Bovine viral diarrhoea virus (BVDV) has emerged as one of the economically important pathogens in cattle populations with a worldwide distribution and causing a complex of disease syndromes. It is a single-stranded RNA virus of the genus Pestivirus in the family Flaviviridae. Two genotypes (1 and 2) of BVDV exist and can be distinguished on the basis of the 5' non-coding region (5' NCR) of the genome using real-time PCR. This technique is more sensitive, specific, less time consuming and has reduced risks of cross contamination of samples compared to a conventional PCR. Limited information exists on BVDV genetic subtypes in South Africa. The aim of this study was to determine the genotypes of BVDV currently circulating in South African feedlots. A total of 279 specimens (219 tissue samples, 59 trans-tracheal aspirates and one blood sample) were collected from dead and living cattle. Pooled homogenates from the same animals were prepared and total RNA was extracted from 200 μl of the homogenates using the RNeasy Mini Kit (Qiagen) as described by the manufacturer. A screening test was performed on the pooled samples and positive pools were investigated individually. The Cador BVDV Type 1/2 RT-PCR Kit (Qiagen, Hilden, Germany) was used for the real-time PCR assay. The PCR was performed on a Lightcycler® V2 (Roche Diagnostics, Mannheim, Germany) real-time PCR machine and the amplified products were detected via fluorescent dyes. The results were read at 530 and 640 nm for BVDV 1 and 2, respectively. Bovine viral diarrhoea virus was detected in a total of 103 samples that included 91 tissue samples, one blood sample and 11 trans-tracheal aspirates. Eighty five of the strains were genotype 1 strains and 18 were genotype 2. These results represent the first documented evidence for the presence of BVDV genotype 2 in South African cattle. / Dissertation (MSc)--University of Pretoria, 2010. / Veterinary Tropical Diseases / unrestricted

Ticks and tick borne-diseases (TBD) are major challenges to cattle production among smallholder farmers in the semiarid areas of South Africa. Nguni cattle have been reported to be resistant to ticks and TBD, however, the mechanisms responsible for the trait are not fully understood. The broad objective of this study was to determine the mechanisms of resistance to ticks in Nguni cattle reared in the semiarid areas of South Africa. Tick infestation levels, body condition scores (BCS), packed cell volumes (PCV) and the molecular prevalence of A. marginale were determined in Nguni (n = 70) and local crossbred (n = 79) cattle reared in the semiarid areas of South Africa. Relationships among skin thickness, hair length, coat score and tick counts were assessed in seven to nine month old Nguni (n = 12) and Bonsmara (n = 12) heifers. As a follow up, cutaneous hypersensitivity responses to unfed larval extracts (ULE) of the ticks Rhipicephalus decoloratus and Rhipicephalus microplus were examined in heifers to determine host immunity to the ticks. Tick counts and inflammatory cell infiltrates in skin biopsies from feeding sites of adult R. microplus ticks in nine-month-old Nguni and Bonsmara heifers were also evaluated. The molecular prevalence of A. marginale was similar in the Nguni (47.7 %) and local crossbred (52.3 %) cattle. Nguni cattle suffered less severe losses from and were more vi resilient to A. marginale infection than local crossbreds. Nguni heifers had lower coat scores, hair length and tick counts than the Bonsmara heifers. The relationship between tick counts and coat score was positive and linear in the Nguni (y = 1.90x – 0.40) and quadratic in Bonsmara (y = -7.98x2 + 12.74x - 3.12) heifers. Bonsmara cattle showed a more intense immediate reaction and no delayed hypersensitivity reaction to ULE of Rhipicephalus ticks. Nguni heifers presented a less intense immediate reaction and a delayed hypersensitivity reaction at 72 h post inoculation with ULE of Rhipicephalus ticks. Reactions to R. decoloratus ULE produced a more intense skin response at all time intervals in both breeds than that of R. microplus. Parasitized sites in Nguni heifers had higher (P < 0.05) counts of basophils, mast and mononuclear cells than those in the Bonsmara heifers. Conversely, parasitized sites in Bonsmara heifers had higher (P < 0.05) neutrophil and eosinophil counts than those in the Nguni heifers. Tick count was negatively correlated (P < 0.05) with basophil and mast cell counts. There was a positive correlation between eosinophil counts and tick counts in both breeds, and between tick counts and mononuclear cell counts in the Bonsmara breed. It was concluded that smooth and short coats, delayed type hypersensitivity and cutaneous basophil and mast cell infiltrations are responsible for increased tick resistance in the indigenous Nguni cattle breed of South Africa. / Ph.D. University of KwaZulu-Natal, Pietermaritzburg 2013.

Background: Neospora caninum is a parasite that causes disease, largely in cattle and dogs. It is a disease of significant interest within New Zealand due to its association with bovine abortion. The economic impact of bovine abortion justifies the development of a bovine vaccine against N. caninum. Aim: To develop and optimise diagnostic procedures for the detection of Neospora from a variety of blood and tissue samples and to isolate a New Zealand strain of Neospora caninum. Methods: A local strain of Toxoplasma gondii and an imported Neospora caninum strain, Nc-Liverpool, were used to optimise tachyzoite growing conditions in bovine endothelial (BE) cells and Vero host cell cultures. A serum study using 112 tissue culture flasks was performed to determine whether foetal bovine serum or horse serum supplemented media provided the optimal growing conditions for Nc-Liverpool tachyzoites. Nc-Liverpool tachyzoites were also used to determine the optimal growth period between passage, and harvest for cryopreservation and cryopreservation conditions. Percoll gradients were also tested using Nc-Liverpool tachyzoites. A known Neospora positive canine sample and murine tissues infected with Toxoplasma, were used during the development of the immunohistochemical diagnostic technique. Antibody concentrations and incubation temperatures were tested to reduce cross-reactivity and increase specific stain intensity. Immunohistochemistry was performed on sections of all tissue samples used for N. caninum isolation and experimentally infected murine tissue. Several PCR techniques were developed, the final PCR used being a combination of the different techniques, which produced a 250kb band. PCR-3 used the NF6/GA1 primer combination for Neospora detection and TF6/GA1 for Toxoplasma detection, additional Mg2+ and an annealing temperature of 55°C were required. Whole tissue was processed via DNA elution whereas cell culture and Percoll purified tachyzoites were used following crude lysis techniques. All bovine and canine tissues used for parasite isolation as well as all experimentally infected mouse tissues were tested for N. caninum using PCR. An immunoblot technique was developed for the detection of N. caninum antibodies in murine blood samples. Lysed Nc-Liverpool tachyzoites were used as antigen with varied results. The primary and secondary antibodies were commercially available and used at concentrations of 1:1,000 and 1:25,000 respectively. BALB/c and CF1 mice were experimentally infected with Toxoplasma gondii and Nc-Liverpool. Forty female BALB/c and 40 female CF1 mice were used in 2 studies to determine the optimal Nc-Liverpool inoculation dose and immunosuppression requirements. Mice were immunosuppressed with 2.5mg of methylprednisolone acetate (MPA) and Nc-Liverpool inoculation ranged from 1.3x106 to 5x103 tachyzoites. Upon death, the brain and blood was harvested from the mouse carcases. Attempts were made to isolate a New Zealand strain of N. caninum from bovine and canine central nervous system (CNS) tissue, and to maintain the parasites in cell culture and by small animal passage, in order to attenuate the parasite strain for use as a live large animal vaccine. Twenty one bovine tissue samples were used for N. caninum isolation attempts, 18 of which were positive for Neospora antibodies using a commercial IFAT. Isolation tissues were purified using a 30% Percoll gradient and inoculated onto 8 cell culture flasks and into 8 immunosuppressed mice (BALB/c and CF1). Results: Nc-Liverpool tachyzoites were found to be viable when grown at 37°C in antibiotic-MEM supplemented with either FBS or ES and grew optimally in FBS despite Neospora antibodies being detected using an IFAT. Passaging cultures at approx. 4 day intervals resulted in the greatest parasite growth. However, cryopreserved parasites should be harvested 2 days post inoculation (PI) for optimal viability. Viable parasites could be isolated using a 30% Percoll gradient and centrifuged at 2,700 x g (3,400 rpm) in a bucket centrifuge for 10 minutes. Tissue cysts could be detected using immunohistochemistry but some degree of cross reaction remained despite optimisation. Cysts were not found in tissues used for isolation attempts or in mouse brains following inoculation with Nc-Liverpool, however cysts were commonly found in mice experimentally infected with T. gondii tachyzoites. PCR-3 was successfully used to detect N. caninum and T. gondii infected tissue and tachyzoites from tissue culture. PCR-3 could detect N. caninum DNA in the brain tissue of 9/24 mice experimentally infected with Nc-Liverpool, even though most mice were culled within 1 week. Although production of N. caninum antigen was only moderately successful, N. caninum antibody detection in mouse blood using one specific antigen batch was reliable and specific. The immunoblot could only detect N. caninum antibody approximately 14 days PI, but was sensitive enough to detect 100% of mice experimentally infected with Nc-Liverpool tachyzoites. PCR-3 strongly correlated with the immunoblot results from 14 days PI. BALB/c mice were found to be far more sensitive to Nc-Liverpool than CF1 mice and developed severe disease at concentrations of approximately 1x106 Nc-Liverpool tachyzoites. Neither BALB/c nor CF1 mice developed peritoneal exudate, irrespective of the parasite inoculation concentration. Despite Neospora DNA being present in the brains of experimentally infected mice, re-isolation and continuous parasite passage from the brains could not be achieved. No mice experimentally infected with either Nc-Liverpool or isolation attempts were found to have brain cysts when tested using immunohistochemistry. Only 1 mouse inoculated with bovine isolation material was found to have a Neospora positive PCR. Through the detection of DNA, antigens and antibodies, parasites were determined to have been present in 10 of the 18 IFAT positive bovine isolation samples, indicating that 55% of calves born to seropositive dams were infected with N. caninum. However, despite numerous attempts to isolate Neospora parasites from naturally infected canine and bovine tissue and culturing using the optimised Nc-Liverpool technique, maintenance of a live culture of a New Zealand strain of N. caninum could not be established. Conclusions: Findings from this study could be used to assist in the maintenance of Neospora caninum and Toxoplasma gondii parasite strains and for detection or diagnosis of these parasites in host tissues.

Background: Neospora caninum is a parasite that causes disease, largely in cattle and dogs. It is a disease of significant interest within New Zealand due to its association with bovine abortion. The economic impact of bovine abortion justifies the development of a bovine vaccine against N. caninum. Aim: To develop and optimise diagnostic procedures for the detection of Neospora from a variety of blood and tissue samples and to isolate a New Zealand strain of Neospora caninum. Methods: A local strain of Toxoplasma gondii and an imported Neospora caninum strain, Nc-Liverpool, were used to optimise tachyzoite growing conditions in bovine endothelial (BE) cells and Vero host cell cultures. A serum study using 112 tissue culture flasks was performed to determine whether foetal bovine serum or horse serum supplemented media provided the optimal growing conditions for Nc-Liverpool tachyzoites. Nc-Liverpool tachyzoites were also used to determine the optimal growth period between passage, and harvest for cryopreservation and cryopreservation conditions. Percoll gradients were also tested using Nc-Liverpool tachyzoites. A known Neospora positive canine sample and murine tissues infected with Toxoplasma, were used during the development of the immunohistochemical diagnostic technique. Antibody concentrations and incubation temperatures were tested to reduce cross-reactivity and increase specific stain intensity. Immunohistochemistry was performed on sections of all tissue samples used for N. caninum isolation and experimentally infected murine tissue. Several PCR techniques were developed, the final PCR used being a combination of the different techniques, which produced a 250kb band. PCR-3 used the NF6/GA1 primer combination for Neospora detection and TF6/GA1 for Toxoplasma detection, additional Mg2+ and an annealing temperature of 55°C were required. Whole tissue was processed via DNA elution whereas cell culture and Percoll purified tachyzoites were used following crude lysis techniques. All bovine and canine tissues used for parasite isolation as well as all experimentally infected mouse tissues were tested for N. caninum using PCR. An immunoblot technique was developed for the detection of N. caninum antibodies in murine blood samples. Lysed Nc-Liverpool tachyzoites were used as antigen with varied results. The primary and secondary antibodies were commercially available and used at concentrations of 1:1,000 and 1:25,000 respectively. BALB/c and CF1 mice were experimentally infected with Toxoplasma gondii and Nc-Liverpool. Forty female BALB/c and 40 female CF1 mice were used in 2 studies to determine the optimal Nc-Liverpool inoculation dose and immunosuppression requirements. Mice were immunosuppressed with 2.5mg of methylprednisolone acetate (MPA) and Nc-Liverpool inoculation ranged from 1.3x106 to 5x103 tachyzoites. Upon death, the brain and blood was harvested from the mouse carcases. Attempts were made to isolate a New Zealand strain of N. caninum from bovine and canine central nervous system (CNS) tissue, and to maintain the parasites in cell culture and by small animal passage, in order to attenuate the parasite strain for use as a live large animal vaccine. Twenty one bovine tissue samples were used for N. caninum isolation attempts, 18 of which were positive for Neospora antibodies using a commercial IFAT. Isolation tissues were purified using a 30% Percoll gradient and inoculated onto 8 cell culture flasks and into 8 immunosuppressed mice (BALB/c and CF1). Results: Nc-Liverpool tachyzoites were found to be viable when grown at 37°C in antibiotic-MEM supplemented with either FBS or ES and grew optimally in FBS despite Neospora antibodies being detected using an IFAT. Passaging cultures at approx. 4 day intervals resulted in the greatest parasite growth. However, cryopreserved parasites should be harvested 2 days post inoculation (PI) for optimal viability. Viable parasites could be isolated using a 30% Percoll gradient and centrifuged at 2,700 x g (3,400 rpm) in a bucket centrifuge for 10 minutes. Tissue cysts could be detected using immunohistochemistry but some degree of cross reaction remained despite optimisation. Cysts were not found in tissues used for isolation attempts or in mouse brains following inoculation with Nc-Liverpool, however cysts were commonly found in mice experimentally infected with T. gondii tachyzoites. PCR-3 was successfully used to detect N. caninum and T. gondii infected tissue and tachyzoites from tissue culture. PCR-3 could detect N. caninum DNA in the brain tissue of 9/24 mice experimentally infected with Nc-Liverpool, even though most mice were culled within 1 week. Although production of N. caninum antigen was only moderately successful, N. caninum antibody detection in mouse blood using one specific antigen batch was reliable and specific. The immunoblot could only detect N. caninum antibody approximately 14 days PI, but was sensitive enough to detect 100% of mice experimentally infected with Nc-Liverpool tachyzoites. PCR-3 strongly correlated with the immunoblot results from 14 days PI. BALB/c mice were found to be far more sensitive to Nc-Liverpool than CF1 mice and developed severe disease at concentrations of approximately 1x106 Nc-Liverpool tachyzoites. Neither BALB/c nor CF1 mice developed peritoneal exudate, irrespective of the parasite inoculation concentration. Despite Neospora DNA being present in the brains of experimentally infected mice, re-isolation and continuous parasite passage from the brains could not be achieved. No mice experimentally infected with either Nc-Liverpool or isolation attempts were found to have brain cysts when tested using immunohistochemistry. Only 1 mouse inoculated with bovine isolation material was found to have a Neospora positive PCR. Through the detection of DNA, antigens and antibodies, parasites were determined to have been present in 10 of the 18 IFAT positive bovine isolation samples, indicating that 55% of calves born to seropositive dams were infected with N. caninum. However, despite numerous attempts to isolate Neospora parasites from naturally infected canine and bovine tissue and culturing using the optimised Nc-Liverpool technique, maintenance of a live culture of a New Zealand strain of N. caninum could not be established. Conclusions: Findings from this study could be used to assist in the maintenance of Neospora caninum and Toxoplasma gondii parasite strains and for detection or diagnosis of these parasites in host tissues.

Background: Neospora caninum is a parasite that causes disease, largely in cattle and dogs. It is a disease of significant interest within New Zealand due to its association with bovine abortion. The economic impact of bovine abortion justifies the development of a bovine vaccine against N. caninum. Aim: To develop and optimise diagnostic procedures for the detection of Neospora from a variety of blood and tissue samples and to isolate a New Zealand strain of Neospora caninum. Methods: A local strain of Toxoplasma gondii and an imported Neospora caninum strain, Nc-Liverpool, were used to optimise tachyzoite growing conditions in bovine endothelial (BE) cells and Vero host cell cultures. A serum study using 112 tissue culture flasks was performed to determine whether foetal bovine serum or horse serum supplemented media provided the optimal growing conditions for Nc-Liverpool tachyzoites. Nc-Liverpool tachyzoites were also used to determine the optimal growth period between passage, and harvest for cryopreservation and cryopreservation conditions. Percoll gradients were also tested using Nc-Liverpool tachyzoites. A known Neospora positive canine sample and murine tissues infected with Toxoplasma, were used during the development of the immunohistochemical diagnostic technique. Antibody concentrations and incubation temperatures were tested to reduce cross-reactivity and increase specific stain intensity. Immunohistochemistry was performed on sections of all tissue samples used for N. caninum isolation and experimentally infected murine tissue. Several PCR techniques were developed, the final PCR used being a combination of the different techniques, which produced a 250kb band. PCR-3 used the NF6/GA1 primer combination for Neospora detection and TF6/GA1 for Toxoplasma detection, additional Mg2+ and an annealing temperature of 55°C were required. Whole tissue was processed via DNA elution whereas cell culture and Percoll purified tachyzoites were used following crude lysis techniques. All bovine and canine tissues used for parasite isolation as well as all experimentally infected mouse tissues were tested for N. caninum using PCR. An immunoblot technique was developed for the detection of N. caninum antibodies in murine blood samples. Lysed Nc-Liverpool tachyzoites were used as antigen with varied results. The primary and secondary antibodies were commercially available and used at concentrations of 1:1,000 and 1:25,000 respectively. BALB/c and CF1 mice were experimentally infected with Toxoplasma gondii and Nc-Liverpool. Forty female BALB/c and 40 female CF1 mice were used in 2 studies to determine the optimal Nc-Liverpool inoculation dose and immunosuppression requirements. Mice were immunosuppressed with 2.5mg of methylprednisolone acetate (MPA) and Nc-Liverpool inoculation ranged from 1.3x106 to 5x103 tachyzoites. Upon death, the brain and blood was harvested from the mouse carcases. Attempts were made to isolate a New Zealand strain of N. caninum from bovine and canine central nervous system (CNS) tissue, and to maintain the parasites in cell culture and by small animal passage, in order to attenuate the parasite strain for use as a live large animal vaccine. Twenty one bovine tissue samples were used for N. caninum isolation attempts, 18 of which were positive for Neospora antibodies using a commercial IFAT. Isolation tissues were purified using a 30% Percoll gradient and inoculated onto 8 cell culture flasks and into 8 immunosuppressed mice (BALB/c and CF1). Results: Nc-Liverpool tachyzoites were found to be viable when grown at 37°C in antibiotic-MEM supplemented with either FBS or ES and grew optimally in FBS despite Neospora antibodies being detected using an IFAT. Passaging cultures at approx. 4 day intervals resulted in the greatest parasite growth. However, cryopreserved parasites should be harvested 2 days post inoculation (PI) for optimal viability. Viable parasites could be isolated using a 30% Percoll gradient and centrifuged at 2,700 x g (3,400 rpm) in a bucket centrifuge for 10 minutes. Tissue cysts could be detected using immunohistochemistry but some degree of cross reaction remained despite optimisation. Cysts were not found in tissues used for isolation attempts or in mouse brains following inoculation with Nc-Liverpool, however cysts were commonly found in mice experimentally infected with T. gondii tachyzoites. PCR-3 was successfully used to detect N. caninum and T. gondii infected tissue and tachyzoites from tissue culture. PCR-3 could detect N. caninum DNA in the brain tissue of 9/24 mice experimentally infected with Nc-Liverpool, even though most mice were culled within 1 week. Although production of N. caninum antigen was only moderately successful, N. caninum antibody detection in mouse blood using one specific antigen batch was reliable and specific. The immunoblot could only detect N. caninum antibody approximately 14 days PI, but was sensitive enough to detect 100% of mice experimentally infected with Nc-Liverpool tachyzoites. PCR-3 strongly correlated with the immunoblot results from 14 days PI. BALB/c mice were found to be far more sensitive to Nc-Liverpool than CF1 mice and developed severe disease at concentrations of approximately 1x106 Nc-Liverpool tachyzoites. Neither BALB/c nor CF1 mice developed peritoneal exudate, irrespective of the parasite inoculation concentration. Despite Neospora DNA being present in the brains of experimentally infected mice, re-isolation and continuous parasite passage from the brains could not be achieved. No mice experimentally infected with either Nc-Liverpool or isolation attempts were found to have brain cysts when tested using immunohistochemistry. Only 1 mouse inoculated with bovine isolation material was found to have a Neospora positive PCR. Through the detection of DNA, antigens and antibodies, parasites were determined to have been present in 10 of the 18 IFAT positive bovine isolation samples, indicating that 55% of calves born to seropositive dams were infected with N. caninum. However, despite numerous attempts to isolate Neospora parasites from naturally infected canine and bovine tissue and culturing using the optimised Nc-Liverpool technique, maintenance of a live culture of a New Zealand strain of N. caninum could not be established. Conclusions: Findings from this study could be used to assist in the maintenance of Neospora caninum and Toxoplasma gondii parasite strains and for detection or diagnosis of these parasites in host tissues.

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a chronic, debilitating enteritis of cattle, other domestic livestock and some wildlife species. JD was first identified in the late 1800s and today it is a worldwide problem in dairy cattle. Heavily infected cows have reduced milk production, a higher risk of removal from the herd and low slaughter value. Several countries have implemented national level control strategies. In New Zealand, JD was first reported in 1912 and today the prevalence of infected dairy herds is thought to be high. To improve our understanding of the epidemiology of JD and to evaluate the feasibility of a national control strategy, four studies were conducted. The first study was a questionnaire based case-control study to identify associations between management practices and the occurrence of clinical JD on farms from four regions of New Zealand. The second study was on the effect of sub-clinical JD on milk production and the risk of removal from the herd in four dairy herds over four milking seasons. The effect of misclassification of disease status on productivity estimates was also studied. In the third study diagnostic test result data from the productivity study was combined with a novel Bayesian regression model to estimate performance of the ELISA and faecal culture tests as a function of covariates and utilising repeated tests on individual cows. Finally, results from these three studies were used to adapt an existing JD simulation model, 'JohneSSim', to represent the epidemiological behaviour of JD in New Zealand dairy herds. Control strategies for the disease were simulated and evaluated based on their cost effectiveness. Of the 427 farmers responding to the questionnaire, 47% had suspected clinical cases of JD in their herd in the preceding 5 years. Only 13% of suspected infected herds had an average incidence of greater than 0.5 cases per 100 cow years at risk. The disease was not considered a serious problem by 20% of herd managers who reported the presence of disease in the preceding 5 years. The presence of Jersey cows in the herd and the purchase of bulls had strong positive associations with the presence of clinical JD. Grazing calves in the hospital paddock, larger herds, the purchase of heifers, and the use of induction were also positively associated with JD. In the productivity study the herd-level prevalence of JD by ELISA and/or faecal culture ranged from 4.5% (95% CI 2.6-6.9) to 14.2% (95% CI 9.2-20.6). Daily milksolids production by JD positive cows was 0.8% (95% CI -6.1%-4.5%) less than that of JD negative cows. However in herd D, JD positive cows produced 15.5%, (95% CI 6.75%-24.2%) milksolids less than JD negative herd mates daily. This equates to a loss of 53kg of milksolids/305 day lactation, or NZD 265/lactation, given a price of NZD 5/kg of milksolids. In herd D only, the annual hazard ratio of removal for JD positive cows was significantly increased. It was 4.7 times and 1.4 times higher in cows older than 5 years and younger than 5 years. The results were insensitive to misclassification. Analysis of the diagnostic test data demonstrated the strengths of our Bayesian regression model. While overall estimates of sensitivity and specificity by this method were comparable to estimates by existing methods, it showed a broad trend of increasing sensitivity in higher parity groups and higher sensitivity in early, relative to late, lactation. It also showed that estimates of prevalence may in fact decline with repeated, relative to single, testing. Our novel approach demonstrated trends that could not be shown by existing methods, but could be improved by application to a larger data set. Simulation showed that control strategies for JD based on either test-and-cull, vaccination, breeding for genetic resistance, or removal of offspring from clinically affected cows, were not cost effective for the average infected herd. Improvement of the hygiene associated with calf management provided the greatest reduction in the within-herd prevalence of JD. While JD is present in a high proportion of New Zealand dairy herds, the incidence of clinical cases is usually low, and most farmers consider it to be of little importance. However, JD causes significant losses in productivity in some herds. The disease would probably be best controlled on a herd-by-herd basis, given the limited success of national-scale control programs for JD in other countries. The education of dairy farmers regarding risky management practices, and the offer of a risk assessment to farmers wishing to control the disease, would provide a combination of wide reaching and targeted approaches, of low cost, for JD control. It seems likely that JD will persist in some capacity in the years ahead, but will remain of minor concern next to major animal health issues, such as infertility and mastitis. Clarification of the effect of genetic strain on the virulence of MAP may help explain differences in the effect of the disease between herds. This knowledge could then be used to further improve the efficiency of JD control.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The epidemiological, clinical and pathological aspects of 128 cases of bovine lymphoma are retrospectively described in this study (1965-2014).Out of the cases were the gender was informed (n=111), 84.7% of affected animals were females and 15.3% were males. Out of the cases were breed was informed (n=108), 63% of affected animals were Holstein cows. The age of affected cows (n=107) varied from 1 to 14 years, with most animals being adults (80.4%) with 5 to 8 years of age (57.9%). The most common clinical sign (n=89) was lymphadenomegaly (74.1%). Gross findings (n=125) included enlargement of lymph nodes in 71.2% of the cases; this finding was classified as localized in 89.6% of the cases and generalized in 10.3% of the cases. A retrospective study of 86 cases of bovine lymphoma classified as with diffuse pattern of distribution and verified by phenotypic (histology) and immunophenotypic (immunohistochemistry [IHC]) is presented. Based on these results, the lymphoma cases were classified by the Working Formulation (WF) of Non-Hodgkin s Lymphomas for Clinical Usage as: diffuse large non-cleaved cell (46.5%), diffuse large cleaved cell (33.7%), diffuse mixed small and large cell (4.6%), diffuse small cell plasmacytoid (7%), immunoblastic (3.5%), diffuse small cell intermediate (2.3%), diffuse small non-cleaved cell (1.2%), and diffuse small non-cleaved cell Burkitt s (1.2%). According to the IHC, 27 out of 86 (31.4%) lymphomas were positive to monoclonal antibody CD79αcy, used to detect B cells, and none were positive for polyclonal antibody CD3, used to detect T cells. Based on this, the B-cell type lymphomas were distributed as follows: diffuse large B-cell lymphoma (81.5%), large cell immunoblastic lymphoma (11.1%), and lymphoplasmacytoid lymphoma (7.4%), according the Revised European-American Classification of Lymphoid Neoplasms (REAL). The results of this retrospective study, similar to what has been described in other parts of the world, allow us to conclude that bovine lymphomas are basically diffuse and predominantly made of intermediate-grade, large cells, with cleaved or non-cleaved nuclei. These lymphomas are due to neoplastic proliferation of B cells and correspond to almost all (92.6%) to what is currently classified as diffuse large B-cell lymphoma by REAL. / Foi realizado um estudo retrospectivo dos arquivos do Laboratório de Patologia Veterinária (LPV) da Universidade Federal de Santa Maria (UFSM) com base nos casos de necropsia e materiais de necropsia de bovinos dos últimos 50 anos (1965-2014). Foram incluídos nesse estudo somente os laudos que possuíam o diagnóstico de linfoma (n=128). Dos laudos que informavam o sexo (n=111), 84,7% correspondiam a fêmeas e 15,3% a machos. Dos laudos em que constava a raça (n=108), a mais prevalente foi a holandesa (63%). Em relação à idade (n=107), houve uma variação entre um e 14 anos. A maioria dos bovinos era adulta (80,4%) e a maior concentração dos casos ocorreu entre 5-8 anos (57,9%). Clinicamente (n=89), linfadenomegalia foi o achado mais frequentemente observado (74,1%). Na necropsia (n=125), 71,2% dos bovinos apresentavam aumento de volume dos linfonodos; essa linfadenomegalia foi classificada como localizada em 89,6% dos casos e generalizada em 10,3% dos casos. Realizou-se também a avaliação fenotípica (histologia) e imunofenotípica (imuno-histoquímica [IHQ]) de 86 casos de linfoma bovino. Com base nestes resultados, os linfomas foram assim distribuídos utilizando-se a classificação proposta pela Working Formulation (WF) of Non-Hodgkin s Lymphomas for Clinical Usage: difuso de grandes células não clivadas (46,5%), difuso de grandes células clivadas (33,7%), difuso de pequenas e grandes células (4,6%), difuso de pequenas células tipo plasmocitoide (7%), imunoblástico (3,5%), difuso de pequenas células tipo intermediário (2,3%), difuso de pequenas células não clivadas (1,2%) e difuso de pequenas células não clivadas tipo Burkitt (1,2%). Na IHQ, 27 dos 86 (31,4%) linfomas foram positivos para o anticorpo monoclonal CD79αcy, utilizado para detecção de linfócitos B, e nenhum caso foi positivo para o anticorpo policlonal CD3, utilizado para detecção de linfócitos T. Com base nestes resultados, os linfomas B foram assim distribuídos utilizando-se a Revised European-American Classification of Lymphoid Neoplasms (REAL): linfoma difuso de grandes células B (81,5%), linfomas imunoblásticos de grandes células (11,1%) e linfomas linfoplasmocíticos (7,4%). Os resultados aqui apresentados permitem concluir que à semelhança do que vem sendo descrito em outras partes do mundo, os linfomas bovinos são basicamente difusos e predominantemente de grau intermediário, de grandes células, com núcleos clivados ou não clivados. Esses linfomas são decorrentes da proliferação neoplásica de linfócitos B e correspondem, em sua quase totalidade (92,6%), ao que atualmente é incluído na REAL como linfoma difuso de grandes células B.

Die vorliegende Analyse diente dem Ziel, Krankheitsschwerpunkte bei Rindern in den Jahren 1968 bis 1999 aus der Sicht der Medizinischen Tierklinik, Leipzig, nach Häufigkeit, Rasse-, Alters-, Jahreszeit- und Geschlechtsdisposition, Behandlungsdauer sowie –erfolg aufzuzeigen. In dem genannten Zeitraum wurden 2295 Rinderpatienten gemäß der Daten in den Kliniktagebüchern unter Berücksichtigung der wechselnden gesellschaftlichen und Besitzverhältnisse ausgewertet. Im Analysezeitraum nahmen Infektionskrankheiten ab, manche, wie z.B. Leukose, Brucellose und Tuberkulose, verschwanden ganz. Auch die Puerperale Hämoglobinurie sowie die Rachitis werden nicht mehr beobachtet. Dafür stieg der Anteil Verdauungsstörungen durch die Dislocatio abomasi beträchtlich an.

Thirty bovine with neoplasms of the upper digestive tract (UDT) associated to the spontaneous consumption of bracken fern (Pteridium aquilinum) were studied. They were from 27 farms, localized in the municipalities of Jaguari (23) and Nova Esperança do Sul (4), Rio Grande do Sul, Brazil. The total bovine population in those farms was 1,090 and large amounts of bracken fern were found in the pastures. Twenty-six were cows e four were castrated males. The age ranged from 3 to 13-years-old. Most of them were 7 to 8-years-old (46,6%). Clinical signs observed in the affected animals were progressive weight loss, absence of ruminal movements, cough, dysphagia, regurgitation, halitosis, diarrhea, and bloat. Less frequent signs were selective appetite, dyspnea, and salivation. Two bovines died and 28 were euthanatized in extremis and submitted to necropsy. The main gross and microscopic findings were found in the same areas of the UDT. They were digestive papillomatosis, transforming papillomas, and squamous cell carcinomas (SCC). Twenty-nine bovines had papillomas of various sizes in several areas of the UDT. The digestive papillomatosis ranged from mild (45%), to moderate (38%), to severe (17%). Three developing phases were observed microscopically in the examined papillomas: an early growing phase, a developing phase, and a regressing phase. The regressing phase was characterized by lymphocytic infiltrates at the base of the papilloma. In 16 cases, the microscopic examination of lesions grossly resembling papillomas (although some were slightly round, with lower or ulcerated finger-like projections) revealed malignant transformation of the papillomas into SCCs. The SCCs were solitary (12/30) or multiple (18/30) and were histologicaly characterized as well, moderately, or poorly differentiated. Grouping the distribution of SCCs of larger extension in the UDT into cranial region (base of the tongue, pharynx/oropharynx, and epiglottis), medial region (esophagus), and caudal region (cardia and rumen), the distribution was cranial in 39%, middle in 16%, and caudal in 45% of the cases. By the same grouping criteria, but considering the total number of times SCCs of varied extensions were diagnosed in the cranial, middle, and caudal regions, the percentages changed to 34%, 26%, and 40%, respectively. Metastases to regional lymph nodes and other organs, like liver and lungs, were observed in 18 cases. Immunohistochemistry for cytokeratin was performed in selected sections of SCCs and metastases, showing strong positive reaction in the well and moderately differentiated SCCs, but weak positive reaction in the poorly differentiated ones. The epidemiological and histomorphological evidences showed in this study are in agreement with the observations that point out the co-carcinogesis between bovine papillomavirus type 4 infection and chemicals of bracken fern in the pathogenesis of the SCCs in the UDT of cattle. / Foram estudados 30 bovinos com neoplasias no trato alimentar superior (TAS) associadas ao consumo espontâneo de samambaia (Pteridium aquilinum) provenientes de 27 propriedades rurais, sendo 23 no município de Jaguari e quatro em Nova Esperança de Sul, Rio Grande do Sul. A população bovina total das 27 propriedades em que ocorreram os casos era de 1.090 bovinos e havia quantidade abundante de samambaia nas áreas de pastoreio dos animais. Vinte e seis bovinos eram vacas e quatro eram machos castrados. A idade variou de três a 13 anos, sendo o maior número de casos entre sete e oito anos (46,6%). Os sinais clínicos observados incluíram emagrecimento progressivo, atonia ruminal, tosse, disfagia, regurgitação, halitose, diarréia e timpanismo. Outros sinais clínicos menos freqüentes foram apetite seletivo, dispnéia e salivação. Dois bovinos tiveram morte espontânea e 28 foram submetidos à eutanásia in extremis e necropsiados. Os principais achados macroscópicos e histológicos observados nos 30 bovinos localizavam-se nos mesmos locais do TAS e consistiam de papilomas, papilomas em transformação para carcinomas de células escamosas (CCEs) e CCEs. Vinte e nove bovinos tinham papilomas de diversos tamanhos, sendo a quantidade variável entre leve (45%), moderada (38%) e acentuada (17%). Nos papilomas examinados microscopicamente, foram observadas três fases de desenvolvimento: a) fase inicial de crescimento; b) fase de desenvolvimento; e c) fase de regressão; essa última era caracterizada por infiltrados linfocitários nos eixos fibrovasculares de sustentação. Em 16 bovinos, o exame histológico de lesões macroscópicas compatíveis com papilomas, porém alguns deles apresentando-se mais arredondados, com projeções digitiformes atenuadas ou ulceradas, revelou a transformação maligna desses papilomas em CCEs. Os CCEs eram únicos (12/30) ou múltiplos (18/30) e variaram quanto ao grau de diferenciação celular entre bem diferenciados, moderadamente diferenciados ou pouco diferenciados. Quando a distribuição dos CCEs de maior extensão foi agrupada em regiões cranial (base da língua, faringe/orofaringe, epiglote), média (terços cranial, médio e caudal do esôfago) e caudal (entrada do rúmen e rúmen) do TAS, observou-se que a localização era cranial em 39% dos casos, média em 16%, e caudal em 45%. Utilizando-se esse mesmo critério de agrupamento, porém considerando o número total de vezes em que CCEs (de extensões variadas) foram diagnosticados nas regiões cranial, média e caudal, os números alteraram-se para 34%, 26% e 40%, respectivamente. Metástases de CCEs para linfonodos regionais e outros órgãos como fígado e pulmão foram observadas em 18/30 bovinos. A técnica de imunoistoquímica para citoqueratina foi realizada em cortes selecionados de CCEs e metástases, observando-se células fortemente positivas nos CCEs bem e moderadamente diferenciados, e fraca imunomarcação nos pouco diferenciados. As evidências epidemiológicas e histomorfológicas relatadas neste estudo reforçam as observações de uma estreita correlação entre a infecção pelo papilomavírus bovino tipo 4, causador da papilomatose digestiva, e a co-carcinogênese química dos princípios tóxicos da samambaia na patogênese dos CCEs do TAS de bovinos.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The pathogenesis of the lesions of the Ramaria flavo-brunnescens poisoning in cattle was studied throughout a retrospective evaluation of selected tissues from nine spontaneous and four experimental cases of the disease. The pathogenesis of lesions observed in the tongue, esophagus, hoof, and tail was investigated analyzing microscopic lesions, histochemical and histochemical-ultrastuctural changes. Histochemical techniques utilized were Masson s Trichrome and Selective Oxidation of Keratin (SOK). The histochemicalultrastuctural study was acomplished throughout the Swift method under transmission electron microscopy. Hair shafts of the tip of the tail were analyzed under polarized light. Lesions of varying degrees of severity were observed. They were more severe in spontaneous than in experimental cases. In the tongue, most microscopic lesions showed keratinization defects, such as loss of the filiform papillae, thinning, irregular stratification, focal lamelar keratinization, and individual cell keratinization (dyskeratosis) in the dorsal epithelium. In the esophagus, there were thinness of superficial epithelium and multifocal ulcers. In the hoof, lesions were in the laminar stratum and characterized by different grades of fusion, shortness, multiple layers of non-keratinized cells in the laminar tip, irregular and discontinuous keratinization with nuclear persistency, individual cell keratinization with citoplasmic vacuolization of keratinocytes of the epidermal laminae. In the skin of the tip of the tail, changes could by separated in follicular wall lesions (affecting the outer [ORS] and the inner root sheets [IRS]) and changes of the hair shaft itself. The main changes observed in the follicular epithelium were disorganization, misalignment, and disceratosis of keratinocytes of the ORS. On tissue sections, main changes in the hair shafts showed irregular contour, tortuousness, and disintegration of shaft. Morphological changes similar to the ones observed on tissue sections and changes in polarizing patterns were seen on polarized light microscopy of hair shafts. Tissue sections stained by Masson s Trichrome technique (with picric acid) revealed defective hard keratinization of filliform papillae, of epidermal lamina of hoof, and of tail tip hair shafts. Sections stained by the SOK technique revealed strong loss of cistine contents, visualized as light staining of these same structures. On the histochemical-ultrastructural study of the hair cuticle, performed throughout the Swift technique under transmission electron microscopy, a low content of cistine was also observed. All changes observed in the keratinized structures studied, mostly in the hard keratin, showed defective keratinization. The morphologic study and the results obtained with SOK and Swift techniques showed that the defective keratinization results of low amounts of sulphur containing amino acids (cystine) in hard keratin structures. This is probably the main pathogenetic mechanism of the lesions observed in R. flavo brunnescens poisoning in cattle. / O estudo da patogênese da intoxicação pelo cogumelo Ramaria flavo-brunnescens em bovinos foi realizado através da avaliação retrospectiva de tecidos selecionados de nove casos espontâneos e quatro casos experimentais. Para a investigação da patogênese das lesões observadas na língua, esôfago, casco e cauda, foram avaliadas as alterações histopatológicas e aspectos histoquímicos e histoquímico-ultra-estruturais das lesões. As técnicas histoquímicas utilizadas foram o Tricrômico de Masson e a oxidação seletiva da ceratina (OSC). O estudo histoquímico-ultra-estrutural foi realizado através da técnica de Swift sob microscopia eletrônica de transmissão. Os pelos da vassoura da cauda foram examinados sob microscopia de luz polarizada. Nos tecidos examinados foram observados diferentes graus e estágios de lesões. Nos bovinos intoxicados espontaneamente as lesões foram mais acentuadas que nos casos experimentais. Na língua, a grande maioria das lesões histopatológicas observadas demonstrou defeitos na ceratinização como desaparecimento das papilas filiformes, adelgaçamento, estratificação irregular, ceratinização lamelar focal e ceratinização individual de células (disceratose) no epitélio de revestimento dorsal. No esôfago observaram-se adelgaçamento do epitélio superficial e úlceras multifocais. Nos cascos havia alterações no estrato laminar, caracterizadas por graus variáveis de fusão, encurtamento, múltiplas camadas de células não-ceratinizadas no topo, ceratinização irregular e descontínua com persistência de núcleos, ceratinização individual e vacuolização citoplasmática de ceratinócitos das lâminas epidérmicas. Na pele da região da vassoura da cauda, as alterações foram divididas em lesões da parede folicular (nas bainhas radicular externa e interna) e alterações nos pelos propriamente ditos. As principais alterações observadas no epitélio folicular foram desorganização, desalinhamento e disceratose de ceratinócitos da bainha radicular externa. Nas seções histológicas, as principais alterações nas hastes pilosas demonstraram contornos irregulares, tortuosidade e desintegração da haste. Alterações morfológicas semelhantes às descritas nas seções histológicas e alterações no padrão de birrefringência foram observadas através do exame polaroscópico dos pelos. Através da técnica do Tricrômico de Masson (com ácido pícrico) observou-se ceratinização dura defeituosa das papilas filiformes linguais, das lâminas epidérmicas do casco e dos pelos da cauda. A técnica da OSC revelou redução marcada do conteúdo de cistina, visualizado como fraca coloração rósea dessas mesmas estruturas. No estudo histoquímico-ultra-estrutural da cutícula pilosa, realizado através da técnica de Swift sob microscopia eletrônica de transmissão, observou-se também um baixo conteúdo de cistina. Todas as alterações observadas nas estruturas ceratinizadas estudadas, mas especialmente nas que sofrem ceratinização dura, revelaram defeitos na ceratinização. Aliando ao estudo morfológico os resultados obtidos através da técnica da OSC e da microscopia eletrônica/técnica de Swift pode-se associar os defeitos na ceratinização a uma redução na quantidade de aminoácidos sulfurados (cistina), principalmente nas estruturas que sofrem ceratinização dura, sendo este provavelmente o principal mecanismo patogenético na intoxicação por R. flavo brunnescens em bovinos.