Anti-enzyme antibodies in rabbits and pigs against Hyostrongylus rubidus (Hassall and Stiles, 1892)

Ether extracts of Hyostrongylus rubidus adult worms isolated on days 14, 35 and 64 of intection were found to contain two isoenzymes of malic dehydrogenase (MDH) and three of acid phosphatase. Sera from rabbits immunized against these extracts and sera from pigs experimentally infected with H. rubidus were tested for their anti-enzyme activity by two different techniques.Sera from rabbits actively immunized with a Day 14 worm extract contained an antibody which complexed only with a slow migrating isoenzyme of acid phosphatase but not with isoenzymes of MDH or acetylcholinesterase (AChE). No antibodies against worm acid phosphates or MDH were detectable by this technique in the sera of pigs which were experimentally infected with H. rubidus.A different technique, however, where the worm extracts were incubated at 60°C either with rabbit anti-H. rubidus serum, or serum from infected pigs, indicated the presence of anti-AChE globulin in both immunized rabbits and infected pigs. When individual immunoglobulins isolated from infected pigs were incubated with the same worm extract it was seen that activity was associated with IgG₁ but not with IgG₂, IgM or IgA. IgG₁ prepared from worm-free pigs did not complex with worm AchE. There was no interaction between the third stage larval AChE and pig IgG₁. Levels of AChE were highest in worms isolated at a period of infection when the hosts immune responses were beginning to manifest themselves and lowest in those worms surviving the population crisis.     

Experimental Trichonema infections in mature ponies

Three of six ponies, 9 and 10 years of age, were each given in excess of 300 000 infective Trichonema larvae experimentally. Although all six ponies had both natural and experimental infections previously, they were virtually parasite-free during the 18-month period immediately preceding experimental reinfection.Appreciable resistance to Trichonema infections was indicated in these mature ponies by a longer prepatency and significantly smaller worm egg outputs than that which occurred when these animals were given comparable infections at a much younger age. Patent infections only developed in two of the three ponies beginning between 17 and 18 weeks post-reinfection with peak egg counts between 200 and 400 eggs per gram (epg) in one and 100 to 150 epg in the other. Within 10 days of reinfection, an abrupt rise in mean circulating eosinophils and an appreciable mean weight loss, which persisted for 2 years, occured in the reinfected but not the control ponies.The findings, particularly of unthriftiness characterized by persistent loss of weight in the reinfected but not the control ponies, demonstrated the pathogenesis of inhibited Trichonema larvae.     

Egg output of Ostertagia circumcincta in sheep given single infections of varying size

The faecal egg counts of groups of sheep given 5 000, 20 000 or 40 000 infective larvae of Ostertagia circumcincta reached a peak at about 28 days after infection; the height of the peak varying directly with the number of larvae given. At 35 days the ratios of total worm burdens and the total eggs in all females amongst the groups were similar. Between 35 and 70 days post infection the faecal egg counts fell to a low level which was roughly the same regardless of the number of larvae administered. At 70 days the two groups of lambs given the two higher doses of larvae had similar worm burdens, but those given 5 000 larvae harboured about half as many. At this time the total eggs in all females was approximately the same in each group. Whatever factor, in addition to worm numbers, which may have limited egg output appeared to be ineffective until some time after the peak egg count was reached.     

Experimental Ostertagia spp. infection of sheep: Populations and lesions in previously worm-free and previously exposed animals

Sheep raised and maintained under worm-free conditions were subjected to one heavy exposure and several low-level exposures of infective Ostertagia spp. larvae. The infections were terminated by treatment with thiabendazole. These sheep and not previously exposed controls were then challenged with 400 000 or 20 000 Ostertagia spp. larvae and killed after a further 8 days. In the previously exposed sheep, worm numbers, rate of worm development, and pathological effect, were greatly reduced. No significant differences were seen between sheep aged 6 and 18 months at the time of challenge.     

Culture requirements of Ascaris suum larvae using a stationary multi-well system: Increased survival,development and growth with cholesterol

Ascaris suum third-stage larvae (L₃) were converted from infected rabbits and cultured in a stationary multi-well plate system. Several different culture media including Mediuim 199, NCTC-135, Minimum Essential Medium-Eagle, Dulbecco's Modified Eagle's Medium, RPMI 1640, McCoy's 5A and Neuman-Tytell mdium were tested to determine which was best for overall larval survival, development and growth. Larvae developed from L₃ to fourth-stage (L₄) in all media tested, but larval survival and the yield and growth of L. were superior in RPMI 1640. Pyruvate is an important medium component because its addition to RPMI 1640 enhanced the yield and growth of L₄ while its removal from DMEM reduced larval survival and the yield and growth of L₄. Cholesterol markedly enhanced larval survival and the yield and growth of L₄ when added to RPMI 1640 as either a soluble supplement in Tween 80 or to a lesser degree, as a component of liposomes. The multi-well culture plate system is a convenient method for determining the effects of different media and changes in media composition on larvae in vitro.     

The effect of γ-radiation on the development of infective larvae of Trichostrongylus colubriformis in guinea pigs and sheep

Infection of guinea pigs with normal Trichostrongylus colubriformis larvae was characterized by an initial logarithmic decline of total worms recovered over the first few days of infection (“loss phase”) followed by a “plateau phase” when worm numbers remained stationary and a third “expulsion phase” when worm numbers again declined logarithmically with time. Irradiation of infective larvae at 50 and 100 kilorads (krad) affected the subsequent pattern of infection by prolonging the initial “loss phase” and contracting the “plateau phase”. The rate of decline of total worms during either the “loss phase” or the “expulsion phase” was not affected by larval irradiation.Few males were recovered at any stage of infection with larvae irradiated at 50 or 100 krad. However, the irradiated larvae (females) which survived continued development at a rate which was similar to that of normal larvae and it was apparent that these parasites were not able to maintain their position in the small intestine, but continued development during migration towards the posterior.The radiosensitivity of male larvae and worm egg production (eggs in utero) over a range of radiation doses (25 to 100 krad) were remarkably similar. On the other hand, female larvae were clearly less sensitive to γ-radiation. These observations suggested that the depressed egg production in females may be due to the absence of males.     

Seasonal patterns of gastrointestinal nematode infection in goats on two Lithuanian farms

Background

This study investigated seasonal changes in naturally acquired gastrointestinal nematode (GIN) infections on two Lithuanian goat farms with different parasite control practices.

Findings

On both farms, nematode faecal egg counts (FEC) and larval cultures were obtained from 15 adult and 10 young goats at bi-weekly intervals from April 2012 to April 2013. Goats on farm A were dewormed with ivermectin (0.3 mg/kg body weight) in October/November 2012, whereas the animals on farm B were left untreated. Thirteen young goats were slaughtered in August/November 2012 and April 2013 and worm burdens in the gastrointestinal tract were enumerated. In goats from both farms, Teladorsagia, Trichostrongylus, Oesophagostomum, Chabertia and Haemonchus were the dominant GIN genera. Herbage contamination with infective third-stage larvae (L₃) peaked in July/August and resulted in high FEC in September/October. Parasitological examination at slaughter showed that Teladorsagia spp. and Haemonchus contortus survived the winter, both in the abomasal mucosa as adults and as early fourth-stage larvae (EL₄). Deworming on farm A significantly reduced FEC, especially of H. contortus, at the start of the grazing period compared with the untreated farm B (P < 0.05).

Conclusions

Goats were heavily infected with several GIN throughout the year. Strategic anthelmintic treatment during housing significantly reduced nematode egg output, in particular by H. contortus, at the start of the grazing season.     

Proceedings in life sciences: Fish diseases : Editor W. Ahne, Third Cooperative Programme of Research on Aquaculture session, 1980, 120 figs., x + 252 pp., DM 89.00 (cloth bound), approx. US $52.60, Springer-Verlag, Berlin-Heidelberg-New York, ISBN 3-540-10406-2

The effect of repeated intradermal injections of small quantities of adult worm extract on the resistance of calves to Cooperia spp. was investigated.A group of five calves injected intradermally was less resistant than a similar group inoculated twice with infective larvae, but more resistant than a group of five calves kept as worm-free controls. The resistance was reflected by worm numbers, worm lengths, inhibition percentage of L4 larvae, number of eggs per female worm, egg production and by antibody titres.     

Parasite population dynamics in pigs infected with Trichuris suis and Oesophagostomum dentatum

The aim of the present study was to investigate the population dynamics and potential interactions between Trichuris suis and Oesophagostomum dentatum in experimentally co-infected pigs, by quantification of parasite parameters such as egg excretion, worm recovery and worm location. Forty-eight helminth naïve pigs were allocated into four groups. Group O was inoculated with 20 O. dentatum L₃/kg/day and Group T with 10 T. suis eggs/kg/day. Group OT was inoculated with both 20 O. dentatum L₃/kg/day and 10 T. suis eggs/kg/day, while Group C was kept as an uninfected control group. All inoculations were trickle infections administered twice weekly and were continued until slaughter. Faecal samples were collected from the rectum of all pigs at day 0, and twice weekly from 2 to 9 weeks post first infection (wpi). Six pigs from each group were necropsied 5 wpi and the remaining 6 pigs from each group were necropsied 10 wpi. The faecal egg counts (FEC) and total worm burdens of O. dentatum were dramatically influenced by the presence of T. suis, with significantly lower mean FECs and worm burdens at 5 and 10 wpi compared to single infected pigs. Furthermore, in the presence of T. suis we found that O. dentatum was located more posteriorly in the gut. The changes in the Trichuris population were less prominent, but faecal egg counts, worm counts 5 wpi (57% recovered vs. 39%) and the proportion of infected animals at 10 wpi were higher in Group OT compared to Group T. The location of T. suis was unaffected by the presence of O. dentatum. These results indicate an antagonistic interaction between T. suis and O. dentatum which is dominated by T. suis.     

Ostertagia ostertagi infections in angora goats

Angora goats grazing on a paddock contaminated with Ostertagia ostertagi and O. circumcincta were found to have infections consisting of equal numbers of both species. Subsequently, goats were infected with O. ostertagi larvae of bovine origin. The infection became patent by day 28 and egg counts reached a plateau about 7 weeks after infection. Worm numbers did not decline during the 12 weeks of the experiment. It was concluded that Angora goats are suitable hosts for O. ostertagi.     

Haemonchus contortus infection in sheep: parasite fecundity correlates with worm size and host lymphocyte counts

Two experiments were conducted to elucidate the timing and nature of the sheep immune response to Haemonchus contortus (Barber's pole worm). The first experiment examined the establishment of H. contortus populations and the immune response by comparing a bolus infection of third-stage larvae in na?ve sheep with a group previously primed by a trickle infection. The second experiment used staggered doses of ivermectin-resistant larvae to compare the development of adult worms during different durations of trickle infection with ivermectin-sensitive larvae. Infections successfully generated pathological signs of haemonchosis such as anaemia. Image analysis software was used to measure the area and perimeter of worms collected at post-mortem, and the number of eggs present in individual adult females (fecundity) was significantly correlated with worm size. A significant inverse correlation was found between blood lymphocyte counts and worm fecundity. The absence of correlation between worm fecundity and other leukocyte and erythrocyte counts highlighted the specificity of the lymphocyte response. This is the first report of a link between haematology profiles and worm fecundity in haemonchosis. The correlation observed between adult worm size and egg content leads to the hypothesis that egg production in H. contortus is limited by immune regulation of worm size and presumably growth. Mean worm size and fecundity declined as sheep received more prolonged trickle infections before necropsy, confirming previous reports that immune responses to adult worms are enhanced by ongoing larval challenge. Immunohistochemical results showed trends consistent with a Th2 (humoral) immune response which has been implicated in reducing nematode burdens in several species.     

Variation of the CD4, CD8, and MHC II cell population in granulomas of immunocompetent and immunosuppressed rabbits in Encephalitozoon cuniculi infection

Encephalitozoon cuniculi (E. cuniculi) is a fungi-related, obligate, zoonotic, spore-forming intracellular eukaryotic microorganism. This emerging pathogen causes granulomas in brain and kidneys of infected individuals. The objective of this study was to detect the distribution of CD4, CD8 and MHCII-positive cells within granulomas in these organs in infected immunocompetent (group A) and infected immunosuppressed (group B) New Zealand white rabbits using immunohistochemistry. In brain, labeled CD4 immune cells were mainly located in the periphery of granulomas in group B. Kidneys of groups A and B, displayed CD4-positive in granulomas and were significant different when compared to brain. CD8 immune cells in brain and kidneys were disseminated in the granulomas in groups A and B; however, no significant difference was observed. MHCII-positive cells were more numerous in brain sections of group B and were significantly different when compared to kidney sections. Granulomas were not observed in control animals of group C and D. In conclusion, we identified CD4-positive cells in both the brain and kidneys of immunocompetent and immunosuppressed animals; CD8-positive cells were more numerous in brain of immunosuppressed rabbits and MHCII cells were more predominant in brain of immunocompetent rabbits. Apparently, the immunosuppression stimulated a change in the cellular phenotype of Th1- to Th2-like granulomas in brain and kidneys by an unknown mechanism. These results increase our understanding of CD4, CD8 and MHCII positive cells within the E. cuniculi granuloma microenvironment and will help in future microsporidian granulomas studies of both immunocompetent and immunosuppressed individuals.     

Strongyle infections in ponies. II. Reinfection of treated animals.

Five of seven ponies whose strongyle worm burdens had previously been removed or markedly reduced by repeated thiabendazole treatments were reinfected with doses ranging from 100,000 to 500,000 small strongyle infective larvae. Reinfection of ponies resulted in the development of clinical signs characterized by abnormal feces, marked loss of weight and delayed shedding of winter hair coats. An abrupt increase in circulating eosinophils occurred during the first three weeks following reinfection. Patent infections developed in all ponies with worm eggs appearing in the feces from 12 to 15 weeks after receiving infective larvae. Worm egg outputs followed a cyclic pattern with approximately four to five peaks in egg output per year. There was an abrupt drop in the high worm egg counts in two untreated ponies approximately two and a half years after reinfection. No worms were recovered in the feces of these animals when they were subsequently treated, suggesting that a depletion in the number of inhibited larvae present in these ponies might have occurred.     

Molecular detection and genotype identification of E. cuniculi from pet rabbits

Encephalitozoon cuniculi is a microsporidian which is frequently reported from rabbits. This microorganism can either ravage rabbit farms or transmit to humans from pet rabbits. This study aimed to investigate the prevalence and the genotype distribution of E. cuniculi among pet rabbits. In this study urine samples were collected from 50 pet rabbits, aged 2 months to 3 years, admitted to teaching veterinary hospital. Four races Lop, Dutch, Mix, and Angora were screened for E. cuniculi. The clinical symptoms were recorded and total DNA was extracted from urine samples. E. cuniculi was identified using amplification of the small subunit ribosomal RNA (ssu rRNA) gene and its genotypes were characterized using PCR/sequencing of the polar tube protein (PTP) gene. Phylogenetic tree was drawn to confirm the characterized genotypes. Out of 50 samples, 41 (82 %) of rabbits were asymptomatic, while nine (18 %) had at least one of symptoms including head-tilt, circling, and ataxia. A statistical correlation was seen between mean age + SD and symptoms (P-value = 0.039). The presence of E. cuniculi was confirmed in 16/50 (32 %) rabbits and all of them were identified as the genotype I. Our findings represented no consistency between E. cuniculi PCR – positive and the presence of symptoms (P-value = 0.318). Our results showed positive correlation between symptoms and age; however, the lack of correlation between PCR results with age may signify the latent infection in younger rabbits. All identified E. cuniculi were the genotype I, which is reported from rabbits and humans, highlighting the zoonotic concern for this genotype, particularly among subjects who keep pet rabbits.     

Self-cure and immunity following infection and reinfection in ovine haemonchosis

Egg expulsion and post mortem examination were used to compare self-cure and resistance to reinfection with Haemonchus contortus in female Merino sheep, AA and BB hemoglobin types. Animals were infected by 500 or 5000 larvae of H. concortus once, or several times, before being challenged by 500 (low dose) or 5000 (high dose) larvae.In AA sheep infected once by 500 larvae self-cure was detected by a challenge of either 500 or 5000 larvae while in multi-infected sheep only a challenge of 5000 L₃ induced the self-cure. In BB sheep a mild self-cure was detected only by a challenge of 5000 larvae.In AA sheep receiving several infections, challenge by 500 larvae was not effective in producing resistance while 5000 L₃ was effective in producing a state of resistance for up to 35 days post challenge.At post mortem examination, a strong state of resistance was evidenced in multi-infected animals by the low number of L₄ and adults present in the abomasum up to the 35th day after challenge.In BB sheep no state of resistance was demonstrated by challenge with either 500 or 5000 L₃, even in multi-infected animals.     

Protection of lambs with a purified metabolite of exsheathed third-stage Haemonchus contortus larvae

Helminth-free lambs (approx 4 months old) were inoculated SC with a purified metabolite of exsheathed third-stage Haemonchus contortus larvae. The metabolite was obtained from in vitro cultivation and was identified as XL3FA1. After 3 periodic XL3FA1 inoculations, lambs were challenge exposed with H contortus. Multiple vaccinations with XL3FA1 did not inhibit the development of worm populations in the lambs; however, worm egg production seemed to be inhibited.     

Stocking rate effects on the worm burdens of Angora goats and Merino sheep

Worm egg counts of yearling Angora or Merino wethers grazing in mixed flocks at either 4/ha or 8/ha were observed over a 5 month period. Based on faecal egg counts infection levels in the sheep increased for the first 3 months and then decreased as the animals developed resistance. Egg counts in the goats did not decrease during the experiment. Post mortem worm counts indicated that there was a significant host species--stocking rate interaction on worm burdens of Haemonchus contortus adults and Ostertagia circumcincta adults and larvae. Goats had more H. contortus and O. circumcincta adults than sheep at the 8/ha stocking rate. At 4/ha goats had fewer O. circumcincta fourth stage larvae than sheep. There was no stocking rate effect on the numbers of Trichostrongylus axei or T. colubriformis. Sheep had more T. axei and fewer T. colubriformis than goats.     

Serological survey of Encephalitozoon cuniculi in farm rabbits in Italy

Rabbit sera (n = 1600) from 40 commercial farms were submitted to a serological screening for Encephalitozoon cuniculi by an enzyme-linked immunosorbent assay (ELISA) and a carbon immunoassay (CIA test). Antibodies anti-Encephalitozoon cuniculi were found in 505/1600 (31.6%) sera analysed, and all the farms (100%) resulted positive. Rabbits older than 4 months showed a significantly higher seropositivity for E. cuniculi (chi-squared test: p < 0.0001) than rabbits under 4 months, E. cuniculi sero-prevalence showed an increasing trend in rabbits within the farm along with the increase in the “number of rabbits on the farm”; however, this trend was not significant (Spearman’s correlation: p = 0.073).The findings of the present study confirm that rabbit is the main reservoir of E. cuniculi; they are of epidemiological relevance and immediate public health importance because of the recognized infectivity in humans by the microsporidium.     

Schistosoma bovis whole egg antigen did not protect Zebu calves against experimental schistosomosis

Six calves were immunized with whole egg antigen of Schistosoma bovis emulsified in Freund's adjuvant. The immune response was monitored by agar-gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA). Using AGID and ELISA, antibodies to whole egg antigen were detected in sera from all immunized calves, but not in sera from control calves. The immunized calves and six control calves were challenged 6 months after the beginning of the immunization with 20 000 cercariae of Schistosoma bovis administered percutaneously to the shaved tail. There was no significant difference between the immunized and the control calves as judged by fecal and tissue egg counts, worm recovery and hematological parameters. There was a lack of association between antibody production and protection.