Detection of Chlamydiaceae DNA in veterinary specimens using a family-specific PCR

Aims: The aim of this work was to develop a rapid molecular test for the detection of the Chlamydiaceae family, irrespective of the species or animal host. Methods and Results: The method described herein is a polymerase chain reaction targeting the 16S rRNA gene of the Chlamydiaceae family, and the results demonstrate that the test reacts with five reference Chlamydiaceae but none of the 19 other bacterial species or five uninfected animal tissues tested. The results also indicate the enhanced sensitivity of this test when compared with conventional culture or serology techniques. This is demonstrated through parallel testing of six real clinical veterinary cases and confirmatory DNA sequence analysis. Conclusions, Significance and Impact of the Study: This test can be used by veterinary diagnostic laboratories for rapid detection of Chlamydiaceae in veterinary specimens, with no restriction of chlamydial species or animal host. The test does not differentiate chlamydial species, and if required, speciation must be carried out retrospectively using alternate methods. However, for the purpose of prescribing therapy for chlamydiosis, this test would be an invaluable laboratory tool.

Diagnosis of a naturally occurring dual infection of layer chickens with fowlpox virus and gallid herpesvirus 1 (infectious laryngotracheitis virus).

An outbreak of acute respiratory disease in layers was diagnosed as being of dual nature due to fowlpox and infectious laryngotracheitis using a multidisciplinary approach including virus isolation, histopathology, electron microscopy and polymerase chain reaction (PCR). The diagnosis was based on virus isolation of gallid herpesvirus 1 (GaHV-1) in chicken kidney cells and fowlpox virus (FWPV) in 9-day-old chicken embryonated eggs inoculated via the chorioallantoic membrane. The histopathology of tracheas from dead birds revealed intra-cytoplasmic and intra-nuclear inclusions suggestive of poxvirus and herpesvirus involvement. The presence of FWPV was further confirmed by electron microscopy, PCR and histology. All FWPV isolates contained the long terminal repeats of reticuloendotheliosis virus as demonstrated by PCR. GaHV-1 isolates were detected by PCR and were shown to have a different restriction fragment length polymorphism pattern when compared with the chicken embryo origin SA2 vaccine strain; however, they shared the same pattern with the Intervet chicken embryo origin vaccine strain. This is a first report of dual infection of chickens with GaHV-1 and naturally occurring FWPV with reticuloendotheliosis virus insertions. Further characterization of the viruses was carried out and the results are reported here.

Bovine cysticercosis-Development of a real-time PCR to enhance classification of suspect cysts identified at meat inspection

Laboratory confirmation methods are important in bovine cysticerosis diagnosis as other pathologies can result in morphologically similar lesions resulting in false identifications. We developed a probe-based real-time PCR assay to identify Taenia saginata in suspect cysts encountered at meat inspection and compared its use with the traditional method of identification, histology, as well as a published nested PCR. The assay simultaneously detects T. saginata DNA and a bovine internal control using the cytochrome c oxidase subunit 1 gene of each species and shows specificity against parasites causing lesions morphologically similar to those of T. saginata. The assay was sufficiently sensitive to detect 1 fg (Ct 35.09 +/- 0.95) of target DNA using serially-diluted plasmid DNA in reactions spiked with bovine DNA as well as in all viable and caseated positive control cysts. A loss in PCR sensitivity was observed with increasing cyst degeneration as seen in other molecular methods. In comparison to histology, the assay offered greater sensitivity and accuracy with 10/19 (53%) T. saginata positives detected by real-time PCR and none by histology. When the results were compared with the reference PCR, the assay was less sensitive but offered advantages of faster turnaround times and reduced contamination risk. Estimates of the assay's repeatability and reproducibility showed the assay is highly reliable with reliability coefficients greater than 0.94. Crown Copyright (C) 2013 Published by Elsevier B.V. All rights reserved.

Fluoroquinolone-resistant extraintestinal pathogenic Escherichia coli, including O25b-ST131, isolated from faeces of hospitalized dogs in an Australian veterinary referral centre

To determine rates of carriage of fluoroquinolone-resistant Escherichia coli and extraintestinal pathogenic E. coli (ExPEC) among dogs in a specialist referral hospital and to examine the population structure of the isolates. Fluoroquinolone-resistant faecal E. coli isolates (n232, from 23 of 123 dogs) recovered from hospitalized dogs in a veterinary referral centre in Sydney, Australia, over 140 days in 2009 were characterized by phylogenetic grouping, virulence genotyping and random amplified polymorphic DNA (RAPD) analysis. The RAPD dendrogram for representative isolates showed one group B2-associated cluster and three group D-associated clusters; each contained isolates with closely related ExPEC-associated virulence profiles. All group B2 faecal isolates represented the O25b-ST131 clonal group and were closely related to recent canine extraintestinal ST131 clinical isolates from the east coast of Australia by RAPD analysis. Hospitalized dogs may carry fluoroquinolone-resistant ExPEC in their faeces, including those representing O25b-ST131.

Infection and pathology in Queensland grouper, Epinephelus lanceolatus, (Bloch), caused by exposure to Streptococcus agalactiae via different routes

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery-reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re-isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high-dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.

Evaluation and histological examination of a Campylobacter fetus subsp. venerealis small animal infection model

Bovine genital campylobacteriosis (BGC), caused by Campylobacter fetus subsp. venerealis, is associated with production losses in cattle worldwide. This study aimed to develop a reliable BGC guinea pig model to facilitate future studies of pathogenicity, abortion mechanisms and vaccine efficacy. Seven groups of five pregnant guinea pigs (1 control per group) were inoculated with one of three strains via intra-peritoneal (IP) or intra-vaginal routes. Samples were examined using culture, PCR and histology. Abortions ranged from 0 to 100 and re-isolation of causative bacteria from sampled sites varied with strain, dose of bacteria and time to abortion. Histology indicated metritis and placentitis, suggesting that the bacteria induce inflammation, placental detachment and subsequent abortion. Variation of virulence between strains was observed and determined by culture and abortion rates. IP administration of C. fetus subsp. venerealis to pregnant guinea pigs is a promising small animal model for the investigation of BGC abortion.

Infection and pathology in Queensland grouper, Epinephelus lanceolatus, (Bloch), caused by exposure to Streptococcus agalactiae via different routes

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery-reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re-isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high-dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.

Ecology of uncultivated oscillospira species in the rumen of cattle, sheep, and reindeer as assessed by microscopy and molecular approaches

The ecology of the uncultured, but large and morphologically conspicuous, rumen bacterium Oscillospira spp. was studied. Oscillospira-specific 16S rRNA gene sequences were detected in North American domestic cattle, sheep from Australia and Japan, and Norwegian reindeer. Phylogenetic analysis of the sequences obtained allowed definition of three operational taxonomic units within the Oscillospira clade. Consistent with this genetic diversity, we observed atypical smaller morphotypes by using an Oscillospira-specific fluorescence in situ hybridization probe. Despite the visual disappearance of typical large Oscillospira morphotypes, the presence of Oscillospira spp. was still detected by Oscillospira-specific PCR in the rumen of cattle and sheep. These observations suggest the broad presence of Oscillospira species in various rumen ecosystems with the level, and most likely the morphological form, dependent on diet. An ecological analysis based on enumeration of the morphologically conspicuous, large-septate form confirms that the highest counts are associated with the feeding of fresh forage diets to cattle and sheep and in two different subspecies of reindeer investigated.

Toxoplasmosis in Indo-Pacific humpbacked dolphins (Sousa chinensis) from Queensland

Objective: To describe the clinical signs, gross pathology, serology, bacteriology, histopathology, electron microscopy and immunohistochemistry findings associated with toxoplasmosis in four Indo-Pacific humpbacked dolphins (Sousa chinensis) that stranded in Queensland in 2000 and 2001. Design: Clinical assessment, gross necropsy, and laboratory examinations. Procedure: Necropsies were performed on four S chinensis to determine cause of death. Laboratory tests including serology, bacteriology, histopathology and transmission electron microscopy were done on the four dolphins. lmmunohistochemistry was done on the brain, heart, liver, lung, spleen and adrenal gland from various dolphins to detect Toxoplasma gondii antigens. Results: Necropsies showed all of four S chinensis that stranded in Queensland in 2000 and 2001 had evidence of predatory shark attack and three were extremely emaciated. Histopathological examinations showed all four dolphins had toxoplasmosis with tissue cysts resembling T gondii in the brain. Tachyzoite stages of T gondii were detected in the lungs, heart, liver, spleen and adrenal gland, variously of all four dolphins. Electron microscopy studies and immunohistochemistry confirmed the tissues cysts were those of Tgondii. All four dolphins also had intercurrent disease including pneumonia, three had peritonitis and one had pancreatitis. Conclusion: Four S chinensis necropsied in Queensland in 2000 and 2001 were found to be infected with toxoplasmosis. It is uncertain how these dolphins became infected and further studies are needed to determine how S chinensis acquire toxoplasmosis. All four dolphins stranded after periods of heavy rainfall, and coastal freshwater runoff may be a risk factor for T gondii infection in S chinensis. This disease should be of concern to wildlife managers since S chinensis is a rare species and its numbers appear to be declining.

Use of Petrifilm(R) 3M to assess coliform numbers on lamb carcasses.

Petrifilm(R) (6410) was used directly on lamb carcasses to enumerate coliforms. 10 sites on 30 carcasses were sampled at each of 4 separate meat processing establishments (works). Coliform counts obtained by this technique were statistically analysed using analysis of variance (ANOVA) to select the optimum sampling sites on the carcass and to assess contamination of the carcass by gut flora at a particular establishment. There was a large variation between sites and between works. In general, works 3 and 4 produced cleaner carcasses than works 2, which in turn was cleaner than works 1. Works 1, 2 and 4 used conventional dressing techniques and works 3 used the inverted dressing method, therefore, the coliform counts found at works 3 and 4 are achievable regardless of dressing technique. Coliform bacteria were most concentrated around the posterior pelvic rim and less prevalent at the carcass extremities. The posterior pelvic rim (sites 3 and 4) had higher (P < 0.05) coliform counts than the exterior ventral flank area (sites 5, 6, 7 and 8), which in turn had higher (P < 0.05) counts than the proximal hind and proximal fore limbs (sites 1, 2, 9 and 10) across all works. With in-line routine testing it is recommended that the majority of carcasses sampled should give coliform counts of <50 cfu/20 cm2 for sites 4 and 8. Reprinted with permission from Journal of Food Protection. Copyright held by the International Association of Food Protection, Des Moines, Iowa, USA. Authors affifiation. J.A.Guthrie & K.J.Dunlop International Food Institute of Queensland, Department of Primary Industries, Rockhampton and G.A.Saunders Veterinary Public Health Division, Livestock and Meat Authority of Queensland, Emerald.

Composition of chemical attractants affects trap catches of the Australian sheep blowfly, lucilia cuprina, and other blowflies

Numbers of Lucilia cuprina (Australian sheep blowfly), Chrysomya spp., and Calliphora spp. blowflies caught on sticky traps baited with various synthetic attractants or a standard liver/sodium sulfide attractant in western Queensland were recorded. Numbers of each genus collected were influenced by the composition of the chemical attractants. Attractant mixtures based on 2-mercaptoethanol, indole, butanoic/pentanoic acid, and a sodium sulfide solution gave 5- to 20-fold higher L. cuprina catches than the liver standard. These blends attracted similar numbers of Chrysomya spp. (0.85–2.7× ) and fewer Calliphora spp. (0.02–0.2× ) compared to the liver standard. These synthetic attractants were more effective and selective for L. cuprina than the standard liver/sodium sulfide attractant, and they can be packaged in controlled-release dispensers to generate constant, prolonged release of the attractant.

Coriza infecciosa: revisión de métodos de diagnóstico y vacunas

La coriza infecciosa es una enfermedad respiratoria aguda de las gallinas domesti- cas causada por la bacteria Haemophilus parugallinarum. Excepcionalmente pueden enfermarse tambien los faisanes y gallinas de Guinea. El H. paragallinarum infecta al ave por via respiratoria y luego de un cor- to periodo de incubation, que varia entre 1 a 3 dias, produce una enfermedad que se manifiesta por inflamacion catarral de los senos paranasales. Este cuadro puede estar asociado a inflamacion de los barbillones, conjuntivitis o queratitis. Los casos de neu- nionia y aerosaculitis son menos frecuentes pero tambien suelen ocurrir en las infeccio- nes puras por estos hemofilos. En las gallinas en produccion causa alta morbilidad, baja o nula mortalidad y una importante perdida en la produccion de huevos, la que generalmente oscila entre 10% y 40%. En pollos parrilleros puede cau- sar un cuadro descrito como «cabeza hin- chada» y ocasionalmente tambien producir septicemia y muerte (48). Esta bacteria ge- neralmente se asocia con otros agentes bacterianos, viricos o parasitarios y cuan- do esto ocurre se agrava el curso de la en- fermedad. Entre los agentes bacterianos mas comunes deben mencionarse los mycoplasinas y las pasteurelas. Cuando H . paragallinarum se asocia con otros agentes esta enfermedad se denomina .«coriza infec- ciosa complicada» (48). En esta recopilacion se aportaran deta- lles sobre la clasificacion, identificacion y serotipificacion del agente causal. Tambien se resumira la informacion disponible sobre nuevos metodos de diagnostico y programas de vacunacion para prevenir esta enferme-dad. A lo largo de esta revision se hara re-ferencia a los hemofilos aviarios que, para el proposito de este trabajo, seran definidos como organisnios gram negativos aislados de aves y que necesariamente requieren factores de crecimiento in vitro. Los dos factores que pueden ser requeridos por los hemofilos para su crecimiento in vitro son hemina (factor X) y/o nicotin-adenin-dinucleirtido (NAD o factor V).

Using simulation techniques to investigate methods to determine resistance of helminths to anthelmintic treatment

The widespread and increasing resistance of internal parasites to anthelmintic control is a serious problem for the Australian sheep and wool industry. As part of control programmes, laboratories use the Faecal Egg Count Reduction Test (FECRT) to determine resistance to anthelmintics. It is important to have confidence in the measure of resistance, not only for the producer planning a drenching programme but also for companies investigating the efficacy of their products. The determination of resistance and corresponding confidence limits as given in anthelmintic efficacy guidelines of the Standing Committee on Agriculture (SCA) is based on a number of assumptions. This study evaluated the appropriateness of these assumptions for typical data and compared the effectiveness of the standard FECRT procedure with the effectiveness of alternative procedures. Several sets of historical experimental data from sheep and goats were analysed to determine that a negative binomial distribution was a more appropriate distribution to describe pre-treatment helminth egg counts in faeces than a normal distribution. Simulated egg counts for control animals were generated stochastically from negative binomial distributions and those for treated animals from negative binomial and binomial distributions. Three methods for determining resistance when percent reduction is based on arithmetic means were applied. The first was that advocated in the SCA guidelines, the second similar to the first but basing the variance estimates on negative binomial distributions, and the third using Wadley’s method with the distribution of the response variate assumed negative binomial and a logit link transformation. These were also compared with a fourth method recommended by the International Co-operation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) programme, in which percent reduction is based on the geometric means. A wide selection of parameters was investigated and for each set 1000 simulations run. Percent reduction and confidence limits were then calculated for the methods, together with the number of times in each set of 1000 simulations the theoretical percent reduction fell within the estimated confidence limits and the number of times resistance would have been said to occur. These simulations provide the basis for setting conditions under which the methods could be recommended. The authors show that given the distribution of helminth egg counts found in Queensland flocks, the method based on arithmetic not geometric means should be used and suggest that resistance be redefined as occurring when the upper level of percent reduction is less than 95%. At least ten animals per group are required in most circumstances, though even 20 may be insufficient where effectiveness of the product is close to the cut off point for defining resistance.

A lack of predatory interaction between rumen ciliate protozoa and Shiga-toxin producing Escherichia coli

Aims: To investigate interactions between rumen protozoa and Shiga toxin-producing Escherichia coli (STEC) and to ascertain whether it is likely that rumen protozoa act as ruminant hosts for STEC. Methods and Results: The presence of stx genes in different microbial fractions recovered from cattle and sheep rumen contents and faeces was examined using PCR. In animals shedding faecal STEC, stx genes were not detected in the rumen bacterial or rumen protozoal fractions. Direct interactions between ruminal protozoa and STEC were investigated by in vitro co-incubation. Rumen protozoa did not appear to ingest STEC, a STEC lysogen or non-STEC E. coli populations when co-incubated. Conclusions: The ruminal environment is unlikely to be a preferred habitat for STEC. Bacterial grazing by rumen protozoa appears to have little, if any, effect on STEC populations. Significance and Impact of the Study: This study indicates that ruminal protozoa are unlikely to be a major factor in the survival of STEC in ruminants. They appear as neither a host that protects STEC from the ruminal environment nor a predator that might reduce STEC numbers.