Targeting the Tick/Pathogen Interface for Developing New Anaplasmosis Vaccine Strategies

Bovine anaplasmosis is a tick-borne hemolytic disease of cattle that occurs worldwide caused by the intraerythrocytic rickettsiae Anaplasma marginale. Control measures, including use of acaricides, administration of antibiotics and vaccines, have varied with geographic location. Our research is focused on the tick-pathogen interface for development of new vaccine strategies with the goal of reducing anaplasmosis, tick infestations and the vectorial capacity of ticks. Toward this approach, we have targeted (1) development of an A. marginale cell culture system to provide a non-bovine antigen source, (2) characterization of an A. marginale adhesion protein, and (3) identification of key tick protective antigens for reduction of tick infestations. A cell culture system for propagation of A. marginale was developed and provided a non-bovine source of A. marginale vaccine antigen. The A. marginale adhesion protein, MSP1a, was characterized and use of recombinant MSP1a in vaccine formulations reduced clinical anaplasmosis and infection levels in ticks that acquired infection on immunized cattle. Most recently, we identified a tick-protective antigen, subolesin, that reduced tick infestations, as well as the vectorial capacity of ticks for acquisition and transmission of A marginale. This integrated approach to vaccine development shows promise for developing new strategies for control of bovine anaplasmosis.     

Seroprevalence of <Emphasis Type="Italic">Anaplasma marginale</Emphasis> and <Emphasis Type="Italic">Babesia bigemina</Emphasis> infections and associated risk factors in Machakos County,Kenya

Anaplasma marginale and Babesia bigemina are important tick-borne pathogens of cattle. A cross-sectional survey was undertaken to determine the seroprevalence of A. marginale and B. bigemina infections and identify associated risk factors on traditional smallholder farms in Machakos County, Kenya. A total of 421 cattle from 127 farms from four divisions in the county were sampled and visited between September and November 2007. The farms were selected by a proportional allocation approach based on the number of farms in the four divisions previously selected by stratified random sampling method. Information on animal and individual farm management variables was obtained using standardized questionnaires. Prevalence of serum antibodies due to A. marginale and B. bigemina pathogens was determined using the enzyme-linked immunosorbent assay (ELISA) technique. The relationship between the seropositivity and associated risk factors was assessed by multivariable analyses using standard logistic regression models. The overall estimation (and their 95% confidence intervals) of A. marginale and B. bigemina seropositivity at the animal level was 53.4% (48.5%, 58.2%) and 40.6% (35.8%, 45.4%), respectively. Two variables, “animal age” and “administrative division,” were significantly associated with the A. marginale seroresponse. Three variables, “animal age” “grazing system” and “administrative division” were significantly associated with the B. bigemina seroresponse. These findings suggest possible indicators of existence of endemic instability for the two infections. The study identifies characterization of environmental suitability for the vectors and how they interact with grazing systems to cause the infections as an area for further studies, for improved understanding of the infections and in designing disease control programs.     

Anaplasma Phagocytophilum - the Most Widespread Tick-Borne Infection in Animals in Europe

The bacterium Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila) may cause infection in several animal species including human. The disease in domestic ruminants is also called tick-borne fever (TBF), and has been known for at least 200 years. In Europe, clinical manifestations due to A. phagocytophilum have been recorded in sheep, goat, cattle, horse, dog, cat, roe deer, reindeer and human. However, seropositive and PCR-positive mammalian have been detected in several other species. Investigations indicate that the infection is prevalent in Ixodes ricinus areas in most countries in Europe. A. phagocytophilum infection may cause high fever, cytoplasmatic inclusions in phagocytes and severe neutropenia, but is seldom fatal unless complicated by other infections. Complications may include abortions, and impaired spermatogenesis for several months. However, the most important aspect of the infection at least in sheep is its implication as a predisposing factor for other infections. Factors such as climate, management, other infections, individual conditions etc. are important for the outcome of the infection. A. phagocytophilum may cause persistent infection in several species. Based on the 16S rRNA gene sequences several variants exist. Different variants may exist within the same herd and even simultaneously in the same animal. Variants may behave differently and interact in the mammalian host.     

Anaplasmosis in cattle in Italy

Bovine anaplasmosis caused by Anaplasma marginale is a disease transmitted by ticks belonging to the Ixodidae family. Southern Italy is considered an endemic zone but environmental and social factors are changing the epidemiology of the disease to expand to previously anaplasmosis-free regions. The available data of published reports of anaplasmosis in Italy together with the data obtained by the National Centre of Reference for Anaplasma, Babesia, Rickettsia and Theileria (C.R.A.Ba.R.T.), allowed to report A. marginale infection in different Italian regions (Lazio, Marche, Campania, Puglia, Basilicata, Calabria, Lombardy, Tuscany, Umbria and Sicily). Cattle are also subject to infection with the related Ixodes ricinus-transmitted pathogen, Anaplasma phagocytophilum that results in reduced milk production in cattle. A. phagocytophilum infect also small ruminants, domestic and wild animals and causes the human granulocytic anaplasmosis. Different studies have been conducted on the presence of A. phagocytophilum in Italy both in the tick vectors and in the wild and domestic reservoirs. Contrary to A. marginale, the prevalence of A. phagocytophilum embraces the whole Italian territory from the Alps to the southern and insular regions.     

Anaplasma phagocytophilum infection in a domestic cat in Finland: Case report

Background

Anaplasmosis is a vectorborne disease caused by the gram-negative bacterium Anaplasma phagocytophilum. This species displays positive tropism to granulocytes and can cause illness in several mammalian species, including cats, dogs, and humans. It is considered as an emerging disease in Europe. The clinical signs are nonspecific and include fever, lethargy, and inappetence. The most typical hematologic abnormality is thrombocytopenia. A tentative diagnosis can be made by detecting intracytoplasmic morulae inside neutrophils. The diagnosis is confirmed by PCR and serology in paired serum samples. A sample for PCR analysis should be taken before treatment. Anaplasmosis is treated with doxycycline.

Case presentation

A feline case of anaplasmosis is presented. The history, clinical presentation, diagnostics, treatment, and follow-up are discussed.

Conclusions

This case indicates that Anaplasma phagocytophilum infects cats in Finland. To provide accurate treatment, anaplasmosis should be listed as a differential diagnosis in cats suffering from acute febrile illness with previous tick exposure.     

Transformation of Anaplasma marginale

The tick-borne pathogen, Anaplasma marginale, has a complex life cycle involving ruminants and ixodid ticks. It causes bovine anaplasmosis, a disease with significant economic impact on cattle farming worldwide. The obligate intracellular growth requirement of the bacteria poses a challenging obstacle to their genetic manipulation, a problem shared with other prokaryotes in the genera Anaplasma, Ehrlichia, and Rickettsia. Following our successful transformation of the human anaplasmosis agent, A. phagocytophilum, we produced plasmid constructs (a transposon bearing plasmid, pHimarAm-trTurboGFP-SS, and a transposase expression plasmid, pET28Am-trA7) designed to mediate random insertion of the TurboGFP and spectinomycin/streptomycin resistance genes by the Himar1 allele A7 into the A. marginale chromosome. In these trans constructs, expression of the fluorescent and the selectable markers on the transposon, and expression of the transposase are under control of the A. marginale tr promoter. Constructs were co-electroporated into A. marginale St. Maries purified from tick cell culture, and bacteria incubated for 2 months under selection with a combination of spectinomycin and streptomycin. At that time, ≤1% of tick cells contained colonies of brightly fluorescent Anaplasma, which eventually increased to infect about 80–90% of the cells. Cloning of the insertion site in E. coli and DNA sequence analyses demonstrated insertion of the entire plasmid pHimarAm-trTurboGFP-SS encoding the transposon in frame into the native tr region of A. marginale in an apparent single homologous crossover event not mediated by the transposase. Transformants are fastidious and require longer subculture intervals than wild type A. marginale. This result suggests that A. marginale, as well as possibly other species of Anaplasma and Ehrlichia, can be transformed using a strategy of homologous recombination.     

Survey of Anaplasmataceae bacteria in sheep from Senegal

Purpose

The authors studied the role of bacteria belonging to Anaplasmataceae family as the causes of acute illnesses of sheep in West Africa.

Methods

We examined and sampled 120 febrile sheep in two regions of Senegal for this study. The DNA extracted from these blood samples was tested by PCR using two pairs of primers (groEL-based and 16S rRNA gene-based).

Results

In 52/120 samples, the microscopic examination revealed intraerythrocytic and/or intraphagocytic spherical inclusions. In 48/52 cases, we succeeded in identifying the bacterial agent: in 38 cases, it was Anaplasma ovis; in six cases, it was Ehrlichia ruminantium; in two cases, Anaplasma phagocytophilum; in one case, Anaplasma platys; and in one case, a yet uncultured Anaplasma sp. closely related to A. phagocytophilum.

Conclusions

Our studies demonstrated the great variety of pathogenic bacteria from the Anaplasmataceae family in the blood of clinically ill sheep. A. ovis was identified unexpectedly often. For the first time, A. phagocytophilum was found in sub-Saharan Africa, and its further epidemiology may be now reconsidered. The roles of canine pathogen, A. platys, and yet undescribed Anaplasma sp. “Badiouré” in ovine pathology should be more closely studied.     

Transmission of Anaplasma marginale from a naturally-infected wild african giant rat (Cricetomys gambianus), waterhouse) to a calf in Nigeria

Blood taken from an African giant rat in Nigeria, transmitted Anaplasma marginale when inoculated into a splenectomised calf. The infection of the calf was sub-clinical and the highest percentage of infected erythrocytes recorded was 6. Anaplasma marginale was first observed in the calf's blood three days after inoculation, but the parasite disappeared suddenly, nine days after inoculation. This is the first experimental demonstration of A. marginale infection in African giant rats in Nigeria, a country where anaplasmosis is endemic in the cattle population. The significance of giant rats being a reservoir host of A. marginale in Nigeria is discussed.     

Management of Wild Ungulate Populations in Italy: Captive-Breeding,Hybridisation and Genetic Consequences of Translocations

Captive-reproduced stocks of some species of ungulates (Artiodactyla), and particularly the red deer (Cervus elaphus), fallow deer (Dama dama), roe deer (Capreolus capreolus) and the wildboar (Sus scrofa) are more or less extensively translocated in Italy, mainly for local reintroductions or restocking of exploited wild populations. However, captive breeding often involves the reproduction of non-indigenous individuals or the production of artificial hybrids. Consequently, translocations of captive-reproduced ungulates are of concern for the conservation of indigenous populations and gene pools. The impact of translocations should be evaluated within the background of the growing knowledge on population genetic and phylogeographic structure of ungulates. Molecular genetic markers are being used to map geographic genetic diversity, and reconstruct the phylogeographic history of natural populations (i.e., in the roe deer). Molecular makers are also used to detect the consequences of domestication and identify hybrids between wild and domesticated populations (i.e., in the wildboar), or to detect inter-specific hybridisation (i.e., between the red deer and wapiti). Hybridisation of wild and domestic pigs, and diffusion of hybrids in nature is widespread in Italy. Admixture of indigenous and non-indigenous roe deer stocks is also widespread. Therefore, conservation and management of indigenous ungulates calls for careful evaluation of captive-reproduced stocks.     

Detection of Chlamydophila abortus in sheep and goat flocks in southern Italy by PCR using four different primer sets

An epidemiological survey was performed to detect the presence of Chlamydophila (C.) abortus and other members of the order Chlamydiales in ovine and caprine flocks with a history of abortion in southern Italy. Four pairs of primers were compared to evaluate their ability to detect Chlamydiales using purified DNA preparations and tissue samples from aborted foetuses with suspected chlamydial infections. As expected, amplification of DNA of the reference strain C. abortus using primer pairs U23F/23Sigr, 16SF2/23R, CTU/CTL and CpsiA/CpsiB produced fragments of about 600 bp, 585 bp, 1000 bp and 300 bp, respectively. The detection limits of the four PCR tests performed on serial DNA dilutions of the C. abortus reference strain were of 10 pg, 0.1 pg, 0.1 pg and 1 fg of DNA, respectively. The most sensitive amplification of DNA extracted from the organ tissues was obtained with primer pairs CpsiA/CpsiB, which detected Chlamydophila spp. DNA in all infected tissue samples. Only C. abortus was identified during the survey. The presence of this agent was confirmed in 3 out of 27 ovine and caprine flocks included in the survey suggesting that abortion due to C. abortus is uncommon in southern Italy.     

Long-term effects of good handling practices during the pre-weaning period of crossbred dairy heifer calves

The aim of this study was to determine whether applying good practices of handling during the pre-weaning period have long-term effects on behavioral and physiological indicators, health status, and average daily gain (ADG) of crossbred Bos taurus × Bos indicus heifer calves. During the pre-weaning period, 98 crossbred of Holstein × Gir heifer calves were allotted into three treatments: (1) good practices of handling + brushing (GPB; n?=?25), (2) good practices of handling (GP; n?=?25), and (3) control (n?=?48). Every 2 months, four evaluation periods (EV₁ to EV₄) were conducted to record data. Behavioral indicators comprised time to drive (TD), flight speed (FS), flight distance (FD), and composite reactivity score (CRS). Physiological indicators of acute stress during handling comprised respiratory and heart rates. Health status comprised data regarding occurrence of most common diseases (i.e., pneumonia and anaplasmosis). Collected data were analyzed by using a linear mixed model for repeated measures, Tukey’s test, and chi-squared procedures. Treatment influenced (P?<?0.05) TD, FS, and FD but not CRS (P?=?0.78). From EV₁ to EV₃, the control calves had the lowest TD. The GPB group had lower FS than the control but did not differ from GP. The GPB group had lower FD means than the other two groups in EV₂, EV₃, and EV₄. No differences (P?>?0.05) due to treatment were observed on heart and respiratory rates, ADG, or occurrence of pneumonia and anaplasmosis. It was concluded that adoption of good practices of handling during pre-weaning period may lead to long-term positive effects.     

Genotypic and phenotypic detection of capsular polysaccharide and biofilm formation in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolated from bovine milk collected from Brazilian dairy farms

Staphylococcus aureus is a pathogen that frequently causes mastitis in bovine herds worldwide. This pathogen produces several virulence factors, including cell-associated adhesins, toxic and cytolytic exoproteins, and capsular polysaccharides. The aim of the present study was to test for the presence of genes involved in capsular polysaccharide production and biofilm formation in S. aureus isolated from bovine mastitis samples collected from 119 dairy herds located in three different Brazilian regions, as well as to assay the production of capsular polysaccharides and biofilm, in vitro. The detection of the cap, icaAD, and bap genes was performed using PCR. The detection and quantification of capsular polysaccharide production was performed using ELISA assays. The ability of the isolates to form a biofilm was examined using the polystyrene surface of microtiter plates. All 159 S. aureus isolates investigated harboured the cap gene: 80 % carried the cap5 gene and 20 % carried the cap8 gene. Sixty-nine percent of the isolates expressed capsular polysaccharide (CP) in vitro, 58 % expressed CP5 and 11 % expressed CP8. All of the isolates harboured the icaA and icaD genes, and 95.6 % of the isolates carried the bap gene. Of the 159 isolates analysed, 97.5 % were biofilm producers. A significant association between the capsular genotype and phenotype and the amount of biofilm formation was detected: cap5/CP5 isolates tended to form more biofilm and to produce a thinner CP layer than cap8/CP8 isolates. The results indicate a high potential for pathogenicity among S. aureus isolated from bovine milk collected from three different regions in Brazil.     

Risk factors for anaplasmosis in dairy cows during the peripartum

Anaplasma marginale is endemic in tropical and subtropical areas around the world. Some studies have suggested that cows during peripartum may present a transient immunosuppression state and development of clinical signs of anaplasmosis. The aim of this study was to investigate the relationship between some risk factors and the seroprevalence of A. marginale in dairy cows during peripartum in Rio de Janeiro, Brazil. The risk factors analyzed in association with the prevalence of antibodies against A. marginale in dairy cows were calving season, reproductive experience, breed standard, tick infestations, stocking density, and milk yield. The antibodies against A. marginale were tested in indirect enzyme-linked immunosorbent assays. A primary screening using a 2 × k contingency table of the exposed variables with the outcomes was performed. All variables for which p?<?0.20 were included in a fixed effects log regression. The risk factors investigated to anaplasmosis were calving (OR 2.61, IC 1.08–7.63), breed standard (OR 3.83, IC 0.08–0.28), reproductive experience (OR 33.7, IC 2.14–5.16), milk yield (OR 3.9, IC 2.24–7.03), Rhipicephalus microplus infestations (OR 10.3, IC 0.05–0.17), and stocking density (OR 22.3, IC 0.05–0.17). Low titers of antibodies against A. marginale during peripartum had been characterized as a period previous to development of clinical anaplasmosis. Thus, studies on anaplasmosis should consider each farm as an epidemiological unit, where environmental and immunological factors may influence the endemic status of the pathogen.     

The natural history of Anaplasma marginale

The intracellular pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), described by Sir Arnold Theiler in 1910, is endemic worldwide in tropical and subtropical areas. Infection of cattle with A. marginale causes bovine anaplasmosis, a mild to severe hemolytic disease that results in considerable economic loss to both dairy and beef industries. Transmission of A. marginale to cattle occurs biologically by ticks and mechanically by biting flies and by blood-contaminated fomites. Both male ticks and cattle hosts become persistently infected with A. marginale and serve as reservoirs of infection. While erythrocytes are the major site of infection in cattle, A. marginale undergoes a complex developmental cycle in ticks that begins by infection of gut cells, and transmission to susceptible hosts occurs from salivary glands during feeding. Major surface proteins (MSPs) play a crucial role in the interaction of A. marginale with host cells, and include adhesion proteins and MSPs from multigene families that undergo antigenic change and selection in cattle, thus contributing to maintenance of persistent infections. Many geographic strains of A. marginale have been identified worldwide, which vary in genotype, antigenic composition, morphology and infectivity for ticks. Isolates of A. marginale may be maintained by independent transmission events and a mechanism of infection/exclusion in cattle and ticks. The increasing numbers of A. marginale genotypes identified in some geographic regions most likely resulted from intensive cattle movement. However, concurrent A. marginale strain infections in cattle was reported, but these strains were more distantly related. Phylogenetic studies of selected geographic isolates of A. marginale, using msp4 and msp1α, provided information about the biogeography and evolution of A. marginale, and msp1α genotypes appear to have evolved under positive selection pressure. Live and killed vaccines have been used for control of anaplasmosis and both types of vaccines have advantages and disadvantages. Vaccines have effectively prevented clinical anaplasmosis in cattle but have failed to block A. marginale infection. Vaccines are needed that can prevent clinical disease and, simultaneously, prevent infection in cattle and ticks, thus eliminating these hosts as reservoirs of infection. Advances in genomics, proteomics, immunology and biochemical and molecular technologies during the last decade have been applied to research on A. marginale and related organisms, and the recent development of a cell culture system for A. marginale has provided a format for studying the pathogen/tick interface. Recent advancements and new research methodologies should provide additional opportunities for development of new strategies for control and prevention of bovine anaplasmosis.     

Identification of bacteria present in ulcerative stomatitis lesions of captive sea turtles <Emphasis Type="Italic">Chelonia mydas</Emphasis>

Anthropogenic activities, predation, and diseases have contributed to a decrease in the sea turtle population in recent years. Ulcerative stomatitis is a condition that occurs in both wild and captive populations. The etiology of this condition is associated with bacteria such as E. coli, Citrobacter diversus, Klebsiella spp., Pseudomonas spp., Flavobacter calcoaceticus, Staphylococcus spp., and Flavobacterium spp. Some of these microorganisms are part of the oral microbiota of turtles, but alterations in the immune response can disturb the homeostatic relationship and cause an increase in the population of microorganisms, which in turn can cause disease. This work presents results on the isolation and identification of bacteria present in ulcerative stomatitis lesions in captive C. mydas turtles. Oral mucosa samples from 20 clinically healthy turtles and ten animals with ulcerative stomatitis lesions were studied. The samples were cultivated in enriched and differential media, and the identification was made using an automated method. The results showed a great diversity of bacteria in animals with ulcerative stomatitis with a higher prevalence of S. lentus and C. braakii was higher (60 and 50%, respectively) than in healthy animals. E. faecium was identified in 40% of diseased animals and 55% healthy animals. Turtles in this study had a diverse oral microbiota, and S. lentus and C. braakii may be involved in the etiopathogenesis of ulcerative stomatitis.     

Prevalence and risk factors associated with <Emphasis Type="Italic">Theileria parva</Emphasis> infection in cattle in three regions of Tanzania

Ticks and tickborne diseases (TBDs) are serious constraints to cattle production in Tanzania and other tropical and subtropical countries. Among the TBDs, East Coast fever (ECF) is the most important as it causes significant economic losses to the cattle industry in Tanzania. However, control of ECF in Tanzania has continued to be a challenge due to inadequate epidemiological information. The main objective of this study was to determine the epidemiological situation of Theileria parva infections in cattle kept under pastoral and agro-pastoral farming systems in Mara, Singida, and Mbeya regions of Tanzania. Blood samples were collected from 648 cattle in the three regions. Genomic DNA was extracted and amplified in a polymerase chain reaction (PCR) using T. parva-specific primers targeting the 104-kD antigen (P104) gene. In addition, information was collected on the possible risk factors of T. parva infection (animal age, region, animal sex, tick burden, tick control method, and frequency of acaricide application). The prevalence of T. parva across the three regions was 14.2%. There was variation in prevalence among the three regions with Mara (21.8%) having a significantly higher (p = 0.001) prevalence than the other regions. Moreover, Mbeya exhibited relatively lower prevalence (7.4%) compared to the other regions. Factors found to be significantly associated with an animal being PCR positive for T. parva were region (p = 0.001) and tick burden (p = 0.003). Other factors were not found to be significant predictors of being PCR positive for T. parva. The present study showed high variation in tick burden and T. parva prevalence across the regions. Therefore, different strategic planning and cost-effective control measures for ticks and T. parva infection should be implemented region by region in order to reduce losses caused by ticks and ECF in the study area.     

<Emphasis Type="Italic">Arcobacter</Emphasis> spp. in fecal samples from Brazilian farmed caimans (<Emphasis Type="Italic">Caiman yacare</Emphasis>, Daudin 1802)

The aim of this study was to perform the identification and molecular characterization of Arcobacter cryaerophilus and Arcobacter butzleri isolated from caiman (Caiman yacare), kept at a production farm, in Brazil. Forty fecal samples were analyzed. After isolation and identification, 21/40 strains of A. butzleri and 19/40 strains of A. cryaerophilus were subjected to PCR for potential virulence gene detection. The results of the PCR showed 38/40 strains positive for the cadF, cj1349, ciaB, and tlyA genes, 39/40 strains positive for the pldA gene, and 40/40 strains positive for the mviN gene. None of the strains presented the irgA gene. Hemagglutinin (hecA gene) and hemolysin (hecB) genes were detected in 21/40 and 16/40 strains, respectively. The SE-AFLP showed a great genetic diversity, but some clonally groups were disseminated in various tanks. These data reveal that the strains presented the same virulence traits described from Arcobacter isolated from food-borne disease in humans.     

Genotypic analysis of virulence genes and antimicrobial profile of diarrheagenic <Emphasis Type="Italic">Escherichia coli</Emphasis> isolated from diseased lambs in Iran

The aim of the present study was to determine the analysis of virulence genes and antimicrobial profile of diarrheagenic Escherichia coli isolated from diseased lambs. Two hundred ninety E. coli isolates were recovered from 300 rectal swabs of diarrheic lambs and were confirmed by biochemical tests. The pathotype determination was done according to the presence of genes including f5, f41, LTI, STI, bfp, ipaH, stx ₁ , stx ₂ , eae, ehlyA, cnf ₁ , cnf ₂ , cdIII, cdIV, and f17 by PCR method. Sixty-six isolates (23.72%) possessed the STI gene and categorized into entrotoxigenic E. coli (ETEC). Nine isolates (3.1%) and five isolates (1.72%) were positive for the cnf1 and cnf2 genes which categorized into necrotoxic E. coli (NTEC). Hundred and seventeen isolates (40.34%) harbored stx ₁ and/or stx ₂ and classified as Shiga toxin-producing E. coli (STEC). Thirteen isolates (4.48%) were assigned to atypical entropathogenic E. coli (aEPEC) and possessed eae gene. Two isolates (0.68%) were positive for ipaH gene and were assigned to entroinvasive E. coli (EIEC). Statistical analysis showed a specific association between eae gene and STEC pathotype (P?<?0.0001). The most prevalent resistance was observed against lincomycin (96.5%) and the lowest resistance was against kanamycine (56.02%), respectively. The high prevalence of STEC and ETEC indicates that diarrheic lambs represent an important reservoir for humans. ETEC may play an important role for frequent occurrence of diarrhea in lambs observed in this region. Due to high antibiotic resistance, appropriate control should be implemented in veterinary medicine to curb the development of novel resistant isolates.     

Frequency of enterotoxins,toxic shock syndrome toxin-1, and biofilm formation genes in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates from cows with mastitis in the Northeast of Brazil

Staphylococcus aureus is among the microorganisms more frequently associated with subclinical bovine mastitis. S. aureus may produce several virulence factors. This study aimed at determining the frequency of virulence factors such as enterotoxins, toxic shock syndrome toxin 1, and ica adhesion genes. In addition, we assessed antimicrobial drug resistance in S. aureus isolated from clinical and subclinical cases of mastitis. A total of 88 cows with clinical or subclinical mastitis were sampled, resulting in 38 S. aureus isolates, from which 25 (65.78%) carried toxin genes, including seb, sec, sed, tst, and icaD adhesion gene. These S. aureus isolates belong to 21 ribotypes and three S. aureus strains belonged to the same ribotype producing ica adhesion gene. Approximately 90% of S. aureus strains obtained in our study demonstrated multiple resistance to different antimicrobial agents. The most efficacious antimicrobial agents against the isolates were gentamicin, amoxicillin, and norfloxacin. Gentamicin was the most efficacious agent inhibiting 78.95% of the S. aureus isolates. The least efficacious were penicillin, streptomycin, and ampicillin. Our results can help in understanding the relationship between virulence factors and subclinical mastitis caused by S. aureus. Further research about diversity of S. aureus isolates and genes responsible for the pathogenicity of subclinical mastitis is essential.     

Comparison of rapid diagnostic tests to detect <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> disseminated infection in bovine liver

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s disease, a chronic enteritis in cattle and other domestic and wild ruminants. The presence of MAP in tissues other than intestines and associated lymph nodes, such as meat and liver, is a potential public health concern. In the present study, the relationship between the results of rapid diagnostic tests of the Johne’s disease, such as serum ELISA, rectal scraping PCR, and acid-fast staining, and the presence of MAP in liver was evaluated. Blood, liver, and rectal scraping samples were collected from 200 slaughtered cattle with unknown Johne’s disease status. ELISA was performed to determine the MAP antibody activity in the serum. Acid-fast staining was performed on rectal scraping samples, and PCR was performed on rectal scraping and liver samples. PCR-positive liver samples were used for mycobacterial culture. Overall, the results of this study demonstrated that MAP can be detected and cultured from liver of slaughtered cattle and rapid diagnostic tests of Johne’s disease have limited value in detecting cattle with MAP infection in liver. These findings show that the presence of MAP in liver tissue may occur in cows with negative results for rapid diagnostic tests and vice versa. Hence, liver might represent another possible risk of human exposure to MAP. Given concerns about a potential zoonotic role for MAP, these results show the necessity to find new methods for detecting cattle with MAP disseminated infection.