الفهرس 
Tick rearingOnly 14 pages are availabe for public view
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Abstract
The argasid tick Ornithodoros erraticus is widely distributed in Egypt and was found to harbor and transmit Borrelia crocidurae, the etiologic agent of the North African relapsing fever. Ticks become infected by feeding upon spirochaetemic animals, then the spirochaetes migrate from the digestive tract to the hemocoele and invade almost all tissues including the reproductive system where they multiply and may be transmitted transovarially to the offspring.
The study of structural and physiological peculiarities of the tick reproduction and interaction between pathogen and vector tick is necessary to understand the basis underlying its success as a vector of diseases. In the present study a laboratory reared female O. erraticus with high infection rate were used to study the effect of borrelial infection on reproduction and proteins involved in egg production. Also, histological localization of the spirochaetes in the infected ovary was investigated.
1 Basic interrelationships of B. crocidurae with female O. erraticus:
• The follow-up and inspection of the level of infection rate in the infected laboratory colony detected the presence of B. crocidurae in 95 – 100% of the examined mated fed females O. erraticus during the period of study.
• Feeding of uninfected female O. erraticus on B. crocidurae infected hamster was associated with the ingestion of the spirochaete in the midgut immediately, then its entry in the haemolymph and salivary gland on the 1st day after feeding (d.a.f) and in the ovary on the 3rd d.a.f. Borrelial infection persisted in these organs mostly till 30 d.a.f in salivary gland and ovary, 40 d.a.f in the gut and 60 d.a.f in haemolymph.
• Transovarial transmission existed in the infected colony where 35 – 52% of the examined unfed larvae in the colony were infected with B. crocidurae during the period of study.
• Feeding of O. erraticus infected females on a clean hamsters resulted in the transmission of infection to the hamster. The percent infection of the hamster gradually increased by increasing the number of feeding infected mated females.
2 Effect of Borrelia crocidurae infection on reproduction of O. erraticus females:
• Infection of female O. erraticus with B. crocidurae caused insignificant increase in the preoviposition period (9.87±0.40 days) and insignificant decrease of the oviposition period (7.60 ±0.46 days) than in the uninfected ones (9.47±0.22 and 8.00±0.72 days, respectively) .
• Borrelia crocidurae infection of female O. erraticus caused significant decrease in fecundity (mean 81.00±5.35 eggs/oviposition/female) and the mean daily egg output (10.92±0.65 eggs) as compared with that of the uninfected females (103.80±6.21 and 15.56±2.66 eggs, respectively).
• Borrelia crocidurae infection of female O. erraticus caused significant decrease in the viability, hatching percent (86.01±2.19%) and prolongation of the incubation period (10.33±0.16 days) as compared with that of the uninfected females (90.46±1.13% and 9.8±0.28 days, respectively).
3 Total protein concentration in the haemolymph of O. erraticus females uninfected and infected with B. crocidurae:
• In the uninfected female O. erraticus, engorgement was followed by a decrease in the level of haemolymph protein concentration immediately and on the next two days after feeding. Then the level of haemolymph proteins gradually increased on the following days to reach maximum on 5 – 9 d.a.f. On 20 d.a.f (end of oviposition) the level of haemolymph protein decreased again to reach the level of the unfed females.
• Changes in the haemolymph total protein concentration of O. erraticus mated females infected with B. crocidurae followed almost the same pattern of the uninfected ones. However, on 3 – 9 d.af the level of haemolymph protein was lower than that of the corresponding days in the uninfected ones. Then the levels of haemolymph protein approached each other on 20 d.a.f in both females.
4 Changes of ovary protein concentration, weight and mature oocytes of O. erraticus female uninfected and infected with B. crocidurae:
• The ovary total protein concentration of uninfected O. erraticus female increased significantly on the 1st d.a.f and the increase continued evidently on the next days to reach maximum level on the 9th d.a.f. The level of ovary total protein concentration largely decreased at the end of oviposition on 20 d.a.f.
• The changes of the ovary weight and mature oocytes number in the uninfected females paralleled the changes of the ovary total protein. These two parameters increased significantly on the 2nd and 3rd d.a.f for the ovary weight and oocytes number, respectively and the increase continued evidently on the following days to reach maximum at the beginning of oviposition (9 d.a.f). Then they declined at the end of oviposition (20 d.a.f).
• Changes in the ovary protein concentration, weight and mature oocytes of the infected female O. erraticus followed the same pattern of that in the uninfected ones. However, the level of ovary total protein concentration was significantly lower in the infected than the uninfected females on the 3rd to the 9th d.a.f. Also the weight of ovary and number of mature oocytes were higher in the uninfected than the infected on 5 – 9 and 3 – 9 d.a.f, respectively. At the end of oviposition (20 d.a.f) the ovary protein level, weight and oocytes number of the uninfected and infected females became similar.
5 Total protein concentration of the eggs uninfected and infected with B. crocidurae:
• Borrelia crocidurae infected females laid eggs with lower total protein concentration (22.53±1.43mg/g tissue) than eggs laid by uninfected ones (26.64±1.26 mg/g tissue).
6 Native protein fractions in female O. erraticus uninfected and infected with B. crocidurae:
• The number and % concentration (amount) of electrophoretically separated native protein in the haemolymph (HL) and ovary varied in the physiological states studied (unfed and fed immediately after feeding ”0 d.a.f”, 3, 6, 9 and 20 d.a.f) of mated female O. erraticus.
• In the HL and ovary of the uninfected female O. erraticus a total of 48 and 78 protein fractions (bands) with molecular weights (MWts) of 60.069 – 282.31 and 17.778 – 313.28 kDa and a number of 7 – 9 and 8 – 16 fractions, respectively, occurred in the different physiological states studied.
• In the HL and ovary of mated female O. erraticus infected with B. crocidurae a total of 51 and 62 protein fractions with MWts of 39.826 – 283.75 and 17 – 228.16 kDa and a number of 6 – 10 and 8 – 14 fractions, respectively, occurred in the different physiological states studied.
7 Egg vitellins and vitellogenic protein fractions in uninfected female O. erraticus:
• Proteins in the freshly deposited eggs of the uninfected mated engorged female O. erraticus were electrophoretically separated into 15 protein fractions or egg vitellins (EVts) with MWts of 18.556 – 309.83 kDa. Vitellogenic protein fractions with more or less similar electrophoretic mobility (MWt of EVt ± 0.1 up to 5 kDa) were identified in the HL (vitellogenins) and ovary (vitellins) of female O. erraticus.
• One fraction of the egg vitellins with the highest MWt (309.83 kDa) and seven fractions with relatively low MWts of 18.556 – 54.7 kDa had corresponding bands (protein fractions) with more or less similar electrophoretic mobility only inside the ovary and were considered as endogenous egg vitellins. The rest of the EVts (7) were considered as exogenous egg vitellins (with MWts of 247.76 – 60.55 kDa) had corresponding bands with more or less similar electrophoretic mobility in the HL (one EVt fraction with MWt of 247.76 kDa) or in HL and ovary (6 EVt fractions with 192.59, 137.41, 100.19, 78.348, 67.609 and 60. 55 kDa).
• The number and sum of percent concentration (% amount) of the vitellogenic protein fractions varied in the different physiological states studied. The total number of vitellins in the ovary (63 Vts) was higher than that of vitellogenins in the HL (30 Vgs).
• The sum of percent concentration of Vg fractions in the HL of mated unfed female (52.17%) decreased immediately after feeding to 42.136%, then increased to 49.823% and 48.327% on 3 and 6 d.a.f, respectively, decreased to 42.47% on the 9th d.a.f to increase greatly on 20 d.a.f (51.146%). However, the sum of percent concentrations of Vts in the ovary increased gradually from 31.839% in the unfed female to 37.244, 46.941% immediately and 3 d.a.f to reach a maximum of 70.153 and 72.638% on 6 and 9 d.a.f, respectively (3 – 9 d.a.f vitellogenesis), and decrease to 26.341% on 20 d.a.f.
8 Egg vitellins and vitellogenic protein fractions in female O. erraticus infected with B. crocidurae:
• Infection of mated female O. erraticus with B. crocidurae reduced the number of egg protein fractions (vitellins) and generally of their corresponding vitellogenic fractions in HL and ovary.
• Proteins in freshly deposited eggs of infected mated engorged females were electrophoretically separated into 9 egg vitellins as compared with 15 EVts in the uninfected females. The observed decrease was mainly a result of disappearance of 4 EVts with high MWt (one endogenous fraction with 309.83 kDa and 3 exogenous fractions with MWts of 247.76, 192.59 and 137.41 kDa). However, the existing 9 EVts in the eggs of the infected female were more or less similar electrophoretically (MWt of 16.276 – 104.23 kDa) to the corresponding counterparts of EVts in the eggs of the uninfected female (MWt of 18.556 – 100.19 kDa).
• Disappearance of the large endogenous EVt (with 309.83 kDa) left no corresponding counterparts in the HL or ovary of the infected female which suggested failure of synthesis of this fraction in the infected female. However, the disappeared 3 large exogenous EVts left behind them corresponding counterparts of Vgs in the HL (246.25, 249.38, 196.25, 193.13,193.13 and 133.75 kDa) of one or more physiological state (s) but not in the ovary. Sometimes these Vgs accumulated in the HL which pointed to impaired uptake by oocytes, and their entry into the ovary and the disappearance from eggs of the infected female O. erraticus.
• In the egg of the infected female O. erraticus all protein fractions with or higher MWt than 39.826 kDa had corresponding bands with more or less similar electrophoretic mobilities in the HL and ovary and were considered as exogenous EVt (Six fractions with MWts of 39.857, 49.778, 61.00, 69.906, 84.038 and 104.23 kDa). Egg protein fractions (EVts) of MWt less than 39.826 had corresponding counterparts only inside the ovary and were considered as endogenous EVts (3 fractions with MWt of 28.857, 23.759 and 16.276 kDa).
• The total number of ovary vitellins (52) was higher than that of HL Vgs (23) in the infected female.
• The sum of percent concentrations of Vg fractions in the HL of mated infected unfed female (39.04%) decreased greatly after feeding and reached 26.594, 20.484% on 3 and 6 d.a.f and 12.188 on 9 d.a.f to DROP to 8.414% on 20 d.a.f. However, the sum % concentrations of Vt fractions in the ovary of infected unfed female (30.785%) and imm.a.f (29.686%) increased to 50.600% on 3 d.a.f to reach maximum of 64.019 and 61.285% on 6 and 9d.a.f, respectively then decreased to 37.164% on 20 d.a.f.
9 Histological studies of the ovary of female O. erraticus and localization of Borrelia inside the ovary of infected females:
• The ovary of female O. erraticus is a single tubular structure which is composed of a central lumen surrounded by a wall of epithelial cells. The oocytes are attached to the ovary through the pedicel cells. The development of oocytes is asynchronous where different developmental stages can be observed. The oocytes can be classified into five developmental stages according to the presence of germ vesicles, cytoplasm appearance, presence of yolk granules and chorion.
• Borrelia crocidurae were localized in the infected ovary of female O. erraticus. Spirochaetes were observed penetrating in the pedicel cells at the points of contact with the oocytes. Also B. crocidurae was observed extracellularly in the ovarian lumen and outside the ovary adhering to the ovarian epithelial wall. Borrelia spirochaetes were also found penetrating the oocytes and yolk spherules. Also, a reduction of the thickness and disappearance of the chorion were occasionally observed.