The present study aimed to investigate the occurrence and molecular identity of Bartonella spp. in Procellariiformes and Magellanic penguins (Spheniscus magellanicus) rescued along the southern and southeastern coasts of Brazil. A total of 52 blood samples from twelve Procellariiformes species and 170 blood samples and 30 spleen samples from Magellanic penguins were collected along the coasts of Santa Catarina and São Paulo states. Following DNA extraction, all samples were screened by conventional PCR targeting the avian β-actin endogenous gene. Successfully amplified samples were further tested by qPCR targeting the 16S-23S ribosomal DNA internal transcribed spacer (ITS) region for Bartonella detection. Positive samples were subjected to additional conventional PCR assays targeting the gltA, ftsZ, groEL, nuoG, pap31, rpoB, ribC, and 16S-23S (ITS) genic regions for molecular characterization. Overall, 56 samples (22.2 %; 56/252) tested positive in the screening, including five blood samples from Procellariiformes (Thalassarche melanophris, n=3; Thalassarche chlororhynchos, n=2) and 51 biological samples (91.1 %; 51/56) from S. magellanicus (42 spleen, 9 blood). Molecular characterization was successful only for S. magellanicus, with amplification of nuoG (14.2 %; 8/56; four spleens, four blood samples), rpoB (12.5 %; 7/56; four spleens, three blood samples), and ITS (12.5 %; 7/56; four spleens, three blood samples). The obtained sequences were closely related to Bartonella henselae previously detected in humans, cats, mongooses, and ticks associated with Passeriformes. In conclusion, Bartonella genotypes circulate in Procellariiformes, and genotypes closely related to B. henselae are present in Magellanic penguins that arrive in the Brazilian coast.

The present study aimed to investigate the occurrence and molecular identity of piroplasmids, haemosporidians and Anaplasmataceae agents in Procelariiformes birds and Magellanic penguins (Spheniscus magellanicus) rescued in different coastal regions of Brazil. Between 2015 and 2022, blood samples (n = 52) from twelve species of Procelariiformes and spleen (n = 170) and blood samples (n = 30) from Magellanic penguins were collected in the coasts of Santa Catarina and São Paulo states, southern and southeastern Brazil, respectively. After DNA extraction, samples positive in a PCR for the avian endogenous β-actin gene were subjected to further PCR assays aiming to detect and molecularly characterize piroplasmids (18S rRNA), haemosporidians (cytochrome B) and Anaplasmataceae agents (16S rRNA, dsb, groEL, gltA, sodB, omp-1, msp-4, msp-5 genes and 23S–5S rRNA intergenic region). As a result, 13/252 (5.1

Abstract Anaplasma marginale is an obligate intraerythrocytic alpha-proteobacterium that causes bovine anaplasmosis, which is responsible for economic losses in the livestock industry worldwide. Although the mechanism of A. marginale infection in tick cell lines has been extensively studied, replication dynamics have not been investigated so far. This study presents the replication kinetics of A. marginale in the RBME-6 tick cell line derived from Rhipicephalus microplus over time. Cell cultures were infected with corpuscles of A. marginale isolated from bovine blood. The replication curve was generated by the number of copies of the msp1β gene obtained by reverse-transcription quantitative PCR (RT-qPCR) using complementary DNA (cDNA) synthesized from RNA. An initial lag phase and an exponential replication phase were recorded. A subsequent death phase was observed, with rapidly decreasing numbers of A. marginale msp1β copies. The surviving population of A. marginale entered a long-term stationary phase from 13 dpi until 29 dpi. Light microscopy was used to monitor the infection in the cells. The propagation kinetics of A. marginale in RBME-6 cells were shown to be essential for guiding future studies aimed at unraveling the crosstalk between this bacterium and its biological tick vector.

This study aimed to evaluate the microbiological quality of coconut water sold from street carts equipped with cooling coils or refrigerated at bakeries in the Grande Vitória Region, Brazil. Additionally, it assessed the phenotypic and genotypic antimicrobial resistance profiles of isolated enterobacteria. The results indicated that coconut water sold at street carts had lower microbiological quality compared to refrigerated samples, as evidenced by significantly higher counts of mesophilic microorganisms. Using MALDI-TOF, the following opportunistic pathogens were identified: Citrobacter freundii, Enterobacter bugandensis, E. kobei, E. roggenkampii, Klebsiella pneumoniae, and Kluyvera ascorbata. Three isolates—E. bugandensis, K. pneumoniae, and K. ascorbata—were classified as multidrug-resistant (MDR). Widespread resistance to β-lactams and cephalosporins was detected, and some isolates were resistant to quinolones, nitrofurans, and phosphonic acids. The gene blaCTX-M-2 was detected in C. freundii, E. bugandensis, E. kobei, and K. ascorbata. However, genes blaNDM, blaKPC, blaCMY-1, and blaCMY-2 were not detected in any isolate. The findings underscore the need to enhance good manufacturing practices in this sector to control the spread of antimicrobial resistance (AMR). To our knowledge, this is the first study documenting the presence of potentially pathogenic enterobacteria in coconut water samples and their associated phenotypic and genotypic AMR profiles.

Zoonotic parasitic diseases in dogs are particularly concerning in regions with low human development indices due to inadequate sanitary services and insufficient environmental and health education. This study aimed to assess the parasitological status of dogs living in households and evaluate their owners’ knowledge about zoonoses. A total of 183 dogs from Rolim de Moura, Rondônia State, were screened for the presence of ectoparasites, and 163 fecal samples were collected for analysis. The results showed that 74.23% (112/163) of the animals had at least one species of endoparasite. The most identified pathogens were Ancylostoma spp. (68.71%, 112/163), Trichuris vulpis (11.66%, 19/163), Toxocara canis (6.75%, 11/163), Cystoisospora canis (4.91%, 8/163), Dipylidium caninum (1.23%, 2/163), and Hammondia/Neospora (0.61%, 1/163). Ectoparasites were observed in 43.17% (79/183) of the evaluated animals, with Rhipicephalus sanguineus found in 31.15% (57/183) and Ctenocephalides felis felis in 20.77% (38/183). Only 11.48% (7/61) of the owners were familiar with the term “Zoonoses.” However, a significant majority (83.61%, 51/61) believed that dogs can transmit diseases to humans. Our findings highlight the prevalence of parasites in the studied area and associated risk factors, underscoring the urgent need for educational interventions to raise awareness about these diseases and their risks to human health.

The consumption of seafood is crucial for food security, but poor hygiene along the food production chain can result in low microbiological quality, posing significant risks for public health and seafood quality. Thus, this study aimed to assess the microbiological quality and antimicrobial sensitivity of E. coli from 69 samples of illegally marketed shrimp and mussels in the Vitória Region, Brazil. These foods exhibited poor microbiological quality due to high counts of mesophilic, psychrotrophic, and enterobacteria microorganisms. While this issue is widespread in this area, shrimp samples displayed higher microbial counts compared to mussels, and fresh mussels had elevated counts of enterobacteria compared to frozen ones. Among the 10 E. coli isolates, none carried the genes blaCTX-M-1, blaCTX-M-2, blaCTX-M-3, blaCTX-M-15, mcr-1, mcr-2, mcr-3, mcr-4, and tet, associated with antibiotic resistance. Phenotypical resistance to tetracycline and fosfomycin was not observed in any isolate, while only 20% demonstrated resistance to ciprofloxacin. Regarding ampicillin and amoxicillin with clavulanic acid, 60% of isolates were resistant, 10% showed intermediate susceptibility, and 30% were sensitive. One isolate was considered simultaneously resistant to β-lactams and quinolones, and none were conserved as ESBL producers. These findings highlight the inherent risks to local public health that arise from consuming improperly prepared seafood in this area.

Rabies is a fatal neglected tropical zoonosis, and its significance for domestic herbivores in the rural cycle is probably associated with rainforest deforestation, livestock, and agricultural expansion. This epidemiological survey aimed to study the occurrence of rabies in bovines and equines in the state of Rondônia, located in the Brazilian's Legal Amazon, between the years 2002 and 2021, correlating these findings with the prophylactic strategies adopted by the local sanitary agency for rabies control. During this period, 201 cases were observed in bovines and 23 in equines. A downward trend in rabies incidence was observed for both domestic herbivores. Rabies did not show a higher occurrence in any specific time of the year, and epidemic periods varied during some years for bovines and equines. Using the Generalized estimating equations (GEE) method, a multiple model approach was obtained with the explanatory variables significantly associated with the decrease in rabies incidence in cattle and horses during the study period: the ratio of treated bats and ratio of vaccine doses sold. Furthermore, the ratio of printed educative material was positively associated with rabies incidence. Despite a decreasing trend in rabies occurrences in this Amazon rainforest area, likely due to the actions taken by the animal sanitary agency, rabies remains endemic and requires monitoring, as well as prophylactic strategies to control this disease.

Rabies is a fatal zoonotic disease, and dogs are the main source for human deaths; however, its importance for domestic herbivores on the rural cycle is increasing, and bat-transmission is probably associated with Amazon rainforest deforestation, livestock and agricultural expansion. This epidemiological survey aimed to study the occurrence of rabies in bovine, equids, and bats in the state of Rondônia, Brazil, between the years 2002 to 2021, correlating these findings to the prophylactic strategies adopted by the sanitary agency for rabies control. During this period, 201 cases were observed in bovine, 23 in equids and three in non-hematophagous bats. A trend for reduction on rabies incidence was observed for both domestic herbivores. Rabies epidemiology had no seasonal pattern and epidemic periods were detected in some years for bovine and equids. The rate of cases per farm was significantly correlated with bats shelter monitoring, number of bats captures, number of vampire bats captured, vampire bats treated by anticoagulant paste, and doses of vaccines sold; all of them negatively correlated. Besides a decreasing trend on rabies occurrence at this Amazon rainforest area, probably due the actions adopted by the animal sanitary agency, rabies remains endemic and requiring monitoring, as well as prophylactic strategies to control this fatal zoonosis.

A globalização e o aumento da densidade populacional levam ao aumento do descarte de produtos gerados pela sociedade. Os resíduos são restos destes produtos que podem ser reciclados, já o lixo não pode ser reutilizado. O descarte inadequado dos resíduos causa impacto ambiental e reflete na Saúde Única. Este estudo teve como objetivo avaliar o nível de conhecimento da população que busca os serviços de saúde da atenção primária e da Unesp-FCAV de Jaboticabal (SP), por meio de um questionário contendo perguntas sobre a temática do lixo e resíduos. No estudo participaram 86 voluntários da atenção primária, com nota satisfatória em 6 das 10 questões. Os 88 voluntários da comunidade acadêmica da Universidade Estadual Paulista “Júlio de Mesquita Filho” (Unesp), Faculdade de Ciências Agrárias e Veterinárias (FCAV) tiveram nota satisfatória em 9 das 10 questões. A formação educacional influencia o nível de conhecimento e conscientização da população sobre o tema do estudo. Medidas educativas são necessárias para conscientizar a população sobre os efeitos dos resíduos na Saúde Única.

Background: Leptospirosis is a contagious disease that affects domestic and wild animals as well as humans. It is caused by infection with some pathogenic species of the genus Leptospira. In Brazil, studies on leptospirosis in capybaras are scarce or nonexistent in some regions, such as the Federal District. The objective of this study was to analyze the presence of DNA of the agent and/or anti-Leptospira spp. antibodies in capybaras.Materials and Methods: Blood samples were collected from 56 free-living capybaras captured in two different sites in the study region. The samples were submitted to hematology and clinical chemistry tests. To identify Leptospira positive samples, a conventional PCR (cPCR) and analysis of anti-Leptospira spp. antibodies by microscopic agglutination test (MAT) were used.Results: No animal showed cPCR amplification of the Lip32 gene, but 41.1% (23/56) of the animals had anti-Leptospira spp. antibodies on MAT. The serovars present were icterohaemorrhagiae (82.61%), copenhageni (65.22%), grippotyphosa (4.35%), and hardjo (4.35%). In the laboratorial tests, differences (p < 0.05) were observed in the biochemical assays of alkaline phosphatase, creatinine, albumin, and globulin. Although these values differed significantly between groups, they all remained within reference range (excluding albumin), and thus there is not enough to infer that this alteration could be caused by Leptospira infection.Conclusions: cPCR using whole blood samples to evaluate Leptospira spp. infection of free-living capybaras was not an efficient tool. The presence of Leptospira seroreactive capybaras shows that the bacteria are circulating in the urban environment of the Federal District.

Leptospirosis is a zoonotic disease that poses a significant threat to human and animal health worldwide. Among different animal species, pigs are known to play a crucial role in the transmission of the pathogenic Leptospira spp. This study aimed to investigate the prevalence of Leptospira spp. infection and associated risk factors in backyard pigs in the state of Paraná, Brazil. A set of 1393 blood samples were collected from pigs on 188 subsistence properties from 136 different municipalities of the Paraná state and tested using the microscopic agglutination test (MAT) to detect antibodies against 24 different Leptospira spp. serovars. The results revealed an overall seroprevalence of 15.87% (221/1393; 95% CI: 13.95–17.78%) for Leptospira spp. antibodies, with Icterohaemorrhagiae, Butembo, and Pomona being the most commonly detected in serovar levels. The lack of rodent control (OR 1.12, 95% CI: 0.63–1.98, p = 0.02) was the only variable associated with disease incidence and was identified as a significant risk factor for Leptospira spp. infection in this context. These findings highlight the urgent need to implement effective control measures, such as improved housing conditions, rodent control, and veterinary assistance, to prevent the spread of this zoonotic disease in backyard pigs in Paraná, Brazil.

ABSTRACT Porcine hemoplasmosis is characterized as a geographically cosmopolitan disease caused by Mycoplasma suis and Mycoplasma parvum. Asymptomatic pigs are considered the focus of hemoplasmosis because they are carriers and reservoirs to new infections. This study aimed to determine the molecular occurrence of porcine hemoplasmas (PH) in the production cycle of technified farrow-to-finished swine herds. For this purpose, 20 swine herds were evaluated, where 501 whole blood samples were collected for qPCR and phylogenetic analyses for hemoplasmas. The epidemiological analysis was performed for the entire population and per the growth stage. The total prevalence for PH was 31.93% (161/501); 95% (19/20) of sampled herds were positive. The occurrence of PH by swine growth stages was nursery (30.47%), growing (31.29%), finishing (26.18%), and slaughter (40.25%). The quantification cycles (Cq) ranged from 3.18- 39.56 and the number of PH 16S rRNA copies per µL of DNA ranged from 5,57 x10-2 to 2.23 x1010. Sequencing and phylogenetic analysis of five selected samples showed 100% identity with M. parvum strain Indiana and two M. parvum sequences from Brazil/Goiás. This is the first report on PH in technified herds in Southeastern Brazil by growth stages.

Anaplasma marginale is a Gram-negative, obligate intraerythrocytic bacterium that causes bovine anaplasmosis. While hard ticks of the genera Dermacentor and Rhipicephalus can be biological vectors, transmitting this pathogen via saliva during blood meals, blood-sucking insects, and fomites play a role as mechanical vectors. Little is known about the interaction between Anaplasma marginale and Argasidae ticks. Among soft ticks, Ornithodoros fonsecai (Labruna and Venzal) and Ornithodoros brasiliensis Aragão inhabit environments surrounding localities where many cases of bovine anaplasmosis have been reported. Ticks of the species O. fonsecai parasitize bats, while O. brasiliensis can parasitize different vertebrate species. Therefore, the present study aimed to feed third-instar nymphs artificially (N3) of O. fonsecai and O. brasiliensis using blood samples obtained from a calf naturally infected with A. marginale and rabbit blood added to A. marginale-containing bovine erythrocytes, to investigate the ability of these nymphs to acquire, infect and transstadially perpetuate this agent. For the artificial feeding system, adapted chambers and parafilm membranes were used. Nymphs of both tick species were submitted to different replications weighed before and after each feeding. Blood samples and molted ticks were submitted to DNA extraction, quantitative real-time PCR for the msp1β gene to detect A. marginale DNA, while a semi-nested polymerase chain reaction for the msp1α gene was performed for genotyping. Using calf blood naturally infected with A. marginale, among the three artificial feeding replications performed with O. fonsecai and O. brasiliensis nymphs, the DNA of A. marginale was detected in both nymphs after 30–50 days of molting. For artificial feeding with rabbit blood added to bovine erythrocytes containing A. marginale, the DNA of this pathogen was also detected in both nymph species. As for the assay for the msp1α gene, strains were found Is9; 78 24-2; 25; 23; α; and β. It was concluded that nymphs (N3) of O. fonsecai and O. brasiliensis could feed artificially through a parafilm membrane using blood from calves and rabbits infected by A. marginale. The DNA of A. marginale was detected in nymphs fed artificially of both tick species studied after molt. However, further studies are needed to confirm transstadial perpetuation in other instars and their host transmission capacity.

•Toxoplasma gondii antibodies were detected in 14.2% (7/49) of insectivorous bats.•Exposure to Patoc and Pomona serovars were detected in insectivorous bats.•Only bats from the genus Molossus and Eumops tested positive for T. gondii and Leptospira spp.•Insectivorous bats are part of the epidemiological chain of T. gondii and Leptospira spp.

ABSTRACT: Bovine fasciolosis is a neglected parasitic zoonosis that causes economic losses to beef production chains due to liver condemnation at abattoirs. Despite the characterization of Bovine fasciolosis in several areas of Brazil, an update on its prevalence in the states of Rio de Janeiro and Espirito Santo is lacking. Thus, fasciolosis prevalence from 2018 to 2021 is presented here according to cattle origin (municipalities of both states). In addition, the associated economic impact on an abbatoir was analyzed through liver condemnation of bovines from both states. The prevalence of fasciolosis in the state of Espírito Santo (10.4%) was significantly higher than Rio de Janeiro (3.6%) (P < 0.01), and resulted in an economic loss of US$12,678.60 for the abattoir. The following municipalities located in the state of Espírito Santo presented prevalences above the mean (10.3%) established in the study: Vargem Alta, Anchieta, Cachoeiro do Itapemirim, Castelo, Atilio Vivacqua, Apiacá and Ibitirama. Thus, majority of these municipalities are considered high-risk areas. Although, there is an aparrent reduction of fasciolosis prevalence, it continues to be a cause of economic losses in abattoirs especially in the state of Espírito Santo. Accordingly, prophylactic measures must still be adopted in cattle farms.

Understanding the diversity and ecology of ectoparasites in wild animals is essential for surveillance of vector-borne diseases. Coatis (Nasua nasua) easily adapt to anthropized areas, favoring close contact with domestic animals and humans, with the possibility of exchange of ectoparasites and pathogens. The present study aimed to identify the diversity of ticks parasitizing coatis from forest urban areas of midwestern Brazil, to evaluate the seasonal dynamics of ticks during the seasons of the year, and to assess the correlation between tick species and gender and age of the sampled coatis. For this purpose, 103 coatis were captured in two Conservation areas, both located in Campo Grande city, Mato Grosso do Sul state, Midwestern Brazil. The animals’ entire body was inspected for the presence of ectoparasites, and ticks were removed for taxonomic identification. In total, 168 captures were performed in both areas during the observational study considering the first capture and recaptures. In total, 2242 ticks were collected: 838 Amblyomma larvae, 1241 A. sculptum nymphs, and 150 A. dubitatum nymphs. Thirteen adult ticks were identified as three males and five females of A. sculptum and two males and three females of A. ovale. While a quantity of Amblyomma larvae was observed in the first months of the year (January, April and May), Amblyomma nymphs showed a higher quantity during the months of July, August, October and November. No statistical difference was observed when comparing mean intensity and prevalence of Amblyomma larvae, nymphs of A. sculptum and A. dubitatum between the two sampled areas, males vs. females and immature vs. mature animals. In conclusion, three tick species were identified parasitizing coatis from forested urban fragments in midwestern Brazil, namely A. dubitatum nymphs, A. sculptum adults and nymphs, and A. ovale adults. Coatis from anthropized areas seem to present tick species diversity lower than those from natural areas. The lack of statistical difference regarding mean intensity and prevalence of Amblyomma larvae and nymphs between males vs. females and immature vs. mature animals might have reflected the gregarious behavior of coatis, since adult males live together with females and offspring outside and inside the mating season, forming large groups of individuals.

In the last decades, the global incidence of arboviruses transmitted by the vector Aedes aegypti has increased dramatically with the increased oh human mobility and urbanization. The study of the mosquito population is of great importance for public health in countries where climatic and environmental conditions are favorable for the spread of these diseases transmitted by A. aegypti. This was a cross-sectional study that assessed causal relationships between why mosquitoes are prevalent in the region studied and socioeconomic variables, practices, knowledge, attitudes, and the effect of the cause in two regions chosen according to vulnerability (São Paulo Social Vulnerability Index). Of the 435 residents interviewed, 95% (413/435) were informed about vector control and claimed to contribute to the day-to-day sanitary practices necessary to combat it, in addition to being able to identify it. Most participants in both regions believed they were at risk, but vector control practices did not match those recorded in the survey. A correlation was found between the population’s level of education and the practices observed. Despite the high levels of knowledge and the perceptions of the interviewed population about A. aegypti, the erroneous behavior of the residents persisted, thus hindering disease prevention and vector control actions, promoting the conditions conducive to the proliferation of the vector, and, consequently, increasing the risk of disease. The study indicated that one of the most effective means to control the A. aegypti vector is a society informed about preventive measures in the surveillance sectors.

ABSTRACT: Bovine cysticercosis (BCC) is an important disease in Brazil due to its detection in abattoirs, resulting in economic losses for cattle farmers, and a public health corcern. Besides its importance, the knowledge about BCC epidemiology and impact remains not fully described in several areas in this country, requiring more studies. Thus, this study focused on establishing BCC prevalence in the regions and municipalities of the state of Espírito Santo from 2017 to 2019, establishing the associated risk factors, and estimating the economic losses for cattle farmers. A set of 2,330 cases of BCC was detected in the 407,529 bovine inspected by Federal Inspection Service (0.57%; C.I. 95% 0.55 - 0.60%) with a higher detection of unviable cysticercus (66.14%). The beef producers lost at least US$153,000.20 due to BCC through this period. The highest risk for BCC infection occurred in Metropolitana (OR = 9.19), Litoral Sul (OR = 7.77), Caparó (OR = 7.44), and Central Sul (OR = 7.19) mesoregions, respectively. The BCC was detected in animals from 67 municipalities and its prevalence increased in areas with high human population density (OR = 1.58;; p = 0.01). In conclusion, BCC is an important disease for beef production chain in the Brazilian state of Espírito Santo and a cause of economic losses, mainly in some areas, and urgently requires the adoption of prophylactic strategies to reduce the ocurrence of this parasite.

ABSTRACT Brucellosis is a zoonotic disease widespread almost all over the world, representing a significant economic and public health problem. Brucella melitensis, B. suis and B. abortus are considered the most pathogenic species for humans. The most virulent species, B. melitensis is endemic in many parts of the world, particularly the biovar 3 in the Mediterranean and Eastern Europe. Some Latin American countries are seriously affected by biovar 1, especially Mexico, Peru and northern Argentina. Furthermore, while Brazil is considered free of this etiologic agent, one recurrent question is whether this infection really does not occur in Brazil or there is a lack of research/data on the subject. To answer this question, this study aims to investigate the occurrence of antibodies against smooth Brucella in goats and sheep in the states of Sergipe, Paraíba, Ceará and Paraíba. All samples were screened by the Rose Bengal test (RBT). The complement fixation (CFT) and the fluorescence polarization (FPT) tests were used as confirmatory tests. There were no positive samples in the confirmatory tests (both CFT and FPT). We, therefore, conclude that this result reinforces the previous knowledge regarding the exotic status of B. melitensis infection in Brazil.

ABSTRACT Leptospirosis affects several animal species, including man. This study aimed to evaluate the prevalence of Leptospira spp. and to identify factors associated with the risk of Leptospira spp. in dairy cattle in the municipality of Ji-Paraná, RO, Brazil, sampled by rural sector, from September 2012 to November 2013. Blood samples from 627 dairy cows were randomly collected from 63 farms belonging to six rural sectors. Leptospirosis was diagnosed by the microscopic serum agglutination technique. Of the 627 animals tested, 255 had anti-Leptospira antibodies (40.48%, 95% CI: 36.64-44.31) and 57 of the 63 studied dairy farms (90.5%, 95% CI: 83.23-97.72) had at least one reactive animal. The results indicate that serovar Hardjo had the highest (12.38%. 95% CI: 10.03-15.18) followed by serovars Shermani, Wolffi, Hebdomadis and Canicola occurrence in dairy cows. Additionally, infection was also associated with abortion occurrences in cows of 36 farms (57.14%) and the presence of dogs roaming free with access to pasture, water and cattle in 47 farms (74.60%). Therefore, free-roaming animals are considered a predisposing factor, highlighting the need for adopting prophylactic measures while raising the awareness from rural producers about the importance and the economic losses that leptospirosis may cause.