Vetennary a~ tmmunolo~

and - ~ unmunopathology E LS EV 1ER Veterinary Immunology and Immunopathology

44 (1994) 1-12

Analysis of leucocytes and lymphocyte subsets for different clinical stages of naturally acquired feline

immunodeficiency virus infection

Catherine Walker*, Paul J. Canfield, Dana N Love

Department o/I~eterlnary Pathology Umversttl' of Svdne~ Svdne~ N S g 2006, 4ustraha

Accepted 21 December 1993

A b s t r a c t

We report alteratmns in leucocytes numbers and lympho%te subset percentages, deter- mined by flow cytometry, for three observed and prevlousb defined clinical stages (asymptomat ic carrier (AC), MDS-rela ted complex (ARC) and AIDS) of naturally oc- curring FIV infection Unstaged FIV-posltl~e cats had significantly lower numbers of total leucoc:~tes (WCC) and neutrophlls, lower percentages of P a n T + and C D 4 + , lower CD4 CD8 ratio and a higher percentage of B cells compared with unstaged FIV-negatlve cats When FIV-posltlve cats were separated into clinical stages and compared with matched FIV-negatlve cats, AC FIV-posltlVe cats had a significantly lower WCC, lower absolute numbers of neutrophlls and 15 mphocytes, lower percentages of CD4 + and CD8 + cells, lower CD4 CD8 ratios and a higher percentage of B cells than health) FIV-negatlve cats ARC FIV-positive cats had lower percentages of PanT + and CD4 + cells, lower CD4 CD8 ratios and higher percentages of B cells than matched FIV-negatl~e cats FIV-posmve cats with AIDS had significantly lower percentages of CD4 + cells than matched FIV-negatl~ e cats Comparisons among the three observed chnlcal stages of FIV-posltl~ e cats showed that AC FIV-posltxve cats had signtficantb lower WCC, significantly lower absolute num- bers of neutrophlls and a slgmficantly lower percentage of CD8 + cells than ARC FIV- positive cats AIDS FIV-posmve cats had a significantly lower percentage of B cells than ~C or ARC FIV-poslt lve cats FIV-posxtive cats had a similar leucocyte response to illness as FIV-negatlve cats but had consistently lower percentages of CD4 + 15 mphocytes Thus,

*Corresponding author

0165-2427/94/$07 00 © 1994 Elsevier Science B V All rights reserved SSDI 0165-2427 ( 94 )05287-3

2 C 14 alker et al / l'etermat ~ lmmunolog~ and hmnunopatholog3 44 (1994) 1-12

m the staging of FIV, a rise in the percentage of CD8 + l)mphocytes could be used to dlstlngmsh between AC and ARC and a fall m the percentage of B l~mphocytes could dlstmgmsh AIDS from AC and ARC

Abbrevlatmns

AC, asymptomatlc carrier, ARC, AIDS-related complex, BSA, bo~me serum albumin EDTA, ethylenedlammetetraacetlc acid, FeLV, feline leukaemm v~rus, FITC, fluorescem lsoth~ocyanate, FIV, fehne ~mmunodeficlenc?y wrus, HIV, human ~mmunodeficlency ~ l- rus, Ig, lmmunoglobulln, PBL, peripheral blood lymphocytes, PBS, phosphate-buffered sahne, PGL, persistent generahsed 1)mphadenopathy, SPF specific pathogen free WCC, white cell count

1. Introduction

Experimental infection with fehne lmmunodeficlency virus (FIV) causes a transient febrile illness and neutropaenla, followed by a lymphadenopathy which may persist for some months Most cats recover to become and remain asymp- tomatic (Yamamoto et al , 1988) Many cats with naturally acquired FIV refec- tion, however, have been reported to have progressive disease leading to an AIDS- like stage characterlsed by weight loss, chronic &sease involving the oral cavity and upper respiratory tract, anorexia, anaemia and behavaoural modifications (Hopper et al , 1989, Friend et al, 1990) In an attempt to describe the progres- sive disease process of natural FIV refection, Ishlda and Tomoda (1990) pro- posed a staging system, largely based on chmcal signs, which has s~mllarltles to that established for human lmmunodeficlency virus (HIV) infection (Centers for Disease Control (CDC), 1986) However, httle attention has been paid to determining whether there are differences in values for laboratory analytes, par- tlcularly lymphocyte subsets, between the chnical stages of FIV infection Such information may increase understanding of the naturally occurring disease pro- cess and, also, validate FIV as a useful animal model for the study of HIV

Differences in lymphocyte subsets between FIV-posltlVe and FIV-negat~ve cats have been reported (Ackley et al , 1990b, Novotney et al , 1990, Barlough et al, 1991, Torten et al, 1991, Hoffmann-Fezer et al, 1992) Whilst these studies have used well-defined groups, they have not examined the influence of clinical staging on alterations in lymphocyte subsets Nor have they utillsed comparable &sease states in FIV-negat~ve cats to determine the relative impact of disease status and FIV status on alterations in lymphocyte subsets

This report describes the general effect of naturally occurring FIV infection on alterations in leucocytes and lymphocyte subsets, and the effect of &sease status on alterations in leucocytes and lymphocyte subsets for cllmcally staged FIV-pos- ltlve cats and for comparably staged FIV-negatlve cats

C H all.et et al / ~ etermarv Imrnunology and Immunopatholog~ 44 (1994) 1-12 3

2. Materials and methods

2 1 Antmals

Male and female pure-bred and domestic cats (1 5 to 18 years old) were ac- cessed through veterinary hospitals or commercial cattenes All cats were FeLV- negative (FeLV-Ag ELISA test - Vxrachek/FeLV, Synblotlcs Corp, San Diego, CA, USA) All cats were tested both with a commercially available FIV-antibody ELISA (CITE, Agntech Systems, Portland, ME, USA) and by western blot anal- ysis to determine FIV status

2 2 Western blot analyst~

FIV (Western Australian isolate, T 91 ) Infected culture fluid (1055 TCIDso ml- 1 ), generously provided by Professor W R Robinson, was centrifuged to re- move cellular debris before centrlfugatlon of the supernatant fluid at 100 000g for 2 h at 4°C to pellet the virus After resuspenslon of the pellet in phosphate- buffered saline (PBS) to 1% of its original volume, the preparation was used directly or layered onto a 25% sucrose in TNE buffer (140 mM NaC1, 10 mM Trls HC1, 1 mM EDTA) cushion and centrifuged for 2 5 h at 218 000g Virus pelleted onto the cushion was removed, and resuspended to 0 1% of the original volume in TNE Both preparations were frozen and thawed, mixed with equal volumes of sample buffer (to a final concentration of 3% sodium dodecyl sul- phate (SDS), 2% mercaptoethanol, 1 M urea, 100 mM Trxs, pH 7 6) and boiled for 3 rain

The FIV polypeptldes were separated on 12 5% w/v SDS-polyacrylamlde gels (Laemmh, 1970) and transferred to nitrocellulose membranes (Towbm et al , 1979), which were then blocked for 3 h with 5% w/v skimmed milk powder in PBS containing 0 5% v/v Tween 20 (PBS/Tween) Individual strips were incu- bated overnight at room temperature with a 1 10 dilution of cat serum prepared m blocking buffer After washing extensively in PBS/Tween, the strips were in- cubated with a 1 1000 dilution of horseradish peroxldase-conjugated goat anti- cat IgG (heavy and light chain specific) antibody (Jackson Immunoresearch Laboratories Inc, West Grove, PA, USA) at room temperature for 4 h The strips were washed extensively in PBS/Tween before colour development with 4-chloro- 1 -naphthol

2 3 Defimtlon of FIVmfectton status

In this report, a cat was considered seropositive if, on western blot, its serum showed a reaction to at least two core proteins, one of which was p24 Most pos- itive cats had three core proteins, most commonly p15, p24 and p65 A cat was classified as seronegatlve when no viral bands were detected after two western blots There was no disparity between ELISA and western blot results

4 C Walker et al / ! etermar) Immunolog) and lmmunopatholog~ 44 (1994) 1-12

2 4 Chnlcalstaglng

On the basis of well-established clinical criteria (Ishida and Tomoda, 1990, Shelton et al, 1990, Sparger, 1993), FIV-posltive cats were placed into asymp- tomatic carrier (AC), persistent generahsed lymphadenopathy (PGL), AIDS- related complex (ARC) and AIDS stages No cat showed clinical signs associated with acute FIV infection AC cats were defined as those cats which presented as clinically normal, with no history of either chronic or intermittent disease PGL was defined as mulnfocal lymph node enlargement ARC cats had a history of persistent or Intermittent chronic problems such as lower urinary tract disease, oral disease, chronic allergic bronchitis, mihary dermatitis, eoslnophihc granu- loma complex, chromc abscesses, smusms, uvems, weight loss or diarrhoea AIDS cats included those with cryptococcos~s or a terminal illness such as lymphosar- coma, lymphoid interstitial pneumoma, myelodysplasm or renal failure

FIV-negatlve cats were placed into matched groups on the basis of similar dis- eases or clinical signs' namely, healthy cats, cats with diseases as defined for ARC ( 'ARC') and cats with diseases as defined for AIDS ( 'AIDS') None of the FIV- negative cats had clinical signs consistent with PGL

2 5 Sample collection

Blood was collected by jugular venipuncture from cats that were non-tranqui1- hzed/non-anaesthetlsed, tranqullhzed with ketamme/dmzapam, or anaesthe- tlsed w~th ketamlne or ketamine/acetylpromazine Generally, samples were col- lected in the morning Into EDTA, stored at room temperature and processed on the same day, with no sample being left more than 72 h before analysis

2 6 Haematology

Total leucocytes (WCC) were determined with a Coulter Counter DN (Coul- ter Electronics Ltd , Luton, UK) Blood films were stained with Giemsa and 100 leucocytes were differentiated for every 10 × 1091- i WCC

2 7 Labelhng ofT lymphocytes wtth monoclonal antibodies

Depending on the WCC, between 25/zl and 100/~1 of whole blood was com- bined in separate tubes with between 0 025pg and 0 05pg of unconjugated mon- oclonal antibodies for feline T cells (PanT+, a CD5-hke molecule, Ackley and Cooper, 1992), T-helper cells ( C D 4 + , Ackley et al, 1990a) and T-cytotoxic/ suppressor cells (CD8 +, Klotz and Cooper, 1986) (Southern Biotechnology As- sociates, Birmingham, AL, USA) Samples were incubated in the dark at room temperature for 20 mm, washed twice with a 0 1% w/v solution of sodium azide in PBS (azlde) and centrifuged at 150g for 5 mln Secondary staining was per- formed by Incubating with FITC (fluorescein isothlocyanate) conjugated goat anti-mouse IgG (ICN Biomedlcals, Costa Mesa, CA, USA) for 20 mln in the

C Walker et al / I'etermarv lmmunolog) and lmmunopathologv 44 (I 994) I- 12 5

dark at room temperature Erythrocytes were lysed in 1 ml of 1X FACS ® Brand Lyslng Solution (Becton Dickinson, Lane Cove, N S W , Australia) for 10 rain at room temperature The samples were then washed and centrifuged twice, sta- bihsed by adding 200#1 of a 1% w/v solution of paraformaldehyde in PBS, and stored at 4°C until analysis A negative control for each sample consisted of a tube without primary antibody

2 8 Labelhng ofB lymphocytes

Bovine serum albumin (BSA) at a concentration of 1% w/v, was added to undiluted FITC-conjugated goat anti-cat IgG (heavy and light chain specific) antibody (Southern Biotechnology Associates, Birmingham, AL, USA) to reduce non-specific binding Depending on the WCC, between 25/~1 and 100#1 of whole blood was washed five times by centrifuging at 150g for 5 min and resuspendlng in azide, to eliminate binding of serum Ig and capping of surface Ig The pellet was combined with between 0 25~tg and 0 5/zg of the anti-cat IgG and incubated at 4°C for 30 mln The sample was lysed as for the T cell samples, washed twice, stablhsed and stored at 4 °C

2 9 Flow cytometrtc analysts

Lymphocyte surface labelling was analysed by a FACScan ® flow cytometer, using either the Consort 30 or Lysls II programs (Becton Dickinson, Lane Cove, N S W , Austraha) Using a dotplot of forward and 90 ° side scatter, a live lym- phocyte gate was constructed and 5000 cells were collected for each antigenic marker Fluorescence histograms of cell numbers versus fluorescence intensity were analysed, and gates delineating the positive regions set against the negative control tube

2 10 Stattsttes and calculatzons

The absolute numbers of neutrophlls and lymphocytes were calculated from the WCC and differential counts Absolute numbers within lymphocyte subsets were derived from the absolute lymphocyte count and the subset percentage ob- served by flow cytometry All results were analysed using Mxnltab statistical soft- ware (Minitab Inc, State College, PA, USA) Differences in mean values were evaluated for statistical significance (P< 0 05 ) by a Student's t-test

3.Results

Of the 93 cats studied, 51% (47/93) were FIV-posltlve After separation of FIV-posxtlve cats into clinical stages, there were 17% (8 /47) AC, 2% (1 /47) PGL, 64% (30/47) ARC and 17% (8 /47) AIDS (Table 1 ) For statistical anal- ysis, the one FIV-posltlVe cat with PGL was included in the ARC group

6 C I4 alker et al / I etermar) bmnunologv and hmnunopatholog)' 44 (1994) 1-12

Table 1 Dxsmbutlon of cats according to FIV mfecuon status and cllmcal stage

FIV-mfecnon Asymptomanc Cllmcal stage status carrier/healthy

Persistent Diseases generahsed associated 15mphadenopath-v ,,vlth ARC (PGL)

Diseases assocmted ~ t h AIDS

FIV-posltl~ e 8 l 30 8 FIV-negatlve 15 0 18 13

Total 23 1 48 2 I

Table 2 Comparison of leucocyte and lymphocyte subset values between FIV-posltlve and FIV-negatl~ e cats

FIV-posmv e FIV-negatlv e P ~ alue

Hhttecellcount 11 14 ~ (6 30) 2 1404 (6 11) 0027* ( n = 9 3 ) Neutrophds ( n = 93 ) percentage 60 3 63 0 0 44 absolute no 6 90 O 00 0 044* L~ mphoc~ tes ( n -- 93 ) percentage 28 1 ( 1 6 1 ) 207 ( 1 4 7 ) 066 absolute no 2 88 (2 71 ) 3 52 (2 39) 0 23 Pan T+ (n--93) percentage 537 (13 2) 635 (16 8) 00025* absolute no 1 65 (1 88) 2 25 (1 66) 0 11 CD4+ (n - -93) percentage 169 (6 37) 323 ( 9 1 7 ) < 0 0 0 0 1 " absolute no 0 50 (0 50) I 11 (0 51 ) < 0 0001" CDS+ 01=93) percentage 203 (10 1) 234 (8 74) 0 12 absolute no 0 75 (1 21) 0 84 (0 65) 0 66 CD4 CD8 raUo ( n = 9 3 ) 0 99 (0 46) 1 58 (0 76) < 0 0001" Bcell ( n = 8 3 ) percentage 41 7 ( 1 8 8 ) 297 ( 1 4 8 ) 00017* absolute no 1 20 (1 08) 1 17 (1 20) 092

l Absolute numbers are expressed as ~ alues × 10 91-1 2Mean (standard devlanon) *Statlsncally slgmficant ( P< 0 05 )

The effect of FIV status on leucocyte values and lymphocyte subsets is pre- sented in Table 2 Data was analysed from cats before they were grouped into clinical stages. FIV-posltlve cats had a significantly lower WCC and absolute numbers of neutrophlls, lower percentages of PanT+ and C D 4 + , lower CD4 + CD8 + ratio and higher percentage of B cells than FIV-negatlve cats

When FIV-posmve cats were separated into clinical stages and compared with FIV-negatlve cats with similar diseases or clinical signs (Table 3), the greatest

Tab

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C

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betw

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FIV

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d FI

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ats

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each

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of F

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fect

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at~

ve

P ~ a

lue

FIV

-po

slt~

ve

FIV

-neg

at~

ve

P va

lue

(n=

8)

(n=

15)

(n

=3

1)

(n=

18

) (n

=8

) (n

= 1

3)

2

CC

N

eutr

ophl

l pe

rcen

tage

ab

solu

te

Lvrn

phoc

yte

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+

perc

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lute

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D4+

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tage

ab

solu

te

CD

8+

perc

enta

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abso

lute

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CD

8 B

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l pe

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tage

abso

lute

7121

(3

13) 2

14

08 (

7 13

)

605

(10

2)

42

6(2

11

)

304

133)

2

33

I 56

)

47 6

14

9)

1 05

(0

66)

17 3

(7

17)

0

36 (

0 23

)

15 0

(3

15)

0

36 (

0 26

) 1

19 (

0 49

)

488

(13

2)

(n=

7)

1 33

(1

05)

13

37

(42

7)

0000

8*

11

94

(67

5)

569

(13

9)

04

9

583

(16

7)

7 75

(3

31)

0 00

6*

6 95

(4

52)

325

(14

5)

07

3

283

(13

4)

41

6(2

25

) 00

34*

29

(2

13

)

614

(16

9)

00

6

535

(12

9)

27

4(1

58)

00

018*

1

86

(20

8~

336

(9 2

6)

00

00

2'

173

(6 6

3)

14

5(0

77

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0001

" 0

57

(05

7)

209

(77

4)

0018

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5 (8

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95

(06

1)

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0(1

19

) 1

79

(07

1)

0028

* 0

97

(04

3)

310

(17

6)

0025

* 43

3 (1

89

) (n

=1

2)

1 72

(1

55)

0 52

1

34 (

1 09

)

64

7

(16

1)

935

60

)

29 4

14

4)

330

3071

682

179)

2

39

1 88

)

33 6

(9

94)

1

12 (

0 88

)

24 9

(6

28)

0

86 (

0 74

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70 (

0 98

)

279

(13

9)

091

(09

3)

021

12

11

(57

4)

14

77

(67

7)

03

5

"~

015

678

(25

8)

67

5

(14

6)

07

9

0 15

9

37 (

6 73

) 9

95 (

5 39

) 0

84

056

212

(23

9)

21

5

(14

5)

09

8

05

6

17

9(1

73

) 3

07

(23

9)

01

7

00

1"

605

(10

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60

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(13

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8

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6(1

88

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59

(12

3)

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7

0 00

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15 "~

(4

77)

29

8

(7 1

4)

0 00

00"

~"

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* 0

37 (

0 44

) 0

78 (

0 56

) 0

16

02

2

49

(1

62

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49

(6

28

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99

~.

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3 0

93

(18

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70

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0

86

(05

5)

12

4(0

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) 01

1

0003

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217

(11

9)

306

(11

1)

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(n=

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(n=

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1 0

13

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8 C Walker et al / Veterinary Immunology and lmmunopathologv 44 (1994) 1-12

Table 4 P values of the differences m leucocyte and l)mphocyte subset values among each cllmcal stage of FIV mfectmn

FIV-posmve FIV-negatlve

AC vs AC ~ s ARC vs Healthy Health) ~ s ARC" ARC AIDS AIDS ~ s 'AIDS" v s

"ARC . . . . AIDS"

kl CC 0 007* Neutrophd percentage 0 65 absolute 0 022* Lymphocyte percentage 0 85 absolute 0 22 PanT+ percentage 0 33 absolute 0 073 CD4 + percentage 1 0 absolute 0 1 CD8 + percentage 0 0081 * absolute 0 064 CD4 CD8 0 28 B cell percentage 0 39 absolute 0 98

0 056 0 94 0 73 0 53 0 79

0 48 0 36 0 15 0 062 0 62 0 074 0 36 0 34 0 22 0 77

0 37 0 39 0 21 0 057 0 45 0 53 0 083 0 23 0 23 0 79

0072 015 023 098 018 057 061 066 0058 016

0 50 0 33 0 87 0 33 0 22 015 028 034 0023" 070

013 048 016 016 085 041 086 073 021 040 0 24 0 63 0 93 0 023* 0 069

0 005* 0 0 l l * 0 57 0 88 0 57 0023* 0000" O l l 0 13 099

*Statistically slgmficant (P< 0 05)

&fferences m leucocyte and lymphocyte subset values were observed between AC FIV-posmve cats and healthy FIV-negatlve cats AC FIV-posltlVe cats had a sig- nificantly lower WCC, lower absolute numbers of neutrophds and lymphocytes, lower percentages ofCD4 + and CD8 + cells, lower CD4 CD8 ratms and a higher percentage of B cells than healthy FIV-negatlve cats ARC FIV-posltlve cats had lower percentages of PanT+ and C D 4 + cells, lower CD4 CD8 ratios and higher percentages of B cells than FIV-negatlve cats with similar &seases or cllmcal s~gns FIV-posmve cats with AIDS had significantly lower percentages of CD4 + cells than FIV-negatlve cats w~th similar &seases

Comparison among the three chmcal stages for FIV-posltlVe cats (Table 4) showed that AC cats had significantly lower WCC, absolute numbers of neutro- phlls and percentage of CD8 + cells than ARC cats The percentage of CD4 + cells was not significantly different among the three groups AIDS FIV-posltlve cats had a significantly lower percentage of B cells than AC or ARC FIV-posmve cats

To assess the effect of 111 health on leucocyte and lymphocyte subset values, without the added variable of FIV infection, the various clinical stages were ex-

c lk alker et al / Vete~man' Immunology and Immunopatholog~ 44 (1994) I-12 9

ammed for the FIV-negatlve cats alone (Table 4) There were no s~gmficant dif- ferences between the chmcal groups except that the CD4 CD8 raho was lower m FIV-negat~ve cats with &seases resembhng AIDS when compared with healthy cats

4. Discussion

The FIV-posltlve cats stu&ed had slgmficantly lower WCC (a result of slgmf- xcantly lower neutrophll and lymphocyte counts) at the AC stage of lnfect~on compared wxth healthy FIV-negatlve cats However, these &fferences were not present at the ARC stage and could not be used to distinguish sick FIV-posltxve from s~ck FIV-negatlve cats This suggests that FIV-posltlve cats are capable of mounting a leucocyte response to dlness in a manner s~mxlar to sick FIV-negatwe cats

Yamamoto et al (1989), Hopper et al (1989) and Shelton et al (1990) re- ported that a percentage of naturally infected cats w~th cllmcal signs of d~sease were leucopaemc, lymphopaenlc and neutropaemc However, these studxes com- pared leucocyte changes m FIV-posltlve cats with values estabhshed for healthy and not sick FIV-negatxve cats Consequently, they did not assess the impact of sickness on leucocyte levels

From this study, it ~s apparent that percentages of PanT + cells can be used to dxstlngulsh between groups of FIV-pos~txve ARC and FIV-negat~ve 'ARC' cats untd the stage of terminal &sease but that they have httle use m &stmgu~shmg among the stages for FIV-posltxve cats Novotney et al (1990) observed the per- centage of PanT + cells to be lower m FIV-poslt~ve cats when compared with both normal, control cats and FIV-negatxve cats with chronic &seases In HIV refection, however, the total T lymphocyte level remains relatively stable, com- pared wxth healthy controls, until the development of AIDS Thxs constancy of PanT+ values is due to a rise m CD8+ cells counterbalancing a fall m CD4+ cells

The percentage of CD4 + lymphocytes was slgmficantly lower m FIV-posmve cats (compared with FIV-negaUve cats) regardless of stage of refection The find- mg that alterahons m T lymphocyte subsets occur prior to the onset of cllmcal &sease ~s s~mdar to that seen xn people with HIV infection, where alterations m lymphocyte subsets occur at seroconvers~on (Glorg~ and Hultln, 1990) How- ever, the finding that the percentage of CD4+ lymphocytes was s~mdar m all stages of FIV refection xs different from that observed in HIV mfecUon, where there is a gradual depletion of CD4 + cells by approximately 10% per year from the time of seroconverslon (Cooper and Lacey, 1988) Barlough et al (1991) found that CD4 + cells were slgmficantly reduced even m cats with short-term ( ~< 10 months) infection whale Ackley et al (1990b) found that even 2 years post infection, despite reduction in CD4 + cells, there was no overt &sease m the FIV- posxhve group The reduction in the percentage of CD4+ cells m FIV-posltlve cats ~s hkely to be a major contributor to the lmmunosuppresslon of FIV mfec-

l 0 C ~all~er et al / ~ etermat v bnmunologv and Immunopathologv 44 (1994) 1-12

tion but, in this study, could not be used to dehneate the stages of FIV infection (as it is with HIV infection)

Direct comparison of FIV-posative cats and FIV-negatlve cats, without consid- eration of clinical stage, showed no significant &fferences in the percentages of CD8 + lymphocytes However, by separation into stages, differences in the per- centages ofCD8 + cells allowed distinction between AC FIV-posltive and healthy FIV-negative cats This distinction was lost with the development of disease This was due to a higher level in the percentage ofCD8 + cells in ARC compared with AC FIV-posltlVe cats and could be used to distinguish between the two stages However, a significant difference was not observed between ARC and AIDS groups This finding is in contrast to that reported for HIV infection, in which the CD8 + percentages increase at seroconverslon, plateau until the AIDS stage of illness and then increase until death (Glorgl and Hultin, 1990)

The finding of significantly lower CD4 CD8 ratios in FIV-posltlVe cats is con- Slstent with many other reports (Ackley et al, 1990b, Novotney et al , 1990, Bar- lough et al , 1991, Torten et al, 1991, Hoffmann-Fezer et al, 1992 ) Barlough et al ( 1991 ) further suggested that a CD4 CD8 ratio of less than one might be an indicator of FIV refection in individual cats However, in our study, significantly lower CD4 CD8 ratios were present for AC and ARC but not for AIDS FIV- positive cats when compared with matched FIV-negatlve cats Twenty-four per- cent (11/46) of the FIV-negative cats had CD4 CD8 ratios less than or equal to one (data not shown) Moreover, the ratio could not be used to distinguish amongst the stages of FIV-posltlve cats or indicate the onset of clinical illness Novotney et al (1990) also found no evidence for a correlation between inverted CD4 CD8 ratios and the onset of illness in FIV-positlve cats

In this study, the percentage of B lymphocytes was significantly higher in AC and ARC FIV-posltive cats when compared with matched FIV-negatlve cats However, AIDS FIV-positive cats had no significant difference in the percentage when compared with matched FIV-negative cats This was due to significantly lower percentages of B lymphocytes in the AIDS FIV-posltlve cats when com- pared with AC or ARC FIV-posltlVe cats

Other workers (Ackley et al , 1990b, Novotney et al , 1990, Hoffmann-Fezer et al , 1992) did not detect higher percentages of B cells for FIV-posltive cats, possibly because of the composition of their groups In our study, when B cell percentages were compared for combined sick FIV-positlve cats with those for healthy FIV-negatlve cats, no significant differences were detected (data not shown)

Another factor that may have produced variant results for B cells in other stud- ies was the method of analysis All other feline studies have used PBL rather than whole blood lysis to enumerate feline B cells However, medical studies suggest that whole blood methods give a truer indication of both T- and B-cell percent- ages (Froland and Natvig, 1973, Reynolds et al , 1992) Brown and Greaves (1974) demonstrated that separation techniques for lymphocytes were selective, resulting in an enrichment of B-cells

Slgmficant differences in B-cell values between HIV-posltive and healthy HIV-

C WalAet et al / I ~etermar)' Immunologv and lmmunopatholog~ 44 (1994) I - 12 11

negative people do not occur (Gmrgl and Hultln, 1990) However, increases in the number of lmmunoglobuhn-secreting lymphocytes early in HIV infection and increasing serum Ig levels throughout all stages of HIV infection suggest poly- clonal B-cell activation (Mlzuma et al , 1987, Shlrai et al , 1992) Increases in serum IgG also have been reported in FIV-posltave cats (Hopper et al , 1989. Ackley et al , 1990b)

The recently redefined classificatmn system for HIV infection is based on a matrix of three ranges of C D 4 + counts, and three chnical categories (CDC, 1992) Despite apparent differences in laboratory data between HIV and FIV, the results from our study suggest that a similar classification method could be established for FIV However, It seems that percentages ofCD8 + and B lympho- cytes rather than C D 4 + lymphocytes are more appropriate markers of immune suppression among stages of FIV infection Nevertheless, levels of CD4 + cells appear to distinguish effectively these stages from FIV-negatlve cats with similar diseases

Acknowledgements

This work was supported in part by a Faculty of Veterinary Science grant The authors thank the Clinical Pathology laboratory for use of their faclhties Thanks also to J Gulnan, M Delfin, K Gallagher, C van der Brink, M Theocharous, J Baka, F Jeltner, V Dermatls and V Hung Nguyen from Cllmcal Immunology, Royal Prince Alfred Hospital for their technical assistance and use of their flow cytometer We are very grateful to C Blxgh and Becton Dickinson for advice and materials for flow cytometry, and Dr J Qulnn, from Westmead Hospital for ad- vice on B-cell staining The FeLV tests were generously supplied by Common- wealth Serum Laboratones Thanks also to the many veterinary practitioners who referred cases to us for this study We are appreciative of the editorial assistance of Professor A Husband

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