Environmental & Forest Biology
Non Technical Summary
The wild turkey (Meleagris gallapavo) is an iconic North American species, an integral part offorest food webs, and an important game bird for sportsmen. Intensive restoration efforts havehelped populations recover substantially from mid-century lows (Tapley 2005), but wild turkeys stillface many threats. According to the New York Department of Environmental Conservation(NYSDEC) wild turkey numbers in the state have steadily declined since 2007. While there are manyreasons for downward trends in wildlife populations, the discovery of Lymphoproliferative DiseaseVims (LPDV) in wild turkeys throughout the eastern United States, including New York, has raisedquestions about the role this vin1s may be playing in these recent fluctuations.Lymphoproliferative Disease Vims is one of three avian retroviruses responsible forneoplastic disease in domestic and wild fowl. Avian leukosis /sarcoma virus (AL V) andreticuloendotheliosis virus (REV) are both C-type retroviruses, for which the most common clinicalsign is tumor formation in chickens and turkeys (Payne, 2000; Davidson, 2008). The tissues andorgans impacted can vary between viruses, and even between individuals infected by the same virus,but the liver and spleen are n:iost commonly affected (Payne, 1998; Payne, 2000). In addition totumor formation, diseased birds may develop non-specific symptoms such ~as mffled feathers,anemia, weight loss, and "depressed" appearance (Dunn, 2013a, 2013b). Mortality rates also varywidely. Subclinical infections of AL V and REV are not uncommon (Payne, 2000) but isolatedoutbreaks of AL V subgroup J have resulted in mortality rates as high as 40% (Hu, 2004). FrequentlyAL V and REV manifest themselves as decreased productivity (weight loss, decreased eggproduction, anemia, and immunosuppression) (Payne, 1998; Payne, 2000). Multiple modes oftransmission occur with AL V and REV, both vertically (mother to offspring) and horizontally (birdto bird), creating challenges for control of these viruses.Lymphoproliferative Disease Virus is also a C-type retrovirus that was first discovered in1978 by researchers in the United Kingdom (Biggs, 1978). The disease associated with the virus ischaracterized by a rapid clinical course prior to death, and gross lesions on a wide range of internalorgans including the spleen, thymus, pancreas, liver, kidneys, gonads, lungs and nerves (Gazit, 1999;Biggs, 1978). Like REV and AL V, many of the clinical signs are non-specific, such as ruffledfeathers, anorexia, or disinclination to move. Although similar in presentation, molecular testingrevealed it to be genetically distinct from both (Biggs, 1978; Gazit, 1979). Furthermore, the virus iscompletely exogenous in turkeys, indicating that it probably did not originate in that species but wastransmitted horizontally from another (still unknown) host (Gazit, 1979).Disease progression in domestic flocks involves the onset of elevated weekly mortality at 8 -10 weeks, with a continual, steady increase (as much as 2% a week) every week until slaughter at 16 -18 weeks (Biggs, 1978, Gazit, 1999). Mortality rates can reach 25% in some flocks (Gazit, 1979).Horizontal transmission between poults has been demonstrated, although the precise mechanism isunclear (Gazit, 1999; Payne, 1998). Age of infection seems to play an important role in how thedisease manifests itself. Poults experimentally infected at 4 weeks old go on to develop neoplasmsinvolving multiple organs, while infection at 1 day old leads to viraemia but no other clinical signs(Gazit, 1999). Experiments have shown that chickens are capable of infection and transmission, butnaturally-occurring LPDV is restricted to turkeys (Ianconescu, 2008; Brown, 2011). Intervention isdifficult because birds do not show signs prior to acute onset of the disease and death can followquickly (Gazit, 1999). Therefore, management in domestic flocks has simply been elimination ofinfected birds (Payne, 1998).In 2009, LPDV unexpectedly appeared in wild turkeys in the United States (Alison, 2014).Eight birds with extensive external lesions on the head, neck, and feet were discovered in thesoutheast- all either dead or moribund (Brown, 2011). The birds were suspected to be sufferingfrom avian pox, but were sent to the Southeastern Cooperative Wildlife Disease Study (SCWDS) inGeorgia for further testing which ultimately revealed LPDV (Brown, 2011). For five of the eightbirds, LPDV was 1-uled to be the primary cause of death, the other i:hree died of secondary causes.(Brown, 2011). By 2012, additional surveillance resulted in a total of 41 LPDV positive birdscollected broadly from Colorado to South Carolina (Alison, 2014). Continued monitoring iscurrently underway in New York and Maine, but preliminary surveys of hunter-harvested birds inother states estimate prevalence to be as high as 45% in adults (Alison, 2014).These findings have raised many new questions about the disease and its etiology in wildbirds. It is unclear how a population can support such high prevalence of a pathogen with apotential 25% mortality rate. The extensive skin lesions that seem to be a hallmark of the disease inwild birds were also not observed in domestic birds (Brown, 2011; Biggs, 1978). However, it isunknown whether this actually represents a new manifestation of the disease or is simply a function of lifespan. Perhaps domestic birds do not develop external lesion because, with an averageslaughter age of 16-18 weeks, they do not live long enough. In addition, studies in domestic birdsalso suggest that LPDV may be able to infect other birds in the order Galliformes (Ianconescu1983). This could have implications for other species of conservation concern in New York, such asthe ring-necked pheasant and the spruce grouse.Asymptomatic infections can make detection of LPDV in living birds difficult. The viruselicits a poor antibody response, making many serum diagnostics ineffective (Patel, 1987). Definitivediagnoses have only been made via Polymerase Chain Reaction (PCR) on tissue samples obtainedpost-mortem, making a reliable blood test highly desirable. Preliminary work by SCWDS on usingPCR to test buffy coat cells (white blood cells) has shown promise as a viable diagnostic method,but small sample size has limited validation. Furthermore, maintaining the integrity of blood samplesfor white cell extraction requires careful handling and can be difficult in the field.Awarded Start Date: 4/1/14Sponsor: NYS Department of Environmental Conservation
Animal Health Component
Research Effort Categories
Goals / Objectives
The objectives of this study are to:1) assess the prevalence and determine the spatial distribution of LPDV infection in wildturkeys throughout New York State;2) develop a reliable blood test for LPDV so that animals can be tested non-lethally;3) use the blood test to diagnose living turkeys and monitor the fate of infected birds.
I. Sample Collection and PCR anafysisa. Bone MarrowIn 2012, approximately 1500 turkey tarsi were collected from hunter harvested birdsthroughout the state of New York. Two-hundred and fifty of them were sent to SCWDS for LPDVdiagnostic testing. Those data, as well as the data from the untested samples, will be included in thisstudy. Additional tarsi from 2013 and 2014 will be added for an estimated total of 3000 tissuesamples. Tarsi collected by the NYSDEC are identified by sex, age, and location hai-vested. They arethen placed in individual ziplock bags and frozen at -20° C until bone marrow removal can becompleted.Bone marrow is removed from the tarsi following a protocol that has been modified slightlyfrom the one established by SCWDS. Prior to collection, all instruments are disinfected using a 10%bleach solution, rinsed with sterile water, dipped in ethanol then flamed (which differs from thebead sterilization used by SCWDS). This procedure is repeated between each sample in order toreduce the chance of cross-contamination. Bone marrow is removed by fractw:ing the tibiotarsuswith heavy-duty shears. Bone rongeurs were used by SCWDS, but these were considered costprohibitiveand it was found that stainless steel pruning shears were an adequate and cheaperalternative. After fracturing the bone, a sample of marrow (25mg) is removed with a sterilized probeor forceps. The sample is placed into an individual screw-top vial and re-frozen in anticipation ofDNA extraction. An additional amount of marrow is collected in a separate 1.5ml Eppendorf tubeand frozen as a tissue archive. The remainder of the tarsus is disposed of according to regulations.Extraction of DNA uses the DNeasy 96 Blood and Tissue Kit (Qiagen) according to themanufacturer's guidelines. Diagnosis ofLPDV employs a polymerase chain reaction (PCR) assay,performed in accordance with the protocol established by SCWDS, using primers based on theIsraeli prototype strain of LPDV that covered a region spanning the p31 and CA genes (5' -ATGAGGACTTGTTAGATTGGTTAC-3', 5'-TGATGGCGTCAGGGCTATTTG-3).Amplifications are performed on a C1000™ Thermal Cycler (BioRad Laboratories, Hercules,California) with initial denaturation at 95 C for 3 min, followed by 35 cycles of 94 C for 30 sec, 56 Cfor 45 sec, 68 C for 90 sec, and a final extension at 68 C for 7 min. Product amplification isevaluated by obse1-vation on a 1 % agarose gel. For initial samples, amplification of LPDV DNA wasconfirmed by DNA sequencing as follows. The remainder of the PCR sample was purified using theE.Z.N.A. Cycle Pure Kit (Omega Bio-Tek, Norcross, Georgia) and DNA was quantified using a DNA spectrophotometer (NanoDrop Technologies Wilmington, Delaware). Direct sequencing wasperformed using the primer :XXX. Reactions were carried out with the ABI BigDye TerminatorCycle Sequencing Ready Reaction I<it v3.1, using the ABI3730xl Genetic Analyzer (AppliedBiosystems, Foster City, California).b. BloodIn January, 2013, the NYSDEC launched a 4-year survival study of wild turkey hensthroughout the state. Tracking methods involve volunteer observers, point counts, radio telemetrydata collection and banding. Bird banding also provides an opportunity to obtain blood samples totest for LPDV and, potentially, to observe the progress of infected birds over a long period of time.Banding will occur from January to March with the goal of trapping approximately 400 turkeys eachyear (1,600 birds total). In December 2013, a pre-season workshop was conducted to orient DECfield crews to the study and provide training on both banding-related procedures (use of the rocketnets, data collection, etc), and avian blood collection using brachia! venipuncture. This training hasallowed field crews to assist in sample collection from around the state, ultimately yielding a highernumber and wider distribution of samples than could have been collected otherwise.Blood is drawn using 20 gauge, 3ml syringes from the brachia! (ulnar) vein. The amount~ollected varies between 1-2 ml, with 0.5 ml being the minimum necessary to maintain sampleintegrity. This portion of the project has been approved under SUNY-ESF IACUC protocol#131102. Once drawn, the blood is immediately transferred to 3ml EDTA tubes and stored in away that prevents freezing until it can be returned to the field office and refrigerated. Teams collectsamples for up to 2 weeks (to minimize shipping and transportation costs) before mailing theircollection to the AHDC where they are refrigerated until they can be transferred to ESF for furtherprocessing. Average time from collection to processing is approximately two weeks, but not inexcess of three. While there is evidence that storing the blood at 4 °C for longer than several daysmay result in a decreased DNA yield (up to 20%), this yield is not believed to be diagnosticallysignificant and therefore refrigeration is considered an acceptable method of long-term storage whenother options are limited (Richardson, 2006).Prior to centrifuging, a blood smear is prepared from each whole-blood sample to inspectfor hemoparasites. Blood smears are fixed in 100% methanol for 1 minute, and then stained withGiemsa stain. To collect buffy coat cells, whole blood is centrifuged for 7 minutes at 1500 x g. Theplasma and buffy coat layers are withdrawn, using a sterilized Pasteur pipette, and placed in a 1.5mlEppendorf tube. This tube is then centrifuged for 5 minutes at 800 x g. A sample of the buffy coat cells (25 μl) is withdrawn and placed in a 1.Sml screw-cap tube for later DNA extraction. Bothsamples are then frozen at -80°C, in accordance with established recommendations, until DNAextraction can be performed (Mychaleckyj, 2011). For samples that have clotted, a sample of clot isretained, but huffy coat extraction is not performed. Testing for the blood samples will beconducted during the summer of 2014 in collaboration with the AHDC molecular lab.II. Band and Radio-Telemetry DataBecause positive diagnoses have only been made in deceased birds, there is no data indicatinghow long infected birds live, what the incubation period is of the disease, and how it may impactreproductive success. Testing live birds that will be banded arid tracked by the DEC provides theopportunity to learn more about the course of the disease in this new population. If a banded birdtests positive for LPDV, and that bird is later harvested by a hunter and reported to the DEC, thiswill provide important baseline information regarding the lifespan of infected birds (i.e., a birdinfected with LPDV is capable of living a minimum ofX months/years with the disease).Information related to the virus's incubation period can also be inferred by band returns. Indomestic flocks the disease was observed to progress rapidly with few clinical symptoms and noexternal lesions (Biggs, 1978). If external lesions are a late-stage symptom, the degree to which theyare present in LPDV-positive birds upon original capture and recapture or harvest, will provide dataregarding the progression of the disease. In addition, the ability to obtain post-mortem tissuesamples from birds that tested positive by blood analysis will provide further evidence eithertowards or against the validation of using blood PCR to diagnose LPDV.Finally, unlike AL V and REV there is currently no data available on the impact LPDV may haveon the reproductive success of domestic and wild hens. Part of the DEC survivorship study entailsfitting birds with radio-transmitters in order to assess their annual reproductive success (Federal Aidin Wildlife Restoration Grant W-173-G). Testing birds that will be tracked in this manner willprovide data on the possible role LPDV plays in clutch size, egg hatchability, and poult survival..