Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4)

A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB) labelled with 6-carboxy-fluorescein (FAM) and VIC for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.     

Use of intralesional cisplatin to successfully treat distal limb haemangiosarcoma in a foal

A 4-month old, 200 kg, grey warmblood colt presented for a firm, non painful mass on the distal medial aspect of the left third metatarsus. Excisional biopsy revealed a diagnosis of haemangiosarcoma. Equine haemangiosarcoma is uncommon and only limited reports of successful treatment are available. The prognosis for survival is therefore considered to be poor. After two separate incidences of recurrence with incomplete excision of the tumour, intralesional treatment with cisplatin without excision or debulking was performed on three separate occasions. Intralesional cisplatin injection was performed at monthly intervals for three treatments. Four years post treatment with cisplatin, the horse remained in remission. This case report describes the diagnostic and treatment challenges for successful treatment of a primary haemangiosarcoma on the distal limb of a warmblood foal using intralesional cisplatin chemotherapy.     

Residues of zeta‐cypermethrin in bovine tissues and milk following pour‐on and spray application

The depletion of zeta‐cypermethrin residues in bovine tissues and milk was studied. Beef cattle were treated three times at 3‐week intervals with 1 ml 10 kg^?1 body weight of a 25 g litre^?1 or 50 g litre^?1 pour‐on formulation (2.5 and 5.0 mg zeta‐cypermethrin kg^?1 body weight) or 100 mg kg^?1 spray to simulate a likely worst‐case treatment regime. Friesian and Jersey dairy cows were treated once with 2.5 mg zeta‐cypermethrin kg^?1 in a pour‐on formulation. Muscle, liver and kidney residue concentrations were generally less than the limit of detection (LOD = 0.01 mg kg^?1). Residues in renal‐fat and back‐fat samples from animals treated with 2.5 mg kg^?1 all exceeded the limit of quantitation (LOQ = 0.05 mg kg^?1), peaking at 10 days after treatment. Only two of five kidney fat samples were above the LOQ after 34 days, but none of the back‐fat samples exceeded the LOQ at 28 days after treatment. Following spray treatments, fat residues were detectable in some animals but were below the LOQ at all sampling intervals. Zeta‐cypermethrin was quantifiable (LOQ = 0.01 mg kg^?1) in only one whole‐milk sample from the Friesian cows (0.015 mg kg^?1, 2 days after treatment). In whole milk from Jersey cows, the mean concentration of zeta‐cypermethrin peaked 1 day after treatment, at 0.015 mg kg^?1, and the highest individual sample concentration was 0.025 mg kg^?1 at 3 days after treatment. Residues in milk were not quantifiable beginning 4 days after treatment. The mean concentrations of zeta‐cypermethrin in milk fat from Friesian and Jersey cows peaked two days after treatment at 0.197 mg kg^?1 and 0.377 mg kg^?1, respectively, and the highest individual sample concentrations were 2 days after treatment at 0.47 mg kg^?1 and 0.98 mg kg^?1, respectively. © 2001 Society of Chemical Industry     

A patch—clamp study of the action of a nitromethylene heterocycle insecticide on cockroach neurones growing in vitro

The mode of action of a nitromethylene heterocycle (NMH) insecticide was studied by patch–clamp techniques using cockroach embryonic cultures as an experimental model. Under whole-cell recordings, this compound elicited inward currents resembling those induced by O-acetylcholine (ACh). The reversal potentials for both ACh and the NMH were similar, suggesting that the inward currents induced by both were carried by the same species of ion. Pharmacological investigations of NMH-induced responses revealed that the insecticidal action of this compound is exerted through agonistic action at the nicotinic acetylcholine receptor. Single-channel studies were also performed to study the interaction of NMH with the nicotinic-receptor-coupled ion channel.     

Distribution and breakdown of DDT in orchard soil

Amounts of DDT and its breakdown products were determined in soil in an apple orchard in Herefordshire. Samples were taken for a number of years (1972–79) after use of the insecticide in the orchard had ceased in 1969. The results were compared with those obtained in an investigation of the same orchard in 1968. From 1968 to 1979, soil residues of pp′-DDT, p′--DDT and pp′--TDE decreased gradually whereas those of pp′--DDE increased, and there were linear relationships between log (concentration) and time. The calculated time for 50% decrease in concentration (Dt₅₀) was 11.7 years for pp′--DDT, 3.3 years for pp′--TDE and 7.1 years for op′--DDT; the time for doubling the concentration for pp′--DDE was 9.1 years. Regression analysis on the two major components (pp′--DDT+pp′--DDE) indicated that the total amount (2.7 mg kg^?1) was not decreasing with time. It was concluded that during a post-spray era, the breakdown of pp′--DDT to pp′--DDE was a significant feature of the persistence of DDT, and that, in contrast to the findings of other workers who sampled when DDT was being used, there were no losses by volatilisation. There was an exponential decrease in the amount of DDT residues with increasing soil depth and approximately 90% was found in the top 10 cm of the undisturbed soil profile.     

The activity of amitraz against infestations of Rhipicephalus appendiculatus

Tick-infested adult cattle were hand sprayed with solutions containing 178 or 250 mg amitraz litre^?1, prepared from a miscible oil concentrate formulation. Both concentrations achieved clearance of infestation within 7 h of spraying. All stages of tick, collected from both sprayings, subsequently died with the exception of two engorged nymphs, which moulted successfully, and six half-fully engorged females, which all laid small batches of eggs. The eggs from the ticks treated at the higher rate failed to hatch. Expulsion of adult Rhipicephalus appendiculatus, applied daily to a single bovine which had been sprayed once at the higher rate, ceased on the 11th day after spraying. All ticks expelled from the animal died within 24 h. First feeding on the animal was visible on day-13 and fully engorged females were first observed 18 days after spraying.     

Effect of tilmicosin on chemotactic, phagocytic, and bactericidal activities of bovine and porcine alveolar macrophages

OBJECTIVE: To evaluate chemotactic, phagocytic, and bactericidal activities of bovine and porcine alveolar macrophages (AM) exposed to tilmicosin. ANIMALS: 12 healthy calves and 12 healthy pigs. PROCEDURES: Lungs were obtained immediately after euthanasia; AM were collected by means of bronchoalveolar lavage and density gradient centrifugation. Chemotactic activity was evaluated by exposing AM to lipopolysaccharide or macrophage inhibitory peptide during incubation with tilmicosin. Phagocytic activity was evaluated by incubating AM with tilmicosin for 24 hours and then with tilmicosin-resistant Salmonella serotype Typhimurium. Bactericidal activity was evaluated by incubating AM with tilmicosin (0, 10, or 20 microg/ml for bovine AM; 0 or 10 microg/ml or 10 microg/ml but washed free of tilmicosin for porcine AM) and then with Mannheimia haemolytica (bovine AM) or with Actinobacillus pleuropneumoniae or Pasteurella multocida (porcine AM). RESULTS: Tilmicosin had no significant effects on chemotactic or phagocytic activities of bovine or porcine AM. The time-course of bactericidal activity was best described by polynomial equations. Time to cessation of bacterial growth and area under the time versus bacterial number curve were significantly affected by incubation of AM with tilmicosin. CONCLUSIONS AND CLINICAL RELEVANCE: Results show that bactericidal activity of bovine and porcine AM was enhanced by tilmicosin, but not in proportion to the reported ability of AM to concentrate tilmicosin intracellularly. With or without exposure to tilmicosin, the time-course of bactericidal activity of bovine AM against M haemolytica and of porcine AM against A pleuropneumoniae or P multocida was too complex to be reduced to a simple linear equation.     

Disseminated Mycobacterium avium infection in a cat

A domestic shorthair cat was presented for lethargy and ataxia. Clinical findings included an abdominal mass, lumbosacral pain, ataxia. Aspirates from the liver and lymph nodes revealed intracellular, negative-staining rods. Treatment for presumptive mycobacterium infection was unsuccessful and the cat was euthanized. Disseminated Mycobacterium avium was confirmed on culture.