Effect of extrinsic incubation temperature on borrelial infection in various organs of Ornithodoros ( O . ) savignyi

Dissemination levels of Borrelia sp. isolated from a natural population of Ornithodoros savignyi in Egypt were assessed in various organs of the infected female tick reared at three different temperatures. Results of the present investigation showed that the extrinsic incubation temperature at which the infected tick was reared (EI) was crucial in affecting borrelial dissemination levels in different organs including gut, salivary glands, coxal organ, ovaries and hemolymph. The increase of EI from 17C to 27C and 37C increased the infection rates (IRs) and mean number (no.) of spirochetes localized in different organs. Also, it enhanced the appearance, prolonged persistence and delayed disappearance of spirochetes in most of the organs tested throughout the period of study (90 days after infective meal). INTRODUCTION The temperature at which the infected tick has been reared may influence the tick competence as a vector of disease and modify the course of infection with pathogens (Injeyan et al., 1971; Young et al., 1979; Young and Leitch, 1981; Dalgliesh and Stewart, 1982). Few studies have shown that the extrinsic incubation temperature (EI) affects survival growth and transmission of lyme disease spirochete, Borrelia burgdorferi, and hence the competence of its ixodid tick vector (Shih et al., 1995). Infection with B. burgdorferi was reduced and undetectable in the gut of Ixodes scapularis nymphs kept at 33P PC and 37 PPC, respectively, as compared to moderate temperature at 27P PC. Furthermore, Barbour (1984) found that lyme disease spirochete B. burgdorferi did not grow when cultured in BSK II medium at temperature in excess of 37PPC. Also, Reisinger (1996) demonstrated that growth of two strains of B. burgdorferi was impaired when cultured at 37PPC and inhibited at 39PPC and 40PPC. In argasid ticks, no studies have been done on the effect of EI or borrelial infection and competence of the vector tick since Hindl investigation (1911) on distribution of B. duttoni in organs of O. moubata kept at 21PPC and 36P PC where spirochetes were found only in organs of ticks kept at 36PPC. Ornithodoros (O.) savignyi is one of seven ornithodorine tick species which had been recorded in Egypt (Hoogstraal & Kaiser, 1958) and was found to be naturally infected and able to transmit a specific Borrelia sp. to mammals (Shanbaky and Helmy, 2000). In evaluating vector competence, knowledge about dissemination and intensity of borrelial infection in the tick organs is essential. Studies on infection of organs involved in transmission to mammals (salivary glands and coxal organs) and to tick offspring (ovaries) are of major importance. The aim of the present study is to evaluate the effect of rearing O. (O.) savignyi at three constant temperatures (17P PC, 27P PC and 37P PC) on infection rates (IRs) with Borrelia sp. and mean numbers (no.) of the spirochetes in different organs and hemolymph of the adult female. MATERIALS AND METHODS Ticks and source: Ornithodoros (O.) savignyi (Audouin), Argasidae, was collected from sand near cattle rearing places in Dahshore, Giza governorate, Egypt. The sand was sieved through paired large and small mesh metal sieves (Gaber et al.


INTRODUCTION
The temperature at which the infected tick has been reared may influence the tick competence as a vector of disease and modify the course of infection with pathogens (Injeyan et al., 1971;Young et al., 1979;Young and Leitch, 1981;Dalgliesh and Stewart, 1982).
Few studies have shown that the extrinsic incubation temperature (EI) affects survival growth and transmission of lyme disease spirochete, Borrelia burgdorferi, and hence the competence of its ixodid tick vector (Shih et al., 1995) Furthermore, Barbour (1984) found that lyme disease spirochete B. burgdorferi did not grow when cultured in BSK II medium at temperature in excess of 37P o PC.
Also, Reisinger (1996) demonstrated that growth of two strains of B. burgdorferi was impaired when cultured at 37P o In argasid ticks, no studies have been done on the effect of EI or borrelial infection and competence of the vector tick since Hindl investigation (1911)  Ornithodoros (O.) savignyi is one of seven ornithodorine tick species which had been recorded in Egypt (Hoogstraal & Kaiser, 1958) and was found to be naturally infected and able to transmit a specific Borrelia sp. to mammals (Shanbaky and Helmy, 2000).
In evaluating vector competence, knowledge about dissemination and intensity of borrelial infection in the tick organs is essential.Studies on infection of organs involved in transmission to mammals (salivary glands and coxal organs) and to tick offspring (ovaries) are of major importance.
The aim of the present study is to evaluate the effect of rearing O. (O.) savignyi at three constant temperatures (17P o on infection rates (IRs) with Borrelia sp. and mean numbers (no.) of the spirochetes in different organs and hemolymph of the adult female.

MATERIALS AND METHODS Ticks and source:
Ornithodoros (O.) savignyi (Audouin), Argasidae, was collected from sand near cattle rearing places in Dahshore, Giza governorate, Egypt.The sand was sieved through paired large and small mesh metal sieves (Gaber et al. 1984).

Uninfected and infected tick colonies:
Field collected uninfected adult ticks were used to start uninfected colonies and were maintained at 27U +U 1P
For IDF antigen slides were prepared from spirochetes obtained from ticks and cultured in modified Kelly's medium (Barbour et al., 1983)

Spirochetes localization and number in tick organs:
Newly moulted uninfected females of O. savignyi were fed on infected hamsters.Counts were made in hemolymph (l µl) and different organs of each of 10 females daily for 10 days and then every 5 days for 60 days after feeding (daf) and then every 10 days till 90 daf.Hemolymph was collected and blood smears were prepared on slides.Gut, salivary glands, coxal organ and ovaries were dissected in a saline solution, squashed on slides, stained and examined microscopically and spirochete number in each organ was counted in ticks at each tested temperature.
The percentage of infection (IR) and mean number (no.) of spirochete in hemolymph and each organ was calculated.Data were analyzed using Chi-square test with the aid of Statistical Package for Social Science (SPSS) version 8.0 for Windows.
The present findings conform to those reported by Burgdorfer (1951), Teravsky (1959) and Nikita (1964Nikita ( & 1965) ) where numbers of the spirochete B. duttoni, B. sogidiana and B. anserim greatly decreased in the gut lumen few days after infection in O. moubata, O. papillipes (tholozani) and Argas persicus, respectively, until disappeared.Migration of the spirochetes from the gut to the hemolymph may contribute to the observed drop in no. of spirochetes in the gut.
Furthermore, Balashov (1972) suggested that later disappearance of spirochetes from gut lumen in ticks is probably associated with the unfavorable changes as ingested blood is gradually digested.
In accordance with the present findings are those of Helmy (2000) where Borrelia sp.IRs in field collected nymphs and adults of O. savignyi were high in summer months and low in winter months during which the highest and lowest temperatures were recorded, respectively.respectively), for longer than two weeks.Although findings of the present study agreed with those of Shih et al. (1995) in the existence of an effect of EI on the level of borrelial infection inside the tick, our data pointed to a general increase of IR and no. of spirochetes and prolongation of persistence of infection inside the gut of the infected O. savignyi by increasing EI of the tick from 17 o C to 27 o C or 37 o C but not from 27 o C to 37 o C.This difference might be attributed to probable biological and physiological differences of borreliae and tick species used in both studies.

Localization of Borrelia sp. in hemolymph:
In hemolymph of adult female O. savignyi, spirochetes were detected in the second and first daf of the tick and persisted for 70, 80 and 90 daf at 17 o C, It is well documented that borreliae as well as other pathogens ingested during tick feeding on infective blood penetrate the gut wall and enter hemolymph to be distributed to other organs (Balashov, 1972& Hoogstraal, 1985).In the present study, the initial increase of spirochete number in HL could be attributed to their migration from the gut to HL as was reported in other tick species (Gaber et al., 1984& Helmy et al., 1996).
However, multiplication of Borrelia sp. in HL might have contributed to their increase.Hodzic et al. (2002) reported that feeding stimulates spirochetes multiplication and their number may increase markedly after tick feeding on either infected or uninfected host.Generally, IR of ticks with spirochetes in HL is dependent on the species of the vector and agent and also by the physiological state of the tick (Balashov, 1972).Hemolymph of argasid tick is known to be rich in important nutrients, fatty acids and amino acids which provide a favorable condition for multiplication of spirochetes (Burgdorfer et al., 1989).Furthermore, tick HL has been reported as a favorable medium which allows survival and intracellular multiplication of other pathogenic organisms such as rickettsiae (Balashov and Daiter, 1965) and piroplasm (Markov & Abromov, 1958).The aforementioned investigations support the present suggestion that migration from the gut and multiplication in HL of Borrelia sp. may explain the initial increase of the IRs and no. of the spirochete in Hl during the first few days after the tick infective meal at the three tested temperatures in the present study.
The early appearance of spirochetes (first daf) in the salivary glands might be attributed to contamination of the gland during feeding on infective blood meal or dissemination from hemolymph.De-Silva and Fikrig (1995) demonstrated that the spirochete, B. burgdorferi had disseminated to the salivary glands in the majority of nymphs of Ixodes scapularis 36-48 hrs after attachment.Piesman et al. (2001) found that spirochete, B. burgdorferi in the tick salivary glands increased more than 17 fold from 1.2 per salivary gland pair before feeding to 20.8 at 72 hrs post attachment.The period of the most rapid increase in number of spirochetes in the salivary glands occurred from 48 to 60 hrs post attachment.
This time period coincided with maximal increase of transmission risk during nymphal feeding.Furthermore, heavy infections with B. duttoni, B. sogidiana and B. anserina were observed in the salivary glands of O. moubata (Giegy, 1951), O. tholozani (Teravsky, 1959) and Argas persicus (Nikitina, 1965), respectively.Varma (1956) reported that the transmission of relapsing fever spirochetes by Ornithodoros ticks was mainly by inoculation of infective saliva and coxal fluid into the bite wound.In the present work, early appearance of spirochetes in the coxal organs of female O. savignyi can be correlated to the production of the coxal fluid which is discharged during feeding and shortly after detachment of the argasid tick species (Balashov, 1972).

Localization of Borrelia sp. in the ovary:
Spirochetes showed their first appearance in the ovary of female O. savignyi on the fourth, third and second daf on infected hamsters at 17P o PC,

27P o
PC, and 37P o PC, respectively (Fig. 5 a & b).The initial appearance was followed by a gradual increase (P < 0.05) of IR and no. of spirochetes in ovaries up to 10 daf at the three-tested temperatures (Fig. 5 (Grun 1950) andB. duttoni (Giegy, 1951) and of Ixodes pacificus with B. burgdorferi (Burgdorfer, et al., 1989) were reported.Zhenqin (1998) burgdorferi could be found intracellularly within ovaries of I. ricinus.We suggest that migration and penetration of spirochetes into ovaries and oocytes of the tick vector are prerequisites of transovarial transmission.Also, it could be considered as a physiological adaptation increasing the potential and competence of the tick as a reservoir and vector of a pathogen (Hoogstraal, 1985).Furthermore, localization of the spirochete in the ovaries may help in recognizing the specificity of the tick species as a vector of a certain B. species particularly in ornithodorine and some argasid ticks (Balashov, 1972& Hoogstraal, 1985).Experimental infection of some argasid (Diab &Soliman, 1977 andZaher et al., 1977) and of some ornithodorine (Gaber et al., 1984 andShanbaky &Helmy, 2000) tick species with borreliae species isolated from other tick vectors resulted in failure of localization of the spirochetes in the gonads and transovarial transmission in the unnatural recipients.
In general, there was no significant difference (P > 0.05) between IR and no. of spirochetes in each organ of O. savignyi adult females kept at 27P The results and discussion of the present study show that the appearance, persistence, IR and no. of the spirochete varied in the different organs examined which conform with other previous investigations (Varma, 1962;Balashov, 1972;Diab & Soliman, 1977;Zaher et al., 1977, andGaber et al., 1984).It was suggested (Burgdorfer, 1951), and experimentally demonstrated (Sarsian, 1959, andGrun &Blatter, 1960) (2002).
A brief summary of hemosporidian agents of farm animals and of their vectors in USSR, Vet., 35: 31-34. Nikitina, R.E. (1964) The relatively high temperature, among other factors prevailed in summer might have contributed to the increased IR's reported in the aforementioned study.Temperature is one environmental factor known to affect persistence of spirochetal infection inside the tick.Shih et al. (1995) demonstrated that the persistence of spirochetal infection of the lyme disease B. burgdorferi in the gut of the nymphal deer tick, Ixodes dammini was reduced or became undetectable by incubation of the ticks at temperatures higher than 27P o PC (33 o C and 37P o PC, 27P o C, and 37P o C, respectively (Fig. 2 a & b).During these periods, IR's and no. of spirochetes in the hemolymph showed an initial period of increase (P < 0.05) in their levels to reach maxima on 7-8 daf (IR = 40-50%, no.= 15.2+7.14 -17.7+8.87),5-9 daf (50-70%, no.= 23.9+6.61-40.8+15.47)and 5-9 daf (IR = 70-80%, no.= 24.4+4.91 -43.5+10.80)at 17P o C, 27P o PC and 37P o PC,respectively.This was followed by a gradual decrease (P < 0.05) of the levels to reach minima (IR = 10, 20 & 10%, no.= 0.2+0.19,0.7+0.52 & 0.1+0.09at 17P o the end of their persistence period.

PC
or reached minima at the end of the persistence period in the gland (90 daf) . Infection with B. burgdorferi was reduced and undetectable in the gut of Ixodes scapularis nymphs kept at 33P o PC.
on distribution of B. duttoni in organs of O. PC.