The detection of anthelmintic resistance in nematodes of veterinary importance

Before revised World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines on the detection of anthelmintic resistance can be produced, validation of modified and new methods is required in laboratories in different parts of the world. There is a great need for improved methods of detection of anthelmintic resistance particularly for the detection of macrocyclic lactone resistance and for the detection of resistant nematodes in cattle. Therefore, revised and new methods are provided here for the detection of anthelmintic resistance in nematodes of ruminants, horses and pigs as a basis for discussion and with the purpose that they are evaluated internationally to establish whether they could in the future be recommended by the WAAVP. The interpretation of the faecal egg count reduction test has been modified and suggestions given on its use with persistent anthelmintics and continuous release devices. An egg hatch test for benzimidazole (BZ) resistance is described. A microagar larval development test for the detection of benzimidazole and levamisole resistance provides third stage larvae for the identification of resistant worms. The sensitivity of these two tests can be increased by using discriminating doses rather than LD(50) values. Details are given of a PCR based test for the analysis of benzimidazole resistance in strongyles of sheep and goats, horses and cattle. Although promising for ruminant trichostrongyles, quantitative determination of gene frequency using real time PCR requires further development before PCR tests will be used in the field. Apart from faecal egg count reduction tests there are currently no satisfactory tests for macrocylic lactone resistance despite the great importance of this subject. Except for treatment and slaughter trials there are no validated tests for fasciolicide resistance or for the detection of resistance in cestodes.     

Genotyping of Toll-like receptor 4, myeloid differentiation factor 2 and CD-14 in the horse: an investigation into the influence of genetic polymorphisms on the LPS induced TNF-alpha response in equine whole blood

The inter- and intra-species differences in the response to lipopolysaccharides (LPS) are well recognised in mammalian species. It has been hypothesized that these differences can be attributed to genetic polymorphisms in the components involved in LPS signal transduction. These components include the cluster of differentiation factor 14 (CD-14), a membrane bound protein on the surface of mononuclear cells that recognises LPS and a receptor complex consisting of Toll-like receptor-4 (TLR-4) and myeloid differentiation factor-2 (MD-2). Sequencing of these three proteins in humans and mice revealed that all three are susceptible to polymorphic alterations, influencing the response to LPS. Previous experiments in the horse showed large inter-individual variations in the response to LPS. With the aim to assess this inter-individual variation, we performed a whole blood assay in 10 healthy horses as a functional assay to study the responsiveness to LPS. In 3 out of the 10 horses, LPS-induced TNF-alpha production was significantly lower compared to the overall mean. Subsequently the entire cDNA sequence encoding for the TLR-4, MD-2 and CD-14 protein was documented for each horse. Although mutations were observed in the sequence of TLR-4, these could not be related to an altered response to LPS in the concentration used in this study, as determined in the whole blood assay. Despite the various mutations found in the TLR-4 receptor protein, no alterations could be found in either the MD-2 or CD-14 gene, which are obviously more conserved structures.     

Canine limbal melanoma: 30 cases (1992-2004). Part 1. Signalment, clinical and histological features and pedigree analysis

OBJECTIVES: (1) To review the signalment, clinical, and histological features of canine limbal melanoma; (2) to perform pedigree analysis on breeds predisposed to limbal melanoma to establish if common ancestry exists; and (3) to investigate if any ancestral relationship exists between canine limbal melanoma and canine anterior uveal melanoma (CAUM). DESIGN: Retrospective study. ANIMALS STUDIED: Thirty dogs with limbal melanoma. METHODS: Medical records of patients were reviewed. Follow-up information was obtained by re-examination of patients or telecommunications with the referring veterinary surgeons or the owners. Pedigrees were analyzed for common ancestry amongst affected dogs. RESULTS The mean age (+/- SD) at diagnosis was 6.2 (+/- 2.75) years with a range from 1 to 11 years. There was a bimodal distribution of ages with a peak at 3-4 years and a peak at 7-10 years. There was no eye predilection or predisposition for sex or coat color. Twenty-five (83%) of the limbal melanomas occurred within a dorsal arc from the dorsomedial to the ventrolateral limbus. Golden retrievers were four times more common in the melanoma group compared to the Animal Health Trust population (P < 0.0001). Labrador retrievers were three times more common in the melanoma group (P=0.01). Pedigree analysis on eight Golden retrievers [limbal melanoma (n=5), CAUM (n=2) and diffuse ocular melanosis (n=1)], revealed a pattern of inter-relatedness consistent with the condition(s) being caused, at least in part, by a genetic mutation(s). A similar level of inter relatedness was evident in six Labrador retrievers (limbal melanoma (n=2) and CAUM (n=4)). In 5/22 cases (23%), histological features suggestive of malignancy were present including intratumor necrosis in 4/22 cases (18%) and cellular atypia in 1/22 cases (5%). CONCLUSIONS: In Golden and Labrador retrievers there is evidence that limbal melanomas, CAUM and ocular melanosis are at least in part heritable and that the same genetic mutation(s) may be causally associated with melanocytic disease at different ocular sites. The same genetic mutation(s) may be present in these two breeds. Histology should be performed on all cases to identify those with greater malignant potential.     

Thermoregulation of the testicle in response to exercise and subsequent effects on semen characteristics of stallions

Stallions (n = 8) were implanted with a thermal sensory device in the muscle of the neck and the subcutaneous tissue of the scrotum and then assigned to either a nonexercise (Non-EX; n = 4) or exercise (EX; n = 4) group. A motorized equine exerciser was used to work EX stallions 30 min/d for 4 d/wk during a 12-wk period from July through October 2010. Temperatures (subcutaneous scrotal, intramuscular neck, and rectal) were recorded at 0, 22, and 30 min after the start of exercise, as well as 60 and 120 min post-exercise. Hourly ambient temperature and relative humidity data were also obtained. Semen was collected at 0, 4, 8, and 12 wk and analyzed for volume, sperm concentration, total sperm numbers, percentages of total and progressively motile sperm, sperm morphologic characteristics, and sperm DNA quality. No effect (P > 0.05) of exercise was observed on any of the measured semen variables. Implantation of thermal sensory devices had no demonstrable acute or chronic effects on the scrotal or neck tissue, indicating that the thermal sensory devices are a safe and effective way to measure subcutaneous scrotal and neck temperatures. At 22 and 30 min of exercise, rectal and neck temperatures increased (P < 0.0001) approximately 1.9 and 2.4°C, respectively, and scrotal temperatures simultaneously increased, although not significantly (P = 0.33), approximately 0.8°C. Correlations existed between scrotal, neck, rectal, and ambient temperatures, with the correlation between scrotal and rectal temperatures being greatest (r(s) = 0.76; P < 0.0001). Although moderate exercise for a short duration in extreme heat and humidity did significantly increase core body temperatures in stallions, scrotal temperatures did not significantly increase, and sperm parameters were unaffected.     

The response of monocyte derived dendritic cells following exposure to a nematode larval carbohydrate antigen

The glycolipid CarLA (carbohydrate larval antigen) is present on the epicuticle of the infective-stage larvae of gastrointestinal nematode parasites infecting livestock. The molecule is lost from the surface of the larvae in the few days post-ingestion by a host animal, and the resulting anti-CarLA antibody response has been demonstrated to be protective in vivo. Both the anti-CarLA response, and anti-parasite immunity in general, are slow to develop, and several months of natural exposure to ingested larvae is required. The current study was designed to provide information on how the anti-CarLA response develops, and focuses on the initial recognition of the molecule by human monocyte derived dendritic cells (mdDC) in vitro. Immunofluorescence and flow cytometry demonstrated that mdDC recognise and internalise both the purified and the native form of CarLA, in the case of the latter once it is shed from the larval surface. However, the recognition of CarLA did not result in classical maturation of DC, while there was only transient or minor up-regulation of CD86, CD83, HLA-DR and CD40. Exposure of mdDC to purified CarLA resulted in the increased production of the pro-inflammatory cytokines IL-6 and to a lesser extent of IL-8 and TNF-α, and a reduced production of the anti-inflammatory cytokine IL-1RA. CarLA therefore has little ability to mature and functionally alter monocyte derived dendritic cell function.     

Prevalence of mange among West African Dwarf sheep and goats and associated haematological and biochemical parameters

This study was carried out to assess the prevalence of mange in sheep and goats in five local government areas (LGAs) of Ogun State in South Western Nigeria. A total of 4,973 sheep and 7,902 goats of West African Dwarf breeds were sampled of which only 4 (0.08%) of sheep and 42 (0.53%) of goats were confirmed positive for mange infestation. In all the LGAs sampled, Odeda LGA had the highest concentration of sheep (39%) and goats (51%) while Abeokuta South had the least percentage of sheep (1%) and goats (1%). All the mange-infested cases in sheep were recorded in just one LGA (Ewekoro) which constituted 0.24% of the population within the location. For goats, three out of five LGAs namely: Obafemi Owode, Ewekoro and Abeokuta North had prevalence of 1.46%, 0.53% and 0.72% respectively. There was no significant (P > 0.05) difference between non-infested and infested animals in terms of packed cell volume (31.79 ± 0.87% vs. 31.41 ± 1.13%), haemoglobin (10.51 ± 0.21 g/100 ml vs. 10.28 ± 0.37 g/100 ml) and red blood cells (8.71 ± 0.29 × 10(12)/l vs. 9.40 ± 0.37 × 10(12)/l). The infested group however showed significantly (P < 0.01) higher white blood cells count (7.60 ± 0.22 × 10(9)/l) than the non-infested animals (6.81 ± 0.17 × 10(9)/l). Neutrophil, eosinophil, basophil and monocyte as well as chloride were not significantly affected by the health status of the animals. There existed significant (P < 0.01) difference between non-infested and infested animals in terms of total protein (5.42 ± 0.16 vs. 4.75 ± 0.20 g/dl), sodium (137.98 ± 1.53 vs. 128.92 ± 1.92 mEq/l) and potassium (4.04 ± 0.14 vs. 3.46 ± 0.18 mEq/l). It can be concluded from this study that the prevalence of mange mite infestation varied with location and was generally low or absent in some of the LGAs considered. Goats were particularly more susceptible to mange infestation than sheep. More concerted effort is needed to control mange in order to avoid spread since it is a contagious disease.     

Outbreak of abdominal distension and obstipation in a C57BL/6J experimental autoimmune encephalomyelitis study

Seventy-four 9-week old female C57BL/6J mice housed in a conventional facility were manipulated to induce experimental autoimmune encephalomyelitis, among which 26 developed clinical signs including lethargy, absence of defecation, and abdominal distension. By gross necropsy examination, there was distension of the cecum and colon with fecal impaction. By histologic examination, there was severe ulcerative and proliferative typhlocolitis. Fecal ELISA confirmed the presence of toxins A and B of Clostridium difficile. Alteration in immune status of the immunocompetent mice, due to stress caused by experimental manipulation or autoimmune disease, may have led to intestinal dysbiosis, followed by opportunistic infections resulting in C. difficile-associated disease. This report brings to light the occurrence of the disease in immunocompetent laboratory mice during experimental manipulations associated with alteration in immune status, and it discusses potential hazards associated with conventional housing within a hospital-associated research institute.     

Impact of regulatory perturbations to disease spread through cattle movements in Great Britain

During the past decade the British livestock industry has suffered from several major pathogen outbreaks, and a variety of regulatory and disease control measures have been applied to the movement of livestock with the express aim of mitigating the spread of infection. The Rapid Analysis and Detection of Animal-related Risks (RADAR) project, which has been collecting data on the movement of cattle since 1998, provides a relatively comprehensive record of how these policies have influenced the movement of cattle between animal holdings, markets, and slaughterhouses in Britain. Many previous studies have focused on the properties of the network that can be derived from these movements--treating farms as nodes and movements as directed (and potentially weighted) edges in the network. However, of far greater importance is how these policy changes have influenced the potential spread of infectious diseases. Here we use a stochastic fully individual-based model of cattle in Britain to assess how the epidemic potential has varied from 2000 to 2009 as the pattern of movements has changed in response to legislation and market forces. Our simulations show that the majority of policy changes lead to significant decreases in the epidemic potential (measured in multiple ways), but that this potential then increases through time as cattle farmers modify their behaviour in response. Our results suggest that the cattle industry is likely to experience boom-bust dynamics, with the actions that farmers take during epidemic-free periods to maximise their profitability likely to increase the potential for large-scale epidemics to occur.