Serological survey for antibodies to Encephalitozoon cuniculi in ownerless dogs from urban areas of Brazil and Colombia

There are 3 strains of Encephalitozoon cuniculi that occur in mammals. Strain III is associated with clinical disease in dogs, although some can be asymptomatic carriers and excrete spores in their urine. Several cases of human E. cuniculi infection caused by strain III have been observed in immunocompromised patients, indicating that E. cuniculi should be considered a zoonotic agent. Encephalitozoon cuniculi can cause fatal disease in maternally-infected or young dogs. Clinical signs in these animals included blindness, encephalitis, retarded growth rate, and nephritis. Encephalitozoon cuniculi has also been associated with primary renal failure in adult dogs. The present study used the direct agglutination test (DAT, cut-off 1:50) and the indirect fluorescent antibody test (IFAT, cut-off 1:10) to examine the prevalence of antibodies to E. cuniculi in dogs from Brazil and Colombia. Using the DAG, 31 (27.4%) of 113 dogs from Brazil and 47 (18.5%) of 254 dogs from Colombia were seropositive. Nine (14.3%) of 63 dogs from Brazil and 18 (35.3%) of the 51 dogs from Colombia were seropositive by indirect immunofluorescent antibody test. These results indicate that dogs from Brazil and Colombia are exposed to E. cuniculi.     

Experimental Encephalitozoonosis in the Blue Fox

Newborn and young pups up to the age of 15 days were exposed to E. cuniculi, either by keeping the pups in cages together with orally inoculated foster-mothers and their offspring, or by oral inoculation with E. cuniculi spores. A majority of pups appeared sero-positive to E. cuniculi with the india-ink immuno-reaction from 35 to 87 days post exposure; spores of E. cuniculi were detected in organs of some of the animals. The non-inoculated pups kept together with the orally inoculated pups became seropositive from 49 to 129 days after the oral inoculations. However, the exposure of newborn and young pups failed to induce clinical encephalitozoo-nosis, and when killed at the time of pelting the body weights and fur quality appeared to be within the normal range in all exposed foxes. No macroscopic lesions were detected in the various organs. Histologically focal interstitial nephritis occurred in the great majority of the seropositive animals. Meningoencephalitis was seen in some of the foxes, whereas slightly thickened walls of some arteries, mainly in the myocardium, were found in a few animals. The lesions of the brain and kidneys seem to be very similar to those seen in chronic cases of rabbit encephalitozoonosis. Polyarteritis nodosa and severe encephalitis and interstitial nephritis with extensive proliferations of plasma cells, which are almost constant findings in cases of clinically diseased foxes, were not detected in any of the subclinically infected animals. Various factors that might be of significance in the pathogenesis of the disease are discussed, and it is concluded that intrauterine infection of the pups via the transplacental route appears to be an essential supposition for the establishment of clinical fox encephalitozoonosis.     

The india-ink immunoreaction: a method for the rapid diagnosis of encephalitozoonosis.

Sera from 37 rabbits were assayed for antibodies against Encephalitozoon cuniculi (Nosema cuniculi) by the india-ink immunoreaction and the indirect fluorescent antibody tests: all animals seropositive to the former were also positive to the latter test. 27 of the rabbits were also tested for skin hypersensitivity and then autopsied. Animals positive to the skin test were also positive to the serological tests. At autopsy 18 of 22 rabbits positive in the immunological tests showed lesions typical of encephalitozoonosis. Sera from 200 rabbits originating from 6 institutes were assayed by the india-ink test: seropositive rabbits were found from all institutes (9.1 to 81.9% incidence), with serum titres ranging from 1:125 to 1:5000. The india-ink test appears to be a rapid and convenient method for diagnosis of encephalitozoonosis in rabbits.     

The eradication of Encephalitozoon cuniculi from a specific pathogen-free rabbit colony.

Encephalitozoan cuniculi was discovered in a large specific pathogen-free rabbit colony during routine quality assurance testing. By using a modified India-ink immunoreaction test we were able to test the entire colony for antibodies to Encephalitozoon cuniculi. The prevalence of the disease was approximately 5%. All seropositive animals were culled, and another test of the entire colony, carried out 4 weeks later, revealed one seropositive rabbit which was also culled. Two subsequent screenings of the whole colony have shown no further seropositive animals.     

Passive protection of mice with the 19 S and 7 S fractions of anti-pertussis sera in experiment.

Protective activity of anti-persussis rabbit and mouse sera and 19 S and 7 S fractions obtained from these sera was investigated in the test of passive protection of mice on a model of pertussis meningoencephalitis. The method of simultaneous intracerebral administration of the serum or fraction with live culture of a virulent B. pertussis strain was used. Hyperimmune rabbit serum containing mercaptoethanol-resistant agglutinins in a high titre was found to have the most pronounced protective effect. Serum of mice, collected 14 days after single immunization of the animals, did not show any protective properties. A small amount of protective activity was observed in the serum collected on the 30th day after a single administration of the vaccine. A sharp increase in the protective activity of the serum was observed after double immunization of mice. Correlation was found between the increase in the titre of agglutinins (in particular of 7 S antibodies) and the protective activity of the serum. Protective properties of 19 S and 7 S fractions isolated from immune rabbit and mouse sera by the method of gel filtration were investigated. Both fractions were found to possess protective properties, but fraction 7 S was more active than fraction 19 S.     

Autogenous Immunity to Endogenous RNA Tumor Virus: Differential Reactivities of Immunoglobulins M and G to Virus Envelope Antigens

The autogenous humoral immune response of mice to their endogenous leukemia virus has been examined in terms of the reactivities of individual classes of antibody present in normal B6C3F(1) serum. Whole serum and the immunoglobulin (Ig) M and IgG fractions of serum from animals of different age groups were compared by radioimmune precipitation assays and viral infectivity neutralization assays. Both IgM and IgG fractions were able to precipitate virus, although not as effectively as whole serum. Virus-specific antibody levels, as well as total antibody concentrations in whole serum, appeared to increase with age. Sodium dodecyl sulfate gel electrophoresis analysis was performed with immune precipitates obtained when whole serum or 19 or 7S fractions from animals of different age groups were reacted with disrupted virus. The 19S antibody fraction reacted with three antigenic determinants on the viral envelope. These antigens have apparent molecular weights of 17,000, 43,000, and 68,000. The last two appear to be glycoproteins and may correspond to the M(2) and M(1) antigens. In contrast, the 7S component reacted only with the 17,000-molecular-weight protein. Neutralization assays against BALB:virus-2, a xenotropic endogenous mouse type C virus, revealed that 19S and whole serum but not the 7S fraction possessed neutralizing activity. These findings indicate that there are differential reactivities of IgM and IgG antibodies in normal serum of B6C3F(1) mice, with respect to both recognition of viral envelope antigens and neutralization of endogenous MuLV. These results are consistent with the hypothesis that the autogenous humoral immune response is a systemic host function that may be important in the regulation of endogenous type C virus expression in vivo.     

Humoral immune response to natural infection with Encephalitozoon cuniculi in rabbits

Sera from 4 generations of a family of rabbits having a natural Encephalitozoon cuniculi infection were investigated. Antibodies were demonstrated in a litter of newborn rabbits and in another litter 11 days old. Histoloigcal examination of the brains and kidneys of these animals failed to reveal lesions attributable to the disease. Sera from a further litter of 2 rabbits, taken at weekly intervals from 4 to 42 weeks of age, revealed a low initial antibody titre which regressed to an undetectable level. After an interval, titres rose and then plateaued to the end of the period of observation. Encephalitozoon cuniculi infections in the dams of all the litters were confirmed by antibody assay and by histology. Parallel titration of samples treated and not treated with 2-mercaptoethanol showed that the india-ink immunoreaction demonstrates only immunoglobulin G.     

Kinetics and Immunoglobulin Nature of the Antibody Response to Mycobacterium tuberculosis

Antibody production after injection of Mycobacterium tuberculosis was studied by use of the single cell plaque assay technique. Maximal numbers of antibody-forming cells were found 9 days after a single injection of M. tuberculosis, about 1 day before the appearance of maximal circulating antibody. Immunoelectrophoretic studies and 2-ME studies on 19S and 7S globulin fractions obtained by gel filtration show that the hemagglutination and hemagglutination-lysis antibody activity is associated with 19S immunoglobulin.     

Evaluation of mouse thymic virus antibody detection techniques

Three serologic test methods for detection of serum antibodies to mouse thymic virus (MTV) were compared, including enzyme-linked immunosorbent assay (ELISA), complement fixation (CF) test and indirect fluorescent antibody (IFA) test. Serum was collected at regular intervals from CD-1 mice inoculated intraperitoneally with approximately 200 infectious doses of MTV, and from uninoculated mice placed in cages with the inoculated animals. The inoculated mice became MTV antibody-positive by all three assay methods at 15 days post inoculation, while the cage-contacts were seropositive by all methods at 30 days after contact. Although the incidence of positive results was similar by all methods, titers measured by ELISA were substantially higher than those measured by CF and IFA tests. Because MTV can cause persistent infections that adversely affect the suitability of mice for research, it is recommended that testing for antibodies to this virus be performed routinely.     

Morphine dependence is associated with changes in neuropeptide S receptor expression and function in rat brain

Neuropeptide S (NPS) is a newly identified ligand for the previously discovered G-protein coupled receptor 154 now named NPSR. Recently, it has been found that NPSR gene expression is altered during ethanol withdrawal. In this study we tried to elucidate if NPSR gene expression is modified in response to morphine withdrawal and its protracted abstinence. To induce opioid dependence Wistar rats were treated for 7 days with morphine. Twelve hours and 7 days after the last morphine administration brains were removed and the expression of NPSR mRNA was analyzed by in situ hybridization (ISH). Successful induction of opioid dependence was confirmed by the naloxone-precipitated withdrawal test 2 h after the last morphine administration. Moreover, 7 days after the last morphine dose animals were checked for signs of anxiety and for intracerebroventricular (ICV) NPS (0.3 and 1.0 nmol) induced anxiolytic effects by elevated plus maze (EPM). Results showed that in morphine treated rats strong somatic signs of naloxone-precipitated withdrawal occurred. ISH data revealed changes in NPSR gene expression in the ventral tegmental area as well as in the basolateral amygdaloid and bed nucleus of stria terminalis at 12 h and 7 days into abstinence, respectively. At 7 days into abstinence post dependent animals showed higher levels of anxiety than controls which were significantly attenuated by NPS. These results demonstrated that morphine dependence induction led to (i) changes in NPSR mRNA expression; (ii) increased anxiety; and (iii) more potent anxiolytic-like effect of NPS.     

Serological Investigation of Granulocytic Ehrlichia Infection in Sheep in Norway

Serum samples of 749 sheep from 75 sheep flocks in Norway, i.e. 361 lambs (6 to 7 months old) and 388 adults (>1.5 year), were analysed for antibodies to Ehrlichia equi. Ten animals from each flock were examined. Seropositive animals were found along the coast of southern Norway from Vestfold to S?r-Tr?ndelag (as far north as 63°38'N). Seropositive sheep were not found in southeast, east or northern Norway. Thirty-two flocks were seropositive, although tick-borne fever had only been diagnosed earlier in half of these. In 78% of the seropositive flocks, more than 80% of the sheep were seropositive. A total of 35.7 % and 36.3 % of lambs and adults were found seropositive, respectively. However, the overall seroprevalence among animals that had been grazing on Ixodes pastures were 0.80 for the lambs and 0.84 for the adults. Mean antibody titres (± SD) (log₁₀) in seropositive lambs and adults were 2.59 (± 0.449) and 2.70 (± 0.481), respectively. No significant differences in either seroprevalence or mean antibody titre between sheep of different ages were obtained in this study. Based on antibodies 94% of sheep flocks on Ixodes pastures were infected with a granulocytic Ehrlichia infection. The association between seropositive flocks and Ixodes infested pasture shows a very high degree of agreement (p < 0.00001). The present study indicates that granulocytic Ehrlichia infection in sheep is underdiagnosed in Norway.     

Plasma progesterone concentrations and fertility of indigenous Small East African goats,bred after treatment with cloprostenol

Twenty non-lactating, indigenous Small East African does and postpubertal doelings were injected 125 μg cloprostenol, PG (i.m.). Animals in oestrus were penned with males every night for 45 days. Jugular blood samples for P4 determination were collected immediately before the cloprostenol injection, 4 days later and twice a week for up to 80 days after the end of the mating season. Of the 19 animals with basal P4 concentration 4 days after PG-treatment, only 4 (20%) conceived within 14 days of the treatment. Starting from 4 days after PG-treatment, 1 (5%), 6 (30%) and 8 (40%) of the remaining 15 animals exhibited a short luteal phase, a luteal phase of normal duration or remained anoestrus for 22.2±2.2 days, respectively. The average interval from introduction of the buck to the fertile oestrus for the 19 animals that eventually became pregnant was 23.2±2.3 days (range 9–45). From conception to midgestation the mean plasma P4 concentration ranged from 2.6±1.2 to 10.8±2.4 ng/ml and between days 25 and 35 of pregnancy these values were higher (P < 0.05) in twin-than in singleton-carrying does.It is concluded that for optimal fertility in does of the Small East African goats treated with cloprostenol, mating should be postponed until the first spontaneous oestrus following such treatment.     

Encephalitozoonosis in pharmacologically immunosuppressed mice

Encephalitozoon cuniculi is a parasite that has been identified as a cause of opportunistic infections in immunocompromised individuals. This study was performed to evaluate E. cuniculi infection in pharmacologically immunosuppressed mice. Mice were immunosuppressed with cyclophosphamide (100mg/kg twice a week, IP) or cyclosporin (10mg/kg daily, IP) and inoculated with 10(7)E. cuniculi spores IP. The E. cuniculi spores were cultivated in MDCK cells. E. cuniculi identification was performed by light microscopy studies using Gram-Chromotrope, Hematoxylin-Eosin and Toluidine blue-fuchsin staining techniques, as well as by PCR at 15, 30 and 45 days post-inoculation (DPI). Cyclophosphamide-immunosuppressed mice have greatly reduced amounts of CD8(+), CD4(+) and CD3(+) T cells and CD19(+) B cells. The cells from these mice were analyzed by FACS and showed acute disseminated and fatal encephalitozoonosis. Mice treated with ciclosporin, which is both antiparasitic and immunosuppressive, have a milder, chronic, non-lethal infection and showed a significant reduction only in CD3(+) and CD4(+) T cell numbers. Our results support the role of CD8(+) T cells in controlling infection by E. cuniculi and show that preventive measures are essential for preventing this zoonosis in individuals undergoing chemotherapy for cancer or other immunosuppressive therapies.     

Application of immunofluorescence to the establishment of an Encephalitozoon cuniculi-free rabbit colony.

Serologic screening of a rabbit breeding colony over a 9-month period showed that all 9-week-old rabbits with Encephalitozoon cuniculi infection were born of E cuniculi-infected does. This observation, obtained from studies on 395 young rabbits, suggested that transmission of infection is either transplacental or the result of close contact soon after birth. On this basis, 16 young healthy rabbits, seronegative to E cuniculi, were isolated and tested at 2-week intervals for antibodies to E cuniculi. In the first 2 months, seven rabbits showed indications of developing antibodies to E cuniculi and were immediately removed from the colony. The remaining rabbits along with their 52 offspring were tested for serum antibodies for a further 16 months and no rabbit became seropositive. Eight months after establishment of the colony, three does, one buck and six 12-week-old rabbits were killed. Macroscopic and extensive histologic and immunofluorescence examinations failed to reveal any evidence of infection with E cuniculi. These results showed that serological screening for E cuniculi infection by immunofluorescence is a simple yet adequate procedure for establishing a rabbit colony free of encephalitozoonosis.     

Structural,ultrastructural and immunohistochemical evidence of testosterone effects and its ablation on the bulbourethal gland of the Artibeus planirostris bat (Chiroptera,Mammalia)

This study evaluated the effects of testosterone in the bulbourethral glands (BG) of the bat, Artibeus planirostris, by performing castration and posterior hormonal supplementation of the animals. The results showed a decrease in testosterone levels in animals 15 days after castration, which induced a small reduction in epithelium height, percentage of AR+ cells, and an increase in the amount of basal cells. This reduction became more severe in groups castrated for longer periods (19 and 22 days), where there was also an increase in apoptotic cells. Moreover, the hormonal supplementation increased testosterone levels (after 3 and 7 days of supplementation), causing a glandular reactivation that increased the epithelium height and AR expression. In conclusion, BG took longer to respond to ablation of testosterone than other reproductive glands, since it showed evident aspects of regression only in animals 22 days after castrated.     

Immune modification of the pathogenesis of Streptococcus uberis mastitis in the dairy cow

Abstract Two groups of 4 cows were vaccinated subcutaneously with live Streptococcus uberis strain 0140J or a surface extract derived from the same strain, at 14 days prior to the cessation of lactation (drying off) and at calving. Both groups also received an intramammary administration of the surface extract 7 days after drying off. A third group of unvaccinated animals acted as controls. Following intramammary challenge of two quarters per cow with the vaccine strain, all quarters on control cows and those vaccinated only with surface extract developed clinical mastitis. However, only 12.5% of challenged quarters on cows which were vaccinated with live bacteria developed clinical mastitis. In addition, the numbers of bacteria in the milk following challenge were 10⁵ times higher from the control and extract vaccinated cows than those which received live vaccine. Serum levels of S. uberis specific IgG₂ were elevated in the animals vaccinated with the live organism when compared to that of either extract-vaccinates or controls, whilst S. uberis specific levels of IgG₁ and IgM were similar in all groups throughout the experiment. Specific antibody levels in milk were unaffected by vaccination. Despite increased levels of IgG₂, no increase in opsonic activity was detected in any serum or milk samples. Peripheral blood lymphocytes from animals vaccinated with live organisms showed a considerable increase in proliferative response to S. uberis antigen in vitro when compared with lymphocytes from control and extract-vaccinated animals. These results suggest that neutrophils and specific opsonising antibody may not form the major defence against infection with S. uberis .     

Prevalence and isolation of Toxoplasma gondii in goats slaughtered for human consumption in the semi-arid of northeastern Brazil

The aim of this study was to determine the seroprevalence of anti-Toxoplasma gondii antibodies and factors associated with infection in goats, and to isolate protozoan strains in tissue samples from seropositive goats that were destined for human consumption in the state of Paraíba, northeastern Brazil. Serum samples from 229 slaughtered goats were tested using the indirect fluorescence antibody test (IFAT), with a cutoff point of 1:64. Epidemiological questionnaires were applied to the producers, to acquire information about the sanitary management used in their herds. Tissue samples from the animals were collected during slaughter, in order to perform bioassays in mice. The seroprevalence found was 21.39% (49/229), with antibody titers ranging from 1:64 to 1:32,768. The municipalities of origin, Patos (OR: 3.047; CI: 1.384–6.706) and Sousa (OR: 3.355; CI: 1.536–7.327), were considered to be factors associated with infection by T. gondii. Thirty-eight bioassays were performed in mice, using tissues from seropositive goats, with an isolation rate of 50% (19/38). There was no correlation between isolation rate and antibody titers. Only one mouse died, at 30 days post-infection, which demonstrated that the strains isolated had low virulence towards mice. It was concluded that there is high seroprevalence in goats in northeastern Brazil, as well as a high percentage of viable tissue cysts in slaughtered animals destined for human consumption. These results demonstrate that there is an imminent one health problem relating to toxoplasmosis, especially in the most populous municipalities in the study (Patos and Sousa), which were identified as factors associated with T. gondii infection in goats.     

The activity of S9-liver fractions from seven species in the Salmonella/mammalian-microsome mutagenicity test

A comparative study was made of the metabolizing activity of microsomal fractions of the liver of the rat, mouse, Chinese hamster, dog, mini-pig, rhesus monkey and baboon, with and without pretreatment of the animals with Aroclor (500 mg/kg i.p.). The activity of the fractions was determined by means of the Salmonella/mammalian-microsome mutagenicity test. The protein content of the S9 fractions was standardized (36.14 mg/ml) to eliminate species-specific and inter-individual differences. Strain TA100 of Salmonella typhimurium was used as test organism. The substances tested, namely benzo[a]pyrene, cyclophosphamide, diethylnitrosamine, β-naphthylamine and o-aminoazotoluene, are all known mutagens or carcinogens belonging to various chemical classes.

The tests made with S9 fractions from animals that had not been pretreated with Aroclor revealed distinct species-specific differences in the metabolizing activity of the fractions and also differences from one substance to another. The fractions from mice and Chinese hamster had only a weakly positive effect with some of the substances, those from the rat and the mini-pig only with 2 and 3 of the 5, resp. The dog-liver fraction gave positive results with all substances except benzo[a]pyrene. The fractions from the baboon and the rhesus monkey had positive to strongly positive effects with all the substances.

In the experiments in which the animals had been pretreated with Aroclor, the S9 fractions from all species produced positive effects with all substances, and the mutagenic effects provoked by various substances, when fractions from untreated animals were used, remained of the same degree or became more distinct. Species-specific differences were no longer discernible.

Demethylase activity in all the S9 fractions used was determined with ethylmorphine. Without prior activation, the fractions from the mouse, Chinese hamster, rat and dog gave highly divergent values. In the fractions from the mini-pig, the baboon and, in particular, the rhesus monkey, the activity was relatively more pronounced. Enzyme induction with Aroclor led to a distinct increase in demethylase activity in the livers of almost all species. Only a very rough quantitative relation was evident between the demethylase activity of the individual S9 fractions and their influence on the substances tested for mutagenicity.     

Changes in abundance of Lactobacillus spp. and Streptococcus suis in the stomach, jejunum and ileum of piglets after weaning

This present study investigated the changes in bacterial community composition, with an emphasis on Lactobacillus spp. and Streptococcus suis populations as potentially beneficial and harmful groups, in the stomach, jejunum and ileum of piglets after weaning (21 days postpartum) by 16S rRNA gene-based methods. Denaturing gradient gel electrophoresis analysis showed that, after weaning, predominant bands related to Lactobacillus spp. disappeared and were replaced by potential pathogenic species, such as Peptostreptococcus anaerobius, Moraxella cuniculi, S. suis and Porphyromonas catoniae. Real-time PCR revealed that the abundances of lactobacilli and Lactobacillus sobrius as a proportion of total bacterial abundance were significantly lower in the stomach, jejunum and ileum of weaned piglets than in 21-day-old piglets. A specific and sensitive real-time PCR assay was developed for quantification of the important pathogen S. suis within gastrointestinal microbiota. The assay showed that S. suis predominated in the stomach samples of weaned piglets with population levels up to 10(7) copies g(-1) digesta, while it was not detected in the stomach before weaning. Streptococcus suis was not dominant in the jejunum and ileum digesta before weaning, but became dominant after weaning, with population levels up to 10(7) copies g(-1) digesta. The results demonstrated for the first time the postweaning dominance of the potentially harmful S. suis in piglet intestine. The results also suggest that the defensive barrier of the stomach can be impaired as S. suis became dominant while the proportion of Lactobacillus populations decreased after weaning, which may further result in an increase of S. suis abundance in the intestine.     

Survey for Encephalitozoon cuniculi in arctic foxes (Alopex lagopus) in Greenland

Wild arctic foxes (Alopex lagopus) from Greenland were tested for antibodies to Encephalitozoon cuniculi with an enzyme-linked immunosorbent assay and a carbon immunoassay. Of 230 tested foxes none was seropositive. This finding contrasts with observations from other arctic areas and absence of rodents in the diet of these arctic foxes is the most likely explanation for absence of E. cuniculi.