J. Protozool., 25(4)., 1978, pp. 497-501 @ 1978 b y the Society of Protozoologlsts
Differences in the Lifecycles Between a Vaccine Strain and an Unmodified Strain of Babesia bovis (Babes, 1889) in the Tick BooPhilus microplus (Canestrid* N. P. STEWART Animal ReJearrh Institute, Yeerongpilly, Brisbane, Queensland, Australia SYNOPSIS. Developmental forms of 2 strains of Babesia bovis (Babes) were studied in the tick vector Boophilus microplus (Canestrini). One strain (designated T) was shown to be infective for the tick, and the other ( N T ) to have lost infectivity for the tick, because of repeated blood passaging in cattle. Parasites of the 2 strains in gut contents of adult female ticks were similar during the first 16 h post-repletion ( P R ) , but thcrcafter their structure differed. From 16-64 h PR, the majority of T strain parasites were spherical and without processes. During the next 32 h elongate forms and vermicules developed. Fission bodies were seen within epithelial cells of the gut by 96 h PR. T-strain parasites in gut contents decreased in number from c 96 h and were difficult to find a t 144 h, the time of the final observation. In contrast, N T strain parasites were plentiful throughout the period of observation. They were predominantly spherical, ranging in diameter from 1.5 to 15 pm. Forms with obvious processes measuring up to 81 pm in length were seen in large nurnbers at seemingly regular intervals from 16-144 h PR, suggesting that a process of development and divisions was being reprated. No vermicules or fission bodies were seen. T-strain, but not N T strain parasites, were seen in hemolymph and ova o f the ticks and in their larval progeny. It is suggested that continuous blood passaging of the N T strain had resulted in selection of parasites incapable of penetrating gut cpittielial cells of the tick. Index Key Words: Babesia bovir ; life cycles; transmissible strain and non-transmissible strain; cattle tick, BooPhilus microplzis.
N Australia, disease in cattle caused by the blood protozoan
I
Babesia bouis (Babes, 1889) is controlled by vaccination ( 2 ) . Strains of B. bovis used to produce vaccine are maintained by repeated blood-passage in splenectomized calves. This procedure attenuates the strains (2),and also causes them to lose infectivity (4, 6 ) for the onr-host tick BooPhilus microplus (Canrstrini) ( 9 ) . The present bvork was undertaken to determine the stage of the life cycle of B. bouiJ in which a vaccine strain and an unmodified strain differ. Because O’Sullivan & Callow ( 6 ) observed no diffrrcnce in the blood forms of 2 such strains in present studies I concentratrd on parasite development in the tick.
MATERIALS AND METHODS Strains of B. bovis.-The 2 strains studied, designated B and T by Dalgliesh & Stcwart (4), were shown by them to be nontransmisrible and transmissible, respectively, by B. microplus. For clarity, thc 2 strains are referred to in this paper as N T (nontransmissible) and T (transmissible) strain. Strain of B. microplus.-The strain used was designated A by Callow & Hoyte ( 1 ), who described its origin. General Procedure.-During the 3 year course of this study, NT strain of B. buuis, which had already been serially blood-passaged 63 times ( 4 ) , was passaged an additional 39 times. O n 5 occasions, after the 63rd, 65th, 70th, 75th and 102nd passages, an attempt was made to infect ticks with the strain by allowing them to feed on acutely infected Uos tauru.s cattle, and microscopic observations were made on parasites ingested by adult female ticks. Since B. microplus normally transmits B. bouis transovarially, transmission tests wrre done by cultivating some of the collected ticks and releasing their larvae on susceptible, splenectomized calves. To serve as a control for these observations, T strain of B. bovis was passaged, by blood inoculation and periodic tick-transmission,
* The work was supported by funds provided by the Australian Meat Research Committee and the Queensland Department of Primary Industries. 497
during the same 3 year period: parasite structurr i n ticks and infectivity for ticks were studied on 6 occasions. Attempts to Infect Ticks.-Cattle were housed individually in moated concrete pens and exposed t o Babpsia-free larval ticks. T h e cattle were inoculated intravenously 14-18 days latrr Tt-ith blood containing either NT or T strain of B. bocis so that parasites were readily detected in thin films of thcir peripheral blood when adult ticks on them were rngorging 18-24 days after larval infestation. Collection, Culture, and Examinntion of Adult Ticks.Adult female ticks that tvere partially to fully engorged w r e c.01lected, incubated a t 28-30 C and > 90% relativc humidity, and examined at 8-h intervals. Initial observations r e \ d c d no differences between parasites in apparently replete ticks plucked from ti h the cattle and those that had fallen from the host \?ittiin before collection. Subsequently, therefore, only ticks taken from the host were examined. Smear preparations of the gut and its contents iverc mndc froni 20 ticks a t each examination, and of hemolyniph froni the siinie number of ticks on days 4 to 12 post-repletion ( P R ). Smears of ova were made before oviposition by dissecting out the ovaries and squashing them between 2 glass slides; aftcr oviposition smears were made in the same way by squashing batches of 100 ova. Smears were air dried a t 50 C, fixed with methyl alcohol, stained with Gicmsi’s stain and examined under oil ininiersion. Examination of Larval Progeny and Infectiuity Tests.-Sonrr of the ova from adult ticks were held at 30 C until hatched. ‘Thc larvae were stored at 14 C and > 90% relative humidity for varying periods n p to 4 weeks until placed on susceptible cattlc. At least 2 g of larvae (- 40,000 ticks) were used at each infestation. Squash preparations were made of 100 larvae t ak m daily from these cattle on days 1-3 after infestation, and prepared as previously described for examination under an oil immersion objective. Thick films of blood taken from thr cattle daily froin day 7 after infestation were examined microscopically for B. bocis. Serum was collected from cattle with no evidence of infection by day 28 after infestation. It was subjected to the indirzct fluorrs-
-
7
498
LIFE CYCLES
OF
cent antibody test ( I F A T ) for babesial antibodies as described by Callow et al. ( 3 ) ,as a further check for the presence of infection.
RESULTS Morpholo,qical Observations
In the 5 attcmpts to infect ticks with the N T strain, parasitrmias in host cattle a t the time of tick collection ranged between 1 ..5-6.00/0. Parasites were plentiful in gut contents of 80-90% of ticks examined. Parasitemias in cattle infected with T strain ranged between 0.2-5.0%, and parasites were observed in only 5-1576 of the ticks examinrd in the 6 experiments. Since the structure did not vary noticeably between experiments, a general description of parasite drvelopment in ticks is given for each strain. I n gcncral, T strain parasites seen were similar to those described by Riek ( 8 ) . Descriptions of these transmissible parasites arc prrsrnted mainly to indicate differences between them and those that had lost this quality. Parasites Obscrucd in Gut of Female Ticks Hefore Repletion,--Intact bovine erythrocytes were seen infrequently in smrars made from gut contents of semi-engorged ticks. With both the T and N T strains, single and paired intraerythrocytic forms, as seen in blood smears of the bovine host, were present, as were single cxtracellular forms. T h e latter were predominantly sphcrical, measuring 1-2.5 pm in diameter; they became more numerous as ticks approached the fully engorged state. Small processes, u p to 0.5 pm in length, were seen occasionally extending from intracellular, and more often from cxtracellular forms, of both strains. As ticks became more engorged, larger processes up to 5 p m in length were associated with intracellular forms of the N T strain but not of the T strain. Occasionally the processrs apprarrd to protrude through the surface of the host cell (Fig. I ) . During the First 24 h of Incubation.-Parasites of various forms were obsrrved in infections with both strains. Many irregularly shaped forms and pyknotic bodies were seen, suggesting that most of the ingested parasites were degenerating. After 8 h of incubation, the predominant and apparently viable form of both strains was spherical, measuring 3-5 p m in diameter, with either a linear band or several dots of chromatin-like material a t the periphery. At that time the processes observed earlier were not present. T h e spherical form persisted for the entire period in infections involving the T, but not the N T strain. With the latter, discrete masses of chromatin-like material formed a t the periphery of the parasites; some of them had a large similar mass a t their center. Toward the end of the period, NT-strain parasitcs appeared to be dividing either by budding (Fig. 2 ) or by simple binary fission (Fig. 3 ) . Division resulted in forms 3-6 pni in dianirter which devrloped processes u p to 22 p m in length (Fig. 4 ) .
Babesia bovis
499
During 24-48 h of Incubation.-T-strain parasites u w e present in small numbers during this period. They appeared to havc changed only slightly from those seen earlier, thr chromatin-likr material having become more central within thr parasites. Parasites of the N T strain were much more numerous, and had oltvioiis processes u p to 5 pni in length. Toward the end of the prriod, spherical forms without processes were also present (Fi During 48-72 h of Incubation.-T-strain parasitcs \ plentiful than a t earlier times. T h e various fornis swn \vew [ ; I ) spherical, u p to 1.5 pin in diameter, similar to thosc sern ear1it.r; ( b ) larger spherical forins 5-6 p m in diametcr with ;I centr:d nucleus; and ( c ) oval forms measuring 0.5 X 2.5 p m . Toivard the end of the period, the appearance of sonic larger spherical forms suggested that they may have bcrn undergoing a process of unfolding, resulting in elongate forms that measurcd 2-2.5X Cj-12 p m (Fig. 6 ) . NT-strain parasites were again more numcrous than those of T strain. Initially, the predominant form ~ Y I Sspherical with ;I large central nucleus. These forms appeared to undergo division, as described earlier (Figs. 2, 3 ) , which again rrsulted in parasitcs with obvious processes. Small numbers of bizarre parasitcs ~ i pto 7 p m across and with large processes were also present. No elongate parasites were seen. During 72-96 h of Incubation.-T-strain parasites wrre nu~iicrous and variable in structure. T w o distinct vcrmicular forms ivrre seen, one of which was much larger than t h r clorigate form sern earlier, being 2.5-4.5 X 16-25 p m (cf. Figs. 6 and 7 ) . I n SOIIIC smears the large number of parasites seen and thr appearance of the verrnicules suggested that these were assuming a circular form (Fig. 8 ) and dividing in the gut to produce man): sinaller, round parasites (Fig. 9 ) . T h e other vermicular form obscived closc~ly resembled those seen later in hernolymph smears (sce Fig. 1 0 ) . It appeared to develop from spherical or oval forms by a pro( unfolding (Fig. 1 I ) . These “unfolding” forms and vcriiiiciiles were generally seen in clusters, suggesting that thry inny hart been released from ruptured epithelial cells of the gut. Toward the end of the period, fission bodies (Fig. 1 2 ) comprised of numerous, irregularly-shaped parasites, each bvith a central nuclcus surrounded by intensely staining cytoplasm, ]\“’re s w n within intact epithelial cclls of the gut. NT-strain parasites were again more numcrous than thosc o f the T strain. T h e NT-strain parasites were prcdominantly round forms u p to 15 pm in diameter, either without procrssrs or \\.it11 processes of varying lengths. NT-strain parasitrs, mrasuring 9 p m in diametcr with processes u p to 81 pin in length, arc sren in Fig. 13. No vermicular forms or fission bodies were found. During 96-144 h of Incubation.-In the T strain, parasites in gut contents were similar in appearanc-e to those described iri tht. preceding section, but became fewer during the period and \\ere difficult to find by 144 h. Fission bodirs wcre see11 for u p t o 120 h. I n contrast, NT-strain parasites rrmaincd plrntiful during the entire period. Most of the forms seen \vcrc siniil;u. t o thost.
-
c Figs. 1-20, [Various stages of Babesia bovis strains T (transmissible) and N T (nontransmissible) in the tick, Boophilus rnicroplu~. .4R, after repletion of the tick. Gienisa’s stain. Except for Figs. 13 and 15, X 1,000.1 1. N T Intraerythrocytic stage before repletion. 2. NT. 24 h AR. Budding. 3. NT, 24 h AR. Binary fission. 4. NT, 24 h AR. A parasite with processes up to 2 2 pm long. 5 . NT, 48 11 AR Parasites without processes. 6. T, 64 h AR. An elongate parasite that appears to develop from a spherical form. 7. T, 96 h AR. A large vermicule in the tick gut contents. 8. T, 96 h AR. A large vermicule becoming spherical in the tick gut contents. 9. T, 96 h AR. Pnrasites believed to develop after division of large vermicules in the tick gut. 10. T, 96 h AR. A small vermicule in the hemolyniph. 11. T , 96 h AR. A parasite in the process of “unfolding” to produce a vermicular form. 12. T , 96 h AR. Fission of the parasites in a gut epithelial cell. 13. NT, 96 h AR. Parasites with processes up to 81 ym in length. X 500. 14. NT, 128-144 h AR. A round parasite. 15. NT, 128-144 h AR. Clumping of parasites. X 500. 16. T , 120 h AR. A parasite in a n ovum taken from the oviduct 17. T, vermicules pointed at both ends in a n ovum after oviposition. 18-20. T, from larvae fed on calves. Note the verniirules, after 24 h feeding (Fig. 18) ; precursors of the fission bodies, after 48 h feeding (Fig. 19) ; and a mature fission body, after 72 h feeding (Fig. 20).
TABLE 1 . Attempts
Parasite strain
iiritted hy ticks.
A T and T strainy of Babesia bovis 63’ Boophilus microplus.
t o tranrnlii
.\ttempt
Blood
xu.
’
iiassaqc‘ No.*
Results of infectivity tests on infested calf? Blood films
IF.XT%
,
iCalves \\?re infested \\it11 larval ~ ~ r i i g r noyf adult ticks that engorgcd on acutely infected cattle.
+ Indirect fluorescent : i r i t i t . d y s N.D. = not
dor1c..
trst.
sti-iicturc of parasitrs of the 2 strains in the gut contents following inyestion b y tlie tick vector was quite different. Second, NTqtr:iiii parasitrs were not dctectcd in any tissues of the ticks exailiincd. whereas diosc o f T strain were seen in epithelial cells of the , ovarian cells of the host tick, and in devrlq i t , h e m o l y ~ n p h nncl opiiig lar\,ar. NT-strain parasites were plentiful in smears of gut c~cintcmt\;,and appcar(d to undrrgo srveral cycles of development ; i n d division. It t h u s seems that the protozoa, although viable \ \ i t h i i i gut contents for m a n y hours after ingestion by ticks, were inc;ip;iblc~ of pcncttntiiiy cpithrlial crlls of the gut and infecting thc nornial vector. In :vner;il, T-strain parasites seen were similar t o those des c r i l x l b y Rick (8),althoiigh 3 additional forms were observed in snicars of gut contcnts. O n e form, a small round organism \\-it11 proressrs, \\-as wen occasionally both inside arid outside red I~lood cells in s m e a i ~ sfrom semi-engorged ticks. T h e procases noted a t that stag? riiight have lieen a n early response Iiy the pamsite to its nc\v eiivironment, but whcther they were a sign of drvclnpinrnt or degeneration could not he deduced. Two other forins not descrilied l q Riek ( 8 ) \\ere a large round or oval form 1 Fig. 1 1 ), \vhich aplicared to br “unfolding” to a verniirulnr slrapr, and a large vcrrniculc (Fig. 7 ) , unlike those wen in herno!I mph snie;irs, Ivliich appt,arrd to divide within the gut contents o f thc tick (Figs. 8. 9 j . Thesc 2 forms niay have bern r e h s e d froin gut cells r u p t u r r d duriiig preparation of the smear, although the), r\.c’Ie n c t w seen rvithin gur cells. Holbrook et 21. ( 5 ) o b s c m d round forms that apparently expanded or unfolded into \ ~ r r n i i c u l ~in~ stheir study of Babrsin cnballi (Nuttall) in the horse tick Dernincenioi- n i t ~ Ncumann, n~ and Rirk ( 7 ) C J I J S ~ ~ similar VC~ f o r n i x for Babt,,irr bigctrfina i n Boophilus w i i c ~ o ~ ~ l z i s . T h e 2 prrdorriinarlt forms of the N‘T strain wrrc round organisms .?-I 2 p i in diarnetcr, arid smaller pnrasites with obvious prcicc~scs(Figs. 4, 5 j. Forms with processes were seen at regular iiitor\.als tliroiiglioiit 144-h period following rt.pletion of tlie tick, :rritl \\(’re present in large numbers a t the final observation. These org;inisnis, nlthough c.;ip;ible of division, \vcre apparently unable caitlier to pent’triite stit crfls thcmsclves, o r to dwclop into forms ;ll)le t l ) do b 0 . I.:loiig;itc forms of the T strain \ v ( w first seen in gut contrnts of ticks incnl)nted f o r ti-t-72 h (Fig. 6 ) . Thtw parasites w i w similar t o the cigar-shaped bodics olxxrved by Rick (8) in ticks incu1);ited for 3 6 h \vhicli lie considrred to be infective for epithelial ct~llsof the gut. N o rlongate forms or verlnic-ides of the N T strain \vcrc seen. Their aiiscnce i n n y cxxplain the inability of inr~ctions ith this strain t o progrt’ss normally in ticks. I t is .;iiggrstccI that contintlous I h o d passaging of R. hovis reaiilts in ;I gradual srlvction of parasites inrapablr of penetratirig gilt epit11cli;il cells of the tick, d u r to an absence of contact with the v m o r . I t \ \ a s rvidrnt from observations 011 a strain of R. h ~ i , i istill infective f o r ticks after 43 blood passqgvs t h a t iii gut c o n t r i i t s of ticks thcrr \vas a higher proportion of parasites with pi-oc.c\srs than in the 1 sti.aiii dtwril)ed h r r r (Stewart Sr Dalgliesh, i1iipuI)liahtd tlntn j . T h e proportion of organisins with obvious p i o c v w ~111;iyI)r a n indrx of infrctivity of the strain fot the tick Y1’f.t
or.
REFERENCES 1. Callo\v LL, Hoyte HMD. 1961. Transmission experiments iisins Babesia bigemina. Theileria mutans, Borrelia sp. and the cattle tick. Boofihilus microp[us. Aust. Vet. 1. 37, 301-90. 2, ~ - _, Mellors LT. 1966. A new vaccine for Babasici nrg~7ifinainfection prepared in splmectoinised calves. Au.rt. V e t . J . 42. 464-65. 3 . --, McGregor W, Parker RJ, Dalgliesh RJ. ‘The imi i i u n i t y of cattle t o Raberin orgentinn after drug sterilisation of infections of varying duration. Azist. V e t . J . 50, 6-11. ~
LIFE CYCLES OF Babesia bovis 4. Dalgliesh RJ, Stewart NP. 1977. Failure of vaccine strains of Babesia bouis to regain Infectivity for ticks during long-standing infections in cattle. Aust. Pet. J. 53, 429-31. 5. Holbrook AA, Anthony DW, Johnson AJ. 1968. Observations on the development of Babesia caballi (Nuttall) in the tropical horse tick Vermacentor nitens Neumann. 1. Protozool. 15, 391-96. 6. O’Sullivan PJ, Callow LL. 1966. Loss of infectivity of a vaccine strain of Babesia argentina for Boophilus microplus. Aust. Pet. J. a, 252-54.
BOOK REVIEW.
501
7. Riek RF. 1964. The life cycle of Babesia bigemina (Smith and Kilborne, 1893) in the tick vector Boophilus microplus (Canestrini). Aust. J . Agric. Res. 15, 802-21. 8. __ 1966. The life cycle of Babesia argentina (Lignieres, 1903) (Sporozoa: Piroplasmidea) in the tick vector Boophilus microplus (Canestrini). Aust. J . Agric. Res. 17, 247-54. 9. Seddon HR. 1952. Diseases of domestic animals in Australia. Part 4. Protozoan and viral diseases. V e p t . Hlth. Sero. Publ., Commonwealth Government Printer, Canberra, Australia.
..
Reissig, J. L., ed. 1977. Microbial Interactions. Vol. 3 of Receptors and Recognition, Series B. Chapman and Hall, London. x 436 pp. $39.50.
+
Contents: Aggregation and Cell Surface Recefitors in Cellular Slime Molds, P. C . Newell; Bacterial Chemotaxis, G. L. Hazelbauer & J. s. Parkinson; Bacterial Receptors for Phages and Colicins as Constituents uf Specific Transport Systems, V. Braiin & K. Hartke; T h e Attachment of Bacteria to the Surfaces of Animal Cells, G. W. Jones; Binding and Entry of D N A in Bacterial Transformation, S. A. Lacks; A Redefinition of the Mating Phenomenon in Bacteria, M. Achtman & R. Skurray; Cell-Cell Interactions During Mating in Saccharomyces cereviseae, T. R. Manney & J. H. Meade; Mating Interactions in Chlaniydomonas, U. W. Goodenough; Cell-Cell Interactions in Ciliates: Evolutionary and Genetic Constraints, D. L. Nanney; A n Ouerviezer, J. L. Reissig. These chapters cover various aspects of chemosensory reception and communication between microorganisms and the microbial environment. Appropriately, emphasis is on the receptors themselves. In the rapidly growing field of microbial sensory-motor physiology, most available information is at the receptor level; in the majority of cases, little is known about sensory transduction mechanisms following receptor stimulation. Of direct concern to
protozoologists are the chapters on cellular slime molds, Chlamgdomonas, and ciliates, though some of the others will be of general interest; we particularly recommend the excellent review of bacterial cheinokinpsis by Hazelbailer & Parkinson. Newell’s detailed discussion of acrasian chemoreception shobvs the maturity of this field. It is a very clear piece of writing, with a fine feeling for quantitative aspects of the subject, now coming to the fore. Goodenough’s review leaves an impression that, despite a great deal of research using sophisticated tools, surprisingly little is known about sensory aspects of Chlamydonionas mating; this chapter is marred a bit by continual, disconcerting reference to unpublished work from the author’s laboratory, \vhile important papers by Gibor and others go unmentioned. Nanney’s review would be a good one to show to a cell biologist or biochemist to get him interestcd in the protozoa: a very clcar exposition, in which historical roots are unearthed, profundities of the subject are well displayed, and current experimental dilemmas are discussed. T h e price is a bit high for students, but microbiologists and others working on chemoreception should find this a useful, stimulating addition to the library. We congratulate the editor on his fine choice of timely topics and interesting authors.--M. LEVANDOWSKY & D. HAUSEK, Haskins Laborntories of Pace University, 41 Park Row, N e w Y o r k hlY 10038, USA.
Differences in the Lifecycles Between a Vaccine Strain and an Unmodified Strain of Babesia bovis (Babes, 1889) in the Tick BooPhilus microplus (Canestrid* N. P. STEWART Animal ReJearrh Institute, Yeerongpilly, Brisbane, Queensland, Australia SYNOPSIS. Developmental forms of 2 strains of Babesia bovis (Babes) were studied in the tick vector Boophilus microplus (Canestrini). One strain (designated T) was shown to be infective for the tick, and the other ( N T ) to have lost infectivity for the tick, because of repeated blood passaging in cattle. Parasites of the 2 strains in gut contents of adult female ticks were similar during the first 16 h post-repletion ( P R ) , but thcrcafter their structure differed. From 16-64 h PR, the majority of T strain parasites were spherical and without processes. During the next 32 h elongate forms and vermicules developed. Fission bodies were seen within epithelial cells of the gut by 96 h PR. T-strain parasites in gut contents decreased in number from c 96 h and were difficult to find a t 144 h, the time of the final observation. In contrast, N T strain parasites were plentiful throughout the period of observation. They were predominantly spherical, ranging in diameter from 1.5 to 15 pm. Forms with obvious processes measuring up to 81 pm in length were seen in large nurnbers at seemingly regular intervals from 16-144 h PR, suggesting that a process of development and divisions was being reprated. No vermicules or fission bodies were seen. T-strain, but not N T strain parasites, were seen in hemolymph and ova o f the ticks and in their larval progeny. It is suggested that continuous blood passaging of the N T strain had resulted in selection of parasites incapable of penetrating gut cpittielial cells of the tick. Index Key Words: Babesia bovir ; life cycles; transmissible strain and non-transmissible strain; cattle tick, BooPhilus microplzis.
N Australia, disease in cattle caused by the blood protozoan
I
Babesia bouis (Babes, 1889) is controlled by vaccination ( 2 ) . Strains of B. bovis used to produce vaccine are maintained by repeated blood-passage in splenectomized calves. This procedure attenuates the strains (2),and also causes them to lose infectivity (4, 6 ) for the onr-host tick BooPhilus microplus (Canrstrini) ( 9 ) . The present bvork was undertaken to determine the stage of the life cycle of B. bouiJ in which a vaccine strain and an unmodified strain differ. Because O’Sullivan & Callow ( 6 ) observed no diffrrcnce in the blood forms of 2 such strains in present studies I concentratrd on parasite development in the tick.
MATERIALS AND METHODS Strains of B. bovis.-The 2 strains studied, designated B and T by Dalgliesh & Stcwart (4), were shown by them to be nontransmisrible and transmissible, respectively, by B. microplus. For clarity, thc 2 strains are referred to in this paper as N T (nontransmissible) and T (transmissible) strain. Strain of B. microplus.-The strain used was designated A by Callow & Hoyte ( 1 ), who described its origin. General Procedure.-During the 3 year course of this study, NT strain of B. buuis, which had already been serially blood-passaged 63 times ( 4 ) , was passaged an additional 39 times. O n 5 occasions, after the 63rd, 65th, 70th, 75th and 102nd passages, an attempt was made to infect ticks with the strain by allowing them to feed on acutely infected Uos tauru.s cattle, and microscopic observations were made on parasites ingested by adult female ticks. Since B. microplus normally transmits B. bouis transovarially, transmission tests wrre done by cultivating some of the collected ticks and releasing their larvae on susceptible, splenectomized calves. To serve as a control for these observations, T strain of B. bovis was passaged, by blood inoculation and periodic tick-transmission,
* The work was supported by funds provided by the Australian Meat Research Committee and the Queensland Department of Primary Industries. 497
during the same 3 year period: parasite structurr i n ticks and infectivity for ticks were studied on 6 occasions. Attempts to Infect Ticks.-Cattle were housed individually in moated concrete pens and exposed t o Babpsia-free larval ticks. T h e cattle were inoculated intravenously 14-18 days latrr Tt-ith blood containing either NT or T strain of B. bocis so that parasites were readily detected in thin films of thcir peripheral blood when adult ticks on them were rngorging 18-24 days after larval infestation. Collection, Culture, and Examinntion of Adult Ticks.Adult female ticks that tvere partially to fully engorged w r e c.01lected, incubated a t 28-30 C and > 90% relativc humidity, and examined at 8-h intervals. Initial observations r e \ d c d no differences between parasites in apparently replete ticks plucked from ti h the cattle and those that had fallen from the host \?ittiin before collection. Subsequently, therefore, only ticks taken from the host were examined. Smear preparations of the gut and its contents iverc mndc froni 20 ticks a t each examination, and of hemolyniph froni the siinie number of ticks on days 4 to 12 post-repletion ( P R ). Smears of ova were made before oviposition by dissecting out the ovaries and squashing them between 2 glass slides; aftcr oviposition smears were made in the same way by squashing batches of 100 ova. Smears were air dried a t 50 C, fixed with methyl alcohol, stained with Gicmsi’s stain and examined under oil ininiersion. Examination of Larval Progeny and Infectiuity Tests.-Sonrr of the ova from adult ticks were held at 30 C until hatched. ‘Thc larvae were stored at 14 C and > 90% relative humidity for varying periods n p to 4 weeks until placed on susceptible cattlc. At least 2 g of larvae (- 40,000 ticks) were used at each infestation. Squash preparations were made of 100 larvae t ak m daily from these cattle on days 1-3 after infestation, and prepared as previously described for examination under an oil immersion objective. Thick films of blood taken from thr cattle daily froin day 7 after infestation were examined microscopically for B. bocis. Serum was collected from cattle with no evidence of infection by day 28 after infestation. It was subjected to the indirzct fluorrs-
-
7
498
LIFE CYCLES
OF
cent antibody test ( I F A T ) for babesial antibodies as described by Callow et al. ( 3 ) ,as a further check for the presence of infection.
RESULTS Morpholo,qical Observations
In the 5 attcmpts to infect ticks with the N T strain, parasitrmias in host cattle a t the time of tick collection ranged between 1 ..5-6.00/0. Parasites were plentiful in gut contents of 80-90% of ticks examined. Parasitemias in cattle infected with T strain ranged between 0.2-5.0%, and parasites were observed in only 5-1576 of the ticks examinrd in the 6 experiments. Since the structure did not vary noticeably between experiments, a general description of parasite drvelopment in ticks is given for each strain. I n gcncral, T strain parasites seen were similar to those described by Riek ( 8 ) . Descriptions of these transmissible parasites arc prrsrnted mainly to indicate differences between them and those that had lost this quality. Parasites Obscrucd in Gut of Female Ticks Hefore Repletion,--Intact bovine erythrocytes were seen infrequently in smrars made from gut contents of semi-engorged ticks. With both the T and N T strains, single and paired intraerythrocytic forms, as seen in blood smears of the bovine host, were present, as were single cxtracellular forms. T h e latter were predominantly sphcrical, measuring 1-2.5 pm in diameter; they became more numerous as ticks approached the fully engorged state. Small processes, u p to 0.5 pm in length, were seen occasionally extending from intracellular, and more often from cxtracellular forms, of both strains. As ticks became more engorged, larger processes up to 5 p m in length were associated with intracellular forms of the N T strain but not of the T strain. Occasionally the processrs apprarrd to protrude through the surface of the host cell (Fig. I ) . During the First 24 h of Incubation.-Parasites of various forms were obsrrved in infections with both strains. Many irregularly shaped forms and pyknotic bodies were seen, suggesting that most of the ingested parasites were degenerating. After 8 h of incubation, the predominant and apparently viable form of both strains was spherical, measuring 3-5 p m in diameter, with either a linear band or several dots of chromatin-like material a t the periphery. At that time the processes observed earlier were not present. T h e spherical form persisted for the entire period in infections involving the T, but not the N T strain. With the latter, discrete masses of chromatin-like material formed a t the periphery of the parasites; some of them had a large similar mass a t their center. Toward the end of the period, NT-strain parasitcs appeared to be dividing either by budding (Fig. 2 ) or by simple binary fission (Fig. 3 ) . Division resulted in forms 3-6 pni in dianirter which devrloped processes u p to 22 p m in length (Fig. 4 ) .
Babesia bovis
499
During 24-48 h of Incubation.-T-strain parasites u w e present in small numbers during this period. They appeared to havc changed only slightly from those seen earlier, thr chromatin-likr material having become more central within thr parasites. Parasites of the N T strain were much more numerous, and had oltvioiis processes u p to 5 pni in length. Toward the end of the prriod, spherical forms without processes were also present (Fi During 48-72 h of Incubation.-T-strain parasitcs \ plentiful than a t earlier times. T h e various fornis swn \vew [ ; I ) spherical, u p to 1.5 pin in diameter, similar to thosc sern ear1it.r; ( b ) larger spherical forins 5-6 p m in diametcr with ;I centr:d nucleus; and ( c ) oval forms measuring 0.5 X 2.5 p m . Toivard the end of the period, the appearance of sonic larger spherical forms suggested that they may have bcrn undergoing a process of unfolding, resulting in elongate forms that measurcd 2-2.5X Cj-12 p m (Fig. 6 ) . NT-strain parasites were again more numcrous than those of T strain. Initially, the predominant form ~ Y I Sspherical with ;I large central nucleus. These forms appeared to undergo division, as described earlier (Figs. 2, 3 ) , which again rrsulted in parasitcs with obvious processes. Small numbers of bizarre parasitcs ~ i pto 7 p m across and with large processes were also present. No elongate parasites were seen. During 72-96 h of Incubation.-T-strain parasites wrre nu~iicrous and variable in structure. T w o distinct vcrmicular forms ivrre seen, one of which was much larger than t h r clorigate form sern earlier, being 2.5-4.5 X 16-25 p m (cf. Figs. 6 and 7 ) . I n SOIIIC smears the large number of parasites seen and thr appearance of the verrnicules suggested that these were assuming a circular form (Fig. 8 ) and dividing in the gut to produce man): sinaller, round parasites (Fig. 9 ) . T h e other vermicular form obscived closc~ly resembled those seen later in hernolymph smears (sce Fig. 1 0 ) . It appeared to develop from spherical or oval forms by a pro( unfolding (Fig. 1 I ) . These “unfolding” forms and vcriiiiciiles were generally seen in clusters, suggesting that thry inny hart been released from ruptured epithelial cells of the gut. Toward the end of the period, fission bodies (Fig. 1 2 ) comprised of numerous, irregularly-shaped parasites, each bvith a central nuclcus surrounded by intensely staining cytoplasm, ]\“’re s w n within intact epithelial cclls of the gut. NT-strain parasites were again more numcrous than thosc o f the T strain. T h e NT-strain parasites were prcdominantly round forms u p to 15 pm in diameter, either without procrssrs or \\.it11 processes of varying lengths. NT-strain parasitrs, mrasuring 9 p m in diametcr with processes u p to 81 pin in length, arc sren in Fig. 13. No vermicular forms or fission bodies were found. During 96-144 h of Incubation.-In the T strain, parasites in gut contents were similar in appearanc-e to those described iri tht. preceding section, but became fewer during the period and \\ere difficult to find by 144 h. Fission bodirs wcre see11 for u p t o 120 h. I n contrast, NT-strain parasites rrmaincd plrntiful during the entire period. Most of the forms seen \vcrc siniil;u. t o thost.
-
c Figs. 1-20, [Various stages of Babesia bovis strains T (transmissible) and N T (nontransmissible) in the tick, Boophilus rnicroplu~. .4R, after repletion of the tick. Gienisa’s stain. Except for Figs. 13 and 15, X 1,000.1 1. N T Intraerythrocytic stage before repletion. 2. NT. 24 h AR. Budding. 3. NT, 24 h AR. Binary fission. 4. NT, 24 h AR. A parasite with processes up to 2 2 pm long. 5 . NT, 48 11 AR Parasites without processes. 6. T, 64 h AR. An elongate parasite that appears to develop from a spherical form. 7. T, 96 h AR. A large vermicule in the tick gut contents. 8. T, 96 h AR. A large vermicule becoming spherical in the tick gut contents. 9. T, 96 h AR. Pnrasites believed to develop after division of large vermicules in the tick gut. 10. T, 96 h AR. A small vermicule in the hemolyniph. 11. T , 96 h AR. A parasite in the process of “unfolding” to produce a vermicular form. 12. T , 96 h AR. Fission of the parasites in a gut epithelial cell. 13. NT, 96 h AR. Parasites with processes up to 81 ym in length. X 500. 14. NT, 128-144 h AR. A round parasite. 15. NT, 128-144 h AR. Clumping of parasites. X 500. 16. T , 120 h AR. A parasite in a n ovum taken from the oviduct 17. T, vermicules pointed at both ends in a n ovum after oviposition. 18-20. T, from larvae fed on calves. Note the verniirules, after 24 h feeding (Fig. 18) ; precursors of the fission bodies, after 48 h feeding (Fig. 19) ; and a mature fission body, after 72 h feeding (Fig. 20).
TABLE 1 . Attempts
Parasite strain
iiritted hy ticks.
A T and T strainy of Babesia bovis 63’ Boophilus microplus.
t o tranrnlii
.\ttempt
Blood
xu.
’
iiassaqc‘ No.*
Results of infectivity tests on infested calf? Blood films
IF.XT%
,
iCalves \\?re infested \\it11 larval ~ ~ r i i g r noyf adult ticks that engorgcd on acutely infected cattle.
+ Indirect fluorescent : i r i t i t . d y s N.D. = not
dor1c..
trst.
sti-iicturc of parasitrs of the 2 strains in the gut contents following inyestion b y tlie tick vector was quite different. Second, NTqtr:iiii parasitrs were not dctectcd in any tissues of the ticks exailiincd. whereas diosc o f T strain were seen in epithelial cells of the , ovarian cells of the host tick, and in devrlq i t , h e m o l y ~ n p h nncl opiiig lar\,ar. NT-strain parasites were plentiful in smears of gut c~cintcmt\;,and appcar(d to undrrgo srveral cycles of development ; i n d division. It t h u s seems that the protozoa, although viable \ \ i t h i i i gut contents for m a n y hours after ingestion by ticks, were inc;ip;iblc~ of pcncttntiiiy cpithrlial crlls of the gut and infecting thc nornial vector. In :vner;il, T-strain parasites seen were similar t o those des c r i l x l b y Rick (8),althoiigh 3 additional forms were observed in snicars of gut contcnts. O n e form, a small round organism \\-it11 proressrs, \\-as wen occasionally both inside arid outside red I~lood cells in s m e a i ~ sfrom semi-engorged ticks. T h e procases noted a t that stag? riiight have lieen a n early response Iiy the pamsite to its nc\v eiivironment, but whcther they were a sign of drvclnpinrnt or degeneration could not he deduced. Two other forins not descrilied l q Riek ( 8 ) \\ere a large round or oval form 1 Fig. 1 1 ), \vhich aplicared to br “unfolding” to a verniirulnr slrapr, and a large vcrrniculc (Fig. 7 ) , unlike those wen in herno!I mph snie;irs, Ivliich appt,arrd to divide within the gut contents o f thc tick (Figs. 8. 9 j . Thesc 2 forms niay have bern r e h s e d froin gut cells r u p t u r r d duriiig preparation of the smear, although the), r\.c’Ie n c t w seen rvithin gur cells. Holbrook et 21. ( 5 ) o b s c m d round forms that apparently expanded or unfolded into \ ~ r r n i i c u l ~in~ stheir study of Babrsin cnballi (Nuttall) in the horse tick Dernincenioi- n i t ~ Ncumann, n~ and Rirk ( 7 ) C J I J S ~ ~ similar VC~ f o r n i x for Babt,,irr bigctrfina i n Boophilus w i i c ~ o ~ ~ l z i s . T h e 2 prrdorriinarlt forms of the N‘T strain wrrc round organisms .?-I 2 p i in diarnetcr, arid smaller pnrasites with obvious prcicc~scs(Figs. 4, 5 j. Forms with processes were seen at regular iiitor\.als tliroiiglioiit 144-h period following rt.pletion of tlie tick, :rritl \\(’re present in large numbers a t the final observation. These org;inisnis, nlthough c.;ip;ible of division, \vcre apparently unable caitlier to pent’triite stit crfls thcmsclves, o r to dwclop into forms ;ll)le t l ) do b 0 . I.:loiig;itc forms of the T strain \ v ( w first seen in gut contrnts of ticks incnl)nted f o r ti-t-72 h (Fig. 6 ) . Thtw parasites w i w similar t o the cigar-shaped bodics olxxrved by Rick (8) in ticks incu1);ited for 3 6 h \vhicli lie considrred to be infective for epithelial ct~llsof the gut. N o rlongate forms or verlnic-ides of the N T strain \vcrc seen. Their aiiscnce i n n y cxxplain the inability of inr~ctions ith this strain t o progrt’ss normally in ticks. I t is .;iiggrstccI that contintlous I h o d passaging of R. hovis reaiilts in ;I gradual srlvction of parasites inrapablr of penetratirig gilt epit11cli;il cells of the tick, d u r to an absence of contact with the v m o r . I t \ \ a s rvidrnt from observations 011 a strain of R. h ~ i , i istill infective f o r ticks after 43 blood passqgvs t h a t iii gut c o n t r i i t s of ticks thcrr \vas a higher proportion of parasites with pi-oc.c\srs than in the 1 sti.aiii dtwril)ed h r r r (Stewart Sr Dalgliesh, i1iipuI)liahtd tlntn j . T h e proportion of organisins with obvious p i o c v w ~111;iyI)r a n indrx of infrctivity of the strain fot the tick Y1’f.t
or.
REFERENCES 1. Callo\v LL, Hoyte HMD. 1961. Transmission experiments iisins Babesia bigemina. Theileria mutans, Borrelia sp. and the cattle tick. Boofihilus microp[us. Aust. Vet. 1. 37, 301-90. 2, ~ - _, Mellors LT. 1966. A new vaccine for Babasici nrg~7ifinainfection prepared in splmectoinised calves. Au.rt. V e t . J . 42. 464-65. 3 . --, McGregor W, Parker RJ, Dalgliesh RJ. ‘The imi i i u n i t y of cattle t o Raberin orgentinn after drug sterilisation of infections of varying duration. Azist. V e t . J . 50, 6-11. ~
LIFE CYCLES OF Babesia bovis 4. Dalgliesh RJ, Stewart NP. 1977. Failure of vaccine strains of Babesia bouis to regain Infectivity for ticks during long-standing infections in cattle. Aust. Pet. J. 53, 429-31. 5. Holbrook AA, Anthony DW, Johnson AJ. 1968. Observations on the development of Babesia caballi (Nuttall) in the tropical horse tick Vermacentor nitens Neumann. 1. Protozool. 15, 391-96. 6. O’Sullivan PJ, Callow LL. 1966. Loss of infectivity of a vaccine strain of Babesia argentina for Boophilus microplus. Aust. Pet. J. a, 252-54.
BOOK REVIEW.
501
7. Riek RF. 1964. The life cycle of Babesia bigemina (Smith and Kilborne, 1893) in the tick vector Boophilus microplus (Canestrini). Aust. J . Agric. Res. 15, 802-21. 8. __ 1966. The life cycle of Babesia argentina (Lignieres, 1903) (Sporozoa: Piroplasmidea) in the tick vector Boophilus microplus (Canestrini). Aust. J . Agric. Res. 17, 247-54. 9. Seddon HR. 1952. Diseases of domestic animals in Australia. Part 4. Protozoan and viral diseases. V e p t . Hlth. Sero. Publ., Commonwealth Government Printer, Canberra, Australia.
..
Reissig, J. L., ed. 1977. Microbial Interactions. Vol. 3 of Receptors and Recognition, Series B. Chapman and Hall, London. x 436 pp. $39.50.
+
Contents: Aggregation and Cell Surface Recefitors in Cellular Slime Molds, P. C . Newell; Bacterial Chemotaxis, G. L. Hazelbauer & J. s. Parkinson; Bacterial Receptors for Phages and Colicins as Constituents uf Specific Transport Systems, V. Braiin & K. Hartke; T h e Attachment of Bacteria to the Surfaces of Animal Cells, G. W. Jones; Binding and Entry of D N A in Bacterial Transformation, S. A. Lacks; A Redefinition of the Mating Phenomenon in Bacteria, M. Achtman & R. Skurray; Cell-Cell Interactions During Mating in Saccharomyces cereviseae, T. R. Manney & J. H. Meade; Mating Interactions in Chlaniydomonas, U. W. Goodenough; Cell-Cell Interactions in Ciliates: Evolutionary and Genetic Constraints, D. L. Nanney; A n Ouerviezer, J. L. Reissig. These chapters cover various aspects of chemosensory reception and communication between microorganisms and the microbial environment. Appropriately, emphasis is on the receptors themselves. In the rapidly growing field of microbial sensory-motor physiology, most available information is at the receptor level; in the majority of cases, little is known about sensory transduction mechanisms following receptor stimulation. Of direct concern to
protozoologists are the chapters on cellular slime molds, Chlamgdomonas, and ciliates, though some of the others will be of general interest; we particularly recommend the excellent review of bacterial cheinokinpsis by Hazelbailer & Parkinson. Newell’s detailed discussion of acrasian chemoreception shobvs the maturity of this field. It is a very clear piece of writing, with a fine feeling for quantitative aspects of the subject, now coming to the fore. Goodenough’s review leaves an impression that, despite a great deal of research using sophisticated tools, surprisingly little is known about sensory aspects of Chlamydonionas mating; this chapter is marred a bit by continual, disconcerting reference to unpublished work from the author’s laboratory, \vhile important papers by Gibor and others go unmentioned. Nanney’s review would be a good one to show to a cell biologist or biochemist to get him interestcd in the protozoa: a very clcar exposition, in which historical roots are unearthed, profundities of the subject are well displayed, and current experimental dilemmas are discussed. T h e price is a bit high for students, but microbiologists and others working on chemoreception should find this a useful, stimulating addition to the library. We congratulate the editor on his fine choice of timely topics and interesting authors.--M. LEVANDOWSKY & D. HAUSEK, Haskins Laborntories of Pace University, 41 Park Row, N e w Y o r k hlY 10038, USA.
