Presence of Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) in southern Kettle Moraine State Forest, Wisconsin, and characterization of strain W97F51.

Lyme disease, caused by Borrelia burgdorferi Johnson, Schmidt, Hyde, Steigerwalt & Brenner; babesiosis, caused by Babesia microti Franca; and human granulocytic ehrlichiosis, caused by Anaplasma phagocytophilum Bakken & Dumler have been reported in Wisconsin, mainly in the endemic areas of the northwestern part of the state. People exposed to blacklegged ticks, Ixodes scapularis Say, from this region can potentially contract one or all of these diseases concurrently. Within the past several years, there have been cases of Lyme disease reported from southeastern Wisconsin, an area that contains deer, mice, and similar vegetation found at regions with high endemicity. However, past white-tailed deer surveys suggested no existence of I. scapularis in southeastern Wisconsin. However, in 1996, we identified questing adult I. scapularis in the southernmost part of the Southern Kettle Moraine State Forest, Walworth County. To determine whether an established population of I. scapularis exists in this region, we performed a detailed survey of the abundance of host-seeking I. scapularis adults and the presence and abundance of subadults feeding on white-footed mice, Peromyscus leucopus Rafinesque. We also tested for possible infections of B. burgdorferi, Ba. microti, and A. phagocytophilum in ticks and B. burgdorferi harbored by mice. In 1997 and 1998, a total of 249 P. leucopus mice and 118 questing adult I. scapularis ticks, in addition to 157 larvae and seven nymphs feeding on mice, were collected and their locations were recorded from the Nordic trails of the Southern Kettle Moraine State Forest. Only one P. leucopus and its attached engorged I. scapularis nymph were infected with B. burgdorferi, whereas none of the engorged larvae attached to mice were infected. However, 4.2% of questing adult I. scapularis were infected with B. burgdorferi. The abundance of questing adult I. scapularis was 1.6 ticks per hour. The prevalence of subadult ticks on mice was 27%, with a mean intensity on infested mice of 2.0. I. scapularis adults were not infected with either Ba. microti or A. phagocytophilum. A unique strain of B. burgdorferi s.l. (W97F51) was discovered, showing 33 nucleotide substitutions and one codon insertion in a 567-bp fragment of the OspB gene, compared with Borrelia bissettii (strain Ca389). The sequences of ospA, ospB, ospC,fla, and rrs genes and the rrf-rrl intergenic spacer region were compared between W97F51 and other B. burgdorferi s.l. species. Although W97F51 was most genetically related to B. bissettii, the genetic identity of W97F51 was less than that of B. bissettii conspecifics. This study documents the existence of an established population of I. scapularis and the presence of B. burgdorferi with a novel strain in southeastern Wisconsin.

Intestinal changes associated with expression of immunity to challenge with Eimeria vermiformis.

To provide more information on the mechanisms involved in the immune inhibition of eimeria infections, NIH mice were adoptively immunized against infection with Eimeria vermiformis by the transfer of mesenteric lymph node cells from primed animals and homologously challenged. Subsequent changes in the architecture and cellular composition of the intestine were compared with those observed in similarly challenged susceptible control mice and correlated with the development of the parasite in the two groups. Actively immunized mice were also examined. In adoptively immunized mice, the development of E. vermiformis was inhibited within 3 days of administering the challenge inoculum. Concurrent changes in the intestine included lymphocytic infiltration, crypt hyperplasia, flattening of the crypt epithelium, and a reduction in the number of Paneth cells. Hyperplasia of goblet and pyroninophilic cells in response to challenge, although accelerated and enhanced in adoptively immunized hosts, occurred after the inhibition of the parasites, and mastocytosis was not observed in these animals, findings which suggest that the activities of goblet, pyroninophilic, and mast cells were not instrumental in reducing the numbers of parasites. The intestines of immunized mice contained fewer intraepithelial lymphocytes at the time of inhibition of the parasites than did those of the controls. The protective effects and intestinal changes described above did not differ appreciably from those seen after challenge of mice that had been immunized by infection.

Variation in susceptibility of inbred lines of chickens to seven species of Eimeria.

The pattern of oocyst production of 8 inbred lines of chickens was compared for each of the 7 species of Eimeria which infect this host. Both the overall numbers and the pattern of oocyst production differed in the inbred lines, but there was no evidence of prolonged cycling of schizogenic developmental stages. Comparison of the numbers of oocysts produced by the different lines indicates that there may be common genetic factors affecting susceptibility to 6 of the 7 species. Surprisingly there appears to be an inverse relationship between susceptibility to E. tenella and susceptibility to the other species: lines which produced most oocysts of E. tenella produced least oocysts of the other species and vice-versa.

A novel method for cloning Eimeria praecox in the chicken.

A new method for producing clones of Eimeria praecox by infection of chickens with a single sporozoite retained within a sporocyst is described.

Eimeria spp. of domestic fowl: the migration of sporozoites intra- and extra-enterically.

Chickens were dosed orally with sporulated oocysts of Eimeria acervulina, E. brunetti, E. maxima, or E. praecox and the subsequent presence, in various tissues, of parasites capable of inducing patent infections was detected by transferring the tissues to coccidia-free recipients. Similar results were obtained with each of the 4 species studied, irrespective of whether initial development occurs in the superficial (E. praecox, E. brunetti) or crypt (E. acervulina, E. maxima) epithelium. Infection was transferable by gut scrapings and liver homogenates at all time intervals (3, 6, 12, 18, 24, and 36 hr postinoculation) studied. Infection was also transferable with blood and with splenic homogenates but not consistently. Transfers made within a short time of the inoculation of donors were more successful in producing patent infections in the recipients. In all transfers the prepatent period was normal for the species. These findings suggest that sporozoites enter the mucosa very shortly after inoculation, and some of them pass to the liver and spleen and then leave these tissues at a somewhat slower rate, possibly to reenter the mucosa. Sporozoites in the lamina propria of the gut were found within host mononuclear cells in all 4 species studied. Most of the cells harbouring E. maxima and some of those with E. praecox were identified as intraepithelial lymphocytes while all others could only be identified as agranular mononuclear cells that were not characteristically macrophages.

A comparative study of two lines of Eimeria tenella attenuated either by selection for precocious development in the chicken or by growth in chicken embryos.

A precocious line (HP) selected from the virulent Houghton (H) strain of Eimeria tenella was found to be attenuated and to have a shorter prepatent time than the parent strain. The second generation schizonts of the HP line were smaller than those of the H strain and contained fewer merozoites. A sub-line of HP, derived from a single oocyst, showed no sign of reversion to virulence after 19 passages without selection for precocity. Similarly, clones (the progeny of single sporozoites) of an attenuated embryo- adapted line (TA) remained avirulent after up to 26 passages in the chicken. In contrast, an uncloned TA line reverted to virulence after 20 passages in chickens. The reproduction in chickens of the HP line was greater than that of the TA line. Chickens kept in wire-floored cages and given small numbers of oocysts of HP or TA were not substantially immune to challenge with the virulent H strain. However, when chickens kept in litter pens were given the HP line they developed a strong immunity to a large challenge with either the H, Weybridge or each of four field strains. In the same conditions, prior inoculation with TA led to only partial resistance against challenge.

Studies on the immunogenicity of seven attenuated lines of Eimeria given as a mixture to chickens.

The ability of attenuated lines of all the seven species of Eimeria which infect the chicken to provide protection against challenge with virulent strains was examined. The parasites were given as a mixture of all seven species to chickens kept on litter in floor pens, by spraying an aqueous suspension of oocysts on to the surface of food in troughs within the floor pens. Treated chickens were challenged 4 weeks later with virulent strains, and changes in weight gain and the presence of gross intestinal lesions were determined. When used in this way, as freshly passaged oocysts or after storage for 3 or 6 months, the consistently good protection afforded by the attenuated lines suggests that they could be applied in the control of coccidiosis in the field.

Eimeria vermiformis: host strains and the developmental cycle.

After confirmatory studies of the endogenous development of Eimeria vermiformis in hybrid mice, a comparison, both qualitative and quantitative, was made of the developmental cycle of the parasite in two strains of host of contrasting susceptibility. No differences were apparent between the resistant, BALB/c, and susceptible, C57BL/6, during the first 5 days of the cycle. Thereafter, more parasites were seen in the C57BL/6, they were more widely distributed along the intestine, and they persisted in the tissues for a considerably greater time. Schizogony continued for longer in the C57BL/6 mice, concurrently with gametogony, and there were probably up to four additional generations of schizonts, which resembled those of the accepted third generation. The kinetics of oocyst production, measured in the feces, reflected events in the intestinal epithelium. The findings are discussed in relation to eimerian life cycles and the immune response.

Host specificity in eimerian coccidia: development of Eimeria vermiformis of the mouse, Mus musculus, in Rattus norvegicus.

The ability of Eimeria vermiformis, a coccidium which normally parasitizes the mouse, to develop in rats was investigated. The Rowett strain (Lac: RNU) rats were euthymic (rnu/+), treated or untreated with cortisone acetate, and athymic (rnu/rnu). E. vermiformis completed its development only in rnu/rnu rats, which passed small numbers of oocysts capable of sporulating and infecting C57BL/6 mice. In the rnu/+ rats, irrespective of cortisone treatment, development appeared to terminate with the completion of the asexual (schizogonic) stages since no gametocytes were recognized in the tissues and no oocysts were detected in the faeces. The findings are discussed with reference to the factors which govern host specificity.

Eimeria praecox: selection and characteristics of precocious lines.

Precocious lines of Eimeria praecox were obtained both from the Houghton laboratory strain and the progeny of a single sporocyst (derived from the Houghton strain) by selection for early development of oocysts in the mature host. The prepatent times of the parasites were reduced from 84 to less than 64 hours. The evolution of the trait for precocious development appeared to be a step-wise process culminating in the depletion of, probably, the fourth generation of schizont. Studies on the precocious line selected from the Houghton strain showed that it had a multiplication rate of less than 10% of its parent strain, yet chickens given small numbers of oocysts were almost completely immune to challenge with either the Houghton strain or any one of five field strains. The precocious line was also slightly less pathogenic. Examination of a sub-line subjected to 10 generations of relaxed passage showed that there was no marked loss of the traits for early prepatent time or for reduced fecundity.

The effect of immunity on the early events in the life-cycle of Eimeria tenella in the caecal mucosa of the chicken.

A comparison was made between the early events (asexual stages) in the life-cycle of Eimeria tenella in specifically immunized and control chickens. Particular attention was paid to the quantitative aspects and to the transport of sporozoites within intra-epithelial lymphocytes (IEL) from the enterocytes of the surface epithelium to the enterocytes of the crypts. There was a moderate decrease in the number of parasites initially seen in the mucosa of the immune birds, suggesting that some of the effects of immunity are exerted before penetration of the surface enterocytes, but the reduction in the numbers of developing parasites was more marked. This latter effect was due, at least partly, to failure of transfer of sporozoites from IELs to crypt enterocytes. These findings are discussed in relation to the efficacy of the immunity induced.

An egg-adapted (attenuated) line of Eimeria necatrix: further studies on its reproduction, pathogenicity and immunogenicity.

An egg-adapted line of Eimeria necatrix has now been passaged 40 times in the chorio-allantoic membranes of embryonated eggs. Between the 21st and 40th passage in eggs the parasite was subjected to a selection for precocious development, and its pre-patent period in this host is now 123-125 h. The parasite continued to adapt to the chorio-allantoic membrane throughout the period of the passages and its development in this tissue caused the growth of the host embryo to be markedly stunted. In chickens, both the reproduction and the pathogenicity of the egg-adapted line decreased with increasing numbers of passages in eggs. A comparison of the immunogenicity of the egg-adapted line and its parent strain in chickens kept on litter, showed that substantially more oocysts of the egg-adapted line had to be given to induce complete protection.

Parasite-host relationships during the development of Eimeria dispersa Tyzzer 1929, in the turkey (Meleagris gallopavo gallopavo) with a description of intestinal intra-epithelial leucocytes.

An electron microscopical study was made on the development of Eimeria dispersa in the small intestine of the domestic turkey. Turkey poults, 10-14 days of age, were inoculated with oocysts and pieces of intestinal tissue were fixed at intervals between 3.5 and 114 h after inoculation. Sporozoites were occasionally seen in enterocytes but more often in 'pale' cells closely resembling lymphocytes. These cells were insinuated between enterocytes and in this study are referred to as intestinal intra-epithelial leucocytes (IEL). Maturation of the first-generation of schizogony occurred in this type of cell. Other generations of schizonts and gametogony occurred in epithelial cells and were characterized by the presence of 'spines' arranged around the parasitophorous vacuole extending into the cytoplasm of the host cell. A limited study of the parasite in the Bobwhite quail, Colinis virginianus, showed that development occurred in similar cells to those of the turkey. The 'spines', which were characteristic of later stages of E. dispersa in the turkey, were also present in this host and seemed to be a characteristic of E. dispersa, not of the host species.

Immunisation against coccidiosis in chickens: tests under simulated field conditions.

Two trials designed to examine the effect of a vaccine against coccidiosis were carried out with commercial replacement layers. The effect of vaccination was evaluated by challenging vaccinated and non-vaccinated chickens. In the first trial chickens vaccinated at 14 days of age were compared with non-vaccinated, medicated chickens fed robenidine at 66 ppm throughout and with unmedicated chickens kept in the same house. In the second trial birds were vaccinated at 7 days of age and compared with non-vaccinated birds on a 'Pancoxin' stepdown programme and with chickens maintained coccidia-free by feeding robenidine at 66 ppm throughout. The vaccine was composed of two attenuated strains of E. mivati and E. tenella, and virulent laboratory strains of three or four other species of Eimeria. Resistance of the chickens to E. acervulina, E. brunetii and E. maxima and partial resistance to the other species developed within 3 weeks. In general, immunity increased and was high against all species, although less complete in the case of E. mivati and, when judged by lesion scores, E. tenella. Vaccination against coccidiosis was more effective, judged by resistance to challenge infection and performance, in terms of food conversion ratio, than the commercially accepted 'Pancoxin' regimen.

Thermal decomposition of propoxyphene during GLC analysis.

The decomposition products of propoxyphene during GLC were prepared on a larger scale by heating propoxyphene hydrochloride in the presence of glass wook. They were identified by mass spectrometry and NMR spectroscopy as two isomeric diphenylbutenes and two isomeric products formed by loss of propionic acid. On-column decomposition of proxyphene can be prevented by injection of the compound in solution in a silylating agent.

Immunological differences in Eimeria maxima: effect of a mixed immunizing inoculum on heterologous challenge.

The immunological differences known to exist between laboratory strains of Eimeria maxima was confirmed. Protection against challenge with different strains or field isolates of the species could be achieved by including small numbers (25 oocysts) of each in the immunizing inoculum. Similar protection was obtained when 4 distinct populations which were allowed to interbreed were used in the immunizing inoculum. This hybrid mixture of E. maxima was used to immunize chickens against challenge with 7 new isolates of E. maxima from poultry houses in different parts of England. The results show that although immunological differences exist within E. maxima good protection against many strains of this species may be achieved by initial infection with the hybrid mixture of E. maxima.

The effect of some anticoccidial drugs on the development of immunity to coccidiosis in field and laboratory conditions.

In a laboratory trial chickens were given immunising doses of six species of Eimeria when they were 2 weeks of age and the effect of medication with monensin or arprinocid from 0-10 weeks was determined by challenge inoculation. Monensin in the food at 60-100 ppm resulted in depression of immunity but concentrations of 40-50 ppm only slightly affected immunity to challenge inoculation. Arprinocid at 50 ppm also greatly reduced the development of resistance to infection but 20 ppm did not. In a field experiment where chickens were exposed only to naturally occurring coccidial populations, the feeding of monensin (45 ppm) or dinitolmide (125 ppm) for 14 weeks did not affect the development of immunity to the indigenous coccidial populations. Monensin-treated birds appeared to be slightly better protected. Chickens treated with monensin reached their target body weight earlier and maintained it better than dinitolmide-treated chickens. Large numbers of oocysts were present in the litter of birds treated with dinitolmide. Oocyst numbers reached a peak at 3 weeks and remained high until the birds were 10 weeks of age. Treatment with monensin at 45 ppm resulted in low oocyst counts in the litter until skip-a-day treatment was initiated. It is concluded that the inclusion of monensin at 40-50 ppm in the food may be suitable for the rearing of replacement chickens.

Eimeria: further studies on the immunisation of young chickens kept in litter pens.

Chickens were inoculated with small numbers of oocysts of attenuated (E. tenella TA and E. mivati MA) and virulent strains of E. acervulina, E. brunetti, E. maxima and E. necatrix under conditions designed to permit reinfection. Chickens were immune to challenge with pathogenic laboratory strains 6 weeks after inoculation with either the homologous strains or attenuated derivatives. Immunity to challenge with heterologous strains was also achieved, except that it was incomplete with E. maxima. To obtain complete protection against heterologous challenge with E. maxima, it was necessary to immunise with several strains. Chickens immunised at 1 or 2 weeks of age showed a stronger immunity than birds immunised at 1 day old when challenged at 6 weeks of age. The effect of inoculating chickens with 0.1-10 times a standard immunising dose was examined. Chickens given 0.1-1 times the standard dose were substantially immunised but body weight gain was reduced with higher doses. The results show that controlled immunisation of 1- or 2-week-old chickens against six species of Eimeria is possible, and that a substantial degree of immunity develops within 2 weeks of exposure. This immunity was effective for at least 18 weeks.

Rejection of Eimeria by foreign hosts.

Oocysts of Eimeria maxima inoculated into guinea fowl do not develop. Infection occurs when intestinal mucosa or liver tissue from guinea fowl given E. maxima is transferred to susceptible chickens. By transferring material at different times after inoculation, it was shown that most of the stages are lost between 6 and 12 h and few, if any, survive to 48 h. Sporozoites of E. tenella had low infectivity after 48 h contact in vitro with peritoneal macrophages from guinea fowls and turkeys. Sporozoites of E. grenieri from the guinea fowl appeared to be destroyed within macrophages taken from chickens.