Infectious coryza is an acute respiratory disease of chickens caused by the bacterium known as Avibacterium paragallinarum, once known as Haemophilus paragallinarum and Haemophilus gallinarum. The greatest economic losses associated with infectious coryza result from poor growth performance in growing birds and marked reduction in egg production in layers. It is generally accepted that Av. paragallinarum is a primary pathogen while the other genus members are either opportunistic pathogens (Av. endocarditidis, Av. gallinarum) or not recognized as pathogens (Av. avium, Av. volantium). Infectious coryza occurs wherever chickens are raised and is a common problem in the intensive chicken industry. The disease also has been reported in other, less intensive situations. The chicken is the natural host for Av. paragallinarum. There are reports that the village chickens of Africa sand Asia are as susceptible to infectious coryza as normal commercial breeds.

Infectious Coryza and Related Bacterial Infections

The gastrointestinal (GI) microbiome of cats and dogs is increasingly recognized as a metabolically active organ inextricably linked to pet health Food serves as a substrate for the GI microbiome of cats and dogs and plays a significant role in defining the composition and metabolism of the GI microbiome The microbiome, in turn, facilitates the host’s nutrient digestion and the production of postbiotics, which are bacterially derived compounds that can influence pet health Consequently, pet owners have a role in shaping the microbiome of cats and dogs through the food they choose to provide Yet, a clear understanding of the impact these food choices have on the microbiome, and thus on the overall health of the pet, is lacking Pet foods are formulated to contain the typical nutritional building blocks of carbohydrates, proteins, and fats, but increasingly include microbiome-targeted ingredients, such as prebiotics and probiotics Each of these categories, as well as their relative proportions in food, can affect the composition and/or function of the microbiome Accumulating evidence suggests that dietary components may impact not only GI disease, but also allergies, oral health, weight management, diabetes, and kidney disease through changes in the GI microbiome Until recently, the focus of microbiome research was to characterize alterations in microbiome composition in disease states, while less research effort has been devoted to understanding how changes in nutrition can influence pet health by modifying the microbiome function This review summarizes the impact of pet food nutritional components on the composition and function of the microbiome and examines evidence for the role of nutrition in impacting host health through the microbiome in a variety of disease states Understanding how nutrition can modulate GI microbiome composition and function may reveal new avenues for enhancing the health and resilience of cats and dogs

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The Effects of Nutrition on the Gastrointestinal Microbiome of Cats and Dogs: Impact on Health and Disease.

Gram-negative bacteria are surrounded by two membranes, called the outer and inner membranes. The space between these membranes, the periplasm, is spanned by a polymeric glycopeptide network, the peptidoglycan. This net has a closely defined mesh size and is attached to the outer and inner membranes by various proteins in the different Gram-negative organisms, leading to an even distance between the two membranes. Proteins that are to be transported to the outer membrane or the extracellular space thus have to cross various obstacles on their way; this process is mediated by a number of highly specialized secretion systems, mostly classified by Roman numerals from type I to type VII (see other chapters in this book for a comprehensive review of these systems). More secretion systems, or new variations of the ones previously described, are still found on a regular basis, making a comprehensive listing almost impossible. The major systems that are either well described and/or widely present in many Gram-negative species can be classified into two general architectures: those that span both membranes and the periplasm as one large secretion complex and those where the individual membranes are crossed using independent (i.e., not physically connected) secretion machineries. Type V secretion systems are part of the latter systems.

Type V Secretion Systems in Bacteria.

Interleukin-12 (IL-12), consisting of p40 and p35 subunits, produces both p70 heterodimer and free p40. p70 is essential for the induction of T-helper 1 (Th1) and cytotoxic T-cell (CTL) immunity, whereas p40 inhibits p70-mediated function. Here, we found that mutations introduced into N-glycosylation sites (N220 of murine p40 and N222 of human p40) reduced secretion of p40 but not p70. Co-immunization of N220 mutant mIL-12 gene with hepatitis C virus (HCV) E2 DNA significantly enhanced long-term E2-specific CD8+ T-cell response and protection against tumor challenge compared with that of wild type. Our results indicate that the ratio of p70 to p40 is important for generating sustained long-term cell-mediated immunity. Thus, the mutant IL-12 could be utilized for the development of DNA vaccines as an adjuvant for the generation of long-term memory T-cell responses.

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Poster Session 4 : Immunology ; Engineering N-glycosylation mutations in IL-12 enhances sustained cytotoxic T Lymphocyte responses for DNA immunization

C hlamydia spp. are obligate, intracellular bacteria that infect humans and animals. Research on these important pathogens has been hindered due to a paucity of genetic tools. We recently adapted a group II intron (GII) mutagenesis platform for creation of ampicillin-selectable gene insertions in C. trachomatis L2. The aims of this study were: (1) to assess the stability of the intron-insertion in an in vivo infection model to gauge the efficacy of this genetic tool for long term animal studies and (2) to expand upon the utility of the method by validating a second selection marker (aadA, conferring spectinomycin resistance) for mutant construction. Intron stability was assessed using a mouse vaginal tract infection model with a C. trachomatis L2 434/Bu incA::GII(bla) mutant. Infections were performed in the absence of selection and isolates shed into the vaginal tract were isolated and expanded in cell culture (also without selection). PCR and inclusion phenotype analysis indicated that the intron was stable for at least 27 days post-infection (at which point bacteria were no longer recovered from the mouse). The aminoglycoside 3′ adenyltransferase (aadA) gene was used to create a spectinomycin-selectable GII intron, facilitating the construction of an incA::GII[aadA] C. trachomatis L2 insertion mutant. Both the GII(aadA) intron and our previously reported GII(bla) intron were then used to create an incA::GII(aadA), rsbV1::GII(bla) double mutant. Mutants were confirmed via PCR, sequencing, inclusion morphology (incA only), and western blot. The stability of the intron-insertion during in vivo growth indicates that the GII-insertion mutants can be used to study pathogenesis using the well-established mouse infection model. In addition, the validation of an additional marker for mutagenesis in Chlamydia allows for gene complementation approaches and construction of targeted, double mutants in Chlamydia. The aadA marker also could be useful for other genetic methods. Collectively, our results expand upon the rapidly growing chlamydial genetic toolkit and will aid in the implementation of studies dissecting the contribution of individual genes to infection.

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Use of aminoglycoside 3′ adenyltransferase as a selection marker for Chlamydia trachomatis intron-mutagenesis and in vivo intron stability

The haemagglutinin of Avibacterium paragallinarum is a trimeric autotransporter adhesin that confers haemagglutination, cell adherence and biofilm formation activities.

Indoxyl sulfate (IS), a protein-bound uraemic toxin, has been found to accumulate in the serum of people with renal diseases and is associated with free radical induction, nephrotoxicity cardiovascular toxicity, and osteoblast cytotoxicity. Although IS has been studied in humans and in experimental models, the role of IS in dogs and cats with kidney disease has not been investigated. A high performance liquid chromatography system was applied to detect plasma IS concentrations in non-azotaemic animals (63 dogs, 16 cats) and in animals with renal azotaemia (66 dogs, 69 cats). The IS levels of azotaemic animals were significantly higher ( P  0.01) than those of non-azotaemic animals (median [IQR] 20.4 (9.5) mg/L vs. 7.2 (8.8) mg/L for dogs; median [IQR] 21 (18.9) mg/L vs. 14.8 (12.3) mg/L for cats). The IS level was significantly correlated with blood urea nitrogen, serum creatinine and phosphate concentrations. Dogs with acute kidney injury had significantly higher IS levels ( P  0.01) than those with chronic kidney diseases (CKD) (median [IQR] 57.7 (40.8) mg/L vs. 17.7 (25.1) mg/L). When CKD was graded using the International Renal Interest Society (IRIS) staging system, IS levels were correlated with CKD severity in both dogs and cats. The IS concentration is directly related to loss of renal function. Further studies are necessary to determine whether measurement of IS provides any additional diagnostic or prognostic information in dogs and cats with kidney disease.

Detection of indoxyl sulfate levels in dogs and cats suffering from naturally occurring kidney diseases.

Chlamydia psittaci, the causative agent of avian chlamydiosis, an important zoonotic disease, infects a wide range of birds. Infected birds, whether symptomatic or asymptomatic, intermittently shed the agent through respiratory and intestinal routes. Therefore, it is essential to investigate the epizootiology of C. psittaci in poultry, pet birds, and wild birds. In this study, cloacal or fecal swabs collected from domestic waterfowl, psittacine birds, Columbidae, and wild birds were used to determine the prevalence of C. psittaci in Taiwan between 2014 and 2017. The C. psittaci infection rate was as high as 34.2% among domestic waterfowl farms. The waterfowl isolates clustered into two groups based on ompA phylogeny: one group (G1-like) clustered with the Polish G1 strains; the other group (waterfowl-TW) clustered near, but independently from, the classical ABE genotype cluster. Separately, 3.1% of parrot samples tested positive for C. psittaci belonging to genotype A. C. psittaci isolates of genotype B were detected in 10.1% of racing pigeons and other Columbidae. Wild bird samples from a wildlife refuge had a 2.2% prevalence rate; among these, two atypical C. psittaci genotypes were detected in samples from a Malayan night heron (Gorsachius melanolophus) and a Taiwan barbet (Megalaima nuchalis). Taken together, our results revealed that the risk of C. psittaci transmission from domestic waterfowl and Columbidae birds to humans could be underestimated, given the high prevalence rates in these birds. Furthermore, the free-range rearing system of waterfowl in Taiwan may promote C. psittaci transmission between poultry and wild birds. Pet birds and racing pigeons, which are in close contact with people, are also possible sources for cross-species transmission. Further studies are necessary to elucidate the virulence, biological and genetic characteristics, and modes of transmission of Taiwanese C. psittaci isolates to facilitate the prevention and control of C. psittaci infection.

Prevalence and Genotyping of Chlamydia psittaci from Domestic Waterfowl, Companion Birds, and Wild Birds in Taiwan.

SUMMARY Avibacterium paragallinarum is the causative agent of infectious coryza, an important respiratory disease of chickens. The capsule is an important virulence determinant of many pathogenic bacteria, but the function of the capsule in Av. paragallinarum is not well defined. In this study, acapsular mutants of Av. paragallinarum were constructed by inactivation of the hctA gene using the TargeTron® gene knockout system. The acapsular mutants were found to have greater hemagglutination activity than did the wild-type strain. Further, acapsular mutants exhibited an increased ability to adhere to DF-1 cells and to form biofilms on abiotic surfaces. Virulence assays showed that acapsular mutants were less virulent than the wild-type strain. Taken together, these results indicated that loss of capsule increases hemagglutination and adhesion activities but decreases the virulence of Av. paragallinarum. These results could be valuable to further elucidate the function of the capsule and the mechanism of pat...

Loss of the Capsule Increases the Adherence Activity but Decreases the Virulence of Avibacterium paragallinarum

Chicken infectious anemia (CIA) is a poultry disease that causes huge economic losses in the poultry industry worldwide. Commercially available CIA vaccines are derived from wild-type chicken anemia viruses (CAVs) by serial passage in cells or chicken embryos. However, these vaccinal viruses are not completely attenuated; therefore, they can be transmitted vertically and horizontally, and may induce clinical symptoms in young birds. In this study, we sought to eliminate these issues by developing a subunit vaccine exploiting the CAV structural proteins, engineering recombinant baculovirus-infected Spodoptera frugiperda (Sf9) cells that contained both the viral protein 1 (VP1) and VP2 of CAV. Moreover, we produced single-chain chicken interleukin-12 (chIL-12) in the same system, to serve as an adjuvant. The recombinant VP1 was recognized by chicken anti-CAV polyclonal antibodies in Western blotting and immunofluorescence assays, and the bioactivity of the recombinant chIL-12 was confirmed by stimulating interferon-γ (IFN-γ) secretion in chicken splenocytes. Furthermore, the ability of the recombinant VP1 to generate self-assembling virus-like particles (VLPs) was confirmed by transmission electron microscopy. Specific pathogen-free (SPF) chickens inoculated with VLPs and co-administered the recombinant chIL-12 induced high CAV-specific antibodies and cell-mediated immunity. Taken together, the VLPs produced by the baculovirus expression system have the potential to be a safe and effective CIA vaccine. Finally, we demonstrated the utility of recombinant chIL-12 as an adjuvant for poultry vaccine development.

Preparation of Chicken Anemia Virus (CAV) Virus-Like Particles and Chicken Interleukin-12 for Vaccine Development Using a Baculovirus Expression System.

Ming-Kun Hsieh

Papers

The haemagglutinin of Avibacterium paragallinarum is a trimeric autotransporter adhesin that confers haemagglutination, cell adherence and biofilm formation activities.

Detection of indoxyl sulfate levels in dogs and cats suffering from naturally occurring kidney diseases.

Prevalence and Genotyping of Chlamydia psittaci from Domestic Waterfowl, Companion Birds, and Wild Birds in Taiwan.

Loss of the Capsule Increases the Adherence Activity but Decreases the Virulence of Avibacterium paragallinarum

Preparation of Chicken Anemia Virus (CAV) Virus-Like Particles and Chicken Interleukin-12 for Vaccine Development Using a Baculovirus Expression System.