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Babesia spp. infection in Boophilus microplus engorged females and eggs in São Paulo State, Brazil
Veterinary Parasitology 130 (2005) 61–67 www.elsevier.com/locate/vetpar
Babesia spp. infection in Boophilus microplus engorged females and eggs in Sa˜o Paulo State, Brazil M.C.S. Oliveira a,*, T.C.G. Oliveira-Sequeira b, J.P. Araujo Jr.c, A.F.T. Amarante b, H.N. Oliveira d a
Embrapa-Pecua´ria Sudeste, Rodovia Washington Luiz, km 234, Caixa Postal 339, CEP 13560-970 Sa˜o Carlos, SP, Brazil b Departamento de Parasitologia, Instituto de Biocieˆncias-Unesp, Botucatu, SP, Brazil c Departamento de Microbiologia e Imunologia, Instituto de Biocieˆncias-Unesp, Botucatu, SP, Brazil d Departamento de Melhoramento e Nutric¸a˜o Animal, FMVZ-UNESP, Botucatu, SP, Brazil Accepted 10 March 2005
Abstract Babesia spp. infections were investigated in Bos taurus Bos indicus dairy cows and calves and in Boophilus microplus engorged female ticks and eggs. Blood samples and engorged female ticks were collected from 25 cows and 27 calves. Babesia spp. was detected in ticks by microscopic examination of hemolymph of engorged female and by squashes of egg samples. Cattle infection was investigated in blood thin smears and by DNA amplification methods (PCR and nested PCR), using specific primers for Babesia bovis and Babesia bigemina. Merozoites of B. bovis (3 animals) and B. bigemina (12 animals) were detected exclusively in blood smears of calves. DNA amplification methods revealed that the frequency of B. bigemina infection in calves (92.6%) and in cows (84%) and of B. bovis in calves (85.2%) and in cows (100%) did not differ significantly (P > 0.05). Babesia spp. infection was more frequent in female ticks and eggs collected from calves (P < 0.01) than from cows, especially in those which had patent parasitemia. Hatching rates of B. microplus larvae were assessed according to the origin of engorged females, parasitemia of the vertebrate host, frequency and intensity of infection in engorged female tick, and frequency of egg infection. Hatching rate was lower in samples collected from calves (P < 0.01) than from cows, and in those in which Babesia spp. was detected in egg samples (P < 0.01). Published by Elsevier B.V. Keywords: Boophilus microplus; Babesia bigemina; Babesia bovis; Cattle; Infection rates
1. Introduction Babesia bovis and Babesia bigemina are the causal agents of bovine babesiosis in Latin America, where * Corresponding author. Tel.: +55 16 2615611; fax: +55 16 2615754. E-mail address: [email protected] (M.C.S. Oliveira). 0304-4017/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.vetpar.2005.03.007
the only known vector is the one-host-tick Boophilus microplus (Guglielmone et al., 1989, 1996). Since the transmission of these protozoa occurs by transovarian route, the rate of Babesia spp. infections in the tick progenies depends both on levels and frequency of female ticks infection. According to previous epidemiological data, the percentage of tick that transmit Babesia spp. to their progeny is low even if they
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become infected on animals in the acute phase of the disease, when parasitemia is high. In this case, the rate of B. bigemina transmission ranges from 20 to 40%, and of B. bovis from 0.5 to 14.5% (Mahoney and Mirre, 1971). Several investigators observed that babesial infection could have a harmful effect on female tick survival and reproductive efficiency, especially in the case of previously free colonies (Riek, 1966; Davey, 1981; Guglielmone et al., 1985). However, it was observed that under natural conditions in tropical regions, infections of B. microplus with Babesia spp. did not interfere with some reproductive traits of the female ticks and therefore it was postulated that there is an adaptive tolerance of ticks to infection in such situation (Guglielmone et al., 1989; Cen-Aguilar et al., 1998). In Brazil, bovine babesiosis occurs endemically in almost the entire country, but no data are available on natural infection of B. microplus or possible effects of infection with Babesia spp. on the reproductive traits of this tick. In the present experiment, rates of B. bovis and B. bigemina infection in young and adult cattle and rates of Babesia spp. infection in engorged B. microplus females and their eggs were assessed, as well as the effect of tick infection on larval hatching rate.
2. Materials and methods 2.1. Animals and sample collection Blood samples and B. microplus engorged female ticks were collected from 52 crossbreds dairy cattle (Bos taurus Bos indicus) reared in an area endemic for babesiosis in the State of Sa˜ o Paulo, Brazil (228010 S and 478530 W). The animals, 27 calves aged 1–4 months and 25 cows older than 3 years, were left without chemical tick control for at least 30 days before collection in order to avoid possible interference with the viability of ticks and of their offspring. The collections were performed from November 2000 to January 2001. Each animal was sampled only once, when blood and B. microplus female ticks were simultaneously collected. Blood samples from the jugular vein and from ear vessels were collected for DNA extraction
and for preparation of thin blood smears, respectively. All the female ticks more than 4.5 mm were collected from each animal to determine the parasite burden. After the counts, 10 fully engorged females tick from each animal were individually placed on hollow polyethylene plates for collection of eggs. 2.2. Blood sample processing Thin blood smears were fixed in methanol and stained with Giemsa for microscopic detection of Babesia and the assessment of parasitemia. Blood DNA was extracted from 300 ml of each sample using the GFXTM Genomic Blood DNA Purification kit (Amersham Biosciences) as recommended by the manufacturer. All DNA samples were submitted to a PCR amplification and only the PCRnegative samples were submitted to a second round of amplification by nested-PCR (nPCR). Specific primer sequences for B. bigemina and B. bovis designed by Figueroa et al. (1993) were used for both PCR and nPCR amplification. The 25 ml PCR reaction mixture consisted of 5 ml of the template DNA, 20 ml master mix containing 2.5 ml of 10 PCR buffer (KCl 500 mM, MgCl2 15 mM, Tris–HCl 100 mM, pH 9.0), 0.5 ml of dNTP mix (10 mM), 1 ml of each specific primer (10 mM) of B. bigemina (BiIA and BiIB) or B. bovis (BoR and BoF), 1.5 u Taq DNA polymerase (Amersham Biosciences) and 14.7 ml of distilled water. Nested-PCR was performed using 2 ml of the PCR products and 23 ml of reaction buffer at the described concentrations, and primers BiIAN and BiIBN for B. bigemina or BoFN and BoRN for B. bovis. The PCR amplification conditions for B. bigemina or B. bovis were as follows: 35 cycles with denaturation at 95 8C for 1 min; extension at 73 8C for 1.5 min. Annealing was performed at 64 8C for 1 min for the B. bigemina primers and at 60 8C for 1 min for B. bovis primers. Final extension was at 73 8C for 5 min. For nPCR reactions, the cycle sequence was identical, except for the annealing temperature, which was 70 8C for B. bigemina primers and 65 8C for B. bovis primers. DNA from pure B. bovis and B. bigemina samples was used as positive control. A negative control containing no DNA sample was included in each reaction. The final PCR products were visualized in 1.5% agarose electrophoresis in
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TBE buffer. B. bigemina-positive samples had bands visible at 278 bp (first PCR) and 170 bp (nPCR), while B. bovis-positive samples showed bands at 356 bp (first PCR) and 291 bp (nPCR). Randomly chosen amplicons were sequenced as an additional control and were confirmed to correspond to Genbank deposit sequences number S45366 (Spel-Aval restriction fragment of B. bigemina) and AF030061 (rap-1 protein of B. bovis).
For determination of larval hatching rates, the other aliquot of eggs from each engorged female was individually placed in a paper envelope and incubated for 40 days in a BOD chamber at 27 1 8C and 85– 86% of relative humidity. After incubation, the envelopes were dipped in 708 alcohol and unhatched eggs and larvae were counted under a stereomicroscope. Hatching rate was expressed as a percentage of eggs hatched into larvae.
2.3. Processing of B. microplus samples
2.4. Statistical analysis
Ten fully engorged B. microplus female ticks from each animal were individually incubated in a BOD chamber at 27 1 8C and 85–86% of relative humidity. On the 15th day of oviposition, a hemolymph sample was collected on a slide by sectioning one of the legs of each engorged female tick (Burgdorfer, 1970). The slides were fixed in methanol, stained with Giemsa and examined under light microscope with a 100 objective. Kinetes were counted on 15 microscopic fields and the results are reported in terms of mean kinetes/field. Only the mass of eggs laid within the sixth and the fifteenth days (Mahoney and Mirre, 1971) was used to prepare the egg smears and to determine the larval hatching rates. Two aliquots of eggs were taken from each egg mass using a spatula. Previous repeated counts accomplished under a stereomicroscope indicated that each aliquot had about 100 eggs. Smears were performed by squashing eggs between two slides (Bu¨ scher, 1988) and, after drying, slides were fixed in methanol, stained with Giemsa and examined under the light microscope with a 100 objective. The identification of the developing forms of Babesia spp. in the tick eggs, as well as in hemolymph, was based on descriptions of Riek (1964, 1966).
Data concerning the examination of blood smears and direct examination of hemolymph of B. microplus female collected from cows and calves were compared by Chi-square test. The Exact Fisher test was used to compare the data for blood smear with data of blood PCR and nPCR, and B. microplus larval hatching rates were analyzed by ANOVA. Regression analysis was applied to samples of engorged female ticks carrying kinetes obtained from calves in order to determine the effect of the number of kinetes detected in the hemolymph on larval hatching rate. All analyses were performed using SAS (1996).
3. Results 3.1. Blood samples Comparison between blood smear and DNA amplification (PCR and nPCR) for diagnosis of Babesia is presented in Table 1. DNA amplification was significantly more sensitive for the diagnosis of both Babesia species in both animal categories than blood smears (P < 0.01). Merozoites of B. bigemina (0.1–0.2% parasitized erythrocytes) and B. bovis (less than 0.1%) were found
Table 1 Comparison of blood smears and DNA amplification (PCR/nPCR) for the diagnosis of Babesia bovis and Babesia bigemina in calves and cows Blood smear
DNA amplification (PCR/nPCR) Calves
Cows
B. bovis
Positive Negative
B. bigemina
B. bovis
B. bigemina
Positive
Negative
Positive
Negative
Positive
Negative
Positive
Negative
3 20
0 4
12 13
0 2
0 25
0 0
0 21
0 4
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exclusively in the blood smears of calves. B. bigemina merozoites were found in 12 animals and B. bovis in 3, with only 1 of then carrying both species. Twenty-two of the 23 calf blood samples positive for B. bovis were detected by PCR and one by nPCR, and of the 25 samples positive for B. bigemina, 8 were detected by nPCR. Nineteen cow blood samples positive for B. bovis were detected by PCR and 6 by nPCR, and 14 of the 21 samples positive for B. bigemina were detected by PCR. The high frequency of B. bigemina DNA in calves (92.6%) did not differ from that in cows (84%) (P > 0.05). Similarly, the rate of B. bovis infection in cows (100%) did not differ (P > 0.05) from that detected in calves (85.2%), and concomitant infection with the two Babesia species was detected in 21 cows and 21 calves. 3.2. B. microplus samples The number of engorged females collected from calves (8–40) was lower (P < 0.01) than the number collected from cows (8–122) but in both animal categories the number of ticks presented a negative binomial distribution, with few animals carrying high infestations. Mean number of kinetes in the hemolymph of engorged female ticks and the frequency of Babesia spp. in egg samples are presented in Table 2. Babesia spp. kinetes were found more frequently (P < 0.01) in the hemolymph of ticks collected from calves (77/ 260) than in those collected from cows (7/236). Of the 77 female ticks carrying Babesia kinetes, 56 were from calves in which Babesia spp. merozoites were detected in blood smears. The mean number of kinetes per microscopic field was also higher (P < 0.01) in females collected from calves (9.7) than in females collected from cows (0.24), but kinetes distribution in female ticks collected in both animal categories was clustered.
The highest frequency of Babesia spp. infection was also detected (P < 0.01) in eggs laid by females that engorged on calves (115/218). Of the 218 egg samples from females collected from cows, only 63 showed Babesia spp. infection. Circular forms with a ring-like appearance of variable size, with a vacuolated cytoplasm stained light blue and the cromatin distributed around the periphery, were the predominant forms of Babesia spp. found in eggs. 3.3. Larval hatching rate Mean hatching rate was significantly lower (P < 0.01) in samples originated from calves (75.5%) than that collected from cows (88.5%), but did not differ (P > 0.05) in samples from calves with or without Babesia merozoites in their blood. Hatching rates analysis according to tick infection revealed no association with the presence of kinetes in female tick hemolymph (Table 3), but regression analysis run to determine the effect of the level of kinetes in hemolymph on larval hatching rate showed that for each additional kinete in hemolymph there was a reduction of 0.57% in hatching rate (P < 0.05).
Table 3 Least squares means (LSM) of hatching rates of Boophilus microplus larvae according to the presence of Babesia spp. (tick infection) in hemolymph of engorged female ticks and eggs Category
Tick infection
Hatching rates (LSM) (%) Hemolymph
Eggs
Calves
Positive Negative
76.7 74.7
76.6 b 83.7 a
Cows
Positive Negative
82.0 88.5
91.1 88.5
Means followed by different letters (a and b) in the same column indicate significant differences (P < 0.01).
Table 2 Mean number of kinetes in hemolymph and frequency Babesia spp. in B. microplus engorged female and eggs from calves and cows
Calves (n = 27) Cows (n = 25)
Mean number of kinetes/field
Frequency of Babesia spp. infection Engorged female ticks (%)
Eggs (%)
9.7 (0.26–21.5) 0.24 (0.06–0.64)
29.6 2.96
52.75 28.89
Minimum and maximum values are given in parenthesis.
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4. Discussion The detection of merozoites exclusively in blood smears from calves and the high frequency of DNA both in calves and cows indicate that the region studied is a stable endemic area for B. bigemina and B. bovis. It was estimated that if at least 75% of the calves are exposed to infection before 9 months of age the state of endemic stability will prevail, and clinical diseases will be rare (Mahoney and Ross, 1972). Since infection was detected in almost all calves including those ones with only 1 month of age, the transmission of the protozoans could occur at levels sufficient to produce infection of young animals during the period when they present greatest resistance. According to Mahoney et al. (1981), the stable epidemiological situation for babesiosis is generated in an endemic area through the constant inoculation of protozoa by the ticks, and in these regions carrier animals represent the largest reservoir of infection for the tick vectors (Joyner and Donnelly, 1979). However, experimental studies have indicated that B. microplus females become infected during the final 24 h of the parasitic phase when there is a patent parasitemia. This is the reason why carrier animals are considered to produce only a small number of infected ticks (Callow, 1968). Since only calves presented patent parasitemia by both Babesia species and infections in cows were detected exclusively by DNA amplification indicating their carrier status, the role of each category of cattle in the induction of vector infection was quantitatively assessed. Data obtained revealed that both the number of engorged female carrying kinetes and the number of infected eggs were greater in female ticks engorged on calves with patent parasitemia. These results confirm previous observations that density of parasites in the blood of vertebrate hosts is important for the establishment of Babesia infection in tick (Riek, 1964, 1966; Callow, 1968; Mahoney and Ross, 1972; Yeruham et al., 2001). Paradoxically, these findings cannot be interpreted as evidence of a greater contribution of young animals in maintaining the endemy, since the number of ticks that engorged on calves was significantly lower than the number of ticks that engorged on cows. These data agree with those obtained by Utech et al. (1978) and Mahoney et al. (1981) who observed that young
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animals were more resistant to tick infection. So, the possibility that young animals are responsible for a higher rate of infection of female ticks is counterbalanced somewhat by the fact that young animals contribute only to 25% of the tick population in the herd (Mahoney et al., 1981). It should also be noticed that, although the female ticks collected from calves produced a higher percentage of infected eggs, one cannot assure that this infection will persist in the larval stage (Cafrune and Aguirre, 1995), also because larval hatching rate was lower in ticks that fed on calves. The higher rates of Babesia spp. infection in eggs than in females that laid them could be a result of further cycles of multiple fission that occurs in eggs (Riek, 1964, 1966), and as observed in previous reports (Mahoney and Mirre, 1971; Cafrune and Aguirre, 1995), indicate that the hemolymph exam is a poor indicator of the level of progeny infection. Transovarian transmission of Babesia represents an effective mechanism for babesial dissemination, but this strategy does not seem to be harmless for the invertebrate host since negative effects of babesia infection on B. microplus have been described by several authors (Riek, 1964, 1966; Davey, 1981; Mangold et al., 1993). According to Hodgson et al. (1992), the effects of Babesia spp. infection depend on the level of parasitemia of the vertebrate host, on the susceptibility of the tick and on the Babesia strain. The effects of Babesia infection on ticks were investigated here by assessing the larval hatching rate according to the following variables: female tick origin, parasitemia of the vertebrate host, frequency of engorged female infection and frequency of infection in the eggs. Regarding the origin, larval hatching rates were lower in samples from female ticks that had engorged on calves. Since young cattle has been reported to be more resistant to tick infestation and since the response of the host can interfere with different physiological functions of the parasites, it is possible that the greater resistance of calves to ticks could also interfere with tick fertility, expressed as a lower larval hatching rate. This is reinforced by the observation that larval hatching rates were not affected by parasitemia of calves or by the presence of kinetes in female ticks that had engorged on these animals. Host resistance reducing parasite fertility is a wellknown phenomena in parasitic nematodes (Coop and Kyriazakis, 2001).
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The absence of a correlation between female tick infection and hatching rate could be due to the clustered distribution of kinetes. Considering that few engorged ticks present a large number of kinetes in the hemolymph, the low hatching rates presented by these ticks have little effect on the overall mean. Although it is largely accepted that primary infection with Babesia has negative effects on various biological parameters of the tick, several authors have observed that, under natural conditions, B. microplus develops an adaptive tolerance to Babesia spp. infection (Guglielmone et al., 1989; Cafrune et al., 1993; Cen-Aguilar et al., 1998) and in such situation ticks would not be significantly affected by infection. If, the data presented corroborate the occurrence of the phenomenon of adaptive tolerance, on the other hand, there are also evidences that infection with Babesia is not fully harmless to ticks, even under conditions of natural infection. Lower hatching rate in Babesia spp. infected eggs laid by females engorged on calves (Table 3) seems to be due a harmful effect of infection on developing larvae. Another piece of evidence was obtained when hatching rates were analyzed only for female ticks carrying kinetes. This analysis showed that for each additional kinete detected in the female hemolymph there was a 0.57% decrease in the larval hatching rate. These results suggest that also under natural conditions, Babesia spp. infection is able to cause subtle damage to ticks.
Acknowledgements To Dr. Raul Henrique Kessler and Dr. Cla´ udio Roberto Madruga (EMBRAPA/CNPGC) who kindly provided the pure B. bovis and B. bigemina samples used as positive control. Grants for this work were from the Fundac¸a˜ o de Amparo a Pesquisa do Estado de Sa˜ o Paulo (FAPESP-00/09103-7).
References Burgdorfer, W., 1970. Hemolymph test: a technique for detection of rickettsiae in ticks. Am. J. Trop. Med. Hyg. 19, 1010–1014. Bu¨ scher, G., 1988. The infection of various tick species with Babesia bigemina, its transmission and identification. Parasitol. Res. 74, 324–330.
Cafrune, M.M., Aguirre, D.H., 1995. Experimental studies of the rate of infection of Boophilus microplus eggs with Babesia bovis. Res. Vet. Sci. 58, 284–285. Cafrune, M.M., Aguirre, D.H., Mangold, A.J., Guglielmone, A.A., 1993. Oviposition in Boophilus microplus infected artificially with Babesia bovis and naturally with B. bovis and Babesia bigemina. Ann. Parasitol. Hum. Comp. 68, 196–198. Callow, L.L., 1968. The infection of Boophilus microplus with Babesia bigemina. Parasitology 58, 663–670. Cen-Aguilar, J.F., Rodrı´guez-Vivas, R.I., Domı´nguez-Alzipar, J.L., Wagner, G.G., 1998. Studies on the effect of infection by Babesia sp. on oviposition of Boophilus microplus engorged female naturally infected in Mexican Tropics. Vet. Parasitol. 78, 253–257. Coop, R.L., Kyriazakis, I., 2001. Influence of host nutrition on the development and consequences of nematode parasitism in ruminants. Trends Parasitol. 17, 325–330. Davey, R.B., 1981. Effects of Babesia bovis on the ovipositional success of the southern cattle tick, Boophilus microplus. Ann. Entomol. Soc. Am. 74, 331–333. Figueroa, J.V., Chieves, L.P., Johson, G.S., Buening, G.M., 1993. Multiplex polymerase chain reaction based assay for the detection of Babesia bigemina, Babesia bovis and Anaplasma marginale DNA in bovine blood. Vet. Parasitol. 50, 69–81. Guglielmone, A.A., Mangold, A.J., Bermudez, A.C., Hadani, A., 1985. Deteccio´ n de merozoitos grandes (vermı´culos) de Babesia en teleoginas de Boophilus microplus alimentadas sobre terneros com distintos nı´veles de parasitemia de Babesia bigemina y Babesia bovis (=Babesia argentina). Rev. Ibe´ r. Parasitol. 45, 303–311. Guglielmone, A.A., Mangold, A.J., Aguirre, D.H., Gaido, A.B., De Olsen, A.A., 1989. The effect of infection by Babesia sp. on some biological parameters of engorged female of Boophilus microplus. Folia Parasitol. 36, 1–6. Guglielmone, A.A., Gaido, A.B., Mangold, A.J., 1996. Light microscopy diagnosis of Babesia bovis and Babesia bigemina kinetes in the haemolymph of artificially infected Boophilus microplus engorged female ticks. Vet. Parasitol. 61, 15–20. Hodgson, J.L., Stiller, D., Jasmer, D.P., Buening, G.M., Wagner, G.G., McGuirre, T.C., 1992. Babesia bigemina: quantitation of infection in nymphal and adult Boophilus microplus using DNA probe. Exp. Parasitol. 74, 117–126. Joyner, L.P., Donnelly, J., 1979. The epidemiology of babesial infection. Adv. Parasitol. 17, 115–140. Mahoney, D.F., Mirre, G.B., 1971. Bovine babesiasis: estimation of infection rates in the tick vector Boophilus microplus (Canestrini). Ann. Trop. Med. Parasitol. 65, 309–317. Mahoney, D.F., Ross, D.R., 1972. Epizootiological factors in the control of bovine babesiosis. Aust. Vet. J. 48, 292–298. Mahoney, D.F., Wright, I.G., Goodger, B.V., Mirre, G.B., Sutherst, R.W., Utech, K.B.W., 1981. The transmission of Babesia bovis in herds of European and Zebu European cattle infested with the tick, Boophilus microplus. Aust. Vet. J. 57, 461–469. Mangold, A.J., Aguirre, D.H., Cafrune, M.M., Echaide, S.T., Guglielmone, A.A., 1993. Evaluation of the infectivity of a vaccinal and a pathogenic Babesia bovis strains from Argentina to Boophilus microplus. Vet. Parasitol. 51, 143–148.
M.C.S. Oliveira et al. / Veterinary Parasitology 130 (2005) 61–67 Riek, R.F., 1964. The cycle of Babesia bigemina (Smith and Kilborne, 1893) in the tick vector Boophilus microplus (Canestrini). Aust. J. Agric. Res. 15, 802–821. Riek, R.F., 1966. The cycle of Babesia argentina (Lignie`res, 1903) (Sporozoa: Piroplasmidea) in the tick vector Boophilus microplus (Canestrini). Aust. J. Agric. Res. 17, 247–254. SAS Institute Inc., 1996. SAS/STAT. User’s Guide, Version 6.11, v.2, fourth ed. Cary, SAS Institute Inc., 842 p.
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Utech, K.B.W., Seifert, G.W., Wharton, R.H., 1978. Breeding Australian Illawarra Shorthorn cattle for resistance to Boophilus microplus. I. Factors affecting resistance. Aust. J. Agric. Res. 29, 411–422. Yeruham, I., Hadani, A., Galker, 2001. The effect of the ovine host parasitaemia on the development of Babesia ovis (Babes, 1892) in the tick Rhipicephalus bursa (Canestrini and Fanzago, 1877). Vet. Parasitol. 96, 195–202.
Babesia spp. infection in Boophilus microplus engorged females and eggs in Sa˜o Paulo State, Brazil M.C.S. Oliveira a,*, T.C.G. Oliveira-Sequeira b, J.P. Araujo Jr.c, A.F.T. Amarante b, H.N. Oliveira d a
Embrapa-Pecua´ria Sudeste, Rodovia Washington Luiz, km 234, Caixa Postal 339, CEP 13560-970 Sa˜o Carlos, SP, Brazil b Departamento de Parasitologia, Instituto de Biocieˆncias-Unesp, Botucatu, SP, Brazil c Departamento de Microbiologia e Imunologia, Instituto de Biocieˆncias-Unesp, Botucatu, SP, Brazil d Departamento de Melhoramento e Nutric¸a˜o Animal, FMVZ-UNESP, Botucatu, SP, Brazil Accepted 10 March 2005
Abstract Babesia spp. infections were investigated in Bos taurus Bos indicus dairy cows and calves and in Boophilus microplus engorged female ticks and eggs. Blood samples and engorged female ticks were collected from 25 cows and 27 calves. Babesia spp. was detected in ticks by microscopic examination of hemolymph of engorged female and by squashes of egg samples. Cattle infection was investigated in blood thin smears and by DNA amplification methods (PCR and nested PCR), using specific primers for Babesia bovis and Babesia bigemina. Merozoites of B. bovis (3 animals) and B. bigemina (12 animals) were detected exclusively in blood smears of calves. DNA amplification methods revealed that the frequency of B. bigemina infection in calves (92.6%) and in cows (84%) and of B. bovis in calves (85.2%) and in cows (100%) did not differ significantly (P > 0.05). Babesia spp. infection was more frequent in female ticks and eggs collected from calves (P < 0.01) than from cows, especially in those which had patent parasitemia. Hatching rates of B. microplus larvae were assessed according to the origin of engorged females, parasitemia of the vertebrate host, frequency and intensity of infection in engorged female tick, and frequency of egg infection. Hatching rate was lower in samples collected from calves (P < 0.01) than from cows, and in those in which Babesia spp. was detected in egg samples (P < 0.01). Published by Elsevier B.V. Keywords: Boophilus microplus; Babesia bigemina; Babesia bovis; Cattle; Infection rates
1. Introduction Babesia bovis and Babesia bigemina are the causal agents of bovine babesiosis in Latin America, where * Corresponding author. Tel.: +55 16 2615611; fax: +55 16 2615754. E-mail address: [email protected] (M.C.S. Oliveira). 0304-4017/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.vetpar.2005.03.007
the only known vector is the one-host-tick Boophilus microplus (Guglielmone et al., 1989, 1996). Since the transmission of these protozoa occurs by transovarian route, the rate of Babesia spp. infections in the tick progenies depends both on levels and frequency of female ticks infection. According to previous epidemiological data, the percentage of tick that transmit Babesia spp. to their progeny is low even if they
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become infected on animals in the acute phase of the disease, when parasitemia is high. In this case, the rate of B. bigemina transmission ranges from 20 to 40%, and of B. bovis from 0.5 to 14.5% (Mahoney and Mirre, 1971). Several investigators observed that babesial infection could have a harmful effect on female tick survival and reproductive efficiency, especially in the case of previously free colonies (Riek, 1966; Davey, 1981; Guglielmone et al., 1985). However, it was observed that under natural conditions in tropical regions, infections of B. microplus with Babesia spp. did not interfere with some reproductive traits of the female ticks and therefore it was postulated that there is an adaptive tolerance of ticks to infection in such situation (Guglielmone et al., 1989; Cen-Aguilar et al., 1998). In Brazil, bovine babesiosis occurs endemically in almost the entire country, but no data are available on natural infection of B. microplus or possible effects of infection with Babesia spp. on the reproductive traits of this tick. In the present experiment, rates of B. bovis and B. bigemina infection in young and adult cattle and rates of Babesia spp. infection in engorged B. microplus females and their eggs were assessed, as well as the effect of tick infection on larval hatching rate.
2. Materials and methods 2.1. Animals and sample collection Blood samples and B. microplus engorged female ticks were collected from 52 crossbreds dairy cattle (Bos taurus Bos indicus) reared in an area endemic for babesiosis in the State of Sa˜ o Paulo, Brazil (228010 S and 478530 W). The animals, 27 calves aged 1–4 months and 25 cows older than 3 years, were left without chemical tick control for at least 30 days before collection in order to avoid possible interference with the viability of ticks and of their offspring. The collections were performed from November 2000 to January 2001. Each animal was sampled only once, when blood and B. microplus female ticks were simultaneously collected. Blood samples from the jugular vein and from ear vessels were collected for DNA extraction
and for preparation of thin blood smears, respectively. All the female ticks more than 4.5 mm were collected from each animal to determine the parasite burden. After the counts, 10 fully engorged females tick from each animal were individually placed on hollow polyethylene plates for collection of eggs. 2.2. Blood sample processing Thin blood smears were fixed in methanol and stained with Giemsa for microscopic detection of Babesia and the assessment of parasitemia. Blood DNA was extracted from 300 ml of each sample using the GFXTM Genomic Blood DNA Purification kit (Amersham Biosciences) as recommended by the manufacturer. All DNA samples were submitted to a PCR amplification and only the PCRnegative samples were submitted to a second round of amplification by nested-PCR (nPCR). Specific primer sequences for B. bigemina and B. bovis designed by Figueroa et al. (1993) were used for both PCR and nPCR amplification. The 25 ml PCR reaction mixture consisted of 5 ml of the template DNA, 20 ml master mix containing 2.5 ml of 10 PCR buffer (KCl 500 mM, MgCl2 15 mM, Tris–HCl 100 mM, pH 9.0), 0.5 ml of dNTP mix (10 mM), 1 ml of each specific primer (10 mM) of B. bigemina (BiIA and BiIB) or B. bovis (BoR and BoF), 1.5 u Taq DNA polymerase (Amersham Biosciences) and 14.7 ml of distilled water. Nested-PCR was performed using 2 ml of the PCR products and 23 ml of reaction buffer at the described concentrations, and primers BiIAN and BiIBN for B. bigemina or BoFN and BoRN for B. bovis. The PCR amplification conditions for B. bigemina or B. bovis were as follows: 35 cycles with denaturation at 95 8C for 1 min; extension at 73 8C for 1.5 min. Annealing was performed at 64 8C for 1 min for the B. bigemina primers and at 60 8C for 1 min for B. bovis primers. Final extension was at 73 8C for 5 min. For nPCR reactions, the cycle sequence was identical, except for the annealing temperature, which was 70 8C for B. bigemina primers and 65 8C for B. bovis primers. DNA from pure B. bovis and B. bigemina samples was used as positive control. A negative control containing no DNA sample was included in each reaction. The final PCR products were visualized in 1.5% agarose electrophoresis in
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TBE buffer. B. bigemina-positive samples had bands visible at 278 bp (first PCR) and 170 bp (nPCR), while B. bovis-positive samples showed bands at 356 bp (first PCR) and 291 bp (nPCR). Randomly chosen amplicons were sequenced as an additional control and were confirmed to correspond to Genbank deposit sequences number S45366 (Spel-Aval restriction fragment of B. bigemina) and AF030061 (rap-1 protein of B. bovis).
For determination of larval hatching rates, the other aliquot of eggs from each engorged female was individually placed in a paper envelope and incubated for 40 days in a BOD chamber at 27 1 8C and 85– 86% of relative humidity. After incubation, the envelopes were dipped in 708 alcohol and unhatched eggs and larvae were counted under a stereomicroscope. Hatching rate was expressed as a percentage of eggs hatched into larvae.
2.3. Processing of B. microplus samples
2.4. Statistical analysis
Ten fully engorged B. microplus female ticks from each animal were individually incubated in a BOD chamber at 27 1 8C and 85–86% of relative humidity. On the 15th day of oviposition, a hemolymph sample was collected on a slide by sectioning one of the legs of each engorged female tick (Burgdorfer, 1970). The slides were fixed in methanol, stained with Giemsa and examined under light microscope with a 100 objective. Kinetes were counted on 15 microscopic fields and the results are reported in terms of mean kinetes/field. Only the mass of eggs laid within the sixth and the fifteenth days (Mahoney and Mirre, 1971) was used to prepare the egg smears and to determine the larval hatching rates. Two aliquots of eggs were taken from each egg mass using a spatula. Previous repeated counts accomplished under a stereomicroscope indicated that each aliquot had about 100 eggs. Smears were performed by squashing eggs between two slides (Bu¨ scher, 1988) and, after drying, slides were fixed in methanol, stained with Giemsa and examined under the light microscope with a 100 objective. The identification of the developing forms of Babesia spp. in the tick eggs, as well as in hemolymph, was based on descriptions of Riek (1964, 1966).
Data concerning the examination of blood smears and direct examination of hemolymph of B. microplus female collected from cows and calves were compared by Chi-square test. The Exact Fisher test was used to compare the data for blood smear with data of blood PCR and nPCR, and B. microplus larval hatching rates were analyzed by ANOVA. Regression analysis was applied to samples of engorged female ticks carrying kinetes obtained from calves in order to determine the effect of the number of kinetes detected in the hemolymph on larval hatching rate. All analyses were performed using SAS (1996).
3. Results 3.1. Blood samples Comparison between blood smear and DNA amplification (PCR and nPCR) for diagnosis of Babesia is presented in Table 1. DNA amplification was significantly more sensitive for the diagnosis of both Babesia species in both animal categories than blood smears (P < 0.01). Merozoites of B. bigemina (0.1–0.2% parasitized erythrocytes) and B. bovis (less than 0.1%) were found
Table 1 Comparison of blood smears and DNA amplification (PCR/nPCR) for the diagnosis of Babesia bovis and Babesia bigemina in calves and cows Blood smear
DNA amplification (PCR/nPCR) Calves
Cows
B. bovis
Positive Negative
B. bigemina
B. bovis
B. bigemina
Positive
Negative
Positive
Negative
Positive
Negative
Positive
Negative
3 20
0 4
12 13
0 2
0 25
0 0
0 21
0 4
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exclusively in the blood smears of calves. B. bigemina merozoites were found in 12 animals and B. bovis in 3, with only 1 of then carrying both species. Twenty-two of the 23 calf blood samples positive for B. bovis were detected by PCR and one by nPCR, and of the 25 samples positive for B. bigemina, 8 were detected by nPCR. Nineteen cow blood samples positive for B. bovis were detected by PCR and 6 by nPCR, and 14 of the 21 samples positive for B. bigemina were detected by PCR. The high frequency of B. bigemina DNA in calves (92.6%) did not differ from that in cows (84%) (P > 0.05). Similarly, the rate of B. bovis infection in cows (100%) did not differ (P > 0.05) from that detected in calves (85.2%), and concomitant infection with the two Babesia species was detected in 21 cows and 21 calves. 3.2. B. microplus samples The number of engorged females collected from calves (8–40) was lower (P < 0.01) than the number collected from cows (8–122) but in both animal categories the number of ticks presented a negative binomial distribution, with few animals carrying high infestations. Mean number of kinetes in the hemolymph of engorged female ticks and the frequency of Babesia spp. in egg samples are presented in Table 2. Babesia spp. kinetes were found more frequently (P < 0.01) in the hemolymph of ticks collected from calves (77/ 260) than in those collected from cows (7/236). Of the 77 female ticks carrying Babesia kinetes, 56 were from calves in which Babesia spp. merozoites were detected in blood smears. The mean number of kinetes per microscopic field was also higher (P < 0.01) in females collected from calves (9.7) than in females collected from cows (0.24), but kinetes distribution in female ticks collected in both animal categories was clustered.
The highest frequency of Babesia spp. infection was also detected (P < 0.01) in eggs laid by females that engorged on calves (115/218). Of the 218 egg samples from females collected from cows, only 63 showed Babesia spp. infection. Circular forms with a ring-like appearance of variable size, with a vacuolated cytoplasm stained light blue and the cromatin distributed around the periphery, were the predominant forms of Babesia spp. found in eggs. 3.3. Larval hatching rate Mean hatching rate was significantly lower (P < 0.01) in samples originated from calves (75.5%) than that collected from cows (88.5%), but did not differ (P > 0.05) in samples from calves with or without Babesia merozoites in their blood. Hatching rates analysis according to tick infection revealed no association with the presence of kinetes in female tick hemolymph (Table 3), but regression analysis run to determine the effect of the level of kinetes in hemolymph on larval hatching rate showed that for each additional kinete in hemolymph there was a reduction of 0.57% in hatching rate (P < 0.05).
Table 3 Least squares means (LSM) of hatching rates of Boophilus microplus larvae according to the presence of Babesia spp. (tick infection) in hemolymph of engorged female ticks and eggs Category
Tick infection
Hatching rates (LSM) (%) Hemolymph
Eggs
Calves
Positive Negative
76.7 74.7
76.6 b 83.7 a
Cows
Positive Negative
82.0 88.5
91.1 88.5
Means followed by different letters (a and b) in the same column indicate significant differences (P < 0.01).
Table 2 Mean number of kinetes in hemolymph and frequency Babesia spp. in B. microplus engorged female and eggs from calves and cows
Calves (n = 27) Cows (n = 25)
Mean number of kinetes/field
Frequency of Babesia spp. infection Engorged female ticks (%)
Eggs (%)
9.7 (0.26–21.5) 0.24 (0.06–0.64)
29.6 2.96
52.75 28.89
Minimum and maximum values are given in parenthesis.
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4. Discussion The detection of merozoites exclusively in blood smears from calves and the high frequency of DNA both in calves and cows indicate that the region studied is a stable endemic area for B. bigemina and B. bovis. It was estimated that if at least 75% of the calves are exposed to infection before 9 months of age the state of endemic stability will prevail, and clinical diseases will be rare (Mahoney and Ross, 1972). Since infection was detected in almost all calves including those ones with only 1 month of age, the transmission of the protozoans could occur at levels sufficient to produce infection of young animals during the period when they present greatest resistance. According to Mahoney et al. (1981), the stable epidemiological situation for babesiosis is generated in an endemic area through the constant inoculation of protozoa by the ticks, and in these regions carrier animals represent the largest reservoir of infection for the tick vectors (Joyner and Donnelly, 1979). However, experimental studies have indicated that B. microplus females become infected during the final 24 h of the parasitic phase when there is a patent parasitemia. This is the reason why carrier animals are considered to produce only a small number of infected ticks (Callow, 1968). Since only calves presented patent parasitemia by both Babesia species and infections in cows were detected exclusively by DNA amplification indicating their carrier status, the role of each category of cattle in the induction of vector infection was quantitatively assessed. Data obtained revealed that both the number of engorged female carrying kinetes and the number of infected eggs were greater in female ticks engorged on calves with patent parasitemia. These results confirm previous observations that density of parasites in the blood of vertebrate hosts is important for the establishment of Babesia infection in tick (Riek, 1964, 1966; Callow, 1968; Mahoney and Ross, 1972; Yeruham et al., 2001). Paradoxically, these findings cannot be interpreted as evidence of a greater contribution of young animals in maintaining the endemy, since the number of ticks that engorged on calves was significantly lower than the number of ticks that engorged on cows. These data agree with those obtained by Utech et al. (1978) and Mahoney et al. (1981) who observed that young
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animals were more resistant to tick infection. So, the possibility that young animals are responsible for a higher rate of infection of female ticks is counterbalanced somewhat by the fact that young animals contribute only to 25% of the tick population in the herd (Mahoney et al., 1981). It should also be noticed that, although the female ticks collected from calves produced a higher percentage of infected eggs, one cannot assure that this infection will persist in the larval stage (Cafrune and Aguirre, 1995), also because larval hatching rate was lower in ticks that fed on calves. The higher rates of Babesia spp. infection in eggs than in females that laid them could be a result of further cycles of multiple fission that occurs in eggs (Riek, 1964, 1966), and as observed in previous reports (Mahoney and Mirre, 1971; Cafrune and Aguirre, 1995), indicate that the hemolymph exam is a poor indicator of the level of progeny infection. Transovarian transmission of Babesia represents an effective mechanism for babesial dissemination, but this strategy does not seem to be harmless for the invertebrate host since negative effects of babesia infection on B. microplus have been described by several authors (Riek, 1964, 1966; Davey, 1981; Mangold et al., 1993). According to Hodgson et al. (1992), the effects of Babesia spp. infection depend on the level of parasitemia of the vertebrate host, on the susceptibility of the tick and on the Babesia strain. The effects of Babesia infection on ticks were investigated here by assessing the larval hatching rate according to the following variables: female tick origin, parasitemia of the vertebrate host, frequency of engorged female infection and frequency of infection in the eggs. Regarding the origin, larval hatching rates were lower in samples from female ticks that had engorged on calves. Since young cattle has been reported to be more resistant to tick infestation and since the response of the host can interfere with different physiological functions of the parasites, it is possible that the greater resistance of calves to ticks could also interfere with tick fertility, expressed as a lower larval hatching rate. This is reinforced by the observation that larval hatching rates were not affected by parasitemia of calves or by the presence of kinetes in female ticks that had engorged on these animals. Host resistance reducing parasite fertility is a wellknown phenomena in parasitic nematodes (Coop and Kyriazakis, 2001).
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The absence of a correlation between female tick infection and hatching rate could be due to the clustered distribution of kinetes. Considering that few engorged ticks present a large number of kinetes in the hemolymph, the low hatching rates presented by these ticks have little effect on the overall mean. Although it is largely accepted that primary infection with Babesia has negative effects on various biological parameters of the tick, several authors have observed that, under natural conditions, B. microplus develops an adaptive tolerance to Babesia spp. infection (Guglielmone et al., 1989; Cafrune et al., 1993; Cen-Aguilar et al., 1998) and in such situation ticks would not be significantly affected by infection. If, the data presented corroborate the occurrence of the phenomenon of adaptive tolerance, on the other hand, there are also evidences that infection with Babesia is not fully harmless to ticks, even under conditions of natural infection. Lower hatching rate in Babesia spp. infected eggs laid by females engorged on calves (Table 3) seems to be due a harmful effect of infection on developing larvae. Another piece of evidence was obtained when hatching rates were analyzed only for female ticks carrying kinetes. This analysis showed that for each additional kinete detected in the female hemolymph there was a 0.57% decrease in the larval hatching rate. These results suggest that also under natural conditions, Babesia spp. infection is able to cause subtle damage to ticks.
Acknowledgements To Dr. Raul Henrique Kessler and Dr. Cla´ udio Roberto Madruga (EMBRAPA/CNPGC) who kindly provided the pure B. bovis and B. bigemina samples used as positive control. Grants for this work were from the Fundac¸a˜ o de Amparo a Pesquisa do Estado de Sa˜ o Paulo (FAPESP-00/09103-7).
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