Neuroanatomical distribution of disease-associated prion protein in cases of bovine spongiform encephalopathy detected by fallen stock surveillance in Japan

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder of cattle characterized by accumulation of the disease-associated prion protein (PrP(Sc)) in the central nervous system (CNS). The immunohistochemical patterns and distribution of PrP(Sc) were investigated in the CNS, brains, and spinal cords of 7 naturally occurring BSE cases confirmed by the fallen stock surveillance program in Japan. No animals showed characteristic clinical signs of the disease. Coronal slices of 14 different brain areas in each case were immunohistochemically analyzed using an anti-prion protein antibody. Immunolabeled PrP(Sc) deposition was widely observed throughout each brain and spinal cord. Intense PrP(Sc) deposition was greater in the thalamus, brainstem, and spinal cord of the gray matter than in the neocortices. The topographical distribution pattern and severity of PrP(Sc) accumulation were mapped and plotted as immunohistochemical profiles of the different brain areas along the caudal-rostral axis of the brain. The distribution pattern and severity of the immunolabeled PrP(Sc) in the CNS were almost the same among the 7 cases analyzed, suggesting that the naturally occurring cases in this study were at the preclinical stage of the disease. Immunohistochemical mapping of the PrP(Sc) deposits will be used to clarify the different stages of BSE in cattle.     

Isolation of Mycoplasma bovoculi from cases of infectious bovine keratoconjunctivitis.

Five outbreaks of infectious bovine keratoconjunctivitis were examined for bacteria and mycoplasmas. Mycoplasma bovoculi was demonstrated in four of the five outbreaks. Other mycoplasmatales were represented by Ureaplasma in one sample. Moraxella bovis and Neisseria ovis were found in all the outbreaks, the former being present in the vast majority of the animals. Transmission experiments with Mycoplasma bovoculi and Moraxella bovis in combination were carried out on four young, colostrumdeprived calves. Mycoplasma bovoculi appeared to have an enhancing effect on the pathogenicity of Moraxella bovis.     

Coxiella burnetii infection is associated with placentitis in cases of bovine abortion.

A positive score on a modified acid-fast (MAF)-stained smear test of fresh placenta was used to identify a group of bovine abortion submissions believed to be infected with Coxiella burnetii. Immunohistochemical (IHC) testing for Coxiella and Chlamydia antigens was performed on 14 MAF smear-positive cases as well as 29 MAF smear-negative cases received during the study period. Nine MAF smear-positive cases as well as 1 MAF smear-negative case were Coxiella-positive via the IHC test. No placentas were positive for Chlamydia antigen. Various histopathologic features were categorized for all placentas and the presence or absence of selected risk categories was also graded for each case. The results between Coxiella IHC-positive cases and Coxiella IHC-negative/MAF-negative cases were compared using Fisher's exact test (P value at 95% confidence). Significant associations were found between Coxiella IHC-positive cases and the presence of placental inflammation (P = 0.0027), placental necrosis (P = 0.012), fetal pneumonia (P = 0.0152), and the visibility of Coxiella-like organisms within trophoblasts on hematoxylin and eosin-stained sections (P < 0.0001). Histopathologic features of Coxiella IHC-positive placentas included infiltration of the chorionic stroma by mononuclear cells, necrosis of chorionic trophoblasts, and focal exudation of fibrin and neutrophils. The results indicate that MAF smears are a good screening tool for the presence of Coxiella in placentas from bovine abortion cases and that the detection of this pathogen in aborted placentas via traditional staining or IHC methods is usually associated with placentitis.     

Molecular subtyping of Staphylococcus aureus isolates from cases of bovine mastitis in Brazil.

Sixty-six isolates of Staphylococcus aureus obtained from milk samples of dairy cows suffering from subclinical mastitis in southern Brazil were analysed by five different molecular typing methods. These included the analysis of plasmid profiles, the analysis of coagulase (coa) gene polymorphisms by PCR amplification of the 3' terminal region of the coa gene, the PCR-based detection of polymorphisms in the X region of the protein A gene (spa), the PCR-directed analysis of variations in the spacer region between 16S and 23S rRNA, and the comparison of pulsed-field gel electrophoretically separated genomic SmaI fragment patterns. The molecular typing methods were supplemented with the biochemical characterization of the isolates and the determination of their in-vitro susceptibility to 14 different antibiotics. All genotypic and phenotypic typing methods were analyzed for their ability to discriminate between the isolates. Macrorestriction analysis proved to be the most discriminatory single method (D = 0.96) followed by rRNA spacer typing (D = 0.85), coa PCR (D = 0.82), and spa PCR (D = 0.80).     

Endometritis in postparturient cattle associated with bovine herpesvirus-4 infection: 15 cases.

Suppurative, ulcerative endometritis associated with bovine herpesvirus-4 (BHV-4) infection was identified in 15 postparturient dairy cows from 5 separate dairies. Characteristic eosinophilic to amphophilic intranuclear viral inclusion bodies were identified within degenerate endometrial lining epithelium and endothelial cells. Bovine herpesvirus-4 was confirmed as the etiology by a combination of fluorescent antibody assays, viral isolation, heminested PCR, ultrastructural examination of the uterus and inoculated tissue culture cells, and negative-stain electron microscopy of tissue culture supernatant. Viral particles measuring 70-95 nm were demonstrated in uterine epithelial and endothelial cells by electron microscopy. Bacteria including Arcanobacterium pyogenes, Escherichia coli, and an alpha-Streptococcus isolate were isolated from all uteri. Bovine herpesvirus-4-associated endometritis has been previously reported in sporadic cases in Europe but has not been previously reported in the United States. Endometritis associated with BHV-4 appears to be an emerging syndrome in Georgia dairy herds.     

Identification and characterization of two bovine spongiform encephalopathy cases diagnosed in the United States.

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy of cattle, first detected in 1986 in the United Kingdom and subsequently in other countries. It is the most likely cause of variant Creutzfeldt-Jakob disease (vCJD) in humans, but the origin of BSE has not been elucidated so far. This report describes the identification and characterization of two cases of BSE diagnosed in the United States. Case 1 (December 2003) exhibited spongiform changes in the obex area of the brainstem and the presence of the abnormal form of the prion protein, PrP(Sc), in the same brain area, by immunohistochemistry (IHC) and Western blot analysis. Initial suspect diagnosis of BSE for case 2 (November 2004) was made by a rapid ELISA-based BSE test. Case 2 did not exhibit unambiguous spongiform changes in the obex area, but PrP(Sc) was detected by IHC and enrichment Western blot analysis in the obex. Using Western blot analysis, PrP(Sc) from case 1 showed molecular features similar to typical BSE isolates, whereas PrP(Sc) from case 2 revealed an unusual molecular PrP(Sc) pattern: molecular mass of the unglycosylated and monoglycosylated isoform was higher than that of typical BSE isolates and case 2 was strongly labeled with antibody P4, which is consistent with a higher molecular mass. Sequencing of the prion protein gene of both BSE-positive animals revealed that the sequences of both animals were within [corrected] the range of the prion protein gene sequence diversity previously reported for cattle.     

Modelling the expected numbers of preclinical and clinical cases of bovine spongiform encephalopathy in Switzerland.

The objective of this study was to model the expected numbers of cattle incubating bovine spongiform encephalopathy (BSE) and the numbers of clinical cases of BSE in the Swiss cattle population between 1984 and 2005. The results were compared with the observed number of clinical BSE cases and with the results of a culling and testing scheme on herdmates of cattle with BSE. The age distribution of the Swiss cattle population, the age-at-death distribution of the first 235 BSE cases and exposure information were used to calculate the expected number of infected cattle in each birth cohort and the resulting numbers of clinical cases and survivors incubating the disease for each year. The model which did not assume any under-reporting of cases fitted the observed epidemic curve of clinical cases reasonably well, and predicted that the Swiss BSE epidemic would come to an end between 2003 and 2005. The age of survivors incubating BSE is increasing. The higher than expected incidence of subclinical cases observed in animals from the culling scheme is most probably the result of the heterogeneous distribution of infected animals and affected herds in the population. The results of the model need to be taken into account when designing surveillance and testing schemes for BSE.     

An immunodiffusion test for detection of bovine viral diarrhea virus antibodies in bovine serum.

An immunodiffusion test (IDT) was developed for detecting bovine viral diarrhea virus antibodies in bovine serum. The antigen utilized in the IDT was prepared from bovine viral diarrhea virus-infected monolayer cultures. Results of the IDT were obtained within 48 hours and correlated with the virus-neutralization test.     

Microimmunodiffusion test for detection of antibodies to infectious bovine rhinotracheitis virus in bovine serum.

A microimmunodiffusion test (MIDT) specific for detection of antibodies to infectious bovine rhinotracheitis (IBR) in bovine serum has been developed. The antigen used in the MIDT was prepared from IBR virus-infected Madin-Darby bovine kidney cells grown in tissue culture. The antigen was stable, and relatively high yields were obtained readily. Results of the MIDT were obtained within 48 hours and agreed with those of the serum-neutralization test.