The essential and non-essential genes of Bovine herpesvirus 1

Bovine herpesvirus 1 (BoHV-1) is an economically important pathogen of cattle associated with respiratory and reproductive disease. To further develop BoHV-1 as a vaccine vector, a study was conducted to identify the essential and non-essential genes required for in vitro viability. Randominsertion mutagenesis utilizing a Tn5 transposition system and targeted gene deletion were employed to construct gene disruption and gene deletion libraries, respectively, of an infectious clone of BoHV-1. Transposon insertion position and confirmation of gene deletion were determined by direct sequencing. The essential or non-essential requirement of either transposed or deleted open reading frames (ORFs) was assessed by transfection of respective BoHV-1 DNA into host cells. Of the 73 recognized ORFs encoded by the BoHV-1 genome, 33 were determined to be essential and 36 to be non-essential for virus viability in cell culture; determining the requirement of the two dual copy ORFs was inconclusive. The majority of ORFs were shown to conform to the in vitro requirements of BoHV-1 homologues encoded by human herpesvirus 1 (HHV-1). However, ORFs encoding glycoprotein K (UL53), regulatory, membrane, tegument and capsid proteins (UL54, UL49.5, UL49, UL35, UL20, UL16 and UL7) were shown to differ in requirement when compared to HHV-1-encoded homologues.

Laboratory studies on Australian isolates of Metarhizium anisopliae as a biopesticide for the cattle tick Boophilus microplus

Thirty-one isolates of Metarhizium anisopliae were bioassayed against the cattle tick (Boophilus microplus). More than half of the isolates showed a high degree of virulence to ticks. Radial growth curves for growth between 20 °C and 40 °C were obtained for all isolates. This information together with information on virulence will be important for the selection of isolates suitable to kill ticks on the surface of cattle. A biopesticide for cattle ticks must kill ticks rapidly at temperatures within the upper end of most isolates' growth curves. It was also found that the time taken to achieve 100% tick mortality in vitro using a virulent isolate could be halved by applying conidia in a 10% oil emulsion. Scanning electron microscopy and light microscopy were used to investigate and compare the germination and penetration of conidia formulated in aqueous and oil formulations. It was found that conidia in both formulations were able to germinate and produce appressoria on the surface of ticks in less than 11 h. Marked weakness within 26 h, followed by extensive hyphal growth on the cuticle characterised the invasion of ticks by M. anisopliae.

Pen studies on the control of cattle tick (Rhipicephalus (Boophilus) microplus) with Metarhizium anisopliae (Sorokin)

Three field trials were conducted over 12 months to assess the pathogenicity of Metarhizium anisopliae to parasitic stages of Rhipicephalus (Boophilus) microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30 °C), good spore production characteristics and ability to quickly kill adult engorged ticks in the laboratory. Spores were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated in the laboratory to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. In each trial the formulation rapidly caused 100% mortality in unengorged ticks that were removed from cattle and cultured in the laboratory. A significant reduction in egg production was recorded for engorged ticks collected in the 3 days post-treatment. However, there was little effect of the formulation on the survival of ticks on cattle, indicating that there is an interaction between the environment of the ticks on the cattle and the biopesticide, which reduces its efficacy against ticks.

Letter to the editor - Name that plant scientifically, please!

The report of Brunfelsia poisoning of dogs in Sydney by Drs Singh, Cowan and Child is very welcome, but it raises one important scientific point that has been further highlighted by the letter from Dr Seavers. Neither in the case report nor in Dr Seavers' letter is the species or cultivar of the plants identified. There are about 40 known Brunfelsia species, a number of which are cultivated in tropical to temperate climate gardens in Australia. It is noteworthy that Dr Seavers speculates about the Brunfelsia plants involved in dog poisonings in Australia, suggesting that 'dwarf' plants may be more poisonous ...

Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitis

In the article 'Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitis' (Milinovich et al., 2007), it is found that with reference to Horse 1, the histological signs of laminitis were first observed at 12 h post-oligofructose administration, and not 30 h as was indicated in the Results section under the subheading 'Induction of Laminitis' and in Fig. 1.

Identification of two distinct bovine parainfluenza virus type 3 genotypes

The partial gene sequencing of the matrix (M) protein from seven clinical isolates of bovine parainfluenza virus type 3 (BPIV-3), and the complete sequencing of a representative isolate (Q5592) was completed in this study. Nucleotide sequence analysis was initiated because of the failure of in-house BPIV-3 RT-PCR methods to yield expected products for four of the isolates. Phylogenetic reconstructions based on the nucleotide sequences for the M-protein and the entire genome, using all of the available BPIV-3 nucleotide sequences, demonstrated that there were two distinct BPIV-3 genotypes (BPIV-3a and BPIV-3b). These newly identified genotypes have implications for the development of BPIV-3 molecular detection methods and may also impact on BPIV-3 vaccine formulations.

Resistance to insect growth regulator insecticides in populations of sheep lice as assessed by a moulting disruption assay

Low-volume, backline applications with the benzoylphenyl urea insecticides triflumuron and diflubenzuron represent in excess of 70% of treatments for the control of sheep lice, Bovicola ovis (Schrank) (Phthiraptera: Trichodectidae), in Australia. Reports of reduced effectiveness from 2003 and subsequent controlled treatment trials suggested the emergence of resistance to these compounds in B. ovis populations. A laboratory assay based on the measurement of moulting success in nymphs was developed and used to assess susceptibility to diflubenzuron and triflumuron in louse populations collected from sheep where a control failure had occurred. These tests confirmed the development of resistance to triflumuron and diflubenzuron in at least two instances, with estimated resistance ratios of 67-94X at LC50.

New insights into the altered adhesive and mechanical properties of red blood cells parasitized by Babesia bovis

Sequestration of parasite-infected red blood cells (RBCs) in the microvasculature is an important pathological feature of both bovine babesiosis caused by Babesia bovis and human malaria caused by Plasmodium falciparum. Surprisingly, when compared with malaria, the cellular and molecular mechanisms that underlie this abnormal circulatory behaviour for RBCs infected with B. bovis have been relatively ignored. Here, we present some novel insights into the adhesive and mechanical changes that occur in B. bovis-infected bovine RBCs and compare them with the alterations that occur in human RBCs infected with P. falciparum. After infection with B. bovis, bovine RBCs become rigid and adhere to vascular endothelial cells under conditions of physiologically relevant flow. These alterations are accompanied by the appearance of ridge-like structures on the RBC surface that are analogous, but morphologically and biochemically different, to the knob-like structures on the surface of human RBCs infected with P. falciparum. Importantly, albeit for a limited number of parasite lines examined here, the extent of these cellular and rheological changes appear to be related to parasite virulence. Future investigations to identify the precise molecular composition of ridges and the proteins that mediate adhesion will provide important insight into the pathogenesis of both babesiosis and malaria.

Encroachment of Echinococcus granulosus into urban areas in eastern Queensland, Australia

Objective: To investigate the prevalence of Echinococcus granulosus in wild dogs (dingos and dingo-domestic dog hybrids) living in and around human habitation on Fraser Island and in townships of the Maroochy Shire, on Queensland's Sunshine Coast, Australia. Design: Wild dogs were humanely killed on Fraser Island and in the Maroochy Shire because they were deemed a potential danger to the public. Their intestines were collected and the contents examined for intestinal parasites. Procedure: Intestines were removed as soon after death as possible, packed in plastic bags and kept frozen until examination. The intestinal contents were washed, sieved and examined microscopically for the presence of helminths, which were identified and counted. Results: Intestines from 108 wild dogs, 7 foxes and 18 Fraser Island dingoes were examined. Echinococcus granulosus was only present in the wild dogs from Maroochy Shire (46.3%) with worm burdens of between 30 and 104,000. Other helminths included Spirometra erinacei, Dipylidium caninum, Taenia spp., Ancylostoma caninum and Toxocara canis. Two specimens of a trematode (Haplorchinae sp.) usually found infecting fish and seabirds were recovered from a Fraser Island dingo. Conclusion: Dingoes on Fraser Island are not infected with E. granulosus and do not pose a hydatid disease public health risk to residents or visitors. However, wild dogs examined from the Maroochy Shire do present a potential hydatid disease public health risk.

Productivity and health effects of anaplasmosis and babesiosis on Bos indicus cattle and their crosses, and the effects of differing intensity of tick control in Australia

Tick fever is an important disease of cattle where Rhipicephalus (Boophilus) microplus acts as a vector for the three causal organisms Babesia bovis, Babesia bigemina and Anaplasma marginale. Bos indicus cattle and their crosses are more resistant to the clinical effects of infection with B. bovis and B. bigemina than are Bos taurus cattle. Resistance is not complete, however, and herds of B. indicus-cross cattle are still at risk of babesiosis in environments where exposure to B. bovis is light in most years but occasionally high. The susceptibility of B. indicus cattle and their crosses to infection with A. marginale is similar to that of B. taurus cattle. In herds of B. indicus cattle and their crosses the infection rate of Babesia spp. and A. marginale is lowered because fewer ticks are likely to attach per day due to reduced numbers of ticks in the field (long-term effect on population, arising from high host resistance) and because a smaller proportion of ticks that do develop to feed on infected cattle will in turn be infected (due to lower parasitaemia). As a consequence, herds of B. indicus cattle are less likely than herds of B. taurus cattle to have high levels of population immunity to babesiosis or anaplasmosis. The effects of acaricide application on the probability of clinical disease due to anaplasmosis and babesiosis are unpredictable and dependent on the prevalence of infection in ticks and in cattle at the time of application. Attempting to manipulate population immunity through the toleration of specific threshold numbers of ticks with the aim of controlling tick fever is not reliable and the justification for acaricide application should be for the control of ticks rather than for tick fever. Vaccination of B. indicus cattle and their crosses is advisable in all areas where ticks exist, although vaccination against B. bigemina is probably not essential in pure B. indicus animals.

Bovine mucopolysaccharidosis type IIIB

Mucopolysaccharidosis IIIB, an autosomal recessive lysosomal storage disorder of heparan sulfate caused by mutations in the α-N-acetylglucosaminidase (NAGLU) gene, was recently discovered in cattle. Clinical signs include progressive ataxia, stumbling gait, swaying and difficulty in balance and walking. These clinical signs are usually first observed at approximately 2 years of age and then develop progressively over the lifespan of the animals. Affected bulls were found to be homozygous for the missense mutation E452K (c.1354G>A). The availability of mutational analysis permits screening for the NAGLU mutation to eradicate this mutation from the cattle breeding population.

Cyanide poisoning in cattle from Dysphania glomulifera (red crumbweed): Using the internet for rapid plant identification and diagnostic advice: Case Report

A 300-strong Angus-Brahman cattle herd near Springsure, central Queensland, was being fed Acacia shirleyi (lancewood) browse during drought and crossed a 5-hectare, previously burnt area with an almost pure growth of Dysphania glomulifera subspecies glomulifera (red crumbweed) on their way to drinking water. Forty cows died of cyanide poisoning over 2 days before further access to the plant was prevented. A digital image of a plant specimen made on a flat-bed scanner and transmitted by email was used to identify D glomulifera. Specific advice on the plant's poisonous properties and management of the case was then provided by email within 2 hours of an initial telephone call by the field veterinarian to the laboratory some 600 km away. The conventional method using physical transport of a pressed dried plant specimen to confirm the identification took 5 days. D glomulifera was identified in the rumen of one of two cows necropsied. The cyanogenic potential of D glomulifera measured 4 days after collection from the site of cattle deaths was 18,600 mg HCN/kg in dry matter. The lethal dose of D glomulifera for a 420 kg cow was estimated as 150 to 190 g wet weight. The plant also contained 4.8% KNO3 equivalent in dry matter, but nitrate-nitrite poisoning was not involved in the deaths.

Comparison of Culture and a Novel 5' Taq Nuclease Assay for Direct Detection of Campylobacter fetus subsp. venerealis in Clinical Specimens from Cattle

A Campylobacter fetus subsp. venerealis-specific 5' Taq nuclease PCR assay using a 3' minor groove binder-DNA probe (TaqMan MGB) was developed based on a subspecies-specific fragment of unknown identity (S. Hum, K. Quinn, J. Brunner, and S. L. On, Aust. Vet. J. 75:827-831, 1997). The assay specifically detected four C. fetus subsp. venerealis strains with no observed cross-reaction with C. fetus subsp. fetus-related Campylobacter species or other bovine venereal microflora. The 5' Taq nuclease assay detected approximately one single cell compared to 100 and 10 cells in the conventional PCR assay and 2,500 and 25,000 cells from selective culture from inoculated smegma and mucus, respectively. The respective detection limits following the enrichments from smegma and mucus were 5,000 and 50 cells/inoculum for the conventional PCR compared to 500 and 50 cells/inoculum for the 5' Taq nuclease assay. Field sampling confirmed the sensitivity and the specificity of the 5' Taq nuclease assay by detecting an additional 40 bulls that were not detected by culture. Urine-inoculated samples demonstrated comparable detection of C. fetus subsp. venerealis by both culture and the 5' Taq nuclease assay; however, urine was found to be less effective than smegma for bull sampling. Three infected bulls were tested repetitively to compare sampling tools, and the bull rasper proved to be the most suitable, as evidenced by the improved ease of specimen collection and the consistent detection of higher levels of C. fetus subsp. venerealis. The 5' Taq nuclease assay demonstrates a statistically significant association with culture (2 = 29.8; P < 0.001) and significant improvements for the detection of C. fetus subsp. venerealis-infected animals from crude clinical extracts following prolonged transport.

Detection of Helicobacter pullorum in meat chickens in Australia

The bacterial genus Helicobacter is a member of the Campylobacteriales in the Epsilonproteobacteria subphylum, and is comprised of organisms that are morphologically similar to Campylobacter. The term ‘campylobacteria’ is used to encompass the genera Campylobacter, Arcobacter, Helicobacter and Anaerobiospirillum. Helicobacter was separated from the genus Campylobacter in 1989. Helicobacter spp have been isolated from gastric tissue or intestinal contents of humans and a wide range of animal species, with some associated with disease...

Molecular and serological detection of A. centrale- and A. marginale-infected cattle grazing within an endemic area

A reverse line blot hybridization (RLB) one-stage nested PCR (nPCR) for Anaplasma centrale and a nested PCR for Anaplasma marginale were used to detect infected cattle grazing within an endemic region in Israel. A novel set of PCR primers and oligonucleotide probes based on a 16S ribosomal RNA gene was designed for RLB detection of both Anaplasma species, and the performance of the molecular assays compared. The immunofluorescent antibody test (IFA) was used to detect antibodies to both Anaplasma species, whereas, a highly sensitive and specific competitive enzyme-linked immunosorbent assay (cELISA) was used to detect antibodies in A. centrale-vaccinated cattle. The RLB and the nested PCR procedures showed bacteremia with sensitivity of 50 infected erythrocytes per milliliter. Up to 93% of the A. centrale vaccinates carried specific antibodies that were detected by cELISA, and up to 71% of the vaccinated cattle were found to be naturally infected with A. marginale according to the PCR and the RLB assays. Nevertheless, no severe outbreaks of A. marginale infection occurred among vaccinated herds in this endemic region. It appears that both, molecular tools and serology are useful for evaluation of the vaccine efficacy. In the light of wide natural field infection with A. marginale, strong recommendations to continue the A. centrale vaccination program regime will continue until a new generation of non-blood-based vaccine will be developed.