Anthelmintic resistance detection methods.

The development of species and populations of parasitic helminths with resistance to one or more anthelmintics is an increasing problem world-wide. The majority of currently available anthelmintics used to control parasitic nematodes of cattle and sheep belong to only three main groups, the benzimidazoles, imidazothiazoles and the avermectins/milbemycins. The successful implementation of helminth control programmes designed to limit the development of resistance in nematode populations depends to some degree on the availability of effective and sensitive methods for its detection and monitoring. A variety of in vivo and in vitro tests have been developed for the detection of nematode populations resistant to the main anthelmintic groups, but each suffers to some degree from reliability, reproducibility, sensitivity and ease of interpretation. This review covers those tests that have been reported and described and highlights some of their strengths and weaknesses.     

BOOK REVIEWS

Book reviewed in this article:
Guide to the Dissection of Domestic Ruminants . RE Habel
Biology and Medicine of Rabbits and Rodents . JE Harkness and JF Wagner     

Phenotypic characterisation of Australian sheep and cattle isolates of Mannheimia haemolytica, Mannheimia granulomatis and Mannheimia varigena

Objective To perform a comprehensive phenotypic characterisation of 35 isolates of bacteria previously identified as haemolytic Pasteurella‐Actinobacillus and obtained from cattle and sheep. Design The 35 isolates that had been obtained from Australian animals, 30 from cattle and five from sheep, were compared with reference strains of the five recognised species of the genus Mannheimia ‐ M haemolytica, M glucosida, M granulomatis, M ruminalis and M varigena. Results Thirty‐four of the isolates could be confidently assigned to three species of the genus Mannheimia. Twenty‐nine were M haemolytica, with 25 being isolated from cattle and four from sheep. All but three of the bovine M haemolytica were isolated from pneumonic lungs. Of the three remaining bovine M haemolytica isolates, one was obtained in pure culture from a bovine milk sample and the other two as part of a mixed flora associated with a middle ear infection of a calf suffering mucosal disease. Of the four ovine M haemolytica isolates, two were isolated in pure culture from milk and two, also in pure culture, from pneumonic lungs. Three bovine isolates were identified as M granulomatis ‐ one from a tongue abscess, one from a jaw abscess and one from a lung showing suppurative bronchopneumonia. Two bovine isolates were identified as M varigena‐ one coming from an udder and the other from a spleen. The available diagnostic records provided no information on whether these isolates were associated with a disease process. The remaining isolate was obtained from an ovine tongue abscess and could not be assigned to a recognised species within the genus Mannheimia. Conclusion The study represents the first time that M haemolytica, M granulomatis and M varigena have been recognised as being present in cattle and sheep in Australia. Veterinary laboratories that encounter Pasteurella‐Actinobacillus‐like organisms from cattle and sheep should attempt as complete a characterisation as possible to help improve our knowledge of the disease potential of these organsims.     

Porcine pleuropneumonia in Australian pigs due to Actinobacillus pleuropneumoniae serovar 5

Book reviewed in this article: Guide to the Dissection of Domestic Ruminants . RE Habel Biology and Medicine of Rabbits and Rodents . JE Harkness and JF Wagner     

Compatibility between livestock databases used for quantitative biosecurity response in New Zealand

AIM: To characterise New Zealand's livestock biosecurity databases, and investigate their compatibility and capacity to provide a single integrated data source for quantitative outbreak analysis.

METHODS: Contemporary snapshots of the data in three national livestock biosecurity databases, AgriBase, FarmsOnLine (FOL) and the National Animal Identification and Tracing Scheme (NAIT), were obtained on 16 September, 1 September and 30 April 2014, respectively, and loaded into a relational database. A frequency table of animal numbers per farm was calculated for the AgriBase and FOL datasets. A two dimensional kernel density estimate was calculated for farms reporting the presence of cattle, pigs, deer, and small ruminants in each database and the ratio of farm densities for AgriBase versus FOL calculated. The extent to which records in the three databases could be matched and linked was quantified, and the level of agreement amongst them for the presence of different species on properties assessed using Cohen's kappa statistic.

RESULTS: AgriBase contained fewer records than FOL, but recorded animal numbers present on each farm, whereas FOL contained more records, but captured only presence/absence of animals. The ratio of farm densities in AgriBase relative to FOL for pigs and deer was reasonably homogeneous across New Zealand, with AgriBase having a farm density approximately 80% of FOL. For cattle and small ruminants, there was considerable heterogeneity, with AgriBase showing a density of cattle farms in the Central Otago region that was 20% of FOL, and a density of small ruminant farms in the central West Coast area that was twice that of FOL. Only 37% of records in FOL could be linked to AgriBase, but the level of agreement for the presence of different species between these databases was substantial (kappa >0.6). Both NAIT and FOL shared common farm identifiers which could be used to georeference animal movements, and there was a fair to substantial agreement (kappa 0.32–0.69) between these databases for the presence of cattle and deer on properties.

CONCLUSIONS: The three databases broadly agreed with each other, but important differences existed in both species composition and spatial coverage which raises concern over their accuracy. Importantly, they cannot be reliably linked together to provide a single picture of New Zealand's livestock industry, limiting the ability to use advanced quantitative techniques to provide effective decision support during disease outbreaks. We recommend that a single integrated database be developed, with alignment of resources and legislation for its upkeep.     

Vesicles Cytoplasmic Injection: An Efficient Technique to Produce Porcine Transgene‐Expressing Embryos

The use of vesicles co‐incubated with plasmids showed to improve the efficiency of cytoplasmic injection of transgenes in cattle. Here, this technique was tested as a simplified alternative for transgenes delivery in porcine zygotes. To this aim, cytoplasmic injection of the plasmid alone was compared to the injection with plasmids co‐incubated with vesicles both in diploid parthenogenic and IVF zygotes. The plasmid pcx‐egfp was injected circular (CP) at 3, 30 and 300 ng/μl and linear (LP) at 30 ng/μl. The experimental groups using parthenogenetic zygotes were as follows: CP naked at 3 ng/μl (N = 105), 30 ng/μl (N = 95) and 300 ng/μl (N = 65); Sham (N = 105); control not injected (N = 223); LP naked at 30 ng/μl (N = 78); LP vesicles (N = 115) and Sham vesicles (N = 59). For IVF zygotes: LP naked (N = 44) LP vesicles (N = 94), Sham (N = 59) and control (N = 79). Cleavage, blastocyst and GFP+ rates were analysed by Fisher's test (p < 0.05). The parthenogenic CP naked group showed lower cleavage respect to control (p < 0.05). The highest concentration of plasmids to allow development to blastocyst stage was 30 ng/μl. There were no differences in DNA fragmentation between groups. The parthenogenic LP naked group resulted in high GFP rates (46%) and also allowed the production of GFP blastocysts (33%). The cytoplasmic injection with LP vesicles into parthenogenic zygotes allowed 100% GFP blastocysts. Injected IVF showed higher cleavage rates than control (p < 0.05). In IVF zygotes, only the use of vesicles produced GFP blastocysts. The use of vesicles co‐incubated with plasmids improves the transgene expression efficiency for cytoplasmic injection in porcine zygotes and constitutes a simple technique for easy delivery of plasmids.     

Ultrasonographic and Endocrine Characterization of Follicular Waves in Llamas with a Special Reference to the Overlapping Phenomenon During Successive Waves

Ovarian follicular growth in non‐mated llamas occurs in successive waves that generally superimpose their origin on the regression of the preceding wave (overlapping), originating prolonged sexual receptivity in the species. The aim of this study was to perform an ultrasonographic and endocrine characterization of individual and successive waves in non‐mated llamas with a special interest on the overlapping phenomenon. Twelve llamas were examined daily by transrectal ultrasonography for at least two consecutive waves. In six females, blood samples were collected daily at the end of each examination. The development of the largest follicle (F) showed a wavelike pattern with a mean duration of 25 days. All waves evaluated were partially overlapped on the preceding wave and emerged at a mean interval of 15.8 ± 0.5 days. This interwave interval determines a mean overlapping degree of 32% of the wave length. Similarly, mean plasma oestradiol‐17β (E₂) concentrations followed a wavelike pattern. However, E₂ concentrations started to decline before the structural regression of the F was observed. Mean basal E₂ concentrations remain higher than 10.9 ± 0.6 pmol/l. In conclusion, follicular activity in non‐mated llamas is characterized by continuous emergence of successive waves that always overlap the preceding wave with variable degrees. E₂ production during the follicular wave is shorter in duration than the morphological development of the F. Finally, the overlapping phenomenon maintains increased plasma E₂ concentrations persistently and this could explain the prolonged periods of sexual receptivity registered in llamas.     

First insights into insecticidal activity against Aedes aegypti and partial biochemical characterization of a novel low molecular mass chymotrypsin‐trypsin inhibitor purified from Lonchocarpus sericeus seeds

BACKGROUND

Arboviroses such as dengue, Zika and chikungunya represent a serious public health issue as a consequence of the absence of approved vaccines or specific antiviral drugs against the arboviruses that cause them. One way to prevent these diseases is by combating the vector mosquito, Aedes aegypti (Diptera), which has serine proteases in the midgut. Protease inhibitors are molecules that can block enzyme activity, impairing digestion and nutrition, which can lead to death. Thus, we purified and characterized a novel chymotrypsin‐trypsin inhibitor (LsCTI) from Lonchocarpus sericeus seeds and investigated its effect upon Ae. aegypti egg hatching, larval development and digestive proteases.

RESULTS

LsCTI showed a single protein band in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE), and the molecular mass determined by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) was 8870.45 Da. Kinetics analyses revealed a noncompetitive type of inhibition and low inhibition constant (K_i) for chymotrypsin (8.24 x 10^‐8 m ). The thermal resistance was remarkable, even at 100 °C for 180 min. The inhibitor concentration required for 50‐percent enzyme inhibition (IC₅₀) of LsCTI was 4.7 x 10^‐7 m for Ae. aegypti midgut larval enzymes. LsCTI did not affect egg hatchability at 0.3 mg mL^‐1, but caused a high larval mortality rate (77%) and delayed development (37%).

CONCLUSIONS

LsCTI is a novel protease inhibitor with remarkable biochemical characteristics and is a potential tool to control Ae. aegypti development. © 2017 Society of Chemical Industry