Differential diagnosis of fowlpox and infectious laryngotracheitis viruses in chicken diphtheritic manifestations by mono and duplex real-time polymerase chain reaction

Abstract

Infectious laryngotracheitis virus (ILTV) and fowlpox virus (FPV) cause diphtheritic lesions in chicken tracheas and can simultaneously infect the same bird. A differential molecular diagnostic test, the duplex real-time polymerase chain reaction, is now reported using ILTV and FPV vaccine viruses and clinical samples from chickens, either uninfected or naturally infected with ILTV or FPV, or with both viruses. The dual virus amplification by real-time polymerase chain reaction was demonstrated to behave similarly to monoplex amplification, in spite of the fact that the real-time exponential amplification plots of the vaccine viruses were more illustrative than those of the clinical samples.

Introduction

Infectious laryngotracheitis is a respiratory disease of poultry caused by Gallid herpesvirus type I (infectious laryngotracheitis virus [ILTV]) (Garcia et al., 2013). The virus can cause severe production losses due to mortality and decreased egg production. The clinical signs range from mild to severe, with mortality rates that can reach up to 70%, depending on the virulence of the infecting virus. The milder form of infectious laryngotracheitis is manifested by respiratory signs, including gasping and expectoration of bloody mucus with nasal discharge, conjunctivitis and reduced egg production. In severe forms the clinical signs include gasping with efforts to inhale, coughing and excretion of bloody mucus in the trachea. Diphtheritic changes caused by ILTV might extend over the entire length of the trachea, resembling clinically the diphtheritic clinical signs caused by fowlpox virus (FPV) (Tripathy & Reed, 2013). ILTV of lower virulence also resembles lesions caused by other respiratory diseases, such as Newcastle disease, avian influenza, infectious bronchitis, fowl adenovirus and Aspergillus spp.

FPV commonly causes disease in commercial chickens, pets and wild birds, accompanied by a drop in egg production. The disease can occur in two forms: the cutaneous form and the diphtheritic form. In the cutaneous form fowlpox manifests itself as discrete nodular proliferative lesions appearing on the non-feathered parts of the bird, while in the diphtheritic form, which is pathologically undistinguishable from lesions caused by ILTV, the tracheal mucous membrane of the upper respiratory tracts as well as the mouth and oesophagus are affected.

Because FPV and ILTV can produce similar tracheal lesions, associated respiratory distress and clinical signs in chickens, the diphtheritic form of fowlpox in chickens must be differentiated from infectious laryngotracheitis. Moreover, simultaneous dual infections with both FPV and ILTV have been described in commercial chickens and are expected to be quite common, because FPV is a ubiquitous virus while ILTV vaccination is extensively used.

Previous studies described the differential virus detection by laborious and relatively insensitive assays, such as immunohistochemistry (Tripathy et al., 1975) and dot-blot hybridization (Fatumbi et al., 1995). In addition, Tadese et al. (2007) described an end-point multiplex polymerase chain reaction (PCR) for the detection of both viruses in chicken tissues, and a multi-assay approach, including virus isolation, histopathology, electron microscopy and PCR, was used by Diallo et al. (2010) to detect both viruses in a natural co-infection.

We now aim to evaluate in retrospect, for the first time, a real-time multiplex PCR (rtPCR) for the simultaneous detection of both ILTV and FPV in diphtheritic tracheal lesions of commercial chicken flocks, which had previously been submitted for diagnosis of each virus separately by end-point PCR. Identical DNA preparations of diphtheritic tracheas were assessed by rtPCR in monoplex configurations for each virus, and as a multiplex assay for the concurrent detection of both viruses.

Materials and Methods

Tracheal sections from chicken flocks with and without typical diphtheritic clinical signs were the source of DNA for amplification. The tracheas were cut on the long axis in order to collect the exudate using a scalpel. Viral DNA was purified from the exudate using the Maxwell® AS1030 Tissue DNA purification kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. The samples used in the present study were previously diagnosed by the established diagnostic assay of TK gene nested end-point PCR for ILTV (Davidson et al., 2009) and PER-PCR for FPV. The PER-PCR amplified a FPV fragment, denoted by “P” in “PER”, the fragment of the env gene was denoted by “E”, and because it belongs to the reticuloendotheliosis virus (REV) group it was denoted by “R” (Davidson et al., 2008). Positive and/or negative DNA samples for ILTV and/or FPV were included in the present study to evaluate the performance of the two rtPCR methods, monoplex for each virus compared with duplex rtPCR using the same samples. Histopathological examination of several samples was performed to support the amplification findings.

The FPV and ILTV commercial vaccine viruses (FPV, Lot 20511011; Phibro Ltd, Airport City, Israel; and ILTV, Lot 1–1041105; Biovac Ltd, Or Akiva, Israel) were extracted directly from the commercial bottle after reconstitution in phosphate-buffered saline using the Maxwell® AS1020 Cell DNA purification kit (Promega, Ltd), according to the manufacturer’s instructions. Taking into account the total number of doses stated by the manufacturer to be included in the bottle, the DNA concentration for amplification was adjusted to 1 dose/1 µl. Moreover, the DNA content in both DNA preparations was comparable, namely 24 µg/ µl.

Real-time polymerase chain reaction primers and probes

The rtPCR primers and probes used for ILTV were based on the thymidine kinase gene to amplify a 108 base pair (bp) amplicon based on the ILTV genomic region reported by Corney et al. (2010) with additional location modifications on the ILTV strain Samberg sequence (accession number DQ522947) as follows, denoting the location changes compared with Corney et al. (2010): forward, 5′-CAAAATGTTCACGGGGAAAGA-3′, 49 bp upstream; reverse, 5′-GAGGCCATGTGCTGGTAAGTAAA-3′, 1 bp upstream; and a dually labelled probe, 5′-CAL Fluor Gold 540-AAACTCGCGACGGTATTGAAA-BHQ1-3′, 20 bp upstream.

The rtPCR primers and probes for the pox virus were based on the 4b gene to amplify a 109 bp amplicon (Hauck et al., 2009) as follows: forward, 5′-TCAGCAGTTTGTTACAAGACA-3′; reverse, 5′-CCATTTCCGTGAATAGAATAGTAT-3′; and a dually labelled probe, 5′-FAM-ATCTCCGCCGTCGCAACTTCC-BHQ1-3′.

Design of multiplex quantitative real-time polymerase chain reaction

The amplification mix at a volume of 20 µl contained 10 µl Mastermix (Quanta BioSciences, Inc., Gaithersburg, MD, USA), 1 µl each of the four primers and two probes, at a concentration of 500 nm for the primers and 250 nm for the probes, and 2 µl each of the two DNA controls, or the samples to be examined. The cycling conditions were 3 min at 95°C, and 40 cycles of 10 sec at 95°C and 45 sec at 60°C. Assays were performed on a StepOne real-time PCR system (Applied Biosystems, Foster City, CA, USA).

Results and Discussion

Evaluation of the rtPCR as mono and duplex amplification of ILTV and FPV vaccine viruses, as compared with end-point PCR

To assess the compatibility and the relative sensitivities of the monoplex and duplex rtPCR amplification in relation to the end-point PCRs for the detection of each virus, the same DNA preparations were used for all amplification combinations. DNA purified from the commercial vaccines was used undiluted, and followed by eight 10-fold dilutions, up to 10⁻⁸ of the initial sample, which corresponded to two vaccine doses.

Table 1 presents the performance of all amplification modes. The end-point PCR for ILTV (Davidson et al., 2009), being a two-step nested amplification, was recorded separately. While the second amplification did not reach an endpoint, reflecting its high sensitivity, the first amplification step was approximately 2 logs less sensitive than the ILTV monoplex rtPCR, and slightly less sensitive than the multiplex rtPCR. It is also noted that for ILTV the monoplex rtPCR resulted in a 1 log higher sensitivity than the duplex rtPCR. In a similar manner, FPV amplification by the end-point PCR and by the monoplex rtPCR was of similar sensitivity, and both were 1 log less sensitive than the duplex rtPCR. The monoplex and duplex real-time amplification of ILTV gave similar results. The amplification of FPV in the duplex configuration seemed to be more sensitive than a monoplex configuration.

Table 1. Evaluation of the rtPCR as mono and duplex amplification of infectious laryngotracheitis and fowlpox virus vaccine viruses as compared with end-point PCR.

	ILTV			FPV		ILTV + FPV
	End-point nested PCR
DNA dilution	PCR-I^a	PCR-II^a	Monoplex rtPCR (CT value)	End-point PCR	Monoplex rtPCR (CT value)	Duplex ILTV–FPV rtPCR (CT value)
10^0b	+	+	12.1	+	20.7	12.4	15.4
10^−1	+	+	15.5	+	23.9	15.7	18.7
10^−2	+	+	18.8	+	27.3	18.9	21.9
10^−3	+	+	22.0	+	30.4	22.0	25.1
10^−4	+	+	25.3	+	33.9	25.6	28.5
10^−5	+	+	28.6	+	37.0	28.8	31.8
10^−6	−^c	+	32.6	−	Negative	32.2	35.3
10^−7	−	+	35.4	−	Negative	Negative	Negative
10^−8	−	+	Negative	−	Negative	Negative	Negative

^a PCR-I and PCR-II are the first and second amplification stages of the nested end-point PCR (Davidson et al., 2009). ^bThe initial DNA amount corresponded in volume to two vaccine doses, according to the manufacturers' recommendations (Phibro Ltd; Biovac Ltd), with losses encountered during the process of DNA purification. ^cEnd-point PCR negative.

The use of rtPCR is advantageous compared with end-point PCR because, in addition to the enhanced sensitivity of detection, it is less laborious and more straightforward.

As a whole, the enhanced sensitivity of the rtPCR was demonstrated when compared with the first-step end-point amplification of ILTV. Moreover, the advantages of employing rtPCR instead of the end-point PCR are evident, and we demonstrated that the simultaneous detection of both ILTV and FPV is feasible.

In contrast to the FPV gene used for the former end-point PCR—which detected the pox virus and the inserted REV long terminal repeat remnants in chickens and turkeys (Davidson et al., 2008), therefore being vulnerable to the REV-insert genomic variations—the present FPV 4b gene amplicon is more conserved and might also detect FPV in wild birds. As the rtPCR for both ILTV and FPV was developed using vaccine viruses as the positive control, the assay would also detect vaccine viruses if present in the tracheas at the time of sampling.

Evaluation of the rtPCR as mono and duplex amplification of ILTV and FPV in clinical samples as compared with end-point PCR

To evaluate the feasibility and suitability of the rtPCR, for use as ILTV and FPV monoplexes or as a duplex rtPCR for both viruses in the differential diagnosis of diphtheritic forms of lesions in domestic chickens, the clinical samples were grouped according to the results of the respective end-point PCRs. All assays were applied to detect the viruses in four groups of DNA purified directly from the bird tracheas: a group negative for both viruses by separate end-point PCRs, a group positive for both viruses, and two groups that were positive for one virus only (Table 2). Sample numbers 24 to 29 were negative by all assays, except for samples 28 and 29 that had marginal cycle threshold (C_T) values by the ILTV monoplex. Sample numbers 13 to 23 were positive by all assays, while samples 1 to 6 and samples 7 to 12 were positive for FPV and for ILTV only, respectively, by all three assays. While the C_T values recorded for ILTV were identical in the mono and in the duplex rtPCR configurations, the C_T values for FPV detection in the duplex configuration were slightly lower than in its mono rtPCR configuration for both the vaccine virus and the field samples, probably due to standard deviations or chemical interaction between the reaction components. In addition, some commercial samples had slightly concave rtPCR amplification plots for FPV in the duplex configuration. However, the C_T values were distinctive between positive and negative clinical samples, demonstrating the feasibility of using the duplex rtPCR for differential diagnosis of the diphtheritic lesions in chickens.

Table 2. Evaluation of the rtPCR as mono and duplex amplification of infectious laryngotracheitis and fowlpox viruses in clinical samples as compared with end-point PCR.

	End-point PCR		Monoplex rtPCR (CT values)		Duplex rtPCR (CT values)
DNA sample	FPV	ILTV	FPV	ILTV	FPV	ILTV
1	+	−^a	12.8	Negative	12.4	Negative
2	+	−	15.5	Negative	16.0	Negative
3	+	−	14.2	Negative	13.9	Negative
4	+	−	13.2	Negative	13.6	Negative
5	+	−	11.8	Negative	12.2	Negative
6	+	−	13.1	Negative	13.8	Negative
7	−	+	Negative	11.6	Negative	11.3
8	−	+	Negative	13.2	Negative	12.9
9	−	+	Negative	16.5	Negative	17.2
10	−	+	Negative	14.3	Negative	14.7
11	−	+	Negative	20.7	Negative	20.7
12	−	+	Negative	21.6	Negative	21.8
13	+	+	25.2	28.0	16.1	29.4
14	+	+	22.0	12.4	17.3	12.3
15	+	+	22.1	12.0	16.5	11.8
16	+	+	29.5	19.9	25.2	20.1
17	+	+	31.1	28.3	30.8	28.5
18	+	+	29.0	14.0	19.8	14.5
19	+	+	29.5	25.9	29.0	26.0
20	+	+	18.3	17.5	18.1	17.2
21	+	+	12.7	12.2	12.1	11.3
22	+	+	12.8	13.5	11.9	11.7
23	+	+	30.9	19.2	23.9	18.6
24	−	−	Negative	Negative	Negative	Negative
25	−	−	Negative	Negative	Negative	Negative
26	−	−	Negative	Negative	Negative	Negative
27	−	−	Negative	Negative	Negative	Negative
28	−	−	Negative	34.2	Negative	Negative
29	−	−	Negative	37.0	Negative	Negative

^a End-point PCR negative.

The histopathological examination of samples 14, 15, 18 and 19 supports the amplification findings, revealing typical lesions of ILTV and FPV in both the organs and on the chorioallantoic membrane of embryonated eggs that were inoculated with the tissue homogenates.

In conclusion, we describe an assay that can amplify both ILTV and FPV simultaneously. This is an important development since the two viruses often cause a dual infection with diphtheritic, clinical manifestations.