Phenotypic and genotypic analysis of benzimidazole resistance in reciprocal genetic crosses of Haemonchus contortus.

Haemonchus contortus is arguably one of the most economically important and ubiquitous parasites of livestock globally and commonly involved in cases of anthelmintic resistance. Here, we performed reciprocal genetic crosses using susceptible (MHco3(ISE)) and multiple anthelmintic resistant (MHco18(UGA2004)) H. contortus isolates. Resultant admixed populations were designated MHco3/18 or MHco18/3, where the lead isolate reflects the origin of the females. Three independent filial generations were generated for each cross, which were subjected to bioassays, molecular approaches and population genetic analyses to investigate the phenotypic and genotypic inheritance of benzimidazole (BZ) resistance at each stage. A panel of microsatellite markers confirmed the success of the genetic cross as markers from both parents were seen in the F1 crosses. Egg hatch tests revealed a stark difference between the two F1 crosses with ED50 estimates for MHco18/3 being 9 times greater than those for MHco3/18. Resistance factors based on ED50 estimates ranged from 6 to 57 fold in the filial progeny compared to MHco3(ISE) parents. Molecular analysis of the F167Y and F200Y SNP markers associated with BZ resistance were analysed by pyrosequencing and MiSeq deep amplicon sequencing, which showed that MHco3/18.F1 and MHco18/3.F1 both had similar frequencies of the F200Y resistant allele (45.3% and 44.3%, respectively), whereas for F167Y, MHco18/3.F1 had a two-fold greater frequency of the resistant-allele compared to MHco3/18.F1 (18.2% and 8.8%, respectively). Comparison between pyrosequencing and MiSeq amplicon sequencing revealed that the allele frequencies derived from both methods were concordant at codon 200 (rc = 0.97), but were less comparable for codon 167 (rc = 0.55). The use of controlled reciprocal genetic crosses have revealed a potential difference in BZ resistance phenotype dependent on whether the resistant allele is paternally or maternally inherited. These findings provide new insight and prompt further investigation into the inheritance of BZ resistance in H. contortus.

Precision worm control in grazing lambs by targeting group treatment based on performance of sentinels.

Given the economic impact of gastrointestinal nematode infection on livestock farming worldwide, and increasing anthelmintic resistance, it is imperative to develop practical, efficient and sustainable control strategies. Targeted selective treatment (TST), whereby anthelmintic treatments are administered to animals individually, based on selection criteria such as weight gain, has been shown to successfully maintain animal productivity whilst reducing the selection pressure for anthelmintic resistance and the economic cost of treatment in experimental and commercial settings. Despite the benefits of the TST approach, the equipment and time required to monitor animals individually make this strategy unsuitable for some farming enterprises. The sentinel group approach aims to maintain the benefits observed using TST whilst reducing these requirements. The study involved two experiments, each following a group of 80 lambs through their first grazing season. Anthelmintic treatment of the whole group was determined by monitoring the weight gain of identified sentinel lambs within it every 2 weeks: when 40% of the sentinel lambs failed to reach their weight gain targets, the whole group was treated. The sentinel lambs consisted of 45% of the group (n = 36) in experiment one and 20% (n = 16) in experiment two. A control group of 20 lambs was co-grazed with the main group during both experiments; in experiment one, the sentinel approach was compared with a TST approach, in which control lambs were treated on an individual basis in response to weight gain. In experiment two, the sentinel approach was compared with conventional prophylaxis, where all lambs in the control group were treated at strategic time points throughout the season (= strategic prophylactic treatment). The sentinel lambs were found to be representative of overall group performance regardless of the proportion of sentinels within the group: they recorded similar growth rates and reached weight gain targets simultaneously at each time point and overall. Live-weight gain was also similar between sentinel and control animals in both experiments. The findings of the current study suggest that monitoring sentinel lambs comprising 20% of a group of grazing lambs is sufficient to determine the need for anthelmintic treatment within the whole group, and that this approach maintains production in line with conventional or TST treatment regimes.

First evidence of Kunjin strain of West Nile virus associated with saltwater crocodile (Crocodylus porosus) skin lesions.

Recently, the Kunjin strain of West Nile virus (WNVKUN ) has been detected using qRT-PCR in belly skin lesions of farmed juvenile saltwater crocodiles. This follows an established association between similar lesions and West Nile virus in American alligators. The lesions present as cutaneous lymphohistiocytic aggregates in the dermal layers of both species. While these lesion do not create an obvious defect on the live crocodile, upon tanning the lesion area collapses and does not uptake the dye evenly, thus reducing its aesthetic appeal. As a result, skins are being rejected jeopardising the economic viability of the Australian crocodile industry. Over 50 skin lesions have since been confirmed as WNVKUN -positive and preliminary evidence of lesion restructuring is presented. Horizontal transmission of WNVKUN by mosquitoes is well-established but other transmission routes, such as ingestion and cloacal shedding, need further evaluation. An infection trial is currently underway to ensure WNVKUN is the causative agent of these skin lesions.

Post-viraemic detection of bovine ephemeral fever virus by use of autogenous lymphoid tissue-derived bovine primary cell cultures.

BACKGROUND: The potential tissue replication sites and specific cell types that support in vivo virus survival beyond the acute phase of bovine ephemeral fever virus (BEFV) infection have not been fully defined in cattle. To clarify the knowledge gap, tissue specimens were tested after collection from an adult steer necropsied 1 week after acute BEF. CASE REPORT: Significant necropsy findings included fibrinoproliferative synovitis in the stifle joints and fibrin clot-laden fluid in serous body cavities. Moderate numbers of infiltrating neutrophils were demonstrated in sections of the prefemoral lymph nodes and haemal node, and lymphoid hyperplasia in the spleen, haemal node and prefemoral lymph nodes. Viral RNA was detected by qRT-PCR in fresh spleen, haemal node, prefemoral lymph node, synovial fluid and in several spleen-derived cell cultures. BEFV was isolated from autogenously derived splenic primary cell cultures 6 days after cessation of viraemia, and characteristic bullet-shaped virions were confirmed by electron microscopy of an ultrathin haemal node section. In sections of the spleen, haemal node and other tissues, immunohistochemistry demonstrated BEFV antigens that were intracellularly associated with probable histiocytic cells. CONCLUSION: BEFV has preferential tropism for bovine lymphoid tissues and the spleen and haemal node may be potential sites for post-viraemic virus replication.

Viral neurotropism, peripheral neuropathy and other morphological abnormalities in bovine ephemeral fever virus-infected downer cattle.

OBJECTIVE: This study assessed the neurotropism of bovine ephemeral fever (BEF) virus (BEFV) and described histomorphological abnormalities of the brain, spinal cord and peripheral nerves that may causally contribute to paresis or paralysis in BEF. METHODS: Four paralysed and six asymptomatic but virus-infected cattle were monitored, and blood and serum samples screened by qRT-PCR, virus isolation and neutralisation tests. Fresh brain, spinal cord, peripheral nerve and other tissues were qRT-PCR-tested for viral RNA, while formalin-fixed specimens were processed routinely and immunohistochemically evaluated for histomorphological abnormalities and viral antigen distribution, respectively. RESULTS: The neurotropism of BEFV was immunohistochemically confirmed in the brain and peripheral nerves and peripheral neuropathy was demonstrated in three paralysed but not the six aneurological but virus-infected animals. Wallerian degeneration (WD) was present in the ventral funicular white matter of the lumbar spinal cord of a paralysed steer and in cervical and thoracic spinal cord segments of three paralysed animals. Although no spinal cord lesions were seen in the steer euthanased within 7 days of illness, peripheral neuropathy was present and more severe in nerves of the brachial plexuses than in the gluteal or fibular nerves. The only steer with WD in the lumbar spinal cord also showed intrahistiocytic cell viral antigen that was spatially distributed within areas of moderate brain stem encephalitis. CONCLUSION: The data confirmed neurotropism of BEFV in cattle and documented histomorphological abnormalities in peripheral nerves and brain which, together with spinal cord lesions, may contribute to chronic paralysis in BEFV-infected downer cattle.

Kinetics of selected plasma cytokines during innate-adaptive immune response transition in adult cattle infected with the bovine ephemeral fever virus.

While virus neutralizing antibodies are known to be variably protective against bovine ephemeral fever (BEF) virus (BEFV) infections, the cytokine events that mediate the nascent adaptive immune response have not been defined in cattle. This study determined the plasma kinetics of IL-2, IFN-γ, IL-6, and IL-10 during the period of innate-immune response transition and evaluated the relationship between the virus neutralizing antibody response and viraemia in BEFV-infected cattle. Plasma from four virus-infected and uninfected negative control animals was tested by cytokine-specific immunoenzymatic assays, viraemia monitored by qRT-PCR, and virus neutralizing antibody titres determined using a standard protocol. Unlike the negative controls, plasma IL-6 and IL-10 were increased in all the virus-infected animals starting several days prior to initiation of viraemia. In one animal, plasma IL-2 and IFN-γ were consistently higher than in the other three virus-infected animals and the negative control mean. The animal with the strongest IL-2 and IFN-γ responses had the shortest viraemia while the heifer with the lowest IL-2/IFN-γ indices demonstrated the longest viraemia. Evidently, increase in plasma IL-6 and IL-10 precedes seroconversion during BEFV infections in cattle suggesting the two cytokines may influence immunological events that pave way to B-cell activation and seroconversion. While there is remarkable variability in IL-2 and IFN-γ expression amongst BEFV-infected animals, increased plasma levels of the two cytokines appear to be associated with a shorter viraemia. Ongoing studies will help define the precise role of T cells in anti-BEFV adaptive immune responses.

Kinetics of pro-inflammatory cytokines, interleukin-10, and virus neutralising antibodies during acute ephemeral fever virus infections in Brahman cattle.

While fever and inflammation are hallmark features of bovine ephemeral fever (BEF), the cytokine networks that underlie the acute phase of the disease have not been empirically defined in cattle. This study characterised the plasma kinetics of proinflammatory cytokines (IL-1ß, IL-6, TNF-α) and IL-10 during acute BEF and elucidated on the relationship between the onset of the virus neutralizing antibody response and resolution of viraemia in natural BEF virus (BEFV) infections in cattle. Plasma from three BEFV-infected and three uninfected cattle was tested for the study cytokines by a cELISA, viraemia monitored by qRT-PCR, and virus neutralizing antibody titres determined using a standard protocol. Unlike the negative controls, plasma concentrations of IL-1ß, TNF-α, IL-6, and IL-10 were consistently increased in the three virus-infected animals. Two of the infected heifers were recumbent and pyrexic on the first day of monitoring and increased cytokine production was already in progress by the time viraemia was detected in all the three infected animals. In all the virus-infected heifers, IL-1ß was the most strongly expressed cytokine, IL-6 and IL-10 manifested intermediate plasma concentrations while TNF-α was the least expressed and demonstrated bi-phasic peaks three and five days after the onset of pyrexia. In two of the BEFV-infected heifers, viraemia resolved on the day of seroconversion while in the other infected animal, viral RNA was detectable up to three days after seroconversion. The present data document variable increase in plasma IL-1ß, IL-6, TNF-α, and IL-10 during natural BEFV infections and the fact that upregulation of all but TNF-α precedes seroconversion. In addition to virus neutralising antibodies, it is likely that cytokine-mediated cellular mechanisms may be required for resolution of viraemia in BEF. Considering the anti-inflammatory properties of IL-10, its upregulation may potentially antagonise the fever response in BEFV-infected cattle.

Gastric adenocarcinoma in a diamond python (Morelia spilota spilota).

CASE REPORT: A 5-year-old captive male diamond python (Morelia spilota spilota) was presented with a 1-month history of regurgitation and anorexia and discrete coelomic distention. Physical examination revealed a firm, immobile mass at approximately two-thirds of the snout-vent length from the front of the head. Ultrasound-guided fine needle aspirate biopsy of the mass in the region of the stomach showed necrosis with bacterial infiltration and possibly neoplastic changes. A gastroscopy was conducted, but showed grossly normal gastric mucosa, confirmed by biopsy. On exploratory coeliotomy, it was confirmed the mass involved most of the stomach wall and occluded the gastric lumen. The mass was completely excised and based on histopathology, a diagnosis of gastric adenocarcinoma was made. The snake was found dead 12 h postoperatively, but no specific cause of death was found on postmortem examination. CONCLUSION: Most cases of adenocarcinoma in snakes go undiagnosed. This case report illustrates that the architecture of gastric masses may lead to false-negative gastric biopsy results in snakes with neoplasia.

Synergistic interactions of the antiretroviral protease inhibitors saquinavir and ritonavir with chloroquine and mefloquine against Plasmodium falciparum in vitro.

The antimalarial activity of several antiretroviral protease inhibitor combinations was investigated. Data demonstrate that ritonavir and saquinavir behave synergistically with chloroquine and mefloquine. These data, and interactions with pepstatin-A, E-64, and bestatin, suggest that human immunodeficiency virus protease inhibitors do not target digestive-vacuole plasmepsins.

Cultured skin fibroblast cells derived from bluetongue virus-inoculated sheep and field-infected cattle are not a source of late and protracted recoverable virus.

A recent hypothesis to explain the recurrence of bluetongue disease after winter seasonal absences of the vector has suggested a role for persistent infection of sheep. This report presents combined independent work from two laboratories investigating the possible recovery of Bluetongue virus (BTV) over a protracted period after infection of both sheep and cattle. Prior to infection with either cell-culture-adapted or non-culture-adapted BTV, sheep were subjected to a preliminary exposure to Culicoides sp. insects, which reportedly facilitates recovery of virus from infected sheep several months post-infection (p.i.). A series of skin biopsies at different intervals p.i. was used to establish skin fibroblast (SF) cultures from which attempts were made to detect virus by isolation and by molecular and immunological methods. Also examined was the effect on virus recovery of additional exposure to Culicoides sp. prior to skin biopsy during the post-inoculation period. A herd of cattle sentinels for surveillance of natural BTV infection in northern Australia was monitored prospectively for seroconversion. Evidence of infection initiated attempted virus recovery by establishing SF cultures. It was found that in both cattle and sheep there was not a protracted period over which BTV could be recovered from SF cultures. The data do not support a general hypothesis that BTV persists in either sheep or cattle.

Structural features of mycorrhizal associations in two members of the Monotropoideae, Monotropa uniflora and Pterospora andromedea.

Species in the subfamily Monotropoideae (family Ericaceae) are achlorophyllous and myco-heterotrophic. They have become highly specialized in that each plant species is associated with a limited number of fungal species which in turn are linked to autotrophic plants. This study provides an updated and comprehensive examination of the anatomical features of two species that have recently received attention with respect to their host-fungal specificity. Root systems of Monotropa uniflora and Pterospora andromedea collected from the field were characterized by light microscopy and scanning electron microscopy. All roots of both species were associated with fungi, each root having a well-developed mantle, paraepidermal Hartig net, and intracellular "fungal pegs" within epidermal cells. The mantle of M. uniflora was multi-layered and numerous outer mantle hyphae developed into cystidia of two distinct morphologies. Large calcium oxalate crystals were present, primarily on the mantle surface. The outer mantle of P. andromedea was more loosely organized, lacked cystidia, and had smaller plate-like as well as cylindrical crystals on the surface and between outer mantle hyphae. Fungal pegs in M. uniflora originated from inner mantle hyphae that penetrated the outer tangential wall of epidermal cells; in P. andromedea, these structures were initiated either from inner mantle hyphae or Hartig net hyphae and penetrated radial walls of epidermal cells. With respect to function, fungal pegs occurred frequently in both host species and, although presumed to be the sites of active nutrient exchange, no direct evidence exists to support this. Differences between these two monotropoid hosts, resulting from the mycorrhizal fungi with which each associates, are discussed.

Bluetongue surveillance methods in an endemic area: Australia.

Surveillance for bluetongue (BT) viruses (BTV) has been carried out in the Northern Territory, Australia since 1980. The number of sites, intensity of sampling and methods of testing have varied during this period. Monthly serology is conducted at a number of sentinel sites and intensive weekly sampling for virus isolation is conducted at the site of highest known arboviral activity. This has enabled the isolation of all eight BTV serotypes identified in Australia. Natural viraemias are between one and eight weeks. No additional serotypes have been isolated since 1986. However, genetic analysis of isolates has shown incursions of viruses of South-East Asian origin in 1992, 1994 and 1995. Trapping for Culicoides spp. has also been carried out at these sites on a regular basis. In recent years, an annual serological survey has supplemented the sentinel herds to more accurately define the BT zones described under OIE guidelines.

Protection of cattle from Culicoides spp. in Australia by shelter and chemical treatments.

Trials were conducted in three regions of Australia to investigate the potential for improvised shelters and chemical treatments to reduce feeding by Culicoides on cattle and thereby minimise the risk of bluetongue transmission during transport of cattle to ports. Various designs and combinations of roofs and walls were placed around penned cattle. Chemical treatments were applied to other penned cattle. Culicoides were collected from the cattle by vacuum samplers or by light traps in the pens. Roofs alone did not consistently reduce the numbers of Culicoides brevitarsis or C. fulvus and increased the numbers of C. actoni collected. Walls alone reduced the numbers of C. wadai but not C. brevitarsis. Roofs and walls in combination reduced the numbers of C. brevitarsis and C. wadai. The chemical treatments 'Flyaway' (a blend of repellents) and fenvalerate reduced the numbers of C. brevitarsis and C. wadai up to 52 h post treatment.

Temporal activity of biting midges (Diptera: Ceratopogonidae) on cattle near Darwin, Northern Territory, Australia.

The activity of nine species of biting midges aspirated from cattle was recorded in the late afternoon, evening and early morning at a site near Darwin, Northern Territory, between March and June in 1999 and 2001. There were no significant differences between the temporal activity patterns for nulliparous and parous females of any species. Nulliparous females dominated collections of all species except Culicoides marksi. C. actoni and Forcipomyia (Lasiohelea) sp., were mostly active during daylight hours while C. peregrinus, C. bundyensis and C. brevipalpis, were nocturnal. Differences in the peak activity of C. brevitarsis were noted between years and occurred slightly earlier than that observed at other sites. C. fulvus, C. marksi and C. oxystoma were generally crepuscular but differed in the length and peak period of activity. C. actoni was four times more active in the evening than in the morning while C. marksi and C. peregrinus, were respectively 2.6 and 3.4 times more active in the morning than in the evening. Numbers of the other six species were not significantly different in the evening and morning. All nine species were collected at least once from cattle shortly after dawn.

Genetic diversity of bluetongue viruses in Australasia.

The authors have characterised the genetic diversity of the bluetongue virus (BTV) RNA segments 3 and 10 from Indonesia, Malaysia and Australia. Analysis of RNA segment 3, which codes for the core protein VP3, showed conserved sequences in the previously defined Australasian topotype, but which further divided into four distinct clades or genotypes. Certain genotypes appeared to be geographically restricted while others were distributed widely throughout South-East Asia. Ongoing surveillance programmes in Australia have identified the movement of Indonesian genotypes into northern Australia and possible reassortment among them. Similarly, analysis of RNA segment 10, which codes for the non-structural protein NS3/3A, showed they were also conserved and grouped into five clades or genotypes, three Asian and two North American/South African.

Bluetongue virus does not persist in naturally infected cattle.

Studies were designed to test if observations by Takamatsu et al. in 2003 were applicable to natural infection of cattle with bluetongue virus (BTV). These observations suggested that ovine gamma delta T-cells could become persistently infected and subsequent midge feeding could induce virus replication. Skin biopsies and blood were collected from 28 cattle naturally infected with BTV-1. Blood samples were processed for virus isolation by embryonated chicken egg inoculation and for serology by BTV competitive enzyme-linked immunosorbent assay and BTV-1 virus neutralisation. BTV-1 was isolated from the blood of all animals and serology confirmed infection with BTV-1. A total of 288 skin biopsies were collected and cultured in the presence of interleukin 2 and epidermal growth factor. Sampling commenced as soon as either serology or virus isolation indicated infection with BTV and continued at weekly intervals for at least eight weeks then monthly for another two months. The natural viraemias in this experiment ranged from one to five weeks. BTV-1 was isolated from only one skin biopsy sample. This sample was collected during the week in which the animal was viraemic. These findings provide compelling evidence that BTV does not persist in gamma delta T-cells in the skin of naturally infected cattle.

Excretion of bluetongue virus in cattle semen: a feature of laboratory-adapted virus.

A series of experiments was conducted over a period of four years and involved both young (2-4 years) and old bulls (5-15 years) that were both naturally and experimentally infected with bluetongue virus (BTV). Several different virus serotypes were studied. In the Northern Territory, young bulls were exposed to natural infection with BTV over three wet seasons. During this time, bulls were infected with BTV-1, BTV-3, BTV-16 and BTV-20. In New South Wales, semen samples were examined from a large group of bulls of mixed ages that were naturally infected with BTV-1. Experimental infections in both young and old bulls (5-8 animals per group) employed both 'wild-type' and laboratory-adapted viruses from serotypes 1 and 23. A total of 41 bulls were included in the studies of natural BTV infection and 52 bulls in experimental infections. There was no evidence of BTV in any of the semen samples collected from naturally infected bulls or experimentally infected young bulls. BTV was detected intermittently in semen from a number of old bulls infected with both laboratory-adapted BTV-1 and BTV-23. These detections occurred during or immediately after the period of detectable viraemia. Virus was also detected in a few semen samples from very old bulls infected with 'wild-type' BTV-23. These samples were collected during the period of viraemia and there was usually evidence of blood in the semen. Viraemia varied in duration between 17 and 38 days. Following immunosuppression, there was no evidence of resurgence of viraemia, or excretion of virus in semen, even in animals in which virus had been previously detected in semen. When the bulls were slaughtered, virus was not detected in any tissues.

Characterisation and monitoring of neutralisation-resistant VP2 phenotypes in BTV-1 isolates from northern Australia collected over a twenty-year period.

Phenotypic profiles of the VP2 protein of isolates of bluetongue virus serotype 1 (BTV-1) collected from Queensland and the Northern Territory, Australia, between 1979 and 1986 were analysed using neutralising monoclonal antibodies (MAbs) raised to the prototype isolate of Australian BTV-1 collected in the Northern Territory in 1979. Two distinct profiles were found. Northern Territory isolates exhibited the prototype profile, yet those from Queensland had a significantly different ('resistant') profile. Nucleotide sequencing of gene segment 2 from both groups of isolates was undertaken. When the nucleotide sequences of isolates from a later period in each State were analysed (1997-2001), all exhibited the 'resistant' profile. Thus, a novel VP2 phenotype, already in existence in Queensland, had supplanted a pre-existing VP2 phenotype in the Northern Territory between the two periods. Furthermore, amino acid differences between the resistant and prototype VP2 proteins were analogous to amino acid substitutions known to be associated with neutralisation resistance. The host immune response may therefore have contributed to selection of the 'resistant' phenotype.

The effectiveness of scalp cooling in preventing alopecia for patients receiving epirubicin and docetaxel.

The aim of this study was to establish the effectiveness of scalp cooling in preventing alopecia for patients with breast cancer who received the trial combination chemotherapy of Epirubicin and Docetaxel. Doubt remains about the general effectiveness of scalp cooling in preventing hair loss for patients receiving chemotherapy. There is very little information available about its specific effectiveness with combinations of Taxanes and Anthracycline drugs. Of the 40 patients who received this drug combination, 10 were included in a pilot study whereas the remaining 30 constituted the main study sample. A randomized controlled study was undertaken whereby the intervention group received scalp cooling via gel cool caps and the control group received no specific preventative intervention. Nurses assessed participants' hair loss using a modified version of the WHO scale at seven time points and also recorded hair loss photographically. Two independent experts rated the photographs using the same scale. Patients self-reported in relation to overall hair loss, hair condition, levels of emotional upset, negativity about appearance, hair re-growth and wig use. Significantly greater hair loss was apparent in the control group during most of the treatment period. However, the level of protection afforded by the cool caps was relatively poor with this chemotherapy combination. The marginal benefits of scalp cooling in this context must be clearly explained to patients.