Rickettsia in Synanthropic and Domestic Animals and Their Hosts from Two Areas of Low Endemicity for Brazilian Spotted Fever in the Eastern Region of Minas Gerais, Brazil

The aim of this study was to understand the current epidemiology of rickettsial diseases in two rickettsial-endemic regions in Brazil. In the municipalities of Pingo D`Agua and Santa Cruz do Escalvado, among serum samples obtained from horses and dogs, reactivity by immunofluorescent assay against spotted fever group rickettsiae was verified. In some serum samples from opossums (Didelphis aurita) captured in Santa Cruz do Escalvado, serologic response against rickettsiae was also verified. Polymerase chain reaction identified rickettsiae only in ticks and fleas obtained in Santa Cruz do Escalvado. Rickettsiae in samples had 100% sequence homology with Rickettsia fells. These results highlight the importance of marsupials in maintenance of the sylvatic cycle of rickettsial disease and potential integration with the domestic cycle. Our data also support the importance of horses and dogs as sentinels in monitoring circulation of rickettsiae in an urban area.

Multiplex semi-nested RT-PCR with exogenous internal control for simultaneous detection of bovine coronavirus and group A rotavirus

Neonatal calf diarrhea is a multi-etiology syndrome of cattle and direct detection of the two major agents of the syndrome, group A rotavirus and Bovine coronavirus (BCoV) is hampered by their fastidious growth in cell culture. This study aimed at developing a multiplex semi-nested RT-PCR for simultaneous detection of BCoV (N gene) and group A rotavirus (VP1 gene) with the addition of an internal control (mRNA ND5). The assay was tested in 75 bovine feces samples tested previously for rotavirus using PAGE and for BCoV using nested RT-PCR targeted to RdRp gene. Agreement with reference tests was optimal for BCoV (kappa = 0.833) and substantial for rotavirus detection (kappa = 0.648). the internal control, ND5 mRNA, was detected successfully in all reactions. Results demonstrated that this multiplex semi-nested RT-PCR was effective in the detection of BCoV and rotavirus, with high sensitivity and specificity for simultaneous detection of both viruses at a lower cost, providing an important tool for studies on the etiology of diarrhea in cattle. (C) 2010 Elsevier B.V. All rights reserved.

Modeling the Dynamics of Viral Evolution Considering Competition Within Individual Hosts and at Population Level: The Effects of Treatment

We consider two viral strains competing against each other within individual hosts (at cellular level) and at population level (for infecting hosts) by studying two cases. In the first case, the strains do not mutate into each other. In this case, we found that each individual in the population can be infected by only one strain and that co-existence in the population is possible only when the strain that has the greater basic intracellular reproduction number, R (0c) , has the smaller population number R (0p) . Treatment against the one strain shifts the population equilibrium toward the other strain in a complicated way (see Appendix B). In the second case, we assume that the strain that has the greater intracellular number R (0c) can mutate into the other strain. In this case, individual hosts can be simultaneously infected by both strains (co-existence within the host). Treatment shifts the prevalence of the two strains within the hosts, depending on the mortality induced by the treatment, which is, in turn, dependent upon the doses given to each individual. The relative proportions of the strains at the population level, under treatment, depend both on the relative proportions within the hosts (which is determined by the dosage of treatment) and on the number of individuals treated per unit time, that is, the rate of treatment. Implications for cases of real diseases are briefly discussed.

In vitro algaecide effect of sodium hypochlorite and iodine based antiseptics on Prototheca zopfii strains isolated from bovine milk

Prototheca zopfii has been considered one of the most important causes of environmental mastitis in Brazil. These algae are refractory to conventional therapy and cause great damage to the mammary gland. The present study evaluated the in vitro algaecide effect of sodium hypochlorite and iodine based antiseptics on 27 P. zopfii strains isolated from the milk of cattle. Low concentrations of sodium hypochlorite (0.0390625-0.15625%) and iodine (0.15625-0.625%) were effective against the isolates. These antiseptics may be recommended for hygiene routines, pre and postdipping and cauterization of bovine mammary glands infected by P. zopfii. (C) 2009 Elsevier Ltd. All rights reserved.

Low genetic diversities of rabies virus populations within different hosts in Brazil

The low rates of nonsynonymous evolution observed in natural rabies virus (RABV) isolates are suggested to have arisen in association with the structural and functional constraints operating on the virus protein and the infection strategies employed by RABV within infected hosts to avoid strong selection by the immune response. In order to investigate the relationship between the genetic characteristics of RABV populations within hosts and the virus evolution, the present study examined the genetic heterogeneities of RABV populations within naturally infected dogs and foxes in Brazil, as well as those of bat RABV populations that were passaged once in suckling mice. Sequence analyses of complete RABV glycoprotein (G) genes showed that RABV populations within infected hosts were genetically highly homogeneous whether they were infected naturally or experimentally (nucleotide diversities of 0-0.95 x 10(-3)). In addition, amino acid mutations were randomly distributed over the entire region of the G protein, and the nonsynonymous/synonymous rate ratios (d(N)/d(S)) for the G protein gene were less than 1. These findings suggest that the low genetic diversities of RABV populations within hosts reflect the stabilizing selection operating on the virus, the infection strategies of the virus, and eventually, the evolutionary patterns of the virus. (C) 2009 Elsevier B.V. All rights reserved.

Correlation between mastitis occurrence and the count of microorganisms in bulk raw milk of bovine dairy herds in four selective culture media

Milk is the normal secretion of the mammary gland, practically free of colostrum and obtained by the complete milking of oil(? or more healthy animals. Mastitis is all inflammatory process of the mammary gland and it may cause alterations in the milk. The present work aimed to verify whether it is possible, by means of the counts of microorganism in the bulk raw milk ill four selective culture media, to establish a correlation with the occurrence of mastitis and therefore, to monitor this disease in bovine dairy herds. The following selective culture media were Used: KF Streptococcus Agar, Edwards Agar, Baird-Parker Agar, Blood Agar plus potassium tellurite. Spearman`s correlation coefficient was calculated in order to compare the Occurrence of mastitis (percentage) in each herd with respective selective culture media counts of microorganisms in bulk raw milk. Thirty-six possibilities were analysed (Tamis and CMT-positive rates were compared with the log-transformed count in four selective Culture media) and there was a negative correlation between Tamis 3 and the Baird-Parker Agar plate count. The total results of microbiological tests showed that there were three correlations of the counts in selective culture media. Fifty-two possibilities were analysed and there was a negative correlation between no-bacteria I-growth mastitis rates and log(10) of KF Streptoccocus Agar plate Count and there were two positive correlations between coagulase-positive staphylococci and log(10) of Baird-Parker Agar plate count and Blood Agar plus potassium tellurite plate count.

Inquiry of antibodies anti-Neospora caninum in dairy cattle, dogs and rural workers of the south-west region of Mato Grosso State

Considering the importance of neosporosis in the animal health and production, the frequency of antibodies to Neospora caninum was evaluated in dairy cattle of the Southwestern region of Mato Grosso State, Brazil, in addition to serum samples obtained from dogs and humans living in the farms. A total of 1036 serum samples were analyzed, from which 932 were from dairy bovine females, 37 from dogs and 67 from humans, from 24 farms and examined by the indirect fluorescent antibody test (IFAT). Reactive human scrum samples were retested by Western-blotting to confirm the results. Antibodies to N. caninum were found in 499 cattle sera (53.5 %), with at least one positive in each farm, 25 dog sera (67.6 %) and seven human sera (10.5 %). There was no significant difference in the number of positive cattle sera according to age group. The results indicate a wide dissemination of N caninum in the studied region.

Crab-eating fox (Cerdocyon thous), a South American canid, as a definitive host for Hammondia heydorni

Hammondia heydorni is a cyst forming coccidia closely related to other apicomplexans, such as Toxoplasma gondii, Neospora caninum and Hammondia hammondi with a two-host life cycle. Dogs and other canids as red foxes (Vulpes vulpes) and coyotes (Canis latrans) may serve as definitive hosts for H. heydorni. Sporulated oocysts are infective for cattle, sheep and goats, which may serve as intermediate hosts. Herein, we describe the ability of crab-eating fox (Cerdocyon thous), a wild carnivore that is commonly found from northern Argentina to northern South America, to serve as definitive host of H. heydorni. The whole masseter muscle and brain from two 2-year-old bovines were collected, minced and pooled together for the fox infection. The bovine pooled tissues were equally administered to four foxes, in two consecutive days. Two foxes shed subspherical unsporulated oocysts measuring 10-15 mu m, after 8 and 9 days post-infection, respectively. One of the foxes eliminated oocysts for 5 days, while the other fox shed oocysts for 9 days. A DNA sample of oocysts detected at each day of oocyst elimination was tested by two PCRs, one of them carried out employing primers directed to the common toxoplasmatiid 18S and 5.8S ribosomal RNA coding genes (PCR-ITS1) and the other based on heat-shock protein 70 kDa coding gene (PCR-HSP70). These samples were also submitted to a N. caninum specific nested-PCR protocol based on a N. caninum specific gene (Nc5-nPCR). All of them were positive by PCR-ITS1 and PCR-HSP70 but negative by Nc5-nPCR. The PCR-ITS1 and PCR-HSP70 nucleotide sequences amplified from the oocysts shed by the foxes revealed 100% identity with homologous sequences of H. heydorni. In conclusion, it is clear that H. heydorni also uses the crab-eating fox as a definitive host. The crab-eating fox is usually reported to live in close contact with livestock in several regions of Brazil. Therefore, it is reasonable to infer that such carnivores may play an important role in the sylvatic and domestic cycles of H. heydorni infection. (C) 2009 Elsevier B.V. All rights reserved.

Effect of Recombinant Bovine Somatotropin on Plasma Concentrations of Insulin-like Growth Factor I, Insulin and Membrane Integrity of Bull Spermatozoa

Contents This study aimed to evaluate the effect of the exogenous recombinant bovine somatotropin (rbST) on plasma concentrations of insulin-like growth factor I (IGF-I), insulin and semen quality of bulls. Twenty bulls (Aberdeen Angus and Brangus) were divided by breed into two groups. Placebo group was injected with NaCl 0.9% (s.c.) and treatment group with rbST (s.c., 500 mg) at days 0 and 14 of the experiment. Immediately after semen collection, blood samples were taken on days 0, 14, 28, 42 and 56 of the experiment. Semen was also collected on day 70 of the experiment. Evaluation of sperm motility was performed at pre-freezing and post-thawing stage, whereas assessment of sperm membrane integrity was performed after freezing and thawing. Analysis of data revealed that the effect of treatment and treatment-by-collection day on plasma concentrations of IGF-I and insulin was not significant. However, mean plasma concentrations of IGF-I and insulin were affected (p < 0.0001) by days of blood sampling. Effect of treatment and treatment-by-collection day on motility of spermatozoa was similar (p > 0.05) at pre-freezing and post-thawing stage. Intactness of plasmalemma and tail membrane of spermatozoa at post-thawing stage was higher (p < 0.05) in rbST-treated group than in control. In conclusion, rbST did not affect plasma concentrations of IGF-I and insulin, however, it did improve post-thaw sperm membrane integrity.

Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation

Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 X 10(6) cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 degrees C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P < 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced. (C) 2010 Elsevier Inc. All rights reserved.

Neither bovine somatotropin nor growth hormone-releasing factor alters expression of thyroid hormone receptors in liver and mammary tissues

Physiological effects of thyroid hormones are mediated primarily by binding of triiodothyronine to specific nuclear receptors. Organ-specific changes in production of triiodothyronine from its prohormone, thyroxine, have been hypothesized to target the action of thyroid hormones on the mammary gland and play a role in mediating or augmenting a galactopoietic response to bovine somatotropin (bST). Additionally, tissue responsiveness to thyroid hormones may be altered by changes in the number or affinity of nuclear receptors for thyroid hormones. In the present study, effects of bST and bovine growth hormone-releasing factor (bGRF) on thyroid hormone receptors in liver and mammary gland were studied. Lactating Holstein cows received continuous infusions of bST or bGRF for 63 d or served as uninfused controls. Nuclei were isolated from harvested mammary and liver tissues and incubated with [(125)I]-triiodothyronine. Treatments did not alter the capacity or affinity of specific binding sites for triiodothyronine in liver or mammary nuclei. Evaluation of transcript abundance for thyroid hormone receptors showed that isoforms of thyroid hormone receptor or retinoid receptor (which may influence thyroid receptor action) expressed in the mammary gland were not altered by bST or bGRF treatment. Data do not support the hypothesis that administration of bST or bGRF alters sensitivity of mammary tissue by changing expression of thyroid hormone receptors.

Effect of Antioxidants During Bovine In Vitro Fertilization Procedures on Spermatozoa and Embryo Development

Increased amounts of reactive oxygen species (ROS) during in vitro fertilization (IVF) may cause cytotoxic damage to gametes, whereas small amounts of ROS favour sperm capacitation. The aim of this study was to investigate the effect of antioxidants [50 mu M beta-mercaptoethanol (beta-ME) and 50 mu M cysteamine (Cyst)] or a pro-oxidant (5 mm buthionine sulfoximine) on the quality and penetrability of spermatozoa into bovine oocytes and on the subsequent embryo development and quality when added during IVF. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes, and mitochondrial function, was diminished (p < 0.05) after 4-h culture in the presence of antioxidants. Oocyte penetration rates were similar between treatments (p > 0.05), but antioxidants adversely affected the normal pronuclear formation rates (p < 0.05). The incidence of polyspermy was high for beta-ME (p < 0.05). No differences were observed in cleavage rates between treatments (p > 0.05). However, the developmental rate to the blastocyst stage was adversely affected by Cyst treatment (p < 0.05). The quality of embryos that reached the blastocyst stage, evaluated by total, inner cell mass (ICM) and trophectoderm cell numbers and ICM/total cell ratio was unaffected (p > 0.05) by treatments. The results indicate that ROS play a role in the fertilizing capacity in bovine spermatozoa, as well as in the interaction between the spermatozoa and the oocytes. It can be concluded that supplementation with antioxidants during IVF procedures impairs sperm quality, normal pronuclear formation and embryo development to the blastocyst stage.

Bovine embryo transfer recipient synchronisation and management in tropical environments

Numerous studies have shown that it is possible to manipulate follicular and luteal dynamics, thereby eliminating the need for oestrus detection in embryo transfer (ET) programmes. Fixed-time ET (FTET) protocols are based on the use of gonadotrophin-releasing hormone (GnRH) and prostaglandin (PG) F or progesterone/progestogen (P4)-releasing devices and oestradiol. The FTET protocols increases the proportion of recipients transferred, and therefore pregnancy rates, compared with the use of PGF followed by ET 7 days after oestrus. Furthermore, the addition of equine chorionic gonadotrophin (eCG) to the P4 and oestradiol-based FTET protocols results in an even higher proportion of recipients transferred, and thus higher pregnancy rates. The beneficial effect of eCG treatment may be related to increased growth of the dominant follicle and increased plasma P4 concentrations during the subsequent luteal phase. In Bos taurus x Bos indicus recipients, pregnancy rates were positively correlated with the diameter of the corpus luteum (CL) and the number of CL at ET. When repeat-breeder Holstein cows were used as recipients, FTET protocols increased number of recipients transferred and pregnancy rates compared with the traditional PGF-based synchronisation protocols. In conclusion, the use of FTET protocols eliminates the need for the detection of oestrus and results in a greater proportion of recipients transferred and satisfactory pregnancy rates. Thus, FTET optimises the use of recipients, reducing labour and animal handling and facilitating the use of ET.

Xenooplasmic Transfer between Buffalo and Bovine Enables Development of Homoplasmic Offspring

Nuclear-mitochondrial incompatibilities may be responsible for the development failure reported in embryos and fetuses produced by interspecies somatic cell nuclear transfer (iSCNT). Herein we performed xenooplasmic transfer (XOT) by introducing 10 to 15% of buffalo ooplasm into bovine zygotes to assess its effect on the persistence of buffalo mitochondrial DNA (mtDNA). Blastocyst rates were not compromised by XOT in comparison to both in vitro fertilized embryos and embryos produced by transfer of bovine ooplasm into bovine zygotes. Moreover, offspring were born after transfer of XOT embryos to recipient cows. Buffalo mtDNA introduced in zygotes was still present at the blastocyst stage (8.3 vs. 9.3%, p = 0.11), indicating unaltered heteroplasmy during early development. Nonetheless, no vestige of buffalo mtDNA was found in offspring, indicating a drift to homoplasmy during later stages of development. In conclusion, we show that the buffalo mtDNA introduced by XOT into a bovine zygote do not compromise embryo development. On the other hand, buffalo mtDNA was not inherited by offspring indicating a possible failure in the process of interspecies mtDNA replication.

Influence of Osteopontin in Bovine Uterine Tube Fluid on Sperm Binding and Fertilization in RCA-1 Lectin-treated Oocytes

This study was conducted to determine the effect of pre-exposure of oocytes to Ricinus communis (RCA-1) lectin and osteopontin (OPN) in uterine tube fluid (UTF) on in vitro sperm-egg binding and fertilization. In vitro-matured bovine oocytes were incubated (39 degrees C, 5% CO(2) in air) for 2 h in the following treatments: (i) 500 mu l of fertilization medium (FM); (ii) 250 mu l of FM with 0.25 ml of non-luteal ampullary uterine tube fluid (NLAUTF); (iii) 250 mu l of FM with 250 mu l of NLAUTF and 4 mu l of RCA-1 lectin; (iv) 250 mu l of FM with 250 mu l of NLAUTF, a rabbit polyclonal antibody (1:200) against purified bovine milk OPN, and RCA-1 lectin; (v) 500 mu l of FM and RCA-1 lectin. Following incubation, oocytes were washed, placed in FM with 2 mu g heparin, and incubated with 1 x 10(5) frozen-thawed spermatozoa per 10 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zona pellucida counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zona pellucida (mean +/- SEM) when oocytes were incubated in treatment 3 (59.0 +/- 5.5) than in treatments 2 (46.4 +/- 5.6), 4 (18.1 +/- 5.4), 5 (33.4 +/- 5.6) or 1 (32.5 +/- 5.6). More oocytes were fertilized when incubated in treatment 3 (91% +/- 3.0) than in 2 (84% +/- 3.0), 4 (40% +/- 3.0), 5 (77% +/- 3.0) or 1 (76% +/- 3.0). As in previous studies, this study suggests that RCA-1 lectin enhances binding of UTF-derived OPN to bovine oocytes, resulting in increased sperm-egg binding and fertilization in vitro and a possible role in fertilization.