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Coxiella burnetii is a highly transmissible intracellular bacterium with a low infective dose that causes Q fever (coxiellosis), a notifiable zoonotic disease distributed worldwide. Livestock are the main source of C. burnetii transmission to humans, which occurs mostly through the aerogenous route. Although C. burnetii is a major abortifacient in small ruminants, it is less frequently diagnosed in aborting cattle. We report a case of C. burnetii abortion in a lactating Holstein cow from a dairy farm producing and selling artisanal cheese directly to consumers in Uruguay, and review the literature on coxiellosis as a bovine abortifacient in South America and as a milk-borne disease. The aborted cow had severe necrotizing placentitis with abundant intratrophoblastic and intralesional C. burnetii confirmed by immunohistochemistry and PCR. After primo-infection in cattle, C. burnetii remains latent in the lymph nodes and mammary glands, with milk being a significant and persistent excretion route. Viable C. burnetii has been found in unpasteurized milk and cheeses after several months of maturing. The risk of coxiellosis after the consumption of unpasteurized dairy products, including cheese, is not negligible. This report raises awareness on bovine coxiellosis as a potential food safety problem in on-farm raw cheese manufacturing and sales. The scant publications on abortive coxiellosis in cattle in South America suggest that the condition has probably gone underreported in all countries of this subcontinent except for Uruguay. Therefore, we also discuss the diagnostic criteria for laboratory-based confirmation of C. burnetii abortion in ruminants as a guideline for veterinary diagnosticians.

Cattle are broadly deemed a source of Coxiella burnetii; however, evidence reinforcing their role in human infection is scarce. Most published human Q fever outbreaks relate to exposure to small ruminants, notably goats. Anti-phase II C. burnetii IgG and IgM were measured by indirect fluorescent antibody tests in 27 farm and veterinary diagnostic laboratory workers to ascertain whether occupational exposure to cattle aborting due to C. burnetii was the probable source of exposure. Four serological profiles were identified on the basis of anti-phase II IgG and IgM titres. Profile 1, characterised by high IgM levels and concurrent, lower IgG titres (3/27; 11.1%); Profile 2, with both isotypes with IgG titres higher than IgM (2/27; 7.4%); Profile 3 with only IgG phase II (5/27; 18.5%); and Profile 4, in which neither IgM nor IgG were detected (17/27; 63.0%). Profiles 1 and 2 are suggestive of recent C. burnetii exposure, most likely 2.5–4.5 months before testing and, hence, during the window of exposure to the bovine abortions. Profile 3 suggested C. burnetii exposure that most likely predated the window of exposure to aborting cattle, while Profile 4 represented seronegative individuals and, hence, likely uninfected. This study formally linked human Q fever to exposure to C. burnetii infected cattle as a specific occupational hazard for farm and laboratory workers handling bovine aborted material.

Infectious diseases are the leading aetiological cause of bovine abortion globally as well as in Uruguayan dairy farms. Studies conducted in this country showed that a proportion of aborted fetuses without diagnosis present histological lesions compatible with the action of an infectious agent. These undiagnosed abortion cases could be due to infectious agents not currently included in the battery of diagnostic tests routinely applied. Infectious agents such as Coxiella burnetii and Chlamydia spp., typically considered to have a minor or relative marginal role in causing abortion in cattle, have never been systematically investigated in Uruguay. This thesis challenged the current diagnostic protocols, seeking evidence supporting the inclusion of these agents to dismiss the number of undiagnosed cases. This work investigated the association of these pathogens with cattle abortion in commercial dairy herds and the potential as zoonotic threats in the local context. The thesis will first introduce the main characteristics of dairy production in Uruguay (Chapter One) and thoroughly review Coxiella burnetii and Chlamydia abortus (Chapter Two). After this, two main sections will be presented. The first section including a systematic review and meta-analysis (Chapter Three), and the second section comprising the observational fieldwork studies conducted out on dairy farms in Uruguay, which includes a cross-sectional study of pooled milk (PM) samples from commercial herds (Chapter Four), a case-series sampling of placentas (Chapter Five), a case-control sampling of aborted and non-aborted dairy animals (Chapter Six), and finally a retrospective cohort evaluation of workers exposed to a local bovine Q fever outbreak (Chapter Seven). A final discussion (Chapter Eight) will consider the main findings' implications and integrate these results into the general body of knowledge. The systematic review and meta-analysis evidenced bulk tank milk (BTM) sampling as a broadly used epidemiological methodology for large-scale investigations of C. burnetii and provided a global framework of the prevalence of C. burnetii in collective milk samples from commercial farms. This study showed a widespread herd-level circulation of C. burnetii in bovine dairy farms by reporting a high overall global prevalence of 37.0% (CI95% 25.2-49.5%). A meta-regression showed the herd size as the most relevant moderator, with the odds of a BTM sample testing positive doubling with every unit increase. This C. burnetii meta-prevalence roles as a benchmark for comparison with the findings of the molecular investigation on PM samples obtained from local dairy farms. The mass-scale molecular evaluation on PM samples evidenced a low incidence (1.7%) of C. burnetii DNA. The findings showed that clinically healthy (asymptomatic) cows might shed the bacterium, raising awareness of Q fever as potential food safety and public health concern considering the C. burnetii survival as a milk-borne pathogen in unpasteurised milk and raw dairy products. Due to the exceptionally high infectivity, low infective doses, and aerosol transmission, the culture of C. burnetii and Chlamydiales should be done on BSL-3 laboratories not currently operating in Uruguay. Bacterial cultivation was not attempted in any of the studies conducted in this thesis. Hitherto the lack of diagnostic tools and the impossibility of doing culture have restricted local epidemiological investigation of these agents. Therefore, developing diagnostic tests to be used routinely in domestic laboratories is imperative to save costs and optimise currently available facilities and work with higher autonomy. For this purpose, a published protocol targeting well-evaluated genes was adapted to provide an available tool for local laboratories. Coxiella burnetii, C. abortus, and C. pecorum were investigated for the first time using an m-PCR in placentas from aborted dairy cows. Coxiella burnetii-DNA was detected and quantified in those samples, which supported this bacterium as an abortifacient agent in Uruguay. No co-infections of these pathogens were found. Evidence supporting Chlamydiales as a source of cattle abortion remain blurred. Coxiella burnetii was detected on the aborted placenta from a cow from an artisanal cheese-producing farm. Consumption of raw milk and dairy products represent a potential source for human infection. This finding underlined that the public health risk posed by C. burnetii should not be neglected and should be emphasised the need for on-farm milk pasteurisation by local artisanal cheesemakers. Molecular investigation of C. abortus in vulvo-vaginal swabs samples showed no evidence of this bacterium neither in aborted nor in control animals. Difficulties in identifying low-grade infection and evaluating a single sample per animal would have constrained the detection. The first attempted studies conducted so far support C. abortus as a no substantial abortifacient agent in cattle from Uruguay. Serological evidence confirmed the local bovine population as a potential reservoir for C. burnetii infection in humans. Anti-C. burnetii phase II IgM and IgG immunoglobulins were detected in a group of farmworkers and laboratory technicians exposed to aborted dairy cattle or aborted materials (fetuses and placenta) by indirect immunofluorescence. Molecular approaches were assessed, optimised and validated on veterinary clinical samples such as aborted placentas, vulvo-vaginal swabs or collective milk samples, providing valuable alternatives beyond the bacterial culture and isolation. The thesis presents original research studies that utilise different epidemiological strategies to search for evidence of an association between the infection by the pathogens and the occurrence of bovine abortion.

The aim of this work was to identify causes of abortion through laboratory investigations in sheep flocks in Uruguay. One hundred cases of abortion, comprising 58 fetuses, 36 fetuses with their placentas, and 6 placentas were investigated in 2015–2021. Cases were subjected to gross and microscopic pathologic examinations, and microbiological and serological testing for the identification of causes of abortion, including protozoal, bacterial, and viral pathogens. An etiologic diagnosis was determined in 46 (46%) cases, including 33 (33%) cases caused by infectious pathogens, as determined by the detection of a pathogen along with the identification of fetoplacental lesions attributable to the detected pathogen. Twenty-seven cases (27%) were caused by Toxoplasma gondii , 5 (5%) by Campylobacter fetus subspecies fetus , and 1 (1%) by an unidentified species of Campylobacter . Fourteen cases (14%) had inflammatory and/or necrotizing fetoplacental lesions compatible with an infectious etiology. Although the cause for these lesions was not clearly identified, T. gondii was detected in 4 of these cases, opportunistic bacteria ( Bacillus licheniformis, Streptococcus sp.) were isolated in 2 cases, and bovine viral diarrhea virus 1 subtype i (BVDV-1i) was detected in another. Campylobacter jejuni was identified in 1 (1%) severely autolyzed, mummified fetus. BVDV-2b was identified incidentally in one fetus with an etiologic diagnosis of toxoplasmosis. Microscopic agglutination test revealed antibodies against ≥1 Leptospira serovars in 15/63 (23.8%) fetuses; however, Leptospira was not identified by a combination of qPCR, culture, fluorescent antibody testing nor immunohistochemistry. Neospora caninum, Chlamydia abortus, Chlamydia pecorum, Coxiella burnetii and border disease virus were not detected in any of the analyzed cases. Death was attributed to dystocia in 13 (13%) fetuses delivered by 8 sheep, mostly from one highly prolific flock. Congenital malformations including inferior prognathism, a focal hepatic cyst, and enterohepatic agenesis were identified in one fetus each, the latter being the only one considered incompatible with postnatal life. Toxoplasmosis, campylobacteriosis and dystocia were the main identified causes of fetal losses. Despite the relatively low overall success rate in establishing an etiologic diagnosis, a systematic laboratory workup in cases of abortion is of value to identify their causes and enables zoonotic pathogens surveillance.

The authors sought to find out whether certain Webometric indexes of a sample of pediatric Web resources, and some tests based on them, could be helpful predictors of their disappearance. The authors performed a retrospective study of a sample of 363 pediatric Websites and pages they had followed for 4 years. Main measurements included: number of resources that disappeared, number of inbound links and their annual increment, average daily visits to the resources in the sample, sample compliance with the quality criteria of 3 international organizations, and online time of the Web resources. On average, 11% of the sample disappeared annually. However, 13% of these were available again at the end of follow up. Disappearing and surviving Websites did not show differences in the variables studied. However, surviving Web pages had a higher number of inbound links and higher annual increment in inbound links. Similarly, Web pages that survived showed higher compliance with recognized sets of quality criteria than those that disappeared. A subset of 14 quality criteria whose compliance accounted for 90% of the probability of online permanence was identified. Finally, a progressive increment of inbound links was found to be a marker of good prognosis, showing high specificity and positive predictive value (88% and 94%, respectively). The number of inbound links and annual increment of inbound links could be useful markers of the permanence probability for pediatric Web pages. Strategies that assure the Web editors' awareness of their Web resources' popularity could stimulate them to improve the quality of their Websites.

Cattle are broadly deemed a source of Coxiella burnetii; however, evidence reinforcing their role in human infection is scarce. Most published human Q fever outbreaks relate to exposure to small ruminants, notably goats. Anti-phase II C. burnetii IgG and IgM were measured by indirect fluorescent antibody tests in 27 farm and veterinary diagnostic laboratory workers to ascertain whether occupational exposure to cattle aborting due to C. burnetii was the probable source of exposure. Four serological profiles were identified on the basis of anti-phase II IgG and IgM titres. Profile 1, characterised by high IgM levels and concurrent, lower IgG titres (3/27; 11.1%); Profile 2, with both isotypes with IgG titres higher than IgM (2/27; 7.4%); Profile 3 with only IgG phase II (5/27; 18.5%); and Profile 4, in which neither IgM nor IgG were detected (17/27; 63.0%). Profiles 1 and 2 are suggestive of recent C. burnetii exposure, most likely 2.5–4.5 months before testing and, hence, during the window of exposure to the bovine abortions. Profile 3 suggested C. burnetii exposure that most likely predated the window of exposure to aborting cattle, while Profile 4 represented seronegative individuals and, hence, likely uninfected. This study formally linked human Q fever to exposure to C. burnetii infected cattle as a specific occupational hazard for farm and laboratory workers handling bovine aborted material.

Background Patients with liver cirrhosis and minimal hepatic encephalopathy (MHE) show mild cognitive impairment and spatial learning dysfunction. Hyperammonemia acts synergistically with inflammation to induce cognitive impairment in MHE. Hyperammonemia-induced neuroinflammation in hippocampus could contribute to spatial learning impairment in MHE. Two main aims of this work were: (1) to assess whether chronic hyperammonemia increases inflammatory factors in the hippocampus and if this is associated with microglia and/or astrocytes activation and (2) to assess whether hyperammonemia-induced neuroinflammation in the hippocampus is associated with altered membrane expression of glutamate and GABA receptors and spatial learning impairment. There are no specific treatments for cognitive alterations in patients with MHE. A third aim was to assess whether treatment with sulforaphane enhances endogenous the anti-inflammatory system, reduces neuroinflammation in the hippocampus of hyperammonemic rats, and restores spatial learning and if normalization of receptor membrane expression is associated with learning improvement. Methods We analyzed the following in control and hyperammonemic rats, treated or not with sulforaphane: (1) microglia and astrocytes activation by immunohistochemistry, (2) markers of pro-inflammatory (M1) (IL-1β, IL-6) and anti-inflammatory (M2) microglia (Arg1, YM-1) by Western blot, (3) membrane expression of GABA, AMPA, and NMDA receptors using the BS3 cross-linker, and (4) spatial learning using the radial maze. Results The results reported show that hyperammonemia induces astrocytes and microglia activation in the hippocampus, increasing pro-inflammatory cytokines IL-1β and IL-6. This is associated with altered membrane expression of AMPA, NMDA, and GABA receptors which would be responsible for altered neurotransmission and impairment of spatial learning in the radial maze. Treatment with sulforaphane promotes microglia differentiation from pro-inflammatory M1 to anti-inflammatory M2 phenotype and reduces activation of astrocytes in hyperammonemic rats. This reduces neuroinflammation, normalizes membrane expression of glutamate and GABA receptors, and restores spatial learning in hyperammonemic rats. Conclusions Hyperammonemia-induced neuroinflammation impairs glutamatergic and GABAergic neurotransmission by altering membrane expression of glutamate and GABA receptors, resulting in impaired spatial learning. Sulforaphane reverses all these effects. Treatment with sulforaphane could be useful to improve cognitive function in cirrhotic patients with minimal or clinical hepatic encephalopathy.

Background: Postoperative cognitive dysfunction affects the quality of recovery, particularly affecting the elderly, and poses a burden on the health system. We hypothesize that the use of sugammadex (SG) could optimize the quality of postoperative cognitive function and overall recovery through a neuroprotective effect. Methods: A pilot observational study in patients undergoing cardiac surgery with Enhanced Recovery After Surgery (ERAS), was designed to compare Sugammadex-treated (SG, n=14) vs. neostigmine-treated (NG, n=7) patients. The Postoperative Quality Recovery Scale (PORS) was used at different times to evaluate patients’ cognitive function and overall recovery. An online survey amongst anesthesiologists on SG use was also performed. Additionally, an animal model study was designed to explore the SG effects on the hippocampus. Results: SG was associated with favorable postoperative recovery in cognitive domains particularly 30 days after surgery in patients undergoing aortic valve replacement by cardiopulmonary bypass and ERACS approach; however, it failed to demonstrate a short-term decrease in length of ICU and hospital stay. The survey information indicated a positive appreciation of the SG recovery properties. The SG reverts postoperative memory deficit and induces the expression of anti-inflammatory microglial markers. Conclusions: The results show a postoperative cognitive improvement by SG treatment in patients undergoing aortic valve replacement procedure by ERACS approach. Additionally, experimental data from an animal model of mild surgery, confirm the cognitive SG effect and suggest a potential effect over glia cells as an underlying mechanism.

Background Hyperammonemia induces neuroinflammation and increases GABAergic tone in the cerebellum which contributes to cognitive and motor impairment in hepatic encephalopathy (HE). The link between neuroinflammation and GABAergic tone remains unknown. New treatments reducing neuroinflammation and GABAergic tone could improve neurological impairment. The aims were, in hyperammonemic rats, to assess whether: Enhancing endogenous anti-inflammatory mechanisms by sulforaphane treatment reduces neuroinflammation and restores learning and motor coordination. Reduction of neuroinflammation by sulforaphane normalizes extracellular GABA and glutamate-NO-cGMP pathway and identify underlying mechanisms. Identify steps by which hyperammonemia-induced microglial activation impairs cognitive and motor function and how sulforaphane restores them. Methods We analyzed in control and hyperammonemic rats, treated or not with sulforaphane, (a) learning in the Y maze; (b) motor coordination in the beam walking; (c) glutamate-NO-cGMP pathway and extracellular GABA by microdialysis; (d) microglial activation, by analyzing by immunohistochemistry or Western blot markers of pro-inflammatory (M1) (IL-1b, Iba-1) and anti-inflammatory (M2) microglia (Iba1, IL-4, IL-10, Arg1, YM-1); and (e) membrane expression of the GABA transporter GAT-3. Results Hyperammonemia induces activation of astrocytes and microglia in the cerebellum as assessed by immunohistochemistry. Hyperammonemia-induced neuroinflammation is associated with increased membrane expression of the GABA transporter GAT-3, mainly in activated astrocytes. This is also associated with increased extracellular GABA in the cerebellum and with motor in-coordination and impaired learning ability in the Y maze. Sulforaphane promotes polarization of microglia from the M1 to the M2 phenotype, reducing IL-1b and increasing IL-4, IL-10, Arg1, and YM-1 in the cerebellum. This is associated with astrocytes deactivation and normalization of GAT-3 membrane expression, extracellular GABA, glutamate-nitric oxide-cGMP pathway, and learning and motor coordination. Conclusions Neuroinflammation increases GABAergic tone in the cerebellum by increasing GAT-3 membrane expression. This impairs motor coordination and learning in the Y maze. Sulforaphane could be a new therapeutic approach to improve cognitive and motor function in hyperammonemia, hepatic encephalopathy, and other pathologies associated with neuroinflammation by promoting microglia differentiation from M1 to M2.

ObjectiveInflammation plays a role in neurological alterations in patients with hepatic encephalopathy (HE). Animal models of HE show neuroinflammation. Treatment with ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), reduces neuroinflammation and restores cognitive and motor function in rats with HE due to portacaval shunts (PCS). This suggests that reducing neuroinflammation would improve neurological status in patients with minimal or clinical HE. NSAID induce kidney damage in patients with cirrhosis and PCS rats and are not suitable for clinical use. It is therefore necessary to look for procedures to eliminate neuroinflammation without inducing secondary effects in the kidney. Inhibition of p38 MAPK is being tested as a therapeutic target in inflammatory diseases and reduces microglial activation. This study aimed to assess whether inhibiting p38 with SB239063 reduces neuroinflammation and improves cognitive and motor function in PCS rats without affecting the kidney.Resultsp38 activity is increased in the brains of PCS rats and treatment with SB239063 reduces microglial activation, as well as inflammatory markers in brain (prostaglandin E2, cyclooxygenase activity, iNOS, IL-1β, TNFα) and blood (prostaglandin E2 and TNFα). PCS rats showed increased ammonia and glutamine in the brain, which was not affected by SB239063. PCS rats showed reduced ability to learn a Y-maze conditional discrimination task, reduced motor activity and impaired motor coordination, as assessed in the rotarod. Treatment with SB239063 completely restored learning ability, motor activity and coordination in PCS rats. SB239063 did not affect creatinine or sodium levels in serum, indicating that it does not induce kidney damage.ConclusionThese findings suggest that reducing neuroinflammation by using inhibitors of p38 would improve the neurological status in HE without inducing secondary effects in the kidney.