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The genetic information for three feline hemoplasmas is limited in Southeast Asia. According to the limited genetic data, this study modified a nested-PCR method targeting the 16S rRNA gene by designing a novel primary forward degenerate primer. Two hundred and thirty-one archived DNA extracts from the blood of client-owned cats with a variety of diseases were used. The modified nested PCR detected feline hemoplasma DNA in 64 of 231 (27.7 %) samples. Sanger DNA sequencing, BLAST, and phylogenetic analyses revealed nine nucleotide sequences of Mycoplasma haemofelis (Mhf) (3.9 %, 9/231), fifty-three nucleotide sequences of Candidatus Mycoplasma haemominutum (CMhm) (22.94 %, 53/231) and two nucleotide sequences of Candidatus Mycoplasma turicensis (CMtc) (0.86 %, 2/231). The phylogenetic analysis demonstrated separate genotypes of 30 DNA sequences of Thai CMhm. In addition, this analysis elucidated distinct genotypes of CMhm in Thai fishing cats (Prionailurus viverrinus). The domestic cat and Thai fishing cat groups were the two major groups separating Thai CMhm genotypes based on the 16S rRNA. One CMhm sequence in Thai fishing cats was also present in domestic cat CMhm genotypes. This result suggests that transmission of CMhm between domestic cats and Thai fishing cats has likely occurred. One Mhf sequence had low genetic identity (82 % similarity). The phylogenetic analysis confirmed that this sequence was still very closely related to Mhf reference sequences. This Mhf-like genotype could be a candidate novel Mhf genotype. This new genetic information for feline hemotropic Mycoplasma provides valuable information for future feline-related clinical studies., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The efficacy of antibody detection tools for all stages of Ehrlichia canis infections and for various genotypes remains unclear. We produced recombinant gp36 (rgp36) antigens from different isolates of Thai E. canis to confirm the immunoreactivities to these recombinant proteins from naturally infected dogs. Sera and blood samples were taken from 21 dogs naturally infected with E. canis and in the clinical stages of acute phase ehrlichiosis. The expression vectors and competent E. coli produced two isolates of rgp36. These two major rgp36s were recognized by the dogs' sera in Western blotting, with both anti-dog IgM and IgG used as secondary antibodies. The two different genotypes of these local recombinant immunoreactive proteins were gp36 subgroup A (isolate 1055) and subgroup B (isolate 533). The Western blot analyses successfully identified both specific IgM and IgG from the dogs' sera. Of all 21 cases, five dogs presented specific IgM, twenty dogs presented specific IgG, and the commercial test used found fifteen seropositive dogs. There were four dogs that presented both specific IgM and IgG. Only one dog presented specific IgM only. This report is the first identification of a specific IgM in dogs in response to acute infections with E. canis . The recombinant gp36 isolates may be useful as potential antigenic material for subsequent serological tests that have a high possibility for differentiating between acute, chronic, primary, and nonprimary infections with E. canis ., (© 2020 The Author(s).)

Background: The Sanger sequencing technique has been questioned and challenged by advanced high-throughput sequencing approaches. Sanger sequencing seems to be an obsolete technology. However, there are still research problems that could be answered using the Sanger sequencing technology. Fastidious obligate anaerobic bacteria are mostly associated with abscesses in animals. These bacteria are difficult to isolate from abscesses and are frequently excluded due to the bias of conventional bacterial culturing., Aim: This study demonstrated the usefulness of a broad-range polymerase chain reaction (PCR) with Sanger sequencing to identify the majority population of bacteria in abscesses from exotic pet animals., Materials and Methods: This study performed a pilot investigation of abscesses from 20 clinical cases (17 rabbits, 2 hedgehogs, and 1 sugar glider) using standard culture methods for both aerobes and anaerobes and broad-range nested PCR targeting the 16S rRNA gene followed by the Sanger sequencing technique., Results: The standard culture and PCR techniques detected bacteria in 9 and 17 of 20 samples, respectively. From the 17 sequencings of the 16S rRNA, 10 PCR products were found to be closely related with obligate anaerobes including Bacteroides spp., Fusobacterium spp., Prevotella spp. Phylogenetic analysis using the rpoB gene revealed that the species for the Bacteroides was thetaiotaomicron and for the Fusobacterium was varium and nucleatum . However, the amplification of the rpoB gene for the Prevotella spp. was unsuccessful. Correlations between the standard culture and PCR techniques were found in 9 (6 positive and 3 negative samples) of 20 samples. Eleven samples were discordant between the standard culture and PCR techniques which were composed of eight samples negative by culture but positive by PCR and three samples had different bacteria by the culture and PCR techniques., Conclusion: According to this study, broad-range PCR combined with Sanger sequencing might be useful for the detection of dominant anaerobic bacteria in abscesses that were overlooked based on conventional bacterial culture., (Copyright: © Duangurai, et al.)

Canine tick-borne bacteria; Ehrlichia canis, hemotropic Mycoplasma spp. and Anaplasma spp., are organisms transmitted by Rhipicephalus sanguineus ticks. However, only a few clinical studies evaluating dogs infected with these organisms and anemia condition have been published. In this study, the potential tick-borne bacteria linked to anemia were investigated in eighty-one blood samples selected from anemic dogs using a broad range nested-PCR of the 16S rRNA gene. Positive results were shown in 12/81 blood specimens (14.81%). Nucleotide sequences from the PCR products were analyzed using BLAST and resulted in identification of Ehrlichia canis (8), Candidatus Mycoplasma haematoparvum (1) and Anaplasma platys (3). Two other PCR assays were used to detect and identify the positive results of these pathogens including a specific PCR for Ehrlichia canis (gp36) and a specific nested-PCR for hemoplasma species (16S rRNA) and the phylogenetic analyses of E. canis and canine hemoplasmas were performed using these two loci. These specific PCRs revealed co-infection of E. canis and Mycoplasma haemocanis in two cases. These two male dogs had presented with jaundice, severe hemolytic anemia, severe thrombocytopenia, leukocytosis, mild azotemia and hepatitis. Ehrlichia canis was detected in a significantly greater number of severe anemia cases (PCV<15%) than moderate or mild anemia cases (PCV 16-29%) (P<0.05) and these severe anemia cases were 7-fold more at risk of having E. canis infections (odds ratio: 7.11, p=0.020). However, no statistical differences were detected between E. canis detection and degrees of thrombocytopenia or leukopenia. From the results of this study, we conclude that the severity of anemia is associated with E. canis infections rather than the severity of thrombocytopenia., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: The One Health (OH) approach, which seeks to bring together human and animal health, is particularly suited to the effective management of zoonotic diseases across both sectors. To overcome professional silos, OH needs to be taught at the undergraduate level. Here, we describe a problem-based learning activity using the OH approach that was conducted outdoors for 3 rd -year veterinary students in Malaysia., Materials and Methods: A total of 118 students, divided into two groups, completed the activity which spanned 1½ days at a deer park adjacent to a wilderness area. Students were asked to evaluate the activity using an online survey that had quantitative and qualitative components., Results: Response rate was 69.5%. The activity was rated excellent by 69.5% and good by 30.4%. Levels of satisfaction were high on a range of criteria. 97.5% of students intended to take action in their studies as a result of what they had learned., Conclusions: Delivery of an outdoor problem-based learning activity using OH approach was very successful in terms of participation, knowledge delivery and understanding, and the willingness of students to integrate OH into their future practice. For the improvement of future programs, the involvement of other disciplines (such as Medical, Biology, Biotechnology, Biomedical, and Public Health) is being considered.

Worldwide there are few isolate collections of the intracellular bacterium Coxiella burnetii, due to the difficulties associated with working with the organism and the scarcity of suitable samples from which to attempt isolation. Particularly lacking are isolates from acute Q fever patients. The aim of this study was to evaluate whether the serum samples taken from patients with confirmed acute Q fever during the early stage of their disease represented a potential source of viable C. burnetii. Isolation was attempted from 65 of these samples by inoculation of the serum into Vero cell culture and was successful in 36 cases (55%). This high success rate was likely due to extended incubation of up to twelve weeks of the inoculated cultures, allowing the growth of the organism to levels detectable by PCR. Retrospective analysis of the time the sera was stored prior to inoculation into culture demonstrated that C. burnetii remained viable for 224 days in samples stored refrigerated and 371 days in samples stored frozen at -20 °C. These results demonstrate that standard serum samples taken from acute Q fever patients are a valuable source of new isolates of C. burnetii, with no special handling of the specimens required to maintain the organism's viability., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A total of 1,136 samples from 289 households in four provinces in northern Laos were subjected to Japanese encephalitis virus (JEV) and dengue virus hemagglutination inhibition (DENV HI). Overall, antibodies to JEV were detected by HI in 620 (54.6%) of 1,136 people; of which 217 (19.1%) had HI activity against JEV only. Antibodies to DENV4 were detected by HI in 526 (46.3%) of 1,136 people; of which 124 (10.9%) had HI activity against DENV4 only. Antibodies to DENV1-3 were detected by HI in 296 (26.1%), 274 (24.1%), and 283 (24.9) of 1,136 people, respectively; of which 7, 1, and 0, respectively, had HI activity against DENV1-3 only. JEV was the most prevalent Flavivirus in Oudomxay, Luangprabang, and Huaphan provinces and DENV4 was the most prevalent in Xiengkhouang province. Seroprevalence for JEV increased with increasing age and wealth and was higher in villages where rice was cultivated in paddy fields and highest for people of Lao-Tai ethnicity., (© The American Society of Tropical Medicine and Hygiene.)

Bushland activity has previously been linked to rickettsial exposure in eastern and central regions of Australia, whereas little is known about the risks in Western Australia. The isolation of Rickettsia gravesii sp. nov. from Amblyomma triguttatum ticks and anecdotal reports of low-grade illness among bush recreationists raised the possibility of rickettsial transmission in the State. This study investigated rickettsial seroprevalence and potential risk of exposure to the spotted fever group rickettsiae in rogainers. Our results showed that rogainers active in the bush had a significantly higher risk of seropositivity (immunofluorescence total antibody titer ≥ 128) for the spotted fever group Rickettsia (odds ratio [OR] = 14.02, 95% confidence interval [CI] = 1.38-142.07) compared with a reference population, the overall seroprevalence in the rogainer group being 23.1%., (© The American Society of Tropical Medicine and Hygiene.)

The parasitic zoonoses human cysticercosis (Taenia solium), taeniasis (other Taenia species) and trichinellosis (Trichinella species) are endemic in the Lao People's Democratic Republic (Lao PDR). This study was designed to quantify the economic burden pig-associated zoonotic disease pose in Lao PDR. In particular, the analysis included estimation of the losses in the pork industry as well as losses due to human illness and lost productivity. A Markov-probability based decision-tree model was chosen to form the basis of the calculations to estimate the economic and public health impacts of taeniasis, trichinellosis and cysticercosis. Two different decision trees were run simultaneously on the model's human cohort. A third decision tree simulated the potential impacts on pig production. The human capital method was used to estimate productivity loss. The results found varied significantly depending on the rate of hospitalisation due to neurocysticerosis. This study is the first systematic estimate of the economic impact of pig-associated zoonotic diseases in Lao PDR that demonstrates the significance of the diseases in that country.

The aim of this work is to investigate the presence of Coxiella burnetii in Perameles bougainville and their ticks on two islands off Western Australia. Haemaphysalis humerosa, Haemaphysalis ratti, and Haemaphysalis lagostrophi were collected from P. bougainville on Bernier and Dorre Islands from 2005 to 2007; only Amblyomma limbatum was collected from humans over the same interval. One of 13 tick samples and 1 of 12 P. bougainville fecal samples were positive for C. burnetii DNA using quantitative polymerase chain reaction. DNA fragments had >99% similarity to published C. burnetii sequences. Three of 35 P. bougainville sera tested positive for anti-C. burnetii antibodies using enzyme-linked immunosorbent assay. C. burnetii was found in P. bougainville feces and a H. humerosa tick on Dorre Island and Bernier Island, respectively. This is the first reported use of enzyme-linked immunosorbent assay for screening of P. bougainville sera. The risk of zoonotic Q fever infection for human visitors to these islands is considered relatively low, however, appropriate precautions should be taken when handling western barred bandicoots, their feces and their ticks on Bernier and Dorre Islands.

Bartonella are arthropod-borne, fastidious, Gram-negative, and aerobic bacilli distributed by fleas, lice, sand flies, and, possibly, ticks. The zoonotic Bartonella species, Bartonella henselae and Bartonella clarridgeiae, which are the causes of cat scratch disease and endocarditis in humans, have been reported from cats, cat fleas, and humans in Australia. However, to date, there has been no report of B. henselae or B. clarridgeiae in Australian wild animals and their ectoparasites. B. henselae and B. clarridgeiae were detected in fleas (Ctenocephalides felis) from red foxes (Vulpes vulpes), an introduced pest animal species in Australia, and only B. clarridgeiae was detected in blood from one red fox. Phylogenetic analysis of the ribosomal intergenic spacer region revealed that the B. henselae detected in the current study were related to B. henselae strain Houston-1, a major pathogenic strain in humans in Australia, and confirmed the genetic distinctness of B. clarridgeiae. The identification and characterization of Bartonella species in red foxes in the Southwest of Western Australia suggests that red foxes may act as reservoirs of infection for animals and humans in this region.

Fleas are important arthropod vectors for a variety of diseases in veterinary and human medicine, and bacteria belonging to the genus Bartonella are among the organisms most commonly transmitted by these ectoparasites. Recently, a number of novel Bartonella species and novel species candidates have been reported in marsupial fleas in Australia. In the present study the genetic diversity of marsupial fleas was investigated; 10 species of fleas were collected from seven different marsupial and placental mammal hosts in Western Australia including woylies (Bettongia penicillata), western barred bandicoots (Perameles bougainville), mardos (Antechinus flavipes), bush rats (Rattus fuscipes), red foxes (Vulpes vulpes), feral cats (Felis catus) and rabbits (Oryctolagus cuniculus). PCR and sequence analysis of the cytochrome oxidase subunit I (COI) and the 18S rRNA genes from these fleas was performed. Concatenated phylogenetic analysis of the COI and 18S rRNA genes revealed a close genetic relationship between marsupial fleas, with Pygiopsylla hilli from woylies, Pygiopsylla tunneyi from western barred bandicoots and Acanthopsylla jordani from mardos, forming a separate cluster from fleas collected from the placental mammals in the same geographical area. The clustering of Bartonella species with their marsupial flea hosts suggests co-evolution of marsupial hosts, marsupial fleas and Bartonella species in Australia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We investigated the role of the western grey kangaroo (Macropus fuliginosus) in the maintenance and transmission of Coxiella burnetii in Western Australia. Sera from 1,017 kangaroos were tested using an indirect enzyme-linked immunosorbent assay (ELISA) for the presence of C. burnetii antibodies. The overall antibody prevalence across 12 locations throughout mid- to southwestern Western Australia was 24.1% (95% CI: 21.6-26.8). Feces from 990 of the same animals were tested using PCR to identify active shedding of C. burnetii in excreta. Coxiella burnetii DNA was detected in 4.1% (95% CI: 3.1-5.6) of samples. Our results suggest that kangaroos are reservoirs for C. burnetii in Western Australia and may contribute to transmission of the organism to domestic livestock and humans.

This is the first extensive study of the prevalence of naturally acquired Salmonella infection in wild-caught kangaroos in Australia. Given the close association between kangaroos, livestock, and humans and the growing popularity of kangaroo meat, it is important to identify epidemiologic factors associated with infection in these marsupials in order to minimize the risk of Salmonella transmission. The overall prevalence of fecal Salmonella in 645 western grey kangaroos (Macropus fuliginosus) sampled across 10 locations in Western Australia was 3.6% (95% CI: 2.3-5.3). Seven Salmonella serovars were identified including Salmonella enterica serovar Muenchen, Kiambu, Rubislaw, Lindern, Champaign, Saintpaul and II 42:g,t:-. Prevalence was significantly associated with rainfall (P<0.05) and was highest in the April-June quarter (P<0.05). There was no association between age or sex and the prevalence of Salmonella in fecal samples. Our results suggest that, while kangaroos are infected with Salmonella in their natural habitat, infection is less common than in hand-reared joeys, pet kangaroos, and macropods raised in captivity. Care should be taken to maintain hygiene during the evisceration, processing, and handling of kangaroos and to adequately cook kangaroo meat prior to consumption to reduce the risk of salmonellosis.

A variety of Bartonella species were detected in two species of ticks and three species of fleas collected from marsupial hosts; brush-tailed bettong or woylie (Bettongia penicillata) and western barred bandicoots (Perameles bougainville) and from a rodent host; Rattus fuscipes in Western Australia. Bartonella species were detected using nested-PCR of the gltA gene and the 16S-23S ribosomal internal transcribed spacer region (ITS), and species were characterized using DNA sequencing of the 16S rRNA, gltA, rpoB, ftsZ genes and the ITS region. Bartonella rattaustraliani and B. coopersplainsensis were detected in Ixodes spp. ticks and fleas (Stephanocircus pectinipes) respectively collected from rodents. Two novel Bartonella species were detected from marsupials; Candidatus Bartonella woyliei n. sp. was detected in both fleas (Pygiopsylla hilli) and ticks (Ixodes australiensis) collected from woylies and Candidatus Bartonella bandicootii n. sp. was detected in fleas (Pygiopsylla tunneyi) collected from western barred bandicoots. Concatenated phylogenetic analysis of all 5 loci clarified the marsupial cluster of Bartonella species in Australia and confirmed the species status of these two Bartonella species in ticks and fleas from woylies and western barred bandicoots, which are classified as threatened species and are vulnerable to extinction., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Bartonella are fastidious, Gram-negative, aerobic bacilli belonging to the Alphaproteobacteria group. In the last ten years, the discovery of new Bartonella species from a variety of mammalian hosts, arthropod vectors and geographical areas has increased. More than 20 species of Bartonella have been identified, of which approximately thirteen are associated with disease in humans and animals. Recently, four novel species of Bartonella were isolated from mammalian hosts in Australia: Bartonella australis from eastern grey kangaroos (Macropus giganteus) and Bartonella rattaustraliani, Bartonella queenslandensis and Bartonella coopersplainsensis from rodents. Bartonella-like organisms have also been detected from Ixodes tasmani ticks collected from koalas (Phascolarctos cinereus). However, very little is known about Bartonella spp. in other marsupials in Australia. We report the identification of a novel Bartonella species detected from fleas (Acanthopsylla jordani) and ticks (Ixodes antechini) collected from a small carnivorous marsupial, Antechinus flavipes (Mardos or Yellow-footed antechinus) in the southwest of Western Australia. New nested-PCRs targeting the gltA gene and the ribosomal ITS region were developed as part of the present study. DNA sequencing of the 16S rRNA, gltA, ftsZ and rpoB genes and the ribosomal ITS region revealed that this detection is a distinct Bartonella species and is related to B. australis isolated from kangaroos. This is the first report of two different possible arthropod vectors in Australia (ticks and fleas) being infected with the same species of Bartonella. We propose the name Candidatus Bartonella antechini n. sp. for the recently characterized organism., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Aims: To determine the optimal DNA extraction method for the detection of Coxiella burnetii including the small-cell variant (SCV) by real-time PCR (qPCR) in clinical samples., Methods and Results: A duplex qPCR detecting two Coxiella burnetii gene targets (com1 and IS1111a genes) was developed. Each target in this PCR had a sensitivity of one copy number per reaction. DNA extraction methods were compared on spiked negative samples and included a silica column kit, a chloroform separation prior to a silica column method and a chloroform/phenol separation and DNA precipitation method., Conclusions: The silica column extraction method was more efficient at recovering C. burnetii DNA, from large-cell and small-cell variants, than a chloroform or chloroform/phenol method. The silica column method was useful on spiked human samples including serum, buffy coat and bone marrow samples., Significance and Impact of Study: This study demonstrated that a simple column kit method is efficient to use for the detection of C. burnetii in clinical samples including the SCV., (© 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)

A survey of ectoparasites on feral pigs identified two commonly occurring ixodid tick species; Amblyomma triguttatum triguttatum and Ixodes australiensis. Molecular screening of A. t. triguttatum and I. australiensis for the presence of Rickettsia species detected the presence of rickettsiae belonging to the Spotted Fever Group (SFG) in 78.4% of screened A. t. triguttatum. None of the screened I. australiensis were positive for rickettsiae. Sequence analysis of the gltA and ompA loci of positive Rickettsia isolates were 100% homologous to the newly described species Rickettsia gravesii sp. nov. BWI-1. Serological screening of feral pigs detected antibodies to SFG Rickettsia in 50% of serum samples tested. These findings suggest that A. t. triguttatum is a vector/reservoir for R. gravesii sp. nov., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Tetanus toxoid (TT) was assessed as a positive marker for avian influenza (AI) virus vaccination in chickens, in a vaccination and challenge study. Chickens were vaccinated twice with inactivated AI H5N2 virus vaccine, and then challenged three weeks later with highly pathogenic AI H5N1 virus. Vaccinated chickens were compared with other groups that were either sham-vaccinated or vaccinated with virus with the TT marker. All sham-vaccinated chickens died by 36 hours postinfection, whereas all vaccinated chickens, with or without the TT marker, were protected from morbidity and mortality following exposure to the challenge virus. Serological testing for H5-specific antibodies identified anamnestic responses to H5 in some of the vaccinated birds, indicating active virus infection.

There is a general consensus that with increasing age a biofilm shows increased resistance to antimicrobials. In this study the susceptibility of 3-, 5- and 7-day-old Salmonella enterica serovar Typhimurium biofilms to disinfectants was evaluated. It was hypothesized that 7-day-old biofilms would be more resistant to disinfectants compared to 3- and 5-day-old biofilms. Biofilms were formed using the MBEC™ system and treated with six chemical disinfectants for 1 and 5 min. Four disinfectants at the highest concentration available showed 100% reduction in viable cells from all ages of biofilms after exposure for 5 min, and ethanol at 70% v/v was the least effective against biofilms, followed by chlorhexidine gluconate (CG). At the recommended user concentrations, only sodium hypochlorite showed 100% reduction in viable cells from all ages of biofilms. Benzalkonium chloride and CG were the least effective against biofilms, followed by quaternary ammonium compound which only showed 100% reduction in viable cells from 5-day-old biofilms. Overall, the results from this study do not display enhanced resistance in 7-day-old biofilms compared to 3- and 5-day-old biofilms. It is concluded that under the conditions of this study, the age of biofilm did not contribute to resistance towards disinfectants. Rather, the concentration of disinfectant and an increased contact time were both shown to play a role in successful sanitization.

The objective of this study was to investigate the prevalence of Coxiella burnetii in two domestic ruminant species (cattle and sheep) and the western grey kangaroo (Macropus fuliginosus) in Western Australia (WA). The IDEXX CHEKiT Q Fever ELISA and CFT were used to test sera from 50 sheep and 329 head of cattle for anti-C. burnetii antibodies and 343 kangaroo sera were tested using an indirect ELISA developed specifically for this study. Faecal or urine samples collected from the same animals were tested with two PCR assays to identify active shedding of C. burnetii in excreta. Only two of the 379 ruminant sera had detectable levels of anti-C. burnetii antibodies according to the ELISA while the CFT did not detect any positive samples. In contrast 115 of the 343 western grey kangaroo serum samples were positive when tested with the antibody-ELISA. The first qPCR assay, targeting the IS1111a element, identified 41 of 379 ruminant and 42 of 343 kangaroo DNA samples as positive for C. burnetii DNA. The second qPCR, targeting the JB153-3 gene, identified nine C. burnetii DNA-positive ruminant samples and six positive kangaroo samples. Sequence comparisons showed high degrees of identity with C. burnetii. Isolation of C. burnetii from faeces was also attempted but was not successful. From the results presented here it appears that domestic ruminants may not be the most significant reservoir of C. burnetii in WA and that kangaroos may pose a significant threat for zoonotic transfer of this pathogen., ((c) 2009 Elsevier B.V. All rights reserved.)

Aims: To compare the susceptibility of a 3-day-old biofilm and planktonic Salmonella to disinfectants at different exposure times. We hypothesize that Salmonella biofilms are more resilient to disinfectants compared to planktonic Salmonella., Methods and Results: The susceptibility of planktonic cells to disinfectants was tested by a modified version of the Council of Europe suspension test EN 1276. Salmonella biofilms were formed using the Calgary Biofilm Device. Results show that 3-day-old Salmonella biofilms are less susceptible to the disinfectants benzalkonium chloride, chlorhexidine gluconate, citric acid, quaternary ammonium compounds, sodium hypochlorite (SH) and ethanol, compared to planktonic Salmonella. Surprisingly, the results also demonstrate that low concentrations of SH were more effective against a 3-day-old biofilm compared to high concentrations of SH., Conclusions: While all the disinfectants evaluated were able to reduce biofilm-associated cells at concentrations and contact times sufficient to eliminate planktonic cells, there were still sufficient viable cells remaining in the biofilm to cause further contamination and potential infection., Significance and Impact of the Study: Protocols for the use of chemical disinfectants need to include biofilm susceptibility testing. There is a requirement for an effective and standardized tool for determining the susceptibility of biofilms to disinfectants.

The protective efficacy of a subunit avian influenza virus H5 vaccine based on recombinant baculovirus expressed H5 haemagglutinin antigen and an inactivated H5N2 avian influenza vaccine combined with a marker antigen (tetanus toxoid) was compared with commercially available inactivated H5N2 avian influenza vaccine in young ducks. Antibody responses, morbidity, mortality, and virus shedding were evaluated after challenge with a Vietnamese clade 1 H5N1 HPAI virus [A/VN/1203/04 (H5N1)] that was known to cause a high mortality rate in ducks. All three vaccines, administered with water-in-oil adjuvant, provided significant protection and dramatically reduced the duration and titer of virus shedding in the vaccinated challenged ducks compared with unvaccinated controls. The H5 subunit vaccine was shown to provide equivalent protection to the other two vaccines despite the H5 antibody responses in subunit vaccinated ducks being significantly lower prior to challenge. Ducks vaccinated with the H5N2 marker vaccine consistently produced antitetanus toxoid antibody. The two novel vaccines have attributes that would enhance H5N1 avian influenza surveillance and control by vaccination in small scale and village poultry systems.

This article discusses the integration of global and veterinary public health issues into the Australian veterinary curriculum. Formal veterinary education in Australia has a history of over 100 years and veterinarians have played a major role in the control of zoonotic and transboundary diseases for an even longer period. Australia is the largest exporter of red meat and live animals in the world. Therefore, educating veterinarians to promote and ensure food safety and animal welfare is prominent in Australian veterinary curricula. Veterinary degrees are accredited to allow Australian graduates to work professionally overseas, including in the United Kingdom and United States of America, and, in recent years, globalisation of the student body at Australian veterinary schools has occurred. For this reason, an appropriately broad curriculum is required to produce graduates who are able to address challenges in veterinary public health throughout the world. A Public Health University Network has been established to harmonise the veterinary public health curricula at the various veterinary schools and to develop the 'Australian veterinary public health philosophy', with its links to global issues and the 'One World, One Health' concept. Finally, conclusions are drawn on the implications of veterinary public health teaching in Australia and the preparation of Australian graduates for the global profession.

An education in veterinary virology should establish a basis for life-long learning and enable veterinary graduates to address professionally the control and eradication of viral diseases, both locally and globally. It is therefore more important that the curriculum focuses on a sound understanding of the nature and behaviour of viruses and their interactions with animal hosts, rather than imparting detailed information on an ever-increasing number of individual viral diseases in a widening range of animal species. Graduate veterinarians should be prepared with a comprehensive knowledge of the nature of viruses and their close dependence on the hosts thatthey infect, as well as a good understanding of pathogenesis, immunology, epidemiology, diagnostic approaches and control options. All these are necessary if the profession is successfully to meet familiar and new challenges in viral diseases in a wide range of host species, under different management conditions, in various geographic areas of the world.

Infection with Toxoplasma gondii is a significant problem in Australian marsupials, and can lead to devastating disease and predispose animals to predation. T. gondii infection in kangaroos is also of public health significance due to the kangaroo meat trade. A moderate seroprevalence of T. gondii was observed in a study of western grey kangaroos located in the Perth metropolitan area in Western Australia. Of 219 kangaroos tested, 15.5% (95%CI: 10.7-20.3) were positive for T. gondii antibodies using an ELISA developed to detect T. gondii IgG in macropod marsupials. When compared with the commercially available MAT (modified agglutination test), the ELISA developed was in absolute agreement and yielded a kappa coefficient of 1.00. Of 18 kangaroos tested for the presence of T. gondii DNA by PCR, the 9 ELISA positive kangaroos tested PCR positive and the 9 ELISA negative kangaroos tested PCR negative indicating the ELISA protocol was both highly specific and sensitive and correlated 100% with the more labour intensive PCR assay.

Feral pigs are recognized as being a potential reservoir of pathogenic microorganisms that can infect domestic pigs and other species. The aim of this study was to investigate whether feral pigs in Western Australia were colonized by the pathogenic enteric bacteria Lawsonia intracellularis, Brachyspira hyodysenteriae and/or Brachyspira pilosicoli. A total of 222 feral pigs from three study-populations were sampled. DNA was extracted from faeces or colonic contents and subjected to a previously described multiplex PCR for the three pathogenic bacterial species. A subset of 61 samples was cultured for Brachyspira species. A total of 42 (18.9%) of the 222 samples were PCR positive for L. intracellularis, 18 (8.1%) for B. hyodysenteriae and 1 (0.45%) for B. pilosicoli. Four samples were positive for both L. intracellularis and B. hyodysenteriae. Samples positive for the latter two pathogens were found in pigs from all three study-sites. A strongly haemolytic B. hyodysenteriae isolate was recovered from one of the 61 cultured samples. Comparison of a 1250-base pair region of the 16S rRNA gene amplified from DNA extracted from the isolate and five of the B. hyodysenteriae PCR positive faecal samples helped confirm these as being from B. hyodysenteriae. This is the first time that B. hyodysenteriae has been detected in feral pigs. As these animals range over considerable distances, they present a potential source of B. hyodysenteriae for any domesticated pigs with which they may come into contact.

Control measures for H5N1 avian influenza involve increased biosecurity, monitoring, surveillance and vaccination. Subclinical infection in farmed ducks is important for virus persistence. In major duck rearing countries, homologous H5N1 vaccines are being used in ducks, so sero-surveillance using H5- or N1-specific antibody testing cannot identify infected flocks. An alternative is to include a positive marker for vaccination. Testing for an antibody response to the marker would confirm approved vaccine use. Concurrent testing for H5 antibody responses would determine levels adequate for protection or indicate recent infection, with an anamnestic H5 antibody response requiring further virological investigation. In this study, we have evaluated the use of a TT marker in ducks given avian influenza vaccination. Wild or domestic ducks were tested for antibodies against TT and all 463 ducks were negative. High levels of TT-specific antibodies, produced in twice-TT vaccinated Muscovy ducks, persisted out to 19 weeks. There was no interference by inclusion of TT in an inactivated H6N2 vaccine for H6- or TT-seroconversion. Thus TT is a highly suitable exogenous marker for avian influenza vaccination in ducks and allows sero-surveillance in countries using H5N1 vaccination.

Strategies for differentiating infected from vaccinated animals (DIVA) require improvement for increased surveillance of avian influenza (AI), where vaccination is employed to control disease. We propose a novel DIVA approach for chickens using tetanus toxoid (TT) as an exogenous marker independent of serotype and relatedness of circulating and vaccine strains. Of 1779 chickens tested from Australia, Hong Kong and China, 100% were seronegative for TT-specific antibodies without vaccination. Tetanus toxoid adjuvanted to mineral oil was immunogenic in chickens. Co-delivery of both TT and inactivated LPAI (H6N2) vaccines in chickens elicited strong TT and influenza-specific antibody responses, which persisted to 53 weeks post-vaccination. Furthermore, immunization with a combined vaccine composed of TT and AI induced high levels of antibodies to both antigens. We conclude that TT is a highly suitable exogenous marker for AI vaccination in chickens allowing simple and effective monitoring of AI vaccination status.

A simple and reliable method was developed to isolate intact epithelial cells from pig and rabbit ilea and these were used to investigate the adhesion of Yersinia enterocolitica. Hydrophobic interaction was eliminated by treating the bacterial culture with 0.8 M tetramethyl urea (TMU). Virulent strains of Y. enterocolitica had significantly greater attachment than avirulent strains but both attached in a linear dose-dependant fashion. Epithelial cells prepared from pig ilea were attached to more readily than those prepared from rabbit ilea.

Campylobacter species are a significant cause of sheep abortion in most sheep-raising countries. In New Zealand, Campylobacter fetus subsp. fetus is the leading cause of diagnosed sheep abortion and the species C. jejuni and C. coli have also been implicated. To date, strain typing information of C. jejuni sheep abortion isolates is limited. The objective of the present study was to genotype C. jejuni isolates cultured from sheep abortions submitted to diagnostic laboratories in New Zealand during the 2000 breeding season, using pulsed-field gel electrophoresis (PFGE). In this study, C. jejuni isolates were cultured from approximately 10% of farms from which Campylobacter species were isolated from sheep abortions in the year 2000. This equated to 25 C. jejuni isolates from 21 farms. These isolates were obtained from the veterinary diagnostic laboratories and strain typed using the molecular typing technique PFGE. Ten distinct PFGE types were identified amongst the isolates. No particular PFGE type was found most frequently amongst these C. jejuni sheep abortion isolates. However, indistinguishable or similar C. jejuni PFGE types were identified from different aborted foetuses from the same flock, consistent with the role of C. jejuni as an infectious cause of abortion in sheep. These strain types were similar or indistinguishable from C. jejuni sheep abortion isolates obtained in 1999 in a smaller study (Mannering, S.A., Marchant, R.M., Middelberg, A., Perkins, N.R., West, D.M., Fenwick, S.G., 2003. Pulsed-field gel electrophoresis typing of C. fetus subsp. fetus from sheep abortions in the Hawke's Bay region of New Zealand. NZ Vet. J. 51, 33-37).

This study is the first confirmation of Rickettsia felis in Australia. The organism was identified from 4 species of fleas obtained from dogs and cats in Western Australia, by using polymerase chain reaction amplification and DNA sequencing of the citrate synthase and outer membrane protein A genes.

Aims: To investigate the epidemiology of Yersinia species in healthy goats in New Zealand, in particular to determine the prevalence of farms with infected goats, the prevalence of infected goats on those farms, the serotypes involved, and potential risk factors for carriage., Methods: A cross-sectional study of the prevalence of Yersinia infection in infected flocks in a study population of thirty commercial goat farms in the Manawatu region of New Zealand., Results: Infection was detected on 60% of farms in an initial study. In a prevalence study on 18 infected farms, the study population comprised 6770 animals (mean of 376, median of 175 and range of 36 to 1295 goats/farm). Of 902 goats (296 < 1 year, 178 1 to 2 years, and 428 > 2 years) sampled from the study population, 135 (73 < 1 year, 21 1 to 2 years, and 41 > 2 years) were excreting Yersinia spp, giving an overall prevalence of 14.97% (95% confidence interval [CI]; 12.8 to 17.4) with individual farm prevalences ranging from 0.0 (+ 7.9) to 58.14% (95% Cl, 43.3 to 71.6). Goats < 1 year were more likely to be infected than 1-2 year and > 2 year old animals (relative risk [RR] = 2.1; 95% Cl, 1.3 to 3.3) and 2.6 (95% Cl, 1.8 to 3.6) respectively), but there was no significant difference between risks for 1 to 2 year and > 2 year goats (RR = 1.2; 95% CI, 0.7 to 2.0). Yersinia enterocolitica was the most common species isolated in the youngest age group, with prevalence declining with increasing age, while other species were more common in the older age groups., Conclusion: Yersinia infections were common in goats in the study region, with younger animals apparently more susceptible to infection and in particular to infection with Y enterocolitica. The prevalence on infected farms appeared to decrease as flock size increased and to increase as stocking rates and the number of paddocks grazed increased.

Objective: To determine the temporal pattern of Yersinia infections in three goat flocks and examine the influence of management and seasonal factors on the incidence of those infections over a 1-year period., Methods: A longitudinal study involving monthly culture of faeces for Yersinia spp. from age groups of randomly selected goats on three farms in the Manawatu region of New Zealand., Results: The incidence of excretion of potentially pathogenic Yersinia (Yersinia pseudotuberculosis and Y enterocolitica biotypes 2, 3 and 5) peaked in winter and fell in summer. In contrast, environmental Yersinia (Y enterocolitica biotype 1A, Y frederiksenii, Y intermedia and Y rohdei) showed no clear pattern of seasonal variation. Pathogenic Yersinia were more prevalent in young animals than in adults, while environmental Yersinia were more prevalent in adults. The same type was isolated from the same animal in two or more successive months in about 20 to 25% of cases, and in the remaining cases there was a gap of at least one month between successive isolations, with many animals yielding a particular type on only a single occasion. A notable difference was that with the potentially pathogenic types, no animal had more than one period of time when it was found to be excreting a particular type, suggesting that immunity develops following exposure. In contrast, it was common for environmental types to be isolated from the same animal throughout the study period. Two goats were suspected to have developed clinical yersiniosis but all remaining infected animals showed no clinical signs of infection., Conclusions: Asymptomatic Yersinia carriage was common in goats in New Zealand, with a clear seasonal and age group pattern of infection with potentially pathogenic types. There was evidence that immunity developed to potentially pathogenic types. This is the first time that Y rohdei has been isolated from goats.