Quantitative PCR monitoring of the effect of azoxystrobin treatments on Mycosphaerella graminicola epidemics in the field

Quantitative PCR and visual monitoring of Mycosphaerella graminicola epidemics were performed to investigate the effect of curative and preventative applications of azoxystrobin in wheat field crops. A non-systemic protectant and a systemic curative fungicide, chlorothalonil and epoxiconazole, respectively, were used as references. PCR diagnosis detected leaf infection by M graminicola 3 weeks before symptom appearance, thereby allowing a clear distinction between curative and preventative treatments. When applied 1 week after the beginning of infection, azoxystrobin curative activity was intermediate between chlorothalonil (low effect) and epoxiconazole. When applied preventatively, none of the fungicides completely prevented leaf infection. There was some indication that azoxystrobin preventative treatments may delay fungal DNA increase more than epoxiconazole at the beginning of leaf infection. Both curative and preventative treatments increased the time lapse between the earliest PCR detection and the measurement of a 10% necrotic leaf area. Azoxystrobin only slightly decreased the speed of necrotic area increase compared with epoxiconazole. Hence, azoxystrobin activity toward M graminicola mainly resides in lengthening the time lapse between the earliest PCR detection and the measurement of a 10% necrotic leaf area. Information generated in this way is useful for optimal positioning of azoxystrobin treatments on M graminicola.     

Safety of moxidectin in avermectin-sensitive collies

OBJECTIVE: To evaluate the safety of moxidectin administration at doses of 30, 60, and 90 microg/kg of body weight (10, 20, and 30 times the manufacturer's recommended dose) in avermectin-sensitive Collies. ANIMALS: 24 Collies. PROCEDURE: Collies with mild to severe reactions to ivermectin challenge (120 mg/kg; 20 times the recommended dose for heartworm prevention) were used. Six replicates of 4 dogs each were formed on the basis of body weight and severity of reaction to ivermectin test dose. Within replicates, each dog was randomly allocated to treatment with oral administration of 30, 60, or 90 microg of moxidectin/kg or was given a comparable volume of placebo tablet formulation. Dogs were observed hourly for the first 8 hours and twice daily thereafter for 1 month for signs of toxicosis. RESULTS: Signs of toxicosis were not observed in any control group dog throughout the treatment observation period. Likewise, signs of toxicosis were not observed in any dog receiving moxidectin at 30, 60, or 90 microg/kg. CONCLUSIONS AND CLINICAL RELEVANCE: The moxidectin formulation used in the study reported here appears to have a wider margin of safety than ivermectin or milbemycin in avermectin-sensitive Collies.     

Detection and quantification of equine herpesvirus-1 viremia and nasal shedding by real-time polymerase chain reaction.

Equine herpesvirus-1 (EHV-1) infection is common in young horses throughout the world, resulting in respiratory disease, epidemic abortion, sporadic myelitis, or latent infections. To improve on conventional diagnostic tests for EHV-1, a real-time polymerase chain reaction (PCR) technique was developed, using primers and probes specific for the EHV-1 gB gene. Amplification efficiencies of 100% +/- 5% were obtained for DNA isolated from a plasmid, infected peripheral blood mononuclear cells (PBMCs), and nasal secretions from infected ponies. The dynamic range of the assay was 8 log10 dilutions, and the lower limit of detection was 6 DNA copies. Fifteen ponies, seronegative for EHV-1, were experimentally infected with EHV-1, and nasal samples were used to quantify shedding of virus by both virus isolation and real-time PCR analysis. Virus isolation identified nasal shedding of EHV-1 in 12/15 ponies on a total of 25 days; real-time PCR detected viral shedding in 15/15 ponies on 75 days. Viremia was quantified using PBMC DNA, subsequent to challenge infection in 3 additional ponies. Viremia was identified in 1/3 ponies on a single day by virus isolation; real-time PCR detected viremia in 3/3 ponies on 17 days. When real-time PCR was used to analyze PBMC DNA from 11 latently infected ponies (documented by nested PCR), EHV-1 was not detected. We conclude that real-time PCR is a sensitive and quantitative test for EHV-1 nasal shedding and viremia and provides a valuable tool for EHV-1 surveillance, diagnosis of clinical disease, and investigation of vaccine efficacy.     

Performance of a foot-and-mouth disease virus reverse transcription-polymerase chain reaction with amplification controls between three real-time instruments.

The foot-and-mouth disease virus (FMDV) is a member of the picornavirus family, possessing an 8-kb single-stranded RNA genome of positive polarity. It is highly contagious among several livestock species and can lead to severe economic consequences, as evidenced by the UK outbreak in 2001. The usage of real-time polymerase chain reaction has facilitated rapid detection of FMDV. Several real-time PCR instruments are available with various capabilities, such as portability and high sample volume analysis. Primers and a dual-labeled TaqMan probe were optimized to detect a single, highly conserved 88-bp segment of the FMDV 3D (RNA polymerase) gene. To increase the confidence of the RT-PCR result, a positive amplification control was synthesized to detect potential false-positive results due to contamination if a wild-type virus is used as positive control. In addition, a preventative measure against false-negative results was developed in which endogenous beta actin mRNA is coamplified by RT-PCR. Assay performance was compared on the LightCycler1.2 (Roche), the SmartCyclerII (Cepheid), and the SDS 7900HT (ABI). These assays successfully identified the FMDV genome and beta actin mRNA from several sources of infected nasal and oral swabs, as well as probang samples.     

Diagnosis and treatment of presumptive postobstructive pulmonary edema in a Florida panther (Puma concolor coryi).

A free-ranging, adult male Florida panther (Puma concolor coryi) was immobilized and evaluated for hematuria following routine capture. Prior to anesthetic recovery, the panther was fitted with a telemetry collar. After an initially quiet recovery, the panther began thrashing in the transport cage, and was again immobilized. Pink foam was evident from the nostrils, and crackles were ausculted over the chest, indicating pulmonary edema. Postobstructive pulmonary edema was diagnosed based on history, clinical signs, radiographic evaluation, and blood gas analysis. The animal was treated intensively for several hours with diuretics, oxygen, and manual ventilation. The panther responded rapidly to therapy and was released back into the wild 48 hr after presentation. Postobstructive pulmonary edema, also called negative-pressure pulmonary edema, may be underrecognized in veterinary medicine. In this case, the telemetry collar, in conjunction with anesthetic recovery in a small transport crate, may have contributed to tracheal obstruction. Wildlife veterinarians and biologists should be aware of the risk of airway obstruction when placing tracking collars, and animals should be continuously monitored during anesthetic recovery to ensure the presence of a patent airway.