Contribution to the diagnosis of Johne's disease in cattle. Comparative studies on the validity of Ziehl-Neelsen staining, faecal culture and a commercially available DNA-Probe test in detecting Mycobacterium paratuberculosis in faeces from cattle

In the present study, 132 selected faecal samples from clinically affected and subclinically infected cattle from dairy herds known to be affected by Johne's disease were investigated for the presence of Mycobacterium paratuberculosis using Ziehl-Neelsen staining, faecal culture and a commercially available DNA-Probe test. The sensitivity was 36.4% for Ziehl-Neelsen staining, 85.6% for faecal culture and 47.7% for the DNA-Probe test. Proving the presence of acid-fast bacteria in 49.3% of the samples from clinically affected cattle and 19.3% of those from subclinically infected cattle, Ziehl-Neelsen staining had the lowest detection rate of the three tests under investigation. Faecal culture showed the highest detection rate of M. paratuberculosis in samples from both clinically affected (84.0%) and subclinically infected (87.7%) animals. The DNA-Probe test showed a positive result in 68.0% of the samples from clinically affected cattle and 21.1% of those from subclinically infected cattle. Ziehl-Neelsen staining proved unreliable in diagnosing Johne's disease. Faecal culture was the most sensitive method for detecting M. paratuberculosis both in clinically affected and subclinically infected cattle. The sensitivity of a commercially available DNA-Probe test has to be enhanced to enable a quick and reliable diagnosis of Johne's disease.     

Persistent hematomas in Japanese black cattle with impaired platelet aggregation function and large granule eosinophils.

In Japanese black cattle with large and long-existing hematomas, platelets was impaired in collagen aggregation function in vitro. There was no statistically significant difference from control animals in the tests of PT (prothrombin time) and PTT (partial thromboplastin time) for extrinsic and intrinsic blood coagulation system. Aside from impaired collagen aggregation function, platelets in the hematoma cattle showed the similar aggregation patterns as the normal cattle, when ADP, serotonin (5-HT), thrombin, arachidonic acid, epinephrine and ristocetin were used as agents for inducing aggregation. Decreased aggregation function as well as impaired collagen-induced release response in platelets suggested the hematoma cattle to be of storage pool disease (SPD). The impaired platelet was postulated to be a main cause of the large and long-existing hematomas. All of the hematoma cattle with impaired platelet functions had the eosinophils in peripheral blood of which granules were fewer and larger than normal ones. These large eosinophil granules were peroxidase positive and periodic acid Schiff (PAS) staining negative as typical eosinophil granules.     

Contribution to the Diagnosis of Johne’s Disease in Cattle.Comparative Studies on the Validity of Ziehl-Neelsen Staining,Faecal Culture and a Commercially Available DNA-Probe® Testb in Detecting Mycobacterium paratuberculosis in Faeces from Cattle

In the present study, 132 selected faecal samples from clinically affected and subclinically infectedcattle from dairy herds known to be affected by Johne’s disease were investigated for the presence of Mycobacterium paratuberculosis using Ziehl-Neelsen staining, faecal culture and a commercially available DNAProbe ® test. The sensitivity was 36.4% for Ziehl-Neelsen staining, 85.6% for faecal culture and 47.7% for the DNA-Probe® test. Proving the presence of acid-fast bacteria in 49.3% of the samples from clinically affected cattle and 19.3% of those from subclinically infected cattle, Ziehl-Neelsen staining had the lowest detection rate of the three tests under investigation. Faecal culture showed the highest detection rate of M. paratuberculosis in samples from both clinically affected (84.0%) and subclinically infected (87.7%) animals. The DNA-Probe® test showed a positive result in 68.0% of the samples from clinically affected cattle and 21.1% of those from subclinically infected cattle. Ziehl-Neelsen staining proved unreliable in diagnosing Johne’s disease. Faecal culture was the most sensitive method for detecting M. paratuberculosis both in clinically affected and subclinically infected cattle. The sensitivity of a commercially available DNAProbe® test has to be enhanced to enable a quick and reliable diagnosis of Johne’s disease.     

Isolation of Babesia divergens from carrier cattle blood using in vitro culture

Babesia divergens, the main causative agent of bovine babesiosis in Western Europe, was isolated from naturally infected cattle. Ninety-six blood samples were examined by means of an in vitro culture technique in sheep erythrocytes: 19 of them were collected from animals in the acute phase of the disease with visible parasitemia on blood smears, while the 77 remaining animals showed no microscopically detectable parasites. B. divergens was cultured from the 19 first blood samples as well as from 31 samples collected from asymptomatic animals. The time period before parasites could be detected in the culture varied in the latter samples from 6 to 20 days. The effects of sampling condition (anticoagulant used) and storage length were tested. A good correlation was obtained between immunofluorescent antibody test and culture, with identical results (positive or negative) for 89.6% of the samples collected from asymptomatic animals. The sensitivity of the in vitro culture method was determined and was about 10 parasites/mL of whole blood from three independent experiments performed with three different isolates, confirming its suitability to detect and culture diverse B. divergens isolates from carrier cattle. The parasites could indeed be isolated 9 months after the acute babesiosis phase in the blood of naturally infected animals. The 50 isolates collected in this study were successfully subcultured, cryopreserved and resuscitated using the same culture medium. The in vitro isolation of B. divergens from asymptomatic carrier cattle was achieved and will allow the analysis of parasite diversity within cattle herds.     

A comparative study of an indirect fluorescent antibody test and an immunoperoxidase monolayer assay for the diagnosis of porcine proliferative enteropathy.

The currently used indirect fluorescent antibody test (IFAT) for the detection of antibodies against porcine proliferative enteropathy (PPE) was compared to an immunoperoxidase monolayer assay (IPMA). Serum samples used in this comparison were collected from 5-week-old pigs on day 0 (pre-experimental challenge) and on days 7, 14, 21, and 28 after oral inoculation with intestinal homogenate from pigs affected by PPE (28 challenged pigs) and sucrose phosphate glutamate solution (2 control pigs). All animals were euthanized 4 weeks after inoculation. Immunohistochemistry staining was applied to formalin-fixed, paraffin-embedded sections of ileum for the detection of Lawsonia intracellularis antigen. The serology results with each method agreed in all samples, except on days 0 and 7 in 1 control animal, which was positive by IPMA, but negative by IFAT. The percentage of agreement between IFAT and IPMA was 98.6%.     

A longitudinal study of Neospora caninum infection on three dairy farms in Brazil

Neospora caninum is an apicomplexan protozoan parasite that is one of the most important infectious causes of abortion in both dairy and beef cattle in many countries. The objectives of this longitudinal study were to determine the prevalence, rates of vertical and horizontal transmission of N. caninum and hazard for culling of N. caninum-seropositive animals in three Brazilian dairy herds. Blood samples from all animals were collected nine times at each of the three farms over a two-year period. Serum was tested for antibodies against N. caninum using the indirect fluorescent antibody test with a cutoff value of 1:100. The percentage of N. caninum-positive samples at each sampling time ranged at Farm I from 3.32% to 11.71%, at Farm II from 3.90% to 22.06% and at Farm III from 3.90% to 22.06%. The number of positive serological reactions varied in relation to the number of repeated samples taken from individual animals at each farm. In all herds, there was a high degree (P<0.05) of association between the N. caninum serological status of dams and daughters. The seropositive conversion rate was estimated as 0.37%, 3.00% and 6.94% per 100 cow-years at Farms I, II and III, respectively. The seronegative conversion rate was estimated as 31.58% and 11.11% per 100 cow-years at Farms I and III, respectively. In all herds, there was no difference (P>0.05) in the culling rate between the cattle that were seropositive cattle and seronegative for N. caninum infection. The results from this study confirm the importance of vertical transmission in the epidemiology of N. caninum. Although a few positive seroconversions indicated horizontal transmission, it does not appear to be the major route of infection for N. caninum.     

The prevalence of types I and II feline coronavirus infections in cats.

The types of feline coronaviruses that are prevalent throughout Japan were determined by competitive enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (MAb) to feline infectious peritonitis virus (FIPV) Type II and neutralizing test using Type II FIPV as challenge virus. A total of 1,079 cat serum samples were tested by indirect fluorescent antibody (IFA) assay for FIPV Type II antigen, all 42 sample from natural cases of FIP, 138 of 647 (21.3%) from cases with some chronic diseases and 57 of 390 (14.6%) from apparently non-diseased cases were positive. Of the 42 cases with FIP, 29 (69%) and 13 (31%) were found to have infection with FIPV Types I and II, respectively. Of the cases with chronic diseases, 111 (80.4%) were shown to have infection with FIPV or FECV Type I, while 14 (10.1%) with FIPV or FECV Type II. All of the 57 apparently non-diseased cases seemed to have been infected with FIPV or FECV Type I. These results indicated that feline coronavirus Type I is more high prevalent in Japan.     

Biochemical characterization of cartilage affected by osteochondritis dissecans in the humeral head of dogs

OBJECTIVE: To determine glycosaminoglycan (GAG) concentration and immunohistochemical staining characteristics of type-I, -II, and -X collagen from cartilage affected by osteochondritis dissecans (OCD) in dogs. ANIMALS: 31 dogs with OCD and 11 clinically normal purpose-bred dogs. PROCEDURE: Cartilage samples were evaluated microscopically, and GAG content was determined. Immunohistochemical staining was performed for type-I, -II, and -X collagen. Sections were subjectively evaluated for location and intensity of staining. RESULTS: Cartilage affected by OCD had a variety of pathologic changes and significantly lower GAG concentrations than did normal cartilage. Normal cartilage had no detectable type-I collagen. For dogs < 9 months of age, cartilage affected by OCD had significantly more type-I collagen but significantly less type-X collagen than did control cartilage. For dogs > 12 months of age, cartilage affected by OCD contained significantly more type-I collagen than did control cartilage. There was a significant negative correlation between immunoreactivity of type-I collagen and that of type-II and -X collagen. A significant positive correlation was found between immunoreactivity of type-II and -X collagen. CONCLUSIONS AND CLINICAL RELEVANCE: Cartilage affected by OCD contains less GAG, more type-I collagen, and less type-X collagen, compared with normal cartilage. A direct correlation between these changes and the etiopathogenesis of OCD was not established.     

Molecular evidence of bovine herpesvirus 1 and 5 in cattle with suspected rabies in Rio Grande do Sul state,Brazil

Rabies and herpetic encephalitis are the main viral infections in bovines with neurological symptoms. Bovine rabies has a high prevalence in Central and South America, while bovine encephalitis associated with herpesvirus is especially important in South America. Viral isolation is the classical way to confirm herpesvirus infection, but molecular evidence of the presence of the virus in affected animals is gaining importance in the diagnosis of the disease in the laboratory. This study investigated the presence of herpesvirus type 1 and 5 (BoHV-1 and BoHV-5) in 182 encephalon of rabies-suspected cattle in Rio Grande do Sul state (RS), Brazil using multiplex real-time polymerase chain reaction (mRT-PCR). The rabies virus was investigated by direct fluorescent antibody assay and intracerebral suckling mouse inoculation. The genomes of BoHV-1 and BoHV-5 were detected in 17% of samples. BoHV-5 and BoHV-1 were detected in 100% and 19% of BoHV positive samples, respectively, indicating the circulation of the pathogens in cattle herds in RS. The high Ct values and the absence of isolation suggest viral latency. Coinfection of herpesvirus and the rabies virus was detected in 28% of samples, although no significant association between pathogens was observed. Rabies was detected in 57.7% of suspected samples, confirming the importance of the disease in the state. Concerning the method by which samples were conserved, no significant difference was observed between the number of positive results in frozen and refrigerated samples.     

Seroprevalence of Neospora caninum infection in dairy and beef cattle in Paraguay

Seroprevalence of Neospora caninum in 879 beef and dairy cattle in different locations of Paraguay was determined by an ELISA. In the survey, 262 (29.8%) cattle were positive to N. caninum, and animals with anti-Neospora antibody titre were observed in all the locations tested in the country. Serum samples taken from a herd that exhibited persistent abortion had the highest percentage of animals being positive to the parasite (17/30, 56.7%). In the same herd, abortion was significantly more likely in animals with high anti-Neospora antibody titre. Immunoblot analysis demonstrated that the banding pattern from positive Paraguayan cattle was similar to that seen with the positive control sample. In conclusion, N. caninum infection is present among Paraguayan beef and dairy cattle, and it may be an important cause of bovine abortion in Paraguay.     

Blood flow parameters associated with udder oedema in Jersey cattle affected with rectovaginal constriction

Udder oedema was studied in eight Jersey cows with rectovaginal constriction (RVC) and three normal Jersey cows. A complete physical examination was performed at least two weeks before parturition, at parturition and two weeks after parturition. Jersey cows with RVC developed udder oedema as early as 14 days before parturition, and condition persisted for three to 10 days following parturition. Mammary blood flow and cranial superficial epigastric venous blood pressure were measured in three Jersey cows with RVC with udder oedema and three normal Jersey cows. The former with udder oedema had a significant decrease in mammary blood flow at parturition compared to the normal Jersey cows (P less than 0.05); there was no significant difference in mammary blood flow between Jersey cows with RVC and normal Jersey cows two weeks before parturition (P greater than 0.05). A significant mean increase in cranial superficial epigastric venous blood pressure occurred at parturition when compared to two weeks before parturition in Jersey cows with RVC (P less than 0.05), but normal Jersey cows had no significant increase in cranial superficial epigastric vein pressure during these two periods of measurement (P greater than 0.05). There was no significant increase in jugular vein blood pressure during the two periods of measurement in Jersey cows with RVC or normal Jersey cows (P greater than 0.05). There was a significant negative correlation between mammary blood flow and cranial superficial epigastric pressure at parturition (r = 0.733, P less than 0.05).     

Failure to demonstrate involvement of antibodies to Acinetobacter calcoaceticus in transmissible spongiform encephalopathies of animals

Acinetobacter calcoaceticus, a soil microbe, contains molecular sequences which resemble those found in neurofilaments of the brain tissue. It was hypothesized that if cattle ingest large amounts of feedstuff containing A. calcoaceticus, they may develop an autoimmune reaction, with consequences of pathological changes associated with transmissible spongiform encephalopathies (TSEs). The hypothesis was tested using a small number of serum samples collected from cattle and it was found that affected individuals had elevated serum antibody levels to this organism. If this finding was substantiated, it would provide a possible means of diagnosing TSEs in vivo. In the present communication, a larger number of cattle, elk and sheep with or without TSEs were tested using A. calcoaceticus whole cell and lipopolysaccharide antigens as well as myelin basic protein (MBP). It was found that antibody levels in normal and affected animals overlapped considerably, thus casting doubt on the usefulness of these antigens as diagnostic tools for TSEs and on the hypothesis of A. calcoaceticus being a cause of TSEs.     

Evaluation of an indirect fluorescent antibody test, enzyme-linked immunosorbent assay and quantification of immunoglobulins in the diagnosis of bovine trypanosomiasis.

An indirect fluorescent antibody test (IFAT), a microscale version of the enzyme-linked immunosorbent assay (microELISA) and determination of IgM levels in serum were assessed for their comparative diagnostic value in the detection of bovine trypanosomiasis. Serum samples from drug-treated N'dama cattle and untreated N'dama and Zebu cattle from Liberia were examined for the presence fo antibodies to trypanosomes. In the untreated Zebu cattle, infections with T. vivax predominated and the prevalence of infection was higher than that found in untreated N'damas in which infections with T. congolense predominated. The proportion of animals which showed serological evidence of trypanosomiasis in the untreated Zebus was slightly higher than that found in the untreated N'damas. The prevalence of infection was low in N'dama cattle which had been treated with diminazene aceturate and homidium chloride but 50% of the animals showed serological evidence of trypanosomiasis. More serologically positive animals were detected by microELISA than IFAT, but both tests were equally sensitive in detecting antibodies in cattle in which trypanosomes were demonstrated by examination of peripheral blood. With both IFAT and microELISA it was necessary to carry out tests using antigens prepared from T. brucei, T. vivax and T. congolense in order to detect all serologically positive animals. Increases in serum IgM occurred in both N'dama and Zebu cattle but the levels were raised in only approximately half of the known infected animals. Overall, more animals gave positive reactions with IFAT and microELISA than showed raised IgM levels.     

An antigen capture test for the detection of cattle viremic with bovine viral diarrhoea virus--a comparison with BVD virus isolation from buffy coat cells in bovine kidney cells.

An antigen capture enzyme immunoassay (EIA) for the detection of bovine viral diarrhoea (BVD) viral antigen in peripheral blood lymphocytes of cattle was used for the screening of 241 animals. The test used a monoclonal antibody directed against a conserved antigenic domain of a nonstructural protein (p125/p80) of pestiviruses for antigen capture. Bound antigen was detected with a pestivirus-specific polyclonal peroxidase conjugate. In parallel the samples were analysed by routine virus isolation procedures based on cell culture. Virus isolation and antigen capture EIA were positive in 54 cases. The latter test scored one additional sample.     

Detection of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> in tissue samples of cattle and buffaloes

Tissue samples were collected at random from cattle (Bos taurus) and buffalo (Bubalus bubalis) from an abattoir of the district of Lahore and were analyzed for the presence of Mycobacterium avium subsp. paratuberculosis and Mycobacterium bovis through acid-fast staining and polymerase chain reaction (PCR). Body condition of animals and diarrhea were recorded. Most of the animals were emaciated. Diarrhea was noticed in 15.6% of buffaloes and 19.2% of cattle. Intestinal pathology was observed in 29% of buffaloes and 32.8% of cattle. Number of mesenteric lymph node (MLN) showing gross lesions was a bit higher (35.6%) in cattle than buffalo (31.2%). Acid-fast staining of tissue scraping smears revealed the presence of acid-fast bacilli (AFB) in 17.4% intestinal and 16.4% MLN tissue samples in buffalo, while in cattle 19.2% intestinal and 17.8% MLN were found positive for AFB. In buffaloes, PCR confirmed 12.8% intestinal and 12.4% MLN positive samples for M. avium subsp. paratuberculosis. However, in cattle, PCR analysis demonstrated 14.2% positive results for M. avium subsp. paratuberculosis in both MLN and intestinal tissue samples. PCR also confirmed M. bovis in 5.8% of cattle and 5% of buffalo MLN and intestinal tissues. PCR positive tissue samples for M. avium subsp. paratuberculosis were from those animals which were emaciated, having diarrhea, and severe gross lesions. AFB were also detected in tissue scraping smears of these animals. It is concluded that infection by various mycobacterium species can be differentiated by PCR, which is not possible by acid-fast staining technique.     

Detection of antibodies against the rabies virus in Korean raccoon dogs (Nyctereutes procyonoides koreensis)

Rabies has been reported in dogs, cattle, and raccoon dogs in Korea, especially in the provinces of Gyeonggi and Gangwon. The aim of this study was to investigate the seroprevalence of the rabies in wild Korean raccoon dogs by using 50 blood samples collected in Gyeonggi and Gangwon provinces. Antibodies against rabies were measured by using a fluorescent antibody virus neutralization test. Of the 50 blood samples tested, 20 raccoon dogs (40%) tested positive. Seropositive animals were distributed in Paju, Yangpyeong, Goyang, Yangju, Yanggu, and Hoengseong cities.     

The occurrence of dermatosparaxis in a commercial Drakensberger cattle herd in South Africa

Dermatosparaxis is a heritable collagen dysplasia causing skin extensibility and fragility. In Belgian Blue cattle this mutation has been described as a 3 base pair (bp) change followed by a 17bp deletion in the gene coding for procollagen 1 N-Proteinase (pNPI). An outbreak in a commercial Drakensberger herd in South Africa followed the introduction in late 2000 of a 3-year-old bull that developed skin lesions in 2001 and was culled in 2002. Some of his offspring were similarly affected, 1 of which was kept as a breeding bull after his sire's death. Two affected calves were referred to the Onderstepoort Veterinary Academic Hospital in October 2005. Detailed examination revealed only skin abnormalities limited to the lateral extremities of the thorax, abdomen and pelvis, viz. either acute lacerations of varying sizes, slow healing defects or thin scars in chronic cases. During a subsequent farm visit, 13 animals with similar wounds were seen in the herd of 146 animals. Electron microscopic examination of skin biopsies revealed haphazard arrangement and loose packing of dermal collagen fibrils within collagen fibres. The fibrils showed size variation and slightly irregular outlines on cross-section, consistent with mild dermatosparaxis. DNA samples of affected calves were analysed using primers designed to amplify the region of the pNPI gene that contained the mutation described in Belgian Blue cattle, but this mutation could not be demonstrated in any of the animals tested. It is concluded that a form of dermatosparaxis with a different gene mutation from that described in Belgian Blue cattle exists in Drakensberger cattle in South Africa. This possibly also explains the milder and more delayed clinical signs and the milder dermal collagen ultrastructural abnormalities.     

Collagen Types in Healing Rabbit Tendons: A Biochemical Assessment

Eight repaired and eight contralateral control superficial digital flexor tendons were collected from rabbits (2–6 kg) 1, 4, and 8 weeks after surgery. Acid soluble and pepsin solubilized collagen fractions were prepared and separated using SDS-PAGE to determine if healing tendons contain detectable amounts of Type III collagen. No biochemical differences between normal and healing tendon collagen content could be detected. Type I collagen was the predominant collagen species found in all 88 samples examined. Alpha chains indicative of Type III collagen were detected in control and experimental tendons of five animals. Alpha chains suggestive of Type IV collagen were detected in control and experimental tendons of eight rabbits.     

Prevalence of Coxiella burnetti infection in wild and farmed ungulates

The aim of this study was to evaluate by serology and PCR analyses the prevalence of Coxiella burnetti infection in ungulates in Spain. Sera were collected from red deer (Cervus elaphus; n=116), roe deer (Capreolus capreolus; n=39), fallow deer (Dama dama; n=13) and cattle (n=79). Sera were tested for anti-C. burnetii antibody detection by means of an immunofluorescence antibody assay (IFA) and C. burnetii DNA was amplified by PCR in samples from ungulates that had antibodies to phase II antigens. Twenty-nine, 15 and 39 percent of the red deer, roe deer and cattle had antibodies against C. burnetii, respectively. None of the fallow deer sera tested positive. Seroprevalence was statistically higher in farmed than in wild red deer and higher in northern than in southern populations, whereas an inverse pattern was observed for the roe deer. Most of the seropositive animals had only anti-C. burnetii phase II antibodies, thus showing the acute nature of infections in the sampled ungulates. These results show that C. burnetii circulates in wild ungulates in Spain and suggest that they can act as pathogen reservoirs for both domestic animals and humans.     

Response of cattle persistently infected with bovine virus diarrhoea virus to bovine leukosis virus

Six cattle persistently infected with bovine virus diarrhoea virus (BVDV) and seronegative, and two control, virus negative seropositive cattle were inoculated with lymphocytes infected with bovine leukosis virus (BLV). The two controls produced a normal immune response to BLV, developing antibodies at four and five weeks after inoculation. Two of the six cattle persistently infected with BVDV developed a strong antibody response by six weeks after inoculation with BLV. Four developed a depressed response to BLV, characterised in three by a 'hooking' reaction in the immunodiffusion test which persisted in successive bleedings but was interspersed occasionally by a weak positive reaction. In one of these animals, a series of 'hooking' reactions was followed by a number of negative results. The fourth animal remained serologically negative until 16 weeks after inoculation when a 'hooking' reaction was observed followed by a series of negative results. BLV was isolated from all the cattle persistently infected with BVDV at 42 or 58 weeks after inoculation regardless of whether the serum samples gave negative, 'hooking', weak positive or positive reactions in the immunodiffusion test. BLV was consistently isolated from the nasal secretions of a steer which was BVDV negative but seropositive. The possibility of decreased immune responsiveness to BLV in animals persistently infected with BVDV should be considered when formulating regulations governing the testing of animals for freedom from BLV.