Lymphatic fluid for the detection of Mycobacterium avium subsp. paratuberculosis in cows by PCR, compared to fecal sampling and detection of antibodies in blood and milk.

Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), can cause considerable economic losses in affected herds. Early diagnosis of JD is hampered by the chronic nature of the disease with a slow subclincal progression. The aim of the present study was to challenge the hypothesis that lymphatic fluid is of diagnostic value in the early stages of the disease. Lymphatic fluid from 122 animals was collected and tested for MAP by nested PCR for IS900 and compared to the results of testing for MAP in feces (culture), blood and milk (ELISA) in 110 of these samples. MAP was detected by PCR in 27.1% of the lymph samples. Agreement between the tests was poor: 6.9% of the lymph positive cows were also positive in all other tests applied, and 69.0% had negative results in fecal culture, blood and milk ELISA. Resampling of 25 cows after 8 to 12 and 16 to 20 months revealed 20.0% lymph positive animals at the first, 5.5% at the second and 27.8% at the third sampling, respectively. Only one cow showed positive lymph-PCR results at more than one sampling date. Lymph-positive cows had a 7.2 times greater likelihood of being culled within 8 to 12 months after sampling, compared to negative cows, mainly due to other health issues than JD. It can be concluded, that lymphatic fluid might be promising for the detection of early MAP-infection in cows, but further studies to elucidate the potential of this diagnostic approach are needed.

The collection of lymphatic fluid from the bovine udder and its use for the detection of Mycobacterium avium subsp. paratuberculosis in the cow.

The objective of the current study was to evaluate the feasibility of lymph collection from the bovine udder and to investigate if the lymphatic fluid might be of diagnostic value in cows infected with Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of paratuberculosis. Lymph fluid collection was attempted from 58 cows, and the reactions of the cows as well as the level of difficulty of the procedure were recorded in 56 animals. Lymph samples (51 in total) were tested for the presence of MAP by nested polymerase chain reaction. Collection of the lymphatic fluid caused no or mild signs of discomfort in 94.6% of the cows; in 51.8% of cows, lymphatic fluid was attained on the first attempt, while sample collection was unsuccessful in 12.1%. Mycobacterium avium subsp. paratuberculosis was detected in 43.1% of all lymph samples. The bacterium was present in 66.7% of cows with clinical Johne's disease, in 42.8% of asymptomatic cows with a positive or suspicious enzyme-linked immunosorbent assay (ELISA) result in blood, and in 38.7% of cows with a negative ELISA result in blood. The present study shows that the procedure was well tolerated by most cows and can easily be performed on farm. The current report of the isolation of MAP from lymph fluid suggests that the present approach could be used for the early detection of Johne's disease in cattle.

Exposure of young dairy cattle to Mycobacterium avium subsp. paratuberculosis (MAP) through intensive grazing of contaminated pastures in a herd positive for Johne's disease.

This study investigated the susceptibility of 1- to 2-year-old cattle to Mycobacterium avium subsp. paratuberculosis (MAP) on pasture previously grazed by infected cattle. The exposure of yearling cattle to pastures contaminated with MAP resulted in infection with MAP, showing that age resistance to infection can be overcome by pressure of infection.

Candidate gene polymorphisms (BoIFNG, TLR4, SLC11A1) as risk factors for paratuberculosis infection in cattle.

Paratuberculosis (Johne's disease) imposes a significant problem to the world dairy and beef industries and today is considered a potential zoonosis. The disease is caused by Mycobacterium avium subsp. paratuberculosis and is characterized by progressive weight loss and profuse diarrhoea. Susceptibility to infection is suspected to have a genetic component, and moderated values for heritability of infection have been reported. Interferon gamma is an inducible cytokine with a crucial role in the innate host response to intracellular bacteria. Toll-like receptors are trans-membrane structures responsible for coordination of innate and adaptive immune responses. The solute carrier family 11 member 1 (SLC11A1, formerly NRAMP1) gene plays an important role in innate immunity, preventing bacterial growth in macrophages during the initial stages of infection. The objective of this candidate gene case-control study was to characterize the distribution of polymorphisms in three candidate genes related to the immune function; interferon gamma (BoIFNG), toll-like receptor 4 (TLR4), and SLC11A1 genes and to test their role as potential risk factors for paratuberculosis infection in cattle. The statistical analysis demonstrated significant differences in allelic frequencies between cases and controls for BoIFNG-SNP(1)2781 and SLC11A1 microsatellites, indicating a significant association between infection and variant alleles. In the analysis of genotypes, a significant association was also found between infection status and BoIFNG-SNP(1)2781 and SLC11A1-275-279-281 microsatellites. However, when variables such as breed and age were included in the multivariate logistic regression analysis, a tendency toward statistical significance for the effect of polymorphisms in the odds of infection was only found for alleles SLC11A1-275 and 279.

Association between CARD15/NOD2 gene polymorphisms and paratuberculosis infection in cattle.

Paratuberculosis represents a major problem in farmed ruminants and at the present is considered a potential zoonosis. The disease is caused by Mycobacterium avium subsp. paratuberculosis, and susceptibility to infection is suspected to have a genetic component. Caspase recruitment domain 15 (CARD15) gene encodes for a cytosolic protein implicated in bacterial recognition during innate immunity. Crohn's disease (CD) is an idiopathic inflammatory bowel disease in humans comparable in many features to bovine paratuberculosis involving an abnormal mucosal immune response. The association between mutations in the CARD15 gene and increased risk of Crohn's disease has been described. The objective of this candidate gene case-control study was to characterize the distribution of three polymorphisms in the bovine CARD15 gene and test their association with paratuberculosis infection in cattle. Three previously reported single nucleotide polymorphisms (E2[-32] intron 1; 2197/C733R and 3020/Q1007L) were screened for the study population (431 adult cows). The statistical analysis resulted in significant differences in allelic frequencies between cases and controls for SNP2197/C733R (P<0.001), indicating a significant association between infection and variant allele. In the analysis of genotypes, a significant association was also found between SNP2197/C733R and infection status (P<0.0001); cows with the heterozygous genotype were 3.35 times more likely to be infected than cows with the reference genotype (P=0.01). Results suggest a role for CARD15 gene in the susceptibility of cattle to paratuberculosis infection. These data contribute to the understanding of paratuberculosis, suggest new similarities with Crohn's disease and provide new information for the control of bovine paratuberculosis.

Attachment of Mycobacterium avium subspecies paratuberculosis to bovine intestinal organ cultures: method development and strain differences.

Mycobacterium avium spp. paratuberculosis (MAP) causes chronic granulomatous inflammation of the intestinal tract in many species of animals, but the mechanisms of disease are poorly understood. Attachment of bacteria to epithelial cells is a critical step in pathogenesis of many mucosal diseases. The goal of these studies was to develop an in vitro method to study attachment of MAP to bovine intestinal epithelial cells. Short-term, bovine intestinal organ cultures were used to show a significant difference in the ability of radiolabelled MAP strains to attach to intestinal epithelium. We found significant differences in the ability of different strains of MAP to attach, but there were no differences in attachment among different regions of the intestinal tract. Examination of acid fast stained tissue sections of organ cultures demonstrated that organisms were located adjacent to mucosal epithelium or within goblet cells. Coating of the organisms with fibronectin, which has been shown to be involved in attachment of many mycobacteria, including MAP, affected the attachment of the MAP strains in different ways, but did not affect the overall attachment of the organisms to different regions of the gastrointestinal tract. This organ culture method should also prove useful for defining the molecular mechanisms of attachment and interactions of MAP with intestinal epithelium.

Generalized osteosclerosis in a cat.

A 12-year-old, neutered male, domestic long-hair cat was evaluated for a 6-month history of inspiratory stertor and epiphora. In computed tomography of the skull and pelvis, and radiographs of the thorax, right femur and stifle there was generalized osteosclerosis, with obliteration of the nasal turbinates and nasolacrimal duct obstruction. The cat also had a large fibrosarcoma involving the right pelvic limb. Osteosclerosis is a rare disorder that is poorly understood but has been described in several species. Various manifestations, potential causes, and radiologic findings of osteosclerosis are discussed.

West Nile virus encephalomyelitis in horses: 46 cases (2001).

OBJECTIVE: To determine signalment, clinical findings, results of diagnostic testing, outcome, and postmortem findings in horses with West Nile virus (WNV) encephalomyelitis. DESIGN: Retrospective study. ANIMALS: 46 horses with WNV encephalomyelitis. PROCEDURE: Clinical data were extracted from medical records of affected horses. RESULTS: On the basis of clinical signs and results of serologic testing, WNV encephalomyelitis was diagnosed in 46 of 56 horses with CNS signs. Significantly more males than females were affected. Increased rectal temperature, weakness or ataxia, and muscle fasciculations were the most common clinical signs. Paresis was more common than ataxia, although both could be asymmetrical and multifocal. Supportive treatment included anti-inflammatory medications, fluids, antimicrobials, and slinging of recumbent horses. Results of the IgM capture ELISA and the plaque reduction neutralization test provided a diagnosis in 43 horses, and only results of the plaque reduction neutralization test were positive in 3 horses. Mortality rate was 30%, and 71% of recumbent horses were euthanatized. One horse that had received 2 vaccinations for WNV developed the disease and was euthanatized. Follow-up communications with 19 owners revealed that most horses had residual deficits at 1 month after release from the hospital; abnormalities were resolved in all but 2 horses by 12 months after release. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings were similar to those of previous WNV outbreaks in horses but provided additional clinical details from monitored hospitalized horses. Diagnostic testing is essential to diagnosis, treatment is supportive, and recovery rate of discharged ambulatory horses is < 100%.

Equine cardiovascular pathology: an overview.

The few data collections that evaluate the involvement of organ systems in horse diseases are in agreement that the locomotor, gastrointestinal and nervous systems are the sites of primary disease in the vast majority of sick horses. When compared with diseases of these organ systems, equine cardiovascular diseases occur infrequently. The most detailed and comprehensive survey of equine cardiac pathology was reported in 1972 by Else and Holmes, who summarized the gross and microscopic cardiac findings from 1500 abattoir horses. This paper reviews the pathology of the cardiovascular diseases typically encountered in horses. Most of the pathological examples are from the files of the Veterinary Medical Teaching Hospital at the University of Florida, encompassing 24 years (1978-2002) and some 6000 equine necropsy cases. Preceding the specific topics are principles of the anatomy and function of the normal equine heart. Pathological entities include equine congenital cardiovascular diseases, acquired diseases of the pericardium, myocardium, endocardium and valves, cardiac neoplasms, and common equine vascular diseases and vascular neoplasms. Extensive use is made of photographs to illustrate the features of individual case examples.

Causes of mortality of free-ranging Florida panthers.

The Florida panther (Puma concolor coryi) is one of the most endangered mammals, with the entire population estimated to consist of only 30-50 adult animals. Between 1978 and 1999, 73 free-ranging Florida panther carcasses were submitted for postmortem evaluation, of which 47 (64%) were radiocollared and 26 (36%) were uncollared cats. Overall, mortality of panthers > 6-mo-old was due to vehicular trauma in 25 (35%), intraspecific aggression in 19 (26%), illegal kill in seven (10%), research activities in two (3%), infectious diseases in two (3%), esophageal tear in one (1%), pleuritis in one (1%), pyothorax in one (1%), aortic aneurysm in one (1%), atrial septal defect in one (1%), and causes of death were undetermined in 13 (18%) due to autolysis. Of the 25 panthers that were killed by vehicular trauma, 20 (80%) died between October and April. This coincides with increased number of winter visitors to south Florida. Among radiocollared panthers, intraspecific aggression was the primary cause of mortality for 19 (41%) dead cats. Of these cats, 16 (84%) were males and 14 (88%) were either less than 3 or more than 8-yr-old. These animals were probably fighting to establish or retain territory. Among the 26 uncollared panthers, vehicular trauma was the primary cause of mortality and was responsible for 16 (62%) deaths. This study documents the causes of mortality and the age, sex, and seasonal mortality trends for both radiocollared and uncollared free-ranging endangered Florida panthers over a 21-yr-period.