Malignant catarrhal fever in pigs and a genetic comparison of porcine and ruminant virus isolates in Finland.

An outbreak of the sheep-associated form of malignant catarrhal fever (MCF) in a Finnish sow herd was diagnosed by histopathology and confirmed by PCR. Several gilts and sows were suffering from high fever and anorexia and had aborted, and six of them had died. Typical signs of lymphoproliferation and vasculitis were observed histologically in several tissues, including the uterus. Ovine herpesvirus-2 (OvHV-2) was detected by PCR in two sows. Sequences of the OvHV-2 tegument protein gene obtained from the sows and from three cases of sheep-associated mcf in Finnish cattle were compared and found to be identical. These are the first confirmed cases of mcf in pigs in Finland.

Parvovirus antibodies in vaccinated gilts in field conditions--results with HI and ELISA tests.

This study was conducted to determine the antibody response for porcine parvovirus (PPV) of 39 gilts in field conditions after vaccination. Gilts from four herds endemically infected with PPV were injected twice with a commercial vaccine of inactivated PPV and Erysipelothrix rhusiopathiae. The PPV antibodies were analysed both with haemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) in order to study the agreement between these methods. The possible association between high-antibody titres and reproductive failure (repeat breeding, culling for infertility, < or = 6 piglets born alive) was also investigated. In these study herds, endemically infected by PPV, most gilts (84.6%) had not seroconverted by the age of 6 months. On-field vaccination resulted in a consistent increase of humoral immunity not exceeding the antibody level of 1 : 512 in the majority of gilts in all herds examined. The agreement between ELISA and HI tests was moderate (Spearman's rho = 0.87, kappa = 0.63). The seroconversion over the level >1:512 by mid-pregnancy was not associated with reproductive failure.

Trichinellosis in farmed wild boar: meat inspection findings and seroprevalence.

A reflection of highly prevalent endemic wildlife trichinellosis is seen in wild boar farming in Finland. During the last five years, 0.7% (15/2265) of wild boars undergoing official meat inspection have been determined to be Trichinella-positive. These findings originate from six different farms. In Finland, T. spiralis and T. pseudospiralis have been discovered in meat inspection of wild boars. ELISA showed 11 out of 99 serum samples (11%) as having specific antibodies for T. spiralis crude antigen. Positive samples were from three out of the thirteen farms from which the sera were available. Most of the positive serum samples (8/11) originated from a farm where trichinellosis was also revealed in meat inspection, the other two seropositive farms were without previous Trichinella records. Over the last few decades, no reports have been made of human trichinellosis acquired in Finland. This indicates both efficient meat inspection as well as public awareness of high-risk foodstuff.

Significance of apoptosis and its relationship to antioxidants after ochratoxin A administration in mice.

A study of the appearance of liver apoptosis after ochratoxin A (OTA) administration was performed in male mice. Administration of OTA twice a week for one or two weeks period results in the occurrence of apoptosis in mice"s liver. The presence of intracellular apoptosis bodies was detected at two weeks after toxin treatment. Light microscopic examination demonstrated the presence of eosinophilic globules, often containing apoptotic bodies. They were found within the cytoplasm of intact hepatic cells. The number of apoptotic bodies was further enhanced at two weeks, resulting in 8 fold increases in liver over the control values. No evidence of cell necrosis could be observed by histological and biochemical analysis at one week. However, centrilobular necrosis was evident at two weeks. The ability of the combined antioxidants: Coenzyme Q 10 (CoQ 10), L-carnitine, Zn, Mg, N-acetyl cysteine, vitamin C, vitamin E and selenium or tamoxifen to intervene in apoptosis induced in livers of mice by OTA was also investigated. The inhibition by these scavengers was more clear in mice treated with OTA for one week than those mice treated for two weeks. Treatment with tamoxifen, known in restoration of tumor suppressor function and on induction of programmed cell death (apoptosis), after OTA administration, had no significant inhibition effect on the incidence of apoptotic bodies in liver. Because hepatic glutathione represents the major defence against toxic liver injury, we studied the activity of tissue reduced glutathione (GSH), known to inhibit apoptosis. Our finding showed that two weeks after treatment, OTA caused a decrease of the GSH activity. However, treatment of mice with the combined antioxidants could enhance hepatic antioxidant/detoxification system, as indicated by increase in hepatic reduced glutathione level. In the light of these results, apoptosis was observed after two weeks of OTA administration. We also suggest that use of the combined antioxidants may be of interest in conditions were certain toxin-mediated forms of cell death and/or apoptosis contribute significantly to toxicity.

Fumonisin B1-induced DNA damage in rat liver and spleen: effects of pretreatment with coenzyme Q10, L-carnitine, alpha-tocopherol and selenium.

Active oxygen radical species are reported to cause organ damage. This study was designed to determine whether oxidative stress contributed to the initiation or progression of hepatic and splenic cell DNA damage induced by fumonisin B1 (FB1) in rats. Another aim was to investigate the protective effects of the antioxidants coenzyme Q10 (CoQ10), L-carnitine, vitamin E (alpha-tocopherol) and selenium against DNA damage in the liver and spleen of rats treated with FB1. Fasted rats were injected intravenously with a single dose of fumonisin B1 at 1.55 mg kg-1 body wt. into the tail vein. Treatment with FB1 led to splenic and hepatic DNA fragmentation in 85% of the test animals. DNA fragmentation was investigated as a critical event in toxic cell death by testing total Ca2+ in liver. FB1 administration caused total Ca2+ in liver to increase within 4 h (204% of control). Measurement of liver enzyme activities showed an increase in aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT). FB1 also markedly decreased splenic and hepatic glutathione (GSH) levels. Pretreatment with CoQ10 (30 mg CoQ10 kg-1 diet) together with L-carnitine (2.8 mg carnitine kg-1 diet), alpha-tocopherol (30 IU vitamin E kg-1 diet) and selenium (1 mg selenium as sodium selenite kg-1 diet), decreased DNA damage and the activities of Ca2+, ASAT and ALAT in the liver. On the other hand, the level of GSH was slightly increased. The CoQ10 alone did not significantly protect against toxic cell death and glutathione depletion caused by FB1. Oxidative damage caused by FB1 may be one of the underlining mechanisms of FB1-induced cell injury and DNA damage.

Effects of intranasal exposure to spores of Stachybotrys atra in mice.

The effects of highly toxic and nontoxic spores of Stachybotrys atra were investigated in mice after six intranasal administrations of 1 x 10(5) and 1 x 10(3) spores in phosphate-buffered saline during a 3-week period. Toxic spores contained the trichothecene mycotoxins, satratoxins G and H, as well as the immunosuppressant stachybotrylactones and -lactams. No trichothecenes were detected in the nontoxic spores, and they contained only minor amounts of stachybotrylactones and -lactams. In mice injected with toxic and nontoxic spores, the platelet count was decreased and leucocyte and erythrocyte counts, hemoglobin concentration, and hematocrit were increased. No IgG antibodies to S. atra were detected in sera of mice exposed intranasally to spores. No histological changes were detected in spleen, thymus, or intestines of mice. The mice receiving 1 x 10(5) toxic spores intranasally developed severe inflammatory changes within both bronchioles and alveoli. Hemorrhage was detected in alveoli. The mice receiving 1 x 10(5) nontoxic spores also developed inflammatory changes in the lungs, but these changes were significantly milder than those in mice receiving toxic spores. The mice receiving 1 x 10(3) toxic spores developed inflammatory changes in the lungs that were less severe than those in the mice receiving 1 x 10(5) toxic spores. No inflammatory changes were detected in the mice receiving 1 x 10(3) of nontoxic spores. The present findings indicate that exposure to S. atra spores containing toxins (satratoxins) can be a significant health risk.

T-2 toxin-induced DNA damage in mouse livers: the effect of pretreatment with coenzyme Q10 and alpha-tocopherol.

Active oxygen species are reported to cause organ damage. This study was therefore designed to determine whether oxidative stress contributed to the initiation or progression of hepatic DNA damage produced by T-2 toxin. The aim of the study was also to investigate the behaviour of the antioxidants coenzyme Q10 (CoQ10), and alpha-tocopherol (vitamin E) against DNA damage in the livers of mice fed T-2 toxin. Treatment of fasted mice with a single dose of T-2 toxin (1.8 or 2.8 mg/kg body weight) by oral gavage led to 76% hepatic DNA fragmentation. T-2 toxin also decreased hepatic glutathione (GSH) levels markedly. Pretreatment with CoQ10 (6 mg/kg) together with alpha tocopherol (6 mg/kg) decreased DNA damage. The CoQ10 and vitamin E showed some protection against toxic cell death and glutathione depletion caused by T-2 toxin. Oxidative damage caused by T-2 toxin may be one of the underlying mechanisms for T-2 toxin-induced cell injury and DNA damage, which eventually lead to tumourigenesis.

Comparison of RT-PCR assay and virus isolation in cell cultures for the detection of bovine viral diarrhoea virus (BVDV) in field samples.

The virus isolation-immunoperoxidase test (IPX) on cell cultures and the reverse transcription-polymerase chain reaction (RT-PCR) assay were compared for the detection of bovine viral diarrhoea virus (BVDV) directly in serum samples. Material for this study consisted of 403 sera originating from cattle in 41 BVDV-infected Finnish dairy herds and one suckler cow herd. The presence of virus was demonstrated in 48 samples by both assays. In addition, two more samples were found to be positive by the RT-PCR assay. Both methods proved to be extremely sensitive, detecting pestiviruses even in high serum dilutions, and thus to be suitable for demonstrating the occurrence of persistently infected (PI) cattle. In conclusion, the RT-PCR method used had the advantage of ascertaining BVDV nucleic acid sequences in samples in which the virus had been inactivated, eg during transport or due to the presence of neutralising antibodies.

Detection of toxigenic Pasteurella multocida infections in swine herds by assaying antibodies in sow colostrum.

Toxigenic Pasteurella multocida is the causative agent of progressive atrophic rhinitis (PAR), a serious respiratory infection of swine. Diagnosis of the disease has hitherto been based on clinical signs, pathologic findings, and subsequent isolation of the agent. The best Finnish pig breeding herds participating in the Finnish Pig Health Scheme have been surveyed for PAR since 1963, and the disease has been eradicated from these herds. In this study, a total of 5,650 colostrum samples from 188 Finnish Pig Health Scheme herds were analyzed with a new serologic screening method: an enzyme-linked immunosorbent assay (ELISA) able to detect antibodies to the toxin of P. multocida (PMT). Although the herds had been continuously controlled for PAR, 1 herd with PMT antibodies was found. The positive reactions in the ELISA were confirmed by isolating the causative organism. The origin of the infection also appeared to be obvious. The serologic ELISA is a suitable method for the detection and screening of toxigenic P. multocida-infected pig herds.

Antibodies against 12 serotypes of Actinobacillus pleuropneumoniae in finnish slaughter sows.

To determine the prevalence of A. pleuropneumoniae serotypes in Finnish pig populations, 692 blood samples of sows were randomly collected from Finnish slaughterhouses. These were assayed with a direct ELISA for 12 Actinobacillus pleuropneumoniae serotypes. The specificity of the ELISA was tested using rabbit antisera against these serotypes. Cross-reactions were detected between serotypes 6 and 8 and between serotypes 1, 9 and 11, and serotype 5 antiserum reacted with serotype 6 antigen, but the other serotypes did not cross-react. When assaying the blood samples serotype 3 and 2 antibodies were found in 51% and 26% of samples, respectively. Other serotypes were found only in smaller numbers. Most of the samples, 61%, had antibodies towards some serotype of A. pleuropneumoniae. Antibodies towards serotypes 2 and 3 were found in pigs throughout Finland.

Detection of canine parvovirus antigens with antibodies to synthetic peptides.

Antibodies produced in rabbits against an 18-amino acid peptide (peptide 1, NSLPQSEGATNFGDIGVP) of capsid protein VP2/residues 292-309 of canine parvovirus (CPV) or against an 18-amino acid peptide (peptide 2, GKRNTVLFHGPASTKGKS) of nonstructural protein NS1/residues 391-409 of CPV identified, in immunofluorescence analysis, viral antigens in canine A 72 cells infected with CPV. Antibodies to peptide 2 also identified viral antigens in bovine cells infected with bovine parvovirus. In western blot analysis, antibodies to peptide 1 and peptide 2 also detected viral antigens derived from blue fox parvovirus, feline parvovirus, mink enteritis virus and raccoon dog parvovirus. The peptide antibodies could be used as convenient tools in diagnosis of infections caused by CPV or closely related viruses affecting cats, minks, blue foxes and raccoon dogs.

Comparison of detection methods for trichothecenes produced by Fusarium sporotrichioides on fodder and grains at different air humidities.

Growth and toxin production of a highly toxic strain of Fusarium sporotrichioides Sherb were studied on oat and wheat grains and on straw under experimental conditions, in which relative humidity (RH) of air was regulated. The materials were incubated at three different RH levels at a range of 84-100%. F. sporotrichioides grew well on oat and wheat grains at RH 97-100% but grew less well at RH 84-88% and on straw. Toxin production was measured with three biological toxicity tests (cytotoxicity test, dermotoxicity test, and yeast cell toxicity test), with chemical analysis, and T-2 ELISA assay. Cytotoxicity and production of trichothecene mycotoxins were detected in all the samples incubated at all three RH levels. On oat and wheat grains, T-2 toxin, neosolaniol, and diacetoxyscirpenol were found, and on straw T-2 toxin, HT-2 toxin, neosolaniol, and T-2 tetraol were determined. In the T-2 ELISA assay, all material samples were found to contain T-2 toxin. The cytotoxicity test was the most sensitive method for detecting biological toxicity of samples inoculated with fungus. The T-2 ELISA assay and chemical analysis were about equally sensitive to detect T-2 toxin in samples.

Evolution of the feline-subgroup parvoviruses and the control of canine host range in vivo.

A related group of parvoviruses infects members of many different carnivore families. Some of those viruses differ in host range or antigenic properties, but the true relationships are poorly understood. We examined 24 VP1/VP2 and 8 NS1 gene sequences from various parvovirus isolates to determine the phylogenetic relationships between viruses isolated from cats, dogs, Asiatic raccoon dogs, mink, raccoons, and foxes. There were about 1.3% pairwise sequence differences between the VP1/VP2 genes of viruses collected up to four decades apart. Viruses from cats, mink, foxes, and raccoons were not distinguished from each other phylogenetically, but the canine or Asiatic raccoon dog isolates formed a distinct clade. Characteristic antigenic, tissue culture host range, and other properties of the canine isolates have previously been shown to be determined by differences in the VP1/VP2 gene, and we show here that there are at least 10 nucleotide sequence differences which distinguish all canine isolates from any other virus. The VP1/VP2 gene sequences grouped roughly according to the time of virus isolation, and there were similar rates of sequence divergence among the canine isolates and those from the other species. A smaller number of differences were present in the NS1 gene sequences, but a similar phylogeny was revealed. Inoculation of mutants of a feline virus isolate into dogs showed that three or four CPV-specific differences in the VP1/VP2 gene controlled the in vivo canine host range.

Actinobacillus pleuropneumoniae serotype-2 antibodies in sow colostrum in Finnish pig-health-scheme herds.

A direct ELISA with phenol-extracted antigen for Actinobacillus pleuropneumoniae serotype 2 was developed. The test was specific when tested with rabbit antisera prepared against different A. pleuropneumoniae serotypes. It had better-than-moderate repeatability and it made a clear distinction between positive and negative samples. A total of 5477 colostrum samples from breeding sows from herds participating in the Finnish Pig-health Scheme were tested using the ELISA test. A total of 1307 positive samples were found in 129 out of 154 herds, thus indicating that most of the disease-control herds in Finland are infected with A. pleuropneumoniae serotype 2. These infections were almost entirely subclinical.

Two dominant neutralizing antigenic determinants of canine parvovirus are found on the threefold spike of the virus capsid.

The 25-nm diameter parvovirus capsid is assembled from 60 copies of a sequence common to the overlapping VP1 and VP2 proteins. Here we examine the epitope specificity's of 28 monoclonal antibodies (MAb) prepared against canine parvovirus (CPV), feline panleukopenia virus (FPV), and raccoon-dog parvovirus or blue (Arctic) fox parvovirus. Comparing the reactivity of those MAb with various MAb-selected escape mutants, or with natural variants of CPV or mink enteritis virus (MEV) which differ at known sequences, showed that the binding of 20 of those MAb was strongly affected by variations of two regions on the threefold spike of the CPV capsid. One region was adjacent to the tip of the threefold spike, and the second was around VP2 residue 300, on the shoulder of that structure. MAb recognizing both antigenic sites efficiently neutralized the virus infectivity and inhibited hemagglutination. Mutations leading to natural antigenic variation have also been observed in both those sites in naturally variant strains of CPV or MEV, suggesting that they are important antigenic structures on these parvoviruses. The bindings of several MAb were not affected by the mutations at those antigenic sites, indicating that they recognized other, and perhaps conserved, structures.

Factors affecting the results of T-2 mycotoxin ELISA assay.

Certain substances in the sample may increase or decrease the reaction between the enzyme and substrate in ELISA assays. During a survey of T-2 trichothecene in food and animal feed 75% of milled grain samples gave a higher O.D. value in competitive T-2 toxin ELISA than the negative control. In samples spiked with small quantities (10 micrograms/kg) of T-2 toxin this type of reaction resulted in underestimates of toxin content. However, the effect was weak and, owing to the high sensitivity of the assay, it did not result in false negative reactions. The low efficiency of the carrier solvent and natural peroxidases in food and feed were considered to be the cause of the inaccurate reactions. A few fermented and processed foodstuffs and feed gave positive results in the T-2 toxin ELISA assay, but verification of the results by gas chromatography (GC) showed that the reactions were false. Certain substances in the samples destroyed or decreased the enzyme activity. False positive reactions can be distinguished from correct ones by retesting the extracts in different dilutions.

The prevalence of Yersinia enterocolitica 0:3 in Finnish pigs and pork.

One hundred and forty seven samples of pig faeces collected from 14 herds located in different parts of Finland were examined for Yersinia enterocolitica biotype 4 serotype 0:3. Twenty six (17.7%) animals and 5 herds (35.7%) were positive. The tonsils of 350 animals from 35 herds with high condemnation figures at meat inspection and the tonsils of 131 animals from 13 herds with low condemnation figures collected from 2 abattoirs in southwest Finland were also examined. The prevalence raes of Y.e. in animals were 38.3% and 31.3% and in herds 74.3% and 61.5%, respectively. The prevalence of Y.e. in herds with the high and low partial condemnation percentages did not differ significantly. No isolation of Y.e. was made from 104 samples of pork and minced pork collected from retail markets in Helsinki and from exporting slaughterhouses.

Pathogenesis of blue fox parvovirus on blue fox kits and pregnant vixens.

To study the pathogenicity of a newly isolated parvovirus of blue fox (Alopex lagopus), pregnant vixens and 43 kits of different ages were experimentally infected with the agent. The kits had no clinical signs of disease, even though most of them excreted virus in their feces, and they responded immunologically to viral exposure. Infection during pregnancy appeared to affect the fetuses. A group of 15 blue fox vixens inoculated with the virus produced a statistically smaller number of kits (78) than did 15 untreated controls (131). Vaccination with a homologous inactivated virus preparation appeared to afford protection against reproductive losses. After infection, 15 vaccinated vixens gave birth to 97 kits, compared with 54 kits born to a similar, non-vaccinated experimental group.