Lymphocyte transformation suppression caused by pyoderma—Failure to demonstrate it in uncomplicated demodectic mange

Comp. lmmun. Microhiol. b!]ect. Dis. Vol. 6. pp. 9- 17. 1983

0147-9571/83/010009-09503.00/0 Copyright © 1983 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

ORIGINAL

LYMPHOCYTE

PAPERS

TRANSFORMATION

BY P Y O D E R M A - - F A I L U R E IT IN U N C O M P L I C A T E D

SUPPRESSION

CAUSED

TO DEMONSTRATE DEMODECTIC

MANGE

OTA BARTA, CELESTEWALTMAN, PRISCILLA P. OYEKAN, RENEE K. MCGRATH a n d THOMAS N . HRIBERNIK Department of Veterinary Microbiology and Parasitology, Clinical Immunology Research Unit and Veterinary Teaching Hospital, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, U.S.A. Abstract Three dogs with demodectic mange uncomplicated by a bacterial infection and 9 dogs with demodectic mange and pyoderma were tested for their lymphocyte response to phytomitogens in vitro and for the presence of the serum's lymphocyte immunoregulatory factors (SLIF) suppressing blastogenesis. None of the 3 dogs with uncomplicated demodectic mange showed any detectable dysfunction of their lymphocytes or presence of the blastogenesis suppressing SLIF. Their lymphocytes generally responded to the mitogens with more blastogenesis than lymphocytes from healthy controls. On the other hand, in the group of 9 dogs with demodicosis c~mplicated by a bacterial infection, high levels of the blastogenesis suppressing SLIF for concanavalin A-sensitive cells were detected in 4 dogs, for phytohemagglutinin-sensitive cells in 2 dogs, and for pokeweed mitogen-sensitive cells in 1 (of only 3 tested) dog. Dysfunction of lymphocytes per se (detected by a decreased blastogenesis in nonsuppressive normal canine and bovine sera) was detected in 3 dogs with demodicosis with pyoderma. The success of the treatment of demodectic mange or the bacterial skin infection did not correlate with the previous presence or absence of the blastogenesis suppressing SLIF. The treatment of pyoderma was less successful in dogs with an increase in blastogenesis of unstimulated cells in fresh normal canine serum over that in autologous serum. All 3 dogs with a detected dysfunction of their lymphocytes either died or were euthanatized as untreatable cases. It is concluded that the development of demodectic mange per se did not cause the appearance of the blastogenesis suppressing SLIF, which was primarily related to the appearance and extent of the secondary bacterial skin infection. K e y words: Dog, demodectic mange, lymphocyte blastogenesis, blastogenesis suppression, serum's blastogenesis regulating factors, pyoderma

SUPPRESSION DE LA TRANSFORMATION LYMPHOCYTAIRE CAUSI~E PAR LA PYODERMITE ET ABSENTE CHEZ LES CHIENS DEMODECIQUES SANS COMPLICATIONS Resum6--Trois chiens d6mod6ciques sans complication bact~rienne et 9 chiens d6mod6ciques avec pyodermite ont 6t6 6xamin6s quant fi la r6sponse de leur lymphocytes fi une stimulation par des phytomitog6nes in vitro et vis /i vis de la pr6sence des facteurs s6riques immunordgulateurs des lymphocytes (SLIF) reprimant la blastogdn6se. Les 3 chiens ddmod6ciques sans complication n'ont pr6sent6 ni dysfonction lymphocytaire ddtectable ni SLIF reprimant la blastogdn6se. Leurs lymphocytes ont r6pondu 5_ la stimulation par des mitog6nes avec une blastog6n6se plus importante que celle des contr61es sains. D'autre part, parmi les 9 chiens avec une d6mod6cie compliqu6e par l'infection bact6rienne, 4 ont eu des taux hauts de SLIF reprimant la blastog6n6se des cellules r+pondant ~ la stimulation par la concanavaline A, 2 pour les cellules r6pondant 5. la stimulation par la phytoh6magglutinine et 1 (sur 3 6xamin6s seulement) pour les cellules rdpondant ~i la stimulation par le pokeweed mitog6n. Le dysfonctionnement des lymphocytes per se (d6termin6 par la r6pression de la blastog6n6se dans le s6rum du chien sain et du f6tus bovin) a ~t6 mis en 6vidence chez 3 chiens avec pyod~mod6cie. Le succ~s du traitement n'a pas 6t6 corr616 la pr6sence ou l'absence de SLIF r6primant la blastog6n6se. Le traitement 6tait plus difficile chez les chiens ayant une blastog6n6se des cellules non stimul6es dans du s6rum frais de chien sain augment6e par rapport 5_ la blastog6n6se dans du s6rum autologue. Les 3 chiens avec dysfonctionnement lymphocytaire sont morts ou ont 6t6 euthanasi6s, le traitement demeurant inefficace. On peut conclure que le d6velopement de la d6mod6cie per se n'6tait pas la raison de

10

OTA BARTAet al. l'apparition de SLIF rdprimant la blastog6nese celle-ci ayant un rapport avec Fapparition et l'extension des infectionscutanndes secondaires. Mots-cl~js: Chien, d6mod6cie, blastogdnese lymphocytaire, suppression de la blastogdn6se, facteurs seriques regulant la blastogdnese, pyodermite

INTRODUCTION D e m o d e x canis has been recognized as a normal inhabitant of canine skin for a number of years [,1, 2]. A clinical disease called demodectic mange (demodicosis, demodicidosis, red mange, follicular mange, acarus mange) is associated with an increased number of D e m o d e x canis in the skin and develops in some dogs for unexplained reasons in many areas around the world [3]. Some authors believe that generalized demodicosis is a disease due to hypersensitivity to mites, bacteria or other factors associated with an increased infestation of the skin with the mites. The hypersensitivity theory was supported by the evidence that (i) histamine levels in the skin of demodectic dogs were found to be 8- to 10-times that of normal dogs; (ii) there is an increased number of mast cells in the skin of diseased dogs; (iii) the histopathology of demodectic skin resembles that of delayed hypersensitivity, including the increased infiltration of mononuclear cells [-4-7]. On the other hand, the absence of delayed hypersensitivity reaction in the skin to a crude demodex antigen in dogs with demodicosis, as compared to the presence of this reaction in normal dogs, challenges the theory of hypersensitivity [-8]. The first experimental indication that immunodeficiency may contribute to the development of demodicosis appeared in a report stating that injections of immunosuppressive antilymphocyte sera supported the development of clinical demodicosis in puppies, the untreated littermates of which remained free of the disease [-9]. A number of subsequent studies reported a suppressed responsiveness of lymphocytes to phytomitogens due to the blastogenesis suppressing serum factors in demodectic dogs [10 13]. The function of the lymphocytes was reported unimpaired if they were cultured in media with healthy dog sera [11 14]. An elevation of alpha- and beta-globulins in the suppressive sera was reported by some researchers [-14]. The suppressive factors were reported recently to be present in the beta-globulin fraction of the serum [,15], whereas earlier they were suggested to be immunoglobulins or antigen antibody complexes [,10] as based on circumstantial evidence. The immunosuppressive theory of the development of demodicosis may be questioned in the light of the discovery that treatment of diseased dogs with levamisol, which is suggested to be a lymphocyte stimulator, was ineffective in killing mites or causing remission of the lesions, though the lymphocyte function was partially restored even in the presence of the blastogenesis suppressing serum [13]. Despite continuing studies on demodectic mange, the role of the immune response in the development of demodicosis remains questionable. This study was undertaken to (i) recheck the correlation between demodicosis and the appearance of the blastogenesis suppressing SLIF, (ii) check the possibility of correlation of the blastogenesis suppressing SLIF appearance with the presence of bacterial skin infection, and (iii) to check if the original presence or absence of the blastogenesis suppressing SLIF has a correlation with the outcome of the disease.

Lymphocytetransformation suppression caused by pyoderma

11

MATERIALS AND METHODS

Animals and treatment Clinical patients of the Veterinary Teaching Hospital of the School of Veterinary Medicine were used in the study. The clinical diagnosis of demodicosis was always confirmed by the finding of increased numbers of mites in skin scrapings. The clinical diagnosis of poyderma was confirmed in most cases by positive bacterial cultivations (see Table 2) and by histopathologic examinations of the skin samples of euthanatized dogs. The dogs with pyoderma were treated with Tribrissen®, * the dogs with demodectic mange were treated with dips in Ronnellt or an experimental mitocidal nonorganophosphate compound. + The doses, the mode of application and the treatment regime were as recommended by the producers of the products. The treatment was applied until the clinical signs disappeared or until euthanasia was elected in dogs not showing an improvement within 3 months or longer, if the owner wished to continue with the treatment. The evaluation of the efficacy of the treatment procedure or products is beyond the scope of this study.

Blood sampling Blood samples were taken from the jugular vein. Samples for lymphocyte transformation were defibrinated by shaking with glass beads. The samples for serum collection were allowed to clot at room temperature for 1 hr and the serum was harvested after centrifugation.

Lymphocyte transformation The lymphocyte transformation test was done as described in detail elsewhere [-16]. In brief, the test was carried out in flat bottom microtiter plates with 2 x 105 viable cells per well. The medium (RPMI 1640)§ was supplemented with 0.03~o L-glutamine, sodium bicarbonate (20 g/1000 ml) penicillin G (100 IU/ml), and streptomycin sulfate (0.1 mg/ml). The medium in wells contained a final concentration of 20~o (v/v) of serum. Each patient's lymphocytes were tested with all of the following serum additions: fresh autologous, heat-inactivated (56°C, 30 min) autologous, fresh 'normal' (nonsuppressive) canine, heat-inactivated 'normal' canine, and heat-inactivated bovine fetal sera. The lymphocytes from a healthy control dog tested simultaneously had the same serum additions as above with the exception that the patient's serum was used instead of the 'normal' canine serum; the autologous serum from the control animal represented the 'normal' canine serum in this case. All sera were filter sterilized before their addition to the media. The total well content was 0.2 ml to which 5 #1 of one of the following mitogens was added to make the following final concentrations of the mitogens in the wells: concanavalin A (Con A)t (5 and 15 #g/ml), phytohemagglutinin (PHA)II (1 and 5 #g/ml), and, in a limited number of tests, pokeweed mitogen (PWM) II (0.2 and 2 #g/ml). The lower concentrations represented the suboptimal, the higher concentrations the optimal doses of mitogens for lymphocytes from a majority of clinically healthy dogs. The optimal and suboptimal * Tribrissen; Wellcome Veterinary Division, Burrough Wellcome Co., Triange Park, NC 27709, U.S.A. t Ronnell; "Ectoral" by Pitman-Moore, Inc., Washington Crossing, NJ 08560, U.S.A.; "Purina Spray and Dip" by Ralston Purina Co., Checkerboard Square, St. Louis. MO 63188, U.S.A. ++Upjohn Company, Kalamazoo, MI 49001, U.S.A. § Grand Island Biological Company, New York, NY, U.S.A. ILSigma Chemicals, St. Louis MO, U.S.A.

12

OTA BARTA et al.

doses are not absolute and vary not only with the batch of mitogen, but also among the individual dogs and their momentary health condition. It was practically impossible to run a dose curve with each of the lymphocytes. With each animal there were 4 control wells used with lymphocytes without additions of the mitogens and 3 wells for each mitogen concentration and the given serum addition. The cells were incubated for 3 days at 37°C in 5~o C O / a t m o s p h e r e , then labeled with 3H-thymidine (1 ~Ci/well) and reincubated for an additional 16 hr. The optimal incubation time may also vary with animals and their health condition, but the selected time was found optimal for most healthy dogs, and it was impossible to test the optimal time for all lymphocytes. After the incubation, the cells were harvested on fiber glass filters using a cell harvester* and the radioactivity of the samples was determined after treatment with Concifluort-toluene scintillation cocktail in a scintillation counter.+ The results were expressed in counts per minute (cpm) for each sample and in the stimulation indices (S.I.) using the following equation: S.I. = cpm of mitogen treated cells cpm of untreated cells grown in the same medium-serum mixture" RESULTS

Variation of lymphocyte transformation results in healthy doys Six clinically healthy dogs were used in our laboratory as donors of 'normal' lymphocytes and serum. Three of these dogs were used in the experiments described below. The variation of results in 26 lymphocyte transformations performed on lymphocytes from 6 dogs is summarized in Table 1. Only data obtained with fresh autologous serum are included in the table to allow for comparison with the data given in Table 2. Heatinactivation changed the results in healthy dogs only insignificantly in the average, but caused a significantly lower cpm in some dogs' lymphocytes. In the light of the results presented in the experimental group (Table 2, dog No. 10104) it cannot be excluded that * Skatron, AS, Lierbyen, Norway. + Concifluor, Mallinckrodt Chemical Works, St. Louis, MO, U.S.A. ~. Analytic 92 Liquid Scintillation System, Model 6892, Searle Analytic, Des Plaines, IL, U.S.A. Table 1. Variation of lymphocyte transformation results in clinically healthy dogs tested in 20"/o autologous serum*

Serum Autologous fresh

Autologous heat-activated

Mitogen None Con A PHA PWM None ('on A PHA PWM

Final concentration (llg m[)

15 5 2 15 5 2

Counts per minute ( x 10 3)+ Minimal Maximal Mean 0 170 430 471 240 0.173 8.9 8.0 I 1.5

0.480 88.1 87.0 57.5 0.487 102.7 80.6 74.3

0.295 67,2 68.6 42.2 0.275 52.5 43.6 40.8

Minimal

Stimulation index+ Maximal

Mean

190 12"7 58

389 269 161

260 186 [22

24 22 31

445 367 286

205 I32 131

* Data from 26 lymphocyte transformations with lymphocytes from 6 dogs tested in RPMI 1640 medium with an addition of 20~o serum. The counts per minute used in the table were obtained with both fresh and heat-inactivated serum in simultaneous tests. t Numbers rounded to the nearest 100 for mitogen stimulated cells. ++The stimulation index wdues are taken from the actual values obtained within the group and were not calculated from the counts per minute in this table.

ND

ND

Staph, mrreus~

Staph. am'eus

NDPf

81157

8080

5514

6802

5485

Demodic. cured. pododerma not Demodic. cured. pododerma not

Cured for over 12 months Cured for over 3 monlhs Cured: relapse w0hin 3 months

No response to earlier treatment: ow ner declined further Sfuph. am'eus Cured for over 12 months Staph. epiderm.¶ No response to Pseudomoml~ tier', treatment Stuph. allr~'lt5 Died uncured w0hin t months for other causes

NDP

0.245 0,209

0.213

(} 117

1.096

0.10~

0252

0.170

0.185

0071

0.136

0490

0105

Without mitogen

18.3 (I.65

0.269

0 1I 9

3~.2

0.037

3.1

76.1

9. I

35.5

108.8

143.5

1655 293

§ Staphylococcus aureus. [I Pseudontonas aeruginosa. ¶ Staphylococcus epidermidis.

ND 0.290

49

69

ND 0.78(I

22 0.92

0.215

0.081

14.6 0.49

I

ND

55 2.1

1,3

1

90 4.4

I

0,7

59 2.3++

48

ND

0.231

0.674

0.124

1.586

102

109

0.260

0.130

Severe demodicosis complicated by generalized pyoderma 797 ND 30 7~ ND

0.4

12

ND

108

0.225

ND

ND

Demodicosis complicated by pyoderma ND 448 673

20

ND

105

27.6

114 2

12.8

0.064

591

290

0.125

Demodicosis complicated by localized pododerma 64.2 ND 502 883 ND

800

ND

204

86.4

31.1

187,5

1 I 1.8

83.3

72.4

44.3

159.5

103.3

172.1

172

14.7

90.3

17.3

1349

95

88.6

52.9

388

80.9

123.6

160

31.2

9.1

527

ND

ND

ND

ND

ND

62. I

ND

ND

ND

ND

Counts per minute ( x 10 31 in media with 20% fresh healthy dog serum Without Con A PHA PWM mitogen (159g ml) 15,ug ml) (21tg ml)

0.139

ND

ND

Uncomplicated demodicosis ND 1569 548

Stimulation index Con A PHA PWM

ND

8(I.4

142.2

57.5

Corrals per minute ( x 10 31 in media ~ith 20". fresh aulologous serum Con A PHA PWM (15 ~g roll (5 ,ug ml) (2 ug ml)

* N D = not determined; the clinical status did not indicate bacterial infections of the lesions. f N D P = no bacterial isolation done, p y o d e r m a determined clinically. + Values determined in media with 20"~a of heat-inactivated autologous serum.

10104

10706

8099

7544

7858

7504

ND*

7321

Outcome of the disease

Cured: relapse within 3 months Staph. moeus Partial response: Kh'hsiella sp. never cured Pseudonlonas cter. cl~mplelely Staph allrelts Cured: relapse v, ithin 3 months

Bacteria isolated

Pa0ent number

Table 2. L y m p h o c y t e transformation results with cells from dogs with d e m o d i c o s i s

88

128

251

118

497

11)6

280

341

2484

743

597

1376

64

134

140

85

422

114

204

298

1260

889

555

250

39

78

ND

ND

ND

ND

ND

479

ND

ND

ND

ND

Stimulation index ('on A PHA PWM

O.

o~ e'~

O

~"

~. pz

a"

"~

14

OTA BARTA et al.

these dogs had at the time of sampling a subclinical infection, most probably viral, which caused the suppression of lymphocyte blastogenesis only mildly and only after the heatinactivation of the serum. Further experiments with pathogen- or germ-free animals may clarify this finding.

Lymphocyte transformation results from dogs with demodicosis Only selected data are presented in Table 2 giving the results obtained with lymphocytes grown in fresh autologous and fresh 'normal' (nonsuppressive) dog serum. Other important results are presented in the text below. All animals were tested before the first treatment in our Hospital. Heat-inactivation of the serum did not change the results significantly with one exception. The fresh serum of No. 10104 caused only a partial suppression of lymphocyte blastogenesis, whereas after heat-inactivation it suppressed the blastogenesis almost completely. The same dog's lymphocytes also transformed poorly in the media with all other serum additions indicating the lymphocyte hypofunctions per se. It is highly probable that the dog suffered from other undiagnosed infections as well. The cases of a significant suppression of lymphocyte blastogenesis by heatinactivated serum (not observed in fresh serum) may be important in detecting some subclinical infections. This is also indicated by some results from our clinically healthy controls in Table 1. Increased blastogenesis in 'normal' serum over that in autologous serum (780, 1586 and 674 cpm, respectively in No. 7504, 7544 and 10706) was detected in all 3 animals responding poorly or not at all to treatment.

Correlation between appearance of demodectic mange and lymphocyte blastogenesis There was no detectable presence of blastogenesis suppressing SLIF or lymphocyte dysfunction (as judged by lymphocyte transformations in bovine fetal serum--data not presented in Table 2--and in 'normal' canine serum) in the dogs with demodectic mange uncomplicated by pyoderma. The S.I. of these dogs exceeded that obtained with lymphocytes from healthy controls when tested in both autologous and homologous sera. This indicates that the lymphocytes per se were rather hyperactive than suppressed. No blastogenesis suppressing SLIF was detected in those dogs when tested on both autologous and homologous (healthy control) canine cells. Despite the absence of blastogenesis suppressing SLIF, the treatment of 1 of the 3 dogs was unsuccessful. Presence of high numbers of mites in skin scrapings and clinical demodectic mange did not cause appearance of blastogenesis suppressing SLIF in these patients.

Correlation between bacterial skin infections and lymphocyte transformation results There was a significant degrees of correlation between the presence and extent of pyoderma and the appearance of blastogenesis suppressing SLIF in our patients. Animals without a clinical pyoderma had no blastogenesis suppressing SLIF. Animals with localized pododermatitis had low levels of the blastogenesis suppressing SLIF as it is obvious from their cpm's (No. 5514 and 6802). The S.I. of No. 5514 were high primarily due to the low background cpm of unstimulated cells. The suppression was mostly expressed on Con A-sensitive cells. The lymphocytes of No. 6802 were slightly less responsive to the stimulation than the cells from the other dog with the same disease as judged from the response in a homologous 'normal' dog serum. However, they were still within the response limits of clinically healthy dog's lymphocytes (Table 1). On the other hand, dogs with extensive generalized pyoderma had usually high levels of the blastoge-

Lymphocytetransformation suppression caused by pyoderma

15

nesis suppressing SLIF (e.g. Nos. 8099 and 10706), which completely suppressed blastogenesis of lymphocytes in the dogs' own serum. The lymphocytes of these dogs responded similarly as from the 'normal' dogs (8099) or within the range of the 'normal' dogs (10706) in a 'normal' serum. The third dog with the diagnosis of generalized pyoderma was treated unsuccessfully before its presentation to our hospital and the owner declined treatment for financial reasons. This dog's lymphocytes reacted unconventionally in that they had abnormally high background cpm of unstimulated cells (No. 7544) when tested in any serum source. The suppression of blastogenesis by this patient serum was relatively mild and again more expressed on Con A-sensitive lymphocytes. Unfortunately, the dog was not checked by bacterial cultivation. Variable levels of the blastogenesis suppressing SLIF were detected in the remaining dogs with clinically diagnosed demodicosis with pyoderma of lesser superficial extent: it ranged from absence of detectable suppression (No. 5485) to a severe suppression of Con A-induced blastogenesis (No. 7858). All dogs in this group had relatively little affected PHA-induced blastogenesis. In all cases the blastogenesis suppressing sera expressed a similar effect on lymphocytes from 'normal' dogs: nonsuppressive sera did not suppress the control lymphocyte blastogenesis, whereas suppressive sera did to an extent similar to that presented on autologous cells.

Correlation between the lymphocyte transformation results and the outcome of the disease There was no detectable correlation between the presence or absence of the blastogenesis suppressing SLIF and the results of treatment. For instance, dog No. 5514 with absence of serum suppressive effects was euthanatized as an untreatable case with demodicosis and a localized pododermatitis, whereas dog No. 8099 with generalized demodicosis and pyoderma, and with a high degree of suppression by its serum before treatment was cured for at least 1 yr after treatment. Histocompatibility restriction of the blastoyenesis suppressin9 SLIF There was not detectable histocompatibility restriction for the action of the blastogenesis suppressing SLIF; blastogenesis of both the patient and the 'normal' control dog cells was suppressed. However, the extent of the suppression was not necessarily identical. Heat-stability of blastogenesis suppressin9 SLI F Blastogenesis suppressing SLIF appearing with pyodermal infections due to Staphylococcus aureus or mixed infections of S. aureus, Klebsiella sp., and Pseudomrmas aeruyinosa was heat-stable at 56°C for 30 min. The results obtained with heat-inactivated sera (not presented) were similar to those obtained with fresh sera (Table 2). The only noticeable exception was discussed earlier and represented not a removal of the suppressive serum effect, but the appearance of it after heat-inactivation. Target cells for blastogenesis suppressin9 SLIF In the cases of strong blastogenesis suppression (Nos. 8099 and 10706) SLIF induced by pyodermas expressed a broad range of suppression affecting all subpopulations of lymphocytes being stimulated by any of the three mitogens, Con A, PHA and PWM. In 2 dogs with a milder form of pyoderma (Nos. 7504 and 7858), the suppression affected primarily the Con A-sensitive cells with a very mild effect on PHA-sensitive cells. In 2

16

OTA BARTA et al.

other dogs (Nos. 6802 and 7544) the suppression was only mild, but still affecting the Con A-sensitive cells slightly more than the PHA-sensitive. Because the dose curve of these sera was not tested in many cases, we cannot determine the dose effect on the final result. DISCUSSION As reported earlier by other authors [-10-14], we have also found a number of demodectic dog sera which had blastogenesis-suppressing activity on canine lymphocytes. However, in studies reported by others [13, 15], as well as our study, some of the patient sera did not contain any detectable blastogenesis suppressing activity. After a careful screening of all available clinical and laboratory data, we could diagnose only 3 dogs as having demodectic mange without bacterial contaminations. None of these dogs had a detectable blastogenesis suppressing SLIF in their sera. Thus we failed to support the statement that a "demonstrated T cell suppression is associated with the large population of mites" [17], or the theory of production of lesions through an immunosuppressive mechanism [8]. On the other hand, in the group of 9 dogs with demodicosis complicated by pyoderma only 1 dog (with a clinically diagnosed mild pyoderma) had undetectable blastogenesis suppressing SLIF. Serum of 2 dogs had a mild blastogenesis suppressing activity; one of them had a localized pododerma, the other clinically diagnosed pyoderma. All other dogs had various levels of blastogenesis suppressing SLIF, 3 of them so severe that the lymphocytes treated with mitogens did not divide at all in media containing their sera. Although bacterial infections of demodectic lesions were indicated in some of the previous studies [14], the blastogenesis suppressing SLIF activity was attributed to the mites [17] rather than to bacteria. Our studies indicate that the mites are not the causative agents for induction of the blastogenesis suppressing SLIF. The bacterial infection secondary to demodectic mange in the present study or juvenile pyoderma without an increase in the number of mites [18] usually lead to development of the blastogenesis suppressing SLIF, always in cases with severe pyodermas. Because mild or local infections caused little blastogenesis suppressing effect in the sera, deeper infiltration of bacteria or bacteremia is probably the prerequisite for the blastogenesis suppressing SLIF appearance. The outcome of treatment of pyoderma with Tribrissen and of demodicosis with anti-mite dips did not correlate directly with the presence or absence of the blastogenesis suppressing SLIF in the serum at the beginning of treatment. One of our animals had a suppressed lymphocyte transformation due to the lymphocyte hypofunction. This has not been reported elsewhere in connection with demodicosis and we also suggest that this hyporesponsiveness to phytomitogens may have been caused by viral or bacterial infections unrelated to the skin lesions, because a number of infections may induce the blastogenesis suppressing SLIF formation [18]. The blastogenesis suppressing SLIF from this animal also differed from the others in that it expressed a stronger activity after heat-inactivation. This was not the feature of the blastogenesis suppressing SLIF in other dogs in this study. By using three different phytomitogens (Con A, a suggested broad T cell stimulator; PHA, a suggested T effector cell stimulator; PWM, a suggested broad B or B and T cell stimulator) in optimal and suboptimal doses we found that in most cases the Con Asensitive cells were the most affected in accordance with other authors [-17]. However, all

Lymphocyte transformation suppression caused by pyoderma

17

three s u b p o p u l a t i o n s of lymphocytes usually showed decreased blastogenesis in highly suppressive sera. The s u b o p t i m a l doses of the mitogens helped in detecting weakly suppressive sera that gave only a m a r g i n a l suppression when o p t i m a l doses were used. The blastogenesis suppresssing S L I F induced by p y o d e r m a s was heat-stable at 56~C for 30 min. It may be n o t e d here that all 3 dogs with demodicosis and p y o d e r m a that responded poorly or not at all to the t r e a t m e n t had increased b a c k g r o u n d blastogenesis of u n s t i m u lated lymphocytes in wells with fresh ' n o r m a l ' dog serum over that o b t a i n e d in fresh a u t o l o g o u s serum. The significance of this p h e n o m e n o n had to be studied in a larger n u m b e r of cases. The significance of the blastogenesis suppressing S L I F in the d e v e l o p m e n t of demodectic m a n g e is highly questionable. O u r results indicate that the serum mediated i m m u n o suppression in demodicosis is due to the secondary bacterial infections a n d is absent in a n i m a l s with u n c o m p l i c a t e d demodectic mange. The finding of h y p o f u n c t i o n of lymphocytes p e r se is rare and usually associated with dogs indicating presence of other infections. Demodicosis obviously developed in dogs with a n o r m a l l y m p h o c y t e responsiveness to p h y t o m i t o g e n s a n d in the absence of any detectable blastogenesis suppressing S L I F in their sera. Acknowledqements The study was supported in part by a grant from the Morris Animal Foundation, Englewood, Colorado, U.S.A. and by the Biomedical Research Development Grant RR-09087 from N.I.H., Bethesda, Maryland, U.S.A.

REFERENCES 1. Koutz, F. R., Demodex folliculorum studies. VI. The internal phase of canine demodectic mange. J. Am. vet. reed. Ass. 131, 45 48 (1957). 2. Yamane, O. and Sako, S., Studies on canine demodicosis. I. Demodectic mite population in normal dogs, J. Jap. vet. reed. Ass. 10, 362-365 (1957). 3. Gaafar, S. M., Demodectic mange. In Current Veterinary Therapy III. (Ed. by Kirk, R.) pp. 261 263 W. B. Saunders, Philadelphia (1968). 4. Copeman, D. B., Histamine in canine demodicosis. M.S. Thesis. Purdue University, Lafayette, Indiana (1965). 5. Krawiec, D. R., Relationship of canine demodicosis to allergic dermititis. M.S. Thesis, Purdue University, Lafayette, Indiana (1973). 6. Gaafar, S. M., Pathogenie de la demodecie canine. Proc. World Assoc. Adv. vet. Parasitol., Lyon. France (1967). 7. Sheahan, B. J. and Gaafar, S. M., Histologic and histochemical changes in cutaneous lesions of capine demodicosis, Am. J. vet. Res. 31, 1241-1243 (1970). 8. Bell, F. G. and Farris, R. A., A discourse and proposal on the genesis of generalized demodectic mange: A theory of production of lesions through an immunosuppressive mechanism, Western Vet. 1, 21 24 (1973). 9. Owen, L. N., Demodectic mange in dogs immunosuppressed with anti-lymphocyte serum, Transplantation 13, 616-617 (1972). 10. Corbett, R., Banks, K., Hinrichs, D. et al., Cellular immune responsiveness in dogs with demodectic mange. Transplant. Proc. 7, 557-559 (1975). 11. Scott, D. W., Farrow, B. R. H. and Schultz, R. D., Studies on the therapeutic and immunologic aspects of generalized demodectic mange in the dogs, J. Am. anita. Hosp. Ass. 10, 233 244 (1974). 12. Scott, D. W., Further studies on the immunologic and therapeutic aspects of canine demodicosis, J. Am. vet. med. Ass. 167, 855 (Abs), (1975). 13. Scott, D. W., Schultz0 R. D. and Baker, E., Further studies on the therapeutic and immunologic aspects of generalized demodectic mange in the dog, J. Am. anita. Hosp. Ass. 12, 203 213 (1976). 14. Hirsch, D. C., Baker, B., Wigger, N. et al., Suppression of in vitro lymphocyte transformation by serum from dogs with generalized demodicosis Am. J. vet. Res. 36, 1591 1595 (1975). 15. Krawiec, D. R. and Gaafar, S. M., Studies on the immunology of canine demodicosis, J. Am. anita. Hosp. Assoc. 16, 669 676 (1980). C.I.M.I.D. 6 / I - - B

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