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Management Practices to Reduce Infectious Laryngotracheitis Virus in Poultry Litter

J. J. Giambrone^*,1, O. Fagbohun and K. S. Macklin^*

^* Department of Poultry Science, and Department of Pathobiology, Auburn University, Auburn, AL 36849-5416

¹ Corresponding author: giambjj{at}auburn.edu

	SUMMARY

TOP SUMMARY DESCRIPTION OF PROBLEM MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS AND APPLICATIONS REFERENCES AND NOTES

 
We developed a natural challenge method using sentinel chickens reared on litter contaminated with infectious laryngotracheitis (ILT) vaccine virus (V) and a nested polymerase chain reaction (PCR) to determine the presence of ILTV in the feces and tracheas of the chickens. In 3 separate experiments, we determined that several commercially available poultry litter treatments (Poultry Guard, Al+Clear, and PLT), heating the litter to 38°C (100°F) for 24 h, and in-house composting for 5 d reduced ILTV below our detection level. This information is of immediate use to members of the poultry industry for controlling ILTV-induced disease in broilers and may reduce other important viral pathogens in poultry houses as well. Poultry companies around the United States are now using our methods for reducing ILTV in their houses.

Key Words: management • infectious laryngotracheitis virus • vaccine • poultry litter

	DESCRIPTION OF PROBLEM

TOP SUMMARY DESCRIPTION OF PROBLEM MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS AND APPLICATIONS REFERENCES AND NOTES

 
Infectious laryngotracheitis virus (ILTV) is a serious respiratory pathogen in chickens worldwide [1–3]. In pullets it is controlled by attenuated vaccines given by eye drops or by a fowl pox recombinant vaccine given by injection [4]. However, in broilers individually applied vaccines are not economically feasible. Less attenuated vaccines must be given in mass by coarse spray or drinking water [5]. Vaccination of broilers against ILTV is expensive, and reactions can cause reduced performance [6]. In addition, the vaccine produced in the United States on a yearly basis is insufficient to vaccinate all the broilers in needed areas.

Severe reactions caused by less attenuated vaccines, which have been back-passed (bird-to-bird transfer) within the same grow-out or subsequent grow-outs in the field, have resulted in vaccination-induced ILTV in broilers [5]. They can develop mild tracheitis and have reduced weight gain and feed efficiency, which are commonly associated with clinical ILTV. To control these ILTV vaccine-induced outbreaks in broilers, improved biosecurity (quarantine of affected flocks), and management practices to reduce the ILTV concentrations in chicken houses are needed.

In-house composting, heating the house, and litter treatments between grow-outs are effective in reducing ammonia and bacteria in the house [7]. The use of these methods and products in reducing ILTV has not been examined under controlled conditions. Heating of ILTV to 38°C for 48 h was found to inactivate the virus [8]. We used this work as a reference point from which to begin our studies.

We explored the use of management techniques, including commercial litter treatments, in-house composting, and heating the litter, to reduce ILTV in fecal material mixed in used pine shavings. To do this, we used a natural challenge method of sentinel chickens placed on used poultry litter that was contaminated with ILTV. A recently developed nested polymerase chain reaction (PCR) [3] determined the presence of the vaccine virus DNA in the fecal material and tracheas of birds exposed to the ILTV-infected and ILTV-treated litter. The nested PCR was 100x more sensitive than regular PCR and was sensitive enough to detect ILTV DNA equivalent to 354 viral copies.

	MATERIALS AND METHODS

TOP SUMMARY DESCRIPTION OF PROBLEM MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS AND APPLICATIONS REFERENCES AND NOTES

 
Vaccine Virus
An embryo-adapted commercial vaccine (LTBLEN) [9] designed for drinking water application was used at 100x the recommended dosage for broilers by the oculonasal route. This route and dosage produced a severe reaction in the birds, simulating a field challenge as observed with vaccinal ILTV and seeding the litter with vaccine virus.

Chickens
A total of 113 specific pathogen-free (SPF) broilers were obtained from an Auburn University flock. Nine birds were used in the first section (litter contamination) and the rest were used for section 2 (litter treatment). All were reared in modified Horsfall Bauer isolation units maintained with filtered air under positive pressure. A small wire mesh was placed over the wire floors of each unit so that 4 to 8 cm of used chicken litter could be added to each unit. All birds were fed a standard corn-soybean broiler starter diet and given water ad libitum. They were reared according to the University’s Animal Care and Use Committee guidelines.

Nested PCR Test
Total DNA was isolated from 25 mg of chicken feces from each group by using an UltraClean Fecal DNA Kit [10] according to the manufacturer’s instructions. Total DNA was isolated from the ILTV vaccine to serve as the positive control. Total DNA was also extracted from the trachea with a DNeasy Kit for tissues [11] according to the manufacturer’s instructions.

A 440-bp fragment of ILTV ICP4 gene was amplified from all samples by using a pair of outer primers: P1 (5'-AGA ACG AGA CAA TCC TCC-3') and P2 (5'-GGT CGG TTC AGT CAG TAA-3'). All PCR were performed in a 50-µL reaction mixture containing 5 µL of DNA, 4 µL of 10x PCR buffer, 2.5 mM MgCl₂, 0.4 mM each deoxy nucleotide 5'-triphosphate, 0.4 pmol/µL of P1, 0.4 pmol/µL of P2, 2.5 units of Ampli Taq [12], and 29.5 µL of nuclease-free water. The amplification reaction was carried out in a GeneAmp PCR system 9700 Thermal Cycler [12] under the following conditions: 94°C for 2 min for 1 cycle, 35 cycles of 95°C for 20 s, 60°C for 1 min, 72°C for 1.5 min, and final product extension in 1 cycle at 72°C for 5 min. A 190-bp nested PCR product was then generated by using internal primers to the 440 bp. The internal primers P3 (5'-CAA GGC GCT CTA ACT GTT CC-3') and P4 (5'-CTC GCG TTT TTG TTC CAG A-3') were designed with the Primer3 program (http://justbio.com). Nested PCR was performed in a 50-µL reaction mixture containing 2.5 µL of PCR product of the 440-bp fragment, 4 µL of 10x PCR buffer, 2.5 mM MgCl₂, 0.4 mM each deoxy nucleotide 5'-triphosphate, 0.2 pmol/µL of P3, 0.2 pmol/µL of P4, 2.5 units of Ampli Taq, and 33 µL of nuclease-free water. The GeneAmp PCR system 9700 thermal cycler was used under the following conditions: 94°C for 2 min for 1 cycle, 35 cycles of 95°C for 20 s, 60°C for 1 min, 72°C for 1.5 min, and final extension at 72°C for 5 min. The PCR products were analyzed by electrophoresis with 1.5% agarose.

Litter Treatments
The ILTV-infected and control litter samples, as determined by PCR, were taken in sealed plastic containers (30 x 40 x 80 cm) to a nearby concrete block building for treatment. Control litter was kept in a separate room from contaminated litter. The house contained 4 rooms and each was 3 x 3 x 4 m. Samples of contaminated litter were either exposed to heat (separate room or a laboratory oven) for various time intervals, composted (placed in a pile), or commercial litter treatments were added and mixed with the litter. Composting and litter treatments were added for 5 d. Composting for 5 d is normally practiced in the industry [7]. Composting was monitored every 6 h by using a temperature probe [6]. The following litter treatments were tested: Al+Clear [13], Poultry Guard [14], and PLT [15]. All were tested at 22.68 kg (50 lb/ton) or 907.2 kg, which is the common level of use in the industry.

Experimental Methods
The methods were divided into 2 sections. The first was to seed the litter with ILTV, and the second included the 3 litter treatment experiments.

Section 1.
Pine shavings were taken from the floor of an Auburn University experimental broiler house, which had experienced several broiler grow-outs. No ILTV vaccine was used in any of the broilers and no ILTV outbreaks had been diagnosed on the farm. This litter was determined free of ILTV by nested PCR. Some of the units contained litter that was contaminated with ILTV vaccine, and this litter was determined to contain ILTV by nested PCR. Other control units were kept free of ILTV vaccine as determined by PCR.

Three 3-wk-old SPF broilers were vaccinated by eye and nose drops with ILTV vaccine. One week later, these birds had mild tracheal rales and conjunctivitis. Three more 4-wk-old SPF birds were added to the same unit. One week later, these contact-exposed birds had similar respiratory signs. Three more 5-wk-old SPF birds were added to the same unit. One week later, these contact-exposed birds showed respiratory signs. All 9 birds were then killed and their tracheas were examined for gross and microscopic lesions. Pulled tracheal swabs from each time period and fecal material at the end of the study were examined for ILTV DNA by nested PCR. All tracheal swabs and fecal material samples were positive for ILTV by PCR.

The presence of ILTV DNA, at levels in the trachea needed to produce a positive PCR (354 viral copies), clinical signs, and gross and microscopic lesions in sentinel birds, could only have come from active viral replication in the trachea. In contrast, the presence of a positive PCR in the feces could have arisen from inactivated viral DNA. Tracheas had gross (increased mucous) and microscopic (lymphocytic infiltration) lesions indicative of moderate tracheitis. Therefore, we presumed that the untreated litter contained live vaccine virus. However, we did not note an increase in virulence of the vaccine virus after successive infections of sentinal birds as measured by clinical disease.

Section 2.
Three litter treatment experiments were conducted. In each experiment, six 1-d-old broilers were used per litter treatment group. At 4 and 5 wk of age, 3 birds each were killed and examined postmortem. Tracheal swabs were pulled from each treatment group at each time period for nested PCR. Sections of trachea and eyelids were taken for histological observation. Lesions were scored based on the amount of lymphocytic infiltration. Sections without lymphocytes were given a score of 0, those containing small amounts of lymphocytes were given a score of 1, and those containing moderate amounts were given a score of 2. No sections seen in this experiment had severe grade 3 lesions (large amounts of lymphocytes) or intranuclear inclusion bodies. These lesions are typically seen in severe ILTV outbreaks in the field, which are characterized by necrotic and or hemorrhagic tracheitis [8]. The lesion scoring is an arbitrary method used by clinicians at the Alabama State Veterinary Diagnostic Laboratory in Auburn, Alabama. This laboratory uses clinical signs, gross and microscopic lesions, and PCR to diagnose ILTV in commercial flocks.

In the first experiment, 7 treatment groups were used. The litter of all groups, except group G, contained ILTV. The litter of group A was not treated, whereas the litter of groups B, C, D, and E was heated in an oven to 38°C for 48, 96, 128, and 144 h, respectively. Oven heating was done to produce a more uniform heating. The litter of group F was composted for 120 h. In the second experiment, 7 groups were used. All groups were placed on litter infected with ILTV, except for group E. Group A had ILTV-infected litter with no treatment. The litter of group B was treated with PLT, that of group C was treated with Poultry Guard, and that of group D was treated with Al+Clear. The litter of groups F and G was heated to 38°C for 24 h in the oven or house, respectively. Experiment 3 was a repeat of the first 2 experiments, except that only the 24-h heating period was used. All groups received infected litter, except group E. Group A did not have treated litter. The litter of group B was treated with PLT, that of group C was treated with Poultry Guard, and that of group D was treated with Al+Clear. The litter of groups F and G was heated to 100°C for 24 h in the oven or house, respectively. The litter of group H was composted for 120 h. No statistical analysis was done in this study, because observations were based on the presence or absence of lesions, clinical signs, or an ILTV DNA PCR band. However, experiment 3 was a replicate of experiment 2, with the addition of another composting treatment originally done in experiment 1.

	RESULTS AND DISCUSSION

TOP SUMMARY DESCRIPTION OF PROBLEM MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS AND APPLICATIONS REFERENCES AND NOTES

 
Results for the 3 experiments are listed in Tables 1 to 3. There were no differences in the results for the 2 time periods (4 and 5 wk of age). Therefore, only results of the 5-wk period are listed. In addition, a positive result (presence of clinical signs or gross or microscopic lesions for any bird) in a treatment group was listed as a positive (+) result. A 2-plus positive (++) microscopic lesion score indicated the presence of a grade 2 lesion score for any bird in the group. No birds placed on ILTV-negative control litter had gross lesions, clinical signs, or PCR-positive results, whereas all birds placed on ILTV-positive litter that was not treated had clinical signs, gross lesions, and PCR-positive results.

To reduce ILTV under industry conditions, one would use windrow composting to kill the virus in the litter and would heat the house to 38°C to kill the virus in other areas of the house above the litter, where levels of organic material would be less. Heating alone may not be effective in reducing the virus in lower levels of deeply built-up litter.

Whether these management techniques would reduce more virulent ILTV pathotypes or other viruses was not determined in these studies. However, the high temperatures reached in composted litter (60°C) would kill all known viral pathogens in poultry [7]. These laboratory pilot studies will serve as a starting point for future field studies using poultry litter obtained from natural outbreaks of ILTV. In addition, swabbing sentinal birds on farms and testing them for PCR where a previous ILTV outbreak had occurred could prove useful in determining whether management strategies were successful in reducing ILTV.

	CONCLUSIONS AND APPLICATION

TOP SUMMARY DESCRIPTION OF PROBLEM MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS AND APPLICATIONS REFERENCES AND NOTES

 

1. A natural challenge method was developed by using sentinel chickens placed on used poultry litter that was contaminated with ILTV as well as a nested PCR test to determine the presence of the vaccine virus in the fecal material and tracheas of exposed birds.
   
2. Commercial poultry litter treatments (Poultry Guard, Al+Clear, PLT), heating the litter to 38°C for 24 h, and in-house composting for 5 d reduced inactivated ILTV.
   
3. These litter treatments, management methods, and swabbing of sentinel birds for nested PCR are now being used in the poultry industry to control vaccinal ILTV outbreaks in broilers and may reduce the incidence and severity of other important viral pathogens as well.
   

	ACKNOWLEDGMENTS

 
We thank Merial Animal Health (Gainesville, GA) for providing the vaccine. We also thank the Alabama Agricultural Experiment Station (Auburn, AL) and US Poultry and Egg Association (Tucker, GA) for their financial support.

	REFERENCES AND NOTES

TOP SUMMARY DESCRIPTION OF PROBLEM MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS AND APPLICATIONS REFERENCES AND NOTES

 

1. Davidson, S., and K. Miller. 1988. Recent laryngotracheitis outbreaks in Pennsylvania broilers. Pages 135–136 in Proc. 37th West Poult Conf., Sacramento, CA. Omni Press, Madison, WI.
2. Linares, J. A., A. A. Bickford, G. L. Cooper, B. R. Charlton, and P. R. Woolcock. 1994. An outbreak of infectious laryngotracheitis in California broilers. Avian Dis. 38:188–192.[CrossRef][Medline]
3. Fagbohun, O., J. J. Giambrone, and K. S. Macklin. 2007. Nested PCR for the detection of infectious laryngotracheitis virus. Southern Conf. Avian Dis., Atlanta, GA, Jan. 22–23. US Poult. Egg Assoc., Tucker, GA.
4. Fulton, R. M., D. L. Schrader, and M. Will. 2000. Effect of route of vaccination on the prevention of infectious laryngotracheitis in commercial egg-laying chickens. Avian Dis. 44:8–16.[CrossRef][Medline]
5. Guy, J. S., H. J. Barnes, and L. G. Smith. 1991. Increased virulence of modified-live infectious laryngotracheitis vaccine virus following bird to bird passage. Avian Dis. 35:348–355.[CrossRef][Medline]
6. Hillbink, F. W., H. L. Oei, and D. J. can Roozelaar. 1987. Virulence of five live virus vaccines against infectious laryngotracheitis and their immunogenicity and spread after eye drop or spray vaccination. Vet. Q. 9:215–225.[Medline]
7. Macklin, K. S., J. B. Hess, S. F. Bilgili, and R. A. Norton. 2006. Effects of in-house composting of litter on bacterial levels. J. Appl. Poult. Res. 15:531–537.[Abstract/Free Full Text]
8. Jordan, F. T. W. 1966. A review of the literature on infectious laryngotracheitis virus. Avian Dis. 10:1–10.[CrossRef]
9. Merial Animal Health Inc., Gainesville, GA.
10. Mo Bio Laboratories, Carlsbad, CA.
11. Qiagen, Valencia, CA.
12. Applied Biosystems, Foster City, CA.
13. Al+Clear, General Chemical Corp., Parsippany, NJ.
14. Poultry Guard, Oil-Dri Corporation, Chicago, IL.
15. PLT, Jones-Hamilton Co., Walbridge, OH.

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