Mycobacteriosis in naturally infected ring-neck doves (Streptopelia ...

24 downloads 354 Views 5MB Size Report
ISSN 0307-9457 (print) ISSN 1465-3338 (online) 08 40443-08 2008 Houghton Trust lid .... College of Veterinary Medicine and Biomedical Sciences, Texas A&M.
Taylor &Franck

v/an PaiIialagv (August 2008) 37(4), 44 4

Taylor & Frafl5 Group

Mycobacteriosis in naturally infected ring-neck doves (Streptopelia risoria): investigation of the association between feather colour and susceptibility to infection, disease and lesions type Miguel D. Saggese l2 * , Ian Tizard' and David N. Phalen"3 ' The Schithot Exotic Bird Health Center, Department of Veierinari' Pathohiologt', College oJ Veierinw-v Medicine and Biomedical .Sciences, Texas A & M Unirersirv, 4467 TA MU College Station, TX 77843-4467, USA. 'College of Veiei'inarr Medicine, Western Unirersiiv of Health Sciences, Pomona. CA 91766. USA, and 3 The Wildlife Health (111(1 Conservation Centre, Unitersiti' of' Sidne y, Camden, Neic South Wales 4475, Australia

Prevalence of infection and disease. the degree of organ involvement and the nature of the lesions were investigated in 11 white and 18 non-white ring-neck doves coming from a flock naturally infected with lvi vcoha cteriuin ui/mn subsp. ac/mn. Lesions were common in the liver, spleen, lung. kidney, intestines, ovary and bone marrow. Overall, 18 out of 29 (62) birds were considered infected with a sequevar of M. arjuni subsp. ar/u/n that contains serotypes 2. 3, 4 and 9. The prevalence of infection in the white doves (36.4 1V) was (77,77). White doves had on average fewer organs affected significantly lower than in the non-white morphs (mean = 3.1) than the non-white doves (mean = 5.9). A diffuse pattern of inflammation in the liver and spleen was observed mainly in non-white doves. Focal or mu]iift)cal granulomatous inflammation of the liver and spleen was predominant in white doves. Genetic mechanisms of immunity to mycobacteriosis may be contributing or determining these differences. There are three basic colour morphs in ring-neck doves—dark or wild type, blond and white—and the alleles coding for colour are sex-linked and located on the sex (Z) chromosome. Female's single sexual chromosomed (ZW) and homozygous males (ZZ) can be white if they carry the white alleles. It is very probable that the gene or genes modulating the immune response to M. ar/unl subsp. ai'/wn infection in these doves could be associated to these loci or at least located in the same (Z) chromosome, as the association with white colour suggests.

Introduction

Mycobacteriosis is a relatively common cause of disease in pet, avicultural. zoo and free-ranging birds (Montali c/cd.. 1976; Tell ci cii.. 2001: Pollock, 2006: Converse, 2007). Most cases of mycobacteriosis are caused by Mi'c'ohaeieriuin aria/n sLibsp, cniiini and Mi'cohacterium genai'c'nse, although other species of mycobacteria such as Mvcobacteriuni iuhercu/osis. Mycobacterium br/ui-

tam, Mi'cohaeternan gorcioncie. Mi'cobaelerniiii iio,,cliwmogenicu/n. Mtc'obacteriuni si/iliac and Micohcicteriwn cciatuni may also infect birds (Hoop ci al., 1996; Tell ci al., 2001: Bertelsen ci al., 2006: Steinmetz ci cii.. 2006: Travis ci cii.. 2007). The pathogenesis of natural

avian mycobacteriosis has been minimally investigated (Hejlicek & Treml. 1995: Cromie ci cii.. 2000: Tell etc/i.. 2001, 2003) and many aspects of the disease remain unclear. Although reports of mycobacteriosis exist for most orders of birds, susceptibility, prevalence of infection and diseases, the degree of organ involvement and the nature of the lesions vary widely (Montali ci al.. 1976: Hejlicek & Treml. 1995; Cromie ci cli.. 2000: Friend,

2001; Tell ci al., 2001: Schmidt ci al., 2003; Pollock, 2006; Converse, 2007). Environmental factors have been

proposed to explain different susceptibility (Montali ci

al.. 1976: Tell ci al., 2001; Pollock, 2006). Malnutrition.

overcrowding, concurrent diseases, and poor hygiene alone or combined are potential stressors that may predispose birds to mycobacteriosis. 1-lowever, environmental factors do not explain completely the different susceptibility of birds to mycohacteriosis. Not all birds housed in the same facilities and under the same management Practices become infected and develop disease (Cromie ci cii.. 1991. 1992: M. D. Saggese, unpublished data) and their response to these different stressors may vary. Variations in the pathology of natural and experimental infection, the degree of organ involvement and the nature of the lesions of mycohacteriosis has been attributed either to characteristics of the agent. to the stage of the disease or to the bird's immune response (Montali ci cii.. 1976: Cromie et al., 2000; Schmidt cicd., 2003). Limited studies suggest that host genetic factors

*To whom correspondence should he addressed. Tel: + 1 909 706 3532. E-mail: [email protected] Received 24 March 2008 ISSN 0307-9457 (print) ISSN 1465-3338 (online) 08 40443-08 2008 Houghton Trust lid DO!: 10,1050 03079450802210655

444 M. D. Sarrgesc c l

play a major role in determining susceptibility to avian mycohacteriosis (Cromic ci al.. 1991. 1992: Eiejlicek & Trernl. 1995). A differential susceptibility to mycohacteriosis, reflected by the number of affected birds and the nature of their lesions, has been demonstrated in two chicken lines challenged with M. at/mn (Gross ci al., 1989). Recently, genetic susceptibility has also been suggested as a predisposing cause of disease and the nature of lesions in captive white winged-ducks and two species of doves (Cromie ci (i!. 1991. 1992: Saggese & Phalen, 2005: Saggese ci al., 2007). Pigeons and doves (Order Columbiformes) are considered particularly susceptible to avian mycobacteriosis (Feldman, 1938; Francis, 1958: Van der Schaf ci al., 1976: Montali ci al., 1976: Pond & Rush. 1981: Bougiouk]is ci al., 2005). Other authors, however. consider some species of pigeon and doves to be highly resistant to experimental and natural infection (H(Jlicck & Treml, 1993, 1995). These contrasting opinions may be explained both by genetic factors as well as by local environmental and other risk factors. In one study, differences in species susceptibility to natural and experimental infection with M. whim were observed in collared doves (Sircpiopelia dccaacto) and turtle doves (Sircptopclia tu;'iur) (Hejlicek & Treml, 1993). The former were found to be highly susceptible while the related turtle doves were resistant (HejlIcek & Trernl, 1993, 1995). Other evidence for a possible genetic component for this different susceptibility comes from a natural outbreak of mycobacteriosis in Texas in 1998. A flock containing diamond doves (Gcopclia cuneala) as well as two different colour morphs (white and wild-type) of ring-neck doves (Strcpiopelia risoria), showed significant differences in organ involvement, distribution and histopathologic lesions and serologic responses (Saggesc & Phalen, 2005). While all three groups of doves presented with microscopic lesions in the liver and the spleen, lesions in the intestines, bone marrow and lungs were found only in wild-type ring-neck doves. Nil ultifocal granulomatous hepatitis was observed in all of the birds, but it granulomatous hepatitis was only observed in the wild-type doves (Saggese & Phalen. 2005). The finding, by polyrnerase chain reaction (PCR) and sequencing of the dnaf gene. of the same M. whim subsp. ai'iwn in these doves and in the shared environment in which these birds were housed suggested that these variations were not due to different organisms or to differences in exposure. Furthermore, the absence of antibodies (as detected by complement fixation) in the white rin g -neck doves compared with seropositivity in wild-type and diamond doves suggested that a different humoral immune response occurred in these birds. This also supports the suggestion that differences in the pathogenesis and immune response were associated with colour. Although the number of birds in this study was small, and several variables were not formally controlled, precluding a statistical analysis, these data together with previous studies (Hejlicek & Treml 1993. 1995) su g gested the possibility that differences in susceptibility and pathology of m y cobacterial infections may occur in ring-neck doves with different coloLir morphs, and points to a possible association between genes coding for feather colour and susceptibility to infection.

Ring-neck doves present in several colour morphs. obtained by natural or artificial selection. The genetics of these colour mutations in these species have been anal y sed (Miller, 2007). Identifying and understanding differences in pathogenesis and susceptibility to nlycobacteriosis in closely related species or strains of birds could assist identification of the genes involved in differential susceptibility and pathogenesis. The occurrence in Texas of a captive Population of' ring-neck doves, including individuals of different colour morphs, suffering from avian mycobacteriosis offered ail to investigate the presentation of this disease in a large number of birds and to look for evidence of differential susceptibility. We hypothesized that lesion type and distribution and susceptibility to infection and disease of naturally infected ring-neck doves would be different in white doves compared with other colour niorphs. Therefore, the main goals of this study were to investigate the prevalence of mycobacterial infection and disease in the different colour morphs found in this flock: to describe and characterize the gross and microscopic lesions in affected birds; to identify the species and sequevar of' mycobacteria involved: and to determine whether an association exists between susceptibility to this infection and disease, lesion characteristics and feather colour morphs of ring-neck doves. Materials and Methods Specimens. Sevent y adult ring-neck doves were obtained from an aviary neat - l-lillshor-o, TexaS. USA in July 2005. These birds came from a flock where more than 60 doves had died during the previous 6 months. M y cobacterral infection had been previousl y confirmed in these birds at the Texas Veterinar y Medical Diagnostic Laborator y (College Station. Texas. USA) and the condition of SOC of the survivin g birds (decreased productivity, weight loss. depi'ession) was consistent with nivcohacleriosis. These birds were all housed in it sited that was open to and surrounded on two sides b y an outdoor flight. There was heavy faecal contamination ion ot the floor Lind perching su faces of the shed. Food and writer were contaminated with faeces. The surviving birds were donated and transferred to an isolation building at the College of Veterinary Medicine and Biomedical Sciences, Texas A&M University (College Station. Texas. USA)- During the following 2 weeks. 29 birds (It white, eight blond, six pied, one orrtn ge, two wildtype and one white-silky coloured) (Oliver . 2005: Miller, 2007) were randomly selected, anaesthetized by intramuscular injection of ketamine and xylazine. and euthanized by exsanguination through eardiocentesis. Doves acre necropsied and samples collected within 4 It of euthanasia. Tissues were collected for PCR using cleaned. autoclaved instruments that had been treated with bleach and forinalin - A different set of instruments was used to collect tissues from each bud and organ to prevent DNA carryover. Samples of the liver, spleen, lung, bone marrow and of other organs with gross lesions were collected and frozen For PCR rind culture. The liver, spleen, mug. trachea. heart. kidney, oesophagus. crop. proventriculus, ventriculus. intestines. gonads, pancreas and skeletal muscle were examined for lesions consistent with myeohaeteriosis. Specimens from these organs were fornialin-fixed rind pit raffin-einbedded and stained with hriematoxylin and eosin and Ziehl Neelsen. Microscopically. inflammatory lesions were scored scniiqurtntitatively as mild. moderate and severe based on the number of inhlainmritory cells within the lesions and the area of tissue affected. I hist opal hological changes compatible with mycohacteriosis were described and classified as focal/multiihcal gramiulomatous. consisting of svehl-defined foci of inflammatory cells..s()iaetines strrrounded b y a rin g of hbm'ous tissue and with (sometimes without) a variable amount of ccii trrrl caseous necrosis, or classified as diffuse g ranulomat ous inflammnatirrn. with it degree of diffuse in llrimmritory cell infiltration without formation of discrete foci or nodules, absence of a fibrous cripsule and little or no

NI 'cohacleriosts in ring-neck doves 445 caseous necrosis. The numbers ol acid-fast bacilli were subjectively graded as none, few. many or massive. Congo red staining was employed to investigate the presence of amyloid in the liver and spleen. Immediately after the birds were euthanized, additional tissue samples were collected using the biopsy techniques employed in live birds. Bone marrow aspirates, using a 20 G needle and 6 ml syringe, were obtained from the distal ulna. A bone marrow smear was made. heat-fixed and the remaining sample saved at SO C. The celomic cavit y was opened aseptically through a inidline ventral approach and it piece of liver approximately (4 mm a 4 nim x 4 mm) was excised from the caudal border of the right lobe. This is the location and size of tissue that would be collected from a live bird of this size during a routine liver biopsy. A small portion was saved for PCR and culture, and the remaining tissue fornialin-fixed for histological examination. The duodenum was identified and aspirated with a 25 G needle and I nil syringe. A portion of the aspirated material was smeared onto a glass slide. heat-fixed and ZichlNeelsen stained. Bone tiiarrow 'aspirates and "biopsied" liver were stained svidi Ziehl Ncelsen, cultured and tested by PCR. Detection of nivcobacteriii in tissues. Swabs from the investigated tissues from all of the doves were inoculated into S ml M iddlebroock 7119 broth (Becton Dickinson. Franklin Lakes. New Jerse y . USA) containing 0.5' (',/v ) gly cerol and 10/ )v/s') oleic acid albumin, and incubated at 39 C followin g myeobacteritl culture standards (Million ci al.. 2007). Cultures svei'e inspected weekl y for microbial growth and examined for the presence of mvcobacterta by Ziehl Neelsen staining. DNA was extracted from all of the investigated tissues using the Puregene 5 Genomic DNA Purification Kit )Gentra Systems. Minneapolis, Minnesota. LI SA) following the instructions of the

manufacturer. PCR screcnnig for mscobacterial DNA was performed using primers TI )5-GGGTGACGCG(G/A )CATGGC('CA-3') and T2 )5'-CCGGTTTCGTCGTA('TCCTJ'-3') for amplification of the 236-base-pair thma.l gene as described by Morita ci al. (2004). The I'CR reaction pmrmtmeters were as follows: one initial cs

dc

of 94 C for 5m , 40

cy cles at 94 C for 3)) sec. 60 C for 45 sec and 72 C for I mm: and an

additional elon g ation step at 72 C for 5 tam. Positive (.11. (it him suhsp. mmi'iuiii) and negalive reactioti control (DNA-RNA-free sterile water)

gross and microscopic lesions. A positive identification of mcobacteria by culture and/or PCR or by the identificatioti of acid-fast organisms in stained tissues or smears defined intcctioti status )infected or uninfected). Based on these criteria, four subcategories were identified: diseased infected, birds with moderate to severe inflammation in one or more organs and positive for mycohacteria: diseased uttinfectcd. birds with mild lesions compatible with mycobacteriosis and negative for mycohacteria: healthy uninfected. birds without lesions and negative for mycobacterut: and healthy infected, birds without lesions and negative for acid-fast bacilli in tissues but positive PCR and/or culture results. Statistical anal ysis. 'rhe associatiott between white and non-white coloured doves together with health and infection status, the number of organs with lesions and the type of inflammation in the spleen and liver was anal ysed using contingency tables and Fischer's exact test after demonstration of normality using Kolmogom'ov Smirtiov. Shapiro Wilk

s and [)'Agostino Peai'soti tests. Test statistics were con idered signifi)) birds had microscopic lesions consistent with mycobacteriosis. Lesions were common in the liver, spleen. lung. kidney, intestines, ovar y and bone marrow (Figure 2). Microscopic lesions of the liver could he divided into two forms that corresponded to the observed gross lesions. The first was a severe granulomatous inflammation characterized by nodules of variable size. They were composed mainl y of histioeytes, lymphocytes and multinucleated giant cells, sometimes with small numbers of plasmacytes (Figure le). These granulomas rarely contained a central zone of necrosis, except for two birds and, in some cases, a thin fibrous capsule surrounded the largest nodules. These nodules tended to be periportal. but also widely scattered in some birds. Many to massive numbers of acid-fast organisms were observed. They were restricted to the central necrotic core of the granulomas. The second form of microscopic inflamma tory pattern in the liver was characterized by a severe diffuse infiltration of the parenchvnia with histiocytes. scattered multinucleated giant cells and, to a lesser degree, lymphocytes and plasmacytes (Figure Id). Thick layers of amyloid were observed between the hepatocytes

and the space of Disse. No or few acid-fast organisms were associated with this inflammation pattern. Two similar patterns of inflammation were also observed in the spleen. The diffuse forni was characterized by infiltration of this organ with large numbers of histiocytes and multinucleated giant cells, with lymphocyte and erythrocyte depletion. Caseous necrosis was observed in the more extensive areas of inflammation. Many to massive numbers of acid-fast organisms were observed in these lesions. The second pattern consisted of mild focal or multilocal histioeytic nodules, in some cases accompanied by multinucleated giant cells. Central necrosis was not common in these birds. No or few acidfast organisms were observed in this form of inflammation. Focal or multi-focal granulomatous pneumonia was observed in seven non-white doves and two white doves, respectively. Two white doves and two non-white doves had mild multifocal, lymphocytic nephritis but necrosis was not a component of the kidney lesions. A severe, diffuse, air saculitis characterized by large amounts of necrotic debris, exudate and severe histiocytic infiltration with massive numbers of acid-fast bacteria was observed in three non-white birds. Moderate to severe multifocal scrosal and mucosal granulomatous enteritis was observed in 13 birds. Mild multi-focal to severe diffuse hone marrow inflammation was observed in II non-white doves, characterized by replacement of normal bone marrow by hystiocytes and multinucleated giant cells. Very mild lesions in the liver. spleen or lung, without detectable mycobacteria in acid-fast stained tissues,

Mycobacteriosis in ring-neck doves 447 100 90 80 a) a)

70 60 50

C

0)

00

CL

C

=

C))

. g

)

5 °

00 00 . 9 > .0 9 .9 ' 0 QO 00

5

White • Non-white

0=

5 5 > Co

Co 00 0) Ui

0

0) D

E E

0) 00

.0

0

Organ examined

Figure 2. Higher prevalence of organs affected in non-while diseased ring-neck doves (S. rlsoria compared nob diseased it/tile ringneck doves. Lans include air sacs. Averages f) . the nwnber of organs affected /r the dif.fr rent colour morphs were statistically significant. as stated in Table 1.

smears, cultures and PCR, were observed in three white birds and one non-white bird. Results of culture and PCR. Overall, 18 out of 29 (62%) birds were considered infected, based on positive results from cultures and PCR and/or by the detection of acidfast bacilli in one or more tissues or smears (Table 1). An amplicon of cxpccied molecular mass (236 base pairs) was amplified by PCR from the liver or spleen from four white and six non-white doves. These sequences were identical and had 1007 identity with the sequevars of Al. at'iwn subsp. aria/n that contains serotypes 2, 3, 4 and 9 (Morita ci ai., 2004). Health and infection status. Based on the presence of gross and microscopic lesions, 21 of 29 (724%) birds were considered diseased. Seventeen birds were considered severely diseased while the four birds that had microscopic lesions but were negative for mycobacteria were considered mildly diseased. With the exception of one bird, all of the 18 infected birds showed microscopic lesions and were considered diseased. This negative dove was considered infected based on positive culture and PCR of liver and spleen samples, but classified as healthy infected in the absence of significant lesions. Differences between white and non-white coloured doves.

The prevalence of infection in the white doves (36.4%) was significantly lower than in the non-white morphs (77.7%). White doves had on average fewer organs affected than the non-white doves. The pattern of inflammation was significantly different between the two groups. The association between diffuse pattern in the liver and spleen was significantly different between white and non-white doves (Table 1). This diffuse pattern of inflammation was rare ill the white doves. No statistically significant differences in the prevalence of disease between white doves arid non-white doves were observed. Discussion

The present study investigates a natural presentation of mycobacteriosis in ring-neck doves. Important observa-

tions are the variability observed in susceptibility to infection, organ distribution and nature of lesions between white and non-white doves. In the presence of identical environmental conditions, these differences may he explained by genetic differences associated with the phenotypic characteristic colour morph. Previous findings in another group of ring-neck doves (Saggese & Phalen, 2005) as well in other species of doves (ll(jlicek & Trcml, 1993) support it genetic cause for these differences. While the age and time of infection for each individual dove was unknown, all were adults kept under identical food and housing conditions. The presentation of this disease. constant exposure and the chronic nature of the lesions of mycohacteriosis together with the sample size make it unlikely that age or time of infection influenced our results. There are several reports of mycobacteriosis in pigeons and doves (Feldman. 1938: Pond & Rush. 1981: Hejlicek & Treml. 1993, 1995: Morita ci al., 1994: Bougiouklis ci al., 2005: Saggese & Phalen. 2005), but the prevalence of infection and/or disease in captive Populations of ring-neck doves have not been previously reported to the author's knowledge. Overall, the prevalence of mycobacteriosis in captive collections of birds is rarely hi gher than 15% (Montali et al.. 1976: Vail der Heyden, 1997; Tell ci al., 2001). Nevertheless, while the prevalence of infection (62%) and disease (72.4%) was considered very high in this flock, the finding that not all the birds were infected suggests that CXOSUC alone under the conditions observed in this flock (overcrowding, poor hygiene) was apparently not or disease in some birds. sufficient to cause infection or The distribution of gross and microscopic lesions in these birds was similar to that previously reported in other birds with mycohacteriosis. The liver, spleen, lung. bone marrow are common sites of mycobacterial infection in birds (Feldman, 1938; Francis. 1958; Thoen, 1997; Tell ci al., 2001: Fulton & Tlloen, 2003; Converse, 2007). Focal or multi-local granulomatous inflammation is the most common form observed in this disease (Montali ci al., 1976: Fulton & Thoen. 2003: Schmidt ci al., 2003). The diffuse pattern of granulomatous inflammation observed in this study is consistent with the non-tuberculoid form described by Tell ci al. (2001),

44' NI D.

S:igg.c CI i/I.

in which diffuse infiltration of the organ with inflammatory cells occurs. The diffuse enlargement observed in 12 doves with diffuse histiocytic and multinucleated giant cells and severe amyloidosis is consistent with this description. A diffuse form of granulomatous inflammation similar to that observed in the non-white doves was previously observed in wild-type ring-neck doves but not in white birds (Saggese & Phalen, 2005). This pattern of inflammation resembles the lepromatous form of granulomatous inflammation observed in human leprosy (Connor ci til., 1997). The presence of this diffuse form contrast with reports in other species of doves, where the tuberculoid or nodular form of granulomatous inflarmilation has been reported (Feldman, 1938: Pond & Rush. 1981; Morita et of., 1994; Bougiouklis CT al., 2005). Alternatively, it has been stated that tubercles rarely develop in Columbiformes with niyeobacteriosis, although specific details were not provided (Ramis ci al.. 1996). Nevertheless, the presence of both types of inflammation in the doves of our series may explain these conflicting opinions. Twelve birds had amyloidosis of the liver. Massive amounts of amyloid were observed in doves with diffuse inflammation, but very little was observed in the birds with multifocal inflammation. Amyloidosis is a pathological condition characterized by the deposition of insoluble fibrillar proteins in various tissues and organs of the body followin g prolonged inflammation or infection (Cotran ci al.. 1999). Amyloid deposits have been reported previously in birds with chronic inflammatory diseases such as mycobacteriosis and aspergillosis. Several forms of amyloid have been described in mammals, but only amyloid AA (amyloid associate) has been found in birds (Landman ci al.. 1998; Cotran ci al., 1999: Schmidt ci al., 2003). Amyloid AA is a product of the proteolytic cleavage of serum AA (SAA), an acute phase-protein produced by hepatocytes (Landman ci al.. 1998). The concentration of SAA in the blood increases within a several hours of the onset of injury, infection, or inflammation. Production of SAA is directly stimulated by the cytokines interleukin- I, interleukin-6 and tumour necrosis factor-a produced in response to tissue injury and inflammation (Petersen ci al.. 2004). The persistent inflammation caused by chronic mycobacteriosis is a probable cause of the deposition of amyloid in these organs (Saggese ci al., 2007). Amyloid was not a feature in the mildly diseased doves with focal or multifocal granuloniatous inflammation. suggesting that an inflammatory process in these birds was insufficient or of too short duration to trigger amyloidosis, supporting the suggestion of an infection arrested at early stages. There were significantly fewer organs with gross and microscopic lesions in the white doves compared with other birds. Lung and intestines were rarely affected in the white doves compared with the non-white doves. These findings suggest that lesion distribution, as well as the severity of the lesion and the type of the lesion, are influenced by factors linked to feather colour. Four doves had mild lesions consistent with a mycohacterial infection but were negative oil tests for the organisms. It is possible that these birds had low levels of mycobactcria in their tissues and that they were not detected. However, it is more likely that these birds had recently overcome a mycobacterial infection through a mild but adequate cell-mediated immune response. It is probable that the mild lesions observed in these four

birds represented a controlled or at least an arrested infection, similar to that seen in other niycobacterial infections (.Juhh ci al.. 1993; Cotran ci al.. 1999; Dannemberg, 2006). It suggests that at least some birds may recover from natural infection. The fact that three out of these birds were white is consistent with other observations made between different colour morphs. A single dove with confirmed m ycobacterial infection of the liver and spleen did not have lesions. The significance of this is unknown, but could represent the early stage of infection that was prior to the onset of detectable lesions or that a lesion was present but not detected. This case shows that, in some circumstances. culture or PCR may be a necessary adjunct to histopathology to detect all infected birds. The present study represents the first attempt to examine the association between feather colour and susceptibility to infection, disease and pathology. The while doves had it prevalence of infection, fewer infected organs affected and a different pattern of inflammation as compared with the coloured doves. These data are consistent with epideniiological and genetic studies showing immune response polymorphism to mycohacterial infections in humans and other mammails (Bellamy. 2005: Dorman ci al.. 2004; Barthel ci al.. 2000; Pan ci al., 2005: Di Pietrantonio & Schurr, 2005) and in different strains of chickens (Hit ci al.. 1997: Bacon ci al.. 2000). Specific genes that have been associated with differing immune responses to mycobacterial infections include those that code for the major histocompatahility receptors, cytokines. T-cell receptors. immunoglobulins and NRAMPI (Zekarias ci al., 2002). The lack of information available about the role of genetics and susceptibility to tuberculosis in birds contrasts to what is known in humans and other mammals (Bellamy & Hill, 998). Both environmental and genetic factors and their interaction influence susceptibility to tuberculosis in humans (Casanova & Abel. 2002: Schurr. 2007), in laboratory animals such as rabbits and mice, and in domestic mammals (Barthel ci al., 2000: Phillips ci al., 2002; Di Pietrantonio & Schurm', 2005: Dannemherg. 2006). For example, in humans infected with M. tuberculosis, less than I 01X of infections progress to clinical disease (Cole ci al.. 2004). Resistance to clinical disease has been linked with ethnic background and race (Bellamy ci al.. 2000: Lim, 2000; Casanova & Abel, 2002; Van Helden ci a/.. 2006). Genes coding for natural-resistance-associated-macrophageprotein 1 (NRAMP1). vitamin D receptor, interferon-'y receptor, interleukin- I. interleukin- 10 and interleukin12, HLA class 11 molecules, Toll-like receptor 2 and tumour necrosis factor-. are all considered to influence immunity to mycobacterial infection. Their deficiency. functional defect or genetic polymorphism has been associated with altered susceptibility to mycohacteriosis (Taflik. 2001: Casanova & Abel. 2002: Phillips ci al.. 2002; Acevedo-Whitehouse ci al.. 2005: Bellamy, 2005: Okada & Shirakawa. 2005; Hill, 2006: Naik, 2006). The chromosome location of some of these genes has been elucidated (Cervino ci of., 2002; Bellamy, 2005; Baghdadi ci al., 2006). Ring-neck doves have been selected for different phenotypic traits, mainly colour, for more than 2000 years, and more than 45 colour morphs are currently recognized by pigeon fanciers (Oliver. 2005). The genetics that govern the colouration of ring-neck doves have

Mycobacteriosis in ring-neck doves 449 been partially deduced by Miller (2007). There are three basic colour morphs in ring-neck doves: dark or wild type, blond and white. Dark is dominant both over blond and white, and blond is dominant over white. The alleles coding for colour are sex-linked, and are located on the sex (Z) chromosome. Females are single sexual chromosomed (ZW or Z-) and males are double sexual chromosomed (ZZ). Females' with a single white gene and hornozygous males will be white. It is very probable that the gene or genes involved in immunity to Al. ariuni subsp. aria/n infection in these doves could be associated with these loci or at least located in the same (Z) chromosome. Acknowledgements

The present study received generous support from The Schubot Exotic Bird Health Center. the Association of Avian \/etetrinarians and the Smokey Mountain Bird Club. The authors are grateful to Patricia Gray. Darrel Styles, Elizabeth Tomaszewski and Debra Turner (The Schuhot Exotic Bird Health Center. College of Veterinary Medicine, Texas A&M), and to David McMurray. Luc Bcrghman, Christine McFarland. Gerald Bratton, Bob Daihaussen, Lisa Tell, Michael Garner, Allison Bradley. Jan Suchodolsky, Taflik Omran. Mellisa Libal, Karen Russell, Y. Ni, Ken Turner, Rosemary Vollmar, Chantail Mukherjee, Sarah Jones, John Edwards. Bill Wigle. Gabriel Gomez. George Stoica. S. Vanhooser, John Roths and student workers at Texas A&M University for their assistance with different aspects of this study. References Acevcdo-Whitehouse. K., Vicente. J., Gortazar, C Hiifle. LI., Fern.indee-de-Mera. I.G. & Amos. W. (2005). Genetic resistance to bovine tuberculosis in the Iberian wild boar. :>Io/ecular Ecolog y. 14, 3209 3217. Bacon. L.D. H wit. H.1). & Cheng. 11.11. (2000)..review of the development of chicken lines to resolse genes determining resistance to diseases. Po,idrr ,Scic,ii'e. 79. 1082 1093. Baghdadi .J.F.. Orlova. M., Alter. A., Ranque. B.. Chentoufi, M.. I,azrak. F. Archane, Mi., Casanova, iL.. Benslimane, A.. Schurr. E. & Abel. L. (2006). An aulosomal dominant major gene confers predisposition to pulmonary tuberculosis in adults. Journal I E.vperioieiito/ 3iiluin.. 203. 1679 1684. Barthel, R., Picdrahita, J.A.. McMurray. D.N.. Payeur. J.. Baea, 0.. Suarez-Guemet. 1-.. Perurnalla, VS., Ficht. l.A.. Templeton. J.W.& Adams. L.G. (2000). Patholo g ic findings and association of limobacteriuoi bori,s infection with the bovine N RAMP I gene in cattle from herds with naturally occurring tuberculosis. ..lou'ru-ii Journal of I 'ierioar r Research. 61. 1140 1144. Bellamy, R. (2005). Genetic susceptibility to tuberculosis. C ioiic.v in (7ie,sr Sieilieiiie. 26, 233 246. Bellamy. R.. Bcyers. N., McAdam, K.R. Russende. C.. Gie, R., Samaai. P., Bester. D,. Meyer. M.. Corrah, I., Collin, M.. Camidge. DR., Wilkinson. L).. 1-loal-Van 1-lelden, I'... Whittle, I IC.. Amos. w.. Van Holden, P. & Hill. A.V, (20)10). Genetic susceptibility to tuberculosis in Africans: a genome-wide scan. Proceedings National . cadenir of ,Sci,ues. t-'S,1. 97. 8005-8009. Bellam. R.J. & I lill. A.V. (1998). Genetic susceptibility to mycobacteria and other infectious pathogens in humans. Current Opinions in ininiwio/ogi. /0. 483 487. Bertelsen. M.F., GrOndahl. C. & Giese. S.B. (2006). Disseminated .tlrcobacteriuni ce/ioum infection in a white-tailed trogoil ( Trogon ririi/i,$). Anon Paiho/ogi. 35. 116 319.

Bougiouklis, P. Brellou, G., Fragkiadaki. E.. lordanidis. P., Vlemmas. 1. & (ieorgopoulou, I. (2005). Outbreak of avian mycobacteriosis in a flock of two-year-old domestic pigeons (Colwnha Jima f donie,stiiii). ,-lrian Diseases. 49. 442 445. Casanova. J.L. & Abel. L (2002). Genetic dissection of immunit y to mycohacteria: the human model. Annual Rerir'ir oflinniullologia, 20. 581-620. Cervino, AC., Lakiss. S., Sow, 0., Bellamy, R., Beyers, N.. float van Helden, E., van Holden, P., McAdam, K.P. & Hill, A.V. (2002). Fine mapping of a putative tuberculosis-susceptibility locus on chromosome 15q]1-13 in African fansilies. /iunian Molecular Genetii:s, Ii, 1599 1603. Cole S. Eiscnach, K.1)., McMurray. C. N. & Jacobs. W.R. Jr. (Eds.) (2004). Tuhercu/os is and the inhere/c Bacillus. Washington, DC: ASM Press. Converse, K.A. (2007). Avian tuberculosis. In N.J. Thomas. D.B. I lunter & C.T. Atkinson (Eds.), infectious Diseases of Wild Birds (pp. 289 302). Ames. IA: Blackwell Publishing. Connor. DI-!.. Chandler, F.W.. Schwartz. D.A.. Mane. H.J. & Lack, F. (Eds.) 119971. Pat/io/ogr of /iifeciioirs Diseases. New York: McGrawHill Co. Cotran. R.S.. Kumar. V. & Collins. T. (1999). Bobbins Pathologic Basis of Diseases. 6111 cdii (pp. 251 259). Philadelphia. PA: WB Saunders Co. Cromie. R.L.. Brown, Mi.. Price. D.J. & Stanford, J.L. (1991). Susceptibility of captive wildfowl to asian tuberculosis: the importance of genetic and environmental factors. Tuhencle. 72. 105 109. Cromic, R.L., Brown. M.J. & Stanford, JL. (1992). The epidemiology of asian tuberculosis in white-w iisged wood duck C airuia nato/ala at The Wildfowl and Wetland Trust. Slimbridge ('mitre (1976--1991). I-I i/d/ow/. 43. 211 214. Cromie, R. L.. Ash, N.J., Brown, Mi. & Stanford. J. 1.. (2000). Avian immune response to lfj-eobcicreriuni oriuui: the wildfowl example. Deii'/opineniol and C'oniparotice bnniunologi . 24. 169-185. Dan nenibcrg. A.M. Jr. (2()()6(. Pathogeni'.ris 0/ Haitian Tuberculosis. in.sight.s from the Rabbit Model. Washington. DC: ASM Press. Di Pietrantonio. T. & Schurr. E. (2005). Mouse models for the genetic studs of tuberculosis susceptibility. Briefoig. s in Fiini-iiooa/ (,eiiooius and Proteo,nis. 4, 277 292. Dornian, S.F., Hatem, CL., Tyagi, S.. Aird. K.. Lopez-Molina. J,. Pitt. ML.. look. B.C.. Dannenherg, AM.. Bishai. W.R. & Manahe. Y.C. y (2004). Susceptibilit to tuberculosis: clues from studies with inbred and outhred New Zealand White rabbits. Infecti on and Ioiniunitr. 72, 17)))) 170S. Feldman. 'A'. H. (1938). Arian Tuberculosis infec tions. Baltimore, MD: Williams and Wilkins. Francis J. )1858). Tuberculosis in ,411oials and Ma,i. A Stud y on ('aui/ia/atii'i' Pioliologr. London: Cassell Press. Friend, NI. (2(1(11). Asian tuberculosis. In M. Friend & J.C. I- ranson (Eds.), Field .t/wii,al of i-I ildilfi' Diseases (pp. 93 98). Madison. WI: US Geological Survey. Biological Resources Division. National Wildlife Health Ccc tci. Fulton. R.M. & Thoen. ('.0. (2003). Tuberculosis. In R.W. Calnek. II.H. Baines. C.W. Beard, L.R. McDougald. Y.M. Sail (Eds(. Diseases of Pou/tnr (pp. 1136 844). Ames: Iowa State Press. Gross. WB.. lalkinlsam, J.D. & Pa yeui'. J.B. (1989). Effect of environmental genetic interactions on Mrcobacteriwn aniwn challenge infection. .4vian Diseases. 33, 411-415. Hejlicck. K. & Treml. F. (1993). Epizootiology and pathogenesis of avian mycobacteriosis in doves (Streptopelia sp.). 14'terniart' Medicine (Praha). 38. 119-328. Hejlleek. K. & Treml. F. (1995). Comparison of the pathogenesis and epizootiologic importance of avian mycohacteriosis in various types of domestic and free-living syntropic birds. Vi'terinarr Medicine

(Praha), 40. 187-194. Hill. A.V.S, (2006). Aspect of genetic susceptibility to human infectious diseases. Annual Rei'icir.s in Genetics, 40. 469-486. Hoop. R.K.. Bottger. E.C. & Pfyffer. G.E. (1996). Etiological agents of mycohacteriosis in pet birds between 1986 and 1995. Journal of Clinical Microbiology../4. 991 992. Hu. J.. Bumstead. N.. Barrow, P., Sehastiani, G.. Olien. L.. Morgan. K. & Malo, D. (1997). Resistance to salmonellosis in the chicken is linked to NRAMPI and TNC. Geoonie Research, 7. 693- 704.

45)) M. D. Saggese ci al. Jubh. K.V.F.. Kenned y . P.C. & Palmer. N. (1993). Pathologr of Doniestic Aniiiials, Vol. 1. 4th edn. New York: Wil Saunders Co. Landman, W.J.M.. Gruys. F. & Gielkens. A.L.J. (1998). Avian amyloidosis. Aiim' Patholog y, 27, 437-449. Lim. T.K. (2000). Human genetic susceptibility to tuberculosis. Annals .4cadenii of Mcilicii,e of Si,i'apore, 29, 298 304. Mahon, CR., Maiiciselis. G. & Lehman, D.C. (2007). Textbook of Diaipto.ciic it(icrobiologi New York- U. WB Saunders Co.

Saggese. NI. I). & Phalen. D.N. (2005). Serological and histological findings in doses with mycobacteriosis. In I'roc-c'ecicu,g.s of clii' 26" Conference ( pp. 71 73). Monterey. A.s.socicclioci of .4c'ian 14'ter/naricimc,v

Miller. W.J. (2007). Genotypes and description of ritigneck plumage colors and eye cover. Available online at http:/Iwww.ringncckdove. com Accessed November 25 2007. Montali. Ri.. Bush, M., Thoen. CO. & Smith. E. (1976). Tuberculosis in captive exotic birds. Journal of the ,' lmerican Veterinarian Medical Association. 169, 920 927. Morita, Y., Arai, M., Nomura. 0., Maruyama. S. & Katsube. Y. (1994). Avian tuberculosis which occurred in air pigeon and pathogenicity of the isolate,. Journal of Veterutarr Medical Sciences,

422. Schmidt. RE.. Recti-ill. D.R. & Phalen ., D. (2003). Pctticoloc,'v of i',! and 4,/arc Birds. Ames, IA: Blackwell Publishing. Schuri'. F. (2007). Is susceptibility to tuberculosis acquired or inherited? Journal ol lucternccl ,t.Ic'di,'ini'. 261. 106 111. Steinmetz, H.W,, Rutz. C., Hoop. R.K., Grest. P. Blev. C.R. & Halt. J. NI. (2006). Possible Ii uman-avian transmission of ,ti,'cohoeterimini tmcherculo,sis in a green-winged macaw (Ara chcloroptc'ra). ,'Inion

56. 585 587. Morita, Y., Maruyama, S.. Kabeya. H.. Nagai, A.. Kozawa. K.. Kato. M.. Nakajima. T.. Mikami. T., Katsuhe, Y. & Kimura. H. (2004). Genetic diversity of the dnaJ gene in the All'c-ohaeteriusn anon, complex. Journal of Medical Microbiology. 53. 813- -817. Carl look forward to genetic tests for tuberculosis Naik. S. (2006). susceptibility and severity? Indian Journal of .t.Ic'diccd Research, 124,

Tell, L.. Woods. L. & Cronuie. R.L. (2001). Mycobactercosis in birds. Revue Sc-is'uit/ficice c-i 'Ii'chnique, 0/fcc-c-' I t , iecscccocsnccl des Epc:ootces. /

379 382. Okada. M. & Shirakawa, T. (2005). Frontier of mycobacterium research-gost vs. mycobacterium. Kekkakcc. 80, 613 629. Oliver. K.\V. (2005). Ringstc'ck Dc,re.c.' A Handbook of Care and Breeding. Newports News. VA: Author's edition. Pan. H.. Yan. B.S., Rojas. M., Shebzukhov. Y.V. Zhou. F-I., Kohzik, L.. Higgins, D.E., Daly, Ml.. Bloom. B.R. & Kramnik. 1. (2005). Iprl gene mediates innate immunity to tuberculosis. ,'Vcctcct'c'. 434. 767-772. Petersen, HI-I.. Nielsen, J.P. & Heegaard. M.H. (2004). Application of acute phase protein measurements in veterinar y clinical chemistry. I i'terinarn Research, 35, 163-187. Phillips. Ci., Foster. CR., Morris. P.A. & Teverson. R. (2002). Genetic and management factors that influence the susceptibility of cattle to Mic-obacieriuni hoii.s infection. Animal health Research Rc'c'u'cc', 3. I 13. Pollock. C.G. (2006). Implications of mycobacteria in clinical disorders. In G.J. Harrison & T.L. Lightfoot (Eds.). Clinical Arian Mecficine ( pp. 681-688). Palm Beach, FL: Spix Publishing. Pond. C.L. & Rush, H.G. (1981). Infection of white earneaux pigeons Cohinibia /ii'ia) with ,i'Ii'cohcicteriunt at/mu. Laharatorr Animals Science. 31. 196 199. Raniis. A.. Ferrer, L., Aranaz. A., Lilbana. F., Mateos, A., Dominguez. L., Pascual, C.. Fernandez-Garayazahal. J. & Collins. M.D. (1996). Mucschactericcni genccrensc' infection in canaries. Arian Drseasc'r. 40, 246-251.

CA, USA. Saggese. M.D., Riggs. G.. Tizard, I., Bratton. G.. Taylor. R. & Phalen, D.N. (2007). Gross and microscopic findings and investigation of the aethiopathogenesis of' mycohacteriosis in a captm\e popumlation ot white-winged ducks ) Cairina .ccic(ic/atci ). Arian Pccchicclicgi. 36, 415

Diseases, 50. 641 645. Taffik . H. (2001). The role of' human genetic factors in susceptibility to tuberculosis. Aria ('iendftccc I i'nc'olano. 52) Suppl 1). lb 18.

1(0 203. Tell. I.., Woods. L.. Foley. i.. Needham. M.L. & Walker, RI. (2003). A model of avian mycobacteriosis: clinical and histopathologic findings in Japanese quail ( Cocccr,ci v coicc,'ciix japcctciea ) intravenously inoculated with Mc'eobicccc'ricsnt ariiuhc Am' ian I)csc'ic.,e.,', 47. 433 443. Thoen. CO. (1997). Tuberculosis. in B.W. C'alnek (Ed.). Diseases of Poultr y 10th edn. Ames: Iowa State Universit y Press. Thompson, J.L).. Gibson. T. Plewniak, iF.....,cnmougin. I-. & Higgins. D.G. (1997). The CLLISTAI, X windows interface: flexible strategies 'or multiple sequence alignment aided b y qualit y analysis tools. !Vui-lew .'tc'lsl Resc'arch, 25, 4876 4882. Travis. E.K.. Jtinge, R.E. & Terrel, S.P. (2007). Infection with thc-c-obat-ceriucn sinciae complex in ('our captive Micronesian kingfishers. Jocmrcucf .cuier0 an I 'c'tc'riuiari' Jhi'chc'c'ccl Assoeiat cccii. 230. 1524 1529. Van der l-Iey dcn. N. (1997) Clinical manifestations of mycohacteriosis in per birds. ,Se,uuciccrs hi -I i'icccc and E.vcciie Pc't,s Mechcuie, 6, IS- 24. Van der Schaaf, tV. Hopmans..l.I.. & Van Beck. J. (1976). M ycohacterial intestinal disease in woodpigeons (Columbia palunihus). T,fdsc'hr D/c'cgc'nec'.skd, 101. 1)11(4 1092. Van Heiden. PD., Moller. M., Babh, C. .. Warren. R., WaIzI, G.. U y s. P. & 1-loal. E. (2006). Tuberculosis epidemiology and human genetics. ,-Vorarit.v Fomc,idcctjoic .S'i',mcpo.siicucm. 2 79. 17 -31 Zekarias. B., Ter Hurne. A.A.. Landman. WI Rebel. J.M.. Pol. J.M. & Grays. E. (2002). Immunological basis of differences in disease resistance in the chicken. I 'oter,ciarr Rc'.c'eorc'hi .33, 109-125.