In vitro bacteriological effect of tri-beveled needle electrolysis against Staphylococcus aureus.

Percutaneous needle electrolysis using tri-beveled needles with a specific protocol (5 mA applied for 25 s) has demonstrated to provoke a clinical reduction of recurrent bacterial infections in mammary fistulas. However, the bactericidal effect of needle electrolysis in this pathology remains theoretical. This in vitro study evaluated the bactericidal effect of this protocol and whether it changed when introducing small variations. Staphylococcus aureus were generated in saline solution (9 Log10 CFU/mL) and treated in three different experiments including the main protocol and introducing variations in needle gauge, intensity, and total dosage, respectively. After 24 h, the viable cell count showed that the protocol had an average reduction of 5 log10 CFU/ml compared to the control group. While variations in needle gauge did not modify this effect, variations in current intensity or dosage did. This study demonstrated that the bacterial effect was greater by increasing either current intensity or total dosage, and it decreased with substantial reductions of these parameters.

Effect of Female Sex Hormones on the Immune Response against Chlamydia abortus and on Protection Conferred by an Inactivated Experimental Vaccine in a Mouse Model.

Mice are valuable models extensively used to test vaccine candidates against Chlamydia abortus and to clarify immunopathological mechanisms of the bacteria. As this pathogen has the ability to reactivate during pregnancy, it is important to deepen the knowledge and understanding of some of the effects of female hormones on immunity and vaccination. This study is aimed at describing the role of sex hormones in the pathology of OEA during chlamydial clearance using ovariectomised mice and also gaining an understanding of how 17ß-oestradiol or progesterone may impact the effectiveness of vaccination. Animals were treated with sex hormones and infected with C. abortus, and the kinetics of infection and immune response were analysed by means of bacterial isolation, histopathology, and immunohistochemistry. In a second phase of the study, protection conferred by an experimental vaccine after hormone treatment was assessed. Oestradiol showed a stimulatory effect on the immune response during infection, with a more efficient recruitment of macrophages and T-cells at the infection site. Furthermore, after vaccination, oestradiol-treated animals showed a stronger protection against infection, indicating that this hormone has a positive effect, stimulating a specific memory response to the pathogen.

Synergistic antimicrobial effect of photodynamic therapy and chitosan on the titanium-adherent biofilms of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa: An in vitro study.

BACKGROUND: To date, no studies on the combined use of photodynamic therapy (PDT) and chitosan against peri-implantitis have been published. The aim of this study was to evaluate the possible synergistic antimicrobial effect of PDT and chitosan on the titanium-adherent biofilms of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. METHODS: A total of 60 titanium discs were included in this study. The discs were randomized into three bacterial contaminations (n = 20 discs per bacterium). After being cultured (incubated for 48 hours) they were randomized again into four different disinfection modalities (n = 5 discs per treatment): control (without treatment), PDT, chitosan 3 mg/mL, and PDT + chitosan 3 mg/mL. After the treatments, the colony forming units (CFU) were measured to determine antimicrobial effects, and field emission scanning electron microscopy (FESEM) was used to study cell morphology and titanium adherence. RESULTS: For all the evaluated bacteria and all the variables studied the order from highest to lowest antimicrobial effectiveness was: PDT + chitosan 3 mg/mL > chitosan 3 mg/mL > PDT > control. Although, all disinfection methods were significantly effective when compared to control, the combined treatment of PDT + chitosan 3 mg/mL had the greatest antimicrobial effect against the three studied bacteria. CONCLUSIONS: The combination of PDT and chitosan has a synergistic antimicrobial effect against the bacteria S. aureus, E. coli, and P. aeruginosa, all closely related to peri-implantitis. However, further in vivo studies are needed because this study provides data based on an in vitro scenario that might not be extrapolated to patients with peri-implantitis.

Galvanic current dosage and bacterial concentration are determinants of the bactericidal effect of percutaneous needle electrolysis: an in vitro study.

Percutaneous needle electrolysis (PNE) is a physiotherapy technique that has been shown to be effective in different pathologies such as tendinopathies or mammary fistula. For many years, theoretical bactericidal and germicidal effects have been attributed to this type of galvanic currents, partly explained by the changes in pH that it generates. However, these effects have not yet been demonstrated. The aim of this study was to evaluate the bactericidal effect and the changes in pH caused by PNE. S. aureus were prepared in two different solutions (TSB and saline solution) and in different concentrations (from 9 to 6 Log10 CFU/mL). Bacteria were treated with three experimental PNE doses to assess bacterial death levels and the changes caused to the pH of the medium. The viable cell count showed that all experimental PNE doses had a bactericidal effect against a high concentration (9 Log10 CFU/mL) of S. aureus in saline solution (p < 0.001). Furthermore, we found that when the concentration of bacteria decreased, a lower dose of galvanic current generated the same effect as a higher dose. Changes in pH were registered only in experiments performed with saline solution. PNE had a bactericidal effect against S. aureus and the level of this effect was mainly modulated by the solution, the bacterial concentration and the dose. Changes affecting pH were modulated by the type of solution and there was no relationship between this and bacterial death.

Evaluation of the Efficacy of a New Commercially Available Inactivated Vaccine Against Ovine Enzootic Abortion.

Ovine enzootic abortion (OEA), caused by Chlamydia abortus, is an economically important disease in many countries. Inactivated vaccines have been used for many years as they induce immunity in sheep, although outbreaks of abortions have been described in vaccinated flocks. In addition, there is a commercially available live attenuated vaccine that provides good protective results. Recently however, reports question the attenuation of this vaccine and associate it with the appearance of outbreaks of OEA in vaccinated flocks. In the present study, a recently commercialized inactivated vaccine (INMEVA®; Laboratorios Hipra S.A., Amer, Spain) has been evaluated using mouse and sheep experimental models. In the mouse models (non-pregnant and pregnant models), the efficacy of INMEVA vaccine has been compared to an unvaccinated control group and to an experimental inactivated vaccine considered as a positive protection control (UMU vaccine). In the non- pregnant model, the UMU vaccine was more effective than the INMEVA vaccine regarding the impact on body weight or the presence of C. abortus in the liver, but both vaccinated groups (UMU and INMEVA) had significantly lower C. abortus in the liver compared to the control group. In the pregnant model in terms of reproductive failures, pups per mouse or the presence of C. abortus in the liver or uterus, no significant differences were found between both vaccines, inducing protection compared to the control group. In the ovine pregnant model, where INMEVA vaccine was compared only to an unvaccinated group, the results indicate that this new commercial vaccine is safe and provides a suitable level of protection against an experimental challenge with C. abortus. A 75% reduction in reproductive disorders, 55% reduction in animals with C. abortus shedding on day of parturition/abortion, and a significant reduction of the average amount of chlamydial shedding from parturition/abortion over the next 21 days was observed, in relation to the infected control group. The results suggest that this vaccine is adequate for the control and prevention of OEA; however, future studies are necessary to elucidate the type of protective immune response that it induces.

Salmonella seroprevalence in wild boar from Southeast Spain depends on host population density.

Salmonellosis is the second most prevalent zoonosis in Europe and it has considerable economic and health implications for its monitoring and control as well as being among the most prevalent pathogens on livestock farms. The wild boar (Sus scrofa) has been identified as a competent host and spreader of Salmonella spp. There has been a significant increase in wild boar population in Europe in recent decades, and it is even present in urban areas. This study evaluates the spatial distribution of the seroprevalence of Salmonella spp. in wild boar from Murcia (Southeast Spain) and its relationship with host-related risk factors (sex, age, location and density). The presence of antibodies against S. Typhimurium and Choleraesuis in 269 serum of wild boars hunted in Murcia between 2015 and 2019 were analyzed using a commercial ELISA test (PrioCHECK porcine Salmonella kit). The seroprevalence were spatially distributed using Kernel function, and wild boar density using Gaussian kernel estimates (spatialEco version 1.1.1). The risk function was estimated as the ratio between the intensity of positive samples and the wild boar density The overall seroprevalence was 19.3% (IC95% 16.9-21.8), showing a significant spatial aggregation. The highest seroprevalence detected was 51.8% (IC95% 42.2-61.5) in a specific area with high risk of infection (76-100%) and was related to the wild boar density. Only marginal differences were detected for sex and age. The use of ELISA combined with QGIS (version 3.6.0) has allowed the identification of areas of Salmonella occurrence associated with high density as risk factor.

Canine distemper virus may affect European wild cat populations in Central Spain.

The main objective of this brief communication is to inform about the exposure to certain pathogens of interest for mesocarnivores in wildcats (Felis silvestris silvestris) that inhabit a human-domestic-wild ecotone located in a Natural Park (Serranía de Cuenca, Central Spain). Blood and mucosal swabs (nasal, conjunctival and rectal) samples were collected from nine alive animals to detect canine distemper virus (CDV), parvovirus (CPV/FPV), feline leukaemia virus (FeLV), feline coronavirus (FCoV), feline immunodeficiency virus (FIV), Leptospira interrogans, Chlamydia felis, Ehrlichia canis, Toxoplasma gondii, and Neospora caninum. ELISA, immunochromatograpy, microscopy agglutination test and PCR assays were used. The results show the first worldwide detection of exposure of wildcats to L. interrogans (3 positive/9 analysed) and the first detection of exposure to CVD (7/9), of carriers ofC felis (2/9) and of fecal spreading of CPV-FPV (2/9) in wildcats in Spain. Exposure to T. gondii and CPV-FPV was detected in 5 of the 9 wildcats analysed, and to FelV in 4 of 9. No FIV, FCoV, Ehrlichia canis and Neospora caninum were detected. The results reveal the circulation of pathogens among the wildcat population studied, but more vigilance is needed for an accurate assessment of the impact of these pathogens on the health status of this population.

Occurrence of Anisakis and Hysterothylacium larvae in commercial fish from Balearic Sea (Western Mediterranean Sea).

This study investigates the occurrence of anisakids and raphidascarids in commercial fish from Balearic Sea (Western Mediterranean). A total of 335 fish including 19 black anglerfish (Lophius budegassa), 33 white anglerfish (L. piscatorius), 129 European hake (Merluccius merluccius), 30 red mullet (Mullus barbatus), and 124 striped mullet (M. surmuletus) were examined using enzymatic digestion. A total of 948 nematode larvae were isolated (prevalence 52.53%) being the highest prevalence observed in striped mullet. Forty-six larvae were identified using molecular analyses which included PCR and sequencing of the 629-bp fragment of mitochondrial cox2 gene region. Anisakis pegreffii (80.43%), A. physeteris (8.69%), Hysterothylacium fabri (6.52%), and A. simplex (4.35%) were detected based on molecular analyses of larvae. Total nematode prevalence was positively correlated with weight, length, condition factor, and maturity stage of the host and also with fishing ground depth. Statistical differences between total nematode prevalence and geographical sector of capture were observed when fishing hauls were grouped according to the abundance of sperm whales or common bottlenose dolphins. The results also corroborate that fishing water depth may play an important role in anisakid and raphidascarid parasitization.

Babesia microti-like piroplasm (syn. Babesia vulpes) infection in red foxes (Vulpes vulpes) in NW Spain (Galicia) and its relationship with Ixodes hexagonus.

Piroplasmosis is caused by several species of protozoa such as the Babesia microti-like piroplasm (Bml), an emerging blood protozoan also known as Theileria annae or Babesia vulpes. Infection by Bml was first reported in dogs in Spain where it is endemic today. Recently, a high prevalence of Bml has been increasingly detected in red foxes (Vulpes vulpes) in European countries. The objective of this study was to determine infection levels of this parasite in foxes from Galicia, NW Spain, and ticks species infestation in these carnivores, where they are so far unknown. Samples of blood, spleen and ticks (if present) were taken from 237 hunted red foxes in the Galicia region. Blood smears were prepared for direct parasite observation, and spleen and tick samples were examined by nested PCR. Prevalences of Bml infection in Galician red foxes were estimated at 72% (171/237) by PCR and 38.23% (26/68) by direct observation. Among 837 ticks collected, the main tick identified was Ixodes hexagonus (present in 82.4% of the foxes) followed by Ixodes ricinus (12.3%), Dermacentor reticulatus (12.3%) and Rhipicephalus sanguineus sensu lato (3.5%). From 34 foxes testing positive for Bml, 616 ticks were collected: positive Bml PCR results were obtained in 55.6% (227/408) of ticks collected from 9 foxes, while the 208 ticks from the remaining 25 infected foxes returned negative PCR results. Given that canine piroplasmosis is endemic in this area, our observations point to the red fox as the main reservoir for Bml infection and the high proportion of I. hexagonus among ticks collected from red foxes suggests its likely role as vectors of B. microti-like piroplasm in this region. Further studies are needed for a better understanding of the link between the wild and domestic life cycles of this piroplasm.

On the possible role of ticks in the eco-epidemiology of Coxiella burnetii in a Mediterranean ecosystem.

Ruminant livestock is the main reservoir of Coxiella burnetii (Cb), but little is known about the role of wildlife and ticks in its epidemiology. The Iberian ibex (Capra pyrenaica, Schinz 1838) population of "Ports de Tortosa i Beseit" (NE Spain) suffers intense tick infestations and low reproduction rates. This study aims to (1) assess the relationship between infection in ibexes (detection of serum antibodies and/or of Cb DNA in tissues) and Cb DNA presence in ticks hosted by the same ibexes; and (2) identify Cb associated risk factors. Between 2011 and 2015, serum (n = 130), spleen (n = 72), lymph node (n = 89) and tick (n = 669) samples from 134 hunter-harvested ibexes were collected. Antibody detection was performed by ELISA and Cb DNA presence was assessed by PCR. Potential risk factors were assessed with regression tree models. Although 30% of the ibexes (39/130; 95%CI, [10%-29.8%]) had antibodies, Cb DNA was detected in only 9.8% of the ibexes (11/112; 95%CI [7.6%-27.25%]). The prevalence of Cb-carrier ticks averaged 10% and exceeded 20% for the genus Haemaphysalis. However, lacking correlation between infection in ibexes and their ticks does not support tick-to-ibex transmission or vice versa. Tree modelling points to host, population and environmental factors as drivers of Cb infection in ticks and suggests connections with the domestic cycle. The percentage of Cb-carrier ticks detected is noteworthy. Along with heavy tick infestations, it suggests vector potential for these tick species, especially for the genera Rhipicephalus and Haemaphysalis. Since vector competence has not been assessed in these tick species, a classic vector role cannot be proposed nor discarded, but promoter factors of vector capacity occur. In addition, the risk of tick-borne infection through tick excreta should not be neglected. While the airborne route is the preeminent route for Cb infection, ticks' contribution to Cb epidemiology deserves further attention.

Development of an in vivo model of Chlamydia abortus chronic infection in mice overexpressing IL-10.

Chlamydia abortus, like other members of the family Chlamydiaceae, have a unique intracellular developmental cycle that is characterized by its chronic nature. Infection of a flock can remain undetected for months, until abortion occurs the following reproductive season but, to date, neither the location nor the mechanisms that maintain this latent phase are fully understood. Studies have shown that IL-10 produced as a response to certain micro-organisms sustains the intracellular survival of pathogens and increases host susceptibility to chlamydial infections. In order to induce a sustained infection C. abortus, transgenic mice that constitutively express IL-10 were infected and the immunological mechanisms that maintain infection in these mice were compared with the mechanisms of a resistant wild-type mouse strain. Viable bacteria could be detected in different tissues of transgenic mice up to 28 days after infection, as analysed by bacterial isolation and immunohistochemistry. Chronic infection in these mice was associated with an impaired recruitment of macrophages, decreased iNOS activity at the site of infection and a more diffuse distribution of inflammatory cells in the liver. This murine model can be of great help for understanding the immunological and bacterial mechanisms that lead to chronic chlamydial infections.

Efficacy, safety and tolerance of imidocarb dipropionate versus atovaquone or buparvaquone plus azithromycin used to treat sick dogs naturally infected with the Babesia microti-like piroplasm.

BACKGROUND: Piroplasmosis caused by the Babesia microti-like piroplasm (Bml) is increasingly being detected in dogs in Europe. Sick dogs show acute disease with severe anaemia associated with thrombocytopenia with a poor response to current available drugs. This study assesses the safety and tolerance of three treatments and compares their efficacy over a full year of follow up in dogs naturally infected with Bml. METHODS: Fifty-nine dogs naturally infected with Bml were randomly assigned to a treatment group: imidocarb dipropionate (5 mg/kg SC, 2 doses 14 d apart) (IMI); atovaquone (13.3 mg/kg PO q 8 h, 10 d)/azithromycin (10 mg/kg PO q 24 h, 10 d) (ATO); or buparvaquone (5 mg/kg IM, 2 d apart)/azithromycin (same dosage) (BUP). Before and after treatment (days 15, 45, 90 and 360), all dogs underwent a physical exam, blood tests and parasite detection (blood cytology and PCR). Clinical efficacy was assessed by grading 24 clinical and 8 clinicopathological signs from low to high severity. RESULTS: Before treatment, most dogs had severe regenerative anaemia (88.13%) and thrombocytopenia (71.4%). On treatment Day 45, clinical signs were mostly reduced in all dogs, and by Day 90, practically all dogs under the ATO or BUP regimen were clinically healthy (76.4 and 88%, respectively). Highest percentage reductions in laboratory abnormalities (82.04%) were detected in animals treated with ATO. Over the year, clinical relapse of Bml was observed in 8 dogs (8/17) treated with IMI. However, on Day 360, these animals had recovered clinically, though clinicopathological abnormalities were still present in some of them. Parasitaemia was PCR-confirmed on Days 90 and 360 in 47.05 and 50% of dogs treated with ATO, 68 and 60.08% with BUP, and 94.1 and 73.3% with IMI, respectively. Even after 360 days, 13.3% of the dogs treated with IMI returned a positive blood cytology result. CONCLUSIONS: IMI showed the worse clinical and parasitological, efficacy such that its use to treat Bml infection in dogs is not recommended. The treatments ATO and BUP showed better efficacy, though they were still incapable to completely eliminate PCR-proven infection at the recommended dose. All three treatments showed good tolerance and safety with scarce adverse events observed.

Theileria annae (syn. Babesia microti-like) infection in dogs in NW Spain detected using direct and indirect diagnostic techniques: clinical report of 75 cases.

BACKGROUND: In north-western Spain, piroplamosis caused by Theileria annae is now recognized as a serious problem because veterinarians, despite being aware of the clinical signs of piroplasmosis, lack the necessary information on its epidemiology or specific diagnostic tools for its management. This, along with the fact that T. annae infection is also refractory to current piroplamosis treatments, prompted this study designed to assess the clinical presentation and diagnosis of this largely unknown parasitic disease in dogs. METHODS: One hundred and twenty dogs in NW Spain suspected clinically of having piroplasmosis were examined and piroplasm species detected by light microscopy (LM) observation of Giemsa-stained blood smears, immunofluorescent antibody test (IFAT), and PCR plus sequencing. RESULTS: Seventy five of the sick dogs were confirmed to be infected with T. annae by PCR (designated "true infection cases"). Intraerythrocytic ring-shaped bodies morphologically compatible with small piroplasms were observed by LM in 59 (57 true infections) of the 120 blood samples. Anti-Babesia antibodies were detected by IFAT in 59 of the 120 sera (55 of which were "true infections"). Using PCR as the reference method, moderate agreement was observed between positive LM vs PCR and IFAT vs PCR results (kappa values: 0.6680 and 0.6017, respectively). Microscopy examination and IFAT were moderately sensitive in detecting the pathogen (76% and 73.3%, respectively). In the 75 cases of "true infection", the most common clinical signs observed were pale mucous membranes, anorexia and apathy. Blood cell counts consistently revealed severe regenerative anaemia and thrombocytopenia in dogs with piroplasmosis due to T. annae. Young dogs (≤3 year) (p = 0.0001) were more susceptible to the disease. CONCLUSION: Microscopy showed moderate diagnostic sensitivity for acute T. annae infection while IFAT-determined antibody titres were low (1/64 to 1/128). The infecting species should be therefore confirmed by molecular tests. Our results suggest that the disease affects dogs in regions of Spain bordering the endemic Galicia area where this piroplasm has not been previously reported (Asturias, northern Spain). Further epidemiological surveys based on serological and molecular methods are required to establish the current geographical range of T. annae infection.

Isolation of Chlamydia abortus from a laboratory worker diagnosed with atypical pneumonia.

BACKGROUND: Identifying the aetiological agent of atypical pneumonia in human can sometimes be a tedious process, especially in cases where Mycoplasma pneumoniae, Legionella species and Chlamydia pneumoniae are ruled out. In such cases, a correct anamnesis of the patient is basic to clarify which pathogens might have produced the infection. For this reason, health professionals including veterinarians and laboratory personnel working with zoonotic pathogens should keep their doctors informed. CASE PRESENTATION: A human case of atypical pneumonia linked to Chlamydia abortus is reported. A 47-year-old male, a veterinarian researcher into chlamydiae, developed respiratory symptoms, breathing problems and high fever. Serological analyses ruled out the involvement of several respiratory pathogens, such as M. pneumoniae, Legionella pneumophila, Rickettsia conorii and C. pneumoniae, and Chlamydia abortus was identified as the possible aetiological agent of the infection. The isolation of C. abortus from the patient's sputum and subsequent molecular analysis confirmed the presence of this microorganism. CONCLUSION: As far as we know, although C. abortus has not been previously described as capable of causing pneumonia in humans, this is the first reported case of atypical pneumonia in which C. abortus is thought to have played an aetiological role.

Ante-mortem diagnosis of caprine tuberculosis in persistently infected herds: influence of lesion type on the sensitivity of diagnostic tests.

Caprine tuberculosis is a major health problem for goats, and an important zoonosis. Eradication programmes using the comparative skin test are being developed to aid in decreasing the prevalence of infection. However, persistent tuberculosis infections are found in herds subjected to eradication programmes. In the present study a commercial IFN-γ release assay and an experimental ELISA based on MPB70, were evaluated as potential ancillary tests to detect infection. The relationship between the three techniques (skin test, IFN-γ release and ELISA) and histopathological lesions was analyzed in 162 goats from herds with persistent tuberculosis infection. The presence of related pathogens (paratuberculosis and pseudotuberculosis) was also studied. The IFN-γ release assay and the ELISA had a higher sensitivity than the comparative skin test (65.3% and 66.3% vs 44.5%) using as a gold standard a combination of histopathological analysis and isolation. Used in combination, ELISA and the skin test detected 89.1% of goats with tuberculosis while a combination of IFN-γ assay and skin test detected 78.2%. The types of macroscopic and microscopic lesions reflected the results of diagnostic tests.

B cells are essential for moderating the inflammatory response and controlling bacterial multiplication in a mouse model of vaccination against Chlamydophila abortus infection.

The use of inactivated vaccines associated with suitable adjuvants has been demonstrated to confer a good level of protection against Chlamydophila abortus. However, the basis of the immune protective response induced by these vaccines has been poorly studied. B cells act as an immune regulatory population during primary infection by C. abortus. Thus, it was considered of interest to study the role of B cells in an infection after immunization with a killed vaccine. For this, C57BL/6 and B-cell-deficient mice were immunized with a killed vaccine against C. abortus using QS-21 as the adjuvant. After challenge, the course of infection was established by analysis of morbidity, C. abortus burden in the liver, and histopathological changes. The immune response induced was studied by real-time PCR techniques. Experiments involving transfer of immune serum from vaccinated or previously infected mice were also carried out. The lack of B cells reduced the protection conferred by the QS-21 adjuvant vaccine. Vaccinated B-cell-deficient mice showed a 1,000-fold-greater bacterial burden in the liver than their wild-type counterparts. Obvious differences existed in the liver, where a severe neutrophilic reaction and extended areas of necrosis were observed with vaccinated B-cell-deficient mice. An analysis of the immune response pointed to a significant increase in inflammatory cytokines and chemokines and the deficient production of transforming growth factor beta. The transfer of antibodies restored the level of protection. This study demonstrates that B cells play a crucial role in controlling C. abortus multiplication and prevent an exacerbated inflammatory response.

Evaluation of Chlamydophila abortus DNA extraction protocols for polymerase chain reaction diagnosis in paraffin-embedded tissues.

Polymerase chain reaction (PCR) has gained increasing importance as a tool for directly demonstrating the presence of Chlamydophila in the placentas of aborted sheep and goats. However, because of the zoonotic potential of the disease, it is advisable to use fixed materials. To evaluate 4 different DNA extraction protocols in paraffin-embedded sections for PCR, previously immunohistochemically diagnosed placental samples from outbreaks of abortions in goats and sheep were used. The samples were also used to evaluate the effect of the duration of fixation in formalin on PCR. A protocol that uses Tris-HCl pH 8.5 with EDTA and subsequent digestion with proteinase K was found to be an easy protocol for obtaining excellent PCR products for Chlamydophila abortus diagnosis from formalin-fixed and paraffin-embedded specimens. It was also found that if samples are fixed in formalin for more than 2 weeks, the PCR technique is affected more adversely than immunohistochemical methods.

Papel de los linfocitos T CD4+ y CD8+ en la protección inducida por diferentes vacunas contra Chlamydophila abortus / Role of T CD4+ and CD8+ lymphocytes in the protection conferred by diffent vaccines against chlamydophila abortus infection

Chlamydophila abortus es el agente etiológico del aborto enzoótico ovino (AEO). Para el control de estainfección, existen en el mercado dos tipos de vacunas: inactivadas y atenuadas. Estudios previos han demostradoque una vacuna efectiva frente C. abortus debe estimular una respuesta inmune específica de tipo Th1,caracterizada por la producción temprana de IFN-γ y la activación de los linfocitos T CD8+. El objetivo de estetrabajo fue estudiar, sobre un modelo muy utilizado para el estudio de la infección clamidial como es el murino,el papel desempeñado por los linfocitos T CD4+ y CD8+ en el desarrollo de una respuesta inmune protectoracontra la infección por C. abortus en animales previamente vacunados. Para ello, se ha utilizado una vacuna atenuadacomercial (1B) y dos vacunas inactivadas experimentales adyuvadas con hidróxido de aluminio (HA) oQS-21 (QS), en ratones que fueron posteriormente deplecionados de linfocitos T CD4+ ó CD8+ unos días antesdel desafío. Para la depleción de los linfocitos T se utilizaron los anticuerpos monoclonales (AcMo) producidospor los hibridomas GK 1.5 (anti-CD4) y 2.43 (anti-CD8). Los resultados de morbilidad y mortalidad mostraronque la depleción de los linfocitos T CD4+ indujo menores pérdidas de peso en los animales vacunados con HAy 1B en el día 4 p.i., y no afectó a la supervivencia de los animales infectados. Cuando los animales fuerondeplecionados de los linfocitos T CD8+, la vacunación impidió la muerte de los animales infectados, ya que enel grupo control no vacunado el 75% de los animales deplecionados habían muerto en el día 8 p.i. Los resultadosde colonización hepática mostraron que la depleción de cualquiera de las dos poblaciones de linfocitos Tprovocó un aumento de la carga clamidial en el hígado que estuvo asociada a un retraso en la resolución de lainfección. Se concluye por tanto, que en la respuesta inmune inducida por la vacunación, independientementede la vacuna empleada, la ausencia de una de las dos poblaciones de linfocitos T CD4+ ó CD8+ no parece seresencial per se para el establecimiento de la respuesta inmune protectora durante la infección

Chlamydophila abortus is the etiological agent of ovine enzootic abortion (OEA). For the control of thisdisease there are two types of commercially available vaccines: inactivated vaccines and attenuated vaccines.Previous studies have demonstrated that an effective vaccine against C. abortus must induce a Th1 immuneresponse involving an early IFN-γ production and CD8+ T cells activation. The aim of the present work was toclarify, using a mouse model, the role of CD4+ and CD8+ T lymphocytes in the establishment of a protectiveimmune response against C. abortus infection in mice previously vaccinated. Mice were vaccinated with acommercially available vaccine (1B) and two experimental inactivated vaccines. The inactivated vaccines wereadjuvated with aluminium hydroxide (HA) or QS-21 (QS) respectively. After vaccination and few days beforethe challenge, mice were depleted of CD4+ and CD8+ T cells using monoclonal antibodies produced by GK1.5 (anti-CD4) and 2.43 (anti-CD8) hybridome cells. In depleted CD4+ T cells mice, morbidity and mortalityresults showed lower weight lost in HA and 1B vaccinated mice at day 4 p.i. while none of infected mice died.In depleted CD8+ T cells mice, no mortality was observe in vaccinated mice while 75% of control non vaccinatedmice succumbed to C. abortus infection at day 8 p.i. The bacterium isolation results showed an increaseclamidial burden in liver in both depletion experiments associated with a slower infection resolution. Thus, invaccinated mice the absence of any of the two lymphocyte T cells subpopulation (CD4+ or CD8+) in not essentialper se in the establishment of a protective immune response during the infection

Genetic and pharmacological inhibition of poly(ADP-ribose) polymerase-1 interferes in the chlamydial life cycle.

Chlamydiaceae are intracellular bacteria responsible for a variety of infections, ranging from asymptomatic to very severe, in humans and animals. We have investigated the role of poly(ADP-ribose) polymerase-1 (PARP-1) in Chlamydophila abortus infection using PARP-1-/- and their littermates PARP-1+/+ mice. Infection was resolved more efficiently by PARP-1-/- than PARP-1+/+ mice. However, the inflammatory response was similar in both strains, suggesting a potential role for PARP-1 in the cross-talk between this microorganism and the host cells. PARP-1-/- fibroblasts showed a 10-fold lower rate of chlamydiae production than PARP-1+/+. Moreover, a strong inhibition of bacterial production was also observed after pharmacological inhibition of PARP-1 activity in McCoy cells. Likewise, PARP-1 inhibition induced a higher level of cell death of infected cells, interfering in this way with the normal bacterial cell cycle. Overall, we identify PARP-1 as a new molecule involved in chlamydial developmental cycle, although the intrinsic mechanisms deserve further studies.

Relationship between the immune response and protection conferred by new designed inactivated vaccines against ovine enzootic abortion in a mouse model.

Chlamydophila abortus is a gram-negative obligate intracellular bacterium and the etiological agent of ovine enzootic abortion (OEA), an economically important disease in many countries. Inactivated vaccines have been reported to induce immunity in ewes and they have been used for many years. However, some outbreaks have been reported in correctly vaccinated flocks, so it is clear that new vaccines are necessary to address adequate protection and to avoid the shedding of the microorganism. This idea lead us to design inactivated vaccines, in a previously established mouse model, evaluating different inactivation procedures and new adjuvants. To assess the protection conferred, the results were analyzed on the basis of clinical signs and the isolation of C. abortus from spleen. These findings were correlated with the immune response induced by the vaccines, as determined by the production of C. abortus-specific IFN-gamma and IL-4 from splenocyte cultures and the detection of IgG isotypes in serum. BEI was found to be the best C. abortus-inactivation procedure. The inactivated vaccines adjuvated with QS-21 (QS) or Montanide 773 (M7) induced the best protection both against homologous and heterologous challenge, with an adequate (Th1-like) immune response. Finally, these selected vaccines were evaluated in a pregnant mouse model, in which they were seen to confer good protection and to avoid the C. abortus persistence in uterus after delivery. With these results, this mouse model could be considered as an adequate tool for selecting and optimizing effective vaccines against OEA.