Brugia pahangi: Immunization with early L3 ES alters parasite migration,and reduces microfilaremia and lymphatic lesion formation in gerbils (Meriones unguiculatus)

Previous studies have shown that intradermally (ID) injected Brugia pahangi L3s migrate through various tissues and into the lymphatics of gerbils in a distinct pattern. Excretory/secretory products (ES) produced at the time of invasion of B. pahangi are likely to be important in this early migration phase of the parasite life cycle in their rodent host. Hence, early L3 ES was collected from 24 h in vitro cultures of B. pahangi L3 larvae and used in immunization experiments to investigate the effect of immunity to early L3 ES on worm migration, survival and development of B. pahangi. Immunization of gerbils with ES in RIBI adjuvant produced antibodies to numerous ES proteins eliciting a strong humoral response to ES and indirect fluorescent antibody (IFA) assay using anti-ES serum recognized the ES proteins on the surface of B. pahangi L3 larvae. Following ES immunization, gerbils were challenged either ID or intraperitoneally (IP) with 100 L3s of B. pahangi and euthanized at 3 or 106 days post inoculation (DPI). Immunization with early ES slowed the migration of ID inoculated L3 at 3 DPI and significantly altered the locations of adult worms at 106 DPI. Immunization did not induce protection in any treatment group. However, immunized animals had significantly fewer microfilariae per female worm suggesting the antigens in ES are important in microfilariae development or survival in the host. The number of lymphatic granulomas was also significantly reduced in ES immunized animals. It is important to note that microfilariae serve as a nidus in these granulomas. Our results shows immunization with early Brugia malayi L3 ES alters the worm migration, affects circulating microfilarial numbers and reduces lymphatic granulomas associated with B. pahangi infection in gerbils.     

Detection of anti-parelaphostrongylus tenuis antibodies in experimentally infected and free-ranging moose (Alces alces)

Confirming Parelaphostrongylus tenuis infection in moose (Alces alces) and other susceptible hosts is difficult. An enzyme-linked immunosorbent assay (ELISA) was developed using the excretory-secretory (ES) products of third-stage P. tenuis larvae (ES-ELISA) and the test applied to serum samples obtained from seven moose calves (5-9.5 mo old) given infective larvae (L3) in doses approximating those likely to be received in nature (3-30 L3). Anti-P. tenuis immunoglobulin G antibodies were detected in all seven inoculated moose during the course of infection until the termination of experiment 61-243 days post-inoculation (DPI). Five animals tested between 16-25 DPI had significant antibody levels, while a sixth animal did not test positive until 46 DPI. The seventh animal was not tested until 199 DPI. Antibody levels remained elevated in all five animals that harbored adult worms at the termination of the experiment. Whereas, antibody levels showed a gradual decline in the two remaining animals, presumably because of death of worms, and antibodies were undetected in one animal at the time of necropsy. The other animal displayed an anamnestic increase in antibody level following a challenge inoculation of infective larvae. Terminal and peak optical density (OD) values detected by ES-ELISA strongly correlated with inoculation dose (r = 0.98, P = 0.02 and r = 0.95, P = 0.04, respectively) among animals harboring adult worms (n = 4) but not significantly with the number of worms recovered postmortem (peak OD, r = 0.82, P = 0.18; terminal OD, r = 0.93, P = 0.07). Unlike the ES products, use of somatic antigens of the adult worm in ELISA did not provide satisfactory results. Antibodies to P. tenuis were detectable by ES-ELISA in two of 21 free-ranging moose from an enzootic area but not from any of 23 animals from a non-enzootic area. The ES-ELISA appears to be a useful test for assessing exposure of moose to P. tenuis.     

Larval migration of Toxocara canis in piglets and transfer of larvae from infected porcine tissue to mice

Visceral larva migrans (VLM), caused by Toxocara canis larvae in humans, animals and birds, is now well documented throughout the world. Seven piglets were infected orally with 5 x 104 embryonated eggs and the migration and distribution of T. canis larvae in the tissues were evaluated. After artificial gastric juice digestion, larval yields at necropsy from different organs and muscles on days 1, 3, 7, 15 and 30 post-infection (DPI) revealed 3.05, 0.97, 0.21, 0.13, 0.05, 0.14% recovery from liver, lungs, heart, kidneys, skeletal muscles and brain tissues respectively, with a total of 2486 (4.97%) recovery from all tissues together. The highest number of larvae 1527 (3.05%) was recovered from the liver throughout the period (1-30 DPI), indicating a special affinity of larvae for the liver. Subsequently five mice were each infected orally with 5 g of infected pig liver and, after necropsy on 10 DPI, 20 +/- 3.62, 17 +/- 5.10, 3 +/- 1.26, 12 +/- 3.92 and 30 +/- 5.69 larvae were recovered from liver, lungs, heart, brain and muscles, respectively. Thus, primarily, the migratory potential and adaptation of T. canis larvae in porcine tissue was examined and, subsequently, their establishment in the second paratenic host, the mouse, has been successful. No influence of host sex on the migratory potential of T. canis larvae was observed. The related pathology caused by migratory larvae and its zoonotic significance through the consumption of raw or undercooked pork has been emphasized.     

Experimental infection of the cockroach Periplaneta americana with Toxocara canis and the establishment of patent infections in pups

The possible role of the cockroach Periplaneta americana in the transmission of Toxocara canis eggs and larvae via faeces and tissue migration was studied. Cockroaches fed with 3 x 105 and 5 x 105 embryonated eggs were found to harbour viable eggs and larvae from days 1 to 5 post-infection (DPI). At necropsy on 5 DPI, eggs and larvae were also recovered from the rectal contents but not from the tissues of cockroaches. In addition patent infections were established in pups fed on infected faeces of cockroaches, with eggs first appearing in the faeces of pups at 38 DPI. Adult worms of T. canis were also recovered at necropsy. Therefore the importance of cockroaches as good mechanical disseminators of ascarid eggs, especially T. canis, is discussed.     

Brugia pahangi: effects of duration of infection and parasite burden on lymphatic lesion severity, granulomatous hypersensitivity, and immune responses in jirds (Meriones unguiculatus)

The effects of Brugia pahangi infection duration and parasite burden on parasite-associated inflammatory and immune responses were determined over a 181-day period in jirds receiving from one to eight inoculations of infective larvae. Multiple infections did not produce a protective resistance to reinfection as determined by adult worm recovery at necropsy. Intralymphatic granulomatous lesions, lymph thrombi, were first seen at 48 days post initial inoculation (DPI). The numbers of lymph thrombi reached peak levels in singly inoculated jirds at 90 DPI and significantly decreased to low levels by 160 DPI. The ratio of lymph thrombi to adult worms recovered from the spermatic cord lymphatics followed a similar pattern. Sizes of renal lymph nodes, which drain lymphatics containing parasites, followed a temporal pattern of increase and decrease similar to that of lymph thrombi numbers. Peak granuloma areas around antigen-coated beads embolized in lungs were seen at 27 DPI. Granuloma areas around antigen-coated beads began to decrease after 69 DPI and reached sizes not significantly different from uninfected controls by 118 DPI. Multiple inoculations of infective larvae and increasing worm burdens did not affect the pattern of granulomatous response to antigen-coated beads. Eosinophilia of singly and multiply infected jirds peaked at 26 DPI. Eosinophilia of singly infected jirds returned to normal levels by 103 DPI but those of multiply infected jirds remained elevated until 160 DPI. Lymph node cell blastogenic responses to antigen were greater than those of splenocytes at all time intervals measured. However, significant differences in stimulation indexes between groups with different infection durations were not seen with either cell type. Antibody responses to somatic adult worm antigen as measured by ELISA reached near peak levels by 48 DPI and remained elevated for the course of the study in all infected jirds. The decrease in lymphatic lesion severity seen in chronically infected jirds temporally corresponds to the decrease in granulomatous reactivity measured around antigen-coated beads embolized in the lungs. This observation suggests that host and/or parasite factors associated with these two phenomena may be similar. Although these decreases may be the result of down-regulated immune responses, corresponding decreases in antibody levels and blastogenesis of lymphocytes stimulated by crude worm extracts were not observed in chronic infections.     

Immunity to Heligmosomoides polygyrus induced by subcutaneous vaccination with post-infection larvae

The objective of this study was to investigate, using the Heligmosomoides polygyrus (= Nematospiroides dubius)-mouse model, whether live post-infection trichostrongylid larvae recovered from the intestinal wall of donor animals and placed subcutaneously would serve as vaccine protecting against oral challenge by third-stage (infective) larvae (L3). Experiments were conducted to determine the effect of number and age of post-infective larvae as well as age and sex of host on vaccination. Vaccinated BALB/cByJ mice were challenged with 30 L3 and total adult worm burdens compared between vaccinated groups and sham-treated controls (greater than 90% infection rates). All mice subcutaneously vaccinated with either five or 10 larvae harbored significantly fewer challenge parasites in their intestines than did sham-treated controls (P less than 0.001). Both young and mature mice were significantly protected against challenge by the subcutaneous larval vaccine. Adult female mice had significantly (P less than 0.05) fewer parasites than adult male mice. The age of the larvae (indicated as the days between infection and harvesting of the larvae) was important in that day-4 or day-6 larvae (L4) were significantly more protective (P less than 0.001) than day-2 (L3) or day-8 larvae (L5-preadult). Reduction in worm burden for young vaccinated animals ranged from 31 to 39% (P less than 0.001) and for mature animals from 88 to 100% (P less than 0.001). Passive transfer to serum resulted in the reduction of worm burdens by 26-40% (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue migration capability of larval and adult Brugia pahangi

Infection with mosquito-born filarial nematodes occurs when hosts are bitten by a vector carrying the infective third stage larvae (L3) of the parasites. These larvae, deposited on the skin by the feeding mosquito, are presumed to enter the skin via the vector-induced puncture wound. Larvae of Brugia spp. must then migrate from the entry site, penetrate various skin layers, and locate a lymphatic vessel that leads to their lymphatic predilection site. We have recently established an intradermal (ID) infection model using B. pahangi and the Mongolian gerbil, allowing us to investigate the migratory capability ofB. pahangi. Larval and adult parasites recovered from the peritoneal cavities of gerbils were capable of establishing an infection following ID (larvae) or subcutaneous (adult) injection. Third and fourth stage larvae both migrated away from the injection site within hours, although data suggest they localize to different lymphatic tissues at 3 days postinfection (DPI). Immature adult (28 day) B. pahangi also migrated away from their SC inoculation site within 7 DPI. Mature (45 day) adult B. pahangi displayed little migration away from the SC infection site, suggesting tissue migration may be limited to developing stages of the parasite.     

Passive transfer of protective immunity to larval Dirofilaria immitis from dogs to BALB/c mice

Protective immunity to larval Dirofilaria immitis has been demonstrated in both the natural host, the dog, and in an experimental host, the mouse. In the present study, sera were collected and pooled from dogs that had been shown to have protective immunity to larval D. immitis. The pooled serum was inoculated into normal BALB/cByJ mice that then were challenged with third-stage larvae (L3) implanted in diffusion chambers. Two weeks postchallenge no significant difference was seen in either parasite survival or growth. Three weeks postchallenge, there was a significant decrease in parasite survival in mice receiving serum from immune dogs. Living larvae recovered at 3 wk postchallenge were significantly shorter than cohorts recovered from control mice. Antibody responses to L3 and forth-stage larvae (L4) surface antigens, to L3 and L4 aqueous soluble antigens, and to an excretory-secretory antigen fraction were measured. Only antibody responses to L3 surface antigens were elevated in the immune serum as compared to controls, thus suggesting a possible role for antibodies with specificity for surface antigens in protective immunity.     

Strongyloides stercoralis: Amphidial neuron pair ASJ triggers significant resumption of development by infective larvae under host-mimicking in vitro conditions

Resumption of development by infective larvae (L3i) of parasitic nematodes upon entering a host is a critical first step in establishing a parasitic relationship with a definitive host. It is also considered equivalent to exit from the dauer stage by the free-living nematode Caenorhabditis elegans. Initiation of feeding, an early event in this process, is induced in vitro in L3i of Strongyloides stercoralis, a parasite of humans, other primates and dogs, by culturing the larvae in DMEM with 10% canine serum and 5mM glutathione at 37 degrees C with 5% CO(2). Based on the developmental neurobiology of C. elegans, resumption of development by S. stercoralis L3i should be mediated, in part at least, by neurons homologous to the ASJ pair of C. elegans. To test this hypothesis, the ASJ neurons in S. stercoralis first-stage larvae (L1) were ablated with a laser microbeam. This resulted in a statistically significant (33%) reduction in the number of L3i that resumed feeding in culture. In a second expanded investigation, the thermosensitive ALD neurons, along with the ASJ neurons, were ablated, but there was no further decrease in the initiation of feeding by these worms compared to those in which only the ASJ pair was ablated.     

The life cycle of Paraquimperia tenerrima: a parasite of the European eel Anguilla anguilla

Previous studies on the life history of the nematode eel specialist Paraquimperia tenerrima (Nematoda: Quimperiidae) have failed to determine whether an intermediate host is required in the life cycle. In the laboratory, eggs failed to hatch below 10 degrees C, hatching occurring only at temperatures between 11 and 30 degrees C. Survival of the free-living second stage larvae (L2) was also temperature dependent, with maximal survival between 10 and 20 degrees C. Total survival of the free-living stages (eggs and L2) is unlikely to exceed a month at normal summer water temperatures, confirming that parasite could not survive the 6 month gap between shedding of eggs in spring and infection of eels in early winter outside of a host. Eels could not be infected directly with L2, nor could a range of common freshwater invertebrate species. Third stage larvae (L3) resembling P. tenerrima were found frequently and abundantly in the swimbladder of minnows Phoxinus phoxinus from several localities throughout the year and were able to survive in this host in the laboratory for at least 6 months. Third stage larvae identical to these larvae were recovered from minnows experimentally fed L2 of P. tenerrima, and eels infected experimentally with naturally and experimentally infected minnows were found to harbour fourth stage larvae (L4) and juvenile P. tenerrima in their intestines. Finally, the whole life cycle from eggs to adult was completed in the laboratory, confirming that minnows are an obligate intermediate host for P. tenerrima.     

Effects of in vitro culture methods on morphological development and infectivity of Strongyloides venezuelensis filariform larvae

The effects of in vitro culture methods on morphological development and infectivity of Strongyloides venezuelensis filariform larvae (L3) to rats were investigated. A significantly higher body length was observed in L3 from filter paper culture (597.3 +/- 32.2 microns) than those in fecal (509.9 +/- 35.0 microns) and nutrient broth culture (503.3 +/- 31.0 microns) (P < 0.05). Larval infectivity was assessed by exposing rats to 1,000 L3 from each culture and worms were recovered from the lungs and small intestines. Recovery rate of these worms did not show any significant difference. A significantly greater body length of adults was recorded in those corresponding to the L3 harvested from filter paper (2,777.5 +/- 204.4 microns) and nutrient broth culture (2,732.5 +/- 169.8 microns) than those corresponding to the L3 obtained from fecal culture (2,600.5 +/- 172.4 microns) (P < 0.05). Although worm fecundity and EPG counts differed among culture methods but worm burdens and course of infection did not. These findings suggest that the methods of cultures have a significant effect on the morphological development of the larvae to the L3 stage, but do not influence the infectivity to rats.     

Infection by the microsporidium Vairimorpha necatrix (Microspora: Microsporidia) elevates juvenile hormone titres in larvae of the tomato moth, Lacanobia oleracea (Lepidoptera: Noctuidae)

The effects of infection by a microsporidium, Vairimorpha necatrix (Kramer), on the endogenous levels of juvenile hormones in tomato moth (Lacanobia oleracea L.) larvae were investigated. Levels of juvenile hormone II (JH II) were 10-fold greater in the infected larvae on day two of the sixth stadium but no significant difference was observed on day seven. Juvenile hormone I (JH I) was also detected in day two and day seven sixth instar infected larvae but was not detected in non-infected larvae. The duration of the fifth and sixth stadia was significantly longer for infected larvae when compared with non-infected larvae. No evidence was found to suggest that supernumerary moults are a feature of infection by V. necatrix in L. oleracea larvae. Experiments were performed to determine whether the elevation in JH levels, which probably prevents pupation, is an adaptive mechanism of the microsporidium for extending the growth phase of the host, thereby allowing increased spore production. A proportion of infected larvae were collected on days 9 and 24 of the sixth stadium and spore extracts prepared from each larva. These days represent the average duration of the sixth stadium required for uninfected larvae to reach pupation, and the average number of days that V. necatrix-infected larvae survive in the sixth stadium before dying from infection. The mean spore yields from infected larvae 24 days into the sixth stadium were significantly higher than the spore yields obtained from day nine sixth instar larvae. The hypothesis that V. necatrix manipulates host endocrinology (i.e. prolong the host larval state to maximise spore yield) is discussed in context with the results obtained.     

Differential expression of the CrV1 haemocyte inactivation-associated polydnavirus gene in the African maize stem borer Busseola fusca (Fuller) parasitized by two biotypes of the endoparasitoid Cotesia sesamiae (Cameron)

Polydnaviruses are rarely studied for their natural variation in immune suppressive abilities. The polydnavirus harboring braconid Cotesia sesamiae, a widespread endoparasitoid of Busseola fusca and Sesamia calamistis in sub-Saharan Africa exists as two biotypes. In Kenya, the western biotype completes development in B. fusca larvae. However, eggs of the coastal C. sesamiae are encapsulated in this host and ultimately, no parasitoids emerge from parasitized B. fusca larvae. Both biotypes develop successfully in S. calamistis larvae. Encapsulation activity by B. fusca larvae towards eggs of the avirulent C. sesamiae was detectable six hours post-parasitization. The differences in encapsulation of virulent and avirulent strains were associated with differences in nucleotide sequences and expression of a CrV1 polydnavirus (PDV) gene, which is associated with haemocyte inactivation in the Cotesia rubecula/Pieris rapae system. CrV1 expression was faint or absent in fat body and haemolymph samples from B. fusca parasitized by the avirulent C. sesamiae, which exhibited encapsulation of eggs. Expression was high in fat body and haemolymph samples from both B. fusca and S. calamistis larvae parasitized by the virulent C. sesamiae, encapsulation in the former peaking at the same time points as CrV1 expression in the latter. Non synonymous difference in CrV1 gene sequences between virulent and avirulent wasp suggests that variations in B. fusca parasitism by C. sesamiae may be due to qualitative differences in CrV1-haemocyte interactions.     

Inhibition of juvenile hormone esterase activity in Lymantria dispar (Lepidoptera, Lymantriidae) larvae parasitized by Glyptapanteles liparidis (Hymenoptera, Braconidae)

Glyptapanteles liparidis is a gregarious, polydnavirus (PDV)-carrying braconid wasp that parasitizes larval stages of Lymantria dispar. In previous studies we showed that parasitized hosts dramatically increase juvenile hormone (JH) titers, whereas JH degradation is significantly inhibited in the hemolymph. Here we (i) quantified the effects of parasitism on JH esterase (JHE) activity in hemolymph and fat body of penultimate and final instars of L. dispar hosts and (ii) assessed the relative contribution of individual and combined wasp factors (PDV/venom, teratocytes, and wasp larvae) to the inhibition of host JHE activity. The effects of PDV/venom was investigated through the use of gamma-irradiated wasps, which lay non-viable eggs (leading to pseudoparasitization), while the effects of teratocytes and wasp larvae were examined by injection or insertion of these two components in either control or pseudoparasitized L. dispar larvae. Parasitism strongly suppressed host JHE activity in both hemolymph and fat body irrespective of whether the host was parasitized early (premolt-third instar) or late (mid-fourth instar). Down-regulation of JHE activity is primarily due to the injection of PDV/venom at the time of oviposition, with only very small additive effects of teratocytes and wasp larvae under certain experimental conditions. We compare the results with those reported earlier for L. dispar larvae parasitized by G. liparidis and discuss the possible role of JH alterations in host development disruption.     

Differential host growth regulation by the solitary endoparasitoid, Meteorus pulchricornis in two hosts of greatly differing mass

Solitary koinobiont endoparasitoids generally reduce the growth of their hosts by a significant amount compared with healthy larvae. Here, we compared the development and host usage strategies of the solitary koinobiont endoparasitoid, Meteorus pulchricornis, when developing in larvae of a large host species (Mythimna separata) and a much smaller host species (Plutella xylostella). Caterpillars of M. separata were parasitized as L2 and P. xylostella as L3, when they weighed approximately 2 mg. The growth of parasitized M. separata larvae was reduced by almost 95% compared with controls, whereas parasitized P. xylostella larvae grew some 30% larger than controls. Still, adult wasps emerging from M. separata larvae were almost twice as large as wasps emerging from P. xylostella larvae, had larger egg loads after 5 days and produced more progeny. Survival to eclosion was also higher on M. separata than on P. xylostella, although parasitoids developed significantly faster when developing on P. xylostella. Our results provide evidence that koinobionts are able to differentially regulate the growth of different host species. However, there are clearly also limitations in the ability of parasitoids to regulate phenotypic host traits when size differences between different host species are as extreme as demonstrated here.     

Density-dependent effects on the survival and growth of the rodent stomach worm Protospirura muricola in laboratory mice

The spirurid nematode, Protospirura muricola, is of intrinsic interest as a rodent model of gastric nematode infections. Since worm burdens can be very heavy in nature, density dependent processes may constrain parasite growth. Laboratory mice (BKW) were exposed to varying doses of infective larvae of P. muricola in the range 5 to 40 third-stage larvae (L3), in four separate experiments in which progressively higher doses were utilized. All mice were culled 60 days after infection and a total of 518 worms (226 male and 292 female worms) was recovered, measured and weighed. Overall survival was 58.9%, but survival declined significantly with increasing dose by approximately 21% (from 66% at 5 L3 per mouse to 52% at 40 L3 per mouse). The length and weight of worms correlated positively in both sexes. Total worm biomass increased linearly with increasing numbers of worms. However, whilst the length and weight of male worms declined with increasing worm burden (8.4 and 24.6% respectively), female worms were less affected, only length showing a significant reduction with increasing parasite burden (16.0%). Therefore, increasing worm burdens impeded growth of P. muricola, but reduction in length and weight were relatively small in relation to the overall size of this nematode. Increasing worm burdens were associated with loss of host weight and reduction in stomach weight and worm burdens in excess of 20 exerted a measurable cost to the host, which in the field, may be associated with loss of overall host fitness.     

Parasitoid-mediated transmission of an iridescent virus

We examined the interaction between an invertebrate iridescent virus (IIV) isolated from Spodoptera frugiperda (J.E. Smith) and the solitary ichneumonid endoparasitoid Eiphosoma vitticolle Cresson. In choice tests, parasitoids examined and stung significantly more virus infected than healthy larvae, apparently due to a lack of defense reaction in virus infected hosts. Parasitoid-mediated virus transmission was observed in 100% of the female parasitoids that stung a virus infected host in the laboratory. Each female parasitoid transmitted the virus to an average (+/-SE) of 3.7+/-0.3 larvae immediately after stinging an infected larva. Caged field experiments supported this result; virus transmission to healthy larvae only occurred in cages containing infected hosts (as inoculum) and parasitoids (as vectors). The virus was highly detrimental to parasitoid development because of premature host death and lethal infection of the developing endoparasitoid. Female parasitoids that emerged from virus infected hosts did not transmit the virus to healthy hosts. We suggest that the polyphagous habits of many noctuid parasitoids combined with the catholic host range of most IIVs may represent a mechanism for the transmission of IIVs between different host species in the field.     

Factors Influencing Survival of Ditylenchus dipsaci (Kühn, 1857) in Soil

Ditylenchus dipsaci larvae survived in soil without a host plant for at least 242 days when held at 15 C and 21 C. Larvae held at 15 C remained infective for 212 days. Moisture levels within both clayey and sandy soils did not appreciably affect recovery of larvae. Active nematodes recovered from soil are not necessarily infective. Temperatures of -12, 0 and 4 C had little adverse effect on larvae in infected leaf tissues in soil. Larvae in soil exposed to 0 C for short periods of time were not affected adversely. Recovery of larvae from sandy soil by Baermann funnels was significantly better at 24 C than at 4 C. Differences in recovery from clay soil were not significant at these temperatures.     

Occurrence and characterization of a nucleopolyhedrovirus from Spodoptera littoralis (Lepidoptera: Noctuidae) isolated in the azores

A nucleopolyhedrovirus (SpliMNPV-Az) was isolated from diseased larvae of Spodoptera littoralis, collected at the Island of S. Miguel in Azores. The virulence of this isolate was tested against S. littoralis larvae in laboratory. LD₅₀ against 2nd and 3rd instars were not significantly different, 1.44 × 10⁴, 3.89 × 10⁴ OBs per larvae, respectively, but both were significantly different from that against 4th instar, which was 61.3 × 10⁴ OBs per larvae. The complete codons sequence of SpliMNPV-Az Polh gene obtained was 750 bp (NCBI GenBank Accession No. AY600451). This sequence was compared to other 38 polyhedrin genes from NPVs and to 6 granulin genes from GVs and resulted to be identical to the sequence of a SpliMNPV previously published, thus indicating that the natural host of SpliMNPV-Az must be S. littoralis. Genetic distances estimated from restriction enzymes profiles showed SpliMNPV-Az is close to the Egyptian SpliMNPV type B, despite some degree of genetic divergence suggested by slight differences observed on PstI profile.