Re-emergence of multi-drug resistant <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Stanley from cattle

During 2009, Salmonella enterica subspecies enterica serovar Stanley isolates were recovered from cattle diagnostic specimens in southern Japan, and the isolates were examined to characterize the genetic determinants involved in this new pathogenicity that associated with mortality in cattle. All the isolates were multi-drug resistance exhibited resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, oxytetracycline, and kanamycin (ACSSuT-Km) encoded by bla _TEM, catA, aadA1, sul1, tet(A), and aphA1 genes, respectively. Class 1 integrons of 1.5-kb size were detected in all MDR isolates. The isolates harboured easily transferable plasmids of ca. 210-kb with the potential of transmitting resistance phenotype and genotype detected in the donor isolates. XbaI-digested PFGE patterns generated two related clusters implicated in the dissemination of multi-drug resistance amongst Salmonella Stanley isolates. An emergence of multi-drug resistant Salmonella Stanley amongst food-producing animals, including cattle is a threat to human health, as resistant isolates may be transmitted to humans through the food chain.     

<Emphasis Type="Italic">Theileria</Emphasis> (<Emphasis Type="Italic">Babesia</Emphasis>) <Emphasis Type="Italic">equi</Emphasis> and <Emphasis Type="Italic">Babesia caballi</Emphasis> Infections in Horses in Galicia,Spain

The control of equine piroplasmosis is becoming increasingly important to maintain the international market open to the horse industry. The purpose of this study was to demonstrate the occurrence of equine piroplasmosis (Theileria equi and Babesia caballi) in Galicia, north-west Spain, and to compare haematological and serum biochemistry parameters between non-parasitaemic horses and horses parasitaemic with T. equi and B. caballi. Sixty serum samples (control group) were taken from healthy horses pastured on two farms, and examined for evidence of equine T. equi and B. caballi infection by indirect fluorescent antibody test (IFAT). Of the 60 samples, 24 (40%) and 17 (28.3%) samples were positive for T. equi and B. caballi, respectively. Twelve (20%) samples were positive for both parasites. Haematology and serum biochemistry were compared between controls and a series of 36 horses clinically affected by T. equi (25) or B. caballi (11). Compared with the healthy group, there was a 43% and 37% decrease in the haematocrit for T. equi and B. caballi infection, respectively. Parasitaemic horses presented an intense anaemia and serum biochemistry signs of liver damage. The anaemia was more severe in T. equi-infected than in B. caballi-infected horses. Our results suggest that equine piroplasmosis is widespread in the region and is a cause for concern.     

Antimicrobial susceptibility and phylotyping profile of pathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Salmonella enterica</Emphasis> isolates from calves and pigs in Minas Gerais,Brazil

The aims of the present study were to determine (i) the profiles of phylogroup and (ii) the antimicrobial susceptibility of pathogenic Escherichia coli strains isolated from calves, and of Salmonella spp. strains isolated from calves and pigs in Minas Gerais State, Brazil. Sixty-one pathogenic E. coli strains and Salmonella spp. (n?=?24) strains isolated from fecal samples of calves and Salmonella spp. (n?=?39) strains previously isolated from fecal samples of growing/finishing pigs were tested. The minimum inhibitory concentration (MIC) using the agar dilution method was determined for nalidixic acid, amikacin, amoxicillin, ampicillin, cefoxitin, norfloxacin, gentamicin, tetracycline, and trimethoprim-sulfamethoxazole. All E. coli isolates were susceptible to amikacin. Tetracycline was the antimicrobial that presented the higher frequency of resistance among E. coli strains, followed by ampicillin, trimethoprim-sulfamethoxazole, amoxicillin, nalidixic acid, norfloxacin, gentamicin, and cefoxitin. E. coli (n?=?61) strains isolated from calves belonged to different phylogroup namely, phylogroup A (n?=?26), phylogroup B1 (n?=?31), phylogroup E (n?=?3), and phylogroup F (n?=?1). Phylogroups B2, C, and D were not identified among the E. coli in the present study. All Salmonella spp. (n?=?24) strains isolated from fecal samples of calves were susceptible to amikacin, amoxicillin, ampicillin, norfloxacin, gentamicin, tetracycline, and trimethoprim-sulfamethoxazole. Resistance to nalidixic acid and cefoxitin was detected in 16.66 and 8.33 % of the Salmonella spp. strains, respectively. Among the Salmonella spp. (n?=?39) strains isolated from fecal samples of pigs, the higher frequency of resistance was observed to tetracycline, followed by amoxicillin, gentamicin, ampicillin, trimethoprim-sulfamethoxazole, nalidixic acid, cefoxitin, and norfloxacin. All strains were susceptible to amikacin. Forty-eight (78.68 %) of the E. coli strains were classified as multidrug-resistant, whereas among Salmonella spp. strains, the percentage of multidrug resistance was 57.14 %, being all multidrug-resistant strains isolated from pigs (92.30 %). The results from the present study indicate a high frequency of antimicrobial resistance among pathogenic E. coli strains isolated from calves and Salmonella spp. strains isolated from pigs and a high rate of susceptibility to most antimicrobials tested among Salmonella spp. strains isolated from calves. Our study highlights the presence of multidrug-resistant strains of E. coli and Salmonella spp. isolated from food-producing animals in Minas Gerais, Brazil.     

Prevalence of thin-walled <Emphasis Type="Italic">Sarcocystis cruzi</Emphasis> and thick-walled <Emphasis Type="Italic">Sarcocystis hirsuta</Emphasis> or <Emphasis Type="Italic">Sarcocystis hominis</Emphasis> from cattle in Iran

Bovine sarcocystosis is caused by Sarcocystis cruzi and is known to cause considerable morbidity and mortality in cattle. This species is distributed worldwide in cattle and is the most prevalent of the Sarcocystis species infecting cattle. There is high infection rate of sarcocyst in cattle in Iran, but to our knowledge, there is no study about identification of Sarcocystis species. This work aimed to survey prevalence of S. cruzi cyst in slaughtered cattle of Isfahan, Iran. In this study, esophageal and diaphragmatic muscles of 100 cattle were collected from Fesaran abattoir of Isfahan and examined for the presence of Sarcocystis spp. cysts macroscopically and microscopically. No macroscopic sarcocysts were found in any of the samples. In light microscopy, 89 out of 100 cattle (89%) had thin-walled cysts of S. cruzi, while 21 out of them (21%) had thick-walled sarcocysts. In addition to light microscopy, ultrastructural features of the thin-walled cyst confirmed the presence of S. cruzi.     

Risk factors for <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subspecies <Emphasis Type="Italic">paratuberculosis</Emphasis> in Fars province (Southern Iran) dairy herds

A cross-sectional study was conducted from March to August 2006 in dairy herds in Fars province, southern Iran to determine the herd-level risk factors for infection with Mycobacterium avium subspecies paratuberculosis (MAP). Statistical analysis using multivariable logistic regression showed that contamination of udders of periparturient cows with manure (OR = 6.4, P = 0.02) and history of having suspected cases of Johne's disease in the herd (OR = 6.7, P = 0.04) were significantly associated with the herd infection status. No relationship between breed, herd size and other management practices with the infection status of the herd were found in this study. Implementing high sanitary measures in the farm, particularly with respect to manure handling and cleaning could be considered as one of the important aspects in controlling disease in the region as well as in the future educational effort.     

Prevalence and antimicrobial resistance profiles of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovars isolated from slaughtered cattle in Bahir Dar,Ethiopia

A study was undertaken from October 2006 to March 2007 to determine the prevalence and antimicrobial resistance patterns of Salmonella serovars. Liver, mesenteric lymph nodes, intestinal content, and carcass swab samples (each n?=?186) were collected from 186 apparently healthy slaughtered cattle at Bahir Dar abattoir. Bacteriological analysis was done according to the International Organization for Standardization (ISO 6579 2002). Isolates were serotyped at Agence Française de Securite Sanitaire des Aliments, Cedex, France. Twenty-eight isolates consisting of Salmonella Typhimurium, Salmonella Newport, Salmonella Haifa, Salmonella Heidelberg, Salmonella Infantis, and Salmonella Mishmarhaemek were identified. Salmonella Typhimurium and Salmonella Newport were most frequently isolated while Salmonella Heidelberg and Salmonella Mishmarhaemek were isolated least. Eleven of the 28 (39.3%) were resistant to one or more of the antimicrobials tested. Resistance was shown to ampicillin, chloramphenicol, gentamycin, norfloxacin, polymyxin-B, streptomycin, tetracycline, and trimethoprim. Four of 11 (36.4%) were multiple antimicrobial resistant. All the isolates tested were susceptible to the antimicrobial effects of gentamycin, norfloxacin, and trimethoprim. Eleven, four, and two isolates of the 28 were resistant to streptomycin, tetracycline, and ampicillin, respectively. All isolates of Salmonella Infantis, Salmonella Typhimurium (except one), and Salmonella Mishmarhaemek were susceptible to the tested antimicrobials. One Typhimurium isolate was resistant to chloramphenicol, streptomycin, and tetracycline. Salmonella Haifa was multiply antimicrobial resistant to ampicillin, tetracycline, and streptomycin. All isolates of Salmonella Heidelberg were resistant to streptomycin. Results of this study indicated high level of carcass contamination with antimicrobial-resistant Salmonella serovars which could pose public health risk; suggests need for hygienic slaughtering operations and proper cooking of meat before consumption. Further detailed studies involving different abattoirs, animal products, food items, and animals on different settings were recommended in the study area.     

Pathological,serological, and virological findings in sheep infected simultaneously with <Emphasis Type="Italic">Bluetongue</Emphasis>, <Emphasis Type="Italic">Peste-des-petits-ruminants</Emphasis>, and <Emphasis Type="Italic">Sheeppox viruses</Emphasis>

In this study, pathological, serological and virological examinations were performed on 15 sheep from a flock of 250 sheep and lambs that suffer from simultaneous naturally occurring BTV, PPRV and SPV outbreaks. SPV was diagnosed macroscopically and histopathologically, BTV was diagnosed by ELISA, and PPRV was diagnosed pathologically and by ELISA. Clinically fever, diarrhea, depression, polypnea, conjunctivitis, lacrimation, rhinitis, erosive stomatitis, edema of eyelids, photophobia, cutaneous eruption with erythematous areas especially noticeable in wool-free parts of the body and axilla lesions evolving into papules were observed. At necropsy, the most effected organs were lungs and gut. Subepicardial hemorrhages were also commonly seen. While typical pox lesions were observed in some lambs, usually fibrinous pleuropneumonia was more prominent lung lesion. SPV and PPRV lesions were seen at the histopathological examination of the lesioned tissues, BT lesions were mild than SPV and PPRV microscopically. Serum and leukocyte samples of 15 animals were examined for PPRV and BTV by ELISA; 5 samples were positive for PPRV and 6 BTV, 4 were positive for both PPRV and BTV simultaneously. One hundred animals died, most were lambs. Mortality rates were 100% in lambs and 80% in the herd.     

Biological control of <Emphasis Type="Italic">Ascaris suum</Emphasis> eggs by <Emphasis Type="Italic">Pochonia chlamydosporia</Emphasis> fungus

Ascaris suum is a gastrointestinal nematode parasite of swines. The aim of this study was to observe Pochonia chlamydosporia fungus on biological control of A. suum eggs after fungus passage through swines gastrointestinal tract. Eighteen pigs, previously dewormed, were randomly divided into three groups: group 1, treated with the fungus isolate VC4; group 2, treated with the fungus isolate VC1 and group 3 did not receive fungus (control). In the treated groups, each animal received a 9 g single dose of mycelium mass containing P. chlamydosporia (VC1 or VC4). Thereafter, animal fecal samples were collected at the following intervals: 8, 12, 24, 36, 48, 72 and 96 h after treatment beginning and these were poured in Petri dishes containing 2% water-agar culture medium. Then, 1,000 A. suum eggs were poured into each dish and kept in an incubator at 26°C and in the dark for 30 days. After this period, approximately 100 eggs were removed from each Petri dish and morphologically analyzed under light microscopy following the ovicidal activity parameters. The higher percentage observed for isolated VC4 eggs destruction was 57.5% (36 h) after fungus administration and for isolate VC1 this percentage was 45.8% (24 h and 72 h) (p > 0.01). P. chlamydosporia remained viable after passing through the gastrointestinal tract of swines, maintaining its ability of destroying A. suum eggs.     

Immunoprotection of recombinant <Emphasis Type="Italic">Eg</Emphasis>.P29 against <Emphasis Type="Italic">Echinococcus granulosus</Emphasis> in sheep

Objective

This study aims to investigate the immunoprotection of recombinant Eg.P29 (rEg.P29) vaccine and analyze the underlying mechanism in sheep.

Methods

Three groups of male sheep were immunized subcutaneously with rEg.P29 and PBS, Freund’s complete adjuvant as controls, respectively. After prime-boost vaccination, the sheep were challenged with encapsulated Echinococcus granulosus eggs. The percentage of protection in sheep was determined 36 weeks after the infection. Humoral immune response was analyzed for specific IgG, IgG1, IgG2, IgM and IgE levels. Moreover, cytokines including interferon (IFN)-γ, interleukin (IL)-2, IL-4,and IL-10 were also evaluated.

Results

Immunization with rEg.P29 induced protective immune responses up to 94.5 %, compared with immunoadjuvant group. The levels of specific IgG, IgG1, IgG2, and IgE as well as IFN-γ, IL-2, and IL-4 significantly increased after two immunizations (P < 0.05); however, the levels of IgM and IL-10 did not show difference.

Conclusion

rEg.P29 showed Immunoprotection and induced Th1 and Th2 immune responses; hence, rEg.P29 is a potential vaccine for E. granulosus infection.

     

Control of <Emphasis Type="Italic">Haemonchus contortus</Emphasis> in sheep using basidiocarps of <Emphasis Type="Italic">Agaricus blazei</Emphasis> Murril

Objective

This study evaluated the effects in vitro and in vivo of Agaricus blazei against Haemonchus contortus in sheep.

Methods

The in vitro efficacy of aqueous extract on egg hatching inhibition (EHI) was investigated and after 72 h incubation with varying concentrations the effects on, blastomeres, embryonated eggs, and first stage larvae (L1) were evaluated. Larval development inhibition (LDI) for dry powder and the aqueous extract were evaluated in fecal cultures of sheep infected with H. contortus. In vivo efficacy was determined by reduction in fecal egg count (FEC). Lambs were treated with powder A. blazei (11.4 g/kg pc) or trichlorfon, or were untreated and the possible toxicity of this fungus was monitored by plasmatic enzyme analysis.

Results

Concentrations equal to and higher than 3.62 mg/mL and of aqueous extract were 100% effective in the EHI test. In the LDI test, LC90 was estimated for 5.66 and 106.0 mg/g fecal culture for aqueous extract and powder, respectively. The mean FEC in lambs 14 days post-treatment with A. blazei powder was significantly lower than observed for the negative control, and the serum levels of aspartate transaminase and alanine transaminase were normal.

Conclusion

The fungi supplementation promotes, respectively, high and moderate anthelmintic efficacy with in vitro and in vivo tests, respectively, suggesting it as an alternative or complementary treatment for haemonchosis in sheep.

     

Experimental infection of <Emphasis Type="Italic">Peste des Petit Ruminant</Emphasis> virus and <Emphasis Type="Italic">Mannheimia haemolytica</Emphasis> A2 in goats: immunolocalisation of <Emphasis Type="Italic">Mannheimia haemolytica</Emphasis> antigens

Nigerian strain of Peste des Petit Ruminant (PPR) virus and Mannheimia haemolytica (MH) biotype A serotype 2, was used successfully to reproduce a concurrent disease in West African Dwarf goats. The development of the various pathological features were studied at regular intervals following infection. The acute inflammatory reaction which had developed by day 3 after initial infection was characterised by flooding of the alveoli by neutrophils, oedema, hemorrhage and syncytial cells together with a moderate bronchial and bronchiolar epithelial necrosis. This progressed to a milder acute broncho interstitial pneumonia with giant cells. At this stage, the mucosal immunity were well developed especially the aggregate form of NALT and more of nodular forms of BALT. The organisms were demonstrated with strong immunostaining in the necrotic center, necrotic alveolar wall, fibrin, serous exudate, and degenerated leukocyte in the alveoli and respiratory airways. The bacterial antigens were observed as a strong immunostaining in the blood vessels of the nasal septum, sinusoid in the liver and interstium of the kidney, cytoplasm of alveolar macrophages, pneumocytes, bronchial and bronchiolar epithelium, in the monocytes in the blood vessels. These findings confirmed the enhancement of MH tropism especially in the respiratory tract, liver and kidney. It also showed that West african dwarf goats are highly susceptible to the intratracheal combined infection of PPR virus and MH. The fact that the infection induces strong mucosal responses, this phenomenon can be explored in Africa with the use of combined PPR virus and MH intranasal vaccines to curtail the menace of pneumonia associated with the combined infection on field.     

Ovicidal activity of seven <Emphasis Type="Italic">Pochonia chlamydosporia</Emphasis> fungal isolates on <Emphasis Type="Italic">Ascaris suum</Emphasis> eggs

The ovicidal effect of the nematophagous fungus Pochonia chlamydosporia on eggs of Ascaris suum was tested under laboratory conditions. A. suum eggs were plated on 2% water–agar with seven fungal isolates (Isol. 5, Isol. 31, Isol. 1, VC1, Isol. 12, Isol. 22 and VC4) and control without fungus. After 5, 7, 10, 14, 15 and 21 days of incubation, approximately 100 eggs were removed from the plates and classified according to the following parameters: type 1, biochemical and physiological effect without morphological damage to the eggshell, type 2, lytic effect with morphological alteration of the eggshell and embryo and type 3, lytic effect with morphological alteration of eggshell and embryo showing hyphal penetration and internal egg colonization. The isolates effectively destroyed A. suum eggs and all types of effects were observed during the experiment. There was no variation in ovicidal capacity (type 3 effect) among the isolates (p > 0.05) throughout the experiment. After 21 days, isolate 5 showed the highest percentages of type 3 effect (58.33%). The results indicated that P. chlamydosporia (Isol. 5, Isol. 31, Isol. 1, VC1, Isol. 12, Isol. 22 and VC4) can destroy A. suum eggs and is, therefore, a potential biological control agent of nematodes.     

Polymorphism of growth hormone receptor (<Emphasis Type="Italic">GHR</Emphasis>) gene in Nilagiri sheep

The allelic variation in the regulatory sequence of growth hormone receptor (GHR) gene influences the growth traits of sheep. A study was carried out to find out the polymorphisms associated with exon 10 of GHR gene and its association with growth traits of Nilagiri sheep. The blood samples were collected from Nilagiri sheep (n?=?103) reared at Sheep Breeding Research Station, Sandynallah, Tamil Nadu, India. DNA was isolated using the phenol-chloroform extraction procedure and eight samples having amplified product of part of exon 10 (895 bp) sequenced. The results indicated transitions of nucleotide G>A at loci G177624A and G177878A. The genotyping frequencies estimated using the tetra-primer amplification refractory mutation system-PCR for GG, GA and AA were 0.262, 0.544 and 0.194, and 0.349, 0.505 and 0.146, respectively. The estimated allele frequencies of G and A nucleotides were 0.5340 and 0.4660, and 0.6015 and 0.3985, respectively, at loci G177624A and G177878A. The effects of both the mutations on growth-related traits viz., birth, weaning (3 months) 6, 9 and 12 months weight in Nilagiri sheep were found to be non-significant. This can be a novel approach to assess growth of sheep using the mutation in GHR gene. Thus, this approach can be useful for further investigation as a molecular marker associated with genetic improvement.     

Methicillin and aminoglycoside resistance in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates from bovine mastitis and sequence analysis of their <Emphasis Type="Italic">mecA</Emphasis> genes

Although methicillin-resistant Staphylococcus aureus (MRSA) were generally isolated from human beings; these agents were recently isolated from various animal species. It has been shown that MRSA isolates are not only resistant to beta-lactam antibiotics, but can also be resistant to the other commonly used antibiotics. In this study, 18 phenotypic methicillin resistant S. aureus isolates from bovine mastitis cases were analyzed by PCR for the presence of mecA gene encoding methicillin resistance and aac(6′)/aph(2″), aph(3′)-IIIa and ant(4′)-Ia genes encoding aminoglycoside resistance. Out of 18 S. aureus isolates (oxacillin MICs, ≥4 μg/ml), 3 were positive for mecA gene. Only one from 3 mecA positive isolates was positive for genes encoding aminoglycoside-modifying enzymes and this isolate carried aac(6′)/aph(2″) in combination with aph(3′)-IIIa gene. The aph(3′)-IIIa gene was detected in 3 isolates. These three isolates carrying the aminoglycoside-modifying enzyme genes were resistant to gentamicin, kanamycin and neomycin. The mecA gene of 3 MRSA isolates was sequenced. All three mecA genes of these isolates were identical to that found in human MRSA strains, except a one-base substitution at nucleotide position 757. From the data presented in this study, it can be concluded that MRSA isolated from bovine mastitis may be originated from human beings, but further studies are needed to investigate the possibility of zoonotic transfer of MRSA.     

Virulence of <Emphasis Type="Italic">Brucella abortus</Emphasis> isolated from cattle and water buffalo

Brucellosis has been documented in domestic water buffalo (Bubalus bubalis) but published literature is limited despite the importance of this species in tropical agricultural systems. The objective of this study was to compare the virulence of Brucella abortus isolates recovered from cattle and water buffalo. Nineteen strains of B. abortus from cattle and domestic water buffalo in Trinidad were intraperitoneally inoculated into BALB/c mice. Spleens were cultured for B. abortus and histopathological severity scores were calculated based on lymphoid depletion, lymphoid necrosis, splenitis, and macrophage accumulation. A general linear model approach was used to estimate the effect of isolate source (cattle versus water buffalo) on virulence. Isolates of water buffalo origin were significantly less virulent in the mouse model based on recovered B. abortus from splenic tissues, spleen/weight ratio, and lymphoid necrosis but not overall histopathological severity scores. Further investigation of isolates recovered from water buffalo might provide the key to the development of procedures for brucellosis control in tropical environments.