Methicillin-Resistant Staphylococcus aureus from Brazilian Dairy Farms and Identification of Novel Sequence Types.

The aim of this study was to investigate the phenotypic and genotypic diversity and anti-microbial resistance among staphylococci of dairy herds that originated from Paraiba State, north-eastern Brazil, a region where such studies are rare. Milk samples (n = 552) were collected from 15 dairy farms. Isolates were evaluated for anti-microbial susceptibility by Kirby-Bauer disc diffusion method. Confirmation of methicillin-resistant Staphylococcus aureus (MRSA) was performed using multiplex PCR targeting mecA and nuc genes in addition to phenotypic assay based on PBP-2a latex agglutination. Clonal relatedness of isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) genotyping. Staphylococci were detected in 269 (49%) of the samples. Among these, 65 (24%) were S. aureus. The remaining 204 isolates were either coagulase-negative staphylococci (n = 188; 70%) or coagulase positive other than S. aureus (n = 16; 6%). Staphylococci were cultured in seven (35%) of the 20 hand swab samples, from which five isolates were S. aureus. The isolates were most commonly resistant against penicillin (43%), ampicillin (38%) and oxacillin (27%). The gene mecA was detected in 21 S. aureus from milk and in one isolate from a milker's hand. None of the isolates were resistant to vancomycin. PFGE findings showed high clonal diversity among the isolates. Based on MLST, we identified a total of 11 different sequence types (STs 1, 5, 6, 83, 97, 126, 1583, 1622, 1623, 1624 and 1625) with four novel STs (ST1622-ST1625). The findings show that MRSA is prevalent in milk from semi-extensive dairy cows in north-eastern Brazil, and further investigation on its extent in various types of milk production systems and the farm-to-table continuum is warranted.

Molecular and biological characterization of first isolates of Hammondia hammondi from cats from Ethiopia.

Toxoplasma gondii oocysts are morphologically and antigenically similar to oocysts of another feline coccidian, Hammondia hammondi. The distinction between H. hammondi and T. gondii is important from an epidemiological perspective because all isolates of T. gondii are potentially pathogenic for humans and animals, whereas H. hammondi is not known to cause clinical disease in any naturally infected intermediate or definitive hosts. In the present report, H. hammondi (designated HhCatEt1 and HhCatEt2) oocysts were found microscopically in the feces of 2 of 36 feral domestic cats (Felis catus) from Addis Ababa, Ethiopia. Oocysts were orally infective to Swiss Webster and gamma interferon gene knockout mice; the inoculated mice developed tissue cysts in their muscles. Laboratory-raised cats fed mouse tissues of infected mice shed H. hammondi oocysts with a prepatent period of 5 days. The DNA extracted from sporulated oocysts reacted with H. hammondi-specific primers, and sequences were deposited in GenBank (accession nos. JX477424, and KC223619). This is the first report of isolation of H. hammondi from cats from the African continent.

Genetic diversity of Toxoplasma gondii isolates from Ethiopian feral cats.

Recent studies indicate greater genetic variability among isolates of Toxoplasma gondii worldwide than previously thought. However, there is no information on genetic diversity of T. gondii from any host in Ethiopia. In the present study, genotyping was performed on viable T. gondii isolates by bioassays in mice from tissues and feces of 27 cats from Ethiopia. Viable T. gondii was isolated from hearts of 26 cats, feces alone of 1 cat, and feces and tissues of 6 cats; in total there were 33 isolates. Genotyping was performed on DNA from cell-cultured derived T. gondii tachyzoites and by using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). Four genotypes were recognized, including ToxoDB #1 (Type II clonal, nine isolates), ToxoDB #2 (Type III, five isolates), Toxo DB #3 (Type II variant, ten isolates), and ToxoDB #20 (nine isolates). Of interest is the isolation of different genotypes from tissues and feces of two cats, suggesting re-infection or mixed strain T. gondii infection. These findings are of epidemiological significance with respect to shedding of oocysts by cats. This is the first report of genotyping of T. gondii from any host in Ethiopia.

An investigation into the seroprevalence of Toxoplasma gondii, Bartonella spp., feline immunodeficiency virus (FIV), and feline leukaemia virus (FeLV) in cats in Addis Ababa, Ethiopia.

Toxoplasma gondii and Bartonella spp. are zoonotic pathogens of cats. Feline immunodeficiency virus (FIV), and feline leukaemia virus (FeLV) are immunosuppressive viruses of cats that can affect T. gondii oocyst shedding. In this study, the prevalence of antibodies to T. gondii, Bartonella spp., FIV, as well as FeLV antigens were determined in sera from feral cats (Felis catus) from Addis Ababa, Ethiopia. Using the modified agglutination test, IgG antibodies to T. gondii were found in 41 (85.4%) of the 48 cats with titres of 1:25 in one, 1:50 in one, 1:200 in six, 1:400 in six, 1:800 in six, 1:1600 in eight, and 1:3200 in 13 cats. Toxoplasma gondii IgM antibodies were found in 11/46 cats tested by ELISA, suggesting recent infection. Antibodies to Bartonella spp. were found in five (11%) of 46 cats tested. Antibodies to FIV or FeLV antigen were not detected in any of the 41 cats tested. The results indicate a high prevalence of T. gondii and a low prevalence of Bartonella spp. infection in cats in Ethiopia.

Isolation of viable Toxoplasma gondii from tissues and feces of cats from Addis Ababa, Ethiopia.

Cats are important in the epidemiology of Toxoplasma gondii because they are the only hosts that excrete environmentally resistant oocysts in feces. In the present study, hearts, serum, and feces from 36 feral cats from Addis Ababa area, Ethiopia, were examined for T. gondii infection. Antibodies to T. gondii were determined with the modified agglutination test (MAT, cutoff 1:25); 33 cats were seropositive. Hearts of all 36 cats were homogenized, digested in pepsin, and bioassayed in mice. Feces were examined for T. gondii oocysts by bioassay in mice. Viable T. gondii was isolated from heart of 26 by bioassay in mice and from 25 seropositive and 1 seronegative cats. Toxoplasma gondii was isolated from feces (oocysts) by bioassay in mice. In total, viable T. gondii was isolated from 27 of the 36 cats, and these isolates were designated TgCatEt1 to TgCatEt27. The high prevalence of T. gondii oocysts in feces of 8 (19.4%) of 36 cats is of high epidemiologic significance. This is the first report of isolation of viable T. gondii from any host in Ethiopia.

Prevalence of Toxoplasma gondii from free-range chickens (Gallus domesticus) from Addis Ababa, Ethiopia.

Prevalence of Toxoplasma gondii in free-range chickens (Gallus domesticus) is a good indicator of the environmental contamination with oocysts because chickens become infected mainly by feeding from ground, feed, or soil contaminated with oocysts. The seroprevalence of T. gondii antibodies in 125 free-range chickens from the Addis Ababa, Ethiopia, was determined. Antibodies to T. gondii were assayed by the modified agglutination test; 48 of 125 (38.4%) chickens were seropositive, with titers of 1:5 in 14, 1:10 in 12, 1:20 in 14, 1: 40 in 3, 1: 80 in 1, 1:160 in 1, 1:320 in 1, and ≥1:640 in 2 chickens. The hearts of 115 chickens were bioassayed for T. gondii infection. Hearts of 72 seronegative (modified agglutination test [MAT] < 1:5) chickens were pooled in 4 groups (20 + 18 + 19 + 15) and fed to 4 T. gondii -free cats; none of these 4 cats shed oocysts in their feces examined 3-21 days after feeding chicken tissues. Hearts of 43 seropositive chickens (MAT ≥ 1:5) were bioassayed individually in mice. Toxoplasma gondii was isolated from only 1 chicken, with a MAT titer of 1:80. This isolate was designated TgCKEt1 and was not pathogenic for outbred mice. Restricted fragment length polymorphism (RFLP) genotyping using 10 loci indicated the TgCKEt1 was ToxoDB polymerase chain reaction-RFLP genotype #1 (Type II clonal). Results of this study indicate very low environmental contamination with T. gondii oocysts around Addis Ababa.

A review of toxoplasmosis in humans and animals in Ethiopia.

Toxoplasmosis caused by the protozoan parasite, Toxoplasma gondii, is a worldwide zoonosis. In this paper published information on toxoplasmosis in humans and other animals in Ethiopia is reviewed. Limited data indicate that the prevalence of T. gondii in humans in Ethiopia is very high, up to 41% of children aged 1-5 years were reported to be seropositive. There is little information on seroprevalence data in pregnant women and no data on congenital toxoplasmosis in children. About 1 million adults in Ethiopia are considered to be infected with HIV with less than one-third likely receive highly active antiviral therapy. Based on a conservative T. gondii seroprevalence of 50%, thousands might die of concurrent opportunistic infections, including toxoplasmosis. However, exact figures are not available, and most serological surveys are not current. Serological surveys indicate up to 79% of goats and sheep have T. gondii antibodies. However, there is no information on losses due to toxoplasmosis in livestock or the presence of viable T. gondii in any host in Ethiopia.

Induction of unspliced c-fos messenger RNA in rodent brain by kainic acid and lipopolysaccharide.

The c-fos transcriptional factor forms an activator protein-1 (AP-1) complex with proteins from the Jun family, which plays an important role in the central nervous system. The responses of AP-1 transcriptional factors induced by kainic acid (KA) treatment have been well studied, although the transcriptional regulation of these KA-induced factors has not been clearly characterized. To investigate the role of different stimuli in controlling of the splicing of c-fos mRNA, we performed reverse transcriptional polymerase chain reaction. The results showed that spliced and unspliced c-fos is present in rat brain following KA treatment and in lipopolysaccharide (LPS)-treated primary mouse cortical brain cell cultures. Furthermore, tyrosine kinase and protein phosphatase inhibitors alter the preponderance of c-fos transcripts following LPS treatment.

Cloning and expression of MP13 gene from rat hippocampus, a new factor related to guanosine triphosphate regulation.

C-Fos and the Fos-related antigens (FRA) are induced by various stimuli. A novel 35-37 kDa FRA was induced much longer after the treatment using kainic acid (KA) and may be very important for neuronal survival after brain damage. To identify this long-term FRA, we have constructed a cDNA library derived from hippocampus after KA treatment and screened it with an antibody highly conserved M-peptide region of FRAs. One gene, MP13, was cloned with a 1662 bp open reading frame and coded for a 554-amino acid protein. MP13 has a leucine zipper region, a glutamine repeat region, and has high similarity to the activator of the small guanosine triphosphate (GTP)ase Rab5. Gel retardation analysis revealed that MP13 functions as a GTP regulation related factor.

Long-term increase of Sp-1 transcription factors in the hippocampus after kainic acid treatment.

Systemic administration of kainic acid (KA), a glutamate receptor agonist, causes robust seizures and has been used as an excellent rodent model for human temporal lobe epilepsy. Recently, we have demonstrated that a single injection of KA increases the steady-state levels of proenkephalin (PENK) mRNA in the rat hippocampus for at least one year. However, the molecular mechanisms underlying this long-term increase in PENK mRNA levels have not been clearly defined. To determine the possible involvement of the Sp-1 transcription factors in this regulation, electrophoresis mobility-shift assays were used to study the expression of Sp-1 factors in the hippocampus after KA treatment. The results showed that there are long-lasting increases in Sp-1 DNA-binding activity. The Sp-1 DNA-binding complexes were only competed by the non-radioactive Sp-1 element and not by ENKCRE2, AP-1 or CRE elements, indicating the specificity of Sp-1 DNA-binding activity. Since the expression of Sp-1 parallels the time course of long-lasting increase in the expression of PENK mRNA and mossy fiber sprouting after KA treatment, we hypothesize that the increase in Sp-1 activity may be associated with the long-term changes in the plasticity of hippocampal function after KA-induced seizures.