Perspectives and challenges in validating new diagnostic technologies.

This paper focuses on several new diagnostic technologies, which are set to dominate the testing landscape in the near future and have applications in animal health diagnostics, namely: next-generation sequencing, assays to detect biomarkers, and point-of-care tests. An example of real-time loop-mediated isothermal amplification validation is also provided. Validating these new technologies presents several challenges, which are addressed in this paper.

Les auteurs s'intéressent à plusieurs nouvelles technologies de diagnostic appelées à occuper, dans un futur proche, une place de choix dans le paysage du dépistage et dont il existe déjà des applications en santé animale, à savoir : le séquençage de nouvelle génération, la détection de biomarqueurs et les tests utilisables sur le lieu des soins. Ils décrivent par ailleurs l'exemple de la validation d'une amplification isotherme à médiation par boucle en temps réel. La validation de ces nouvelles technologies présente un certain nombre de difficultés, que les auteurs examinent en détail.

Los autores se centran en varias tecnologías de nuevo cuño que están llamadas a dominar el panorama de las pruebas de diagnóstico en un futuro próximo y que tienen aplicaciones de diagnóstico en sanidad animal, a saber: la secuenciación de próxima generación, los ensayos de detección de marcadores biológicos y las pruebas practicadas en el lugar de consulta. También ofrecen un ejemplo de validación de una técnica de amplificación isotérmica mediada por bucles en tiempo real. La validación de estas nuevas tecnologías presenta varias dificultades, que los autores examinan en estas líneas.

Emerging and endemic zoonotic diseases: surveillance and diagnostics.

In this paper, the authors: (a) list methods used to diagnose zoonotic diseases in humans and animals; (b) identify between-species differences in diagnostic approaches, providing commentary on the benefits that might arise from simultaneous interpretation of data from human and animal health surveillance systems; and (c) reiterate the importance of using species-specific, validated diagnostic tests for surveillance and disease outbreak investigations. Emerging and endemic zoonotic diseases are likely to provide a continued threat to global health in the short- to medium-term future. A good deal of knowledge about the drivers of infectious disease emergence has been developed based on numerous examples from the recent past. Sharing of diagnostic resources across human and animal health sectors, sharing of human and animal health surveillance data, development of skills in the interpretation of that data and awareness of issues related to the validation and interpretation of diagnostic test data are necessary prerequisites for an effective endemic disease surveillance system. A good understanding of the epidemiological patterns of endemic disease will allow human and animal health professionals be able to more quickly detect the presence of emerging disease threats.

Après avoir répertorié les méthodes utilisées pour diagnostiquer les maladies zoonotiques chez l'homme comme chez les animaux, les auteurs définissent les différentes approches diagnostiques suivant les espèces considérées et commentent les avantages qui pourraient découler d'une interprétation simultanée des données par les systèmes de surveillance en santé animale et en santé publique ; il réitèrent ensuite l'importance de recourir à des tests diagnostiques validés et spécifiques de l'espèce considérée dans le cadre de la surveillance et des enquêtes suite à l'apparition d'un foyer. Les maladies zoonotiques émergentes et endémiques représentent potentiellement une menace continue pour la santé mondiale à court et à moyen terme. Les facteurs favorisant l'émergence des maladies infectieuses sont désormais beaucoup mieux connus grâce aux enseignements tirés de nombreux exemples récents. Le partage des ressources diagnostiques entre les secteurs de la santé humaine et animale, les échanges des données de la surveillance sanitaire réunies par les deux secteurs, le renforcement des compétences en matière d'interprétation des données et la sensibilisation aux problématiques de la validation et de l'interprétation des données générées par les tests de diagnostic sont des conditions préalables à la mise en place d'un système efficace de surveillance des maladies endémiques. Une bonne compréhension des profils épidémiologiques des maladies endémiques permettra aux professionnels de la santé humaine et animale de détecter plus rapidement la présence de menaces émergentes.

Los autores proceden aquí a: a) relacionar los métodos empleados para diagnosticar enfermedades zoonóticas en personas y animales; b) señalar las diferencias que existen entre los distintos planteamientos de diagnóstico según la especie de que se trate, comentando asimismo las ventajas que podrían derivarse de la interpretación simultánea de los datos de los sistemas de vigilancia sanitaria y de los de vigilancia zoosanitaria; y c) reiterar la importancia que reviste el uso de pruebas de diagnóstico no solo validadas, sino también adaptadas específicamente a cada especie, para las labores de vigilancia y estudio de brotes. Lo más probable es que a corto y medio plazo las enfermedades zoonóticas, ya sean emergentes o endémicas, sigan constituyendo una amenaza para la salud mundial. Gracias a numerosos ejemplos del pasado reciente se ha ido constituyendo un buen conocimiento de los factores que propician la aparición de enfermedades infecciosas. Para disponer de un eficaz sistema de vigilancia de enfermedades endémicas hay una serie de requisitos previos indispensables: utilización compartida de los recursos de diagnóstico entre los sectores de la salud humana y la sanidad animal; intercambio de los datos de vigilancia sanitaria y de vigilancia zoosanitaria; adquisición de competencias para interpretar esos datos; y buen conocimiento de las cuestiones ligadas a la validación de pruebas de diagnóstico y a la interpretación de los datos que arrojan. Si los profesionales de la salud humana y la sanidad animal conocen debidamente los patrones epidemiológicos de las enfermedades endémicas, estarán en condiciones de detectar con más celeridad la presencia de enfermedades emergentes que constituyan una amenaza.

The Hendra virus vaccine: perceptions regarding the role of antibody titre testing.

OBJECTIVES: To elucidate veterinarians' and horse owners' perceptions towards the use of Hendra virus (HeV) antibody titre testing and how it influences veterinary advice. METHODS: Six semi-structured phone interviews were conducted with veterinarians who have submitted samples for HeV antibody titre testing. Interviews were recorded, transcribed and thematically analysed to identify and report common themes within the data. RESULTS: Veterinarians are predominantly using the titre tests as an alternative to vaccination due to clients' fear of vaccine reactions. The high cost of titre testing, the difficulty interpreting titre results and a lack of titre test recognition by authorities were the major barriers reported to using this test. Some veterinarians detailed difficulties communicating titre test procedures and results to their clients. The majority of veterinarians accepted titres of 64 or greater as evidence of protective immunity and would rely on those results for 12 months. However, there was discrepancy of these values and the level of confidence veterinarians had in interpreting the results of HeV antibody titre tests varied. CONCLUSION: This study has provided an overview of the attitudes of horse owners and veterinarians towards HeV antibody titre testing. Although evidence for HeV vaccination titres as an indication of protective immunity is still inadequate, it will assist veterinarians in interpreting and communicating titre results.

The first five days: field and laboratory investigations during the early stages of the equine influenza outbreak in Australia, 2007.

Until August 2007, Australia was one of only three countries internationally recognised to be free of equine influenza (EI). This report documents the diagnosis of the first cases of EI in Australian horses and summarises the investigations that took place over the next 5 days. During that time, a multifocal outbreak was identified across eastern New South Wales and south-eastern Queensland. The use of an influenza type A pan-reactive real-time reverse transcription polymerase chain reaction allowed rapid confirmation of suspect cases of EI.

Isolation and characterisation of an H3N8 equine influenza virus in Australia, 2007.

Before 2007, equine influenza had never been diagnosed in Australia. On 22 August 2007, infection was confirmed in horses at Eastern Creek Animal Quarantine Station near Sydney. The virus subsequently isolated (A/equine/Sydney/2888-8/2007) was confirmed by sequence analysis of the haemagglutinin (HA) gene as an H3 virus of the variant American Florida lineage that is now referred to as Clade 1. The HA sequence of the virus was identical to that of a virus isolated from a contemporaneous outbreak in Japan and showed high homology to viruses circulating in North America.

Infection and disease in reservoir and spillover hosts: determinants of pathogen emergence.

Infection and disease in reservoir and spillover hosts determine patterns of infectious agent availability and opportunities for infection, which then govern the process of transmission between susceptible species. In this chapter, using the zoonotic agents Hendra virus and Nipah virus as examples, the pathogenesis of infection in various species including the wildlife reservoirs and domestic spillover hosts is reviewed with an emphasis on the aspects of pathogenesis which contribute to the dissemination of infection. Through these discussions, the emergence of these zoonotic agents is explored.

Experimental Nipah virus infection in pteropid bats (Pteropus poliocephalus).

Seventeen grey-headed fruit bats (Pteropus poliocephalus) were inoculated subcutaneously with an isolate of Nipah virus derived from a fatally infected human. A control group of eight guinea-pigs was inoculated intraperitoneally with the same isolate in order to confirm virulence. Three of eight infected guinea-pigs developed clinical signs 7-9 days post-inoculation. Infected fruit bats developed a subclinical infection characterized by the transient presence of virus within selected viscera, episodic viral excretion and seroconversion. A range of histopathological changes was observed within the tissues of infected bats. Nipah virus was excreted in bat urine while neutralizing antibody was present in serum. This intermittent, low-level excretion of Nipah virus in the urine of bats may be sufficient to sustain the net reproductive value of the virus in a species where there is regular urine contamination of the fur, mutual grooming, and where urine droplets are a feature of the environment.

Effects of single amino acid substitutions at the E residue in the conserved GDNE motif of the Nipah virus polymerase (L) protein.

Nipah virus (NiV) is an emergent zoonotic paramyxovirus. The L proteins of most paramyxoviruses contain a GDNQ motif, thought to be part of the catalytic site for polymerase activity. Conversely, NiV L has GDNE in this position. We substituted the E residue with eight different amino acid residues and examined the effect on L function in an in vitro replication assay. Our results demonstrated that NiV L functioned with similar efficiency with either GDNE or GDNQ, but polymerase activity was severely reduced or abolished when a structurally destabilising residue (such as K, P or G) was introduced at this site.

Development of a fluorogenic RT-PCR assay (TaqMan) for the detection of Hendra virus.

A rapid and sensitive one-tube RT-PCR assay using a fluorogenic (TaqMan) probe was developed to improve the diagnosis of Hendra virus (HeV) infection. The TaqMan assay was developed to rapidly and specifically identify Hendra virus. The sensitivity of the new TaqMan-based PCR assay compared favourably with conventional RT-PCR. The major advantage of the TaqMan-based assay was the speed of diagnosis with results available within minutes of completing the PCR, and within 4 h of receiving the specimen. This test greatly reduces the chance of false positives through the elimination of second-round PCR and the requirement for agarose gel. Recombinant primer controls consisting of the Hendra virus primer sequence flanking a rodent GADPH probe sequence and recombinant probe controls consisting of the rodent GADPH primer sequence flanking the Hendra virus probe sequence were designed, cloned and transcribed in vitro to generate RNA. This has alleviated the requirement for viral RNA to be used as positive controls, thus reducing the chance of producing a false positive, at the same time eliminating the biosafety risk associated with handling live virus. This assay will provide a rapid diagnosis of future outbreaks of Hendra virus.

Molecular characterization of the polymerase gene and genomic termini of Nipah virus.

In 1998, Nipah virus (NV) emerged in peninsular Malaysia, causing fatal encephalitis in humans and a respiratory disease in swine. NV is most closely related to Hendra virus (HV), a paramyxovirus that was identified in Australia in 1994, and it has been proposed that HV and NV represent a new genus within the family Paramyxoviridae. This report describes the analysis of the sequences of the polymerase gene (L) and genomic termini of NV as well as a comparison of the full-length, genomic sequences of HV and NV. The L gene of NV is predicted to be 2244 amino acids in size and contains the six domains found within the L proteins of all nonsegmented, negative-stranded (NNS) RNA viruses. However, the GDNQ motif found in most NNS RNA viruses was replaced by GDNE in both NV and HV. The 3' and 5' termini of the NV genome are nearly identical to the genomic termini of HV and share sequence homology with the genomic termini of other members of the subfamily Paramyxovirinae. At 18,246 nucleotides, the genome of NV is 12 nucleotides longer than the genome of HV and they have the largest genomes within the family Paramyxoviridae. The comparison of the structures of the genomes of HV and NV is now complete and this information will help to establish the taxonomic position of these novel viruses within the family Paramyxoviridae.

Emerging viral diseases of Southeast Asia and the Western Pacific.

Over the past 6 years, a number of zoonotic and vectorborne viral diseases have emerged in Southeast Asia and the Western Pacific. Vectorborne disease agents discussed in this article include Japanese encephalitis, Barmah Forest, Ross River, and Chikungunya viruses. However, most emerging viruses have been zoonotic, with fruit bats, including flying fox species as the probable wildlife hosts, and these will be discussed as well. The first of these disease agents to emerge was Hendra virus, formerly called equine morbillivirus. This was followed by outbreaks caused by a rabies-related virus, Australian bat lyssavirus, and a virus associated with porcine stillbirths and malformations, Menangle virus. Nipah virus caused an outbreak of fatal pneumonia in pigs and encephalitis in humans in the Malay Peninsula. Most recently, Tioman virus has been isolated from flying foxes, but it has not yet been associated with animal or human disease. Of nonzoonotic viruses, the most important regionally have been enterovirus 71 and HIV.

Hopelessness and perfectionism.

In a sample of 39 undergraduates, hopelessness scores were associated with two aspects of perfectionism, concern over mistakes and doubts over performance.

Isolation of Hendra virus from pteropid bats: a natural reservoir of Hendra virus.

Since it was first described in Australia in 1994, Hendra virus (HeV) has caused two outbreaks of fatal disease in horses and humans, and an isolated fatal horse case. Our preliminary studies revealed a high prevalence of neutralizing antibodies to HeV in bats of the genus PTEROPUS:, but it was unclear whether this was due to infection with HeV or a related virus. We developed the hypothesis that HeV excretion from bats might be related to the birthing process and we targeted the reproductive tract for virus isolation. Three virus isolates were obtained from the uterine fluid and a pool of foetal lung and liver from one grey-headed flying-fox (Pteropus poliocephalus), and from the foetal lung of one black flying-fox (P. alecto). Antigenically, these isolates appeared to be closely related to HeV, returning positive results on immunofluorescent antibody staining and constant-serum varying-virus neutralization tests. Using an HeV-specific oligonucleotide primer pair, genomic sequences of the isolates were amplified. Sequencing of 200 nucleotides in the matrix gene identified that these three isolates were identical to HeV. Isolations were confirmed after RNA extracted from original material was positive for HeV RNA when screened on an HeV Taqman assay. The isolation of HeV from pteropid bats corroborates our earlier serological and epidemiological evidence that they are a natural reservoir host of the virus.

Newly discovered viruses of flying foxes.

Flying foxes have been the focus of research into three newly described viruses from the order Mononegavirales, namely Hendra virus (HeV), Menangle virus and Australian Bat Lyssavirus (ABL). Early investigations indicate that flying foxes are the reservoir host for these viruses. In 1994, two outbreaks of a new zoonotic disease affecting horses and humans occurred in Queensland. The virus which was found to be responsible was called equine morbillivirus (EMV) and has since been renamed HeV. Investigation into the reservoir of HeV has produced evidence that antibodies capable of neutralising HeV have only been detected in flying foxes. Over 20% of flying foxes in eastern Australia have been identified as being seropositive. Additionally six species of flying foxes in Papua New Guinea have tested positive for antibodies to HeV. In 1996 a virus from the family Paramyxoviridae was isolated from the uterine fluid of a female flying fox. Sequencing of 10000 of the 18000 base pairs (bp) has shown that the sequence is identical to the HeV sequence. As part of investigations into HeV, a virus was isolated from a juvenile flying fox which presented with neurological signs in 1996. This virus was characterised as belonging to the family Rhabdoviridae, and was named ABL. Since then four flying fox species and one insectivorous species have tested positive for ABL. The third virus to be detected in flying foxes is Menangle virus, belonging to the family Paramyxoviridae. This virus was responsible for a zoonotic disease affecting pigs and humans in New South Wales in 1997. Antibodies capable of neutralising Menangle virus, were detected in flying foxes.

Manganese and iron interrelationship in the chick.

Experiments were conducted to investigate the Mn-Fe interrelationship in the chick. Graded levels of Fe and two levels of Mn were added to a corn-soybean meal diet (157 mg Mn/kg; 372 mg Fe/kg) and to an Fe-deficient casein-dextrose diet containing 15.4 mg Mn/kg and 5.0 mg Fe/kg. Dietary Fe had little effect on the Mn status of the chick, regardless of the level of Fe supplementation. Conversely, Mn supplemented at 1,000 mg/kg reduced blood hemoglobin concentration, but only when the dietary Fe level was at or below the chick's requirement. Levels of supplemental Fe up to 2,500 mg/kg had no effect on performance of chicks fed the corn-soybean diet, but a supplemental level of 5,000 mg Fe/kg diet reduced both weight gain and bone ash concentration. These data suggest that the interaction between Mn and Fe in the chick is a unidirectional phenomenon, in which excess Mn impairs Fe utilization but excess Fe does not antagonize Mn.

Efficacy of a manganese-protein chelate compared with that of manganese sulfate for chicks.

Based on bone manganese (Mn) accumulation during a 14-day feeding period. Mn bioavailability in a Mn-proteinate product was judged to be not significantly different than that provided by MnSO4.H2O. Bone and bile Mn concentrations were reduced markedly when 10% wheat bran was added to a phytate and fiber-free casein-dextrose diet that contained 1,000 mg/kg supplemental Mn from either MnSO4.H2O or the Mn-proteinate.

Mechanism of the tissue manganese-lowering effect of corn, soybean meal, fish meal, wheat bran, and rice bran.

Experiments were conducted with young chicks to investigate the mechanism by which various feed ingredients reduce the bioavailability of inorganic Mn. Although corn, soybean meal (SBM), fish meal (FM), wheat bran (WB), and rice bran (RB) contain significant quantities of Mn, these feed ingredients when added to a casein-dextrose diet have been shown to reduce Mn deposition in key tissues (i.e., bone, pancreas, and gallbladder). Studies reported herein indicate that reduction in tissue Mn concentration due to feed ingredient supplementation resulted from reduced Mn absorption, rather than from enhanced Mn excretion. Feedstuffs exerted their effect primarily at the gut level, although some Mn binding may also have occurred in body fluids. Fractionation of each feed ingredient indicated that the neutral detergent fiber (NDF) fraction of WB and a corn-SBM mixture accounted for virtually all of the Mn-binding capabilities of these ingredients. In contrast, ash fraction was responsible for tissue Mn-lowering capabilities of FM. Both NDF and ash fractions of RB significantly lowered tissue Mn concentrations, but not to the same extent as that caused by RB itself. A significant portion (11 to 27%) of the total quantity of copper was found in the NDF fraction of all feed ingredients. Over 12% of the magnesium and iron in FM was contained in the NDF fraction, while 20% of the magnesium in WB and 15% of the iron in RB was likewise present in the NDF fraction. Very little K, Zn, or Mn was associated with the NDF fraction of any feed ingredients. All Ca in WB but less than 1% of Ca in RB were present in the NDF.

Efficiency of manganese absorption in chicks fed corn-soy and casein diets.

Experiments were conducted with young chicks to quantify the absorption efficiency of manganese (Mn) using tibia Mn uptake as the response criterion. Chicks in experiment 1 were fed a corn-soybean meal diet and either injected intraperitoneally or crop intubated twice daily with three levels of inorganic Mn provided as MnSO4 X H2O. After 14 d, chicks were killed, and intact tibias were removed to facilitate calculation of bone Mn concentration. Excellent straight-line fits (bone Mn concentration as a function of Mn administered) allowed estimation of gut absorption efficiency of Mn by slope-ratio methodology. Absorption efficiency of Mn was calculated to be 1.71% for chicks fed the corn-soy diet. Identical methodology was employed in experiment 2 wherein chicks were fed a phytate- and fiber-free casein-dextrose diet. An absorption efficiency of 2.40% was estimated for chicks fed this diet. Hence, absorption efficiency of inorganic Mn was 40% greater in chicks fed the purified diet than in those fed the conventional corn-soy diet.