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Most of our understanding of the ecology and evolution of avian influenza A virus (AIV) in wild birds is derived from studies conducted in the northern hemisphere on waterfowl, with a substantial bias towards dabbling ducks. However, relevant environmental conditions and patterns of avian migration and reproduction are substantially different in the southern hemisphere. Through the sequencing and analysis of 333 unique AIV genomes collected from wild birds collected over 15 years we show that Australia is a global sink for AIV diversity and not integrally linked with the Eurasian gene pool. Rather, AIV are infrequently introduced to Australia, followed by decades of isolated circulation and eventual extinction. The number of co-circulating viral lineages varies per subtype. AIV haemagglutinin (HA) subtypes that are rarely identified at duck-centric study sites (H8-12) had more detected introductions and contemporary co-circulating lineages in Australia. Combined with a lack of duck migration beyond the Australian-Papuan region, these findings suggest introductions by long-distance migratory shorebirds. In addition, on the available data we found no evidence of directional or consistent patterns in virus movement across the Australian continent. This feature corresponds to patterns of bird movement, whereby waterfowl have nomadic and erratic rainfall-dependant distributions rather than consistent intra-continental migratory routes. Finally, we detected high levels of virus gene segment reassortment, with a high diversity of AIV genome constellations across years and locations. These data, in addition to those from other studies in Africa and South America, clearly show that patterns of AIV dynamics in the Southern Hemisphere are distinct from those in the temperate north., Competing Interests: The authors have declared that no competing interests exist.

The number of reptilian viruses detected are continuously increasing due to improvements and developments of new diagnostic techniques. In this case we used primary cell culture and qPCR to describe the first Australian Chelonia mydas papillomavirus. Commercial chelonian cell lines are limited to one cell line from a terrestrial turtle (Terrapene Carolina). To establish primary cultures from green turtles (Chelonia mydas), turtle eggs were collected from Heron Island, Queensland, Australia. From day 35 of incubation at 29°, the embryos were harvested to establish primary cultures. The primary cell cultures were grown in Dulbecco's Modified Eagle Medium, 90% and foetal bovine serum, 10%. The cells became uniformly fibroblastic-shaped after 15 passages. The growth rate resembled that of cells originating from other cold-blooded animals and the average doubling time was ∼5 days from the 20th passage. Karyotyping and molecular analysis of mitochondrial DNA D-loop gene were carried out for cell authentication. The primary cell cultures were screened to exclude mycoplasma contamination. Two primary cell lineages were found to be susceptible to Bohle iridovirus. The primary cell cultures were used to screen samples from green turtles foraging along the East Coast of Queensland for the presence of viruses. Homogenates from eight skin tumour samples caused cytopathic effects and were confirmed by qPCR to be infected with papillomavirus., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Sea turtle fibropapillomatosis (FP) is a disease marked by the proliferation of benign but debilitating cutaneous and occasional visceral tumors, likely to be caused by chelonid alphaherpesvirus 5 (ChHV5). This study presents a phylogeny of ChHV5 strains found on the east coast of Queensland, Australia, and a validation for previously unused primers. Two different primer sets (gB-1534 and gB-813) were designed to target a region including part of the UL27 glycoprotein B (gB) gene and part of UL28 of ChHV5. Sequences obtained from FP tumors found on juvenile green turtles Chelonia mydas (<65 cm curved carapace length) had substantial homology with published ChHV5 sequences, while a skin biopsy from a turtle without FP failed to react in the PCRs used in this study. The resulting sequences were used to generate a neighbor-joining tree from which three clusters of ChHV5 from Australian waters were identified: north Australian, north Queensland, and Queensland clusters. The clusters reflect the collection sites on the east coast of Queensland with a definitive north-south trend. Received October 22, 2016; accepted May 7, 2017.

Background: The Balimo region in Papua New Guinea has previously been identified as melioidosis-endemic with a predilection for children. Where health resources are scarce, seroepidemiology can be used to assess exposure to Burkholderia pseudomallei and therefore risk of acquiring melioidosis., Methods: Logistic regression was used to determine associations between indirect haemagglutination assay (IHA) seroreactivity with environmental and demographic/cultural factors to aid in determining risk factors associated with exposure to B. pseudomallei in children., Results: Of the 968 participants, 92.9% (899/968) were children, representing the majority of the community school population in the immediate Balimo region. Of these, 24.6% (221/899) were seropositive. Bathing in the lagoon (OR=2.679), drinking from the well or lagoon (OR=1.474), and being a member of the Siboko (OR=1.914) or Wagumisi (OR=1.942) clans were significantly associated with seropositivity. In the multivariate analysis, drinking from a well or lagoon (OR=1.713), and the Siboko (OR=2.341) and Wabadala (OR=2.022) clans were associated with seropositivity., Conclusions: This study in children supports observations that interactions with groundwater in this region are risk factors in acquiring melioidosis. Public health measures intended to limit this exposure may help reduce the risk of acquiring melioidosis in this remote community. Associations with clan structure may provide more cultural specific insights, however this requires further elucidation., (© The Author 2014. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

As most newly characterized emerging infectious diseases are considered to be zoonotic, a modern pre-eminence ascribed within this classification lies clearly within the viral taxonomic realm. In particular, RNA viruses deserve special concern given their documented impact on conservation biology, veterinary medicine and public health, with an unprecedented ability to promote an evolutionary host-pathogen arms race from the ultimate infection and immunity perspective. However, besides the requisite molecular/gross anatomical and physiological bases for infectious diseases to transmit from one host to another, both viral pathogens and their reservoirs/vectors exploit a complex anthropological, cultural, historical, psychological and social suite that specifically defines the phylodynamics within Homo sapiens, unlike any other species. Some of these variables include the ecological benefits of living in groups, decisions on hunting and foraging behaviours and dietary preferences, myths and religious doctrines, health economics, travel destinations, population planning, political decisions on agricultural product bans and many others, in a homo-sociome memetic complex. Taken to an extreme, such complexities elucidate the underpinnings of explanations as to why certain viral zoonoses reside in neglected people, places and things, whereas others are chosen selectively and prioritized for active mitigation. Canine-transmitted rabies serves as one prime example of how a neglected viral zoonosis may transition to greater attention on the basis of renewed advocacy, social media, local champions and vested international community engagement. In contrast, certain bat-associated and arboviral diseases suffer from basic ignorance and perpetuated misunderstanding of fundamental reservoir and vector ecology tenets, translated into failed control policies that only exacerbate the underlying environmental conditions of concern. Beyond applied biomedical knowledge, epidemiological skills and biotechnical abilities alone, if a homo-sociome memetic complex approach is also entertained in a modern transdisciplinary context, neglected viral zoonosis may be better understood, controlled, prevented and possibly eliminated, in a more holistic One Health context., (Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Migratory birds may introduce highly pathogenic H5N1 avian influenza from Southeast Asia into Australia via North Queensland, a key stopover along the East Asian-Australasian Flyway, with severe consequences for trade and human health. A 3-year repeated cross sectional study on the epidemiology of avian influenza in Australian nomadic wild aquatic birds was conducted in this potential biosecurity hotspot using molecular and serological techniques. Avian influenza virus subtypes H6 and H9 were commonly present in the studied population. It is likely that one of the H6 viruses was newly introduced through migratory birds confirming the perceived biosecurity risk. The matrix gene of another H6 virus was similar to the Australian H7 subtypes, which suggests the reassortment of a previously introduced H6 and local viruses. Similarly, a H9 subtype had a matrix gene similar to that found in Asian H9 viruses suggesting reassortment of viruses originated from Australia and Asia. Whilst H5N1 was not found, the serological study demonstrated a constant circulation of the H5 subtype in the sampled birds. The odds of being reactive for avian influenza viral antibodies were 13.1(95% CI: 5.9-28.9) for Pacific Black Ducks over Plumed Whistling Ducks, highlighting that some species of waterfowl pose a greater biosecurity risk. Antibody titres were slightly higher during warm wet compared with warm dry weather. Routine surveillance programmes should be established to monitor the introduction of avian influenza viruses from Asia and the interactions of the introduced viruses with resident viruses in order to better detect emerging pathogens in aquatic birds of North Queensland. Surveillance should be targeted towards highly susceptible species such as the Pacific Black Duck and carried out during favourable environmental conditions for viral transmission such as the wet season in northern Australia., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A cross sectional survey was conducted involving 354 farm poultry workers on 85 randomly selected commercial poultry farms in high density poultry farm areas in Pakistan to estimate the sero-prevalence of H5, H7 and H9 and to identify the potential risk factors for infection with the avian influenza virus. A haemagglutination inhibition test titre at 1:160 dilution was considered positive, based on WHO guidelines. The estimated sero-prevalence was 0% for H5, 21.2% for H7 and 47.8% for H9. Based on a generalized linear mixed model, the significant risk factors for H7 infection were area, type of farm and age of poultry worker. Risk of infection increased with the age of poultry workers. Compared with broiler farms, breeder farms presented a greater risk of infection (odds ratio [OR]=3.8, 95% confidence interval [CI]: 1.4, 10.1). Compared with the combined Khyber Pakhtunkhwa Province and Federal area, North Punjab had higher observed biosecurity measures and presented a lesser risk of infection (OR=0.3, 95% CI 0.1, 0.9). Biosecurity should therefore be enhanced (especially in breeder farms) to reduce the occupational risks in poultry farm workers and to decrease the risk of emergent human-adapted strains of AI H7 and H9 viruses., (Copyright © 2014 Elsevier B.V. All rights reserved.)

To investigate the origins, evolution and patterns of spread of HPAI H5N1 outbreaks in Bangladesh, we performed a phylogenetic reconstruction analysis using Bayesian methods. The analysis was conducted using 81 hemagglutinin (HA) gene sequences from the H5N1 viruses isolated in Bangladesh from 2007 to 2011, together with 264 publicly available HA sequences of clade 2.2, 2.3.2 and 2.3.4 retrieved from GenBank. Our study provides evidence that clade 2.2.2 viruses that caused outbreaks in Bangladesh were lineages independent from the viruses introduced earlier into India. Furthermore, the Bangladesh clade 2.2.2 descendents subsequently spread to India and Bhutan. This has implications for avian influenza control in southern Asia suggesting multiple routes of entry of the virus including one pathway that spread to neighboring countries via Bangladesh., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We applied a competitive enzyme-linked immunosorbent assay for the detection of antibodies for influenza A in equine sera to their detection in sera from wild aquatic birds. Suboptimal results were obtained for the optical density (OD) of the monoclonal antibody (MAb) control and reproducibility between duplicate analyses in the initial assessment. It was therefore necessary to modify the assay to deliver increased reliability and reproducibility while maintaining adequate sensitivity. We optimized reagent concentrations to obtain optimal OD values (close to 2) for the monoclonal antibody control and used 2, 2'-Azino-bis: 3-Benzthiazoline-6-Sulphonic Acid as an alternative chromogen to potentially reduce variability in duplicate analyses. The original assay was compared with the optimized versions, with and without post coating, for the detection of avian influenza viral antibodies in 240 sera obtained from wild plumed whistling ducks. A separate analytical sensitivity study on diluted positive field sera of plumed whistling ducks and a test of antigen stability after post coating were also performed. Some quantitative differences were detected between the original and modified assays. The original assay recorded higher percentage inhibition results which were potentially indicative of increased sensitivity. However, when reagent concentrations were increased in the original assay to the same levels as used in the modified versions, there were no quantitative differences for practical purposes. The original assay produced a median (OD) value of 0.81 for the (MAb) controls that is at the limit of acceptability. By contrast, the modified assays always produced acceptable optical density values for MAb controls. Our overall results indicated the modified assays were potentially more reliable (OD values close to 2), and of adequate sensitivity compared to the original assay in the detection of avian influenza viral antibodies in wild bird sera. Although further optimization of antigen and MAb concentrations should also be considered to increase the sensitivity of a modified assay, while maintaining acceptable optical density values for the MAb control. Post coating had a minimal quantitative effect on the results and stabilized the plates for 214 days. We therefore recommend the incorporation of post coating.

Infectious diseases are common causes of significant morbidity and mortality events of wild aquatic birds (WABs) worldwide. Reports of Australian events are infrequent. A 3-yr passive surveillance program investigating the common causes of morbidity and mortality of WABs was conducted at Billabong Sanctuary near Townsville, North Queensland, from April 2007 to March 2010. Forty-two carcasses were obtained and evaluated by clinico-pathologic, histologic, bacteriologic, and virologic (molecular) examinations. Morbidity and mortality were sporadic and more commonly observed in chicks and juvenile birds in April than other months of the year. Morbid birds were frequently unable to walk. Hemorrhagic lesions and infiltration of lymphocytes in various organs were the most common findings in dead birds. Identified bacterial diseases that could cause bird mortality were colibacillosis, pasteurellosis, and salmonellosis. Salmonella serotypes Virchow and Hvittingfoss were isolated from an Australian white ibis (Threskiornis molucca) chick and two juvenile plumed whistling ducks (Dendrocygna eytoni) in April 2007. These strains have been previously isolated from humans in North Queensland. A multiplex real time reverse transcriptase-PCR (rRT-PCR) detected Newcastle disease viral RNA (class 2 type) in one adult Australian pelican (Pelecanus conspicillatus) and a juvenile plumed whistling duck. No avian influenza viral RNA was detected from any sampled birds by the rRT-PCR for avian influenza. This study identified the public health importance of Salmonella in WABs but did not detect the introduction of the high pathogenicity avian influenza H5N1 virus in the population. A successful network was established between the property owner and the James Cook University research team through which dead birds, with accompanying information, were readily obtained for analysis. There is an opportunity for establishing a long-term passive disease surveillance program for WABs in North Queensland, an important region in Australian biosecurity, thus potentially significantly benefitting public health in the region and the country.

Wild aquatic birds (WABs) are considered as reservoir hosts for Newcastle disease viruses (NDVs) and may act as vectors for transferring these viruses to poultry, causing outbreaks of disease. A 3-year epidemiological study was conducted on WABs of north Queensland from April 2007 to March 2010. Swab and fresh moist faecal samples of WABs were screened to detect Newcastle disease viral (NDV) RNA by one-step real time reverse transcriptase polymerase chain reaction (rRT-PCR) in multiplex primers, targeting the matrix gene. The potential reactor samples in rRT-PCR were processed for sequencing of the different NDV genes using conventional PCR. The overall NDV RNA prevalence was 3.5% for live bird samples (N=1461) and 0.4% for faecal samples (N=1157). Plumed whistling ducks (PWDs) had a higher prevalence (4.2%) than Pacific black ducks (PBDs) (0.9%) (χ(2) test, p=0.001). Univariate and multivariate logistic regression analyses were used to estimate the association between the proportion of reactor and non-reactor NDV RNA samples of PWDs and potential risk factors. The odds of reactor samples were 2.7 (95% Confidence Interval 1.5-4.9) times more likely in younger than older ducks (p=0.001) (data set B, multivariate analysis). Both NDV RNA class-one and class-two types were identified in samples of WABs (12 and 59, respectively) (Supplementary Table 1). Phylogenetic analysis of the matrix gene identified two reactor sequences of class-one type NDV RNA (PWD-48 and 55) which were closely related to the sequences of Australian Ibis and duck isolates (Fig. 2). Another reactor sample sequence was determined as class-two type NDV RNA (PWD-46, avirulent) based on analysis of the matrix and fusion genes which was more similar to the sequences of Australian I-2 progenitor virus and vaccine strain virus (Figs. 3 and 4). Our findings of higher prevalence in PWDs along with confirmation of class-one and class-two type NDV RNAs will significantly contribute to the design of surveillance programs for NDVs in northern Australia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Tabanid flies in Australia are potential vectors of the parasite Trypanosoma evansi which causes the animal disease surra. It is endemic to most of south-east Asia and could enter Australia, but evaluation of the potential impact of a surra incursion requires identification of the major hosts of Australian tabanids. This study investigated the natural pattern of feeding and host preference by tabanid flies of Townsville, north Queensland by identification of ingested blood in trap-caught tabanids using ELISA. The assays were developed for identification of horse, cow, macropod and pig blood meals. Macropods were the most frequent food source for each of six major tabanid species in the area. This did not vary with location for one species, Tabanus pallipennis, despite macropod densities being lower than other hosts such as cattle and horses in some locations. Feeding patterns on other hosts generally depended on availability and density of animals. All tabanid species fed on at least three of the host species tested and mixed meals were also commonly encountered, suggesting a level of opportunistic feeding in addition to a preference for macropods. Some of the blood meals detected were possibly from previous gonotrophic cycles. The results indicate that all tabanid species examined could potentially transmit surra and all the host types investigated could be affected, but macropods face the highest transmission risk., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Introduction: Contact investigations conducted in the United States of persons with tuberculosis (TB) who traveled by air while infectious have increased. However, data about transmission risks of Mycobacterium tuberculosis on aircraft are limited., Methods: We analyzed data on index TB cases and passenger contacts from contact investigations initiated by the U.S. Centers for Disease Control and Prevention from January 2007 through June 2008., Results: Contact investigations for 131 index cases met study inclusion criteria, including 4550 passenger contacts. U.S. health departments reported TB screening test results for 758 (22%) of assigned contacts; 182 (24%) had positive results. Of the 142 passenger contacts with positive TB test results with information about risk factors for prior TB infection, 130 (92%) had at least one risk factor and 12 (8%) had no risk factors. Positive TB test results were significantly associated with risk factors for prior TB infection (OR 23; p<0.001). No cases of TB disease among passenger contacts were reported., Conclusion: The risks of M. tuberculosis transmission during air travel remain difficult to quantify. Definitive assessment of transmission risks during flights and determination of the effectiveness of contact-tracing efforts will require comprehensive cohort studies.

We recently characterized three novel alphaviruses isolated from mosquitoes captured in New South Wales, Australia. Initial cross-neutralization studies revealed antigenic similarity to the Sindbis virus (SINV)-like Whataroa virus (WHAV), heretofore found only in New Zealand. Nucleotide sequence analysis showed that the WHAV-like viruses shared >99% nucleotide sequence similarity with each other, and 96-97% similarity with prototype WHAV. Enzyme-linked immunosorbent assay reactions of a panel of monoclonal antibodies to SINV showed that the novel WHAV-like viruses displayed identical binding patterns and were antigenically distinct from all SINV isolates examined. Although these viruses displayed a similar binding pattern to prototype WHAV, three monoclonal antibodies discriminated them from the New Zealand virus. Our results suggest that these novel alphaviruses are antigenic variants of WHAV and represent the first reported isolations of this virus from outside New Zealand. The monoclonal antibodies used in this study will be useful for typing new SINV and SINV-like isolates.

Little is known about the process whereby the emetic toxin (or cereulide) of Bacillus cereus is produced. Two cereulide-producing strains of B. cereus were cloned and sequenced following polymerase chain reaction (PCR) amplification with primers that were specific for conserved regions of non-ribosomal peptide synthetase (NRPS) genes. The cloned regions of the B. cereus strains were highly homologous to conserved regions of other peptide synthetase nucleotide sequences. Primers were designed for two variable regions of the NRPS gene sequence to ensure specificity for the emetic strains. A total of 86 B. cereus strains of known emetic or non-emetic activity were screened using these primers. All of the emetic strains (n=30) displayed a 188 bp band following amplification and gel electrophoresis. We have developed an improved method of identifying emetic strains of B. cereus and provided evidence that cereulide is produced by peptide synthetases.

A simple and inexpensive method of antigen preparation by ultrafiltration was investigated using the V4 strain of Newcastle disease virus. The antigen designated XM300 was used in an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Newcastle disease virus in chicken serum. The assay was evaluated using both experimental and field sera, as well as reference control reactor and non-reactor sera. Antigen prepared by the ultrafiltration method was compared with antigen prepared by ultracentrifugation and the ultrafiltration antigen was found to react specifically with Newcastle disease virus antiserum in this ELISA system. This antigen preparation technique is also suitable for use in developing countries. The ELISA provides an excellent method for measuring antibodies in the early stages of infection in serum samples from experimentally infected chickens. More than 14.58 % of the total serum samples which failed to be recognized as reactors by the conventional haemagglutination inhibition test were detected in the ELISA.

A panel of 10 monoclonal antibodies to a hypervirulent fowl adenovirus (FAdV) strain 398A was produced. Monoclonal antibodies (mAbs) were characterized for their isotype, neutralizing activity, ability to capture viral antigens, immunoblotting of viral polypeptides, competitive inhibition with chicken antisera and their reaction pattern with other FAdVs in indirect immunoperoxidase. Eight out of 10 mAbs reacted strongly in indirect immunoperoxidase staining with most of the FAdVs isolated from inclusion body hepatitis in Australia. One of these mAbs (6E1) was found to specifically react with the strains presumably characterized as hypervirulent FAdVs. IgG(2a) was the predominant sub-isotype. Two out of 10 mAbs neutralized the homologous strain of virus and six captured their target antigen onto a plastic surface. Chicken anti-serum to FAdV strain 398A inhibited the reaction of the seven mAbs that bound with high affinity in a blocking competitive enzyme-linked immunosorbent assay. This panel of mAbs can be used to improve diagnostic assays, study pathogenesis and carry out strain identification.

Five monoclonal antibodies (MoAb) were produced to the envelope (E) protein of Kunjin (KUN) virus. Three of these were specific for the KUN/West Nile complex, and two reacted with epitopes that were common to the flavivirus family. These MoAb defined at least four distinct epitopes on the E protein of KUN virus. Eight MoAb were also produced to unidentified proteins of Kokobera (KOK) virus. Seven were specific for KOK, while the remaining antibody cross-reacted with Stratford virus and an unregistered flavivirus CS946. The application of these MoAb to arboviral surveillance is discussed.

Previous studies have shown that antibodies produced against strategic flavivirus epitopes play an important role in recovery and immunity. Definition of the conformation and location of these epitopes and the degree of their conservation among flaviviruses is important to understanding the humoral response to flavivirus infection. In this study we have examined epitopes recognized by 14 monoclonal antibodies (MAbs) produced to the envelope (E) and non-structural (NS1) proteins of Murray Valley encephalitis virus (MVE). These antibodies were analysed for specificity, neutralization, haemagglutination inhibition (HI) and competitive binding. We have identified six distinct epitopes on the E protein which are located in four non-overlapping domains. MAbs to epitopes in one domain neutralized virus, were specific for MVE and Japanese encephalitis virus, and reacted with epitopes resistant to reduction. Two other E domains, one specific to MVE and the other shared by all flaviviruses, also contained neutralization sites and were stabilized by disulphide bonds. The fourth domain on E was conserved among the flaviviruses, sensitive to SDS denaturation and did not induce neutralizing antibody. Studies with MVE NS1 MAbs revealed that they were mostly type-specific, unreactive with conserved epitopes, and unreactive in HI and neutralization tests. The six epitopes identified on NS1 did not overlap and represent antigenic domains either resistant or sensitive to reduction. Immunoblotting of viral proteins in MVE-infected C6/36 cells revealed two distinct forms of NS1 and high Mr proteins of 97K and 108K that represented disulphide-linked heterodimers of E and NS1.

Eighteen monoclonal antibodies were produced against three Australian bovine enterovirus serogroups. Based on their binding patterns to 14S subunits, natural empty capsids and virions of 4 selected viral isolates (K2577, SL305, 66/27, J2129) in two types of quantitative ELISAs, the 18 antibodies were shown to belong to four groups. Due to the specific reaction of monoclonal antibodies, the four groups likely react with four epitopes. The first epitope was shown mainly on the surface of isolate K2577. The second and the third epitopes were common epitopes shared by all four isolates. These epitopes presented equally well on 14S subunits, natural empty capsids and virions. The fourth epitope was present mainly on the natural empty capsids and shared by most, but not all isolates. The four epitopes seem to be conformation dependent epitopes.

A blocking enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against bovine enterovirus (BEV) in bovine sera. In this ELISA, bovine serum samples were allowed to react with captured viral antigens (by specific chicken IgG), before the addition of specific mouse IgG for measuring non-occupied viral epitopes. The ELISA was slightly more sensitive and required a shorter time period than traditional serum neutralisation (SN). Among the 871 bovine serum samples tested so far, the titres produced by this assay had a significant correlation with those recorded by SN. The ELISA could be used as an alternative assay for SN in a large-scale BEV antibody investigation.

Six goats were inoculated with Brucella ovis. Two goats were inoculated with infected semen by the intratesticular route and 2 each by installation of the semen on to the nasal or preputial epithelium. All goats produced antibody responses as measured by an enzyme-linked immunosorbent assay (ELISA) procedure and the serums of 5 goats reacted in complement fixation tests for B. ovis. The 2 goats inoculated by the intratesticular route and one receiving B. ovis instilled intranasally subsequently excreted B. ovis in their semen. The possibility of natural transmission is discussed.

An enzyme-linked immunosorbent assay (ELISA) for the detection of Brucella ovis specific antibody in ram serum was compared with the currently employed complement fixation test (CFT). Rabbit anti-sheep IgG coupled to horseradish peroxidase was used as the antibody-enzyme conjugate and 2,2'-azino-di[3-ethylbenzthiazolin sulphonate (6)] as the substrate. The ratio of the optical density at 414 nm for positive and negative control sera (P/N ratio) was used to optimise the parameters of the test. Ram serum samples (16,527) were tested using ELISA and CFT (warm and cold) over a one year period. The ELISA was more sensitive and provided a more reliable measure of B ovis specific antibody than did the CFT. Implications of employing ELISA as the sole test in an eradication scheme are discussed.

A randomised double-blind controlled trial compared three-day and 10-day courses of amoxycillin (25 mg/kg daily) in children with otitis media. Seventeen doctors from five centres admitted 84 children between the ages of 2 and 10 years. Symptoms and signs were measured on admission to the trial, on day 3, and on day 15. Mother's observations were recorded daily for 10 days. Audiograms were performed at four and 12 weeks after the end of the trial. The treatment groups showed little difference in the speed of resolution of symptoms and signs, the numbers of primary treatment failures, or the frequency of recurrent ear infections. There were no complications in either group. Most children with otitis media can probably be successfully and safely treated with no more than a three-day course of amoxycillin providing their progress is reviewed about the fifth or sixth day after treatment started. This policy could save over 1 million pounds annually in antibiotic costs.