Arrested development of Trichostrongylus tenuis as third stage larvae in red grouse

Worms were counted in red grouse (Lagopus lagopus scoticus) in north east Scotland in March, April, August, September, November and December 1986. Immature worms occurred as third (L3) and fourth (L4) stage larvae throughout. Two kinds of evidence suggest that L3 larvae spent much of the winter in a state of arrested development. First, recently acquired L3 larvae could be distinguished by the fact that they retained outer L2 cuticles for a few days; these were present only in the August and September samples. Secondly, the mean proportion of larvae (L3 and L4) present as L3 was less than 15 per cent in August and September, but increased to 92 per cent by December and then decreased to 71 per cent by March and April. During March and April the proportion of L4 increased as overwintering L3 resumed their development.     

Observations of the sheath extension of the third stage, infective larvae of Trichostrongylus rugatus

The length of the sheath extension of third stage (L(3)), infective larvae of Trichostrongylus rugatus is reported. The total length and length of the sheath extension of L(3) recovered from the faeces of sheep originating from two localities in South Australia were measured. The total length of larvae was 615-722 microm and the length of the sheath extension was 46-56 microm (mean 51.5). The larvae of T. rugatus comprised up to 72% of the larvae recovered. Worm free sheep were infected with larvae from the two localities and L(3) were recovered following culture of faeces from these experimentally infected animals. L(3) measured 640-746 microm in total length with a sheath extension of 46-56 microm (mean 51.4). Adult nematodes recovered from gastro-intestinal tracts were identified as T. rugatus. The length of the sheath extension used in conjunction with the total length is a characteristic that enables confident differentiation of infective larvae of T. rugatus from other Trichostrongylus species infecting sheep in southern Australia and Haemonchus contortus.     

Winter survival of third stage Trichostrongylus colubriformis larvae: effects on infectivity and fecundity of adult worms

Two groups of six lambs were fed with grass cut from two plots which had been contaminated by spreading Trichostrongylus colubriformis larvae. One plot received larvae in December, the other in April. The post mortem examination results showed that the infectivity of overwintering larvae was significantly higher than that of fresh larvae. An inverse relationship was demonstrated between infectivity of larvae and egg production of adult worms developed from these larvae. The results are compared with those obtained under laboratory conditions.     

Effects of four tropical tanniniferous plant extracts on the inhibition of larval migration and the exsheathment process of Trichostrongylus colubriformis infective stage

The anthelmintic (AH) effect of Acacia pennatula, Leucaena leucocephala, Lisyloma latisiliquum and Piscidia piscipula was evaluated in the infective larvae (L(3)) of Trichostrongylus colubriformis. Different concentrations of lyophilized extracts were tested using the larval migration inhibition (LMI) test. An inhibitor of tannins (the polyvinyl polypyrrolidone [PVPP]) was used to verify whether these compounds were responsible for the AH effects. Then, the effect of extracts on larval exsheathment was examined by observing the exsheathment process at 10-min intervals for 70 min. The LMI test showed a dose-dependant AH effect for A. pennatula, L. leucocephala and L. latisiliquum (P<0.01), but not for P. piscipula. The restoration of L(3) migration to values similar to those of controls after the addition of PVPP, indicates that tannins are involved in AH effects. Trichostrongylus colubriformis exsheathment was partially or totally blocked by the four plants extracts. Tropical tanniniferous plants evaluated in the current study may have potential as AH for the control of T. colubriformis if in vivo investigations indicate useful effects.     

Climate and the distribution of intestinal Trichostrongylus spp. of sheep

The relative prevalences of Trichostrongylus vitrinus, T. colubriformis and T. rugatus in sheep in south-west Western Australia were examined for evidence of associations with climatic conditions in different localities. The region has a mediterranean-type climate with hot, dry summers and cool, wet winters. The intestinal Trichostrongylus spp. populations in sheep sampled comprised 50% T. vitrinus, 38% T. colubriformis and 12% T. rugatus. The prevalence of T. vitrinus was negatively correlated with mean autumn, winter and spring temperatures of a locality. The prevalence of T. colubriformis was positively correlated with mean autumn, winter and spring temperatures. The prevalence of T. rugatus was not correlated with temperature of any season, but was negatively correlated with mean annual rainfall and length of growing season of a locality. There were suggestions of association of amount of rainfall of a locality and prevalence of T. rugatus was not correlated with temperature of any season, but was negatively correlated with mean annual rainfall and length of growing season of a locality. There were suggestions of association of amount of rainfall of a locality and prevalence of T. colubriformis and of differences in seasonal prevalence of T. vitrinus, but the significance of these was obscured by confounding of some climatic factors. The differences in prevalence of the three Trichostrongylus spp. at different locations was attributed to differences in preferred ambient conditions for development and survival of the free-living stages.     

Inhibition of the development of Trichostrongylus spp. as third stage larvae in sheep

In non-lactating Frisian ewes slaughtered in September and November 1975 and in ewes slaughtered in March/April 1976 at lambing, the majority of Trichostrongylus axei, T. vitrinus and T. colubriformis found were inhibited third stage larvae (L₃). However, some larvae at various stages of development were found in the animals slaughtered at lambing. In contrast, in lactating ewes slaughtered 6 weeks after lambing in April 1976, almost all Trichostrongylus spp. appeared to be adults. All ewes grazed a pasture heavily contaminated with gastro-intestinal nematodes from June 1975 until at least 2 weeks before necropsy.In lambs on the pasture from April or June 1975, and slaughtered in September and November 1975 and March 1976 after at least 2 weeks of isolation, only small proportions of the Trichostrongylus spp. populations appeared to be L₃. Similar results were obtained from tracer lambs kept at pasture from August 1975 to March 1976. The implications of these findings are discussed.     

Scanning electron microscopic features of the ovine interdigital sinus

This article describes the scanning electron microscopic (SEM) features of the ovine interdigital sinus. The lumen was filled with a dense secretory material and quite a number of hairs embedded in the luminal content. For SEM purposes, the sinus was divided into three parts: base, body and neck. At the cut surface, the wall exhibited significant folds which were almost absent in the base, the very short blind end of the sinus. The wall had three layers: epidermis, dermis and fibrous capsule. Stratified epithelium with a prominent keratin layer faced the lumen. The inner surface was similar to the skin surface; however, it was coarser due to folds. The fibrous capsule was composed mainly of dense connective tissue, constituting the outermost layer of the wall. The dermis contained common skin structures including sebaceous glands, hair follicles, arrector pili muscles and apocrine glands. Sebaceous glands appeared as groups of bubbles if they were not collapsed. Apocrine glands generally appeared as a group of coiled tubules. They frequently exhibited apocrine blebs, which is a feature of apocrine secretion. SEM was able to locate some secretory vesicles in the lumen of apocrine tubules which is frequently filled by secretory content. Thus, the apocrine tubules exhibited classical features of apocrine secretion.     

Effect of condensed tannins on egg hatching and larval development of Trichostrongylus colubriformis in vitro

The effects of condensed tannins extracted from seven forages on the viability of the eggs and first stage (L1) larvae of the sheep nematode Trichostrongylus colubriformis were evaluated in in vitro assays. The extracts of condensed tannins were obtained from Lotus pedunculatus (LP), Lotus corniculatus (LC), sulla (Hedysarum coronarium), sainfoin (Onobrychus viciifolia), Dorycnium pentaphylum (DP), Dorycnium rectum (DR) and dock (Rumex obtusifolius). Extracts containing 200 to 500 microg/ml reduced the proportion of eggs that hatched. The larval development assay was used to evaluate the effect of the extracts on the development of either eggs or L1 larvae to L3 infective larvae. Development was allowed to proceed for seven days by which time the larvae in control incubations had reached the infective L3 stage. Extracts containing 200 microg/ml from LP, DP, DR or dock prevented egg development, and only 11, 8 and 2 per cent of the eggs developed to L3 larvae with extracts from LC, sulla and sainfoin, respectively. When the concentration was 400 microg/ml no eggs developed to L3 larvae. The addition of the extracts after hatching also inhibited the development of L1 to L3 larvae; 200 microg/ml extracted from LP, LC, sulla, sainfoin, DP, DR and dock resulted in only 14, 18, 17, 15, 14, 16 and 4 per cent of L1 larvae developing to the L3 stage compared with 85 per cent for controls, and 400 microg/ml further reduced the development of L1 larvae. Statistical analyses showed that when the extracts were added before hatching they were significantly (P<0.001) more effective at inhibiting the larval development than when they were added after hatching. The condensed tannins from dock had the greatest inhibitory effect on egg development followed by the tannins from DR, sainfoin, DP, LP, sulla and LC.     

Immunization of sheep against the larval stage of Taenia multiceps

A trial with 200 ewes and 127 lambs showed that vaccination of lambs with 2 doses of Oncosphere Secretory Antigen (OSA) 4 weeks apart, at either 4-8 and 8-12 weeks or at 12-16 and 16-20 weeks of age, was equally effective in protecting them against infestation with the larval stage of Taenia multiceps. In this trial, the lambs of ewes, vaccinated with OSA when they were 90 and again 120 days pregnant, were as susceptible to infestation as the lambs of untreated control ewes. Another trial to provoke passive immunity in lambs gave inconclusive results. In a 3rd trial 2 doses of regular OSA and 2 doses of freeze-dried OSA protected all the lambs in each group, while a single dose of regular OSA protected 9 out of 10 lambs against cerebral lesions.     

Influence of adjuvants on immunity in rabbits vaccinated with infective larval somatic proteins of Trichostrongylus colubriformis

Adult New Zealand rabbits were vaccinated at 2-week intervals with three doses of 100 micrograms of infective larval somatic proteins (L3SP) administered subcutaneously with Freund's complete adjuvant (FCA) or beryllium hydroxide and then challenged orally with 10,000 L3. Groups of rabbits immunized orally with three doses of 5000 or 2000 L3 served as vaccination controls. Intestinal worm burdens on Day 21 after challenge revealed that beryllium hydroxide effectively potentiates the protective immunogenicity of L3SP. The level of protection obtained using the beryllium adjuvant (94.8%) was nearly as high as that in rabbits immunized with three doses of 5000 L3 (99.8%). Rabbits vaccinated using FCA showed very poor immunity (29.5%). Local and systemic antibody levels detected by radioimmunoprecipitation tests using 125I-L3SP showed very little correlation with the degree of protection. The beryllium hydroxide-treated group demonstrated significantly higher bile IgA antibody levels than other experimental rabbits. FCA-treated rabbits developed a much higher serum precipitating antibody response, detectable using gel double diffusion tests, than the beryllium group. Also, mucosal IgA antibody levels detected on Day 21 after challenge were significantly higher in the FCA group than in other groups.     

Field evaluation of a mixture of albendazole sulphoxide and levamisole against Ostertagia and Trichostrongylus spp in sheep

The efficacy of a mixture of albendazole sulphoxide and levamisole, 3.6 and 8.25 mg/kg respectively, at single and double dose rates, was compared with the recommended dose rate of each anthelmintic alone. The comparison was conducted on groups of 6 to 14-week-old lambs on 22 farms, 16 of which had evidence of multiple resistance to benzimidazole and levamisole. A single dose of the mixture reduced mean egg counts by 95% on half the farms with multiple resistance and on all the remaining farms. Consequently, the mixture should be included in an assessment of effective anthelmintics on farms to determine its value for nematode control. A double dose rate of mixture was effective on all but 4 farms. Reductions caused by the mixture were due to the additive actions of the drugs on 18 of 22 farms; synergistic action was noted on only 3 farms. It was concluded that the mixture of albendazole sulphoxide and levamisole offered many farmers an effective anthelmintic for use in preventive control programs. Recommendations for such programs include annual rotation of effective anthelmintics as a means of delaying selection for drug resistance.     

Prevalence and epidemiological features of ovine enzootic abortion in Lithuania

The aim of performed study was to determine the level of enzootic abortion (EA) in sheep breeding farms in different districts of Lithuania, to determine differences in clinical signs and infection frequency between various age groups, and to evaluate the sensitivity and specificity of complement fixation test for antibodies detection and indirect immunofluorescence for antigen detection in sheep chlamydiosis. The clinical, serological and immunological tests in sheep farms were performed in 2004 and 2005. Comparing different age groups of sheep revealed that the lowest number of infected sheep was registered in animals younger than 18 months (23.1%, antibodies titre 3.191 log2, P<0.05) and highest in animals aged 18 to 24 months (53.8%, antibodies titre 4.224 log2, P<0.001). In sheep aged more than 3 years, titre of antibodies was significantly reduced. The majority of infected sheep which aborted (86.4%) was registered in 18-24 month age group. Furthermore, in sheep which aborted the infection level was 2.5-fold higher as compared to sheep which didn't abort. Analysis of smears from patological material by indirect FAT revealed that 54.5% of animals were positive to Chlamydophila abortus infection. The highest prevalence of chlamydia (66.7%) was registered in placentas of sheep which aborted.     

Inhibited development of cyathostominae in the horse in the early third stage

Inhibited early third stage larvae of Cyathostominae were found in the digested mucosa of the large intestine of 12 yearling, female Shetland ponies, which were used in two grazing experiments. The ponies were killed in late autumn. In some ponies the inhibited larvae were very abundant and in most animals the majority of the Cyathostominae populations consisted of these early third stage larvae, suggesting that the phenomenon has an epidemiological significance.     

Measurement in vitro of a larval migration inhibitory factor in gastrointestinal mucus of sheep made resistant to the roundworm Trichostrongylus colubriformis

Lambs were challenged by dosing with 2500 T. colubriformis larvae per day for 34 weeks, rested for 4 weeks and then re-challenged with the infective larvae for a further 10 weeks. A technique for the measurement of inhibition of larval migration from agar gels was used to investigate the antiparasitic activity of mucus, obtained from the small intestine and abomasum of the lambs, at the end of the re-challenge period. Measurements were also made on ileal digesta samples collected at different times during the development of the initial resistance, the rest period and the re-challenge period, and on faeces collected during the re-challenge infection. The mucus from the small intestine and abomasum paralysed and inhibited larval migration from agar gels significantly more (P less than 0.01 and P less than 0.05, respectively) than corresponding mucus from parasite-free control animals. This inhibitory activity was also detected (P less than 0.01) in the ileal digesta of infected animals from Week 6 of primary dosing. The magnitude of the inhibition in the ileal digesta increased with time during the infection period and was again detected during re-infection (P less than 0.01). It was not detectable in resistant sheep towards the end of the rest period. Slight inhibitory activity was detected in faeces after 2 weeks of re-infection. These observations suggest that substances secreted into the lumen of the small intestine of infected animals are responsible for resistance against ingested larvae.     

The effect of ivermectin treatment against inhibited early third stage, late third stage and fourth stage larvae and adult stages of the cyathostomes in Shetland ponies and spontaneous expulsion of these helminths.

A controlled and critical test on the efficacy of ivermectin against larval and adult stages of the cyathostomes was carried out in six yearling castrated male Shetland ponies. The ponies grazed together as one group from 3 May to 4 October 1990, after which they were housed. Three ponies were treated with ivermectin on 29 October while the others served as controls. The shedding of helminths in the faeces was followed in all ponies until necropsy on 14 November. Comparison of worm counts of both groups before and after necropsy showed no evidence for an effect of ivermectin against inhibited early third stage larvae (EL3) and mucosal late third stage (LL3) and fourth stage larvae (L4). However, a high, but not 100%, efficacy was observed against adults and lumenal L4. A remarkable observation was the high incidence of spontaneous expulsion of L4 and adult populations of some species in two of the untreated ponies.     

Gross morphological features of the lung and air sac in the Japanese quail

This study was conducted to reveal the morphological characteristics of the lung and air sacs in Coturnix coturnix japonica (Japanese quails). Ten quails were allocated into two groups. Tracheas of 5 quails with neoprene latex and 5 quails with methylmetacrylate were injected to fill the trachea and air sacs. Latex embalmed animals were stored in 10% formaldehyde solution for two months. Animals given methylmetacrylate were maserated in 30% potassium hydroxide at 40 degrees C for two days. Lungs were located in the dorsal part of the thorax and very close to the thoracic vertebrae and ribs. Shorter than the dorsal border, the ventral border lied between the 3rd and 6th ribs. Cervical, clavicular, cranial thoracic, caudal thoracic and abdominal sacs were identified. These sacs had connection with the 3rd, 4th and 5th lateroventral and 4th mediolateral bronchi. Saccus cervicalis was located on the left and right portions of the vertebrae cervicales et thoraricae with a pronounced communication ventromedially. However, the cervical sac aeration of only all cervical vertebrae was present in this study. Humerus was a non-aerated bone. Pneumatic foramen was absent and did not aerate the sternum. Cranial thoracic sac connected to the 1st, 2nd and 4th medioventral bronchi and gave no diverticulum for aeration. Cranial thoracic sac received air through the 4th medioventral and the 1st and 2nd lateroventral bronchi. Left and right abdominal air sacs paramedially produced diverticulum femorale, but this diverticulum did not enter the femur.     

Characterization of monoclonal antibodies against ovine Sarcocystis spp. antigens by immunoblotting and immuno-electron microscopy

Six monoclonal antibodies were raised in mice against purified cytozoite extracts of Sarcocystis gigantea and S. tenella from sheep. Each monoclonal antibody was evaluated for specificity by enzyme immunoassay, immunoblotting and immuno-electron microscopy using homologous and heterologous antigenic preparations. All six monoclonal antibodies exhibited good species-specificity when reacted against crude soluble cystozoite antigens in enzyme immunoassays. However, only two monoclonal antibodies (IgM and IgG2a) exhibited reactivity in Western blots against specific protein bands. Both reacted against S. gigantea antigens of 100,000, 43,000 and 39,000 molecular weight. Neither monoclonal antibody reacted against the heterologous species S. tenella. Ultrastructural studies performed with colloidal-gold conjugated antisera revealed that both monoclonal antibodies reacted against antigens located around micronemes and amylopectin granules in S. gigantea cystozoites. Another monoclonal antibody (IgGI) reacted only against microneme determinants in S. tenella cystozoites. In contrast, polyclonal sheep and rabbit immune sera cross-reacted against a wide range of cystozoite antigens.     

Development of a new monoclonal antibody to ovine chimeric IgE and its detection of systemic and local IgE antibody responses to the intestinal nematode Trichostrongylus colubriformis

The J558L cell line, previously transfected with the ovine Cepsilon gene, was induced to secrete a chimeric IgE protein composed of the ovine heavy chain and a mouse light chain with MW of approximately 80 and 26 kDa, respectively. After purification, the chimeric protein was used to immunise BALB-c mice and monoclonal antibodies (mAbs) were generated. The mAb 2F1, which had greatest anti-IgE activity in preliminary screens, was chosen for further characterisation and an examination of systemic and local IgE responses to the intestinal nematode, Trichostrongylus colubriformis. The chimeric IgE protein was not recognised in enzyme linked immunosorbent assay (ELISA) by mAbs raised against ovine IgG1, IgG2, IgA or IgM. However, 2F1 was highly specific to the chimeric IgE protein, and did not cross-react with ovine IgG1, IgG2 or IgA. Western blot analysis also showed that 2F1 and secretory IgA (sIgA) did not cross-react, and that 2F1 and the anti-IgA mAb identified different MW bands from colostrum (approximately 200 and 400 kDa, respectively). 2F1 bound to mucosal mast cells (MMC) isolated from the intestines of lambs infected with T. colubriformis, but cultured bone marrow-derived mast cells (BMMC) required prior incubation with the chimeric IgE protein for this binding to occur. Distinctive staining of plasma cells and putative mast cells were observed using 2F1 on immunohistological sections of mesenteric lymph node and jejunum.ELISA incorporating 2F1 was able to detect >0.4 ng chimeric protein. Total IgE in ovine colostrum and intestinal homogenates was quantified using a capture ELISA, with known amounts of chimeric protein used to produce a standard curve. Colostrum from outbred Merino ewes had 0.55-11.05 ng ml(-1) total IgE, and their lambs, at necropsy after infection with a total of 18,000 T. colubriformis infective larvae over a 9-week period, had 45-620 ng g(-1) total IgE in intestinal tissue. Compared to genetically susceptible lambs, antigen-specific levels of IgE were significantly higher in genetically resistant lambs after infection with 4500 T. colubriformis infective larvae (TcL3) per week for 9 weeks (161.4 versus 44.8 geometric mean titres; P=0.043). In western blots, distinctive bands (19-21 and 27 kDa) from T. colubriformis larval antigen were differentially recognised by IgE, as identified by 2F1, in intestinal homogenates from genetically resistant animals.These results have demonstrated the value of 2F1 for quantification of IgE responses in samples derived from ovine fluids and tissues using ELISA, western blots and immunohistology. In this respect, it recognises native ovine IgE and does not require pre-treatment of the sample with denaturing agents or ammonium sulphate.