THE POTENTIAL ROLE OF ORAL PH IN THE PERSISTENCE OF TRICHOMONAS GALLINAE IN COOPER'S HAWKS (ACCIPITER COOPERII)

Trichomoniasis, caused by the protozoan Trichomonas gallinae, affects a variety of species worldwide including avivorious raptors (e.g., Cooper and Petty 1988). Birds of prey usually contract trichomoniasis when they consume members of the family Columbidae, the primary hosts for T. gallinae (Stabler 1947). Clinical signs of trichomoniasis typically occur in the upper digestive tract and range from excess salivation to severe oral and esophageal lesions that can prevent the ability to eat and drink, leading to death (Samour and Naldo 2003). The effects and prevalence of trichomoniasis in free-living raptor populations have not been widely studied, but existing information suggests that the disease has a higher prevalence in young birds (e.g., Boal et al. 1998; Rosenfield et al. 2002). For example, high levels of infection or mortality from trichomoniasis have been observed in nestlings in multiple populations of Northern Goshawks (Accipiter gentilis; e.g., Wieliczko et al. 2003; Krone et al. 2005) and Bonelli's Eagles (Hieraaetus fasciatus; e.g., Real et al. 2000), although prevalence of the disease in other age groups in these populations was not assessed.

Differential susceptibility to trichomoniasis among individuals in different age groups was documented in Cooper's Hawks (Accipiter cooperii) nesting in Tucson, Arizona (Boal et al. 1998). In that population, 85% of nestling hawks had T. gallinae in their oral cavity, whereas only 1% of breeding-age hawks were infected, even though both age groups were believed to be similarly exposed (Boal et al. 1998). A similar pattern of infection among age groups was observed in two populations of Cooper's Hawks in the north-central US, although prevalence of infection was much lower (5.2% and 0% of nestlings and breeding age hawks infected, respectively; Rosenfield et al. 2009).

There are at least two potential explanations for the difference in susceptibility to trichomoniasis between nestling and breeding age Cooper's Hawks. First, hawks exposed to T. gallinae as nestlings could develop immunity to the disease, if they survive the initial infection. Rock Pigeons (Columba livia), in some instances, can become asymptomatic carriers of T. gallinae (Stabler 1954), but the “nature of this immunity is not understood” (Stabler 1954, p. 387). For example, pigeons that lose their long-term latent infection of T. gallinae can reacquire the trichomonads upon reexposure, suggesting that immunologic resistance is not permanent, and may be part of a host-parasite dynamic (Read 1970; for T. vaginalis, Singh et al. 2009). Resistance to T. gallinae has not been demonstrated in bird species outside the family Columbidae (Kocan and Amend 1972) and it is unknown if raptors can become asymptomatic carriers (Kietzmann 1988).

A second potential explanation for differential susceptibility to trichomoniasis among Cooper's Hawks is that nestlings and breeding hawks differ in some physiologic trait that affects survival of T. gallinae in the oral cavity, where clinical trichomoniasis is most readily exhibited in raptors (Samour et al. 1995). Trichomonads are generally sensitive to environmental pH (T. gallinae: e.g., Cailleau 1935; T. gallinae and T. vaginalis: Read 1957; T. vaginalis: Garber and Bowie 1990; Tritrichomonas foetus: Daniel 1948), and T. gallinae thrives when pH is between 6.5 and 7.5 (optimum 7.2; e.g., Read 1957), but dies rapidly when pH is 4.5 (Cailleau 1935). Boal et al. (1998) speculated that a lower pH in the crops of breeding hawks, compared to that of nestlings, could hinder the persistence of T. gallinae, but no information exists on the pH of the digestive systems of raptors.

Our overall goal was to evaluate whether oral pH (hereafter pH) could play a role in the persistence of T. gallinae in Cooper's Hawks. Specific objectives were to 1) ascertain whether pH of Cooper's Hawks differs among age groups and 2) clinically evaluate the prevalence of T. gallinae in Cooper's Hawks in Tucson, Arizona.

We conducted the study in metropolitan Tucson, Arizona, from February to August 2010 and February to July 2011. We measured the pH of all sampled Cooper's Hawks and assessed the presence of T. gallinae in a subsample of males and females from the three age groups: nestlings (14–28 days old [d.o.]), fledglings (55–120 d.o.), and members of breeding pairs (>1 yr old). We sampled fledglings in addition to nestlings and breeding hawks because mortality from trichomoniasis is reduced when hawks approach independence (50+ d.o.; Boal and Mannan 1999). We used a bal-chatri trap to capture fledglings, and either a bal-chatri or a dho-gaza trap (Bloom et al. 2007) to capture breeders. Nestlings were captured by hand in their nests. We distinguished male and female hawks by the diameter of the tarsometatarsus (Hill 1944), and marked each hawk with a U.S. Geological Survey aluminum leg band for individual identification. All hawks sampled were from nests identified in a long-term study of population dynamics of urban-nesting Cooper's Hawks (Mannan et al. 2008). All field methods followed protocols approved by the University of Arizona Institutional Animal Use and Care Committee (Protocol 10-153).

We measured the pH of each hawk by holding a microelectrode (Cole-Parmer combination BNC micro-electrode, Cole-Parmer, Vernon Hills, Illinois, USA) under the ventral plane of the tongue until the reading on a digital field meter (Oakton pHTester 10 BNC, Oakton Instruments, Vernon Hills, Illinois, USA) stabilized (approximately 30 sec). The microelectrode was stored in 4.0 pH buffer (The Lab Depot, Inc., Dawsonville, Georgia, USA) and rinsed with distilled water between samples. We calibrated the microelectrode to three points (pH  =  4.0, 7.0, and 10.0) at least once a day prior to sampling, and tested it in the buffer solutions periodically while sampling to ensure accuracy.

We clinically tested for T. gallinae (Cover et al. 1994) on a subsample of hawks of each sex from each sample age group. We tested the first 20 fledglings and breeders of each sex that were trapped. For nestlings, we selected at random a male and female from each nest to test. If all nestlings in a given nest were of a same sex, we tested two individuals selected at random. For all tested hawks, we swabbed the oral cavity and crop with a dry, cotton-tipped applicator, and immediately placed the swab in a vial containing 6 mL of modified Diamond's medium (Hardy Diagnostics, Santa Maria, California, USA). Swabs were introduced to the vial, swirled for 5–10 sec, and the tip of the applicator was expressed on the side of the vial before removal. We stored the vials at 37 C (Grabensteiner et al. 2010).

After the vials were incubated for 24 hr, we checked for T. gallinae by aseptically removing 30 µL of the modified Diamond's medium and examining it under a microscope at 100× to 400× power (Cover et al. 1994). We identified T. gallinae by morphologic characteristics (Stabler 1947). If we did not find T. gallinae on the first examination, we continued to incubate the vial and examined additional 30-µL samples at 48 hr and 72 hr (e.g., Boal et al. 1998). We considered the sample negative for T. gallinae if we observed no trichomonads by 72 hr (Boal et al. 1998).

We used analysis of variance to explore the relationship between pH and age groups by modeling the main effects of year, age, sex, and an interaction between age and sex. All residuals were assessed for normality and outliers; no transformations or exclusions were made. We then used linear contrast within the model to assess any differences between the means of the three age groups. For this analysis, we excluded data for nestlings that had been recaptured and sampled as fledglings, as the samples were not independent measures. We used a paired t-test to compare the change in pH in the subsample of hawks for which we had repeated measures. All statistical tests were conducted in JMP 9.0.2. (SAS Institute Inc., Cary, North Carolina, USA).

We sampled 375 Cooper's Hawks over the 2010 and 2011 breeding seasons. In 2010, we captured 105 nestlings (64 males, 41 females), 32 fledglings (17 males, 15 females), and 66 breeders (39 males, 27 females). In 2011, we captured 78 nestlings (41 males, 37 females), 40 fledglings (19 males, 21 females), and 54 breeders (33 males, 21 females). We recaptured 15 nestlings after fledging in 2010 and another 20 in 2011. None of the breeders from 2011 were previously captured in 2010.

The pH of Cooper's Hawks in our sample (n = 375) was influenced by year (F = 98.77, df = 1,1; P<0.001) and the interaction of age and sex (F = 5.01, df = 2,2; P = 0.007). The influence of age group (F = 104.20, df = 2,2; P<0.0001) was much larger than that of sex (F = 1.35, df = 1,1; P = 0.245). After accounting for the interaction between sex and age group, the average pH in hawks in 2010 was 0.54 units less acidic than in 2011 (6.70±0.036 [SE] and 6.16±0.037, respectively). After accounting for the effect of year and the crossed effect of sex, the average pH of breeders (n = 120, pH = 6.12±0.059) did not differ from that of fledglings (n = 72, pH = 6.05±0.066; F = 0.0027, df = 1,368; P = 0.9588). The average pH of nestlings (n = 183, pH = 6.83±0.033), after accounting for sex and year, was 0.73 units, or 7.3 times, less acidic than that of fledglings and breeders combined (F = 202.78, df = 1,368; P <0.0001; Fig. 1). We recaptured 35 hawks as fledglings that had been sampled as nestlings, and in this group the pH of nestlings was also, on average, 0.73 units, or 7.3 times less acidic after they had fledged (t = 8.02, P <0.0001).

In 2010 and 2011, 15.7% and 16.3% of nestlings, respectively, were positive for T. gallinae (51 nestlings were tested in 2010 and 49 in 2011). None of the breeders (n = 40 in 2010, n = 40 in 2011) or fledglings (n = 32 in 2010, n = 40 in 2011) were positive for T. gallinae in either year. The pH of nestlings that were positive for T. gallinae did not differ from the average of all sampled nestlings (F = 0.03, df = 1,182; P = 0.95).

Fluid in the oral cavity in fledgling and breeding Cooper's Hawks was over seven times more acidic than that in nestlings. We speculate that the change in pH between nestlings and fledglings is a product, or byproduct, of one of many physiologic changes that take place during early development of birds (Sturkie 1965). Although oral pH has not been studied in raptors, the pH of the digestive system in other bird species (e.g., gizzards of domestic chickens [Gallus domesticus]) can change as birds mature until about 13 wk of age (Vonk et al. 1946). However, little is known about the underlying mechanism that would cause a change of pH during maturation. It seems unlikely that the pH change we observed in Cooper's Hawks as they matured was the result of exposure to T. gallinae, as less than 17% of nestlings had T. gallinae in their oral cavity in this study. We do not know what caused the difference in pH of hawks between 2010 and 2011, but in both years pH of nestlings was consistently less acidic than that of both fledglings and breeders. Additionally, we cannot explain the crossed effect of age and sex on pH, but after accounting for the effect of sex, the effect of age on pH remained statistically significant. It also seems unlikely that a change in diet caused the difference in pH between nestlings and fledglings and adults. Nestlings and fledglings are fed by the adults (primarily Mourning, White-winged, and Inca doves [Zenaida macroura, Zenaida asiatica, Columbina inca]; Estes and Mannan 2003) so hawks in all three groups consume the same prey throughout the period of interest.

Trichomonas gallinae thrives when pH is near 7.2, but declines in both growth potential and viability in more acidic conditions (Read 1957). We speculate that the relatively acidic oral conditions found in fledglings and breeders may be one factor that discourages growth and invasion of T. gallinae in the oral cavity of hawks in these age groups. Although T. gallinae is capable of attaching to cells of the host within 90 min (in vitro testing on erythrocytes of chickens; De Carli and Tasca 2002), it requires 19–24 hr to start the process of invasion of the epithelial lining of the palatal-esophageal junction of the oral cavity, and 48–72 hr to cause substantial pathologic desquamation of the surface of the oral cavity (Kietzmann 1993). It is possible that the environmental conditions in the oral cavity of fledglings and breeders could suppress proliferation and invasion by the trichomonads. For example, pH may have an effect on the hemolytic activity of T. gallinae on erythrocytes (De Carli and Tasca 2002), as has been observed in T. vaginalis (e.g., Garber et al. 1989). When pH is at an optimal level for T. vaginalis (5.5–6.5; Read 1957), the trichomonads secrete a substance that facilitates invasion of host epithelial cells (Fiori et al. 1996). When pH is above or below the optimum, the ability of T. vaginalis to secrete this substance declines rapidly (Fiori et al. 1996). If, as De Carli and Tasca (2002) suggest, T. gallinae has a mechanism for invasion similar to that of T. vaginalis, T. gallinae may secrete hemolytic factors when at nearly optimal pH, which is within the range of pH measured in the oral cavity of nestling Cooper's Hawks, but be less able to secrete hemolytic factors in more acidic conditions.

There are likely multiple factors that influence whether an individual hawk acquires trichomoniasis. Level of virulence of the strain of T. gallinae is known to cause variation in the presentation of clinical disease (Stabler 1948). There also remains the potential for an immunologic response to T. gallinae in raptors, but this possibility has not been investigated. Additionally, although trichomoniasis occurs far more frequently in nestlings, other age groups of raptors also sometimes develop clinical trichomoniasis. In cases of infected adults, and where a definitive diagnosis was made, there is some evidence suggesting that these individuals were more susceptible to infection due to physical stress (e.g., from injury, migration, or captivity) or due to a weakened immune system (e.g., Samour et al. 1995; Willette et al. 2009). Finally, we observed a range of pH values within each age group. In breeders, for example, approximately 34% (±0.05) of individuals had a pH within the optimal growth range of T. gallinae (pH 6.5–7.5). This may be a partial explanation as to why some older birds are susceptible to trichomoniasis, although it is not a commonly observed occurrence.

The prevalence of infection of T. gallinae we observed in nestlings differed from that reported by Boal et al. (1998). Prevalence of T. gallinae is variable over time, perhaps even cyclic as it is in columbids (e.g., Kietzmann 1988), so it is reasonable to hypothesize that the pattern of infection in avivorious raptors may also cycle.

The difference in pH between age groups in Cooper's Hawks is a new discovery meriting further study, in particular as to how it relates to the susceptibility of raptors to T. gallinae infection.