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Enteric Pathogens in MonkeysROBERT C. GOOD, BESSIE D. MAY, AND TOSHIO KAWATOMARI
National Centerfor Primate Biology, University of California, Davis, California 95616
Received for publication 9 October 1968
From 1964 to 1967, 6,646 monkeys, representing 10 primate species, were ex-amined for Shigella and Salmonella infections upon arrival at the NationalCenter for Primate Biology. Of these animals, 12% were infected with Shigella, and75% of the Shigella isolates were S. flexneri 4. The incidence of Salmonella infec-tions decreased from 12 to 3% during the period of study. Epidemiological studies ofanimals in the colony for 90 days or more indicated no seasonal variation in theoccurrence of Shigella and Salmonella. Many of the isolates from incoming monkeysas well as from laboratory-conditioned animals were resistant to chloramphenicol,dihydrostreptomycin, and tetracycline. The possible operation of drug-resistancefactors in these infections is discussed.
In the adaptation of nonhuman primate speciesto laboratory conditions, major efforts must bedirected toward containment of enteric pathogens,specifically Shigella species which are chieflyresponsible for morbidity and mortality incolonized monkeys. The last major survey ofenteric pathogens in newly imported Macacamulatta and M. fascicularis (M. cynomolgus) wasmade in 1955 by Schneider et al. (12). Since thattime, areas of animal trapping have changed andtransportation has been facilitated so that dif-ferences in infection rates and bacterial pathogenswould be expected. Therefore, studies wereundertaken to determine the enteric and respira-tory pathogens in newly imported animals and tofollow their occurrence in animals colonizedat the National Center for Primate Biology be-tween January 1964 and December 1967. Thisreport deals with the incidence of Shigella andSalmonella species in the 10 major groups ofmonkeys represented in the colony; studies re-lating to the antibiotic susceptibility of the iso-lates are also described. Subsequent reports inthis series will deal with prophylaxis and therapyof both enteric and respiratory infections innonhuman primates.
MATERIAS AND METHODSSource and identification of primate species. The
majority of animals studied in the 1964-1965 periodwere received after being conditioned by the im-porters. In 1966 and 1967, most monkeys were ob-tained directly from their country of origin. Thespecies examined were: Aotus trivirgatus (SouthAmerica); Cercocebus atys and Cercopithecus aethiops(Africa); M. fascicularis, M. mulatta, M. nemestrina,M. radiata, M. speciosa, Presbytis cristatus, and P.entellus (Southeast Asia).
Specimen collection. Upon arrival, each animalwas weighed, tatooed, tuberculin-tested, and ex-amined for the presence of enteric pathogens. Speci-mens for bacteriological examination were taken byinserting a moistened cotton swab 7.5 cm into therectum. The swab was rotated to contact the liningof the rectum, and then was placed in Sach's bufferedglycerol-saline holding solution (3). When sampleswere to be held for less than 24 hr, they were refriger-ated; for longer periods of storage, they were frozen.
Bacteriological procedures. The procedures forisolation and identification of bacterial pathogenswere based on those of Edwards and Ewing (3).Swabs were used to inoculate Eosin Methylene BlueAgar (EMB) and Salmonella-Shigella agar (SS)plates, and were then placed in tetrathionate broth.After 24 hr of incubation, tetrathionate broth cul-tures were streaked on Brilliant Green Agar plates.All plates were examined after 24 hr of incubation,and colonies resembling Salmonella and Shigellawere selected to inoculate Triple Sugar Iron Agarslants. These cultures were incubated for 18 to 24hr, and those showing an alkaline slant and produc-ing acid or acid and gas (with or without hydrogensulfide) in the butt were examined for hydrolysis ofurea, indole production, utilization of citrate, anddeamination of phenylalanine. Cultures giving typicalreactions were then examined with Shigella or Salmo-nella typing sera (Difco). Identification of representa-tive isolates was confirmed in the laboratories ofMildred Galton or W. H. Ewing, National Com-municable Disease Center, Atlanta, Ga.
In addition to specimens examined at the time of ananimal's arrival, rectal swabs were taken periodicallyduring and after the 3-month laboratory conditioningperiod. At necropsy, swabs were made of areas ofthe ihtestine which showed evidence of gross patho-logical change.The antibiotic susceptibility of isolates was deter-
mined on Trypticase Soy Agar by use of the disctechnique. Only sensitivity discs (BBL) with high
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ENTERIC PATHOGENS IN MONKEYS
antibiotic concentrations were used to determineresistance, i.e., chloramphenicol, 30 Mg; cephalothin,30 pg; dihydrostreptomycin, 10 Mg; furazolidone, 100,Mg; neomycin, 30 Mug; novobiocin, 30 Mug; polymyxinB, 300 units; and tetracycline, 30 ug. When isolatesfrom an animal gave a mixed sensitivity pattern,only the resistant isolate was counted for inclusion inthe tables.
RESULTS
Enteric pathogens in animals entering colony.Specimens were examined from a total of 6,646monkeys representing the 10 primate speciesentering the colony during the period of study.The number of animals of each species receivedand the number of each infected with eitherSalmonella or Shigella are shown in Table 1.To indicate changes in infection rate during theperiod of study, isolations made in 1967 havebeen separated from earlier years covered inthe tables. The data in Table 1 show that A.trivirgatus and P. cristatus were not heavily in-fected upon arrival. During the 1964-1966 period,20% of newly imported P. entellus were infectedwith enteric pathogens, Salmonella being theprimary isolate. Shigella species have been iso-lated only rarely from the two species of langur,P. entellus and P. cristatus, perhaps owing totheir unique digestive tract, which includes acompartmented stomach (1).
Overall, a marked drop in infection rate wasfound during 1967. However, the data in Table 2show that this was due to a decline only in theisolation of Salmonella species. In the 1964-1966 period, S. anatum was isolated from 9%of the animals on arrival. In the fall of 1966,infections with this bacterial species began adecline which persisted through 1967. However,
even when the number of infected animals was ata peak, little clinical disease was attributable toS. anatum and none to S. stanley. These organ-isms were isolated less often as the animals be-came adapted to the laboratory environment.
Isolations of Shigella species did not declineduring the period of study, and 12% of incominganimals were infected with this group of patho-gens. S. dysenteriae 2, S. sonnei, and S. flexneri1, 2, and 6 were isolated at low but constantfrequencies. With the exception of S. flexneri2, these strains rarely caused clinical signs ofdisease in monkeys. However, the more commonS. flexneri 4 infections were most often associatedwith diarrhea or dysentery in the host.A summary of enteric pathogens found in
monkeys in direct and importer-conditionedshipments is presented in Table 3. A strikingreduction in Salmonella infections in 1967 isevident; however, the percentage of Shigellainfections was constant over the period of study.During 1964-1966, there was little difference ininfection rate between animals received directlyfrom the country of origin and those which hadbeen importer-conditioned. However, the latterwere in good health on arrival and were betterable to adapt to the laboratory environment.
Antibiotic susceptibility of enteric pathogensisolated from monkeys entering the colony. Theantibiotic susceptibility patterns of Salmonellaisolates did not vary markedly during the first3 years of study; however, a trend toward sus-ceptibility developed in the few isolates found inthe 1967 period. Comparisons of susceptibilityof S. anatum and S. stanley isolates from directand importer-conditioned shipments are givenin Table 4. Results for only four of the eight
TABLE 1. Monkeys infected with Salmonella or Shigella species at time of entry into quarters
1964-1966 1967
Primate species No. of monkeys No. of monkeys___Percentage Percentage
antibacterial agents tested are shown in Tables4, 5, and 6, since practically all of the isolateswere susceptible to furazolidone, polymyxin B,and cephalothin, but resistant to novobiocin.As the data in Table 4 indicate, the number ofS. anatwn isolates resistant to chloramphenicolwas greater among isolates from direct ship-ments than among the isolates from conditionedanimals. This finding cannot be explained be-cause histories of animals prior to arrival in ourcolony are unknown. Nevertheless, it is dis-quieting to find that about 80% of Salmonellaisolates from monkeys in direct shipments were
TABLE 2. Enteric pathogens isolated frommonkeys at time of arrival in quarters
v Number of monkeys examined in the 1964-1966period = 5,076.bNumber of monkeys examined in 1967 = 1,570.
resistant to dihydrostreptomycin and tetra-cycline.
In 1967, S. flexneri 4 isolates were susceptibleto a greater number of antibiotics than in theearlier years studied (Table 5). Even so, it wouldbe of interest not only to determine the sourceof S. flexneri 4 in imported animals but also tofind the reservoir of antibiotic-resistant strains,particularly that significant fraction which isresistant to chloramphenicol. The data in Table 6show that 24% of all S. flexneri 4 isolates fromdirectly imported animals were chloramphenicol-resistant. It is significant that about 40% ofisolates were resistant to dihydrostreptomycinand tetracycline and 13% were resistant to neo-mycin. As expected, a greater percentage ofShigella isolates from importer-conditioned ani-mals were resistant to these antibiotics. Thesusceptibility pattern of S. sonnei isolates is in-cluded in this table to represent the differencesthat exist between susceptibility of S. flexneri4 and other Shigella isolates.
Ampicillin susceptibility of 104 S. flexneri 4isolates was determined in a special study in whichsensitivity discs prepared with 10 ,ug of the drugwere used. Seventy-two cultures were susceptibleto ampicillin, and 32 were resistant. Of the 72ampicillin-sensitive cultures, 36 were resistant tochloramphenicol. Of the 32 ampicillin-resistantcultures, 31 were also resistant to chlorampheni-col. These findings indicate that a significantnumber of chloramphenicol-resistant S. flexneri4 infections can be treated with ampicillin.Monthly incidence of Salmonella and Shigella
infections. The incidence of enteric pathogens inthe entire colony was determined by routinesurveys of animals on arrival, at various inter-vals during and after the laboratory conditioningperiod, and by examination of sick and deadanimals. During the 36-month period between
TABLE 3. Isolations of Salmonella and Shigella species from monkeys in direct and conditioned shipments
1964-1966 1967
Bacterial pathogen Type of shipment No. of monkeys No. of monkeys Percen-Percentage _-tage
January 1964 and December 1966, a mean of 8%of the total colony was shedding enteric patho-gens in any 1-month period. Shigella and Salmo-nella species were each shed by approximately5% of the colony. In 1967, only 5% of themonkeys were shedding these pathogens, con-tinuing the decrease which started from a highof 12% in 1964 and declined to 6% in 1966.Shigella isolates fell from an average monthlyincidence of 9% in 1965 to 4% in 1966, but roseto 5% in 1967. The incidence of S. flexneri 4has not changed markedly and is currently 81%of all Shigella isolates.The mean population of the colony during
each of the months of study is shown in Fig. 1.
The monthly incidence of Shigella infections isshown in Fig. 2. As would be expected, the inci-dence of infection in the total colony rose withthe influx of animals in late 1965. At this time,the monkeys were moved from quarters whichdid not have adequate isolation facilities to a newarea which provided a greater measure of pro-tection against cross-infection. Therefore, during1966 and 1967 infection rates in the laboratory-conditioned colony did not fluctuate when newanimals were introduced into the quarters. Peaksof infection in the laboratory-conditioned colonywere caused by carriers and animals completingtheir 3-month period of quarantine.
Confirmation is given in Fig. 3 that Salmonellaisolations declined in mid-1966 and were inci-dental in 1967, particularly in the laboratory-conditioned colony. A test of methods and mediaproved that this was a true reduction in the in-fection rate with Salmonella species.
Variations in antibiotic susceptibility spectra ofS. flexneri 4 isolates. Susceptibility patterns ofS. flexneri 4 strains isolated from animals uponarrival, during the first, second, and third monthsof residence, and after longer than 3 monthsin residence are presented in Table 7. Duringthe first 3 months, strains resistant to chloram-phenicol, dihydrostreptomycin, and tetracyclinewere isolated with increasing frequency. Thegreatest percentage of antibiotic-resistant isolateswas obtained from animals in quarters betweendays 30 and 89. Susceptibility to cephalothin didnot change markedly, and the isolates remainedsusceptible to furazolidone and polymyxin B.The increased occurrence of chloramphenicol-
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GOOD, MAY, AND KAWATOMARI
TABLE 6. Antibiotic susceptibility of Shigella species isolatedfrom monkeys in directand conditioned shipments
resistant strains is compatible with the use ofthis drug for treatment of infections. However,resistance to tetracycline and streptomycin can-not be explained on the same basis because theseantibiotics were rarely used for treatment ofShigella infections.On the basis of antibiotic susceptibility S.
flexneri 4 isolates usually fell into one of twogroups. The first group was resistant only tonovobiocin, and the second group showed multi-ple-drug resistance, i.e., resistance to chloram-phenicol, dihydrostreptomycin, novobiocin, andtetracycline with a varying response to neo-mycin.
Salmonella infections. As shown in Table 2,S. anatwn and S. stanley account for the majorityof Salmonella isolates to date. However, during
1964 and 1965, S. cholerae-suis var. Kunzendorfwas isolated regularly from blood of animalsduring and after the laboratory-conditioningperiod, even though this isolate was rarely foundin cultures made from rectal swabs. No defin-able clinical syndrome was associated with thefinding of this pathogen, but it was often iso-lated from monkeys experimentally infected withmalaria. Since the organism could be eliminatedby penicillin therapy, infection did not constitutea threat to the colony even though 1 to 2% ofmonkeys harbored the pathogen. In early 1966,the infection rate began to decline, and sincemid-1966 few isolations have been made.
Other occasional isolates have been S. typhi-murium, S. typhimurium var. copenhagen, S.hvittingfoss, S. oslo, S. enteritidis, S. cairo, S.
FIG. 1. Mean monthly monkey population of total colony (solid line) and laboratory-conditioned colony (brokenline) during the period of study. The laboratory-conditioned colony represents those monkeys in quarters 90 daysor more.
O
1052 J. BACrERIOL.
.t
ol.
III
IIII
IIII
III
I
III
IIII
ENTERIC PATHOGENS IN MONKEYS 1053
FIG. 2. Percentage of mean total colony (solid line) and laboratory-conditioned colony (broken line) infectedwith Shigella species during each month of study.
amz0
4
0
U,4Az
hia.w
I 1964 1 1965
FiG. 3. Percentage of mean total colony (solid line) awith Salmonella species during each month of study.
newport, S. weltevreden, S. adelaide, S. cholerae-suis, S. infantis, S. java, S. heidelberg, S. san-
diego, S. senftenberg, S. cubana, S. bredeney,S. kentucky, S. eimsbuettel, and an Arizona1,4:1,2,5.
DISCUSSIONThe data presented show the common inci-
dence of enteric bacterial pathogens in newlyimported as well as laboratory-conditionedmonkeys. The two species of langurs and the onespecies of South American monkey were the onlyanimals in our colony that were not regularlyinfected with Shigella. In contrast to the resultsof the earlier study by Schneider et al. (12),the principal Shigella isolate in the present in-vestigation was S. flexneri 4 rather than S.flexneri 2. However, S. anatum was the principalSalmonella species isolated in both studies.Reduced incidence of Salmonella infection in the
1 1966 1 1967 1
and laboratory-conditioned colony (broken line) infected
past 2 years cannot be explained because his-tories of animals prior to arrival are unknown.M. mulatta has been imported in rather large
numbers from India. Since many of these animalslive near human habitations, they would be ex-pected to carry the enteric pathogens whichpredominate in the human population. However,reports by Panda and Gupta (10) on Shigellaincidence and by Nath et al. (8) on Salmonellaincidence in the Indian population do not sub-stantiate this hypothesis. Although recent studiesof the incidence of infection in wild rhesusmonkeys have not been made, a study by Car-penter and Cooke (2) in wild C. aethiops andPapio doguera in Africa failed to detect entericpathogens. The source of infection, then, stillremains unknown. One may conjecture that in-fection with S. flexneri 4 is perpetuated in ex-
porters' compounds, or that nonhuman primates
VOL. 97, 1969
*) I Iz0I- 1i49 "i
aC4
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GOOD, MAY, AND KAWATOMARI
TABLE 7. Antibiotic susceptibility of Shigella flexneri 4 isolates from monkeys in colony
Monkeys Yielding S. flexneri 4
Time in quarters
Antibiotic TotalOn arrival Day 1-day 29 Day 30-day 89 > 90 days
No .Per No. Per No. Per No. Per No. PerN. cent cent cent cent cent
represent a uniquely susceptible host for thispathogen. Many of the animals which appearhealthy on arrival carry enteric pathogens, sothat 2 to 3 weeks after arrival the combinedstress of transport plus acclimatization to a newenvironment and diet provide the conditions foran outbreak of shigellosis. Owing to nonspecificityof the typing sera, many of the isolates weremistakenly referred to as S. flexneri 3 during theearly part of this study, but better absorption ofthe commercial typing sera has resulted in identifi-cation of the organism as S. flexneri 4b.Newly imported monkeys yielded a signifi-
cant number of isolates resistant to more thanone antibiotic and presented a therapeutic prob-lem. Strains resistant to chloramphenicol wereusually resistant to streptomycin and tetracylineand on occasion were also resistant to neomycin.Even though the resistance to chloramphenicoland tetracycline could be expected to occurafter treatment with chloramphenicol (13), thedata presented in Table 7 indirectly point to theoperation of drug-resistance factors. Experi-ments by Kasuya (7), Guinee (5), and Walton(14) have demonstrated that drug resistance canbe transferred in vivo. Further studies such asthose by Salzman and Klemm (11) have indi-cated that this system is operable in humaninfections. The introduction and spread of S.flexneri with transferable drug resistance hasbeen reported by Hardy et al. (6) in a well-studied group of nonhuman primates. In their
laboratories, multiple drug resistance could betransferred from the S. flexneri isolate to Escher-ichia coli by conjugation. Preliminary studies inour laboratory have shown that multiple drugresistance can be transferred in vitro from E.coli to S. flexneri isolated from rhesus monkeys.Transfers between strains isolated from monkeysindicate that the potential is present, but experi-ments to prove in vivo transfer have not beensuccessful. Nevertheless, the advantage acquiredby the recipient enteric pathogen creates a newtherapeutic problem by virtue of the multipleantibiotic resistance.The incidence of Salmonella and Shigella in
both newly imported and laboratory-conditionedanimals was not related to seasonal changes.The principal fluctuations in infection rates inthe total colony were associated with arrival ofnew shipments. Invariably, diarrhea and dysen-tery with increased isolations of enteric patho-gens occurred 2 to 3 weeks after arrival, pre-sumably a delayed reaction to the stress ofshipment. Fluctuations in the infection rates ofanimals in the postconditioning period wereusually associated with the inadvertent intro-duction of carriers into a clean group.Therapy of monkeys showing clinical signs of
shigellosis must be aimed at elimination of theenteric pathogen plus supportive therapy tocombat acute dehydration. Chloramphenicolis the best drug for eradication of Shigella, par-ticularly when used in conjunction with furazoli-
1054 J. BAcTEiuoL.
ENTERIC PATHOGENS IN MONKEYS
done or cephalothin. In cases of chloramphenicol-resistant infection, neomycin in conjunction withfurazolidone or cephalothin has been used withsome success. Ampicillin has been reported tobe effective therapy for shigellosis in children(9), but this compound has not been used in ourquarters for treatment. Further therapeuticstudies of chloramphenicol-resistant infections areanticipated.Those animals which carry S. flexneri 4 with-
out showing signs of disease are a source of in-fection to cagemates. Even with routine exami-nations, many of these carriers go undetected.Elimination of the carrier state has been ac-complished in many cases by the antibioticregimens described above, but whether this issimply the reduction of numbers of bacilli belowthe detection level or represents total eliminationis not clear even though repeated examinationsfor enteric pathogens have been negative. Ingang cages, all monkeys must be treated pro-phylactically to rid the entire group of the in-fectious agent. The use of a vaccine such as thatdescribed by Formal et al. (4) may provide theultimate means of protection of nonhumanprimates against infection with Shigella.
ACKNOWLEDGMENTS
We thank Leon H. Schmidt, Director, National Center forPrimate Biology, University of California, Davis, for his con-tribution to certain phases of this work. We also acknowledge thetechnical assistance of M. Fowlks, F. Jones, C. Scott, J. Utz,and R. Fleishman, and particularly the aid of A. Pratt, whocooperated in computer programming.
This investigation was supported by Public Health Servicegrant FR-00169.
LITERATURE CITED
1. Bauchop, T., and R. W. Martucci. 1968. Ruminant-likedigestion of the langur monkey. Science 161:698-699.
2. Carpenter, K. P., and E. R. N. Cooke. 1965. An attempt tofind shigellae in wild primates. J. Comp. Pathol. 75:201-204.
3. Edwards, P. R., and W. H. Ewing. 1962. Identification ofEnterobacteriaceae. Burgess Publishing Co., Minneapolis.
4. Formal, S. B., T. H. Kent, S. Austin, and E. H. LaBrec. 1966.Fluorescent-antibody and histological study of vaccinatedand control monkeys challenged with Shigella flexneri. J.Bacteriol. 91:2368-2376.
5. Guinee, P. A. M. 1965. Transfer of multiple drug resistancefrom Escherlchla colU to Salmonella typhimurium in themouse intestine. Antonie van Leeuwenhoek J. Microbiol.Serol. 31:314-322.
6. Hardy, P. H., J. R. Lindsey, and E. C. Melby. 1968. Transfer-able multiple drug resistance in Enterobacteriaceae fromnonhuman primates. I. Development and characteristics ofresistance in Shigella flexnerl. Johns Hopkins Med. J.123:29-37.
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8. Nath, M. L., J. B. Shrivastav, S. K. Sethi, and J. Singh. 1966.Salmonella pattern in India. Indian J. Med. Res. 54:512-516.
9. Nelson, J. D., and K. C. Haltalin. 1967. Broad-spectrumpenicillins in enteric infections of children. Ann. N.Y. Acad.Sci. 145:414-422.
10. Panda, G. K., and S. P. Gupta. 1964. Shigella serotypes inUttar Pradesh. Indian J. Med. Res. 52:235-240.
11. Salzman, T. C., and L. Klemm. 1967. Transferable drugresistance (R factors) in Enterobacterlaceae: relationshipto nosocomial infections. Antimicrobial Agents andChemotherapy-1966, p. 212-220.
12. Schneider, N. J., E. C. Prather, A. L. Lewis, J. E. Scatterday,and A. V. Hardy. 1960. Enteric bacteriological studies in alarge colony of primates. Ann. N.Y. Acad. Sci. 85:935-941.
13. Szybalski, W., and V. Bryson. 1952. Genetic studies onmicrobial cross resistance to toxic agents. I. Cross resistanceof Escherichia coli to fifteen antibiotics. J. Bacteriol.64:489-499.
14. Walton, J. R. 1966. In vivo transfer of infectious drug re-sistance. Nature 211:312-313.