Paula Menzies

Mastitis of sheep and goats.

This article presents an overview of the recent research into ovine and caprine mastitis. The common clinical presentations of mastitis in these species are reviewed, as are the important etiologic agents and their significance. The interpretation of somatic cell counts and surrogate tests, factors that affect somatic cell count levels, and association of somatic cell count levels with productivity are reviewed. Investigations into the treatment and prevention of mastitis and milking equipment function are discussed, and comments are made on the public health implications of extra label drug use and the consumption of unpasteurized milk.

Analysis of the immunodominant antigens of Corynebacterium pseudotuberculosis

Antibodies to seven antigens in a whole cell lysate of Corynebacterium pseudotuberculosis ranging in molecular mass from 22 to 120 kilodaltons (kDa) were present in sera of 40 sheep and goats infected with C. pseudotuberculosis. Three antigens of about 120, 68, and 31.5 kDa in size were consistently detected with sera from all animals and twenty-two sera had antibodies to 64, 43, 40, and 22 kDa antigens. None of these antigens were detected by sera from 160 sheep in a C. pseudotuberculosis-free research flock. An NaCl extract of C. pseudotuberculosis cells contained one major protein of about 31.5 kDa and four minor proteins of 68, 64, 43, and 22 kDa in molecular mass as shown by Coomassie Blue staining. Immunoblot analysis demonstrated that the three immunodominant antigens identified in the whole cell extract were contained in the NaCl extract. The 31.5-kDa protein was purified from the NaCl extract by fast-protein liquid chromatography gel filtration to near homogeneity. The purified 31.5-kDa protein showed phospholipase D activity as indicated by synergistic hemolysis with Rhodococcus equi factors and sphingomyelinase activity. The 31.5-kDa protein reacted with antibodies in serum from a sheep naturally infected with C. pseudotuberculosis. This serum also had phospholipase D neutralizing activity. On the basis of its molecular mass, biological activity, N-terminal amino acid sequence analysis, and immunoreactivity, the 31.5-kDa protein was identified as the phospholipase D exotoxin of C. pseudotuberculosis.

A systematic review and meta-analysis of factors associated with anthelmintic resistance in sheep

BACKGROUND Anthelmintic drugs have been widely used in sheep as a cost-effective means for gastro-intestinal nematode (GIN) control. However, growing anthelmintic resistance (AHR) has created a compelling need to identify evidence-based management recommendations that reduce the risk of further development and impact of AHR. OBJECTIVE To identify, critically assess, and synthesize available data from primary research on factors associated with AHR in sheep. METHODS Publications reporting original observational or experimental research on selected factors associated with AHR in sheep GINs and published after 1974, were identified through two processes. Three electronic databases (PubMed, Agricola, CAB) and Web of Science (a collection of databases) were searched for potentially relevant publications. Additional publications were identified through consultation with experts, manual search of references of included publications and conference proceedings, and information solicited from small ruminant practitioner list-serves. Two independent investigators screened abstracts for relevance. Relevant publications were assessed for risk of systematic bias. Where sufficient data were available, random-effects Meta-Analyses (MAs) were performed to estimate the pooled Odds Ratio (OR) and 95% Confidence Intervals (CIs) of AHR for factors reported in ≥2 publications. RESULTS Of the 1712 abstracts screened for eligibility, 131 were deemed relevant for full publication review. Thirty publications describing 25 individual studies (15 observational studies, 7 challenge trials, and 3 controlled trials) were included in the qualitative synthesis and assessed for systematic bias. Unclear (i.e. not reported, or unable to assess) or high risk of selection bias and confounding bias was found in 93% (14/15) and 60% (9/15) of the observational studies, respectively, while unclear risk of selection bias was identified in all of the trials. Ten independent studies were included in the quantitative synthesis, and MAs were performed for five factors. Only high frequency of treatment was a significant risk factor (OR=4.39; 95% CI=1.59, 12.14), while the remaining 4 variables were marginally significant: mixed-species grazing (OR=1.63; 95% CI=0.66, 4.07); flock size (OR=1.02; 95% CI=0.97, 1.07); use of long-acting drug formulations (OR=2.85; 95% CI=0.79, 10.24); and drench-and-shift pasture management (OR=4.08; 95% CI=0.75, 22.16). CONCLUSIONS While there is abundant literature on the topic of AHR in sheep GINs, few studies have explicitly investigated the association between putative risk or protective factors and AHR. Consequently, several of the current recommendations on parasite management are not evidence-based. Moreover, many of the studies included in this review had a high or unclear risk of systematic bias, highlighting the need to improve study design and/or reporting of future research carried out in this field.

Principles of Mastitis Treatment in Sheep and Goats

This article indicates the principles for treatment of mastitis in ewes/does and explains the reasons why treatment may occasionally fail. It presents the principles for administration of antimicrobial agents at drying off of the animals. Finally, it addresses the risk of antimicrobials present in milk when improper withdrawal periods are used and the issues around testing for inhibitors before putting the milk into in a farms tank.

Evaluation of an enzyme-linked immunosorbent assay using an Escherichia coli recombinant phospholipase D antigen for the diagnosis of Corynebacterium pseudotuberculosis infection

Abstract Specificity has been a problem with tests for Corynebacterium pseudotuberculosis , probably due to cross-reaction with nonspecific antigens that are contained in crude preparations of antigen. An ELISA has been developed for the detection of antibodies to the phospholipase D (PLD) exotoxin of C. pseudotuberculosis . Antigen preparation differs from other PLD-ELISA tests in that the source of PLD antigen is an Escherichia coli recombinant containing a plasmid bearing pld . When sera from known positive and negative sheep and goats were tested, specificity and sensitivity were sufficient to allow for commercial application of the test. At the optical density cutpoint 0.080, the sensitivity was 86.3% and the specificity was 82.1 %. It is suggested that the cutpoint can be adjusted to increase test sensitivity or specificity, depending on its intended use. A receiver-operator characteristic (ROC) curve was used to demonstrate the accuracy of the ELISA at different cutpoints. Test reliability was excellent at a value of 0.944. This test would have useful commercial application in aiding in the detection of potentially infected small ruminants for purposes of eradication of caseous lymphadenitis (CLA) from low prevalence flocks and for the screening of animals purchased into flocks free of CLA.

Prevalence and distribution of gastrointestinal nematodes on 32 organic and conventional commercial sheep farms in Ontario and Quebec, Canada (2006-2008).

In order to characterize the epidemiology of sheep gastrointestinal nematodes in organic and conventional flocks in Canada, a longitudinal study was carried out from May 2006 to March 2008 on 32 purposively selected farms in Ontario (ON) and Quebec (QC): 8 certified organic (CO), 16 non-certified organic (NCO), and 8 conventional (C) farms. On each farm, 10 ewes and 10 female lambs were selected. Farm visits were undertaken monthly during the grazing season, and twice in the winter. At each visit, individual fecal samples were taken, and pasture samples were obtained during the grazing season. In addition, body condition score was recorded for all sheep. Fecal egg counts per gram of feces (EPGs) were determined for all fecal samples, and infective larvae (L(3)) were identified in fecal samples (lambs and ewes separately) and pasture samples from farms. Necropsies of 14 lambs from 7 of the 23 Ontario farms were performed at the end of the grazing season in 2006. The mean EPG for year 1 (May 2006 to March 2007) was 181 (range=0-9840) and 351 (range=0-18,940) for the ewes in ON and QC, respectively, and for the lambs was 509 (range=0-25,020) and 147 (range=0-3060) for ON and QC, respectively. During year 2 (April 2007 to March 2008), the mean EPG was 303 (range=0-21,160) and 512 (range=0-22,340) for the ewes in ON and QC, respectively, and for lambs was 460 (range=0-26,180) and 232 (range=0-8280) for ON and QC, respectively. Although the overall mean EPGs were not remarkably high, there were months of higher EPG such as May-June for ewes and July-August for lambs in both provinces. Pasture infectivity was highest in May-June and September. There was a general trend for the CO farms to have lower mean EPG than NCO and C farms. Fecal cultures demonstrated that the most predominant nematode genera were Teladorsagia sp., Haemonchus sp. and Trichostrongylus spp. Pasture infectivity was highest during June-July (984 L3/kg DM) in ON farms and September (mean=436 L3/kg DM) in QC farms during year 1. In year 2, the highest peak was during October in ON (mean=398 L3/kg DM) and July in QC (239 L3/kg DM). Trichostrongylus axei and Trichostrongylus colubriformis were the species most frequently identified from necropsies (36.44% and 38.26%, respectively) at the end of the grazing season in 2006, with Haemonchus contortus and Teladorsagia circumcincta being the next most commonly identified.

Assessment of benzimidazole resistance in Haemonchus contortus in sheep flocks in Ontario, Canada: comparison of detection methods for drug resistance.

In 2011, a field study was conducted to assess drug resistance of gastro-intestinal nematodes in sheep flocks in Ontario, Canada. Benzimidazole resistance in Haemonchus contortus was assessed by genetic analysis of eggs; measurement of resistant allele percentages at codons 167, 198 and 200 in the β-tubulin gene was determined on pools of H. contortus eggs using pyrosequencing. Susceptibility to benzimidazoles in gastro-intestinal nematodes was also determined using a Faecal Egg Count Reduction Test (FECRT) and a Larval Development Assay (LDA). In total, 16 farms were assessed with the genetic test. Based on resistant allele frequencies, all of the farms (16/16) tested had benzimidazole resistance in H. contortus; the overall percentage of benzimidazole-resistant H. contortus (estimated prior to treatment using the Hardy-Weinberg formula) was 68.5%. The FECRT and LDA were performed on 11 and 13 farms, respectively. Resistance to fenbendazole was detected on 100% (11/11) of the farms where the FECRT was performed. The LDA revealed the presence of thiabendazole resistance in H. contortus in 92% (12/13) of the farms. Estimated percentages of resistant parasites in H. contortus populations obtained with the two biological tests and the genetic test were compared. The results of the genetic test were in agreement with the biological tests and confirmed that benzimidazole resistance in H. contortus is present in Ontario sheep flocks. Differences between the different methods of drug resistance detection are discussed in terms of cost, time and sampling.

Control of Important Causes of Infectious Abortion in Sheep and Goats

This article summarizes control measures for the most common causes of abortion in North America, New Zealand, the United Kingdom, and Europe. When dealing with an abortion outbreak in a flock or herd, diagnostic investigation is critical to assuring that any future control measures are effective and worthwhile. Biosecurity is an important consideration for any abortion control program, and should be promoted regardless of whether an abortion problem exists in the flock. Many of the infectious agents that cause abortion in small ruminants are also zoonotic pathogens, and producers should be educated to avoid risk to themselves and their families.

An interferon-gamma assay for diagnosis of Corynebacterium pseudotuberculosis infection in adult sheep from a research flock

The optimal method of control of caseous lymphadenitis of sheep caused by Corynebacterium pseudotuberculosis is eradication of infection by identification and removal of infected carrier animals. Current serological approaches to identification of infected sheep are generally hampered by low sensitivity and specificity of available tests. The objective of this study was to develop a whole blood assay for detection of C. pseudotuberculosis-infected sheep, based on detection of IFN-gamma response to whole cell C. pseudotuberculosis antigens, and to determine the reliability of the assay. A commercially available bovine interferon-gamma assay enzyme-linked immunosorbent assay was used and the test optimised using experimentally infected sheep. The assay was also tested on known CLA-negative sheep. Setting a IFN-gamma optical density cut-off at 0.100 as positive under the conditions used, the test detected C. pseudotuberculosis experimentally infected sheep over a 450-day period with a reliability of 95.7%. It identified known non-infected sheep with a reliability of 95.5%. Repeated vaccination of three uninfected sheep with a commercially available bacterin-toxoid vaccine did not interfere with the assay. The IFN-gamma response of sheep whole blood to C. pseudotuberculosis antigens offers promise for use in a test-and-removal approach to eradication of CLA in sheep.

Full-length genome sequence analysis of enzootic nasal tumor virus reveals an unusually high degree of genetic stability.

Enzootic nasal tumor virus (ENTV) is a betaretrovirus of sheep (ENTV-1) and goats (ENTV-2) associated with neoplastic transformation of epithelial cells of the ethmoid turbinate. Confirmation of the role of ENTV in the pathogenesis of enzootic nasal adenocarcinoma (ENA) has yet to be resolved due to the lack of an infectious molecular clone and the inability to culture the virus. Very little is known about the prevalence of this disease, particularly in North America, and only one full-length sequence is available for each of ENTV-1 and ENTV-2. In order to understand the molecular evolution of ENTV-1, the full-length genome sequence of ten ENTV-1 proviruses derived from clinical samples of ENA isolated from conventionally reared sheep in Canada and the United States was determined. The North American ENTV-1 (ENTV-1(NA)) genomes shared greater than 96% sequence identity with the European ENTV-1 sequence (ENTV-1(EU)). Most of the amino acid differences were found in Orf-x, which in the corresponding ENTV-1(EU) genome is truncated by 44 amino acids. Apart from Orf-x, the long terminal repeat (LTR) is where the majority of differences between ENTV-1(NA) and ENTV-1(EU) reside. Overall, there was an unusually high degree of amino acid conservation among the isolates suggesting that ENTV-1 is under stabilizing selection and K(a)/K(s) ratios calculated for each of the viral genes support this hypothesis. The unusually high degree of genetic stability of the ENTV-1 genome enabled us to develop a hemi-nested PCR assay for detection of ENTV-1 in clinical samples. Additionally, multiple nasal tumor cell clones were established and while most had lost the provirus by passage 5; one polyclonal line retained the provirus and attempts are being made to culture these cells. These tumor cells, the first of their kind, may provide a system for studying ENTV-1 in vitro. This work represents an important step in the study of ENTV and sets the foundation for the construction of an infectious molecular clone of ENTV-1.