Analysis of the value of copper erythrocyte concentration measurement in the diagnosis of copper deficiency in bovines.

BACKGROUND: A reliable and practical method for assessing Cu status in live animals is not available. Blood Cu levels may not accurately reflect the true Cu status of the herd, and can over-predict Cu status during stress and inflammation. On the other hand, assessment of liver Cu is the most reliable indicator of Cu stores, but it is an invasive procedure that requires specialized training. The aim of this study was to evaluate the usefulness of Cu levels in red blood cells to determine the Cu status, with special emphasis in their correlation with erythrocyte Cu, Zn superoxide dismutase enzyme activity (ESOD), in bovines with Cu deficiency induced by high molybdenum and sulfur levels in the diet. METHODS: Three similar assays were performed, with a total of twenty eight calves. The Cu-deficient group (n = 15) received a basal diet supplemented with 11 mg of Mo/kg DM as sodium molybdate, and S as sodium sulfate. The control group (n = 13) received a basal diet supplemented with 9 mg of Cu/kg DM as copper sulfate. Samples of blood and liver were taken every 28-35 days. Cu levels were measured in liver (expressed as µg/g DM), plasma (expressed as µg/dl), and erythrocytes (expressed as µg/g Hb) by flame atomic absorption spectroscopy. Superoxide dismutase (SOD1) activity was determined in red blood cells and was expressed as IU/mg hemoglobin. InfoStat Statistical Software 2020 was used for the statistical analysis. Cu levels in plasma, red blood cells and liver, and ESOD activity were analyzed by ANOVA. The correlation between erythrocyte Cu levels and the rest of the parameters were analyzed by Pearson Correlation test. Unweighted Least Squares Linear Regression of SOD1 was developed. The autocorrelation between the monthly measurements was also determined by Durbin-Watson test and autocorrelation function. RESULTS: The assays lasted 314-341 days, approximately. Levels indicative of Cu deficiency for bovines were detected at 224 days (23 ± 11.6 µg/g DM) for liver Cu concentration; and at 198 days (55 ± 10.4 µg/dl) for plasma Cu concentration, in Cu-deficient animals. Liver and plasma Cu values indicative of Cu deficiency were not observed in the control group. Pearson Correlation test indicated that all indices of Cu status used in this study were significantly correlated. The highest value was obtained between ESOD and red blood Cu (0.74). There was a significant correlation between red blood Cu and plasma Cu (0.65), and with hepatic Cu (0.57). ESOD activity showed a similar significant positive correlation with liver Cu concentrations and with plasma Cu (0.59 and 0.58, respectively). CONCLUSION: The extremely low levels of liver and plasma Cu, the ESOD activity, erythrocyte Cu levels, and the periocular achromotrichia observed in the Cu-deficient animals showed that the clinic phase of Cu deficiency was reached in this group. The ESOD activity and erythrocyte Cu levels showed a strong association, indicating that the values of erythrocyte Cu may serve as an effective tool in assessing Cu status and diagnose a long-term Cu deficiency in cattle.

Early-stage findings in an experimental calf model infected with Argentinean isolates of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is an important pathogen that causes granulomatous enteritis known as Johne's disease or paratuberculosis (PTB). In this study an experimental model of calves infected with Argentinean isolates of MAP for 180 days was used to provide more data of the early PTB stages. Calves were challenged by oral route with MAP strain IS900-RFLPA (MA; n = 3), MAP strain IS900-RFLPC (MC; n = 2) or mock infected (MI; n = 2), and response to infection was evaluated through peripheral cytokine expression, MAP tissue distribution and histopathological early-stage findings. Specific and varied levels of IFN-γ were only detected at 80 days post-infection in infected calves. These data indicate that specific IFN-γ is not a useful indicator for early detection of MAP infection in our calf model. At 110 days post-infection, TNF-α expression was higher than IL-10 in 4 of the 5 infected animals and a significant decrease of TNF-α expression was detected in infected vs. non-infected calves. All calves challenged were identified as infected by mesenteric lymph node tissue culture and real time IS900 PCR. In addition, for lymph nodes samples, the agreement between these techniques was almost perfect (κ = 0.86). Colonization of tissues and levels of tissue infection varied between individuals. Evidence of early MAP dissemination to extraintestinal tissues such as the liver was detected by culture in one animal (MAP strain IS900-RFLPA). In both groups microgranulomatous lesions were observed predominantly in the lymph nodes, with giant cells present only in the MA group. In summary, the findings described herein may indicate that local MAP strains induced specific immune responses with particularities that could suggest differences in their biological behavior. Further studies should be carried out in order to obtain an in-depth understanding of the influence of MAP strains in host-pathogen interactions and the outcome of disease.

First report of phaeohyphomycosis caused by Phialophora americana in a domestic cat from Argentina.

Case summary: A 10-year-old male neutered domestic shorthair cat from Quilmes (Province of Buenos Aires, Argentina) presented at the Infectious Diseases and Parasitology Unit with a hyperpigmented nodule of 5 cm diameter on the nasal plane with a small ulceration of more than 1 year's evolution. A scaly and hyperpigmented alopecic lesion of 3 cm in diameter was found on the lower edge of the tail. The patient was under immunosuppressive therapy with corticosteroids for lymphoplasmacytic duodenitis. Samples of the lesion present on the nasal plane were taken under a surgical procedure. In the wet mount preparations, pigmented irregular hyphae were observed. They developed dark colonies when cultured on Sabouraud medium. On micromorphology, structures compatible with Phialophora species were identified. PCR and sequencing of ITS (ITS1-5.8S-ITS2) confirmed Phialophora americana as the etiologic agent. A therapeutic scheme that included a combination of itraconazole oral solution (1.5 mg/kg PO q12h) with terbinafine (30 mg/kg PO q24h) was indicated for a period of 10 months. The patient died of complications resulting from its underlying disease. Relevance and novel information: As far as the authors are aware, this is the first study to report P americana as an etiologic agent of phaeohyphomycosis in cats. In this case study, the species was identified using molecular tests.

Decrease lysyl oxidase activity in hearts of copper-deficient bovines.

BACKGROUND: Lysyl oxidase (LOX) is a metalloenzyme that requires Cu as a cofactor and it is responsible for the formation of collagen and elastin cross-linking. The objective of this work was to measure the LOX enzyme activity in the heart of bovines with Cu deficiency induced by high molybdenum and sulfur levels in the diet. METHODS: Eighteen myocardial samples were obtained from Cu-deficient (n = 9) and control (n = 9) Holstein bovines during two similar assays. The samples were frozen in liquid nitrogen and stored at -70 °C to measure enzymatic activity. A commercial kit was used, following producer instructions. RESULTS: The results showed that LOX activity from the hearts of Cu-deficient bovines is 29 % lower than the ones of control bovines, being this difference statistically significant (p = 0.03). CONCLUSION: To our knowledge, this is the first report that determined LOX enzymatic activity in bovine heart of Cu-deficient animals. The microscopic alterations found in these animals in our previous work, could be explained by a diminished LOX activity. The results are in agreement with other authors, who found a relationship between LOX activity and dietary Cu intake. The information provided by this work could help to clarify the pathogenesis of cardiac lesions in cattle with dietary Cu deficiency.

A participatory surveillance of marsh deer (Blastocerus dichotomus) morbidity and mortality in Argentina: first results.

BACKGROUND: In an era of unprecedented socio-ecological changes, managing wildlife health demands high-quality data collection and the engagement of local communities. Blastocerus dichotomus, the largest South American deer, is Vulnerable to extinction mainly due to habitat loss. Diseases have been recognised as a potential threat, and winter mortality has been historically described in marsh deer populations from Argentina. Field difficulties have, however, prevented in-depth studies of their health status. RESULTS: Between May 2014 and April 2017, we investigated marsh deer morbidity and mortality in the two largest populations in Argentina. We collected data by means of a passive surveillance system that involved a network of researchers, field partners (veterinarians, park rangers, and local community), and decision makers. We sampled marsh deer during as well as outside mortality events. A total of 44 marsh deer with different body condition scores were evaluated. We obtained haematology and biochemistry values from animals with good body condition score. Marsh deer with poor body condition had a high burden of the ticks Amblyomma triste and Rhipicephalus microplus. Vector-borne agents such as Theileria cervi, Trypanosoma theileri, Trypanosoma evansi, Ehrlichia chaffeensis, Anaplasma platys, Anaplasma odocoilei, Anaplasma marginale, and Candidatus Anaplasma boleense were also found. Haemonchus spp., Ostertagia spp., and Trichostrongylus spp. were the most frequent gastrointestinal parasites in deer with poor body condition. A Multiple Correspondence Analysis reinforced a possible association of winter period with lower body score condition, high tick loads, infection with E. chaffeensis, and presence of harmful gastrointestinal parasites. CONCLUSIONS: Our approach allowed the establishment of a participatory surveillance network of marsh deer morbidity and mortality in Argentina. We report and analyse the first data obtained opportunistically within the framework of this network, providing information on the infectious and parasitic agents in marsh deer populations. The occurrence of Fasciola hepatica and Leptospira interrogans serovar pyrogenes is reported for the first time in wild marsh deer from Argentina. Our data will be useful to improve the interpretation of future mortality events. The field implementation of a surveillance network is key to a holistic approach to wildlife diseases.

First characterization of a canine parvovirus causing fatal disease in coatis (Nasua nasua).

A canine parvovirus (CPV)-like virus was detected by PCR and isolated from dead coatis in Argentina. Analysis of the full-length genome sequence revealed that it resembled CPV-but also contained a mutation in the VP2 protein (Arg377Ser) that has not been described previously. This is the first report of a CPV-like virus producing clinical disease in coatis. Genetic similarity to CPV-2c viruses detected in Brazil suggests a strong relationship between these viruses. Although the pathogenic potential of CPV- and feline panleukopenia virus (FPV)-like strains in wild animals is still not completely understood, this study highlights the importance of parvoviruses as a threat to wildlife if proper conditions are present.

Biochemical and Morphological Alterations in Hearts of Copper-Deficient Bovines.

Copper deficiency is an important disease of cattle that produces several clinical signs and lesions, due to alterations in copper-dependent enzymes. One of the organs affected by this deficiency is the heart (falling disease), but nevertheless, these cardiac lesions have not been extensively studied in bovines. The aim of this work was to propose a possible pathogenic mechanism for cardiac lesions in cattle affected by copper deficiency. Because of the possible existence of oxidative distress caused by low levels of copper-zinc-superoxide dismutase and cytochrome oxidase, ultrastructural and histological lesions have been evaluated in the heart of bovines in which a Cu deficiency had been induced using high molybdenum and sulfur levels in the diet. Our results indicated that copper deficiency produces significant damage in myocardium with high levels of lipid oxidation and a significant reduction in copper-zinc-superoxide dismutase activity leading to an oxidative distress situation. However, cytochrome oxidase activity was not significantly reduced. Histological observation revealed a significant increase in the amount of connective tissue, enlarged basement membranes of myocytes, and numerous Anichkov cells, in the hearts of deficient animals. Ultrastructural observation showed a significant enhancement in the mitochondrial volume density, with presence of lesions such as swelling and cristae disruption. We conclude that copper deficiency in bovines causes morphological lesions in the heart due to an oxidative damage produced by copper-dependent enzyme alterations.

Fatal canine encephalitozoonosis in Latin America, first report.

Encephalitozoon cuniculi is an obligate, intracellular microsporidian organism capable of establish infection in a wide variety of animals. In carnivores it may cause a sporadic, severe disease in the first few months of life, which usually culminates with the death of the animal. The objective of this study was to report a natural fatal case of encephalitozoonosis in a puppy from Argentina. Clinical signs included reduced appetite, depression, vocalizing, weight loss, weakness, convulsions and recumbency. No significant gross lesions were noticed at necropsy. Microscopically, severe, diffuse, lymphocytic encephalitis was seen. Large cytoplasmic vacuoles containing spores, morphologically compatible with E. cuniculi, were present within endothelial cells of brain and kidney, in renal tubular epithelium and hepatocytes. Encephalitozoon cuniculi DNA was detected by PCR in the kidney. Antibody titers to E. cuniculi in serum from the surviving puppies and the dam were ≥1:200. This report contributes to our understanding of neurologic disease in puppies. Encephalitozoonosis should be considered as a differential diagnosis in cases of fatal encephalitis in puppies.

Immunological findings associated with Argentinean strains of Mycobacterium avium subsp. paratuberculosis in bovine models.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of ruminant paratuberculosis. The aim of this study was to evaluate the biological behavior of different Argentinean strains of MAP in two bovine infection models: macrophage (in vitro) and calf (in vivo) through the evaluation of early immune responses at the peripheral and local levels. Two MAP strains (A and C) were selected taking into account the different patterns of TNF-α and IL-10 secretion displayed by infected bovine macrophages in vitro. Two groups of calves were infected with 250mg of total wet weight live MAP: strain A infected group (MA, n=3), strain C infected group (MC, n=2). Another group of animals was mock-infected (MI, n=3). Infection was confirmed by MAP culture of feces and microscopic observation of granulomatous lesions in the gut tissue. All infected calves showed positive results in the DTH skin test. A significant increase in peripheral CD4CD25(+) cells in MC group on day 150 was detected. The specific cellular immune response developed allowed the identification of the infection as early as 30days in the MA group. However, the percentage of CD8CD25(+) cells was significantly increased on day 120 in MC group. Significant differences between groups in proliferation and cellular responses were also detected in ileocecal lymph node samples. In summary, the strains of MAP employed herein induced differential immune responses in peripheral cells, in the proliferative responses and in cell functionality at the local level. Our findings support the hypotheses that the in vitro behavior displayed by macrophages could be a tool to identify differences among MAP strains infecting bovines and that the host-pathogen interactions occurring upon infection are dependent on the strain of MAP involved.

Brucella cyclic ß-1,2-glucan plays a critical role in the induction of splenomegaly in mice.

Brucella, the etiological agent of animal and human brucellosis, is a bacterium with the capacity to modulate the inflammatory response. Cyclic ß-1,2-glucan (CßG) is a virulence factor key for the pathogenesis of Brucella as it is involved in the intracellular life cycle of the bacteria. Using comparative studies with different CßG mutants of Brucella, cgs (CßG synthase), cgt (CßG transporter) and cgm (CßG modifier), we have identified different roles for this polysaccharide in Brucella. While anionic CßG is required for bacterial growth in low osmolarity conditions, the sole requirement for a successful Brucella interaction with mammalian host is its transport to periplasmic space. Our results uncover a new role for CßG in promoting splenomegaly in mice. We showed that CßG-dependent spleen inflammation is the consequence of massive cell recruitment (monocytes, dendritics cells and neutrophils) due to the induction of pro-inflammatory cytokines such as IL-12 and TNF-α and also that the reduced splenomegaly response observed with the cgs mutant is not the consequence of changes in expression levels of the characterized Brucella PAMPs LPS, flagellin or OMP16/19. Complementation of cgs mutant with purified CßG increased significantly spleen inflammation response suggesting a direct role for this polysaccharide.

Bactericidal activity of lachrymal secretion and complement system in copper deficient bovines.

Copper deficiency in humans and animals has been related to increased susceptibility to infections. Neutrophils are one of the most studied components of the immune response; however, to the best of our knowledge, other defenses of the innate immune system have not been analyzed in copper-deficient animals. Our previous studies in copper-deficient bovines have shown increased susceptibility to infectious keratoconjunctivitis, an ocular disease caused by Moraxella bovis. The objective of this work was to evaluate the bactericidal activity of lachrymal secretion and complement system, two main mechanisms of the innate immune response against M. bovis, in copper-deficient cattle. Our results indicate that copper deficiency has no effect on bactericidal activity of complement system and lachrymal secretion against M. bovis in calves. Other components of local and systemic ocular defense mechanisms that could explain the increased susceptibility to infectious keratoconjunctivitis observed in copper-deficient bovines should be investigated.

Diagnostic value of copper parameters to predict growth of suckling calves grazing native range in Argentina / Valor diagnóstico dos parâmetros de cobre para prever o crescimento de bezerros lactantes em pastagem nativas na Argentina

A study was conducted to evaluate the predictive diagnostic value of different copper (Cu) parameters as indicators of average daily gain (ADG) in growing calves. The effects in calves of cow Cu supplementation in the last one-third gestation period were also evaluated. Five supplementation trials, with a total of 300 calves, were carried out. Two groups of 30 calves were randomly assigned to each trial, one group was parenterally supplemented (SG) and the other was not supplemented (NSG). Trials began when calves were three-month-old and ended at weaning time. At each sampling calves were weighed and blood was taken to determine Cu concentrations in plasma, Whole Blood (WB), Red Cells (RC) and Packed Cell Volume (PCV). Liver samples from six animals of each group were taken both at the beginning and at the end of the trial. In two trials the mothers of the SG received Cu supplementation at the last one- third gestation period. Four of the five trials exhibited low ADG in the NSGs. In these groups, plasma Cu concentration decreased rapidly before low ADG was detected, which occurred with values remaining below 25µg/dl. The decrease of RC Cu concentration was considerably slow. WB showed an intermediate position. PCV in the SGs was higher than in the NSGs in all trials. Cow supplementation was insufficient to generate a liver storage able to last after calves reached the 3 months of age. These data could be useful to predict the risk of low ADG in grazing calves.

Foi realizado um estudo para predisser o valor diagnóstico de diferentes parâmetros de cobre (Cu) como indicadores de ganho médio diário (ADG) na criação de bezerros. Também foram avaliados os efeitos da suplementação com Cu nas vacas no último terço da gestação. Cinco ensaios de suplementação, com um total de 300 bezerros, foram realizados. Dois grupos de 30 bezerros foram atribuídos aleatoriamente em cada proba, um grupo foi parenteralmente suple mentado (SG) e o outro não foi suplementado (NSG). Os ensaios começaram quando os bezerros tinham três meses de idade e terminou ao tempo do destete. Em cada ensaio os bezerros foram pesados e mostras do sangue foi tomada para determinar as concentrações Cu no plasma, sangue total (WB), eritrócitos (RC) e hematócrito (PCV). Amostras de fígado foram colhidas em seis animais de cada grupo tanto no início quanto no final do ensaio As mães do SG receberam suplementação de Cu no último terço da gestação em dois ensaios. Quatro dos cinco ensaios apresentavam baixa ADG nos NSGs. Nesses grupos, a concentração plasmática de Cu diminuiu rapidamente antes que a baixa do ADG fossei detectada, o que ocorreu com os valores abaixo 25 µg/d. A diminuição da concentração de RC Cu foi bastante lenta. WB mostrou uma posição intermédia. PCV nos SGs foi mais elevado do que nos NSGs em todos os ensaios. A suplementação das vacas foi insuficiente para gerar um armazenamento hepático capaz de durar até os 3 meses de idade dos bezerros. Estes dados poderiam ser úteis para prever o risco de baixa ADG em bezerros em pastagem.

Moraxella bovis pathogenicity: an update.

Moraxella bovis is the etiologic agent of infectious bovine keratoconjunctivitis, the most important ocular disease affecting cattle worldwide. The severity of the cases varied from eyes that exhibited mild signs to severe clinical cases with profuse lacrimation, conjunctival swelling, corneal opacity, and ulceration. Although the mortality is low, there is a high morbidity and important economic loss in terms of significant reduction in production. This paper examines aspects such as the pathogenesis of the disease and the mechanisms by which this unique bacterium is able to disrupt the corneal epithelium and cause infection.