Abstract
Trichomonas gallinae, a flagellated protozoan, adversely affects the avian livestock, particularly the columbids. It causes an infection called necrotic ingluvitis and the disease can range from asymptomatic to lethal. The parasite affects the anterior gastrointestinal tract of the bird, forms granulomatous lesions, and blocks the oesophagus, making the bird starve, thus leading to death. The virulence of the strain varies with some strains causing up to 75% mortality. In UK, the infection was first recognized as an emerging infectious disease of British finches in summer of 2005. The sick dead birds exhibited signs of malaise such as lethargy and fluffed-up plumage, frequently in combination with dysphagia. A frequent contact of birds feeding at garden feeding stations and opportunities for novel inter-specific contacts at shared feeding sources may provide an increased opportunity for the parasite to infect the naïve host species. Not many molecular studies have been performed to investigate the degree of genetic diversity and cross-transmissibility between different isolates of T. gallinae. There is still limited data on T. gallinae sequences in the UK and therefore additional research is essential to improve understanding of molecular epidemiology of the pathogen.
Introduction
Trichomonas gallinae is a pathogen that affects the avian livestock and poses a threat to species of columbids (pigeons and doves). Columbids are the birds known to be largely affected by T. gallinae, but the parasite was also found to affect a wide range of avian families (Silvanose, 1998; Baker, 1996; McDougald, 2000). The pathogen is a flagellated protozoan that causes the lethal disease known as trichomoniasis (da Silva, 2007). This pathogen is ubiquitous; and the infection can range from asymptomatic to lethal (Grabensteiner, 2010). The parasite lives and survives in the anterior gastrointestinal tract of the bird and forms granulomatous lesions in the tract, which can block the lumen of the oesophagus (Borji, 2011). This leads to severe starvation, ultimately leading to death of the bird (Borji, 2011). The virulence of strain can vary and the spectrum can be wide (Stabler and Kihara, 1954). Some strains cause more than 75% mortality (da Silva, 2007). If the strain is highly virulent, a bird can succumb to infection in just two weeks’ time after being inoculated with a single organism; whereas, with an avirulent isolate, a bird may fail to even seroconvert after being inoculated with 1 * 106 organisms (Stabler and Kihara 1954). Long back, almost six decades ago, Stabler et al demonstrated that clinically normal pigeons can harbor both avirulent and virulent isolates, but naive doves and pigeons who were administered a mixture of virulent and avirulent isolates succumbed to infection (Stabler and Kihara 1954). Virulence also depends on factors such as previous exposure to pathogen (protective immunity) and individual immunocompetence (Stabler and Braun, 1975). Some researchers believe that nonpathogenic strains do not cause clinical disease, but they can be responsible for producing immunity in the infected bird (Stabler 1954; Cooper 1988; Cole 1999). However, strains those are pathogenic cause fibronecrotic and caseous lesions in the mouth, pharynx, and the esophagus, which blocks the food the bird consumes, making it starve and ultimately leads to death. In some birds, there can be a secondary bacterial infection leading to death (Stabler, 1954; Mesa, 1961).
Infection with T. gallinae can be recognized as an emerging infectious disease that is a threat to the wildlife, livestock, and also humans. Critically endangered wildlife populations or geographically isolated populations are usually threatened (Robinson, 2010).
In the UK, the disease primarily affects the columbids such as the pigeons and the doves, sometimes also birds of prey those feed on pigeons and doves that are infected. It is commonly known as “canker” in UK, while if it occurs in the birds of prey, it is termed as “frounce” (Holmes, 2005). It was first noticed in UK in the summer of 2005 (Pennycott, 2005). To be more precise, the first case of the disease in a British finch was in April 2005 followed by small number of mortality incidents, unusually peaking during the period of September to November 2005 (Pennycott, 2005). In summer of 2006, the greenfinch mortality reports increased dramatically with a total of 1054 trichomoniasis incidents recorded. These incidents comprised 50% of all reported incidents of garden bird sickness and death during this period. The sick and dead birds were observed at affected sites in close vicinity to garden bird feeding stations. These birds exhibited non-specific signs of malaise such as lethargy and fluffed-up plumage, frequently in combination with dysphagia (Robinson, 2010).
Columbidae, particularly the domestic pigeon, is known to be T. gallinae’s main host. The parasite is also well known in birds of prey (Stabler 1954), in gallinaceous birds (Pennycott 1998), and in psittacine birds (Baker 1986), but columbiforms are considered to be the main host and reservoir of this parasite (Mehlhorn, 1992). Data on prevalence of the parasite found on columbids varies greatly, from 5.6% cited by Schulz in 2005 to 95% in white-winged doves cited by Conti (1981) in the US. In Spain, as reported by Hfle in 2004, an outbreak of the organism affected 15% of wintering woodpigeons. In Australia, McKeon et al found a prevalence of 49% in domestic pigeons compared to 46% in wild birds. The parasite is reported throughout the year, but outbreaks are mostly seen in spring, summer, and autumn (Cole, 1999; Gerhold, 2007). The variation in age of the birds, the species, and the season explains the difference in prevalence between the studies. Virulent strains of the organism have had large economic impacts through the loss of avian livestock and have posed problems for the wild avian species too (Holmes, 2005).
Duff et al described outbreaks of trichomoniasis in the Wood Pigeon’s in UK (Duff, 2003). The birds had severe lesions and the mortality rate was high. In most studies on Rock Pigeons, juvenile birds were most frequently the sufferers as compared to adult birds (Munoz, 1995; Ostrand, 1995). However, a study by Villuana in Spain showed higher prevalence of the parasite in adult birds, which may be due to game bird feeders and water points in the south of the country. The infected juveniles in this study had a larger bursa of Fabricius, which is an indicator of activation of immune defenses against a current infection (Villuana, 2006). Raberg and Gyfle, in separate studies showed that stress due to strenuous exercise and social stress due to crowding during migration have been the reasons for increased susceptibility of birds to infestation as well as to reactivation of latent disease (Raberg, 1998; Gyfle, 2000).
Some researchers have also found a gender related difference in susceptibility to disease with males being generally more susceptible to the disease (Alexander, 1988; Møller 1998; Blanco 2001).
Disease transmission and pathogenesis
The infection causes a condition called as necrotic ingluvitis. This disease was first recognized as an emerging infectious disease of British finches in 2005, as reported by Pennycott. It caused epidemic mortality; and there was a significant decline of greenfinch and chaffinch populations in the subsequent years (Robinson, 2010). At that time, why there was an infection was not fully understood, but most likely it was due to genetic or environmental factors (Lawson, 2011). A frequent contact between birds feeding at garden feeding stations (Kirkwood, 1998) and opportunities for novel inter-specific contacts at shared feeding sources suggest that recently increased provisioning of wild birds might have an increased opportunity for the parasite to spill over to naïve host species (Jones, 2008). In addition to this, there could have positively been a genetic change within the endemic strain of T. gallinae, thus expanding the range of susceptible host species and leading to an epidemic in the finches (Lawson, 2011). By 2007, breeding populations of greenfinches and chaffinches in the geographic region of highest disease incidence had decreased by 35% and 21% respectively, representing death of almost more than a half million birds (Robinson, 2010).
The disease, necrotic ingluvitis, typically extends to full thickness of esophageal wall and often involves adjacent connective tissue. These findings are revealed on a post mortem examination. The post mortem examination also reveals concurrent soiling of the beak and facial plumage with saliva and food, and the birds are typically thin or shrunken (Robinson, 2010).
In general, transmission of the parasite occurs when the adult birds feed their younger ones, but it may also occur through food in feeders and water (Kocan, 1969). The severity of the lesion and the subclinical manifestation depends on the immune status of the host bird and the pathogenecity of the strain (Cooper, 1988; Mesa, 1961). Some pathogenic strains, occasionally, can cause lesions in the visceral organs, leading to death.
According to Cooper (1988) and further by Boal (1998), in birds of prey, the development of the disease by nestlings was directly related to the consumption of pigeons (Cooper & Petty 1988, Boal, 1998). Columbiforms are increasingly consumed by large accipiterine raptors in the Iberian Peninsula (Del Hoyo, 1994), since typical prey species such as the Rabbit Oryctolagus cuniculus and Red-legged Partridge Alectoris rufa have been reduced noticeably by disease, changes in land-use and hunting pressure (Villafuerte, 1994; Gortazar, 2002). This puts nestlings of highly endangered species such as the Spanish Imperial Eagle Aquila adalberti or the Bonelli’s Eagle Hieraaetus fasciatus at risk from trichomoniasis (Höfle, 2000). The parasite cannot survive outside the body of the host for a longer time and is vulnerable to desiccation (GBHi, n.d.).
Molecular epidemiology
Not many molecular studies have been performed to investigate the degree of genetic diversity and cross-transmissibility between different isolates of T. gallinae.
One recent study by Grabensteiner in Austria, isolated 63 clonal cultures of the parasite obtained from 17 birds belonging to five different avian species. The data showed that the amount of different strains causing the disease is actually more complex. The complexity was complicated by identification of genetically different strains with some potentially different Trichomonas species, coexisting within one individual bird (Grabensteiner, 2010).
In another recent study, Anderson et al. (2009) investigated the molecular epidemiology of T. gallinae infecting northern Californian passerine species using ribosomal DNA sequence. They found no variation with sympatric free-ranging columbiform species or raptors. The researchers highlighted communal feeding and water sources, such as bird feeders and bird baths in suburban habitats, as a potential route for the inter-species spread of the parasite and suggested that localized spill-over from infected columbiforms to house finches is most likely to have occurred at these sites (Anderson, 2009).
One study was conducted in the oceanic island of Mauritius on one of the world’s rarest pigeons, the Pink Pigeon (Columba mayeri) (Da Silva, 2007). It is believed that this pigeon suffers high levels of nestling/ fledging mortality from trichomoniasis. The population of pink pigeons has fluctuated between 300 to 370 birds since the year 2000 and a major mortality factor among these birds was known to be trichomoniasis (Swinnerton, 2005). In this study, isolates were collected from pink pigeons along with another widespread species, the Madagascar turtle-dove (Streptopelia picturata). The study used molecular techniques to confirm the parasites and then investigated the genetic variability using Random Amplified Polymorphic DNA (RAPD) analysis. The Mauritian isolates were found to be similar to T. gallinae of the Brazilian G7 strain sequence (Kleina, 2004), and differed at three bases from the other T. gallinae isolate available from the Rivolta 1878 strain. There was no sequence variation between isolates when comparison of the 5.8S region of rDNA was done and surrounding internally transcribed spacer regions (ITS) showed no sequence variation between isolates or with an unrelated but previously sequenced T. gallinae isolate (Genbank). However, the RAPD analysis of the isolates revealed considerable genotypic variation between isolates, which appeared to correlate with geographic distribution and host species, suggesting inter-species transmission and rapid host adaptation by the parasite. This study was the first evaluation of T. gallinae genotypic heterogeneity; and it found no evidence of multiple subspecies of the organism in Mauritian columbids (Da Silva, 2007).
A very recent study by Lawson looked for evidence of genetic heterogeneity in the parasite causing necrotic ingluvitis in British passerines, columbids, and raptors (Lawson, 2011). It analyzed DNA extracted from the infected lesions or T. gallinae cultures isolated from 17 British bird species (comprising seven Fringillidae (finches), three Accipitridae (hawks), one Turdidae (blackbird), two Columbidae (pigeons), one Prunellidae (dunnock), one Paridae (tit), one Passeridae (sparrow), one Strigidae (owl)). To carry out the study, the researchers employed improved platform-based multilocus typing tools as well as the hydrogenosomal Fe-hydrogenase gene as a single marker locus for fine-typing. Sequence data from columbiform T. gallinae isolates collected before, and following, the emergence of finch trichomoniasis were identical to those for T. gallinae from the other British birds. The reference strain T. gallinae differed from the British isolates with one substitution and one deletion, but still grouped with the British isolates with a high bootstrap value. A variety of complementary molecular methods were used to investigate the genetic heterogeneity of T. gallinae in British avifauna, analysis of which showed no evidence for multiple strains being present within the newly infected population, indicating the emergence of a clonal strain as the etiologic agent of the epidemic (Lawson, 2011). This is in contrast to some other studies in some other parts of the world e.g., USA (Gerhold, 2008), Spain (Sansano- Maestre, 2009), Austria (Grabensteiner, 2010) or Mauritius (Da Silva, 2007). The study found no change in the sequence data over time (2005–2009), by geographical region or by species amongst the British isolates. T. gallinae isolates from finches collected from sites with high levels of mortality (>20 dead birds) had the same sequence data as all other isolates, and there was no evidence for genotypic strain variation that might relate to virulence in British passerines (Lawson, 2011). Although the identification and extent of genetic diversity within the source T. gallinae population is unknown, the clonal nature of the passerine epidemic strain suggests that it has arisen recently from a bottleneck (Sprat and Maiden, 1999), such as would be represented by a single spillover event.
Feeding garden birds is a popular pastime among the British community and this just provides an opportunity for species mixing and unusually high or prolonged congregations of birds at feeding stations. In the US, only 43% of households regularly feed birds (Martinson, 2003), but it is close to 75% in the UK (Cowie, 1988). There is an upward trend in the use of gardens by columbids due to intensification of arable cultivation, which is responsible for promoting increased overwinter survival and spillover from farmland to garden habitats (Gibbons, 1993). This may have led to increased mixing of Fringillidae and Columbidae species in garden habitats, providing increased opportunities for cross-species parasite spread. Besides, the popular activity of feeding birds in the UK can affect every aspect of bird ecology, ranging from daily survival to large-scale migration. Generally, feeding generates positive effects, but there may be negative impact such as predation pressure and transmission of the disease (Robb, 2008).
There is still limited data on T. gallinae sequences in the UK and therefore additional research is essential to improve understanding of molecular epidemiology of the pathogen.
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