Brucella Canis Infection Research Articles

The risk of emerging infections in pets

Emerging infections in pets are a worry for many public health professionals, with Brucella canis infection, the tapeworm Echinococcus multilocularis and heartworm Dirofilaria immitis all in the spotlight. The importation of animals is a big risk factor for these conditions, as is the travel of pets abroad with their owners.

Detección de infección por Brucella canis en perros de raza Pit Bull en Turquía

Brucella canis infection is an often neglected but important zoonotic disease. This study aims to determine its seroprevalence in Pit Bull dogs from the Western Region of the Turkish Anatolian Peninsula. In the Province of Manisa, 2 mL blood samples were taken from the antebrachial region of 35 Pit Bull dogs using sterile K2EDTA (3.6 mg) blood tubes, and the samples were analyzed using both the mercaptoethanol (ME) microagglutination test and B. canis–specific PCR techniques. Of the 35 dogs tested by 2–ME RSAT, 13 (37.14%) tested positive and 22 (63%) tested negative. Of the 13 dogs that tested positive for 2–ME RSAT, 8 (22.85%) were female, and 5 (14.28%) were male. Subsequent PCR analysis of all samples revealed that 7 (20%; 7/35) of the samples that tested positive for 2–ME RSAT were actually B. canis–specific PCR positive. These findings suggest that B. canis is present in Pit Bull dogs, although they provide a general idea of the disease's prevalence of the disease in the region. Multicentre studies with larger numbers of cases in different groups of Pit Bulls, such as healthy, patient and risk groups, are needed to provide comprehensive evidence.

Gene expression of Toll-like receptors, cytokines and a nuclear factor and cytokine secretion in DH82 canine macrophage cells infected with Brucella canis

Canine brucellosis caused by Brucella canis infection occurs mainly in dogs, and is a zoonotic disease that also has the possibility of infection in humans. Many studies have been conducted to understand the immunopathological mechanism of B. canis infection. However, the precise immune mechanism remains to be elucidated because compared to other Brucella spp., B. canis has different immune evasion mechanisms. In this study, gene expression levels of Toll-like receptors (TLRs) and TLR-associated molecules and cytokine production were analyzed to figure out the roles of immune-related host factors in B. canis infection. Time-dependent gene expression of TLRs (1−10) and TLR-related molecules (TNF-α, IL-5, IL-23, CCL4, CD40 and NFκ-B) and release of Th1, Th2 and Th17-related cytokines (IFN-γ, IL-1β, IL-4, IL-6, IL-10 and IL-17A) were investigated in DH82 canine macrophages infected with B. canis. Time-dependent induction of TLRs 3, 7 and 8 was observed, and TLR 7 had the highest expression level (p <0.05). The expression levels of all TLR-related genes were significantly increased after infection. In particular, the expression of the CCL4 and IL-23 genes was highly induced. The amounts of IL-1β, IL-6 and IL-10 were significantly increased by B. canis infection, but the amounts of IL-4 and IL-17A were not. The production of IL-1β and IL-6 was the highest at 24 hr after B. canis infection (p <0.05). This study demonstrates that TLRs 3, 7 and 8 are prominent sites of to immune response induction with the production of related cytokines and a nuclear factor in DH82 cells infected with B. canis. These results suggest a sequential immune mechanism of B. canis infection, involving TLRs, cytokines and their associated factors.

First seroprevalence and molecular identification report of Brucella canis among dogs in Greater Cairo region and Damietta Governorate of Egypt.

Given the rise in stray and imported dogs in Egypt over the past 5 years, it is surprising that no report of Brucella canis infection in dogs or humans has been documented in Egypt's published papers. This study aimed to detect the presence of antibodies against the rough (B. canis) and smooth Brucellae among dogs in Egypt and to characterize the Brucella species circulating in dogs. Blood samples (n = 449) were collected from owned and stray dogs in the Greater Cairo region (n = 309) and Damietta governorate (n = 140). The apparent, true, and total seroprevalence of canine brucellosis caused by B. canis infection were calculated using the 2-mercaptoethanol tube agglutination test (2-ME TAT) and rapid slide agglutination test (RSAT). We used the rose Bengal test (RBT) and the buffered acidified plate antigen test (BAPAT) to check the serum samples from dogs for the presence of antibodies against smooth Brucellae. Three polymerase chain reaction (PCR) assays - Bruce-ladder PCR, B. canis species-specific PCR (BcSS-PCR), and Abortus Melitensis Ovis Suis (AMOS)-PCR - were used to determine the Brucella species in the buffy coats of the serologically positive dogs. The overall apparent and true prevalence of B. canis infection in dogs were estimated to be 3.8% and 13.2%. The estimated true prevalence in stray dogs (15%) was higher than in owned dogs (12.5%). The BAPAT and the RBT using smooth antigens revealed that 11 (2.4%) and 9 (2%) were positive. Bruce-ladder PCR targeting eryC, ABC, and Polysaccharide deacetylase genes was able to identify B. canis in nine out of 17 buffy coat samples. AMOS-PCR identified the eight undetermined Brucella species by Bruce-ladder PCR as Brucella abortus (n = 4) and Brucella melitensis (n = 4). To exclude the presence of Brucella suis, a one-step species-specific BcSS-PCR was performed and specifically amplified all B. canis DNA (n = 9) the same as did the Bruce-ladder PCR. To the best of our knowledge, this is the first report of B. canis detection in dogs in Egypt. Molecular identification of B. abortus and B. melitensis in the Egyptian canines highlights the role of stray dogs in brucellosis remerging in Brucellosis-free dairy farms. Brucella canis infection can be diagnosed specifically with the one-step BcSS-PCR. The obtained results set-an-alarm to the veterinary authorities to launch plans to control this disease in dogs.

Are Italian-Polish veterinarians and breeders prepared to control an outbreak of Brucella canis infection in dogs?

Brucella canis infection is one of the most important causes of infertility in dogs and is a zoonosis for which no effective treatment or vaccines exist. It is not a mandatory notifiable disease. Following an increase of cases in Europe and worldwide, an investigation was performed to evaluate how much Italian and Polish veterinarians and breeders know about canine brucellosis and understand their perceptions of this infection. For this reason, two questionnaires were prepared, in Italian and Polish. Eighteen Italian and Polish veterinarians, specialists in canine reproduction, responded to the first survey and 44.4% of them affirmed having diagnosed canine brucellosis at least once in their clinical practice, and different perceptions emerged regarding the infection in the two countries. The second survey was completed by 145 Italian and Polish breeders; the disease was completely unknown to 22.8% of them, whereas 2.1% had diagnosed infection by B. canis in their kennels. In conclusion, knowledge of B. canis infection differs between these countries, with extremes ranging from diagnosed cases to complete underestimation of the presence of the problem. However, based on international data and reporting of a recent large outbreak in Italy, awareness of this contagious infectious disease and its management must be increased.

Canine brucellosis in Costa Rica reveals widespread Brucella canis infection and the recent introduction of foreign strains

Brucellosis is a prevalent disease in Costa Rica (CR), with an increasing number of human infections. Close to half of homes in CR have one or more dogs, corresponding to ∼1.4 million canines, most of them in the Central Valley within or near the cities of San José, Heredia, and Alajuela. From 302 dog sera collected from this region, 19 were positive for Brucella canis antigens, and five had antibodies against smooth lipopolysaccharide, suggesting infections by both B. canis and other Brucella species. B. canis strains were isolated in the Central Valley from 26 kennel dogs and three pet dogs, all displaying clinical signs of canine brucellosis. We detected three recent introductions of different B. canis strains in kennels: two traced from Mexico and one from Panama. Multiple locus-variable number tandem repeats (MLVA-16) and whole-genome sequencing (WGSA) analyses showed that B. canis CR strains comprise three main lineages. The tree topologies obtained by WGSA and MLVA-16 just partially agreed, indicating that the latter analysis is not suitable for phylogenetic studies. The fatty acid methyl ester analysis resolved five different B. canis groups, showing less resolution power than the MLVA-16 and WGSA. Lactobacillic acid was absent in linages I and II but present in linage III, supporting the recent introductions of B. canis strains from Mexico. B. canis displaying putative functional cyclopropane synthase for the synthesis of lactobacillic acid are phylogenetically intertwined with B. canis with non-functional protein, indicating that mutations have occurred independently in the various lineages.

Brucella canis infection in dogs – A neglected zoonosis

Brucellosis is one of the most important zoonotic diseases worldwide that leads to serious economic loss and public health issues. Among infections due to various Brucella species, presently the most neglected zoonoses worldwide is the infection caused by Brucella canis organism. It serves as one of the major causes of reproductive failures and related diseases in canines, and has the potential to infect humans. In addition, infection due to B. canis is a crucial public health concern issue due to increasing interaction of the humans with dogs. In dogs, the disease is manifested with both symptomatic and asymptomatic infections. In humans, it is associated with febrile condition with usually non-specific symptoms. Moreover, lack of proper diagnostic facilities and misconceptions regarding its prevalence, often renders the disease underdiagnosed. The treatment of infection takes into account the relapse frequency of the disease and is employed in only selective cases. At present, no vaccine is available commercially for the prophylaxis of the B. canis infection, so it is the need of the hour to develop improved diagnostic methods as well as vaccines to combat the disease and associated zoonotic risk.

Diagnosis of human and canine Brucella canis infection: development and evaluation of indirect enzyme-linked immunosorbent assays using recombinant Brucella proteins

Brucella canis, a Gram-negative coccobacilli belonging to the genus Brucellae, is a pathogenic bacterium that can produce infections in dogs and humans. Multiple studies have been carried out to develop diagnostic techniques to detect all zoonotic Brucellae. Diagnosis of Brucella canis infection is challenging due to the lack of highly specific and sensitive diagnostic assays. This work was divided in two phases: in the first one, were identified antigenic proteins in B. canis that could potentially be used for serological diagnosis of brucellosis. Human sera positive for canine brucellosis infection was used to recognize immunoreactive proteins that were then identified by performing 2D-GEL and immunoblot assays. These spots were analyzed using MALDI TOF MS and predicted proteins were identified. Of the 35 protein spots analyzed, 14 proteins were identified and subsequently characterized using bioinformatics, two of this were selected for the next phase. In the second phase, we developed and validated an indirect enzyme-linked immunosorbent assays using those recombinant proteins: inosine 5′ phosphate dehydrogenase, pyruvate dehydrogenase E1 subunit beta (PdhB) and elongation factor Tu (Tuf). These genes were PCR-amplified from genomic DNA of B. canis strain Oliveri, cloned, and expressed in Escherichia coli. Recombinant proteins were purified by metal affinity chromatography, and used as antigens in indirect ELISA. Serum samples from healthy and B. canis-infected humans and dogs were used to evaluate the performance of indirect ELISAs. Our results suggest that PdhB and Tuf proteins could be used as antigens for serologic detection of B. canis infection in humans, but not in dogs. The use of recombinant antigens in iELISA assays to detect B. canis-specific antibodies in human serum could be a valuable tool to improve diagnosis of human brucellosis caused by B. canis.

Brucella ovis mutant in ABC transporter protects against Brucella canis infection in mice and it is safe for dogs.

Background/ObjectivesVaccination is the most important tool for controlling brucellosis, but currently there is no vaccine available for canine brucellosis, which is a zoonotic disease of worldwide distribution caused by Brucella canis. This study aimed to evaluate protection and immune response induced by Brucella ovis ΔabcBA (BoΔabcBA) encapsulated with alginate against the challenge with Brucella canis in mice and to assess the safety of this strain for dogs.MethodsIntracellular growth of the vaccine strain BoΔabcBA was assessed in canine and ovine macrophages. Protection induced by BoΔabcBA against virulent Brucella canis was evaluated in the mouse model. Safety of the vaccine strain BoΔabcBA was assessed in experimentally inoculated dogs.ResultsWild type B. ovis and B. canis had similar internalization and intracellular multiplication profiles in both canine and ovine macrophages. The BoΔabcBA strain had an attenuated phenotype in both canine and ovine macrophages. Immunization of BALB/c mice with alginate-encapsulated BoΔabcBA (108 CFU) induced lymphocyte proliferation, production of IL-10 and IFN-γ, and protected against experimental challenge with B. canis. Dogs immunized with alginate-encapsulated BoΔabcBA (109 CFU) seroconverted, and had no hematologic, biochemical or clinical changes. Furthermore, BoΔabcBA was not detected by isolation or PCR performed using blood, semen, urine samples or vaginal swabs at any time point over the course of this study. BoΔabcBA was isolated from lymph nodes near to the site of inoculation in two dogs at 22 weeks post immunization.ConclusionEncapsulated BoΔabcBA protected mice against experimental B. canis infection, and it is safe for dogs. Therefore, B. ovis ΔabcBA has potential as a vaccine candidate for canine brucellosis prevention.

Seroprevalence of Brucella canis in dogs and at-risk humans in Jordan

Brucella canis infection is a neglected zoonotic disease and its seroprevalence in dogs and at-risk humans has not been previously studied in several countries including Jordan. The main aim of this study was to determine the seroprevalence and identify risk factors of B. canis infection in police, breeding and stray dogs and in at-risk humans in Jordan. A total of 169 sera samples from apparently healthy dogs and 185 samples from apparently healthy people (85 from dog handlers and 100 from the general population) were tested in the study. Antibodies against B. canis were tested using the canine D-Tec&lt;sup&gt;®&lt;/sup&gt; CB Rapid Slide Agglutination Test (RSAT) kit with secondary 2-mercaptoethanol (ME-RSAT). Overall, 8.3% of the dog sera samples tested positive to antibodies against B. canis, and 37.8% of stray dogs tested positive. Seroprevalence was higher in male dogs than in females. Furthermore, none of the tested human samples was positive to antibodies against B. canis. There was a significant association between seropositivity and the type of dog. The study reports preliminary findings that suggest the presence of B. canis among stray dogs in Jordan. Thus, preventive measures should be taken to control the transmission of this pathogen from stray dogs to other dogs and humans as well.

Canine Brucellosis: Insights Into the Epidemiologic Situation in Europe.

Brucella canis is one of many responsible pathogens of discospondylitis in dogs and infections require specific management. Little is known about the epidemiologic situation in Europe. The purpose of the study was to get insights into the occurrence of brucellosis in dogs in Europe. The database of a European veterinary laboratory was screened for Brucella positive samples. Additionally, medical records of a veterinary hospital in Germany were screened for diagnosis of discospondylitis and brucellosis. The laboratory received samples from 20 European countries for Brucella testing in dogs: 3.7% of submitted samples were Brucella spp. PCR-positive (61/1,657), and Brucella canis antibodies were identified in 5.4% of submitted samples (150/2,764). Brucella spp. PCR-positive samples originated from Spain (11.1% of submitted samples), Poland (6.7% of submitted samples) and rarely from Italy and France. Samples with Brucella canis antibodies originated from 13 European countries (Sweden, Belgium, Austria, Switzerland, Italy, Finland, Germany, Denmark, Hungary, Norway, Poland, France, Netherlands). Young dogs (0–24 months) had a 5.4-fold increased risk of PCR positive samples. The supplementary medical records search identified four young female dogs (7–30 months) with Brucella canis discospondylitis in Germany. The four dogs had been imported to Germany from Eastern European countries (Moldavia, Romania, Macedonia). In conclusion, infection with Brucella canis needs to be considered in dogs in Europe and diagnostics for Brucella canis infection appear indicated in young dogs with discospondylitis.

Tissue distribution and cell tropism of Brucella canis in naturally infected canine foetuses and neonates

Brucella canis infection is an underdiagnosed zoonotic disease. Knowledge about perinatal brucellosis in dogs is extremely limited, although foetuses and neonates are under risk of infection due to vertical transmission. In this study, immunohistochemistry was used to determine tissue distribution and cell tropism of B. canis in canine foetuses and neonates. Diagnosis of B. canis in tissues of naturally infected pups was based on PCR and sequencing of amplicons, bacterial isolation, and immunohistochemistry, whose specificity was confirmed by laser capture microdissection. PCR positivity among 200 puppies was 21%, and nine isolates of B. canis were obtained. Tissues from 13 PCR-positive puppies (4 stillborn and 9 neonates) presented widespread immunolabeling. Stomach, intestines, kidney, nervous system, and umbilicus were positive in all animals tested. Other frequently infected organs included the liver (92%), lungs (85%), lymph nodes (69%), and spleen (62%). Immunolabeled coccobacilli occurred mostly in macrophages, but they were also observed in erythrocytes, epithelial cells of gastrointestinal mucosa, renal tubules, epidermis, adipocytes, choroid plexus, ependyma, neuroblasts, blood vessels endothelium, muscle cells, and in the intestinal lumen. These results largely expand our knowledge about perinatal brucellosis in the dog, clearly demonstrating a pantropic distribution of B. canis in naturally infected foetuses and neonates.

Proinflammatory response of canine trophoblasts to Brucella canis infection.

Brucella canis infection is an important cause of late-term abortion in pregnant bitches. The pathophysiological mechanisms leading to B. canis–induced abortion are unknown, but heavily infected trophoblasts are consistently observed. As trophoblasts responses to other pathogens contribute to placental inflammation leading to abortion, the aim of the present study was to characterize the cytokine response of canine trophoblasts to B. canis infection. To achieve this, trophoblasts isolated from term placenta of healthy female dogs were infected with B. canis, culture supernatants were harvested for cytokine determinations, and the load of intracellular viable B. canis was determined at different times post-infection. Additionally, cytokine responses were assessed in non-infected trophoblasts stimulated with conditioned media (CM) from B. canis-infected canine monocytes and neutrophils. Finally, cytokine response and bacteria replication were assessed in canine placental explants infected ex vivo. B. canis successfully infected and replicated in primary canine trophoblasts, eliciting an increase in IL-8 and RANTES (CCL5) secretion. Moreover, the stimulation of trophoblasts with CM from B. canis-infected monocytes and neutrophils induced a significant increase in IL-8, IL-6 and RANTES secretion. B. canis replication was confirmed in infected placental explants and the infection elicited an increased secretion of TNF-α, IL-8, IL-6 and RANTES. This study shows that canine trophoblasts produce proinflammatory cytokines in response to B. canis infection and/or to stimulation with factors produced by infected monocytes and neutrophils. These cytokines may contribute to placental inflammation leading to abortion in B. canis-infected pregnant bitches.

Brucella canis infection in dogs from commercial breeding kennels in Brazil.

Canine brucellosis caused by Brucella canis is a neglected zoonosis worldwide and is a leading cause of reproductive failure in dogs, often causing substantial economic losses in breeding kennels. This study aimed to investigate the occurrence of B.canis infection in dogs of commercial breeding kennels located in São Paulo State, Brazil. A total of 753 dogs (183 males and 570 females) from 38 commercial kennels were clinically examined, and blood samples were collected for brucellosis diagnosis through blood culture. The association between clinical manifestations suggestive of brucellosis and positive results through blood culture was determined. Of the 753 dogs tested, 166 (22.0%) had at least one clinical sign suggestive of brucellosis and 158 (20.9%) had positive blood cultures. Seventy-two dogs had positive blood culture and had at least one clinical sign suggestive of brucellosis, while 91 dogs showed at least one clinical manifestation suggestive of brucellosis although blood culture was negative. Of the 38 kennels, 16 (42.1%) had at least one positive dog. The prevalence of infection in each kennel varied from 3.8% to 62.6%. Abortion/stillbirth, failure to conceive and enlargement of lymph nodes were significantly associated with brucellosis in female. No association of clinical signs and positive results in blood culture was observed in males. None of the kennels has been carrying out programmes to control brucellosis, and the sale of infected dogs was considered a common practice yielding risks to the public health, in view of the zoonotic potential of the infection.

Genetic Polymorphism Characteristics of Brucella canis Isolated in China

In China, brucellosis is an endemic disease typically caused by Brucella melitensis infection (biovars 1 and 3). Brucella canis infection in dogs has not traditionally recognized as a major problem. In recent years however, brucellosis resulting from Brucella canis infection has also been reported, suggesting that infections from this species may be increasing. Data concerning the epidemiology of brucellosis resulting from Brucella canis infection is limited. Therefore, the purpose of this study was to assess the diversity among Chinese Brucella canis strains for epidemiological purposes. First, we employed a 16-marker VNTR assay (Brucella MLVA-16) to assess the diversity and epidemiological relationship of 29 Brucella canis isolates from diverse locations throughout China with 38 isolates from other countries. MLVA-16 analysis separated the 67 Brucella canis isolates into 57 genotypes that grouped into five clusters with genetic similarity coefficients ranging from 67.73 to 100%. Moreover, this analysis revealed a new genotype (2-3-9-11-3-1-5-1:118), which was present in two isolates recovered from Guangxi in 1986 and 1987. Second, multiplex PCR and sequencing analysis were used to determine whether the 29 Chinese Brucella canis isolates had the characteristic BMEI1435 gene deletion. Only two isolates had this deletion. Third, amplification of the omp25 gene revealed that 26 isolates from China had a T545C mutation. Collectively, this study reveals that considerable diversity exists among Brucella canis isolates in China and provides resources for studying the genetic variation and microevolution of Brucella.

The vaccine candidate BLSOmp31 protects mice against Brucella canis infection

Canine brucellosis represents a major reproductive problem worldwide and it is considered a zoonotic disease. New approaches are therefore urgently needed to develop an effective and safe immunization strategy against Brucella canis. In the present study, BALB/c mice were subcutaneously immunized with the recombinant chimera rBLSOmp31 formulated in different adjuvants. The different strategies induced a vigorous immunoglobulin G (IgG) response, with high titers of IgG1 as well as IgG2. Besides, spleen cells from rBLSOmp31-immunized mice produced gamma interferon and IL-4, suggesting the induction of a mixed Th1–Th2. Vaccination with rBLSOmp31-IFA formulation provided the best protection levels comparable with that given by control vaccines. None of the immunization strategies induced serological interference in diagnosis. Hitherto, this is the first report that a recombinant vaccine confers protection against B. canis in mice.

Recent trends in human Brucella canis infection

There is little information in the literature regarding the clinical progress of brucellosis in patients affected by other diseases. We report Brucella canis human infection link to Gaucher's disease and Guillain Barré syndrome and discuss complications observed in a case with infective endocarditis. The three cases described came from areas of socio-economic deprivation and scarce epidemiological information where the healthcare personnel did not even consider such diagnosis. The growth of large urban populations deprived from basic services has created a new set of global health challenges. Changes in the urban environment due to slum communities' expansion have resulted in increased dog populations in the peridomiciliary environment. Eleven laboratory employees working with the strains found and their identification were examined. Sanitary authorities should focus on the zoonotic aspect of B. canis considering the dramatic increase of canine roamers near urban centers.

Brucella canis infection in dogs attended in veterinary clinics from patos, Paraíba state, Brazil

Brucella canis infection in dogs attended in veterinary clinics from patos, Paraíba state, Brazil

Brucella canis infection in dogs attended in veterinary clinics from patos, ParabaĪBA state, Brazil.

To determine the frequency of anti-Brucella canis antibodies in dogs attended in veterinary clinics from Patos, Paraíba State, Brazil, as well as to identify risk factors and to isolate and identify the agent, 193 dogs were used. Agar gel immunodiffusion test (AGID) was used to detect B. canis antibodies in sera. Isolation of B. canis was carried out in blood and bone marrow from seropositive animals. Six animals tested seropositive in AGID, resulting in a frequency of 3.11%. B. canis was isolated from bone marrow of one seropositive animal, with confirmation by PCR. Lack of cleaning of the dog’s environment was identified as risk factor (odds ratio = 7.91). This is the first report of isolation of B. canis in dogs from the Northeast region of Brazil.

Brucella canisendophthalmitis in 3 dogs: clinical features, diagnosis, and treatment

To describe historical, clinical and diagnostic features of dogs with Brucella canis endophthalmitis and the response to medical therapy. Three dogs with naturally acquired B. canis endophthalmitis. Dogs were treated symptomatically with topical ophthalmic anti-inflammatories and a novel antimicrobial protocol that included doxycycline, enrofloxacin, rifampin and streptomycin. All dogs presented with chronic or recurrent uveitis in the absence of overt systemic disease. Clinical ophthalmologic abnormalities were unilateral in each dog and included mild-to-moderate anterior uveitis, iris hyperpigmentation, marked vitreal infiltrates, and multifocal chorioretinitis. Dogs were diagnosed with canine brucellosis serologically and by blood culture (n = 2 dogs) or polymerase chain reaction of aqueous humor and blood (n = 1 dog). Active ocular inflammation resolved in all dogs during treatment, with preservation of vision in 2 dogs. Following treatment, B. canis could not be cultured from blood samples and serological values declined with seronegativity achieved in all dogs after a median of 96 weeks (range: 36-112 weeks) of therapy. Brucella canis infection should be included in the differential diagnosis for dogs with intraocular inflammation, regardless of previous history or neuter status. This is the first report of apparently successful medical therapy of canine brucellosis with ocular involvement.