Avian Infections Caused by Species of Pasteurellaceae an Update

Avian infections caused by species of Pasteurellaceae An update

M. Bisgaard*, H. Christensen, A.M. Bojesen and J.P. Christensen

Department of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, 4 Stigbøjlen, DK-1870 Frederiksberg C, Denmark

* [email protected] or [email protected] ; telephone +45 35 28 27 18; fax +45 35 28 27 57

Introduction The family Pasteurellaceae Pohl 1981 was conceived to accomodate a large group of Gram- negative chemoorganotrophic, facultatively anaerobic, and fermentative bacteria including the genera Pasteurella (Trevisan, 1887), Actinobacillus (Brumpt, 1910), Haemophilus (Winslow et al. , 1917), and many other groups of organisms that exhibited phenotypic and genotypic relationships with these genera (Pohl, 1979; 1981). Several new genera and species have been reported since the taxonomy of the family was reviewed by Bisgaard (1995b). Presently the family includes 10 genera, 60 named species in addition to numerous, not yet named taxa. In addition, the number of genomospecies representing genotypical distinct species without sufficient phenotypic diversity to allow separation and naming, has increased. Serious problems are associated with isolation and identification of these organisms due to difficulties of growing and characterising them which often result in weak or false negative test results just as the use of diffent media and indicators prevent comparison of results obtained. In addition, the use of commercial kits often results in unreliable results (Inzana et al ., 2000). For the same reasons several examples of misidentification have been reported (eg. Boot & Bisgaard, 1995; Blackall et al ., 1997; 2002) and the use of reference strains is highly recommended just as strains should be sent to reference laboratories in case molecular methods are not available (Bisgaard, 1993, Christensen & Bisgaard, 2003; 2004). The epidemiology of most species has remained unclear. However, host specificity has been reported for several species while others seem to be associated with many different hosts (Bisgaard, 1993). Unfortunately, many publications do not allow unambiguous statements as to the species dealt with for reasons stated above just as knowledge on less pathogenic taxa has been based on systematic recordings of origin of isolates only (Bisgaard, 1993). In addition, recent observations with species obtained from several hosts seems to indicate the existence of host-related subclones (Davies et al . 2004).

Genus Pasteurella The genus Pasteurella previously included nine named species and two unnamed taxa (Christensen & Bisgaard, 2003). Reclassification of the avian cluster 18 as Gallibacterium, (Christensen et al., 2003a), Volucribacter (Christensen et al ., 2004b) and Avibacterium (Blackall et al ., 2005), however, limited the genus to include P. multocida, P. canis, P. stomatis, P. dagmatis and the unnamed species B of which only P. multocida is regarded as a major pathogen for birds. Based upon genotypic similarity P. multocida was recently reclassified to include biovar 2 variants of Av. avium and P. canis (Christensen et al., 2004a). Recent studies also documented the existence of two new species-like taxa of Pasteurella related to P. multocida, and special care should be taken in the identification of Pasteurella - like organisms (Christensen et al., 2005). Although it has been known for decades that strains of P. multocida differ considerably in their virulence for fowl very little is known about virulence factors. In particular, information on the molecular basis of virulence is lacking. Christensen & Bisgaard (1997) reviewed virulence factors including capsule, endotoxin and other toxins, outer membrane proteins (OMP), heat shock proteins and enzymes. An update on virulence factors was subsequently reported by Christensen & Bisgaard (2003). Briefly, only the dermonecrotic toxin has been found directly involved in virulence of P. multocida while other genes are characterized as putative virulence genes. The epidemiology of P. multocida was reviewed by Christensen & Bisgaard (2003). Although very diverse (Petersen et al ., 2001; Davies et al ., 2003) outbreaks are normally caused by a single clone under industrial conditions. Longitudinal studies on the stability of an outbreak clone, however, remains to be investigated. Demonstration of the same clone in three outbreaks of fowl cholera in wild web-footed birds in Denmark in 1996 and 2001 and Sweden

in 1998 seems to indicate clonal stability (Christensen et al., 1998; unpublished results). Whether clones associated with outbreaks in one animal species also affect other animal species remains to be investigated in more detail. Comparison of OMP profiles of bovine isolates with those of avian, ovine and porcine strains has shown that the majority of respiratory tract infections in each of these species are caused by different strains of P. multocida . However, the presence of a small number of strains in more than one host species suggests that transmission between different host species also should be considered (Davies et al ., 2004). Identification and typing of P. multocida including DNA-based methods were reviewed by Blackall & Mifflin (2000) and Christensen & Bisgaard (2003). Non-specific PCR (Mifflin & Blackall, 2001) and in situ detection (Mbuthia et al., 2001) previously reported with biovar 2 of Av. avium and P. canis has subsequently been explained by Christensen et al. (2004a). However, other problems have been indicated (Christensen et al., 2005). Only minor progress has been observed during the past decade as to treatment and prophylaxis of fowl cholera and the poultry industry is still waiting for a break through as to the use of defined strains of P. multocida for use as vaccines (Christensen & Bisgaard, 2003). A break through, however, depends on the identification of specific virulence factors.

Genus Avibacterium A major subcluster within the avian 16S rRNA cluster 18 including four named species that are well known to veterinary bacteriologists, [H.] paragallinarum, P. gallinarum, P. avium, P. volantium as well as the unnamed P. sp. A was recently shown to form a monophyletic group with a minimum of 96.8% 16S rRNA sequence similarity. As this group also could be separated phenotypically from all other recognized and named taxa within Pasteurellaceae a new genus, Avibacterium , has been proposed for this group (Blackall et al., 2005). While all of the named species are routinely encountered in the investigation of upper repiratory tract disease of birds, only Av. paragallinarum is regarded as a primary pathogen, the causative agent of infectious coryza, an economically important disease of chickens (Blackall, 1999). An overview of infectious coryza including diagnostic options have been published by Blackall (1999). Briefly, two biovars of Av. paragallinarum have been reported both of which are causing infectious coryza (Mouahid et al ., 1992). After the emergence of the NAD- independent biovar 2 in South Africa (Horner et al., 1995) this biovar has subsequently been reported from Mexico (Garcia et al ., 2004). Serotyping of biovar 2 isolates have demonstrated the existence of all three serovars of Page although the majority belong to Page serovar A (Mifflin et al., 1995; Bragg et al., 1997; Garcia et al., 2004). Horner et al . (1995) suggested that biovar 2 isolates may cause air sacculitis more commonly than the classic biovar 1 isolates do. As for P. multocida a range of virulence factors including HA antigens (Yamaguchi et al ., 1993) and capsule (Sawata et al., 1985) in addition to iron sequestration (Ogunnariwo & Schryvers, 1992) and crude polysaccharide (Iritani et al., 1981) have been suggested for Av. paragallinarum . Molecular typing methods and serological tests were updated in the last edition of Diseases of Poultry by Blackall & Matsumoto (2003) as were treatment and vaccination. Despite routine vaccination with the currently available commercial vaccines, including trivalent vaccines, outbreaks of infectious coryza have been reported from different countries in South America, the US and Africa. Subsequent investigations on the virulence of the nine serovar reference strains of Av. paragallinarum indicate that virulence differences exist. The highest clinical score was obtained for serovar C-1 while the lowest was obtained with serovar C-4 (Soriano et al ., 2004a). Cross-protection studies with the nine serovars confirmed that serogroups A, B, and C represent distinct immunovars and cross-protection among all four serovars of

serogroup A was observed while evidence of a reduced level of cross-protection was noticed for serogroup C (Soriano et al., 2004b). A tetravalent oil adjuvant vaccine containing an additional serotype B isolate appeared immunogenic against all isolates after one vaccination (Jacobs et al ., 2003). Av. paragallinarum is a delicate organism which is inactivated rather rapidly outside the host. Depopulation of infected or recovered flocks which represent the main source of infection therefore represent an effective method of eradication. Since all in all out production is not used in developing countries infectious coryza might have a much greater impact in these countries than in developed countries. Within the order Galliformes, Av. gallinarum has been reported associated with lesions in Gallus domesticus , Meleagris gallopavo and Numida meleagris . However, only isolates from chickens in addition to a single turkey isolate have been confirmed by genetic analysis such as ribotyping and/or 16S rRNA gene sequence comparison (Christensen et al., 2002). A single isolate from a pheasant which did not produce acid from maltose and dextrin was subsequently reported to show 99.5% 16S rRNA gene sequence similarity to Av. gallinarum (Christensen et al., 2003b). Identification of the same clone of Av. gallinarum from 14 outbreaks of acute respiratory disease in turkeys within a time period of two months suggests a common source of infection and a primary rather than secondary role of Av. gallinarum in the outbreaks (Bisgaard et al., 2005). Isolates from rats identified as Av. gallinarum by conventional phenotypic tests belonged to a different ribotype cluster and showed only 93% 16S rRNA similarity with Av. gallinarum (Christensen et al ., 2002). For the same reasons these organisms have been allocated a new genomospecies tentatively designated taxon 47. V-factor requirering strains obtained from sinusitis in partriges and pheasants and isolates from endocarditis in chickens represent new species candidates of Avibacterium tentatively designated taxon 49 and taxon 50, respectively. Finally, isolates from the respiratory tract of pheasants (Petersen et al ., 1998; Christensen et al ., 2003b) have been allocated with Avibacterium .

Genus Actinobacillus The taxonomy of organisms classified with Actinobacillus was recently reviewed with focus on classification and identification including molecular based characterization, typing and identification (Christensen & Bisgaard, 2004). The revised definition of Actinobacillus sensu stricto only includes a single avian taxon, formerly known as avian haemolytic Actinobacillus -like organisms which presently is designated taxon 26. These organisms include four biovars which appeared heterogeneous on species level by DNA:DNA hybridizations (Piechulla et al., 1985). Taxon 26 has been obtained from sinusitis, conjunctivitis and septicaemia in web-footed birds in Europe and the US (Bisgaard, 1993).

Genus Gallibacterium A new genus, Gallibacterium , which incorporates organisms once known as avian Pasteurella haemolytica, Actinobacillus salpingitidis and Pasteurella anatis has recently been proposed by Christensen et al. (2003a), since these taxa form a monophyletic unit with similarities above 95% on the basis of 16S rRNA sequence comparison and they are unrelated with other genera of the family Pasteurellaceae . Similarities between strains of Av. paragallinarum and strains of Gallibacterium ranged between 92.7 and 94.8% while similarities of between 92.2 and 93.8% were obtained to Volucribacter and the unnamed taxa 2, 3 and 34 of Bisgaard. Three species of Gallibacterium, G. anatis and genomospecies 1 and 2 have been reported so far (Christensen et al., 2003a). A total of 21 different plasmid sizes varying between 1.9 and 14.5 kb was demonstrated, the significance of which remains to be investigated.

Disease manifestations, pathogenesis and epidemiology of Gallibacterium has been reviewed by Bojesen (2003). Although neither specific lesions nor syndromes have been associated with Gallibacterium species most reports seem to indicate a predilection for the reproductive tract resulting in salpingitis, oophoritis, peritonitis and sepsis. However, pericarditis, perihepatitis and upper respiratory tract lesions have also been reported according to Bojesen (2003). A new emerging disease of chickens affecting the reproductive tract of layers caused by G. anatis was recently reported from Mexico (Vazques et al., 2003). The severity of lesions and loss in egg production varied according to the different biovars of G. anatis involved (Gonzales et al., 2003). The nature of Gallibacterium infections is poorly understood. Experimental studies with Gallibacterium have shown contradictory results (Bisgaard, 1977; Gerlach, 1977; Mushin et al., 1980) which might be explained as a result of differences in virulence or factors related to the host. Recent in vivo studies in heterophil depleted and normal 15-weeks-old brown layers, however, resulted in lesions similar to previous descriptions of natural infections (Bojesen et al., 2004b).Speculations on possible virulence factors have included RTX toxin and capsule (Bojesen, 2003), both of which have been demonstrated in other Pasteurellaceae . Investigations on the prevalence and transmission of haemolytic Gallibacterium spp. in Danish chicken production systems with different levels of biosecurity showed that the prevalence proportions were highly influenced by the production system and found to be significantly associated with the biosecurity level observed for the flocks. Flock infections resembled an all or none type of colonization, and no evidence of vertical transmission was observed (Bojesen et al., 2003b). Subsequent investigations on clonality indicated that only a single clone was apparent in the flock. One subcluster contained only tracheal isolates while the other primarily included cloacal isolates (Bojesen et al ., 2003a). In addition to chickens Gallibacterium spp. have been isolated from lesions in turkeys, pheasants, partriges, web- footed birds and psittacine birds (Bisgaard, 1993; Christensen et al., 2003a). Isolation and identification depend on classical procedures. Inbucation should always take place under micro-aerophilic conditions since some strains only grow under these conditions at primary isolation. In situ hybridization based upon a probe targeting 16S rRNA has been reported (Bojesen et al ., 2003c). The probe specifically detects Gallibacterium and proved also useful for detection of Gallibacterium in formalin-fixed tissues. A bacterin including biovars 1, 2 and 4 accounting for 95% of the outbraks in Mexico has been developed by Boehringer Ingelheim Vetmedica, Mexico (Gonzales et al., 2003; Vasques et al., 2003).

Genus Volucribacter Due to restrictions and legislation placed on importation of wild caught birds into the European Union supplying the growing market for pet birds increasingly depends on the success of avicultural breeding programmes. Most taxa of the family Pasteurellaceae investigated so far appear to be commensals of freeliving hosts. However, high density and confined rearing facilities developed for breeding in captivity of pet birds might change the balance between host and parasite assumed to exist on mucous membranes under normal conditions, and result in disease. Most members of the family Pasteurellaceae seem to have lost most of the genomic information of their free-living ancestors during adaptation to an obligate parasitic life. During the same process host adaptation and disease potential for the host seem to have evolved. For the same reasons characterization of Pasteurellaceae obtained from diseases of “new hosts” includes the potential of dealing with a new species. Characterization of Pasteurellaceae from psittacine birds thus resulted in description of a new taxon 33 (Bisgaard et al., 1999) associated with lesions of the respiratory tract and septicaemia. Subsequent

attention to the existence of this taxon resulted in 13 additional strains and a final classification of taxon 33 as Volucribacter psittacicida and Volucribacter amazonae (Christensen et al., 2004b). Since these organisms have been unknown to other laboratories isolates have only been obtained in European countries so far, including Belgium, Germany and Denmark. Symptoms as well as lesions associated with these infections have been unspecific and it should be underlined that septic conditions in psittacine birds due to these organisms only might include a slightly swollen liver and spleen in addition to minor vascular disturbances. Methods and characters used for identification of these organisms at genus and species level have been reported by Christensen et al. (2004b).

Unnamed taxa of Pasteurellaceae of veterinary importance Organisms obtained in pure culture from salpingitis and peritonitis in ducks were initially reported as atypical Actinobacillus lignieresii (Bisgaard, 1975). Subsequent investigations of additional isolates from ducks, geese and pigeons classified these organisms as taxon 2 and taxon 3 (Bisgaard, 1982). The importacne of these organisms in salpingitis in web-footed birds was subsequently confirmed by Bisgaard (1995a) who also extended the host and disease spectrum of these organisms to include partriges, pheasants and psittcine birds (Bisgaard, 1993; 1995a; Bisgaard et al ., 1999). AFLP typing of selected strains (Bojesen et al. , 2005) representing existing biovars recently confirmed previous protein profiling (Bisgaard et al., 1993), DNA:DNA hybridization results (Piechulla et al., 1985) and 16S rRNA sequence data (Blackall et al., 2005) suggesting that these taxa represent a new genus of Pasteurellaceae associated with birds. The original publication on taxon 14 included strains from upper respiratory tract lesions in ducks, turkeys and pigeons (Bisgaard & Mutters, 1986). Subsequent publications extended the hosts to include geese and peafowl (Bisgaard, 1993) and pheasant (Christensen et al., 2003b) and clustered these organisms with taxon 32 obtained from respiratory tract lesions in pigeon hawks and taxon 40 obtained from the respiratory tract of seagulls (Christensen et al ., 2003b).

In conclusion the present review has documented the complexity and diversity of organisms classified with Pasteurellaceae . Future research should aim at a more safe basis for diagnosis of these organisms based upon molecular methods, since an unambiguous diagnosis is a prerequisite for improving our knowledge and understanding of the complexity of bacteria- host interactions and the expected diversity of virulence designs of these organisms. Although responsible for some of the most common and devastating diseases in animals and in spite of these infections have been known for decades, mechanisms of colonization, survival and multiplication and invasion of the host have remained incompletely understood, not to mention virulence factors. By tradition the upper respiratory tract has been considered the natural habitat of Pasteurellaceae , an environment where nutrients are in short supply compared to the gastrointestinal tract. Natural selection in this environment most likely have favoured a host-bacteria relationship leading to a certain degree of host specificity in the first place. Metabolic talents allowing these organisms to establish a commensal life with the host, however, remains to be elucidated. Subsequent development of a disease potential might also depend on a complicated interaction between bacteria and host. Several genes might be involved in this process in addition to host factors. For the same reasons designed/random mutagenesis presently might represent a troublesome way forward. Fundamental work on the identification of surface proteins and lipopolysaccarides behind the recently observed existence of host specific clones within the same species and the phylogeny of the genes behind might form a better foundation for subsequent seach for virulence factors. Development of experimental models to target immune cells in different hosts with clones

from different animal species is also highly needed (Bojesen et al ., 2004a) as are investigations on the metabolism of nutriens available on the surface of mucous membranes.

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