Comparison of different methods for diagnosis of porcine proliferative enteropathy

The objectives of this study were: (1) to compare 2 methods of serology; (2) to compare 3 histologic techniques; and (3) to compare 2 methods of detecting shedding in pigs experimentally challenged with Lawsonia intracellularis. The sensitivities of these tests were determined by the detection of infection. Forty 5-week-old pigs were inoculated on day 0 with intestinal homogenate from pigs with proliferative enteropathy (PE). Clinical evaluation was done on day 7 and daily from day 14 to 28 postinoculation. Fecal shedding of L. intracellularis was monitored by use of polymerase chain reaction (PCR) analysis and immunoperoxidase staining at 7-day intervals. Serum was obtained on days 0 and 28 for serologic testing by glass slide and tissue culture indirect fluorescent antibody tests. At euthanasia on day 28, gross intestinal lesions were evaluated and ileum samples collected for histologic analyses. Ileal histologic sections from each animal were stained by hematoxylin and eosin, Warthin-Starry silver stain, and immunohistochemistry (IHC). Of the 40 pigs, 36 had gross lesions typical of PE at necropsy. The percentage of agreement between the 2 serologic methods was 94.4%. Immunoperoxidase stain of fecal smears was more sensitive than PCR for detecting fecal shedding, especially on day 21 (89.5% and 60.5%, respectively) and day 28 (59.4% and 37.5%, respectively) post-inoculation. The IHC stain was much more sensitive for detecting infection than the routinely used hematoxylin and eosin and Warthin-Starry silver stains. In conclusion, in experimentally infected pigs, both serologic methods were appropriate techniques for detecting infection. For fecal samples, PCR has low sensitivity. Immunohistochemistry is the best diagnostic tool for formalin-fixed samples.     

Additional evaluation of undiagnosed bovine abortion cases may reveal fetal neosporosis

Diagnosis of Neospora caninum associated bovine abortion is often made by histological examination of fetal tissues, although this procedure is not very sensitive. One hundred and forty-four undiagnosed bovine abortion cases were evaluated for N. caninum associated bovine abortion using a revised diagnostic protocol. As a result, 12 (8.3%) of these previously undiagnosed bovine abortion cases were definitively diagnosed as N. caninum positive. The 12 new positive cases included 7 that had exhibited histological lesions, and 5 that had not exhibited histological lesions when examined prior to this study. None of the 12 cases had been immunohistochemically (IHC) stained prior to this study. Also, IHC staining implemented during this research revealed tachyzoites without associated histological lesions in at least 1 tissue from 69 (47.9%) of 144 aborted bovine fetuses, and positive IHC staining of cytoplasmic N. caninum antigen in macrophages in at least 1 tissue from 44 (30.6%) of 144 aborted bovine fetuses. These results demonstrate the necessity for more aggressive evaluation of bovine fetuses for neosporosis.     

Comparison of diagnostic procedures for porcine leptospirosis.

Kidneys and matched serum samples were obtained from 368 pigs slaughtered at three Victorian abattoirs, and originating from 42 farms. Macroscopic lesions (white spots) were observed on 102 of the kidneys. Serum samples were tested by the microscopic agglutination test (MAT) and by an IgM enzyme immunoassay (EIA). Kidneys were cultured for leptospires, examined histologically after Warthin-Starry silver staining and after immunogold silver staining (IGSS), and tested for leptospiral DNA by DNA hybridization. Forty-four infected pigs were identified by culture or immunogold silver staining of kidneys or by high MAT titres (greater than or equal to 1024). Infection was demonstrated in 7.5% of visibly normal kidneys, in 23.5% of kidneys with white spots, and in 48% of kidneys with large white spots, of 1 cm diameter or greater. The apparent (maximum) sensitivities of diagnostic procedures for detecting infection were as follows: MAT (at a titre of either 64 or 1024) 95%; IgM EIA 82%; culture 61%; presence of white spots 55%; IGSS 52%; presence of large white spots 30%; Warthin-Starry silver staining 20%. IGSS, Warthin-Starry staining and DNA hybridization all appeared to be highly specific. Of 22 kidney sections identified as positive by IGSS, 13 showed intact leptospires, and these kidneys were all culture-positive. Nine others showed leptospiral antigen in the kidney tubules but no intact leptospires. Only five of these kidneys were culture-positive.     

Leptospira interrogans serovar hardjo is not a major cause of bovine abortion in Victoria

The aim of this study was to determine whether evidence could be obtained of foetal infection with Leptospira interrogans serovar hardjo in aborted foetuses collected from dairy farms. Material from 197 abortions occurring over a wide area of Victoria was collected over 3 years. None of 195 foetal kidney cultures or 7 cultures from membranes was positive for leptospiral organisms. Immunogold silver staining for leptospires was performed on sections of kidneys, lungs or heart from 156 foetuses, with negative results. Evidence of transient leptospiral infection in 11 of 123 foetuses was obtained by foetal heart blood serology. Two isolates of L. interrogans serovar hardjo were obtained from the urine of milking cows. These strains were examined by restriction endonuclease analysis and both were shown to be of the genotype Hardjobovis, as have been all Australian isolates studied so far. It appears that foetal infection with serovar hardjo is not associated with any substantial proportion of bovine abortions in Victoria, in contrast to the situation in Northern Ireland. The apparent absence from Victoria of the pathogenic genotype Hardjoprajitno is a possible explanation.     

The importance of bovine neosporosis for abortion in Switzerland

The protozoan parasite Neospora caninum is one of the most important infectious abortion causes in cattle worldwide. In a Swiss case-control-study we investigated 113 abortion problem farms and 113 control farms. It was possible to detect N. caninum in 21% of 242 investigated aborted fetuses by PCR upon brain samples. Eighty-four % of mothers aborting N. caninum positive fetuses were serologically positive for this parasite. The seroprevalence for N. caninum of all aborting mothers was 44%. Within a period of 3 to 12 months 4,505 cattle from the participating farms were investigated serologically at two different time points. Strong fluctuations of the specific antibody concentration could be observed, resulting in a conversion to seronegativity in the second blood sample in 39% of formerly seropositive animals. Eighteen months after the end of the study, 42 case- and 42 controlfarms were questioned about their present abortion-status. Analysis of the questionnaire revealed that 80% of the former abortion problem farms observed an improvement of the situation. However, in 43% of those farms no preventive measures had been taken and no significant differences in hygiene, forage and animal turnover could be observed between case- and control-farms. Additionally, no significant differences were detected concerning risk factors such as the presence of farm dogs and the disposal procedure for placental and fetal material.     

Single tube nested PCR for the detection of Toxoplasma gondii in fetal tissues from naturally aborted ewes.

A single tube nested polymerase chain reaction (PCR) assay targeting the multicopy 18S-5.8S rRNA internal transcribed spacer (ITS1) region has been developed for the diagnosis of Toxoplasma gondii-induced abortion in ovine fetal tissues. In all, 145 ovine fetal samples including brain, spleen, lung, liver, kidney, placenta and fetal fluids from 53 fetuses and stillborns of 32 farms in Northern Spain were analyzed. Thirty-six samples belonging to nine fetuses and one stillborn lamb were T. gondii PCR-positive. Although T. gondii DNA was amplified from different types of tissues, brain was the tissue with the highest detection rate. All animals that had histopathological lesions associated to T. gondii infection were positive by PCR. In addition, four fetuses whose histological examination was hindered by autolysis were PCR-positive. Results obtained by PCR and indirect fluorescent antibody test (IFAT) showed good correspondence, demonstrating the diagnostic value of the two techniques. However, PCR has the advantage over serology in its ability to diagnose T. gondii infection at earlier stages of gestation when the fetus is not yet immunocompetent and in lambs that have taken colostrum. Once other abortifacient agents are ruled out, PCR detection of the ITS1 region in fetal tissues is a valuable and relatively rapid technique for the diagnosis of ovine abortion caused by T. gondii.     

Immunohistochemical assay for detecting estrogen receptors in canine mammary tumors.

OBJECTIVE: To determine the estrogen receptor (ER) content of canine mammary gland tumors by use of immunohistochemical (IHC) examination of formalin-fixed sections. SAMPLE POPULATION: 21 mammary gland tumors from 20 adult dogs. PROCEDURE: ER were detected in formalin-fixed tissues, using an avidin-biotin alkaline phosphatase IHC assay and were quantified on fresh-frozen tumor samples, using a modified dextran-coated charcoal (DCC) assay. RESULTS: 7 of 21 tumors had visually detectable nuclear ER by use of IHC staining, whereas 8 of 21 tumors were positive for ER by use of the DCC assay. The ER-positive cells in 5 IHC-positive tumors were epithelial cells with histologic criteria of early malignancy. The remaining 2 ER-positive tumors detected by use of IHC had ER-positive mast cells within areas of connective tissue around the tumor. CONCLUSIONS: Immunohistochemistry is an additional method for detection of ER in canine mammary tumors. The major advantage of this type of assay is that it may be performed on formalin-fixed tissues, and individual ER-positive cells may be identified. Discovery of ER-positive mast cells by use of IHC is of concern, particularly if the ER status of a tumor is based on DCC results alone. CLINICAL RELEVANCE: Because most canine mammary tumors are fixed in formalin prior to histologic evaluation, an IHC assay that identifies ER-positive cells is desirable. Adjunctive antiestrogen therapy could be administered to dogs with ER-positive tumors.     

Diagnostic survey of bovine abortion with special reference to Neospora caninum infection: importance, repeated abortion and concurrent infection in aborted fetuses in Southern Brazil

The protozoa Neospora caninum is an important cause of bovine abortion world-wide. The objective of this survey was to determine the distribution pattern of infectious abortion in Southern Brazil with special reference to N. caninum infection. A total of 161 bovine aborted fetuses from 149 farms were analysed during a 1.5 year period. The cause of abortion was identified in 51.5% of cases. Overall, 23% (37/161) of the fetuses were considered to be infected with N. caninum. Bacterial infection accounted for 17.4% (28/161) of cases, fungal infection for 3.1% (5/161) of cases and viral aetiology for 1.8% (3/161). Six fetuses had concurrent infection with N. caninum and Leptospira spp. Data from 111 fetuses and the respective aborted cows were analysed to investigate the association between previous abortion and current N. caninum infection. The prevalence of N. caninum-infected fetuses from cows with and without a history of previous abortion was 44% (11/25) and 24.4% (21/86), respectively. Cows aborting a N. caninum-infected fetuses were 2.4 times more likely to have aborted previously than cows aborting for other reasons (95% CI of odds ratio=0.9-6.8, P=0.06).     

Study on the epidemiology of equine arteritis virus infection with different diagnostic techniques by investigating 96 cases of equine abortion in Hungary

The occurrence of equine arteritis virus (EAV) induced equine abortions was studied with different laboratory methods during a 3-year period. Tissue samples from 96 aborted equine foetuses or newborn foals were collected from 57 farms located in different parts of Hungary. Virus isolation, polymerase chain reaction (PCR), immunohistochemistry and serology were used for the detection of EAV infection. The overall seroprevalence of EAV infection in mares was 65%. EAV induced abortion was diagnosed in eight (8.3%) cases from six (10.5%) herds. Abortion was sporadic in all herds except for one, where epidemic abortion happened. Fetal serology in six (75%) cases, the virus isolation in one (12.5%) case whereas PCR in all of the four investigated cases were positive. The virus could be observed with immunohistochemistry in seven (87.5%) cases mostly in the spleen followed by other organs and the allantochorion. In conclusion, PCR and immunohistochemistry seem to be the most sensitive and useful tests for the diagnosis of EAV induced equine abortion.     

Serologic, virologic, and histopathologic observations of encephalomyocarditis virus infection in mummified and stillborn pigs

Stillborn and mummified swine fetuses from swine farms experiencing reproductive problems were investigated for evidence of infection with encephalomyocarditis (EMC) virus by fetal serology, virus isolation, and histopathologic examination. Fetal sera or thoracic fluids of 478 abnormal fetuses collected during January through December 1987 were tested for the presence of antibody specific to EMC virus. Of 478 samples tested, 175 (36.6%) had EMC virus serum neutralizing antibody titers of 1:64 or greater. The samples positive for EMC virus antibody were obtained from 38 swine farms located in 9 states in the United States. In addition to serologic observations, tissue samples of some abnormal fetuses were examined for the presence of virus and histopathologic lesions. The EMC virus was isolated in 1 case from the fetuses of an aborted litter. The isolate was serologically identical to a reference EMC virus. Nonsuppurative encephalitis and myocarditis were observed in the fetal samples collected from 2 different herds. Thoracic fluid of 1 stillborn pig with lesions was positive for EMC virus antibody at a titer of 1:512. We suggest that a widespread reproductive problem recently experienced in several major swine-producing areas of the United States may have been caused by EMC virus infection.     

Detection of leptospires in tissue using an immunoperoxidase staining procedure

An immunoperoxidase technique for the localisation of leptospires in sections of formalin fixed paraffin embedded kidney tissue is described. The procedure utilises a two-layered antibody sandwich with rabbit anti-leptospiral immunoglobin. Using antiserum to specific leptospiral serovars the presence and distribution of specific serovar in the tissue could be determined. The technique was also used to detect leptospires of given serovars in smears made from infected tissues and fluids. There was good correlation between culture results and results of the immunoperoxidase staining method on kidneys infected with leptospires. The diagnostic possibilities of the technique on formalin fixed tissue specimens are discussed.     

Histochemical and immunohistochemical evidence of a bacterium associated with lesions of epizootic bovine abortion.

Epizootic bovine abortion (EBA), a tick-transmitted disease of pregnant cattle grazing foothill pastures, is a major cause of reproductive failure in California and adjacent states. Affected fetuses develop a chronic disease, resulting in late-term abortion or premature calving. Despite investigations spanning 50 years, to the authors' knowledge, the etiologic agent of EBA has not yet been isolated from affected fetuses or the tick vector. The diagnosis of EBA is based on gross and microscopic lesions. Recently, documentation that the etiologic agent is susceptible to antibiotics and identification of a unique 16S deltaproteobacterial rDNA gene sequence in 90% of thymus tissues from aborted fetuses have supported the role of a bacterial infection as the cause of EBA. To determine whether bacteria could be detected in the tissues, histochemical staining and immunohistochemical procedures were used on formalin-fixed, paraffin-embedded tissues. Use of a modified Steiner silver stain revealed small numbers of intracytoplasmic bacterial rods in 37 of 42 thymic samples from EBA-affected fetuses. Improved detection was achieved by use of immunohistochemical staining with serum from EBA-affected fetuses that resulted in detection of numerous bacterial rods in the cytoplasm of histiocytic cells in the thymus from all 42 EBA-affected fetuses. Immunohistochemical examination of additional tissues from 21 field and experimental EBA cases revealed positively stained intracytoplasmic bacterial rods in many organs with inflammatory lesions. Use of the modified Steiner stain and immunohistochemical staining of tissues from negative-control fetuses failed to reveal organisms. To the authors' knowledge, this is the first report to document morphologic evidence of a bacterium associated with the lesions of EBA.     

Incidence of leptospiral abortion in Brazilian dairy cattle

We examined dams' paired serum samples and foetal kidneys (histopathologically and attempting leptospiral cultures) from 120 Brazilian Holstein abortions from 10 herds near Sao Paulo, Brazil, in 1996. Leptospiras was isolated from 15 foetuses. The Leptospira interrogans serovar hardjo was obtained from four, pomona from three and wolffi from eight foetuses. Ten of these 15 foetuses showed a four-fold rise in titres between the day of abortion and the second samples taken 15 days later. Fifty-seven other foetuses had histologic evidence of leptospires; 27 of their dams (47%) had four-fold rises in titres. In total, 72 of 120 aborted foetuses had evidence of leptospiral infection.     

Encephalitis in aborted bovine fetuses associated with Bovine herpesvirus 1 infection.

Brain tissue from 12 aborted bovine fetuses submitted to the Wisconsin Veterinary Diagnostic Laboratory revealed histologic lesions that consisted of glial nodules and variable degrees of mononuclear inflammation, microhemorrhage, neuronal necrosis, and cerebral cortical cavitation. A diagnosis of Bovine herpesvirus 1 (BHV-1) abortion had been made in all of these cases through multiple testing modalities. Brain tissue from 8 of the 12 fetuses was immunohistochemically stained with a monoclonal antibody specific to BHV-1, and, in 5 fetuses, there was positive intralesional staining of neurons, glial cells, and endothelial cells. This preliminary data suggested that herpesviral infection of brain tissue led to the described neurologic lesions. BHV-1 was then amplified from brain tissue in all 12 of the fetuses and was confirmed by partial sequencing of the thymidine kinase and glycoprotein C genes. To the authors' knowledge, neurologic lesions have not previously been described in BHV-1-infected fetuses, nor has BHV-1 previously been identified in bovine fetal brain tissue. The neurologic histopathology attributed to BHV-1 infection in these cases overlaps with the neurologic lesions produced by Neospora caninum, a common etiologic agent of bovine abortion. Therefore, when bovine fetal neurologic lesions are found, both etiologies should be considered and then distinguished by using additional diagnostic tools.     

An improved immunohistochemical diagnostic technique for canine leptospirosis using antileptospiral antibodies on renal tissue.

The purpose of this study was to compare the immunoreactivity in canine renal tissues stained with antisera specific for 3 leptospiral antigens and those processed with traditional staining methods. In addition, immunoglobulin staining was done on tissues with immunoreactivity to leptospiral antigens. Formalin-fixed renal sections from 12 dogs with chronic interstitial nephritis suspected or proven to have leptospirosis (6 dogs with silver-stained leptospires and 6 dogs in which silver-stained leptospires were not detected) were used. Antibodies consisted of a monoclonal antibody to Leptospira kirschneri serovar grippotyphosa lipopolysaccharide (LPS) and 2 polyclonal antibodies to outer membrane proteins, including OmpL1, a leptospiral porin, and LipL41, an outer membrane lipoprotein. The murine monoclonal antisera against LPS (F71C2-1) had the most abundant and consistent immunoreactivity. Immunoreactive areas were present in 6 of 6 sections positive by silver staining and included extracellular granular debris in intertubular areas, debris in macrophages, organisms in tubular lumina, and cytoplasmic granules in tubular epithelia. Antisera with specificity for the outer membrane proteins OmpL1 and LipL41 detected only intact organisms in tubular lumina. Immunoreactivity to OmpL1 (polyclonal 338) occurred in 4 of 5 sections positive by silver staining, but immunoreactivity to LipL41 (polyclonal 813) occurred in only 1 of 6 silver-positive sections. Each of the kidney sections in which leptospiral antigens were detected by immunohistochemistry also was positive by silver staining. Sections negative by silver staining were also negative by immunostaining. Although immunohistochemistry did not enhance sensitivity, amplification of signal by secondary antibody and hematoxylin counterstaining improved the ease of diagnosis and allowed better evaluation of tissue morphology than did silver staining methods. IgG was the most abundant immunoglobulin. IgG immunoreactivity occurred predominantly in plasma cells within interstitial infiltrates. Interstitial infiltrates contained abundant immunoreactivity to LPS, but immunoreactivity to OmpL1 and LipL41 was not noted.     

High prevalence of chlamydial (Chlamydophila psittaci) infection in fetal membranes of aborted equine fetuses

Seventy-seven cases of equine abortion from 49 Hungarian farms that occurred between 1998 and 2000 were investigated for the presence of chlamydiae by immunohistochemistry, PCR and/or MZN staining. Evidence of the presence of these bacteria was obtained in 64 cases (83.1%) from 41 (83.7%) different farms. Partial ompA gene sequencing of PCR products revealed that the agent was Chlamydophila psittaci. Based on the findings of microbial diagnosis, pathology and case history, chlamydial infection was considered to be the most likely cause of abortion in at least 11 (14.3%) cases. In the remaining 53 Chlamydophila-positive cases, either other bacterial and viral agents (n = 22 or 28.6%) as well as non-infectious factors (n = 14 or 18.2%) were identified as more probable primary causes of disease, or the role of chlamydiae remained unclear because lesions in fetuses and fetal membranes were absent (n = 17 or 22.1%). When chlamydial antigen was detected in aborted equine placental tissue using immunohistochemistry it was seen only in the chorionic epithelial cells, but not in other parts of the fetal membranes nor in any of the fetal tissues. In conclusion, chlamydial infection of the genital tract should be considered a possible factor in equine reproductive disorders.     

The role of Neospora caninum and Toxoplasma gondii in spontaneous bovine abortion in Argentina

The aim of the present work was to evaluate the role of Neospora caninum and Toxoplasma gondii infections in spontaneous bovine abortions in Argentina. Based on histopathological results, 70 presumptive cases of apicomplexan protozoal abortion from a total of 666 cases of spontaneous bovine abortion submitted to the National Institute of Agrarian Technology, Balcarce, from 1999 to 2007 were included in this study. N. caninum infection was diagnosed by an indirect fluorescent antibody test (IFAT), by immunohistochemistry (IHC) and by nested-PCR. T. gondii infection also was diagnosed by nested-PCR. DNA from fetuses was extracted primarily from CNS tissues. Heart, liver, muscle and/or placenta were processed when nervous tissue was not available. Sixty-six (9.9%) fetuses were positive by at least one technique (IFAT, IHC or nested-PCR) for N. caninum infection. Overall, there was poor agreement among results obtained by these diagnostic techniques. In contrast, no Toxoplasma-infection was detected in any aborted bovine fetus.     

A farming systems study of abortion in dairy cattle on the Atherton Tableland: 2. The pattern of infectious diseases

The role of infectious agents on dairy farms on the Atherton Tableland in tropical north Queensland was studied as part of a comprehensive investigation into the causes of bovine abortion. The prevalence of antibody in serums collected from 7 herds whose annual abortion rates ranged from 3% to 21% were as follows: Leptospira hardjo 49.9% (426/853), L. pomona 0.4% (3/851), bovine virus diarrhoea (BVD) 33.7% (35/104). Infectious bovine rhinotracheitis virus (IBR) 11.5% (12/105), Akabane virus 92.2% (95/103), Aino virus 62.1% (64/103), Chlamydia psittaci 3.1% (37/1004), Brucella abortus 0% (0/851), and Toxoplasma gondii 0% (0/105). Testing of serums against a wide range of leptospiral serotypes indicated that reactions occurring in the Hebdomadis and Sejroe serogroups were probably cross reactions with L. hardjo. Infection with L. hardjo and Akabane virus occurred prior to first mating and contact with Aino virus occurred during first pregnancy. Infection with BVD and IBR viruses was sporadic. The pathology and microbiology of 32 aborted foetuses from 24 Tableland herds (10 from the group of 19 farms under more intense study) were performed. Lesions associated with a Sarcocystis-like agent were present in 6, leptospires in 1, suspected toxic hepatosis in 2 and purulent bronchopneumonia (Staphylococcus aureus) in 1 foetus. No diagnoses were made in the remaining 22 foetuses (69%). Evidence for a common infectious cause of abortion in the population was inconclusive.     

Immunohistochemical identification of Campylobacter fetus in natural cases of bovine and ovine abortions

An immunohistochemistry (IHC) procedure for the detection of Campylobacter fetus antigens using an avidin-biotin complex technique was performed on formalin fixed bovine and ovine fetal tissues from 26 natural cases of Campylobacter spp. abortion (four ovine and 22 bovine). The species of Campylobacter isolated included C. fetus ssp. venerealis from 13 bovine fetuses, C. fetus ssp. fetus from two ovine and one bovine fetus, Campylobacter jejuni from seven bovine fetuses, Campylobacter lari from two ovine fetuses and an unspeciated Campylobacter species in one bovine fetus. Histologic lesions identified in the aborted fetuses included placentitis, serositis, pneumonia, gastroenteritis, hepatitis and encephalitis. Campylobacter fetus antigens were identified by IHC in 13 of 13 bovine fetuses from which C. fetus ssp. venerealis was isolated and in two of two ovine fetuses from which C. fetus ssp. fetus was isolated. The IHC stains were negative in tissues from seven bovine fetuses from which C. jejuni was isolated, one bovine fetus infected with C. fetus ssp. fetus, one bovine fetus infected with the unspeciated Campylobacter and two ovine fetuses infected with C. lari. In positive cases, the IHC stain most frequently identified bacteria in the lung and gastrointestinal tract. The C. fetus IHC procedure performed on formalin fixed tissues is a practical tool for the diagnosis of natural cases of ovine and bovine abortion caused by C. fetus.