Problems can arise when vaccines and wild strains of a chicken herpesvirus recombine.

Identification of fowl adenovirus (FAdV) serotypes is of importance in epidemiological studies of disease outbreaks and the adoption of vaccination strategies. In this study, real-time PCR and subsequent high-resolution melting (HRM)-curve analysis of three regions of the hexon gene were developed and assessed for their potential in differentiating 12 FAdV reference serotypes. The results were compared to previously described PCR and restriction enzyme analyses of the hexon gene. Both HRM-curve analysis of a 191-bp region of the hexon gene and restriction enzyme analysis failed to distinguish a number of serotypes used in this study. In addition, PCR of the region spanning nucleotides (nt) 144 to 1040 failed to amplify FAdV-5 in sufficient quantities for further analysis. However, HRM-curve analysis of the region spanning nt 301 to 890 proved a sensitive and specific method of differentiating all 12 serotypes. All melt curves were highly reproducible, and replicates of each serotype were correctly genotyped with a mean confidence value of more than 99% using normalized HRM curves. Sequencing analysis revealed that each profile was related to a unique sequence, with some sequences sharing greater than 94% identity. Melting-curve profiles were found to be related mainly to GC composition and distribution throughout the amplicons, regardless of sequence identity. The results presented in this study show that the closed-tube method of PCR and HRM-curve analysis provides an accurate, rapid, and robust genotyping technique for the identification of FAdV serotypes and can be used as a model for developing genotyping techniques for other pathogens.

High‐frequency phase and antigenic variation of homologous lipoprotein haemagglutinins has been seen in both the major avian mycoplasma pathogens, Mycoplasma synoviae and Mycoplasma gallisepticum. The expression and, hence, antigenic variation of the pMGA gene family (encoding these lipoproteins in M. gallisepticum) is controlled by variation in the length of a trinucleotide repeat motif 5′ to the promoter of each gene. However, such a mechanism was not detected in preliminary observations on M. synoviae. Thus, the basis for control of variation in the vlhA gene family (which encodes the homologous haemagglutinin in M. synoviae) was investigated to enable comparison with its homologue in M. gallisepticum and with other lipoprotein gene families in mycoplasmas. The start point of transcription was identified 119 bp upstream of the initiation codon, but features associated with control of transcription in other mycoplasma lipoprotein genes were not seen. Comparison of three copies of vlhA revealed considerable sequence divergence at the 3′ end of the gene, but conservation of the 5′ end. Southern blot analysis of M. synoviae genomic DNA revealed that the promoter region and part of the conserved 5′ coding sequence occurred as a single copy, whereas the remainder of the coding sequence occurred as multiple copies. A 9.7 kb fragment of the genome was found to contain eight tandemly repeated regions partially homologous to vlhA, all lacking the putative promoter region and the single‐copy 5′ end of vlhA, but extending over one of four distinct overlapping regions of the 3′ coding sequence. Examination of sequential clones of M. synoviae established that unidirectional recombination occurs between the pseudogenes and the expressed vlhA, with duplication of pseudogene sequence and loss of the corresponding region previously seen in the expressed gene. Expression of the 5′ end of two variants of the vlhA gene showed that they differed in their reaction with monoclonal antibodies specific for this region. These data suggest that the control of vlhA antigenic variation in M. synoviae is achieved by multiple gene conversion events using a repertoire of coding sequences to generate a chimeric expressed gene, with the greatest potential for variation generated in the region encoding the haemagglutinin. Thus, completely distinct mechanisms have been adopted to control antigenic variation in homologous gene families.

Microarrays were used to identify changes in gene expression associated with Candida albicans biofilm development. Two biofilm substrates (denture and catheter), and two C. albicans strains for each substrate, were tested to remove model- and strain-dependent variability from the overall dataset. Three biofilm developmental phases were examined: early (6 h), intermediate (12 h), and mature (48 h). Planktonic specimens were collected at the same time points. Data analysis focused primarily on gene expression changes over the time-course of biofilm development. Glycolytic and non-glycolytic carbohydrate assimilation, amino acid metabolism, and intracellular transport mechanisms were important during the early phase of biofilm formation. These early events increase intracellular pools of pyruvate, pentoses and amino acids, which prepare the biofilm for the large biomass increase that begins around 12 h of development. This developmental stage demands energy and utilizes specific transporters for amino acids, sugars, ions, oligopeptides and lactate/pyruvate. At mature phase (48 h), few genes were differentially expressed compared with the 12 h time point, suggesting a relative lack of initiation of new metabolic activity. Data analysis to assess biofilm model-specific gene expression showed more dynamic changes in the denture model than in the catheter model. Data analysis to identify gene expression changes that are associated with each strain/substrate combination identified the same types of genes that were identified in the analysis of the entire dataset. Collectively, these data suggest that genes belonging to different, but interconnected, functional categories regulate the morphology and phenotype of C. albicans biofilm.

Infectious laryngotracheitis (ILT) has been identified in most countries around the world and remains a threat to the intensive poultry industry. Outbreaks of mild to moderate forms of ILT are common in commercial layer flocks, while sporadic outbreaks of ILT in broiler flocks have also been recognized as an emerging problem in several countries. Examination of viral isolates using restriction fragment length polymorphism of polymerase chain reaction (PCR-RFLP) from individual ILTV genes has suggested that some of these outbreaks were caused by vaccine strains. In this study, PCR-RFLP of a number of ILTV genes/genomic regions including gE, gG, TK, ICP4, ICP18.5, and open reading frame (ORF) B-TK was used to examine a number of historical and contemporary Australian ILTV isolates and vaccine strains. PCR-RFLP of gE using restriction endonuclease EaeI failed to distinguish between any of the isolates including the vaccine strains. PCR-RFLP of gG, TK, and ORFB-TK using restriction endonucleases MspI and FokI, respectively, divided all the isolates into two groups. PCR-RFLP of ICP18.5 and ICP4 using restriction endonuclease HaeIII separated the isolates into three different groups with some field isolates only able to be distinguished from vaccine strains by PCR-RFLP of ICP18.5. A combination of groupings including gG, TK, ICP4, ICP18.5, and ORFB-TK PCR-RFLP classified the ILTV isolates under investigation into five different groups with most isolates distinguishable from vaccine strains. Results from this study reveal that to achieve reliable identification of strains of ILTV, the examination of multiple gene regions will be required, and that most of the recent ILT outbreaks in Australia are not being caused by vaccine strains.

Previous studies in our laboratory using a combination of polymerase chain reaction and restriction fragment length polymorphism have identified at least five different genotypes of infectious laryngotracheitis virus (ILTV). However, the virulence of these classes of ILTV was not investigated. In this study, five groups (16 birds each) of 3-week-old specific pathogen free chickens were inoculated via the intratracheal route with 10³ median embryo infected dose of five different strains of ILTV. Three further groups of chickens were inoculated similarly with the vaccine strains SA2 and A20 or with sterile phosphate-buffered saline (PBS) for comparison. Four days post-inoculation, clinical signs were monitored for scoring, and eight chickens from each group were subsequently euthanized, weighed and subjected to pathological and histopathological examinations. The remaining birds were monitored for clinical signs and mortality until 21 days post-inoculation. All groups inoculated with ILTV strains showed moderate to severe clinical signs 4 days after inoculation. The strain Q1-96 caused only minimal breathing symptoms with a median score that was not significantly different to that of the group inoculated with PBS, but was significantly different to those of the groups inoculated with other ILTV strains. The strain Q1-96 caused severe photophobia and conjunctivitis with a median score that was significantly higher than those of all other groups except for the group inoculated with the strain N3-04. All ILTV strains caused a significant reduction in weight gain when compared with the group inoculated with PBS. The strain Q1-96 caused an average weigh loss of 14% that was significantly higher than those of other ILTV strains. The strains S2-04 and Q1-96 induced only minor microscopic tracheal lesions while all the other ILTV strains, including the vaccine strains A20 and SA2, induced moderate to severe microscopic tracheal lesions. Median scores for microscopic tracheal lesions were well correlated with the number of viral genomes detected in trachea. The results revealed that there is considerable variation among ILTV strains in their tropism for trachea or conjunctiva. In addition it was revealed that ILTV strains with high affinity for conjunctiva can severely affect weigh gain. The ILTV numbers and microscopic lesions in trachea were not found to be reliable indicators of virulence since they are not necessarily correlated with mortality rate in ILT. Des études antérieures conduites dans notre laboratoire utilisant une réaction de polymérisation en chaîne et de polymorphisme de taille des fragments de restriction (PCR-RFLP) avaient identifié au moins 5 génotypes différents de virus de la laryngotrachéite infectieuse aviaire (ILTV). Cependant, la virulence de ces classes d'ILTV n'avait pas été investiguée. Dans cette étude, 5 groupes de 16 poulets SPF âgés de 3 semaines ont été inoculés par voie trachéale avec 10³ EID₅₀ de 5 souches différentes d' ILTV. Trois autres groupes de poulets ont été inoculés de façon similaire avec les souches vaccinales SA2 et A20 ou avec une solution de tampon phosphate (PBS) stérile pour comparaison. Quatre jours après l'inoculation, les symptômes ont été relevés et leur intensité enregistrée, huit poulets de chaque groupe ont ensuite été euthanasiés, pesés et soumis à des examens pathologiques et histopathologiques. Les sujets restants ont fait l'objet d'un suivi clinique et la mortalité a été enregistrée jusqu'à 21 jours après l'inoculation. Tous les groupes inoculés avec les souches d'ILTV ont présenté des symptômes modérés à graves 4 jours après l'inoculation. La souche Q1-96 a entraîné seulement des symptômes respiratoires minimums avec un score moyen qui n'était pas significativement différent de celui du groupe inoculé avec le PBS, mais était significativement différent de ceux des groupes inoculés avec les autres souches d'ILTV. La souche Q1-96 a causé une photophobie sévère et une conjonctivite avec un score médian qui a été significativement supérieur à celui des autres groupes à l'exception du groupe inoculé avec la souche N3-04. Toutes les souches d'ILTV ont entraîné une diminution significative du gain de poids comparé au groupe inoculé avec le PBS. La souche Q1-96 a entraîné une perte de poids moyenne de 14% qui a été significativement supérieure à celle des autres souches d'ILTV. Les souches S2-04 et Q1-96 ont induit seulement quelques lésions trachéales microscopiques mineures alors que toutes les autres souches d'ILTV incluant les souches vaccinales A20 et SA2 ont induit des lésions trachéales microscopiques modérées à sévères. Les scores moyens des lésions trachéales microscopiques sont bien corrélés au nombre de génome viral détecté dans la trachée. Les résultats ont révélé qu'il existait une variation considérable parmi les souches d'ILTV dans leur tropisme pour la trachée ou la conjonctive. De plus, il a été révélé que les souches d'ILTV possédant une forte affinité pour la conjonctive peuvent gravement affecter le gain de poids. Les nombres de ILTV et les lésions microscopiques au niveau de la trachée ne se sont pas révélé être des indicateurs fiables de la virulence puisqu'ils ne sont pas nécessairement corrélés avec le taux de mortalité dans la ILT. In den verhergehenden Studien in unserem Labor waren mittels einer Kombination aus Polymerasekettenreaktion und Restriktionsfragmentlängenpolymorphismus (PCR-RFLP) mindestens fünf verschiedene Genotypen des Virus der infektiösen Laryngotracheitis (ILTV) identifiziert worden. Die Virulenz dieser ILTV-Genotypen war jedoch nicht untersucht worden. In dieser Studie wurden fünf Gruppen von jeweils 16 dreiwöchigen SPF-Hühnerküken intratracheal mit je 10³ EID₅₀ von fünf verschiedenen ILTV-Stämmen inokuliert. Zum Vergleich wurden drei weiteren Hühnergruppen auf ähnliche Weise die Vakzinestämme SA2 und A20 oder steriler Phosphatpuffer (PBS) verabreicht. Vier Tage nach der Inokulation wurden die klinischen Symptome erhoben und bewertet. Anschließend wurden aus jeder Gruppe 8 Küken euthanasiert, gewogen und einer pathologisch-anatomischen und pathohistologischen Untersuchung unterzogen. Bei den verbliebenen Tieren wurden bis zum 21. Tag nach der Inokulation auftretende klinische Symptome und die Mortalität protokolliert. Alle mit den ILTV-Stämmen inokulierten Gruppen zeigten am 4. Tag nach der Inokulation mittel- bis hochgradige klinische Symptome. Der Stamm Q1-96 verursachte nur minimale Atemwegssymptome mit einem gemittelten Bewertungsgrad, der sich nicht signifikant von dem der mit PBS inokulierten Gruppe unterschied, der aber signifikante Unterschiede zu denen der Gruppen aufwies, die mit den anderen ILTV-Stämmen infiziert worden waren. Der Stamm Q1-96 induzierte jedoch eine hochgradige Photophobie und Konjunktivitis deren mittlerer Schweregrad signifikant höher war als der der anderen Gruppen außer dem der mit dem Stamm N3-04 inokulierten Gruppe. Alle ILTV-Stämme führten im Vergleich zu der mit PBS inokulierten Gruppe zu einer signifikanten Reduktion der Gewichtszunahmen. Der Stamm Q1-96 verursachte einen mittleren Gewichtsverlust von 14 %, der signifikant über denen der anderen ILTV-Stämme lag. Die Stämme S2-04 und Q1-96 induzierten nur geringgradige mikroskopische Trachealläsionen, während alle anderen ILTV-Stämme einschließlich der Vakzinestämme A20 und SA2 mittel- bis hochgradige mikroskopisch sichtbare Läsionen in der Trachea hervorriefen. Der mittlere Schweregrad dieser Läsionen korrelierte gut mit der Anzahl der in der Trachea nachgewiesenen Virusgenome. Diese Ergebnisse zeigen, dass bei den ILTV-Stämmen erhebliche Unterschiede hinsichtlich ihres Tropismus für Trachea oder Konjunktiva bestehen. Außerdem wurde offensichtlich, dass ILTV-Stämme mit Affinität für die Konjunktiva erhebliche Auswirkungen auf die Gewichtszunahmen haben können. Es wurde festgestellt, dass die Anzahl der ILTV-Partikel sowie die mikroskopischen Läsionen keine zuverlässigen Virulenzindikatoren sind, da sie nicht unbedingt mit der Mortalitätsrate bei einer ILT korrelieren. Estudios anteriores realizados en nuestro laboratorio utilizando una combinación de la reacción en cadena de la polimerasa y polimorfismo de la longitud de de los fragmentos de restricción (PCR-RFLP) identificaron al menos 5 genotipos distintos del virus de laringotraqueítis infecciosa (ILTV). Sin embargo, no se estudió la virulencia de estos tipos de ILTV. En este trabajo, 5 grupos de pollos SPF de 3 semanas de vida (16 en cada grupo) se inocularon vía intratraqueal con 10³ EID₅₀ de ILTV de 5 cepas distintas. Otros tres grupos de pollos se inocularon de manera parecida con las cepas vacunales SA2 y A20 o con solución salina fosfatada tamponada estéril para comparar. Cuatro días post inoculación, se valoraron los signos clínicos mediante puntuación, y a continuación ocho pollos de cada grupo se sacrificaron, se pesaron, y se les realizaron exámenes patológicos e histopatológicos. En las aves restantes se valoraron los signos clínicos y la mortalidad hasta 21 días post inoculación. Todos los grupos inoculados con cepas de ILTV mostraron signos clínicos moderados a graves a los 4 días post inoculación. La cepa Q1-96 sólo causó síntomas respiratorios leves con una valoración media que no se diferenció significativamente de la de los grupos inoculados con PBS, pero sí de la de los grupos inoculados con otras cepas de ILTV. La cepa Q1-96 causó fotofobia grave y conjuntivitis con una valoración media significativamente mayor que la del resto de grupos a excepción del grupo inoculado con la cepa N3-04. Todas las cepas de ILTV causaron una disminución significativa en la ganancia de peso en comparación al grupo inoculado con PBS. La cepa Q1-96 produjo una pérdida de peso media del 14% que fue significativamente mayor que la del resto de cepas de ILTV. Las cepas S2-04 y Q1-96 sólo indujeron lesiones microscópicas leves en la tráquea mientras que el resto de cepas de ILTV, incluyendo las cepas vacunales A20 y SA2, produjeron lesiones moderadas a graves en tráquea. Se observó una buena correlación entre las valoraciones medias de las lesiones traqueales y el número de copias de genoma vírico detectado en la tráquea. Los resultados revelaron que hay una variación considerable del tropismo hacia tráquea o conjuntiva entre las cepas de ILTV. Además se mostró que las cepas de ILTV con elevada afinidad por la conjuntiva pueden afectar gravemente la ganancia de peso. El número de ILTV y las lesiones microscópicas traqueales no fueron indicadores fiables de la virulencia dado que no se correlacionaron necesariamente con la tasa de mortalidad.