Telmo Nunes

Genomic and phenotypic characterization of Escherichia coli isolates recovered from the uterus of puerperal dairy cows

The role of Escherichia coli in the pathogenesis of the puerperal uterine infection of the cow is largely unknown. It is proposed that E. coli favors the persistence of Arcanobacterium pyogenes and gram-negative bacteria that are pivotal to the establishment of the infection. Here, we report the genomic and phenotypic characteristics of 72 E. coli isolates recovered from the uterus of dairy cows with normal puerperium (n = 12; 35 isolates) or clinical metritis (n = 18; 37 isolates), in an attempt to identify characteristics that are related to the establishment of uterine infection. We evaluated DNA fingerprints generated by repetitive element sequence-based PCR, phylogenetic grouping, the presence of 15 virulence factor genes, in vitro biofilm formation and its relationship to curli fimbriae expression, and cellulose production. We found a wide genetic diversity (40 clonal types), including types common to normal puerperium and clinical metritis cows (n = 6), as well as types specific to normal puerperium (n = 14) or clinical metritis (n = 20) cows. Isolates were assigned to phylogenetic groups B1 (58%), A (31%), and D (11%). Only 4 virulence factor genes were detected (hlyE, hlyA, iuc, and eaeA). In vitro biofilm formation was significantly affected by culture medium and incubation temperature. Curli fimbriae expression and cellulose production, although related to biofilm formation, were not required for it. None of the evaluated E. coli characteristics were significantly related to the establishment of the uterine infection. In conclusion, data presented in this paper indicate that E. coli isolates recovered from the uterus of puerperal cows present a wide genetic diversity, do not belong to a known pathogenic group, and have a low potential of virulence and persistence. This corroborates the putative role of the bacterium in the pathogenesis of the puerperal uterine infection of the cow.

Bluetongue virus serotype 1 outbreak in the Basque Country (Northern Spain) 2007-2008. Data support a primary vector windborne transport.

Background Bluetongue (BT) is a vector-borne disease of ruminants that has expanded its traditional global distribution in the last decade. Recently, BTV-1 emerged in Southern Spain and caused several outbreaks in livestock reaching the north of the country. The aim of this paper was to review the emergence of BTV-1 in the Basque Country (Northern Spain) during 2007 and 2008 analyzing the possibility that infected Culicoides were introduced into Basque Country by winds from the infected areas of Southern Spain. Methodology/Principal Findings We use a complex HYSPLIT (Hybrid Single-Particle Lagrangian Integrated Trajectory) model to draw wind roses and backward wind trajectories. The analysis of winds showed September 28 to October 2 as the only period for the introduction of infected midges in the Basque Country. These wind trajectories crossed through the areas affected by serotype 1 on those dates in the South of the Iberian Peninsula. Additionally meteorological data, including wind speed and humidity, and altitude along the trajectories showed suitable conditions for Culicoides survival and dispersion. Conclusions/Significance An active infection in medium-long distance regions, wind with suitable speed, altitude and trajectory, and appropriate weather can lead to outbreaks of BTV-1 by transport of Culicoides imicola, not only over the sea (as reported previously) but also over the land. This shows that an additional factor has to be taken into account for the control of the disease which is currently essentially based on the assumption that midges will only spread the virus in a series of short hops. Moreover, the epidemiological and serological data cannot rule out the involvement of other Culicoides species in the spread of the infection, especially at a local level.

In vitro isolation and seroprevalence of Toxoplasma gondii in stray cats and pigeons in Lisbon, Portugal

Oral contamination with Toxoplasma gondii oocysts shed by cats into the environment has been linked to severe outbreaks of human toxoplasmosis. Pigeons (Columba livia) are highly susceptible to oral infection with oocysts and indirectly indicate soil contamination, since they feed from the ground. A seroprevalence study was performed on cats and pigeons captured in the city of Lisbon. Serum samples collected from 1507 pigeons captured at 64 feeding sites and 423 stray cats were screened for antibodies anti-T. gondii using a commercial direct agglutination test. Seroprevalence in pigeons was 2.6% (39/1507) (95% CI: 1.9-3.5%) and 37.5% (24/64) of pigeon flocks sampled showed to be infected with T. gondii. The proportion of infected pigeons within seropositive flocks ranged between 4.8% and 21.1%. Among cats, seroprevalence was 44.2% (187/423) (95% CI: 39.5-49.1%). Isolation of T. gondii from animal tissues was attempted by in vitro assay. Inoculation of brain homogenates from 20 pigeons and 56 cats into Vero cell cultures allowed isolation of T. gondii from 13 pigeons (65%) and 15 cats (26.8%). Inoculation of muscle homogenates (heart and limbs) prepared by acid-peptic digestion from a subset of 15 cats resulted in the recovery of T. gondii from 10 cats (66.7%).

Eimeria infections in goats in Southern Portugal

Coccidiosis caused by Eimeria species is a major form of intestinal infection affecting intensively and semi-intensively reared goats. The province of Alentejo is the main goat-producing area in Portugal. Therefore, all 15 Serpentina goat farms in Alentejo were analyzed regarding the occurrence and diversity of Eimeria species. Fecal samples obtained from 144 animals (52.1% dairy goats, 47.9% pre-pubertal goats) were examined using the modified McMaster technique to determine the number of oocysts per gram of feces. Eimeria spp. oocysts were present in 98.61% of the fecal samples and, overall, nine different Eimeria species were identified. The most prevalent species were E. ninakohlyakimovae (88%) and E. arloingi (85%), followed by E. alijevi (63%) and E. caprovina (63%). The average number of oocysts shed was significantly lower in dairy goats than in pre-adult animals. Astonishingly, no clinical signs of coccidiosis were observed in any of the animals examined, even though they were shedding high numbers of oocysts and were infected with highly pathogenic species. Thus, implementation of routine diagnostic investigation of the occurrence and diversity of caprine Eimeria species may be a useful tool for determination and better understanding of their potential economic impact on goat herds in southern Portugal.

Efficacy of medical grade honey in the management of canine otitis externa – a pilot study

BACKGROUND The high prevalence of antimicrobial resistance within otic pathogens has created a need for alternative therapies of otitis externa (OE). Evidence suggests that medical grade honey (MGH) may be effective against drug-resistant pathogens. HYPOTHESIS/OBJECTIVES The efficacy of a commercial MGH compound was assessed in an open clinical trial. We hypothesized that it would be an effective alternative to conventional treatments. ANIMALS Client-owned dogs (n = 15) with a confirmed diagnosis of infectious OE were enrolled in this pilot study. METHODS Dogs were prescribed MGH (1 mL daily per ear) until cure was achieved or for a maximum of 21 d. Evaluation was based on weekly clinical scores, cytological progression and owner assessments of pruritus. Swab samples were submitted for culture and susceptibility testing. MGH was tested for biocidal activity against the bacterial isolates. RESULTS Medical grade honey promoted rapid clinical progress, with 70% of dogs achieving clinical cure between days 7 and 14 and over 90% having resolved by Day 21. There was a decrease in clinical scores throughout the duration of the trial (P < 0.001) and owner-assessed pruritus also decreased significantly (P < 0.05). In vitro assays of the biocidal activity of MGH showed activity against all bacterial isolates, including meticillin-resistant strains of Staphylococcus pseudintermedius (MRSP) and other species of drug-resistant bacteria. CONCLUSION AND CLINICAL IMPORTANCE Medical grade honey was successful in both clinical and laboratory settings, thus demonstrating its potential of becoming an alternative treatment for canine OE.

Efficacy of proactive long-term maintenance therapy of canine atopic dermatitis with 0.0584% hydrocortisone aceponate spray: a double-blind placebo controlled pilot study

BACKGROUND Long-term remission between flares of canine atopic dermatitis (CAD) can be difficult to achieve. Therefore, additional strategic forms of treatment are needed in order to target flare prevention. The concept of proactive therapy is recommended in the European guidelines for the treatment of human atopic eczema. OBJECTIVES To evaluate the efficacy of a proactive treatment regimen with a 0.0584% hydrocortisone aceponate (HCA) spray for CAD. ANIMALS Client-owned dogs with spontaneous atopic dermatitis (AD) (n = 41). METHODS This pilot study was conducted as a randomised, placebo-controlled, double-blinded clinical trial with an end-point of treatment failure. Dogs were treated once daily to remission, then randomly assigned to receive either the HCA spray (n = 21) or a placebo (n = 20) spray on two consecutive days each week. All dogs were on appropriate flea control. No topical or systemic anti-inflammatory or antimicrobial agents were permitted. Intention-to-treat analysis was used. RESULTS At Day 0, all the dogs were in remission or had mild AD based on their Canine Atopic Dermatitis Extent and Severity Index, version 3 (CADESI-03) scores. The time to relapse was significantly higher in the HCA group (median 115 d; range 31-260 d) compared to the placebo group (median 33 d; range 15-61 d) (P < 0.0001). No adverse events were attributable to the HCA spray. Four dogs were lost to follow-up and four were withdrawn after receiving prohibited medication. CONCLUSIONS AND CLINICAL IMPORTANCE These results indicate that proactive long-term therapy of CAD with an HCA spray administered on two consecutive days each week is effective and well-tolerated.

Spatial and temporal distribution of Culicoides species in mainland Portugal (2005-2010). Results of the Portuguese Entomological Surveillance Programme.

Bluetongue virus (BTV) is transmitted by Culicoides biting midges and causes an infectious, non-contagious disease of ruminants. It has been rapidly emerging in southern Europe since 1998. In mainland Portugal, strains of BTV belonging to three serotypes have been detected: BTV-10 (1956-1960), BTV-4 (2004-2006 and 2013) and BTV-1 (2007-2012). This paper describes the design, implementation and results of the Entomological Surveillance Programme covering mainland Portugal, between 2005 and 2010, including 5,650 caches. Culicoides imicola Kieffer was mostly found in central and southern regions of Portugal, although it was sporadically detected in northern latitudes. Its peak activity occurred in the autumn and it was active during the winter months in limited areas of the country. Obsoletus group was present at the highest densities in the north although they were found throughout the country in substantial numbers. Culicoides activity occurred all year round but peaked in the spring. A generalized linear mixed model was developed for the analysis of the environmental factors associated with activity of the species of Culicoides suspected vectors of BTV in the country. For C. imicola Kieffer, the most important variables were month, diurnal temperature range (DTR), the number of frost days (FRS) and median monthly temperature (TMP). For the Obsoletus group, the most important factors were month, diurnal temperature range (DTR), and linear and quadratic terms for median monthly temperature (TMP). The results reported can improve our understanding of climatic factors in Culicoides activity influencing their distribution and seasonal pattern.

Immediate after birth transmission of epidemic Salmonella enterica Typhimurium monophasic strains in pigs is a likely event

Sir, Salmonellosis remains an important foodborne disease to date and Salmonella enterica subsp. enterica serovar 4,[5],12:i:2, also known as monophasic Salmonella Typhimurium, has been rising as a cause of infection in humans and is widely spread among certain animal populations, namely in the pig reservoir. – 3 Little is known on the dynamics of transmission of this serovar and, as such, our aim in this study was to determine the likelihood of immediate after birth transmission of monophasic Salmonella Typhimurium from sow to piglet. At one large industrial pig herd in Portugal, 10 sows and 7 piglets from each sow’s litter were randomly chosen and sampled at birth. Salmonella was isolated according to the protocol described in ISO 6579:2002 Annex D and serotyped based on the Kauffmann–White–Le Minor scheme. The genus and the absence of the second-phase flagellar antigen fljB were confirmed by PCR, as recommended by the European Food Safety Authority (EFSA) Panel on Biological Hazards. To determine the likelihood of a sow carrying monophasic Salmonella Typhimurium transmitting Salmonella to her offspring, Fisher’s exact test was employed using R software (http://www.r-project.org). Susceptibility to 17 antimicrobials was determined using the disc diffusion and broth microdilution methods (VetMIC Stordjur, National Veterinary Institute, Uppsala, Sweden), and interpreted according to CLSI guideline M31-A3. CLSI M100-S21 susceptibility criteria were used for nalidixic acid. For neomycin, recommendations from the veterinary working party of the Antibiogram Committee of the French Society for Microbiology were followed. All isolates were screened for blaTEM, blaSHV, blaOXA-1, blaCTX-M, aadA, tet(A), tet(B), floR, sul1, sul2, dfrA1, qnrA, qnrB and qnrS using PCR. Plasmid extraction was performed using standard protocols and the size was determined after linearization with S1 exonuclease. Clonality was assessed by PFGE with XbaI restriction, according to the Pulsenet protocol. The patterns were analysed by BioNumerics software using the unweighted pair group method with arithmetic mean and the Dice similarity coefficient. The definition of a pulsed-field type cluster was based on a similarity cut-off value of ≥80% and a subtype on a value of ≥97%. A total of 10 sows and 70 piglets were sampled, yielding 6 sows and 17 piglets positive for Salmonella 4,[5],12:i:2. Among the positive animals, three sows and one or more of the respective piglets carried monophasic Salmonella Typhimurium (named as families A, B and D) (Table 1). All offspring from the other three positive sows were negative and one positive piglet descended from a sow negative for monophasic Salmonella Typhimurium. All Salmonella 4,[5],12:i:2 isolates from sows and their respective piglets were further characterized. Two resistance patterns were identified: 17 strains were resistant to ampicillin, neomycin, nalidixic acid, sulphonamides, streptomycin and tetracycline, and 2 strains were additionally resistant to amoxicillin/clavulanate. As for the resistance genes identified, all strains harboured blaTEM, tet(B) and sul2, except for a single piglet isolate that was negative for sul2. The plasmid linearization yielded a single small plasmid of 5.6 kbp for all isolates. All isolates belonged to a unique pulsed-field type cluster. Furthermore, 11 strains shared a similar profile representing a subtype with .97% similarity when a 2.5% band tolerance setting was used (see Figure S1, available as Supplementary data at JAC Online). There was a higher probability of a sow positive for monophasic Salmonella Typhimurium having a positive offspring with the same serovar (OR1⁄416.10, P1⁄40.001). Accordingly, the probability of a piglet having monophasic Salmonella Typhimurium was 16 times higher if it belonged to a positive sow. Several researchers have reported a high Salmonella prevalence in sows and their respective piglets early in life. Beyond the food safety risk when the sow ultimately enters the food chain, information on vertical transmission from the sow to her offspring is scarce. One study reported the contradictory fact that Salmonella serovars isolated from a sow often differed from those isolated from her piglets. In this study, the similarity of the resistance patterns, the antimicrobial resistance genes detected, the plasmid profiles and the PFGE types support the transmission of monophasic Salmonella Typhimurium from sow to piglet during or immediately after birth as a means of Salmonella colonization of the newborn’s gut. To the best of our knowledge, this constitutes the first report of mother-to-piglet horizontal transmission of monophasic Salmonella Typhimurium in pigs. The antimicrobial resistance core profile was similar to

Understanding the inguinal sinus in sheep (Ovis aries)—Morphology, secretion, and expression of progesterone, estrogens, and prolactin receptors

Post-parturient behavior of mammalian females is essential for early parent–offspring contact. After delivery, lambs need to ingest colostrum for obtaining the related immunological protection, and early interactions between the mother and the lamb are crucial. Despite visual and auditory cues, olfactory cues are decisive in lamb orientation to the mammary gland. In sheep, the inguinal sinus is located bilaterally near the mammary gland as a skin pouch (IGS) that presents a gland that secretes a strong-smelling wax. Sheep IGS gland functions have many aspects under evaluation. The objective of the present study was to evaluate sheep IGS gland functional aspects and mRNA transcription and the protein expression of several hormone receptors, such as progesterone receptor (PGR), estrogen receptor 1 (ESR1), and 2 (ESR2) and prolactin receptor (PRLR) present. In addition, another aim was to achieve information about IGS ultrastructure and chemical compounds produced in this gland. All hormone receptors evaluated show expression in IGS during the estrous cycle (follicular/luteal phases), pregnancy, and the post-partum period. IGS secretion is rich in triterpenoids that totally differ from the surrounding skin. They might be essential substances for the development of an olfactory preference of newborns to their mothers.

Morphological Aspects and Expression of Estrogen and Progesterone Receptors in the Interdigital Sinus in Cyclic Ewes

Many species that belong to Artiodactyls order show an interdigital sinus (IS), as it occurs in sheep, in all four extremities. These are considered to be scent glands responsible for sexual communication having strong attractiveness to mature males at the peak of the breeding season. The aim of this study was to evaluate, in IS in cyclic ewes, the microscopic and ultrastructure (scanning and transmission electron microscopy) anatomy, secretion composition, and mRNA and protein expression of estrogen receptors α and β and progesterone receptors. Glandular sebaceous structures occupy a superficial area of the pouch. The other glands present in the IS show a coiled tubular structure and tall and polyhedral secretory cells with irregular luminal surface resulting from the secretory process. Protein and mRNA gene transcription studies were performed to determine the presence of ER (α and β) and P4r in IS. At the follicular phase, IS cell populations analyzed using flow cytometry expressed higher levels of ERβ compared with ERα (P < 0.05), whereas no difference was observed between them in the luteal phase. The IS amount of secretion was the highest in the follicular phase compared with luteal phase (P < 0.05) or pregnancy (P < 0.001).To the best of our knowledge, for the first time, the presence of ER (α and β) within the IS was demonstrated. As estrogen action is mediated by specific receptors in target cells, the presence of these receptors in IS might be needed to trigger signaling pathways involved in conspecific chemical (sexual) communication attributed to this area. Microsc. Res. Tech. 77:313–325, 2014.