With the rapid expansion in both the food fish farming and ornamental fish sector over the past years, an increasing number of microorganisms has been identified as important fish pathogens, including Flavobacterium columnare (F. columnare), the causative agent of columnaris disease. This bacterium affects both cultured and wild freshwater fish including many susceptible commercially important fish species such as rainbouw trout and carp. F. columnare infections may result in skin lesions, fin erosion and gill necrosis, with a high degree of mortality, leading to severe economic losses. Especially in the last decade, various research groups have performed studies aimed at elucidating the pathogenesis of columnaris disease, leading to significant progress in defining the complex interactions between the organism and its host. Despite these efforts, the pathogenesis of columnaris disease hitherto largely remains unclear, compromising the further development of efficient curative and preventive measures to combat this disease. Besides elaborating on the agent and the disease it causes, Chapter 1 aims to summarize these pathogenesis data emphasizing the areas meriting further investigation. The specific aims of the thesis can be found in Chapter 2. These were determining the antimicrobial susceptibility pattern of F. columnare by using reliable screening methods on a representative number of F. columnare isolates collected worldwide (Chapter 3), to develop an infection model generating gill lesions typical for columnaris disease in carp and rainbow trout and to classify F. columnare field isolates regarding virulence (Chapter 4) and to study the pathogenesis of columnaris disease with the gill tissue in carp and rainbow trout, adopting both highly and low virulent isolates (Chapter 5). Up until now, only a limited number of effective preventive measures against columnaris disease are available, including an attenuated immersion vaccine registered for use in channel catfish and largemouth bass in the US only. Other countries hence rely upon other aids to combat the disease, amongst which antimicrobial agents. The possibility exists though that harmful bacteria can acquire resistance to previously effective antimicrobial agents, thereby giving rise to uncontrolled bacterial disease outbreaks. Hence, antimicrobial susceptibility monitoring is a necessary prerequisite to inform the veterinary practitioners on the therapeutic value of antimicrobial agents enabling them to make an informed choice. Despite the importance of columnaris disease, the antimicrobial susceptibility pattern of F. columnare is not well-studied. Therefore, 97 bacterial isolates of F. columnare were collected worldwide between 1987 to 2011 from 17 fish species of both the aquaculture and ornamental fish sector, to test their in vitro antimicrobial susceptibility towards 12 antimicrobial agents, as described in Chapter 3. This study is the first in its kind in view of the high number and mixed origin of F. columnare isolates in terms of fish species, year of isolation and geographical area. The broth microdilution technique was utilized for reliable testing of the fastidious organisms. None of the isolates displayed acquired resistance to florfenicol, gentamicin, ormetoprim-sulfadimethoxin and trimethoprim-sulfametoxazole. Acquired resistance to chloramphenicol, nitrofuran, oxytetracycline, flumequin, oxolinic acid and enrofloxacin is reported for the first time in F. columnare. Furthermore, acquired resistance towards erythromycin and ampicillin was detected. The isolates displaying acquired resistance originated from Vietnamese catfish or ornamental fish species except for two isolates coming from wild channel catfish in which acquired resistance was encountered towards oxytetracycline only. Moreover, fifty percent of the resistant isolates from ornamental fish were assigned as multiple resistant. The results obtained in this study indicate less prudent use of antimicrobials, especially in the ornamental fish industry and therefore urges to limit their use and to focus on preventive measures or alternatives for treatment of columnaris disease. The modern aquaculture industry hence needs alternative preventive practices or treatments which allow the use of a “green” label, as environmental awareness grows and urges to help maintain a high animal welfare as well as a healthy environment, resulting in better production and higher profits. Crucial in the development of such techniques in combatting columnaris disease, is the understanding of the pathogenesis of this disease, starting at the initial phase of colonization: the induction of lesions, amongst which the important but understudied gill lesions. The need for research in this neglected field of aquatic veterinary medicine, more specifically with regard to gill lesions caused by columnaris disease in carp and trout, formed the basis for investigating the gill pathology in these fish species by firstly developing an in vivo infection model inducing gill lesions (Chapter 4). The gill tissue of carp and rainbow trout fry were exposed to different F. columnare isolates by means of immersion challenge. The fish were allowed to swim for 90 min in a suspension of one out of five (carp) or six (trout) available F. columnare isolates (approximating 1-7 x 108 CFU/ml, contact infection). For carp, four isolates (0401781, 0901393, 10009061-1 and 10012573/2) resulted in 100% mortality within 12 h, assigning these as highly virulent (HV). The other isolate (CDI-A) gave around 5% mortality within 18 h, being assigned to the low virulent (LV) group. From 100% of the moribund carp, F. columnare cells could be retrieved from the gill tissue. In trout, two isolates (B259 and JIP P11/91) were able to induce mortality in 100% of the fish, being assigned to the HV-group. One trout isolate (LVDJ (D7461)) was able to kill around 50% of the fish and was hence designated as moderately virulent. The other three isolates (H2, Coho 92 and JIP 44/87) with which the challenge resulted in a mortality rate of less than 10%, got assigned a LV-label. Reisolation of F columnare cells from the gill tissue of moribund trout varied from 67% of the inoculated fish in one LV-isolate to over 90% in the fish challenged with the moderately or HV-isolate. Both macro- and microscopic examination of the affected gills revealed a different distribution pattern of the lesions as induced by the HV- F. columnare isolates in carp compared to trout. The gills of the affected carp showed a diffuse distribution of the lesions, affecting the filaments of all gill arches bilaterally. At least half of the gill tissue was destroyed, with large parts of the filaments replaced with necrotic debris entangled with massive clusters of F. columnare bacterial cells, enwrapped in an eosinophilic matrix. In trout, the distribution pattern of the gill lesions was more focal, mostly located unilaterally and only present in the filaments of the two first gill arches. Scanning and transmission electron microscopic observations of the affected gills pictured long, slender bacterial cells attained in an extracellular matrix and in close contact with the destructed gill tissue. This is the first study to reveal gill lesions typical for columnaris disease at a macroscopic, light microscopic and ultrastructural level in both carp and rainbow trout following challenge with F. columnare, opening ample research opportunities regarding pathogen-gill interaction. Subsequently, in Chapter 5, the colonization pattern in the course of time of F. columnare isolates of different virulence and their interaction with the gill tissue were investigated adopting the infection model as described in Chapter 4. For this purpose, carp and trout were exposed to a HV- or LV-isolate and sacrificed at predetermined times post-challenge. Histopathological and ultrastructural examination of carp and trout inoculated with the HV-isolate disclosed the bacterial cells invading the gill tissue spreading from the gill filament tips to the basis. In both carp and trout, significantly higher bacterial cell counts could be retrieved from the gill tissue after an inoculation with the HV- compared to the LV-isolate. Another important parameter investigated in the theme of virulence, was the way the pathogen can escape the host’s inflammation response adopting apoptosis. In both carp and trout, TUNEL-staining showed a significant increase in the number of apoptotic cells in gill sections of fish exposed to the HV-isolate compared to control animals. In trout, the caspase-3 staining affirmed these results. In carp, caspase-3 staining could moreover display significantly more apoptotic cells in the gill tissue of fish challenged with the HV-isolate compared to the LV-isolate and between the LV-isolate inoculated fish and the control animals. Apoptosis was mostly seen in epithelial cells. Trends in gill mucus cell histochemistry included an increase of neutral mucin production in carp, with a significant increase in PAS-positive productive cell numbers following challenge with the HV-isolate compared to the control animals. H&E- and PAS/AB- stained sections depicted basophilic bacteria embedded in an eosinophilic matrix, and bacteria immediately surrounded by a PAS-positive matrix enveloped by AB-positive mucins, respectively. These findings strongly point towards biofilm development. Most bacterial cells of the HV-isolate were seen surrounded with outer membrane vesicles. Another investigated cell type are the eosinophilic granular cells (EGC), or so-called stress cells. While not perceived in trout, EGC were demonstrated in all carp gills investigated using both H&E- and PAS-staining. Mobilization and degranulation of this cell type was however only noted in the gill tissue of carp challenged with the bacterial cells, making this the first study to report this feature. In the general discussion part, the results of the different studies are summarized and discussed. Additionally, future research perspectives are provided to further elucidate possible virulence factors of F. columnare. |
