Farm level and geographic predictors of antibiotic use in Sri Lankan shrimp farms

Black tiger shrimp Penaeus monodon farming is important for Sri Lanka's rural development plans. Consumer confidence is critical for the development and maintenance of export and domestic shrimp markets. Public concern about the use of antimicrobial drugs and chemicals on shrimp farms, however, could threaten market access. We sought to identify high-risk areas and farm-level risk factors for antimicrobial use to inform the core messages and strategic placement of extension programs to help farmers develop best management practices for antimicrobial use. We undertook a survey of 603 operating farms within the Puttalam district over 42 weeks. Lower stocking density and early harvest were associated with a lower risk of antimicrobial use, whereas standard management practices, including water treatment, feed supplements, probiotic use, pond fertilizing, disinfectant use, and pesticide use, were associated with increased risk. Spatial cluster detection found three significant clusters of antimicrobial-using farms. Antimicrobials were more likely to be used in areas with lower farm density. Some of our counterintuitive findings are discussed from a socioecological perspective. A comprehensive understanding of why antimicrobials are used on shrimp farms requires an evaluation of the physical, epidemiological, and socioeconomic factors.     

Pyrosequencing analysis of the beta-tubulin gene in Spanish Teladorsagia circumcincta field isolates

Benzimidazole (BZ) resistance in gastrointestinal nematodes has been associated with single nucleotide polymorphisms (SNPs) at codons 200, 167 and 198 in the beta-tubulin isotype 1 gene and, recently, these SNPs have also been found in macrocyclic lactone (ML) resistant strains of Haemonchus contortus. On this basis, we have studied the same putative SNPs in Spanish Teladorsagia circumcincta field isolates by pyrosequencing. Single L3 (infective 3rd stage larvae) from five sheep flocks were tested after confirming their BZ susceptibility and degree of ivermectin (IVM) resistance. According to the Faecal Egg Count Reduction Test (FECRT) one flock was classified as IVM susceptible, another one was resistant, and the rest had a suspicion of resistance to IVM. DNA extraction was carried out on 598 single L3 and 56% of these were identified as T. circumcincta after the amplification of a species-specific ITS2 fragment. The number of L3 analyzed for the SNPs 198/200 was 255 and for the SNP 167 was 187. Results clearly indicate no resistance-associated SNPs were present at any codon, before or after treatment. Therefore, all T. cicumcincta L3 were designated as susceptible homozygous genotypes for all SNPs. The absence of the mutations in these populations would argue against resistance haplotypes being present in the parasite population prior to drug treatment, at least in Spanish T. circumcincta.     

In vitro screening of plant lectins and tropical plant extracts for anthelmintic properties

Lectins are plant secondary metabolites (PSM) found in many forages and which may confer anthelmintic properties to gastrointestinal parasites through disrupting the development of parasitic larvae throughout its life cycle. In experiment 1, the ability of the plant lectins jacalin (JAC), concanavalin A (Con A), phytohemagglutinin E2L2 (PHA-E2L2), phytohemagglutinin L4 (PHA-L4), phytohemagglutinin E3L (PHA-E3L), kidney bean albumin (KBA), Robinia pseudoacacia agglutinin (RPA), Maackia amurensis lectin (MAA), Maclura pomifera agglutinin (MAA), Dolichos biflorus agglutinin (DBA), wheat germ agglutinin (WGA) and Galanthus nivalis agglutinin (GNA) to disrupt the feeding of the first stage larvae (L(1)) of the sheep gastro-intestinal nematodes (GIN) Teladorsagia circumcincta, Haemonchus contortus and Trichostrongylus colubriformis was investigated using a larval feeding inhibition test (LFIT). Only PHA-E3L, WGA and Con A had a potent effect on disrupting larval feeding of all of the three species of GIN investigated. The lectin concentration required to inhibit feeding in 50% of L(1) (IC50) was 7.3±1.2, 8.3±1.4 and 4.3±1.7 μg/ml for PHA-E3L; 59.1±32.4, 58.7±11.9 and 8.1±7.0 μg/ml for Con A and 78.9±11.2, 69.4±8.1 and 28.0±14.1 μg/ml for WGA for T. circumcincta, H. contortus and T. colubriformis larvae, respectively (P=0.006). The addition of the lectin inhibitors fetuin, glucose/mannose or N-acetylglucosamine for PHA-E3L, Con A and WGA, respectively, caused an increase in the proportion of larvae that had fed at all concentrations for PHA-E3L only. In experiment 2, the effect of extracts from the tropical plants Azadiractha indica, Trichanthera gigantea, Morus alba, Gliricidia sepium and Leucaena leucocephala on the feeding behaviour of H. contortus L(1,) was examined. A. indica, T. gigantea and M. alba failed to inhibit 50% of larvae from feeding at concentrations up to 10mg plant extract per ml. In contrast, both G. sepium and L. leucocephala demonstrated a dose-dependent effect on larval feeding with respective IC50 estimates (mean±s.e.) of 0.015 mg/ml ±0.001 and 3.465 mg/ml ±0.144, effects which were partly reversed by the inclusion of either the tannin inhibitor polyethylene glycol or the lectin inhibitor Fetuin. These studies demonstrate that plant lectins can have an inhibitory effect on the feeding behaviour of first stage larvae of ovine GIN in vitro. Moreover they also provide novel evidence that lectins may contribute to the anthelmintic properties of some tropical forage plant extracts, such as G. sepium and L. leucocephala.     

Development of Immunologic Assays to Measure Response in Horses Vaccinated with Xenogeneic Plasmid DNA Encoding Human Tyrosinase

Xenogeneic plasmid DNA constructs have been developed and optimized for immunotherapies targeting cancer in both humans and dogs. Specifically, plasmid vectors containing the tumor antigen tyrosinase have demonstrated immunoreactivity and clinical benefit in the treatment of melanocytic tumors in these species. Overexpression of tyrosinase has also been noted in equine melanocytic tumors, supporting its role as a valid tumor antigen in the horse. Vaccination with plasmid constructs containing tyrosinase may thus have translational immunoreactivity in the treatment of equine melanomas. Here, we describe a methodology that is highly sensitive and specific for the detection of both humoral and cell-mediated immunoreactivity against tyrosinase in equine patients. These antigen-specific immunoassays are used to measure the humoral and cell-mediated responses in a cohort of horses vaccinated with xenogeneic plasmid DNA encoding human tyrosinase. Serum humoral responses were measured using standard enzyme-linked immunosorbent assay technique against the full-length recombinant human tyrosinase protein. Peripheral blood mononuclear cells were collected from vaccinated horses and stimulated with tyrosinase-specific peptides. Cell-mediated responses were then measured using a novel quantitative real-time-polymerase chain reaction technique to determine resultant interferon-γ expression. All horses developed significantly positive humoral and cell-mediated immune responses compared with their individual prevaccination values. No adverse reactions or signs of autoimmunity were detected. Vaccination with xenogeneic plasmid DNA expressing tyrosinase appears to elicit tumor antigen-specific reactivity and should be evaluated in a larger cohort of horses with melanocytic tumors.     

Descriptive epidemiology of joint injuries in Thoroughbred racehorses in training

Reasons for performing study: No large scale epidemiological studies have previously quantified the occurrence of carpal, metacarpo‐ and metatarsophalangeal (MCP/MTP) joint injuries in Thoroughbred racehorses. Objectives: To develop an objective classification system for carpal and MCP/MTP joint injuries and estimate the incidence of these injuries in young Thoroughbreds in flat race training. Methods: In a prospective cohort study, data on daily exercise and veterinary‐diagnosed carpal and MCP/MTP joint injuries were collected from Thoroughbreds monitored since starting training as yearlings, for up to 2 years. Cases were classified in one of 4 categories: 1) localised to a carpal or MCP/MTP joint based on clinical examination and/or diagnostic analgesia; no diagnostic imaging performed; 2) localised to a carpal or MCP/MTP joint based on clinical examination and/or diagnostic analgesia; radiographs taken but no abnormalities detected; 3) evidence of abnormality of subchondral bone and/or articular margin(s) on diagnostic imaging and 4) evidence of discontinuity of the articular surface on diagnostic imaging. Incidence rates and rate ratios were estimated using Poisson regression, adjusting for trainer‐level clustering. Results: A total of 647 horses from 13 trainers throughout England contributed 7785 months at risk of joint injury. One‐hundred‐and‐eighty‐four cases of carpal (n = 82) or MCP/MTP (n = 102) joint injury were reported in 165 horses and classified in Category 1 (n = 21), Category 2 (n = 21), Category 3 (n = 72) or Category 4 (n = 70). The overall joint injury rate was 1.8 per 100 horse months (95% CI = 1.2, 2.8); rates did not differ significantly between 2‐ and 3‐year‐olds but females sustained Category 1 injuries at triple the rate of males (P = 0.03). Joint injury rates differed significantly between trainers (P<0.001) and there was trainer variation in anatomical site and severity of injury. Conclusions and potential relevance: Carpal and MCP/MTP joint injuries are an important cause of morbidity in Thoroughbred racehorses. Identification of modifiable risk factors for these injuries may reduce their incidence.     

Magnetic resonance imaging diagnosis of intervertebral disc disease and myelomalacia in an American black bear (Ursus americanus)

A 23-yr-old black bear (Ursus americanus) was examined because of paralysis of unknown duration. The precise onset of clinical signs was unknown as a result of seasonal torpor. The bear was immobilized and transported to a university veterinary teaching hospital for further evaluation and treatment. Radiography revealed increased mineral opacity and ventral bridging across vertebral segments T8-11. Magnetic resonance imaging demonstrated dorsal and ventral compression of the spinal cord at T8-9. Given the bear's advanced age, the unknown duration of spinal cord compression, unknown presence of deep pain perception, and thus an unknown prognosis for surgical success, euthanasia was elected. Postmortem examination revealed severe spondylosis deformans from T7 to L3 and dorsal extradural extruded disc material in the area of T8-9. Histopathology demonstrated the dorsal horns of the spinal cord at T9 were replaced by foamy macrophages extending into the dorsal and lateral funiculi of the white matter compatible with focal, severe, chronic myelomalacia. This is the first report of intervertebral disc disease and myelomalacia diagnosed using MRI in a large carnivore.     

Biological control of horse cyathostomin (Nematoda: Cyathostominae) using the nematophagous fungus Duddingtonia flagrans in tropical southeastern Brazil

The viability of a fungal formulation using the nematode-trapping fungus Duddingtonia flagrans was assessed for the biological control of horse cyathostomin. Two groups (fungus-treated and control without fungus treatment), consisting of eight crossbred mares (3–18 years of age) were fed on Cynodon sp. pasture naturally infected with equine cyathostome larvae. Each animal of the treated group received oral doses of sodium alginate mycelial pellets (1 g/(10 kg live weight week)), during 6 months. Significant reduction (p < 0.01) in the number of eggs per gram of feces and coprocultures was found for animals of the fungus-treated group compared with the control group. There was difference (p < 0.01) of 78.5% reduction in herbage samples collected up to (0–20 cm) between the fungus-treated group and the control group, during the experimental period (May–October). Difference of 82.5% (p < 0.01) was found between the fungus-treated group and the control group in the sampling distance (20–40 cm) from fecal pats. During the last 3 months of the experimental period (August, September and October), fungus-treated mares had significant weight gain (p < 0.01) compared with the control group, an increment of 38 kg. The treatment with sodium alginate pellets containing the nematode-trapping fungus D. flagrans reduced cyathostomin in tropical southeastern Brazil and could be an effective tool for biological control of this parasitic nematode in horses.     

Nutritional sensitivity of resistance to Trichostrongylus colubriformis in lactating ewes

It has been suggested that the periparturient breakdown of immunity to parasites has a nutritional basis. Our overall hypothesis is that it results from a prioritized scarce metabolizable protein (MP) allocation to reproductive functions (e.g. milk production) rather than to immune functions. We have earlier shown that the relationship between MP supply, milk production and resistance to the abomasal nematode Teladorsagia circumcincta provides support for this hypothesis. Because nutritional sensitivity of resistance to parasites may differ between parasite species, we used the same nutritional protocol to test the overall hypothesis with a different parasite. Thus, five levels of dietary MP, ranging from 0.60 to 1.2 times assumed requirements, were offered for 4 weeks post-parturition to twin-rearing Greyface ewes, experimentally infected with the small intestinal nematode Trichostrongylus colubriformis. We hypothesized that the initial increments of MP supply would increase milk production without affecting the degree of breakdown of immunity whilst later increments would reduce the degree of breakdown of immunity. Indeed, the data suggest that the first two increments of MP supply increased milk production, whilst final worm burdens were reduced from the second increment onwards. MP supply did not affect mucosal mast cell concentration but increased globule leukocyte concentration. These results support the overall hypothesis that scarce MP allocation is prioritized to milk production over immune functions. In addition, the contrast between effects of MP supply on resistance to T. colubriformis and to T. circumcincta supports the view that periparturient immunity to small intestinal nematodes may be less sensitive to nutrient scarcity than periparturient immunity to abomasal nematodes.