Experimental cerebrospinal elaphostrongylosis (Elaphostrongylus rangiferi) in goats: II. Pathological findings.

Pathological findings in 17 goat kids inoculated with 100-1,000 infective larvae of Elaphostrongylus rangiferi and autopsied 21-154 days post inoculation (p.i.) are reported. The lungs, heart, diaphragm, liver and kidneys contained small foci of necrosis or fibroblastic scarring and interstitial infiltrates of inflammatory cells. The lungs also contained many parasitic granulomas. Infarcts were observed in the myocardium and kidneys. Accumulations of inflammatory cells and granulomas were seen in the peri- and epineurium of the spinal nerve roots, connective tissue of the spinal ganglia, dura and epidural tissue of the cord, choroid plexus of the brain and leptomeninges of the entire central nervous system (CNS). Within nerve fascicles there were endoneural cell infiltrates, axon and myelin sheath degenerations and granulomas. The CNS parenchyma contained foci of traumatic encephalomyelomalacia, microgliosis, secondary axon degeneration, perivascular cuffs and granulomas. Sections of intact nematodes were found in the subarachnoid spaces, brain ventricles, central canal of the spinal cord and CNS parenchyma. Pathological findings from individual animals are compared to the clinical signs described in a separate paper. The development, migration and pathogenesis of E. rangiferi in goats are discussed.     

Experimental elaphostrongylus rangiferi infection in calves and lambs.

In two lambs and two calves infected experimentally with infective larvae of Elaphostrongylus rangiferi, lymphohistiocytic and eosinophilic cell infiltrations in the leptomeninges and lymphohistiocytic granulomas in the perineurium of the cauda equina of the CNS were demonstrated. These histopathological changes are similar to the lesions usually demonstrated in reindeer with elaphostrongylosis. No parasites were found, however, in the CNS or other organs.     

Cerebrospinal elaphostrongylosis in dairy goats in northern Norway.

Ten carcasses and three vertebral columns from north Norwegian dairy goats, which had been killed due to clinical signs of severe neurologic disease, were received for necropsy. Pathological examination revealed nematodes and nematode ova in the central nervous system (CNS) of nine goats. Worms found by gross examination were identified as Elaphostrongylus rangiferi Mitskevich, 1960. Focal traumatic encephalomyelomalacia, apparently caused by migrating worms, perivascular cuffing, eosinophilic leptomeningitis and perineural infiltrations and granulomas, could be demonstrated in CNS sections from all 13 animals examined. Clinical signs reported were initial pruritus followed by motor weakness, lameness, paresis, reduced vision, circling, abnormal head position, bulging eyes and scoliosis. The disease occurred from September to January in regions with a considerable migrant reindeer population. It was concluded that the reported outbreaks of neurologic disease represented seasonal occurrence of cerebrospinal elaphostrongylosis caused by Elaphostrongylus rangiferi, the elaphostrongyloid nematode of reindeer (Rangifer tarandus tarandus).     

Cerebrospinal elaphostrongylosis in sheep in northern Norway.

Pathologic examination of four sheep from northern Norway exhibiting neurologic signs of paresis, paralysis and vestibular system disease revealed nematodes in the central nervous system (CNS). The worms were identified as Elaphostrongylus rangiferi Mitskevich, 1960, the elaphostrongylid nematode of reindeer (Rangifer tarandus tarandus). Microscopic lesions found in the CNS were focal traumatic encephalomyelomalacia caused by migrating worms, eosinophilic meningitis and choroiditis, lymphohistiocytic and nematode granulomas, and perineural infiltrations. The disease occurred in November and December 1990, after the sheep had been on pasture in areas frequented by considerable numbers of reindeer.     

Experimental Elaphostrongylus cervi infection in moose (Alces alces)

An 8-week-old male moose calf was inoculated with 360 infective third-stage larvae (L3) of E. cervi. The calf started to expel first-stage larvae (L1) of E. cervi in faeces 63 days after inoculation. The highest faecal larval count of 1,920 L1 per gram faeces was recorded 133 days post inoculation. Clinically, intermittent lameness, mild ataxia and general stiffness were observed over a 3 months’ period from day 75 after inoculation. The symptoms were moderate, faded gradually and were not seen during the last three weeks of the observation period. The calf had a good appetite and the bodyweight increased continuously throughout the experiment. On day 202 after inoculation the calf was euthanized and autopsied. Adult E. cervi were found in the epidural space of the central nervous system (CNS) and in skeletal muscles. Oedema, haemorrhages, discolouration and extensive inflammatory reactions were observed in the fat and loose connective tissue of the epidural space between the 5 th cervical vertebra and cauda equina. Nematodes or lesions indicating nematode infestation could not be demonstrated in the leptomeninges or in the neural parenchyma of the CNS. Numerous eggs and larvae of E. cervi associated with moderate pathological changes were observed in the lungs.     

Diagnosis of Elaphostrongylus cervi infection in New Zealand red deer (Cervus elaphus) quarantined in Canada, and experimental determination of a new extended prepatent period.

A modified Baermann assay was used to recover dorsal-spined, first stage larvae of Elaphostrongylus cervi from feces and lungs of red deer (Cervus elaphus elaphus) from three of four herds imported from New Zealand into Canadian quarantine facilities. Tests done on a series of fecal collections showed that larval output from infected red deer was low and sporadic, casting doubt on the efficacy of the Baermann assay to detect all infected individuals in the herds. The animals had passed repeated preembarkation Baermann tests for E. cervi in New Zealand. Seven larvae recovered from these red deer were used to establish a patent infection in a naive red deer. The prepatent period was 206 days and larval shedding was intermittent. Elaphostrongylus cervi is a foreign animal parasite in continental North America, which could become irrevocably established if it were introduced. The data reported indicates that there is currently no reliable method for the detection of E. cervi infection.     

Efficacy of ivermectin in oral drench and paste formulation against migrating larvae of experimentally inoculated Parascaris equorum

Twenty-one mixed-breed pony foals, reared and maintained under parasite-free conditions, were used to test the efficacy of ivermectin in oral drench and paste formulations (200 micrograms/kg) against 11-day-old migrating larvae of Parascaris equorum. Three replicates of 4 foals and 3 replicates of 3 foals were formed on the basis of age. Foals in replicates of 4 were randomly allocated to be indicators, or to receive vehicle (control) or ivermectin paste or ivermectin liquid. Foals in replicates of 3 were randomly allocated to receive vehicle or ivermectin paste or ivermectin liquid. The recovery of larvae from the lungs, liver, and small intestines of the indicator foals showed that 99.9% of the larvae were in the lungs 11 days after inoculation (day 0 of treatment). The recoveries of larvae from lungs and small intestines of controls at 25 days after inoculation indicated that all larvae had migrated to the small intestine by this time. The mean length of larvae recovered from the lungs (11 days after inoculation) was 0.87 mm the mean length of those recovered from the small intestine (25 days after inoculation) was 3.65 mm. Using larvae recovered from small intestinal contents for calculations, ivermectin in both formulations was 100% effective against 11-day P equorum (P less than 0.01, compared with control group geometric mean of 1498.4).     

Lethal cerebrospinal elaphostrongylosis in a reindeer calf.

Lethal elaphostrongylosis in a reindeer calf is described. The calf showed signs of abnormal behaviour, mental confusion and reduced vision due to lesions in the brain parenchyma caused by migrating mature Elaphostrongylus rangiferi. Traumatically caused malacia and secondary axon degeneration were observed in all brain areas. Nematodes were found in the skeletal muscles, epidural space of the spinal cord, and in the subdural spaces and leptomeninges of the cord and brain. Developing nematode ova were sectioned along migratory tracks in the brain, in the subdural space of the cord, epidural adipose tissue, perineurium of the spinal nerve roots and in the lungs.     

Some observations on the adaptation of Eimeria tenella (local isolates) sporozoites on chicken embryos through chorioallantoic membrane

Eimeria (E.) tenella (local isolate) sporozoites were adapted on the chorioallantoic membrane (CAM) of 10-12 days chicken embryos and completed its life cycle in 6~7 days at 39℃ and 70 per cent humidity. Only 23 embryos (4.6%) were found dead from 1~4 day post inoculation of sporozoites with mild lesions on CAM with no gametocytes but few sporozoites in chorioallantoic fluid (CAF). On 5~7 day post inoculation, 432 embryos (86.4%) were found dead with severe haemorrhages on CAM and CAF contained uncountable number of gametocytes. After seven days post inoculation, 45 embryos (9%) were found to be alive. Some oocysts were also detected in the CAF on 6~7 days post inoculation. In the histological sections of the CAM, there were abundant small dark colored rounded bodies of gametes; distributed extensively in tissues of CAM on 5~7 days post inoculation of sporozoites. In some cases, cluster of small mature and immature relatively large bodies were seen in increasing numbers on 5~6 days post inoculation.     

Experimental Corynebacterium pseudotuberculosis infection in lambs

Lambs were inoculated IV with 3.2 X 10(3) colony forming units (CFU) to 3.2 X 10(6) CFU of Corynebacterium pseudotuberculosis from a 6-hour broth culture supplemented with 0.1% sorbitan monooleate. After 28 days, multiple abscesses were observed in the lungs and lymph nodes. The number of abscesses in the lungs correlated with the inoculation dose. Two lambs given 10(5) CFU and 10(6) CFU died. Multiple abscesses occurred in other lambs given 10(6) CFU to 10(4) CFU and few abscesses occurred in lambs given 10(3) CFU. Corynebacterium pseudotuberculosis was isolated from lung abscesses, inoculation site abscesses, and lymph node abscesses, but not from normal tissues. Because this procedure consistently induced abscesses in the lungs, we believe it will be a suitable challenge system for studies on the prevention of caseous lymphadenitis in sheep.     

Single-strand conformation polymorphism (SSCP) analysis as a new diagnostic tool to distinguish dorsal-spined larvae of the Elaphostrongylinae (Nematoda: Protostrongylidae) from cervids

Single-strand conformation polymorphism (SSCP) was used to genetically differentiate morphologically indistinguishable first-stage larvae (L(1)) of the six species of elaphostrongyline nematodes. A partial fragment (317-336bp) of the first internal transcribed spacer (pITS-1) plus 5' flanking region (76bp of the 18S gene) of the nuclear ribosomal DNA (rDNA) was amplified from individual L(1) of known identity and subjected to SSCP. The results showed that the four species of elaphostrongylines found in North American cervids, Parelaphostrongylus tenuis, P. andersoni, P. odocoilei and Elaphostrongylus rangiferi, could be distinguished from one another based on their distinct (i.e. species-specific) SSCP profiles. In addition, E. alces, a species that occurs in moose in Fennoscandinavia, also had a distinct SSCP profile with respect to the other species of elaphostrongylines. However, the SSCP profiles of E. cervi could not be distinguished from those of E. rangiferi because of a lack of interspecific sequence differences in this region of the ITS-1. The distinct SSCP profiles for the other species were consistent with the interspecific differences in ITS-1 sequences, which ranged from 2 (between P. tenuis and P. andersoni) to 59bp (between genera). The pITS-1 SSCP approach was also used to identify unknown elaphostrongyline L(1) from different hosts and localities in North America. The ability to distinguish between L(1) of the four elaphostrongyline species that occur in North American cervids has important diagnostic and epidemiological implications.     

Larvae of Elaphostrongylus cervi in the deer of Scotland

Protostrongylid larvae were recovered from the faeces or lungs of red deer (Cervus elaphus), roe deer (Capreolus capreolus) and reindeer (Rangifer tarandus) in Scotland during 1981. Typical protostrongylid first-stage larvae were also recovered from possible intermediate hosts, the grey field slug (Agriolimax reticulata) and the white-soled slug (Arion fasciatus). All these protostrongylid larvae were microscopically identical to those of the nematode Elaphostrongylus cervi. Despite careful search, adult E cervi were not found, but it is concluded that infection with E cervi is widespread in Scottish deer.     

Pulmonary lesions in guinea pigs experimentally infected with Actinobacillus pleuropneumoniae (A.p.) serovar 1

Pathological studies were carried out on the lungs of guinea pigs intratracheally inoculated with 4.6 x 10(6-8) colony forming units (CFU)/head of Actinobacillus pleuropneumoniae serovar 1. All animals in the highest dose group died within 24 hr post inoculation (hpi) and showed pulmonary lesions being hemorrhagic in nature while all animals in the lowest dose group were killed as scheduled at 11 days post inoculation (dpi) and showed only hyperplasia of peribronchial lymphoid tissues. In the middle dose group, two died within 24 hpi, two died at 9 dpi, and the remaining one was killed at 11 dpi. Two guinea pigs which died at 9 dpi showed fibrinonecrotic pleuropneumonia which is the most characteristic acute pulmonary lesion in swine, and has not yet been reproduced in laboratory animals up to the present time. This suggests that guinea pigs may be a useful laboratory animal for studying the pathogenesis of Actinobacillus pleuropneumoniae infection in swine.     

The effect of radiation on the viability and migratory ability of second-stage larvae of Toxocara canis in mice

Larval counts were made on mice 2 days after oral inoculation with X-ray (0-320 Krad) or of gamma ray (0-6 Mrad) irradiated eggs containing second-stage Toxocara canis larvae. The majority of the larvae irradiated with 0-40 Krad were recovered from the liver and lungs, while most of the larvae irradiated with 80 or 160 Krad remained in the digestive tract, mainly in the stomach and the proximal half of the small intestine. Only a small number of the 320 Krad irradiated larvae was recovered from the mice. No significant difference was observed in the viability of irradiated larvae incubated in vitro up to 13 days after irradiation. However, a substantial percentage of the 160 and 320 Krad-irradiated larvae hatched during that period. Very few larvae were recovered from the digestive tract of mice inoculated with eggs irradiated with 0.5 Mrad, and only one and four larvae were recovered from the liver and lungs of a mouse. No visceral larval migration was observed in mice inoculated with 1 Mrad-irradiated eggs. The minimum lethal radiation dose for second-stage T. canis larvae in eggs is proposed to be 1 Mrad.     

桑天牛幼虫感染白僵菌后的组织病理学研究

桑天牛(Apriona germari)幼虫被球孢白僵菌(Beauveria bassiana)Bb00菌株感染后,虫体颜色及外部形态发生相应的变化。组织切片观察表明:白僵菌Bb00菌株处理后24 h,分生孢子即可以附着于桑天牛幼虫体表,48 h即可以萌发并侵入幼虫体表,使内表皮分解;菌丝进入血腔后开始增殖并向侵入点附近的脂肪、肌肉、气管等组织入侵。随着菌丝在幼虫体内的增殖,各组织器官遭到不同程度的破坏,4 d后感染幼虫死亡,5 d后菌丝突破体表在虫体外形成菌丝层并有分生孢子产生。     

Neospora caninum infected the alimentary tract of nude mice and was transmitted to other mice by intraperitoneal inoculation with the intestinal contents

Neospora caninum (BT-2 strain) that originated from the brain of a Holstein calf was serially passaged through 10 generations of BALB/c nude mice by intraperitoneal inoculation. Histological examination of the mice revealed that numerous clusters of tachyzoites appeared in the pancreas, stomach and small intestine as well as in the central nervous system (CNS) and skeletal muscles. Intestinal contents of the infected mice were inoculated intraperitoneally into uninfected nude mice and 3 of the 17 inoculated mice showed clinical signs at post inoculation days 3 to 10. The present experiments demonstrated a proliferation of N. caninum tachyzoites in the mucosa of the alimentary tract and pancreas of the nude mice and the intestinal contents of the mice were infective to other nude mice.     

Bacteriologic and histologic features in mice after intranasal inoculation of Brucella melitensis

OBJECTIVE: To characterize effects of intranasal inoculation of virulent Brucella melitensis strain 16M in mice. ANIMALS: Female Balb/c mice, 6 to 8 weeks old. PROCEDURE: Studies were designed to elucidate gross morphologic lesions, bacterial burden in target organs, and histologic changes in tissues following experimental intranasal inoculation of mice with B melitensis 16M, which could be used to characterize a model for testing vaccine efficacy. RESULTS: Measurable splenomegaly was evident at 3 and 7 weeks after inoculation. A demonstrable increase in splenic colony-forming units (CFU) from infected mice increased over time with increasing dose when comparing inocula of 10(3), 10(4), and 10(5) CFU. Recovery of brucellae from the lungs was possible early in infection with 10(1), 10(3), and 10(5) CFU, but only the group inoculated with 10(5) CFU consistently yielded quantifiable bacteria. At a dose of 10 CFU, few organisms were located in the spleen. Bacteria were recovered up to 140 days after inoculation in mice given 10(3) CFU. At an inoculum of 10(5) CFU, bacterial counts were highest early in infection. Histologic examination of tissues revealed an increase in white pulp and marginal zone in the spleen and lymphohistiocytic hepatitis. CONCLUSION AND CLINICAL RELEVANCE: Changes in the spleen and liver increased with increases in dose and with increased time following intranasal inoculation with B melitensis 16M. Surprisingly, histologic changes were not observed in the lungs of inoculated mice.     

Experimental tuberculosis in red deer (Cervus elaphus)

This study was designed to investigate experimental Mycobacterium bovis infection of red deer (Cervus elaphus). Three intravenously inoculated deer (dose 10 microg-1000 microg) developed miliary tuberculosis of the lungs and all died within 28 days of being infected. No clinical illnesses were observed in four subcutaneously (dose 1 microg-1000 microg) and three intratracheally (dose 10 microg-100 microg) inoculated deer. At the conclusion of the experiment six weeks post inoculation, these seven animals reacted to 2 mg/ml of bovine purified protein derivative. The principal lesions in the intravenously inoculated deer were in the lungs which had multiple foci of necrosis containing very large numbers of acid fast bacilli. A gradation of changes was seen in the subcutaneously inoculated deer. The animal receiving the 1 microg dose only had lesions at the injection site and the draining prescapular lymph node. Deer receiving higher doses also had histopathological changes in the lungs and liver. Microscopic changes in the intratracheally infected animals were restricted to the thoracic cavity. The ability of the deer to controlled infection was related to the route of inoculation.     

Effect of formalin fixation on the immunohistochemical detection of PRRS virus antigen in experimentally and naturally infected pigs.

The purpose of this study was to determine the effect of formalin fixation on the immunohistochemical detection of porcine reproductive and respiratory syndrome (PRRS) viral antigen in lungs of experimentally and naturally infected pigs. In separate trials, five 24-day-old pigs and six 10-day-old pigs were housed as separate groups in isolation and inoculated intranasally with 10(5.5) TCID50 of an isolate of PRRS virus (PRRSV; P129). The older and younger pigs were euthanatized at 7 and 10 days post inoculation (dpi), respectively. At necropsy, all pigs had gross and microscopic lung lesions typical of PRRS, and PRRSV was isolated from all pigs. To insure uniform fixation, lungs from each pig were cut into 1-cm-thick slices and immersed into 10% neutral-buffered formalin. After fixation in formalin for 8 hours or 1, 2, 3, 5, 6, 8, 10, and 15 days, 3 lung sections from some or all pigs were processed for histological examination using routine methods. Immunohistochemical staining for PRRSV antigen was positive at the following times (days unless otherwise stated) after fixation (percentage of pigs staining positive for PRRSV in parentheses): 8 hours (100); 1 (100); 2 (100); 3 (80); 5 (33); and 6, 8, 10, and 15 (0-all negative). To further evaluate the effects of formalin fixation on PRRSV immunodetection, 31 field cases of PRRS were selected for immunohistochemistry (IHC). Over a 3-month period, submitted cases were selected from the Purdue University Animal Disease Diagnostic Laboratory, W. Lafayette, Indiana, for IHC if 1) the clinical history included respiratory disease, 2) PRRSV was isolated from lung and/or serum from the submitted pigs or tissues, 3) at least 1 section of lung fixed in 10% neutral-buffered formalin was submitted for IHC, and 4) the duration of fixation could be accurately determined from the case history. Of the 31 PRRSV-infected pig cases meeting the selection criteria, 23 were fixed in formalin for 4 days or less. Twenty-one of these 23 (91%) were positive by IHC. Two of 8 cases fixed for greater than 4 days (25%) were positive by IHC. In practical terms, 1-day shipping of fixed samples to a laboratory followed by routine tissue processing within a laboratory should not adversely affect immunohistochemical detection of PRRS viral antigen. But a delay in shipping or processing of more than 2 days could reduce or prevent the detection of PRRS viral antigen by IHC.     

The early pathogenesis in chicken inoculated with non-pathogenic serotype 2 Marek's disease virus.

A newly cloned serotype 2 Marek's disease virus (MDV), strain ML-6, was inoculated via the nasal cavity in specific-pathogen-free chicks to examine early virus replication and the expression of Marek's disease (MD)-related antigens. Following inoculation, viral intracellular antigens (VIAs) were detected in lymphoid organs (bursas and spleens) between 5 and 14 days post inoculation (PI), in feather follicles between 14 and 30 days PI, and in lungs at 3 days PI by the immunohistopathological staining of avidin-biotin-peroxidase complex method. But, very few VIAs were expressed in the thymuses between 5 and 14 days PI. However, MD tumor-associated surface antigens were not detected in any organs. Viruses were isolated from separated spleen cells at 14 and 30 days PI. Fluorescent antibodies of convalescent sera were also detected after 10 days PI. As most of the VIAs were detectable in B-cells in bursas and spleens. B-cells were considered to be the main first target cells for the serotype 2 MDV infection.