Classification of Marek's disease viruses according to pathotype: philosophy and methodology

Figures & data

Table 1. Pathotyping of six new isolates by the ADOL assay^a

Trial	Isolate	% MD in chickens vaccinated with:			Pathotype	Virulence rank
		None	HVT	HVT+SB-1
1	670	100.0	45.2*	3.1	vv	24.1
	685	100.0	52.9*	6.3	vv	29.6
	686	100.0	100.0*	57.6*	vv+	78.8
	JM/102W	100.0	5.9
	Md5	100.0		14.7
2	690	100.0	63.6*	39.4	vv	51.5
	691	97.1	52.9*	5.9	vv	30.3
	692	100.0	97.1*	50.0	vv	73.5
	JM/102W	84.8	19.4
	Md5	100.0		44.1
Historic controls	JM/102W	92.8	22.3	8.7
	Md5	99.8	82.8	28.0
^aThe standard ADOL pathotyping assay (Witter, 1997) was applied to the typing of six new field isolates (trials 1 and 2). Line 15×7 chickens were the F1 progeny of line 15I5 males and line 71 females. The dams received MD vaccine of all three serotypes (Witter, 1987) so that progeny chicks were considered positive for maternal antibodies (ab+). Groups of 17 chickens were vaccinated at hatch with 2000 PFU of the respective vaccine (none, HVT or HVT+SB-1), challenged with 500 PFU of the respective challenge virus at 5 days post vaccination, and held in modified Horsfall–Bauer isolators under negative air pressure until the conclusion of the trial, 8 weeks post challenge. The per cent (%) MD responses are based on the number of dead or killed chickens with gross MD lesions; data are pooled from two replicate trials. Virulence rank was calculated as 100 – mean % MD response (HVT and HVT+SB-1 groups). The table also includes mean response data from 10 earlier trials (Witter, 1997) for comparative purposes. * Response values greater than that for the appropriate control virus response (underlined) in the same column, as determined by interaction chi-square analysis (Steel & Torrie, 1960).

Table 2. Challenge of CVI988-vaccinated chickens does not discriminate between vv and vv+ pathotypes^a

Isolate	Previously published data				New data
	Pathotype	Virulence rank	Bivalent (HVT+SB-1)		CVI988
			% MD	% Protection	% MD	% Protection
679B	vv	39.4	9.1	91	0.0	61
595	vv	61.0	46.7	53	34.0	83
615K	vv	62.5	35.5	63	17.6	80
677	vv	67.8	41.9	58	38.2	57
611	vv	70.5	47.1	50	15.2	83
643P	vv	72.0	44.1	56	26.5	71
Md5	vv	57.9	33.1	67	14.7	85
Mean			36.8A	62A	20.9A	74A
645	vv+	79.0	57.6	42	21.2	76
584A	vv+	82.5	67.6	32	17.6	80
675A	vv+	84.8	69.7	30	21.9	76
652	vv+	86.5	72.7	27	30.3	66
671A	vv+	86.8	73.5	26	8.8	90
660A	vv+	88.7	64.7	35	23.5	74
648A	vv+	91.0	81.8	18	21.2	74
Mean			69.7B	30B	20.7A	77A
^aSeven MDV strains of the vv pathotype and seven MDV strains of the vv+ pathotype were evaluated. Previous data on these strains obtained with bivalent (HVT+SB-1) vaccinated 15×7 ab+ chickens are listed (Witter, 1997). New data were obtained for each virus by challenge of line 15×7 ab+ chickens at 5 days post vaccination with 2000 PFU of CVI988 (see Table 1). The CVI988 strain was originally obtained from Rhone Merieux through Select Laboratories (Witter et al., 1995). The % MD through 8 weeks post challenge was calculated as in Table 1. The % protection is calculated as described in Witter et al. (1995). Groups included 17 chickens. Two replicate trials were conducted and the data were pooled. Means in same column identified by different uppercase letters differ (P<0.05).

Table 3. Pathogenicity of selected vv+ isolates in chickens vaccinated with bivalent or CVI988 vaccines^a

Year isolated	Isolate	Vaccine	n	MD+	% MD mortality	% total MD	% protection	C/B ratio
1999	686	CVI988	33	10	18.2	30.3	69	>10
		HVT+SB-1	34	31	17.6	91.2	6
		None	33	33	97	100
1999	690	CVI988	34	4	2.9	11.8	88	1.1
		HVT+SB-1	34	8	0	23.5	76
		None	34	34	23.5	100
1997	677	CVI988	31	4	6.5	12.9	87	1.6
		HVT+SB-1	34	15	0	44.1	54
		None	32	31	56.3	96.9
1995	648A	CVI988	34	7	17.8	20.6	79	2.5
		HVT+SB-1	32	21	3.1	65.6	32
		None	33	33	87.9	100
1994	645	CVI988	34	4	0	11.8	88	3.1
		HVT+SB-1	33	23	0	69.7	28
		None	34	34	0	100
1990	584A	CVI988	34	7	2.9	20.6	79	2.0
		HVT+SB-1	34	20	9.1	58.8	39
		None	34	34	70.6	100
	None		10	0	0	0
Summary
1997 to 1999		CVI988	98	18	9.2	18.4	81	1.8
		FC126/2+SB-1	102	54	5.9	52.9	45
		None	99	98	58.6	99
1990 to 1995		CVI988	102	18	6.9	17.8	82	2.5
		FC126/2+SB-1	99	64	4	64.6	33
		None	101	101	52.5	100
^aTo determine whether recent MDV isolates of the vv+ pathotype have increased virulence for CVI988-vaccinated chickens, three vv+ isolates (1997 to 1999) and three vv+ isolates (1990 to 1995) were used to challenge 15×7 ab+ chickens that were vaccinated at hatch with the respective vaccine (none, CVI988, and HVT+ SB-1). Vaccine and challenge doses were 2000 and 500 PFU, respectively. MD lesion responses were measured through 8 weeks post challenge. Each treatment group was 17 chickens; two replicate trials were conducted and the data were pooled. Data are presented for each virus, and also as pooled data for each group of viruses. The C/B ratio, calculated as the % protection by CVI988 divided by the % protection by HVT+SB-1 vaccines, is presented.

Table 4. Discrimination between vv and vv+ isolates by challenge of different lines of chickens at 3 weeks of age^a

Table 5. Pathotyping data in SPAFAS ab– chickens using standard ADOL assay protocol: correlations and best fit pathotype^a

Virus	15×7^b		% MD			% mortality
	Pathotype	Virulence rank	HVT vaccinated	Bivalent vaccinated	Not vaccinated	Not vaccinated	Virulence rank	Best fit
JM/102W	v	17	17.6	5.9	76.5	29.4	15
Md5	vv	56	23.5	18.8	88.2	70.6	24
648A	vv+	84	35.3	41.2	100.0	100.0	38
571A	v	19	5.9	7.7	81.3	18.8	8	v^c
549A	vv	54	47.1	23.5	94.1	94.1	35	vv+^d
660A	vv+	89	64.7	47.1	100.0	100.0	56	vv+^e
Correlation coefficient^f			0.82	0.98	0.90	0.94	0.92
^aChickens obtained from SPAFAS, Inc. were utilized in the ADOL pathotyping assay (see Table 1) with the following modifications. The chickens were from non-vaccinated parents (ab–). Three prototype control strains were used: JM/102W, Md5 and 648A representing the v, vv and vv+ pathotypes, respectively. Only one replicate trial was conducted and each group contained about 17 chickens which were vaccinated at hatch, challenged at 6 days post vaccination and terminated 8 weeks post challenge. Bivalent vaccine, HVT+SB-1.
^bData from earlier trials with replicate lots of 17 chickens per treatment.
^cGood fit with JM/102W by all criteria.
^dFits between Md5 and 648A by most criteria, assigned vv+ on basis of HVT and virulence rank.
^eGood fit with 648A by all criteria, exceeds 648A by some criteria.
^fCompared against virulence rank value determined in 15×7 chickens by an excel program.

Table 6. Pathotyping data in Hy-Vac line TK ab– chickens using standard ADOL assay protocol: correlations and best fit pathotype^a

Virus	15×7^b		% MD			% mortality
	Pathotype	Virulence rank	HVT vaccinated	Bivalent vaccinated	Not vaccinated	Not vaccinated	Virulence rank	Best fit
JM/102W	v	17	5.9	0.0	41.2	11.8	7
Md5	vv	56	11.8	11.8	88.2	41.2	13
648A	vv+	84	50.0	18.8	100.0	94.1	34
571A	v	19	29.4	26.7	47.1	23.5	68	v^c
549A	vv	54	11.8	11.8	100.0	100.0	13	vv^d
660A	vv+	89	35.3	31.3	100.0	94.1	33	vv+^e
Correlation coefficient^f			0.62	0.48	0.91	0.86	−0.09
^a Chickens of Hy-Vac line TK ab− were utilized in a pathotyping assay as described for Table 5. Data are based on single lots of 17 chickens vaccinated at hatch, challenged at 6 days post vaccination and terminated 8 weeks post challenge. Bivalent vaccine, HVT+SB-1.
^bData from earlier trials with replicate lots of 17 chickens per treatment.
^cGood fit with JM/102W in not vac chickens, but exceeds JM/102W by other criteria.
^dGood fit with Md5 by most criteria, but % mortality most closely resembles that of 648A.
^eGood fit with 648A by most criteria.
^fCompared against virulence rank value determined in 15×7 chickens by an excel program.

Table 7. Two-step experimental design for pathotyping by best fit method

Test	Lot	Chicks	Chicken strain	Vaccine 2000 PFU	Virus challenge			Test duration
					Strain	PFU	Age
A^a	1	10	SPF, ab−	None	Virus 1^c	500	1 day	56 pcd
	2	10			JM/102W	500	1 day
	3	10			Md5	500	1 day
	4	10			None	500	1 day
B^b	1	14	SPF, ab−	HVT	Virus 1^c	500	5 pvd	56 pcd
	2	14			JM/102W	500	5 pvd
	3	14			Md5	500	5 pvd
	4	14			648A	500	5 pvd
	5	14			None	500	5 pvd
	6	14		HVT+S2	Virus 1^c	500	5 pvd
	7	14			JM/102W	500	5 pvd
	8	14			Md5	500	5 pvd
	9	14			648A	500	5 pvd
	10	14			None	500	5 pvd
^aTest method A: compare mortality and tumour response to controls. Viruses that resemble JM/102W more closely than Md5 are designated as pathotype v. Viruses that induce no response are classified as nonpathogenic. Other viruses are tested further (Test B). pvd, post vaccination day; pcd, post challenge day.
^bTest method B (viruses with greater virulence than JM): compare mortality and tumour resonse to controls. Consider data from both vaccinated lots. Strains that best resemble JM/102W are classified as pathotype v. Strains that best resemble Md5 are classified as pathotype vv. Strains that best resemble 648A are classified as pathotype vv+. For strains with measurable virulence but less than that of JM, revise design to include JM/102W and CU-2 as prototype controls (omitting Md5 and 648A).
^cAdditional viruses are tested by inserting additional treatment groups.

Table 8. Lymphoid organ weights in chickens inoculated with serotype 1 MDVs^a

Pathotype	Strain	15×7 (ab−)			P2a (ab−)
		Number of trials	Bursa weight	Thymus weight	Number of trials	Bursa weight	Thymus weight
v	JM/102W	3	74.0	62.7	5	71.8	47.6
	571A	2	67.0	62.5	3	74.0	60.3
	596A	2	63.0	59.0	3	76.3	59.7
	617A	2	56.5	61.5	1	108.0	80.0
vv	Md5	3	66.0	61.7	5	65.0	53.4
	RB1B	0			1	62.0	43.0
	549A	2	57.0	58.5	3	68.3	53.7
	587A	2	60.5	59.5	3	67.3	70.3
	595	0			2	52.5	35.0
	643P	0			2	66.0	61.5
	653A	2	47.5	41.5	1	75.0	51.0
vv+	584A	0			2	66.0	51.5
	610A	2	57.5	63.5	1	88.0	66.0
	645	2	49.0	44.5	3	74.3	50.3
	648A	0			2	64.5	46.0
	648B	2	55.5	57.5	1	70.0	55.0
	651	0			2	68.0	59.5
	660A	0			2	57.0	46.0
	776	0			2	67.0	65.5
Group means
v	4	4	65.1A	61.4A	4	82.5A	61.9A
vv	7	4	57.8A	55.3A	7	65.2A	52.6A
vv+	8	3	54.0A	55.2A	8	69.4A	55.0A
^aTrials were conducted at Cornell University using previously described techniques (Calnek et al., 1998). Chickens were inoculated at 14 days of age with about 500 PFU of virus. Organs were harvested 8 days post inoculation, weighed and converted to relative weights. Data are expressed as the percent of control relative weights. Six trials were conducted; three with P2a ab− chickens, one with 15×7 ab− chickens, and two with both chicken types. Not all viruses were tested in each trial (see table for number). The number of chickens per treatment group varied from three to eight (trials 1 and 2) and from nine to 10 (trials 3 to 6). The RB1B stock was from Cornell. All other stocks were supplied by ADOL. Means followed by different uppercase letters within the same column re different (P<0.05) by Bonferroni t test (Games, 1977).

Table 9. Relationship between virulence and lymphoid organ weight depression^a

Virus	Pathotype	Virulence rank	Bursa weights (% of control)				Thymus weights (% of control)				Non-vaccinated mean
			Non-vaccinated		HVT vaccinated		Non-vaccinated		HVT vaccinated
			15×7	TK	15×7	TK	15×7	TK	15×7	TK
571A	v	19.5	67.6	80.7	95.2	95.3	46.6	39.2	73.6	83.0	58.5
596A	v	28.5	39.5	83.5	93.1	99.2	34.9	50.2	75.5	83.6	52.0
653A	vv	42.5	49.5	60.2	90.9	106.1	26.8	57.8	69.2	118.2	48.6
595	vv	61.0	50.2	76.9	101.0	120.1	44.1	36.6	78.2	86.9	52.0
610A	vv+	79.5	56.1	52.6	85.5	113.7	30.5	76.1	72.3	107.8	53.8
676	vv+	85.0	44.1	75.9	99.5	110.6	39.7	69.9	85.2	67.4	57.4
648B	vv+	87.0	57.7	63.9	81.4	113.6	54.6	64.3	75.6	65.9	60.1
660A	vv+	89.0	50.3	59.6	95.2	122.0	34.0	57.5	76.4	107.4	50.4
Correlation coefficient^b			−0.12	−0.59	−0.21	0.84	0.09	0.66	0.43	−0.10	0.14
^aEight MDV strains, representing all three pathotypes and selected to include viruses that appeared to differ for immunodepressive properties, were used. Groups of chickens of line 15×7 ab− and Hy-Vac line TK ab− that differed in MD susceptibility were either vaccinated at hatch with 2000 PFU of HVT or were left unvaccinated. All chickens were challenged at 7 days post vaccination with 500 PFU of the respective MDV strain. Non-challenged control lots for each vaccine and chicken type were also used. Chickens were killed 8 days post challenge. Body weight, relative bursa weight and relative thymus weight were determined for each chicken as described (Witter et al., 1997). The trials were conducted in four replicates; two replicates with 15×7 ab− chickens and two replicates with TK ab− chickens. Each treatment group contained about eight chickens. Weights are expressed as a per cent of control weight. The non-vaccinated mean is the mean of all non-vaccinated values, including both organs and both chicken strains. A small proportion of non-vaccinated, MDV-challenged chickens had normal organ weights and differed from other treated chickens in the same group. These were considered to be non-responders due to errors in inoculation and the data were excluded.
^bCompared with virulence rank values (determined from earlier trials).

Table 10. Relation between pathotype and neuropathotype^a

Pathotype	Number of strains	Number of strains classed as neuropathotype^b
		A	B	C
v	6	5	1	0
vv	13	1	4	8
vv+	10	0	0	10
^aData from Gimeno et al. (2002).
^bNeuropathotype classification: A, variable classical transient paralysis (TP), low TP with mortality before 15 days (acute TP), low persistent neurological disease (neurological signs starting at 19 days or later) (PND); B, hi TP, low acute TP, high PND; C, high TP, high acute TP, high PND.

Table 11. Relationship between pathotype and virus load

Trial	Virus strain	Pathotype	Chicken strain	Days post challenge	PFU/10^6 leukocytes	Real-time reverse transcriptase PCR
						ICP4/LAT	gB
A^a	JM-16	v	P2a	4 to 8	14.4±2.7
	RB1B	vv	P2a	4 to 8	41.2±5.5
	RK1	vv+	P2a	4 to 8	30.3±7.3
B^b	JM-16	v	N2a	1 to 3		0.017	0.045
	RK-1	vv+	N2a	1 to 3		0.008	0.012
	JM-16	v	N2a	4 to 5		0.483	0.577
	RK-1	vv+	N2a	4 to 5		0.736*	0.752
	JM-16	v	N2a	6 to 10		0.039	0.196
	RK-1	vv+	N2a	6 to 10		0.316*	0.509*
C^c	R2/13	Virulent	15×7 ab−	14	137
	R2/22	Virulent	15×7 ab−	14	127
	R2/23	Avirulent	15×7 ab−	14	19
	R2/29	Avirulent	15×7 ab−	14	31
^aData from Calnek et al. (1998). Data are pooled from 30 birds/group over 4 to 8 days post inoculation. Leukocytes were obtained from spleens.
^bData from Schat and coworkers (Yunis et al., 2004). RNA was harvested from spleens between 1 and 10 days post infection (d.p.i.) and the levels of ICP4/LAT and gB transcripts were measured by real-time reverse transcriptse-PCR. Data are the mean number of ICP4/LAT and gB transcripts during the prelytic (1 to 3 d.p.i.), lytic (4 to 5 d.p.i.) and latent (6 to 10 d.p.i.) period. The values were normalized for cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript copies. * Significantly different at P<0.05 between JM-16 and RK-1 for the same period post infection.
^cData from Witter (1991). Data are from four clones of Md11/75C/R2 that vary in virulence. Leukocytes were obtained from buffy coat preparations.

Table 12. Suggested serotype 1 prototype strains for best fit pathotyping

Strain	Pathotype	Virulence rank	Clone	Passage	ATCC number	Neuropathotype	Reference
JM/102W	v	17.8	Yes	11 to 13 DEF	VR-585	A	Sevoian et al. (1962), Stephens et al. (1976)
Md5	vv	55.7	No	5 to 7 DEF	VR-987	B	Witter et al. (1980)
648A	vv+	80.9	No	6 to 8 DEF	VR-1576	C	Witter (1997)

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