Bruno Penna

Leptospirosis as the most frequent infectious disease impairing productivity in small ruminants in Rio de Janeiro, Brazil.

Despite the importance of small ruminants breeding in developing countries, milk/meat productivity remains unsatisfactory. Infectious diseases, such as leptospirosis, brucellosis, and small ruminant lentiviruses (SRLVs), contribute to this scenario. The objective of the present study was to determine the role of each of these diseases in the productivity of small ruminants breeding in Rio de Janeiro, Brazil. In goats, 343 samples were tested for leptospirosis, 560 for Brucella abortus, and 506 for caprine arthritis-encephalitis (CAE), whereas in sheep, 308 samples were tested for leptospirosis, 319 for B. abortus, 374 for Brucella ovis, and 278 for Maedi-Visna (MV). Regarding leptospirosis, 25.9% of goats and 47.4% sheep were seroreactive, with serovar Hardjo the most prevalent in both species. Anti-B. abortus agglutinins were found in 0.7% of all samples, exclusively in goats. In relation to SRLVs, 8.6% of goats and 3.2% of sheep samples were positive for CAE and MV, respectively. Leptospirosis was the major infectious problem in the small ruminants sampled and may contribute to impaired productivity of these animals.

Isolation of Leptospira interrogans Hardjoprajitno from vaginal fluid of a clinically healthy ewe suggests potential for venereal transmission.

A total of 15 adult ewes from one flock known to be seroreactive for leptospirosis was studied. Urine and vaginal fluid were collected from each animal to test for the presence of leptospires using bacterial culture and conventional PCR methods. One pure culture of Leptospira sp. was obtained from the vaginal fluid sample of a non-pregnant ewe. The isolate was characterized by DNA sequencing of the rrs and secY genes, variable-number of tandem-repeats (VNTR) analysis and serogrouping, and the isolate was typed as Leptospira interrogans serogroup Sejroe serovar Hardjo type Hardjoprajitno. This report indicates the presence of viable Leptospira in the vaginal fluid of a ewe, suggesting the potential for venereal transmission of leptospires in sheep.

Progestin-impregnated intravaginal sponges for estrus induction and synchronization influences on goats vaginal flora and antimicrobial susceptibility

The objective was to characterize vaginal bacteria, their antimicrobial sensitivity, and the incidence of vaginitis, in goats before and after insertion of intravaginal sponges containing progesterone. Sponges were inserted in 37 Saanen goats and removed after 6, 9 or 12d (G6, G9 and G12). At sponge removal, all goats had clinical signs of vaginitis. Sampling was conducted just before sponge insertion and at 0, 24, 48, and 72 h after sponge removal. Vaginal secretions were subjected to standard bacteriological procedures, including isolation of bacteria, subculture, and determination of sensitivity to antimicrobials (gentamicin, cefalotin, tetracycline, ciprofloxacin, trimethoprim-sulfamethoxazole, amoxicillin and clavulanic acid, penicillin G and cefoxitin). Ciprofloxacin, gentamicin, tetracycline and trimethoprim-sulfamethoxazole were the most effective for coliforms (100% sensitivity), whereas ciprofloxacin, gentamicin and tetracycline were the most effective for cocci (100, 98.6 and 97.2% sensitivity, respectively). In contrast, the least effective antimicrobials were cefalotin for the coliforms, and penicillin for the cocci (37.5 and 64.4% sensitivity, respectively), regardless of duration of implant presence and interval from implant removal to sampling. In conclusion, insertion of intravaginal progestin-impregnated sponges induced clinical vaginitis in goats. Members of Staphylococcus genus were the most frequently recovered species of the vaginal samples cultured, and all isolates were resistant to several antimicrobials.

Antimicrobial susceptibility of Staphylococci isolated from naturally occurring canine external ocular diseases

OBJECTIVE The purpose of this study was to describe the species distribution and antimicrobial susceptibility patterns of Staphylococci isolated from naturally occurring canine ocular diseases. SAMPLES AND PROCEDURES: Samples obtained from 68 dogs with signs of external eye disease were processed for isolation of bacteria of the genus Staphylococcus sp. Isolates were identified by biochemical reactions and tested for susceptibility to 11 antimicrobials of six classes. RESULTS Forty (58.8%) samples yielded Staphylococcus sp. in pure culture. Coagulase-positive Staphylococci were most common and Staphylococcus intermedius was the most frequent (45%) species, followed by S. aureus (22.5%) and the coagulase-negative species S. epidermidis (20%), and S. simulans (12.5%). Resistance to at least one drug was observed in 92.5% of the isolates, and multidrug resistance was a common finding (72.5%). The most effective drugs against Staphylococcus strains isolated from extra-ocular canine ocular diseases were ceftiofur and cefalexin. CONCLUSION This study highlights the presence of Staphylococcus sp. genus in naturally occurring extra-ocular canine ocular disease and the emergence of resistant strains to common antimicrobial drugs. It also emphasizes the need for bacterial culture with species identification and susceptibility testing in order to choose the appropriate antimicrobial therapy.

Usage of Leptospira spp. local strains as antigens increases the sensitivity of the serodiagnosis of bovine leptospirosis.

Leptospirosis is a zoonotic disease that occurs worldwide, particularly in tropical countries. In livestock the agent is responsible for reproductive problems such as infertility and abortion. Serogroup Sejroe, particularly serovar Hardjo, prevails in cattle in several regions. The microscopic agglutination test (MAT) is the current method for diagnosing leptospirosis. It has been proposed that the inclusion of local strains could detect a larger set of seroreactive animals. In that context, the aim of the present study was to evaluate if the usage of local strains as antigens increases the sensitivity of the serodiagnosis of bovine leptospirosis. Blood and urine samples were collected from 314 bovines from several herds randomly selected in a slaughterhouse in Rio de Janeiro, Brazil. Serological diagnosis was made with MAT using a 21 reference-strains panel (MAT21). Additionally, 12 local strains (MAT33) were included as antigens. PCR was performed with the urine samples and it was positive on 71 out of 222 samples (31.9%). MAT21 identified as seroreactive 173 (55.1%) out of the 314 animals studied, with Sejroe the most common (38.1%). In MAT33, 204 (65.0%) animals were seroreactive with a significant increase on seroreactivity (9.9%). In conclusion, MAT presented with a significant increase of sensitivity when local strains were used as antigens. Among the local strains, 2013_U152 (KP263062) (serogroup Shermani) and 2013_U280 (KP263069) (serogroup Grippotyphosa) showed to be more antigenic.

Differences between seroreactivity to leptospirosis in dairy and beef cattle from the same herd in Rio de Janeiro, Brazil.

We would like to share our 20-year experience with diagnosis of leptospirosis in cattle in the state of Rio de Janeiro, Brazil. Bovine leptospirosis is associated with abortion, lowered fertility, and decreased milk production in cattle, causing substantial economic losses (Faine et al. 2000). Under tropical conditions, the disease is common; indeed, it is endemic in some tropical regions (Lilenbaum and Souza 2003). Herd management may affect the overall seropreva-lence of the disease and the distribution of serovars, and the prevalence is generally higher in dairy than beef cattle (Faine et al. 2000). However, there is limited understanding regarding seroprevalence and transmission of pathogenic Leptospira in herds comprised of both dairy and beef cattle. A brucellosis-free herd located in the state of Rio de Janeiro, Brazil, was investigated. The land had minimal slope, and was subject to frequent flooding, particularly during summer. The herd consisted of both dairy cattle (206 cows, Holstein-Gir cross), managed in a semi-intensive system, and beef cattle (226 animals, Nelore), managed extensively. Serum samples were obtained from all 432 adult cows (aged 2–7 years). Samples were examined for leptospiral antibodies by the microscopic agglutination test, using live antigens grown in liquid medium (EMJH). There were 28 serovars of antigens, encompassing 20 serogroups of Leptospira. Every serum sample with agglutinating activity at 1:100 dilution had titres further tested by twofold serial dilutions. The antigen with the highest titre was considered the infective serovar (Faine et al. 2000). Overall, there were 252/432 (58.3%) positive reactions, with 170 (82.5%) and 82 (36.3%) of the dairy and beef cattle, respectively, seropositive (P<0.0001). Therefore, a dairy cow was 8.29 times more likely (odds ratio) of being seroreactive than a beef cow. Hardjo was the predominant serovar in both groups (74.6% of seropositve cattle), while weak reactions were also observed against Wolffi (19.8%) and Bratislava (5.6%). The overall seroprevalence was very similar to that previously reported in Brazil (Lilenbaum and Souza 2003) or Tanzania (Schoonman and Swai 2010). Hardjo was significantly more common in dairy than beef cattle (P<0.0001), while the incidental serovars, as Wolffi and Bratislava, were more common on beef than dairy cattle (P<0.005). Noteworthy, we observe that seroreactiv-ity against incidental serovars, as Wolffi or Bratislava, were not only less frequent but also concentrated in low titres, what suggest that they represent exposure rather than infection, while anti-Hardjo titres were more frequent and higher (Table 1). Considering those results, Hardjo …

In vitro antimicrobial susceptibility of staphylococci isolated from canine pyoderma in Rio de Janeiro, Brazil

The study aimed to determine the etiology and antimicrobial resistance of staphylococci isolated from canine pyoderma. Samples were obtained from dogs with pyoderma and isolates were identified by biochemical reactions and tested for susceptibility to 15 antimicrobials. Thirty nine staphylococci isolates were obtained, and S. pseudintermedius was the most frequent (47.4%). All isolates showed resistance to at least one drug and 77.1% were multiresistant. The most effective drug was oxacillin. The study reports the alarming antimicrobial resistance of Staphylococcus isolated from canine pyoderma samples.

Carriage of methicillin susceptible and resistant Staphylococcus schleiferi among dog with or without topic infections.

After reading with great interest the recent publican entitled ‘‘Nasal carriage of Staphylococcus schleiferi m healthy dogs and dogs with otitis, pyoderma or th’’ (May et al., 2012), we would like to share with e readers our experience with staphylococci from gs with or without topic infections in Rio de Janeiro, azil. Canine topic infections are very common, and very ten determined by staphylococci (Penna et al., 2010). agulase-positive staphylococci (CPS), particularly Staylococcus pseudintermedius, are well-known opportustic pathogens that represent a huge concern in terinary medicine (Feng et al., 2012), while coaguse-negative staphylococci (CNS) has been increasingly cognized as potentially aggressive (Penna et al., 2010; ay et al., 2012). We studied 354 adult (>1 year) dogs, being 100 nares of althy, 93 with pyoderma and 161 with otitis externa, all ithout the use of any antimicrobial agent in the last 30 ys. Samples were collected with sterile swabs and ocessed according to current bacteriological methods, as eviously described (Penna et al., 2010). For all the phylococcal isolates, testing for methicillin resistance as performed using the disk diffusion method (CLSI, 11). Independently of the disk test results, detection of e mecA gene was conducted in all staphylococcus lates, following Zhang et al. (2005). Staphylococci were recovered from 283 samples. From e healthy dogs’ nares, 89 isolates were obtained, stributed between 34 CPS (31 S. pseudintermedius and ree S. aureus), and 54 CNS (28 S. schleiferi schleiferi and 26 her species). From pyoderma, 72 isolates were obtained, ing 37 CPS (33 S. pseudintermedius and four S. aureus), d 35 CNS (23 S. schleiferi schleiferi and 12 other species). relation to the otitis externa, CPS were recovered from of the samples, being 52 S. pseudintermedius and 39 S. hleiferi (Table 1). Methicillin resistance could be observed (phenotypilly) in 38 staphylococcal isolates, being 36 confirmed notypically (22 CPS and 14 CNS). From those, 15 were m nares, 14 from otitis and seven from pyoderma. In gard to the species distribution, methicillin-resistant S. eudintermedius (MRSP) was the most frequently isolated (17), followed by methicillin-resistant S. schleiferi schleiferi (MRSS), obtained from nine samples. Although slightly higher than those recently reported (May et al., 2012), our findings agree with other studies, what reinforces the importance of S. schleiferi in both clinically normal dogs or with topical infections (Griffeth et al., 2008). In relation to the methicillin resistance, it is consensus that MRSP represents the most frequent isolate from dogs (Feng et al., 2012), what agree with our findings. Nevertheless, although May et al. (2012) reported only one MRSS isolate, in our study it was the second most frequent MRS species. It represents an unexpected result, since few reports regarding methicillin resistance in CNS in canine hosts are available (Griffeth et al., 2008). It was particularly surprising the high rate (44.1%) of MRS obtained from healthy dogs’ nares, what suggests the extensive distribution of those microorganisms among the canine population. In conclusion, this communication reinforces the importance of S. schleiferi in healthy dogs as well as in those with otitis and/or pyoderma. The present study also highlights the presence of methicillin resistance among CNS, particularly in S. schleiferi isolates.

In vitro antimicrobial resistance of Pseudomonas aeruginosa isolated from canine otitis externa in Rio de Janeiro , Brazil

Isolates of Pseudomonas aeruginosa (167) were obtained from 528 samples of canine otitis externa, identified by biochemical reactions and tested for susceptibility to 10 antimicrobials. The most effective drug was ciprofloxacin. The study reports alarming resistance among P. aeruginosa isolated from canine otitis externa samples in Rio de Janeiro, Brazil.

Rapid diagnostic of leptospirosis in an aborted bovine fetus by PCR in Rio de Janeiro, Brazil.

Polymerase chain reaction (PCR) has been increasingly d for the diagnosis of leptospirosis in animal samples. group has recently demonstrated that serology, ough an acceptable tool to screen for bovine leptossis on a herd basis, is not a good predictor for detecting ction on an individual basis (Otaka et al., 2012). refore, we would like to add information to that ment sharing with the readers a recent successful case iagnostic of leptospirosis in an aborted bovine fetus by in Rio de Janeiro, Brazil. Bovine leptospirosis is an important disease of the roductive sphere and usually leads to abortion, lowered ility, birth of weak calves, stillbirth or neonatal rtality, fetal mummification and decreased milk protion, causing substantial economic losses. Cattle act as intenance host of serovar Hardjo, which is the most mon agent of the disease in many countries (Hernán-Rodrı́guez et al., 2011). In relation to its diagnosis, bacterial culturing is orious and present low sensitivity; serology is worlde employed but does not confirm the infection in an ividual level; Therefore, molecular tools such as PCR e been increasingly been employed for the individual gnosis of leptospirosis as a reproductive disease in mals (Pinna et al., 2011). In July 2012, a dairy herd of 29 Jersey cows housed in ered free-stall barns with concrete floor, brucellosis for at least five years and with no specific cination against leptospirosis presented three cases bortions and two fetal mummifications in a period of ee months. Leptospirosis was suspected and seroloal test (Microscopic agglutination test – MAT) using live serovars of Leptospira as antigens was performed all the 29 adult cows. In this meantime, one cow sented signs of preterm labor, and an abortion of a five-month fetus was directly collected in a plastic bag (with no contact to soil in order to avoid contamination) immediately necropsied. Samples of liver, kidneys and gastric juice were collected and processed for both bacterial culturing (EMJH media-DIFCO, for sixteen weeks) and PCR. Briefly, PCR was performed by extraction of DNA with the Promega Wizard SV kit genomic DNA Purification System and amplification targeting on primers LipL32_45F (50 AAG CAT TAC TTG CGC TGG TG 30) and LipL32_286R (50 TTT CAG CCA GAA CTC CGA TT 30) (Stoddard et al., 2009). Four cows (13.8%) were MAT-positive with titers 100, all of them against serovar Hardjo. Although all bacterial culturing were negative, PCR was negative for kidneys and liver samples, but successfully detected leptospiral DNA from the gastric juice of the aborted calf. Although PCR has been described as a useful tool for the diagnosis of bovine leptospirosis (Hernández-Rodrı́guez et al., 2011; Otaka et al., 2012), it is not often employed, particularly to abortion samples. In this study, while bacterial culturing was negative, PCR could rapidly and efficiently confirm the infection. In conclusion, PCR for diagnosis of leptospirosis in the gastric juice of abortions may be used for definitive diagnosis and should be more often employed for the diagnosis of suspected cases of leptospirosis in cattle abortions.