Effect of extrinsic incubation temperature on transstadial and transovarial transmission of Borrelia sp . in Ornithodoros ( O . ) savignyi and infectivity to vertebrate host

Effects of three extrinsic incubation (EI) temperatures on vector competence of Ornithodoros savignyi to Borrelia sp. isolated from a natural population of the tick in Egypt were investigated. The EI temperature influenced the efficiency of Borrelia sp. transmission by the female O. savignyi to hamsters. An EI temperature for 27C was optimum for successful transmission of Borrelia sp. where the highest infection rates (IRs) in hamsters occurred. Generally, the IRs in hamsters decreased by lowering the EI temperature to 17C or rising it to 37C in each of the corresponding experimental cases (number and postinfection period of the tick feeding per hamster) tested. Ornithodoros savignyi kept at 27C and 37C maintained borrelial infection transstadially from nymph 1 to 7 and adults. The transstadially infected stages transmitted the spirochetes to hamsters with higher IRs at 27C than 37C. Transovarial transmission of Borrelia sp. occurred during the first and second (at higher rates) gonadotrophic cycle in the infected female ticks kept at 27C and 37C. However, no molting or oviposition occurred at 17C.

High temperature affects the competence of the vector ticks by affecting the persistence of the pathogen in the vector sp. of Lyme disease and relapsing fever.Shih et al. (1995) demonstrated that the persistence of spirochetal infection of Lyme disease B. burgdorferi in nymphal deer tick Ixodes dammini is reduced by incubation at temperature higher than 27 o C for longer than two weeks and spirochetes became undetectable at four weeks thereafter.Increase of temperature more than 27 o C has been found to stimulate or inhibit synthesis of certain proteins in the spirochete B. burgdorferi (Cluss and Boothby, 1990;Schwan et al., 1995;Obonyo et al., 1999 andRamamoorthy andScholl, 2001).In addition, Lyme disease spirochete, B. burgdorferi, did not grow when cultured at temperature in excess of 37 o C (Barbour, 1984).In contrast to findings of Shih et al,. 1995, the increase of the extrinsic incubation (EI) temperature of rearing the female O. Savignyi infected with Borrelia sp.(mostly of relapsing fever) from 17 o C to 27 o C and 37 o C increased the mean number (no.), infection rate (IR) and prolonged persistence of the spirochetes in different organs of the vector tick throughout 90 days after the infective blood meal (Helmy et al., 2009).
The sand tampan, O. (O.) savignyi, collected from different localities in Egypt has been found to be naturally infected with Borrelia sp.(Shanbaky andHelmy, 2000 andHelmy, 2000b) and is capable of experimentally acquiring and maintaining the spirochetal infection by transstadial and transovarial transmission in the tick population and transmitting it to the vertebrate host (Shanbaky and Helmy, 2000).These three biological processes are important criteria which must be considered in evaluating the efficiency of the tick as a reservoir for Borrelia and its competence as a vector of a disease (Hoogstraal, 1985).
The present work investigates the effect of three extrinsic incubation temperatures (17 o C, 27 o C and 37 o C) on the ability of female O. savignyi to maintain the infection with Borrelia sp.within developing life stages and generations by transstadial transfer and transovarial transmission, respectively.Also, the ability of the tick to transmit infection to vertebrate host at each tested temperature at different intervals following the infective blood meal.

MATERIALS AND METHODS Tick colonies:
Ornithodoros (O.) savignyi (Audouin), Argasidae, was collected from sand where cattle rest at Dahshore, Giza governorate, Egypt.Field collected uninfected adults were used to start uninfected colonies and were maintained at 27+1 o C, 75% RH and 16 hrs light day.Ticks were fed on hamster, Mesocricetus auratus, as described by Helmy (2000).Infected colonies started by feeding clean ticks on laboratory infected hamsters which had been fed upon by naturally infected O. savignyi.Infected colonies were kept at three different temperatures of 17 o C, 27 o C and 37 o C.

Transstadial transfer of Borrelia sp. and transmission to uninfected hamsters:
About 300 uninfected nymph (N 1 ) O. savignyi were fed on hamsters infected with Borrelia sp.The engorged N 1 were collected and a sample of ten ticks were examined individually 1 hr after infective blood meal.Each N 1 was separately crushed in saline, stained with Fontana stain and/or DF technique and examined microscopically to ensure that spirochetes had been obtained with the infective blood meal.
After the remaining N 1 had molted to N 2 , 10-20 infected N 2 were fed on each of five uninfected hamsters.
The hamster blood was examined for spirochetes 4-7 days later.This step was repeated for each subsequent nymphal instar and adult stages.Samples of ten ticks of each subsequent instar and adult stage were tested for spirochetes.A drop of hemolymph (HL) was collected from broken tarsus of each tick after feeding and smeared and stained with Fontana stain and /or DF technique and examined microscopically for spirochetes transstadial transfer (TST).The aforementioned steps were repeated for ticks kept at each of the three tested temperatures.
Transovarial transmission of Borrelia sp. in the tick: Laboratory reared virgin uninfected females of O. savignyi were fed on hamsters infected with Borrelia sp.Each of ten infected parent females (P 1 ) was placed individually in polyethylene tube with an uninfected recently fed male and observed for oviposition (first gonadotrophic cycle).When eggs hatched, 10 F1 larvae from each female were examined individually for spirochetes.After 30 days following first blood meal, females were offered a second but a clean blood meal, and observed for oviposition (second gonadotrophic cycle).
Transovarial transmission (Tov) and filial infection rates (IRs) were calculated for females and their larval progeny (Hoogstraal, 1985) during first and second gonadotrophic cycle.These steps were repeated for tick kept at each of the three tested temperatures.

Transmission of Borrelia sp. infection from tick to hamster:
After 30, 60 and 90 days post feeding infective blood meal, different numbers (1, 3, 6 and 10 ticks) of adult female O. savignyi infected with Borrelia sp.isolated from the tick natural populations were fed on each of five clean uninfected hamsters.Four to seven days post feeding the tick, blood samples from each hamster was examined for spirochetes as described before.Those steps were repeated for each of the tested three temperatures.

Statistical analysis:
The percentages of the different life stages of the tick and that of Borrelia sp.infected blood samples collected from the vertebrate host were determined and compared using Chisquare test with the aid of statistical Package of Social Science (SPSS) version 8.0 for Windows.

RESULTS AND DISCUSSION
Effect of EI temperature on transstadial transfer of Borrelia sp. in O. savignyi and subsequent transmission to hamster: At 17 o C N 1 failed to molt, however at 27 o C and 37 o C the infected N 1 molted and transmitted spirochetes to 60% and 70% of the subsequent nymphal life stage (N 2 ) respectively (Table 1).Female O. savignyi failed to oviposit at 17 o C, however 60% and 70% of infected females kept at 27 o C and 37 o C transmitted Borrelia sp.transovarially to their eggs and about 15% and 23% of the emerged larvae were infected with Borrelia during the first gonadotrophic cycle (Fig. 1).A second but a clean blood meal induced higher (P < 0.05) transovarial (100% and 90%) and filial (37.5% and 40%) IRs in the same females during the second gonadotrophic cycle than that in the first gonadotrophic cycle at the two tested temperatures.There was no significant difference (P > 0.05) between each of the percent of transovarial transmission or filial IRs of F 1 larvae of O. savignyi when parent females were kept at 27 o C and 37 o C during each of the first and second gonadotrophic cycle.Similar data were obtained by Gaber et al. (1984) and who found that infected female O. erraticus transmitted spirochetes, B. crocidurae transovarially to their eggs and F 1 larvae in higher percentages during the second gonadotrophic cycle than the first cycle.These findings suggested that spirochetes number progressively increases during the successive egg developmental cycles of the tick.
The dispersal of borrelial infection in various organs of O. savignyi (Helmy et al., 2009) and ability of the tick to transmit the spirochete transovarially and transstadially (at 27 o C and 37 o C and to transmit the spirochete while feeding on uninfected hamsters at the temperatures tested in the present work prove the capacity of this tick species to serve as vector and reservoir (Hoogstraal, 1985) of Borrelia sp. in Egypt.Furthermore, this suggestion is supported by the fact that the three tested temperatures are most prevailing temperatures during the year (Helmy 2000b) in Giza governorate, Egypt where the tick was collected.In accordance with the present findings the field data of Helmy (2000b) in the same locality where high prevalence of Borrelia sp.infection was found in field collected nymphs and adults of O. savignyi during summer months while low infection rates were found in winter months during which the highest and lowest temperatures were recorded, respectively.

Effect of EI temperature on tick transmission of Borrelia sp. to hamsters:
Adult female O. savignyi infected with Borrelia sp.transmitted the infection to hamsters fed upon at the three EI temperatures tested in the present study (Table 2).At each temperature, Borrelia IRs in hamsters gradually increased (P < 0.05) by increasing the number of infected ticks feeding on each hamster from six to ten and from one to ten at 17 o C and 27 o C or 37 o C, respectively.Generally, IRs in hamsters gradually decreased (P < 0.05) by increasing the time passed after the ticks had the infective blood meal from 30 to 90 days (Table 2).
The present results agree with those of Piesman and Sinsky (1988) who found that efficiency of transmitting B. burgdorferi in I. scapularis had increased by increasing the number of feeding infected ticks per host animal.Also Lane et al. (1994) found that the plasma antibody titer to B. burgdorferi increased from 1 : 128 to 1 : 256 in mice exposed to feeding by one or two infected nymphs, respectively, of I. pacificus.Varma (1956) found that nymphs and young adults of O. turicata transmitted the infection with B. turicata better than old adults.He attributed this to a decrease in intensity or death of spirochetes in the salivary glands of old adults.Nikitina (1965) stated that F 1 larvae of infected female A. persicus rarely transmitted B. anserina because there were few spirochetes in the larvae.She believed that after larvae had fed and digested the meal, spirochetes multiplied and their numbers in nymphs increased to a point of causing infection in fowl hosts.Crippa et al. (2002) demonstrated that transmission of B. burgdorferi from I. ricinus nymphs to mouse increased with duration of tick attachment.Balashov (1972) suggested that spirochetes within the tick body need to reach a threshold level to be able to infect vertebrate.
At each temperature tested in the present work, the gradual decrease of IRs in hamsters exposed to feeding of infected ticks after different time intervals following tick infective blood meal (30 to 90 days) suggested a decrease of efficiency in transmitting Borrelia sp. from O. savignyi to hamster as the post infection period of the tick was prolonged.This decrease might be attributed to the general decrease of IRs and no. of spirochetes or their disappearance (Helmy et al., 2009) from the tick organs (Helmy et al., 2009) during the delayed intervals of tick post infection (60 -90 days); specially in organs responsible for transmitting infection to vertebrate host (salivary glands and coxal organ).
In  (Helmy et al., 2009).Therefore, the observed decrease of Borrelia sp.transmission to hamster at 37 o C less than 27 o C might be attributed to a probable change or interference with production of borrelial protein associated with transmission of spirochetes to the vertebrate host.Reciprocal expression of two main outer surface (lipo) proteins OspA and OspC occurs as B. burgdorferi is transmitted from its vector tick, I. scapularis, into mammalian host (Schwan et al., 1995).
It was suggested that synthesis of OspC by spirochete during tick feeding may play an essential role in the capacity of B. burgdorferi to successfully infect mammalian host including human.A shift to higher temperatures (34 o C and 37 o C) appears to be one important stimulus for triggering the induction of OspC (Schwan et al., 1995 andYang et al,. 2000).
Contraversy between effects of temperature on transmission of spirochetes to vertebrate host might be attributed to differences between tick and borreliae species used in the aforementioned studies.For better understanding of the effects of temperature on transmission of Borrelia from ticks to vertebrate hosts further investigations are required on the outer surface and other proteins of borreliae not only in argasid ticks but also in ixodids other than I. scapularis.commission 2. ed.The Williams and Wilkins Company.Baltimore 289 pp.Crippa, M.; Rais, O. and Gern, L. (2002).
Investigation on the mode and dynamics of transmission and infectivity of Borrelia burgdorferi

Fig. 1 :
Fig. 1: Transovarial and filial infection rates of Borrelia sp. in O. savignyi during the 1 st & gonadotrophic cycle of the parent female kept at 27 and 37ºC

Table ( 1
): The percentage of transstadial transfer of Borrelia sp.* infection in nymphal and adult O. Borrelia sp. in O. savignyi.However, results of Borrelia sp.transmission to hamster were in accordance of those by Shih et al. (1995) on b. burgdorferi where they found that incubation of I. dammini nymphs at temperature higher than 27 o C for two weeks or longer were not permissive for the transmission of the spirochetes to mice.

Table ( 2
): Effect of number and post infection time of female O. savignyi kept at three different temperatures on transmission of Borrelia sp. to uninfected hamsters.