H-tetrazole-5- acetamide and structurally related tetrazole amide derivatives.

A series of tetrazole amide derivatives of (+/-)-2-dodecyl-alpha-phenyl-N-(2,4,6-trimethoxyphenyl)-2H-tetrazole-5- acetamide (1) was prepared and evaluated for their ability to inhibit acyl-CoA: cholesterol O-acyltransferase (ACAT) in vitro and to lower plasma total cholesterol in vivo. For this series of compounds, our objective was to systematically replace substituents appended to the amide and tetrazole moieties of 1 with structurally diverse functionalities and assess the effect that these changes have on biological activity. The ensuing structure-activity relationship (SAR) studies identified aryl (7b) and heteroaryl (7f,g) replacements for 2,4,6-trimethoxyphenyl that potently inhibit liver microsomal and macrophage ACAT in vitro and exhibit good cholesterol lowering activity (56-66% decreases in plasma total cholesterol at 30 mg/kg), relative to 1, when compared in the acute rat model of hypercholesterolemia. Replacement of the alpha-phenyl moiety with electron-withdrawing substituents (13e-h), however, significantly reduced liver microsomal ACAT inhibitory activity (IC50 > 1 microM). This is in contrast to electron-donating substituents (13ij,m-q), which produce IC50 values ranging from 5 to 75 nM in the hepatic microsomal assay. For selected tetrazole amides (1, 7b, 13n,o), reversing the order of substituents appended to the 2- and 5-positions in the tetrazole ring (36a-d), in general, improved macrophage ACAT inhibitory activity and provided excellent cholesterol-lowering activity (ranging from 65% to 77% decreases in plasma total cholesterol at 30 mg/kg) in the acute rat screen. The most potent isomeric pair in this set of unsubstituted methylene derivatives (13n and 36a) caused adrenocortical cell degeneration in guinea pigs treated with these inhibitors. In contrast, adrenal glands taken from guinea pigs treated with the corresponding alpha-phenyl-substituted analogs (7b and 36c) were essentially unchanged compared to untreated controls. Subsequent evaluation of 7b and 36c in a rabbit bioassay showed that both compounds and/or their metabolities were present in plasma after oral dosing. Unlike 7b and 36c, compound 1 and related 2,4,6-trimethoxyanilides (13j, 30c,d) showed poor oral activity in the rabbit bioassay. Nevertheless, in cholesterol-fed rabbits, both systemically available (7b, 36c) and poorly absorbed inhibitors (1, 36d) were more effective in lowering plasma total cholesterol than the fatty acid amide CI-976.

Inhibitors of acyl-CoA:cholesterol O-acyltransferase. 17. Structure-activity relationships of several series of compounds derived from N-chlorosulfonyl isocyanate.

Several series of acyl-CoA:cholesterol O-acyltransferase inhibitors were prepared by the stepwise addition of nitrogen, oxygen, and sulfur nucleophiles to N-chlorosulfonyl isocyanate. The (aminosulfonyl)ureas 3-44 were the most potent inhibitors in vitro, with several compounds having IC50 values < 1 microM. Although the other series of compounds were not as potent in vitro, many compounds did display good in vivo activity in cholesterol-fed rats. Several of the oxysulfonyl carbamates (including CI-999, 115) showed excellent lipid-lowering activity in the chronic in vivo screen, demonstrating significant cholesterol lowering in a pre-established hypercholesterolemic state.

Inhibitors of acyl-CoA:cholesterol O-acyltransferase. 11. Structure-activity relationships of several series of compounds derived from N-(chlorocarbonyl) isocyanate.

Five series of compounds (4-9) derived from N-(chlorocarbonyl) isocyanate have been synthesized and evaluated for their ability to inhibit the enzyme acyl-CoA:cholesterol O-acyltransferase and lower plasma cholesterol levels in cholesterol-fed rats. Structure-activity relationships indicate that the imino dicarboxylates (6 and 7) and the oxycarbonyl thiocarbamates (8) are the most potent and efficacious series. In these series, the combination of a 2,6-diisopropylphenyl group and an aliphatic alkyl group with a chain length between 6 and 14 carbon atoms gives good activity in vitro and in vivo. In addition, a hydrogen donor is required to maintain good in vitro activity, and the acidic proton on the central nitrogen in these series appears to be important for in vivo activity.

Influence of intravenously administered lidocaine on cerebral blood flow in a baboon model standardized under controlled general anaesthesia using single-photon emission tomography and technetium-99m hexamethylpropylene amine oxime.

The baboon under general anaesthesia as a model to assess drug-induced cerebral blood flow changes (delta CBF) using single-photon emission tomography (SPET) offers great in vivo possibilities but has to comply with demands on control of anaesthesia-related influencing factors, such as PaCO2 changes. The model sought in this study and described here allows control of PaCO2, in the baboon under thiopentone anaesthesia by ventilation, and was evaluated for the functional dependence of delta CBF vs delta PaCO2, using SPET technetium-99m hexamethylpropylene amine oxime (HMPAO) and the split-dose method together with controlled ventilation. During the experiment the model was validated for normal reactivity to PaCO2 changes, and subsequently applied to investigate the mechanisms (still uncertain) of CBF increase known to follow administration of the local anaesthetic lidocaine. Six baboons received 6 mg/kg lidocaine intravenously. CBF was measured between two consecutive SPET acquisitions (split-dose method) respectively relating to HM-PAO distributions in the brain before and after the injection of lidocaine. Meanwhile the animals were maintained at constant respiratory rate and volume. The results indicate that the correlation between delta CBF and the ensuing fall in PaCO2 deviated from the baseline pattern from the model and confirmed a cerebrovascular contribution to the lidocaine-induced CBF increase. This agreed well with mean and systolic blood pressure changes and heart rate.

Inhibitors of acyl-Coa:cholesterol acyltransferase. 4. A novel series of urea ACAT inhibitors as potential hypocholesterolemic agents.

We have synthesized a series of N-phenyl-N'-aralkyl and N-phenyl-N'-(1-phenylcycloalkyl)ureas as inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). This intracellular enzyme is thought to be responsible for the esterification of dietary cholesterol; hence inhibition of this enzyme could reduce diet-induced hypercholesterolemia. For this series of compounds, the in vitro ACAT inhibitory activity was improved by increasing the bulk of the 2,6-substituents on the phenyl ring. Additionally, we found that spacing of the aromatic rings was critical for ACAT inhibitory activity. A phenyl ring five atoms away from the requisite 2,6-diisopropylphenyl moiety was optimal for in vitro activity. Substitution alpha to the N'-phenyl moiety enhanced in vitro potency. In the case of phenylcycloalkyl ureas, ACAT inhibitory activity was independent of the size of the cycloalkyl ring. From this series of analogs, compound 25, which had excellent in vitro potency for inhibiting ACAT, was found to lower plasma cholesterol by 73% in vivo when administered in the diet at 50 mg/kg in an animal model of hypercholesterolemia. In this model, compound 25 lowered plasma cholesterol dose dependently and was as efficacious as the Lederle ACAT inhibitor CL 277082.