Vol. 29, No. 12JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1991, p. 2720-27230095-1137/91/122720-04$02.00/0Copyright © 1991, American Society for Microbiology

Group G Streptococcal Lymphadenitis in RatsBRIAN F. CORNING,t JAMES C. MURPHY, AND JAMES G. FOX*

Division of Comparative Medicine, Massachusetts Institute of Technology,Cambridge, Massachusetts 02139

Received 10 June 1991/Accepted 23 September 1991

Group G streptococci which have been isolated from the oral fora of rats are also normal inhabitants of thehuman skin, oropharynx, gastrointestinal tract, and female genital tract. This group of streptococci can causea wide variety of clinical diseases in humans, including septicemia, pharyngitis, endocarditis, pneumonia, andmeningitis. Ten days after oral gavage with 7,12-dimethylbenz[a]anthracene, 12 of 22 two-month-old, female,outbred, viral-antibody-free rats presented with red ocular and nasal discharges and marked swelling of thecervical region. Various degrees of firm, nonpitting edema in the region of the cervical lymph nodes andsalivary glands as well as pale mucous membranes and dehydration were observed. Pure cultures ofbeta-hemolytic streptococci were obtained from the cervical lymph nodes of three rats that were necropsied. Arapid latex test system identified the isolates to have group G-specific antigen. These streptococcal isolatesfermented trehalose and lactose but not sorbitol and inulin and did not hydrolize sodium hippurate or bileesculin. A Voges-Proskauer test was negative for all six isolates. Serologic tests to detect the presence ofimmunoglobulin G antibody to rat viral pathogens and Mycoplasma pulmonis were negative. Histopathologicchanges included acute necrotizing inflammation of the cervical lymph nodes with multiple large colonies ofcoccoid bacteria at the perimeter of the necrotic zone. To our knowledge, this is the first report of naturallyoccurring disease attributed to group G streptococci in rats.

The most common streptococcal species encountered inrats is alpha-hemolytic Streptococcus pneumoniae, whichoften colonies the oral cavity. In cases of overt clinicaldisease caused by S. pneumoniae, bacteremia and bron-chopneumonia often develop and may lead to pulmonaryconsolidation, fibrinopurulent pleuritis, pericarditis, andperitonitis (10).Group G beta-hemolytic streptococci have also been iso-

lated from the oral flora of rats (13). Their location in the oralcavity presented an opportunity for this organism to be usedto experimentally induce valvular endocarditis in rats (13).After the presence of periodontal disease and catheter-induced endocarditis was confirmed, tooth extractions wereperformed. Group G streptococci as well as other organismswere isolated from blood cultures drawn immediately aftertooth extraction. Group G streptococci were cultured fromthe aortic valves of 15 of 18 animals with experimentalendocarditis (13).Asymptomatic pharyngeal carriage of group G strepto-

cocci is found in up to 23% of humans (15). These bacteriaare normal inhabitants of the human skin, oropharynx,gastrointestinal tract, and female genital tract. However,group G streptococci can cause a wide variety of clinicaldiseases in humans, including septicemia, pharyngitis, en-docarditis, pneumonia, and meningitis (6). They are also themajor streptococcal type isolated as commensal flora fromthe skin and mucosa of dogs (2) but can also cause ab-scesses, dermatitis, polyarthritis, mastitis, and other genitalinfections (8).

Contagious streptococcal lymphadenitis caused by groupG beta-hemolytic streptococci in laboratory cats has beenreported (7, 17). Cervical lymphadenitis, also caused by

* Corresponding author.t Present address: Comparative Research Center, Rush-Presby-

terian-St. Luke's Medical Center, 1653 West Congress Parkway,Chicago, IL 60612.

group C streptococci, in horses (18), swine (11), and guineapigs (14) has been documented. To our knowledge, naturallyoccurring disease attributed to group G streptococci in ratshas not been reported. The purpose of this report is todescribe an outbreak of group G streptococcal cervicallymphadenitis in a colony of rats treated with the carcino-genic agent 7,12-dimethylbenz[a]anthracene (DMBA).

MATERIALS AND METHODS

A group of 24 two-month-old, female, outbred, viral-antibody-free rats were obtained from a commercial vendorfor the purpose of studying the efficacy of a chemotherapeu-tic agent used to treat mammary carcinomas. Animals werehoused in an American Association for the Accreditation ofLaboratory Animal Care-approved facility in groups of twoto four in polycarbonate cages (19 by 10 1/2 by 8 in. [1 in. =2.54 cm]) with Micro-Isolator (Lab Products, Inc., May-wood, N.J.) lids. They received an alpha-tocopherol-freesynthetic diet, high in unsaturated fat, for the duration of theexperiment. Water was provided ad libitum. Hardwood chipbedding was used and was changed twice weekly.

Eleven days after arrival, all rats were orally administered20 mg of DMBA in a sesame oil base by gavage. The samegavage needle was used for each rat. The procedure wasperformed under the observation of trained personnel andwas without incident. Two rats were found dead, one eachon days one and five following oral gavage. Necropsy ofthese animals was not performed.Ten days after oral gavage, 12 of the 22 treated animals

had red ocular and nasal discharges and marked swelling ofthe cervical region. Various degrees of firm, nonpittingedema in the region of the cervical lymph nodes and salivaryglands of several rats were observed. Pale mucous mem-branes and dehydration in some rats were also observed.Two of the most severely affected rats were submitted fornecropsy.Four to five days after clinical signs were first observed,

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GROUP G STREPTOCOCCAL LYMPHADENITIS IN RATS

all rats showed significant reduction in the amount of cervi-cal swelling. Food and water consumption appeared normal.One anorectic, thin rat was euthanatized and submitted fornecropsy. There have been no subsequent deaths or appar-ent recrudescence of symptoms.

Pharyngeal cultures were taken from all remaining rats ofthe affected group and from two technical personnel 7 daysafter symptoms were first observed. Fourteen days afterclinical signs in the DMBA-treated rats were noted, pharyn-geal cultures were taken from twelve rats on other experi-mental protocols, including two sentinel rats, housed in thesame room. All rats were of the same strain, originating fromthe same vendor.

Diagnostic studies. Three rats were submitted for completenecropsy. They were anesthetized with carbon dioxide, bledvia cardiac puncture, and euthanatized with an overdose ofC02. Samples of all body organs and tissues were placed inneutral buffered 10% formalin for histopathologic evalua-tion. The tissues were processed by conventional methodsand embedded in paraffin; sections were cut at 5 ktm andstained with hematoxylin and eosin. Selected tissues werestained with Giemsa and Gram's stains.Serum was evaluated by utilizing an enzyme-linked im-

munosorbent assay for detection of immunoglobulin G anti-body to the following agents: Sendai virus, sialodacryoad-enitis virus, pneumonia virus of mice, reovirus 3,Mycoplasma pulmonis, Toolan H-1 virus, and Kilham ratvirus.Samples of heart blood, cervical lymph node, and pharyn-

geal swabs were collected aseptically for microbiologicalexamination, placed onto sheep blood agar plates and intoTrypticase soy broth, and incubated at 35°C. Cultures thatproduced mixed flora were subcultured onto phenylethanolmedia selective for the isolation of gram-positive cocci;isolates from this media were then transferred to blood agarplates. Streptococcal group-specific antigens were identifiedwith a rapid latex test system (Wellcome Diagnostics, Dart-ford, England).

Biochemical tests were performed with six isolates ofstreptococci. Isolates 1 to 3 were cultured from the affectedlymph nodes of the three rats which were necropsied. Theremaining isolates, of pharyngeal origin, were selected atrandom from other, DMBA-treated, asymptomatic ratshoused in the same room. Fermentation of trehalose, sorb-itol, lactose, and inulin; hydrolysis of sodium hippurate andbile esculin; and production of acetylmethylcarbinol (Voges-Proskauer) were examined.

Antimicrobial susceptibility tests were performed with thesame six isolates of group G streptococci. Amoxicillin withclavulinic acid (10 Fg), chloramphenicol (30 ptg), sulfameth-oxazole-trimethoprim (25 Fg), cephalothin (30 p.g), erythro-mycin (15 ptg), gentamicin (10 Fg), bacitracin (10 U), neo-mycin (30 pig), and polymyxin (300 U) were employed in thedisc method of Bauer et al. by using discs prepared by DifcoLaboratories, Detroit, Mich. (1).

RESULTS

Pure cultures of beta-hemolytic streptococci were ob-tained from the cervical lymph nodes of the three rats thatwere necropsied. The rapid latex test system identified theseisolates to have group G-specific antigen.Gross postmortem findings from the first two rats nec-

ropsied were similar. These included multiple petechiae andecchymoses in the subcutaneous tissues of the thorax andabdomen, and a suffuse hemorrhage in the medial muscula-

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FIG. 1. Ventral view of the cervical region of a rat infected withgroup G streptococci. Swollen, hemorrhagic cervical lymph nodesare confluent with the salivary glands.

ture of the right rear leg of one rat. Sublumbar and mesen-teric lymph nodes were swollen and congested. Adrenalglands were also congested. Cervical lymph nodes and thesalivary glands in the cervical area were swollen and hem-orrhagic and adhered to one another, forming a single firmmass (Fig. 1). Gross abnormalities in the third rat, in additionto inanition, were limited to congestion of the cervical andinguinal lymph nodes.

Histopathologic changes in the first two rats includedacute necrotizing inflammation of the cervical lymph nodes,with multiple large colonies of coccoid bacteria at theperimeter of the necrotic zone (Fig. 2). The inflammatoryprocess extended to the adjacent salivary glands and con-nective tissue, where it was characterized by edema, fibrindeposition, and a predominantly polymorphonuclear cellinfiltration. Edema and mild polymorphonuclear cell infiltra-tion extended throughout the subcutaneous tissues of thehead. Because of thrombosis of the adrenal vein, one adrenalgland of one rat had extensive necrosis and hemorrhage thatdestroyed all but a small portion of the medulla and a thinrim of subcapsular cortex. Histopathologic changes in thethird rat were limited to accumulations of hemosiderin in thecervical lymph nodes.Group G beta-hemolytic streptococci were isolated from

the pharynges of 16 of 22 rats from the same experimentalgroup. Pharyngeal samples from 3 of 12 rats housed in thesame room but not from this experimental group (no DMBAadministered) were also positive for this organism. Pharyn-

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2722 CORNING ET AL.

FIG. 2. Hematoxylin and eosin stain showing acute necrotizing inflammation of a cervical lymph node (N), due to group G streptococcusinfection, with colonies of bacteria at the perimeter of the necrotic zone (arrowheads). The inflammatory process extends to the perinodalconnective tissue (C) and adjacent salivary glands (S). Bar = 120 ,um.

geal samples taken from the sentinel rats in the room andfrom personnel handling the affected animals were negative.

Serologic tests to detect the presence of immunoglobulinG antibody to rat viral pathogens and M. pulmonis werenegative for the three rats necropsied. Heart blood culturesfrom the necropsied rats were also negative.

All six streptococcal isolates fermented trehalose andlactose but not sorbitol and inulin and did not hydrolyzesodium hippurate or bile esculin. All isolates were negativefor the Voges-Proskauer reaction in which Streptococcusanginosus was employed as a positive control. Antimicrobialsusceptibility tests for the same isolates of group G strepto-cocci showed susceptibility to amoxicillin with clavulinicacid, bacitracin, cephalothin, chloramphenicol, and erythro-mycin. All isolates were resistant to gentamicin and neomy-cin and partially or completely resistant to polymyxin B andtetracycline. Four of the six isolates were resistant tosulfamethoxazole-trimethoprim, and two were susceptible.

DISCUSSION

Since first classified by Lancefield (12), group G strepto-cocci have been isolated from dogs (8); cats (7); nonhumanprimates (21); cattle, sheep, and swine (11); and mice (20).Streptococcus canis is the proposed official name for groupG beta-hemolytic streptococci of animal origin (4). Theinformation from biotyping and DNA hybridization studiesof animal and human group G streptococci indicates thatthey are not identical and probably do not have a highzoonotic potential (3, 16). The biochemical test results forthe six isolates examined are identical to those publishedelsewhere (18).

All rats in the room were from the same vendor but arrivedat the facility at different times over a 10-month period. Itseems unlikely to find 73% of the DMBA-dosed rats natu-rally harboring this organism, considering the 25% preva-lence noted for other rats from the same vendor. However,the lack of documentation in the literature identifying groupG streptococci in rats may also reflect a reluctance to

serogroup beta-hemolytic streptococci routinely in the pastbecause of the lengthy procedures involved. Readily avail-able kits now permit fast, reliable serogrouping.With the prevalence of group G beta-hemolytic strepto-

cocci in rats not currently known, it is only speculativewhether all of the affected animals were harboring theorganism at the time of the oral gavage procedure. Alterna-tively, the gavage needle may have served as a fomite fortransmission. Stress of the experimental protocol, potentialscarification of the oral mucosa at the time of the gavageprocedure in the presence of the group G streptococci, andthe powdered synthetic diet also may have contributed to theinitiation and progression of the cervical lymphadenitis.Trauma of the oral mucosa was not a determining factor forsuccessful infection of cats with group G streptococci (17) orfor the induction of cervical lymphadenitis in guinea pigswith group C streptococci (14). The most frequently docu-mented portal of entry of group G streptococci in humans isthe skin, although the upper respiratory tract can also serveas a site (6). It is interesting that the cervical lymphadenitiswas self-limiting in the majority of these rats, unlike strep-tococcal lymphadenitis in cats (17) and guinea pigs (14).The induction of periodontal disease in rats is frequently

accomplished by feeding them a soft synthetic diet high incarbohydrates (13). The synthetic diet used for these ratsmay have contributed to the proliferation of the streptococciin the gingival tissue.

Administration of DMBA may have played an importantrole in the pathogenesis of the disease. Oral administrationof DMBA in sesame oil to 50-day-old female Sprague-Dawley rats results in the rapid induction of breast cancer;this model is influenced by endocrine control, as is thedisease in human females (19). The side effects or toxicreactions associated with this compound may have ac-counted for the two unexplained deaths shortly after thegavage procedure, as well as for the pale mucous membranesseen in several of the rats with lymphadenitis. Importantly,DMBA has been reported to induce leukopenia as well ascarcinoma from a single 20-mg feeding (9). The leukopenic

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stage lasted approximately 10 days in the rats before signs ofrecovery from this symptom were evident (9). The clinicaldisease observed in this outbreak corresponded to the ex-pected duration of the leukopenic response previously notedfor DMBA-treated rats (9). A decrease in resistance to thestreptococcal infection occurring in the leukopenic rats mayhave contributed to the development of the cervical lym-phadenitis and may provide an explanation for the transientnature of the clinical symptoms. Increased susceptibility tostreptococcal disease as a result of neutropenia in alcoholicswith folate deficiency has been identified (6).Group B beta-hemolytic streptococcus was isolated from

one animal in the surveillance group. Commonly associatedwith mastitis in livestock, group B Streptococcus agalactiaehas also been isolated and has been known to cause diseasein humans, dogs, and mice (6, 8, 20). Its importance in theoral flora of rats is unknown.The oral flora cultured from rats, or other species, may

vary depending on the place of origin or environmentalfactors. Under the appropriate conditions, opportunisticpathogens can cause disease in the host. Although group Gstreptococci have previously been identified as normal oralflora in rats, they can under certain conditions cause cervicallymphadenitis and should be included in the differentialdiagnosis, along with sialodacryoadenitis virus, as a cause ofcervical swelling in rats (10). Experimental transmissionstudies with the streptococcus group G isolates from thisoutbreak are needed to elucidate the pathogenic potential ofthis organism in the immunocompetent rat.

ACKNOWLEDGMENTS

This work was supported in part by Public Health Service grantsRR01046 and RR07036-03 from the Division of Research Resources,National Institutes of Health.We thank Dorothy Green, Christine Zanella, and Kat Weeks for

their assistance with this report.

REFERENCES1. Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck.

1966. Antibiotic susceptibility testing by a standardized singledisk method. Am. J. Clin. Pathol. 45:493-496.

2. Biberstein, E. L., C. Brown, and T. Smith. 1980. Serogroups andbiotypes among beta-hemolytic streptococci of canine origin. J.Clin. Microbiol. 11:558-561.

3. Clark, R. B., J. F. Berrafati, J. M. Janda, and E. J. Bottone.1984. Biotyping and exoenzyme profiling as an aid in thedifferentiation of human from bovine group G streptococci. J.Clin. Microbiol. 20:706-710.

4. Devriese, L. A., J. Hommez, R. Kilpper-Balz, and K.-H. Schle-ifer. 1986. Streptococcus canis sp. nov.: a species of group Gstreptococci from animals. Int. J. Syst. Bacteriol. 36:422-425.

5. Ganaway, J. R. 1976. Bacterial, mycoplasma, and rickettsial

diseases, p. 122-125. In J. E. Wagner and P. J. Manning (ed.),The biology of the guinea pig. Academic Press, Inc., New York.

6. Ginsburg, I. 1986. Streptococcus, p. 242-253. In A. I. Braude,C. E. Davis, and J. Fierer (ed.), Infectious diseases and medicalmicrobiology. The W. B. Saunders Co., Philadelphia.

7. Goldman, P. M., and T. D. Moore. 1973. Spontaneous Lance-field group G streptococcal infection in a random source catcolony. Lab. Anim. Sci. 23:565-566.

8. Greene, C. E. (ed.). 1990. Infectious diseases of the dog and cat,p. 599-610. The W. B. Saunders Co., Philadelphia.

9. Huggins, C., L. C. Grand, and F. P. Brillantes. 1961. Mammarycancer induced by a single feeding of polynuclear hydrocarbons,and its suppression. Nature (London) 189:204-207.

10. Jacoby, R. O., P. N. Bhatt, and A. M. Jonas. 1979. Viraldiseases, p. 286. In H. J. Baker, J. R. Lindsey, and S. H.Weisbroth (ed.), The laboratory rat, vol. I. Academic Press,Inc., New York.

11. Kraftz, G. E., and H. W. Dunne. 1965. An attempt to classifystreptococci isolates from domestic animals. Am. J. Vet. Res.26:951-959.

12. Lancefield, R. C. 1933. A serological differentiation of humanand other groups of hemolytic streptococci. J. Exp. Med.57:571-595.

13. Moreillon, P., C. D. Overholser, R. Malinverni, J. Bille, andM. P. Glauser. 1988. Predictors of endocarditis in isolates fromcultures of blood following dental extractions in rats withperiodontal disease. J. Infect. Dis. 157:990-995.

14. Murphy, J. C., J. I. Ackerman, R. P. Marini, and J. G. Fox.1991. Cervical Iymphadenitis in guinea pigs: infection via intactocular and nasal mucosa by Streptococcus zooepidemicus. Lab.Anim. Sci. 41:251-254.

15. Rolston, K. V. I. 1986. Group G streptococcal infections. Arch.Intern. Med. 146:857-858.

16. Simpson, W. J., J. C. Robbins, and P. P. Cleary. 1987. Evidencefor group A-related M protein genes in human but not animal-associated group G streptococcal pathogens. Microb. Pathog.3:339-350.

17. Swindle, M. M., O. Narayan, M. Luzarraga, and D. L. Bobbie.1980. Contagious streptococcal lymphadenitis in cats. J. Am.Vet. Med. Assoc. 177:829-830.

18. Timony, J. F., J. H. Gillespie, F. W. Scott, and J. E. Barlough(ed.). 1988. Hagan and Bruner's microbiology and infectiousdiseases of domestic animals, 8th ed., p. 182. Comstock Pub-lishing Association, Ithaca, N.Y.

19. Williams, G. M., and J. H. Weisburger. 1986. Chemical carcin-ogens, p. 107. In C. D. Klaassen, M. O. Amdur, and J. Doull(ed.), Casarett and Doull's toxicology-the basic science ofpoisons, 3rd ed. Macmillan Publishing Co., New York.

20. Williford, C. B., and J. E. Wagner. 1982. Bacterial and mycoticdiseases of the integumentary system, p. 62. In H. L. Foster,J. D. Small, and J. G. Fox (ed.), The mouse in biomedicalresearch, vol. Il. Academic Press, Inc., New York.

21. Wilson, G. S., and A. A. Miles (ed.). 1964. Topley and Wilson'sprinciples of bacteriology and immunity, p. 720. The Williams &Wilkins Co., Baltimore.

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