Occurrence and molecular characterization of Bartonella spp. and hemoplasmas in neotropical primates from Brazilian Amazon

Little is known about the prevalence and genetic diversity of Bartonella spp. and hemoplasmas in nonhuman primates (NHP). The present study aimed to investigate the occurrence of and assess the phylogenetic position of Bartonella spp. and hemoplasma species infecting neotropical NHP from Brazilian Amazon. From 2009 to 2013, a total of 98 blood samples from NHP belonging to the Family Cebidae were collected in the island of São Luís, state of Maranhão, of which 87 NHP were from Wild Animal Screening Center (CETAS) in the municipality of São Luís, and 11 (9 Sapajus sp. and 2 Saimiri sciureus) were from NHP caught in the Sítio Aguahy Private Reserve. DNA samples were screened by previously described PCR protocols for amplifying Bartonella spp. and Mycoplasma spp. based on nuoG, gltA and 16S rRNA genes, respectively. Purified amplicons were submitted to sequencing and phylogenetic analysis. Bacteremia with one or more Bartonella spp. was not detected in NHP. Conversely, 35 NHP were PCR positive to Mycoplasma spp. The Blastn analysis of seven positive randomly selected sequencing products showed percentage of identity ranging from 95% to 99% with other primates hemoplasmas. The Maximum Likelihood phylogenetic analysis based on a 1510 bp of 16S rRNA gene showed the presence of two distinct clusters, positioned within Mycoplasma haemofelis and Mycoplasma suis groups. The phylogenetic assessment suggests the presence of a novel hemoplasma species in NHP from the Brazilian Amazon.     

Genetic diversity and hematological and biochemical alterations in Alouatta primates naturally infected with hemoplasmas in Brazil

Mycoplasma spp. and Bartonella spp. are Gram-negative bacteria transmitted by arthropod vectors that infect red blood cells of several mammal species. This study investigated the occurrence and genetic diversity of hemoplasmas and Bartonella spp. in 68 howler monkeys kept in captivity in São Paulo, a southeastern state in Brazil. In addition, possible hematological, biochemical and electrophoretic changes of serum proteins associated with the occurrence of hemoplasmas and Bartonella spp. in captive primates were also investigated. The cPCR results showed that all sampled howler monkeys were negative for Bartonella spp. based on the gltA gene. The cPCR results indicated that 18 (26.47%) non-human primates (NHP) were positive for hemoplasmas based on the 16S rRNA gene. Monocyte and lymphocyte counts were higher in hemoplasma-positive howlers (P < 0.05). Platelet counts decreased in nonhuman primates (NHP) positive for hemoplasmas (P < 0.05). The results from the blood serum proteinogram and biochemistry analyses were not significantly different between NHPs positive and negative for hemotrophic mycoplasmas. Phylogenetic analysis using Bayesian Inference (BI) based on the 16S rRNA gene positioned the obtained sequences close to ‘Candidatus Mycoplasma kahanei’. The analysis of sequence diversity of the 16S rRNA gene showed that 5 different genotypes are circulating in NHP in Brazil and in the world; besides, a clear separation between the sequences of hemoplasmas that infect NHP of the Sapajus and Alouatta genus in Brazil was found, probably corresponding to two different species. The pathogenic potential of this hemoplasma species in NHP should be further investigated.     

Laboratory diagnosis of malaria in nonhuman primates

Abstract: Nonhuman primates (NHPs) are commonly used for biomedical research because of the high level of gene homology that underlies physiologic similarity to human beings. Malaria parasites of the genus Plasmodium cause one of the most frequent parasitic diseases of NHPs originating from tropical and subtropical areas and as such represent a significant research confounder. Malaria in NHPs presents a diagnostic challenge especially to those laboratories that see no more than a few malaria cases per year in NHPs. The accurate and timely diagnosis of malaria infection in NHPs facilitates the appropriate treatment of individuals infected with the malaria parasites. Conventional microscopy based on the examination of Giemsa‐stained thick and thin blood films remains the mainstay of laboratory diagnosis of malaria infection because of the high diagnostic sensitivity and specificity and also the capability for Plasmodium species identification and parasite counts. This procedure is recognized as technically difficult and time‐consuming, requiring considerable training to obtain the necessary skills. In the past few years, efforts to replace the traditional but tedious reading of blood films have led to different techniques for the detection of malaria parasites, including fluorescence microscopy, detection of intraleukocytic hemozoin or malaria pigment using automated blood cell analyzers, immunochromatographic rapid diagnostic tests based on malaria antigen detection, and PCR assays. These techniques offer new approaches for diagnosing malaria in NHPs. This review focuses on the available laboratory diagnostic tools for malaria in NHPs.     

Presence and diversity of mixed avian Plasmodium spp. infections in introduced birds whose distribution overlapped with threatened New Zealand endemic birds

ABSTRACT

Aims: To determine the presence of infection and co-infection of Plasmodium lineages in introduced birds at translocation sites for the North Island saddleback (Philesturnus rufusater), to investigate their role as Plasmodium spp. reservoirs.

Methods: Blood samples were collected from introduced bird species, with a special focus on blackbirds (Turdus merula) and song thrushes (Turdus philomelos), at six locations in the North Island of New Zealand that were the origin, or translocation sites, for North Island saddleback. Where available, blood smears were examined, and blood samples were tested using nested PCR with subsequent sequence analysis, for the presence of Plasmodium spp.

Results: Of the 55 samples tested using PCR analysis, 39 (71%) were positive for Plasmodium spp., and 28/40 (62%) blood smears were positive for Plasmodium spp. Overall, 31 blood samples were from blackbirds with 28/31 (90%) samples positive for Plasmodium spp. Six distinct avian Plasmodium lineages were identified, including three cosmopolitan lineages; Plasmodium vaughani SYAT05 was detected in 16 samples, Plasmodium matutinum Linn1 in 10 samples and Plasmodium elongatum GRW6 in eight samples. Mixed infections with more than one lineage were detected in 12 samples. Samples from two Australian magpies (Gymnorhina tibicen) were positive for Plasmodium. sp. lineage MYNA02, previously not identified in New Zealand.

Conclusions and clinical relevance: This is the first report from New Zealand in which specific Plasmodium spp. mixed infections have been found in introduced birds. Co-infections with several cosmopolitan Plasmodium lineages were identified, as well as the first report in New Zealand of an exotic avian Plasmodium sp. lineage, in Australian magpies. Whilst the role of introduced birds in maintaining and spreading pathogenic avian malaria in New Zealand is unclear, there is a potential infection risk to native birds, especially where distributions overlap.     

Malaria in cynomolgus monkeys used in toxicity studies in Japan

Plasmodium spp. protozoa cause malaria and are known to infect humans and a variety of animal species including macaque monkeys. Here we report both our experience with malaria recrudescence in cynomolgus monkeys (Macaca fascicularis) in a toxicity study and the results of a survey on Plasmodium infection in cynomolgus monkeys imported to Japan for laboratory use. A cynomolgus monkey from the toxicity study presented with severe anemia and Plasmodium protozoa in erythrocytes on a thin blood smear and was subsequently diagnosed with symptomatic malaria. In this animal, congestion and accumulation of hemozoin (malaria pigment) in macrophages were noted in the enlarged and darkly discolored spleen. As a follow-up for the experience, spleen sections from 800 cynomolgus monkeys in toxicity studies conducted between 2003 and 2013 were retrospectively examined for hemozoin deposition as a marker of Plasmodium infection. The origin of the animals included Cambodia, China, Indonesia, and Vietnam. Hemozoin deposition was confirmed in 44% of all examined monkeys. Monkeys from Indonesia showed the highest incidence of hemozoin deposition (approx. 80%). A high prevalence of Plasmodium infection in laboratory monkeys was also confirmed with polymerase chain reaction (PCR) by using Plasmodium genus-specific primers. Although Japan is not a country with endemic malaria, it is important to be aware of the prevalence and potential impact of background infection with Plasmodium spp. and recrudescence of symptomatic malaria in imported laboratory monkeys on pharmaceutical toxicity studies.     

An outbreak of avian malaria in captive yellowheads/mohua (Mohoua ochrocephala)

CASE HISTORY: Eight mohua, or yellowheads (Mohoua ochrocephala), were held in a large open aviary over the summer months of 2003–2004, following their capture for captivebreeding purposes. Two birds died of transportation trauma shortly after arrival, one became ill and died a month later, and another four died within a 2-week period in February 2004. The eighth bird also became ill at this time but survived for a year following treatment with chloroquine and doxycycline.

CLINICAL AND PATHOLOGICAL FINDINGS: The affected birds were depressed, lethargic and dyspnoeic. Necropsy of three birds showed a slightly pale and swollen liver and spleen. Impression smears of the liver of one bird revealed schizonts resembling Plasmodium spp. within the cytoplasm of many hepatocytes, which was confirmed histopathologically. Similar protozoal organisms were seen within splenic histiocytes and pulmonary endothelial cells of 5/6 birds. Electron microscopy identified these as protozoal schizonts containing merozoites of similar size and structure to those of Plasmodium spp.

DIAGNOSIS: The birds were infected with a protozoal haemoparasite resembling Plasmodium spp.; asexual stages within hepatocytes and endothelial cells of the lung and spleen were typical of this organism.

CONCLUSIONS AND CLINICAL RELEVANCE: The mohua captured from west Otago were highly susceptible to avian malaria as they came from an isolated population that was likely to be naïve and have had no previous contact with this organism. The birds were probably infected by bites from mosquitoes feeding off local populations of blackbirds subsequently found to be infected with Plasmodium spp.     

Morphological and Morphometric Description of Female Reproductive Tract of Sapajus apella (Capuchin monkey)

Forest destruction has progressively hampered the survival of many species, and this is why it is so important to study of the lives of primates in captivity. This study aimed to describe the morphological aspects of the female reproductive tract of Sapajus apella. We used five animals obtained from the National Primate Center, Ananindeua – PA. The ovaries were paired, compact and symmetrical and had a smooth surface. The uterine tubes were bilateral and convoluted in adult animals and straight in young individuals. The uterus was simple and located in the pelvic region. The vagina was a long structure due to the position of the uterus. The external genitalia were located in the urogenital perineum and consisted of dark pigmented labia majora and labia minora, a vaginal vestibule as long as the vagina and a well‐developed clitoris. The results showed that the genitals of S. apella resemble those of other Neotropical primates.     

Additivity of standardized ileal digestibility of amino acids in mixed diets containing multiple protein sources for growing pigs fed three crude protein levels

This study was conducted to investigate the effect of dietary crude protein (CP) levels of semi‐purified diets on the additivity of values for standardized ileal digestibility (SID) of amino acids (AA) in mixed diets from multiple protein sources for growing pigs. A total of 28 barrows (initial BW, 66.4 ± 1.3 kg) were surgically fitted with simple T‐cannulas at the distal ileum and assigned to a replicated 14 × 4 incomplete Latin square design with 14 diets and 4 periods. The 14 experimental diets consisted of a nitrogen‐free diet; a corn‐based diet (80 g CP/kg); nine semi‐purified diets containing soya bean meal (SBM), canola meal (CM) or corn distillers dried grains with solubles (cDDGS), each type (protein source) of semi‐purified diets supplied 80, 120 or 160 g CP/kg, respectively; three mixed diets based on corn, SBM, CM and cDDGS formulated to contain 120, 160 and 200 g CP/kg respectively. Pigs were fed each of the 14 diets during a seven‐day period, and ileal digesta were collected from 08:00 a.m to 6:00 p.m on day 6 and 7. Chromic oxide was added as an indigestible marker. Results indicated that the SID of CP and AA were not affected by CP levels (p > .05). Values for SID of AA were additive (p > .05) with the exception of His and Lys; Arg and Lys; Arg, Lys, Thr, Asp, Cys and Gly in the mixed diets containing 120, 160 and 200 g CP/kg respectively (p < .05). In conclusion, additivity of SID values of AA in the mixed diets at different CP levels was not affected by the CP levels of semi‐purified diets for growing pigs. Therefore, it is recommended that SID values of AA should be used to formulate practical diets containing multiple ingredients for pigs.     

Effect of cellobiose supplementation on in vitro fermentation activity and bacterial numbers of porcine inocula

In this in vitro study, the modified Hohenheim gas test (HGT) was applied to determine fermentation activity and bacterial composition of pig's faecal microbial inoculum using different concentrations of cellobiose. Incubation procedures included normal buffered and osmotic stress conditions (elevated medium salinity). After 24 hr of fermentation, production of gas, ammonia and short‐chain fatty acid (SCFA) was measured, and the gene copy numbers of total bacteria, Lactobacillus spp., Bifidobacterium spp., Roseburia spp., Clostridium Cluster IV spp. and Enterobacteriaceae were analysed using real‐time polymerase chain reaction. There was a significant reduction in gas production after 24 hr when comparing osmotic stress conditions with normal buffered conditions. Under osmotic stress, increasing cellobiose concentrations linearly increased gas production (p < .001), while ammonia, acetic acid and isobutyric acid concentrations decreased (p < .001, p = .012, p = .035 respectively). Under normal buffered conditions, Roseburia spp. gene copies linearly increased with increasing cellobiose concentrations (p = .048). Lactobacillus spp. and Bifidobacterium spp. numbers were higher under osmotic stress (p < .001) compared to normal conditions. Results might point towards a positive impact of cellobiose supplementation on gut health especially under osmotic stress conditions.     

Giardia duodenalis in primates: Classification and host specificity based on phylogenetic analysis of sequence data

Giardia duodenalis colonizes the gastrointestinal tract of a wide range of hosts, including humans and other primates. It is grouped into eight different Assemblages and, beyond that, into a number of sub‐Assemblages, defined ad hoc on the basis of genetic differences; these various groups are often considered to be associated with a specific restricted host range. The aim of this study was to use publicly available genotyping data to investigate the relatedness of human and non‐human primate (NHP) Giardia isolates in order to evaluate the usefulness of current taxonomic classification and to assess whether there is potential for zoonotic transmission between humans and NHP. Our final data set consisted of sequence data from 165 isolates, 111 from NHP and 54 from humans. Assemblages were well defined, but sub‐Assemblages across Assemblage B were not resolved. Although sub‐Assemblages AI and AII were resolved, the terms were not found to capture any useful molecular or host/deme properties. In the phylogenetic tree, NHP isolates were scattered among human isolates across Assemblages A and B, and were even found in Assemblage E. We conclude that there does not appear to be significant molecular distinction between human and NHP Giardia isolates across these four molecular markers. Thus, on the basis of these markers, we cannot exclude a risk for zoonotic and anthropozoonotic transmission of Assemblages A and B isolates, irrespective of sub‐Assemblage classification. We further evaluated the relative merit of the four genes used in genotyping studies. The tpi, gdh and bg genes gave relatively congruent tree topologies, but the SSU gene did not resolve Assemblages according to the current classification. Future genotyping efforts should aim for multilocus or whole‐genome approaches and, in particular, use of the SSU gene as the sole marker should be avoided when possible.     

Detection of Toxoplasma gondii in Crassostrea spp. oysters cultured in an estuarine region in eastern Amazon

The aim of the present study was to detect DNA of Toxoplasma gondii in Crassostrea spp. oysters cultured in the state of Pará, Brazil. A total of 400 oysters were directly collected from a fixed rack system. Gills, gastrointestinal tract (GIT) and intervalvular liquid were separated and grouped into pool samples of 10 animals, resulting in 40 samples each of gills, GIT and intervalvular liquid. DNA extraction was performed using a commercial kit, and T. gondii DNA was detected by nested PCR using the primers Toxo3 and Toxo4, which produced an amplification product of 155 bp of the T. gondii gene B1. Nucleotide sequencing was performed for positive samples, and the obtained sequences were identified by comparison with sequences in GenBank. The DNA of T. gondii was detected in 5.8% (7/120) of the pool samples, of which 7.5% (3/40) was in the GIT, 5% (2/40) in the gills, and 5% (2/40) was in the intervalvular liquid. The obtained sequences presented 100% identity and overlap with T. gondii DNA sequences. This is the report of detection of T. gondii DNA in oysters from genus Crassostrea spp. originating from the state of Pará, eastern Amazon.     

Brucella abortus is Prevalent in Both Humans and Animals in Bangladesh

To determine the role of different Brucella (B.) spp. in Bangladesh, 62 animal samples and 500 human sera were tested. Animal samples from cattle, goats and sheep (including milk, bull semen, vaginal swabs and placentas) were cultured for Brucella spp. Three test‐positive human sera and all animal samples were screened by Brucella genus‐specific real‐time PCR (RT‐PCR), and positive samples were then tested by IS711 RT‐PCR to detect B. abortus and B. melitensis DNA. Only B. abortus DNA was amplified from 13 human and six animal samples. This is the first report describing B. abortus as the aetiological agent of brucellosis in occupationally exposed humans in Bangladesh. Of note is failure to detect B. melitensis DNA, the species most often associated with human brucellosis worldwide. Further studies are required to explore the occurrence of Brucella melitensis in Bangladesh.     

Meat Juice Serology and Improved Food Chain Information as Control Tools for Pork‐Related Public Health Hazards

The seroprevalence of Salmonella spp., pathogenic Yersinia spp., Toxoplasma gondii and Trichinella spp. was studied in 1353 finishing pigs from 259 farms that were allocated according to farm types: large fattening farms (≥1000 pig places), small fattening farms (< 1000 pig places) and farrow‐to‐finish farms. The antibodies were analysed with commercial ELISA kits in meat juice samples that were collected at Finnish slaughterhouses. Salmonella antibodies were rare (3% of pigs, 14% of farms) when the cut‐off optical density (OD) value 0.2 was used. Antibodies to pathogenic Yersinia spp. and T. gondii were detected in 57% of pigs and 85% of farms (OD ≥0.3) and in 3% of pigs and 9% of farms (OD ≥0.15), respectively. No antibodies to Trichinella spp. were detected (OD ≥0.3). The European Food Safety Authority (EFSA) considers Salmonella spp., Yersinia enterocolitica, T. gondii and Trichinella spp. as the most relevant biological hazards in the context of meat inspection of pigs. The seroprevalence of these important zoonotic pathogens was low in Finland, except that of Yersinia. The seroprevalence of Toxoplasma was significantly higher in pigs originating from small‐scale fattening farms (P < 0.05). Strong positive correlation was observed at the animal level between Salmonella and Yersinia seropositivity and between Salmonella and Toxoplasma seropositivity (P < 0.05). We suggest that these results reflect the level and importance of biosecurity measures applied on the farms. Meat juice serology at slaughter is a useful tool for targeting measures to control these pathogens. The information obtained from analyses should be used as part of the food chain information (FCI).     

Urban Prevalence of Listeria spp. and Listeria monocytogenes in Public Lavatories and on Shoe Soles of Facility Patrons in the European Capital City Vienna

The aim of this study was to determine the prevalence of Listeria spp. and Listeria monocytogenes (L. monocytogenes) in urban public lavatories and on shoe soles of facility patrons in a European capital city. More than 91% of all municipal public lavatories in Vienna close to public hubs were included in this study. Overall, 373 swab samples of public lavatories and shoes of facility patrons were enriched, according to ISO 11290‐1. Listeria monocytogenes isolates were subtyped using pulsed‐field gel electrophoresis. A total of 24 samples were positive for Listeria spp., yielding an overall prevalence of 6.4% (24/373). Listeria monocytogenes was found in 2.1% (8/373) of all samples. Swabs from lavatories in parks, container lavatories and lavatories at markets had the highest prevalences of 20.7% (6/29), 20% (2/10) and 12.5% (1/8) Listeria spp., respectively. These detection rates were statistically significantly higher than those associated with lavatories in shopping centres (P = 0.003, P = 0.002, P = 0.02) and at public transport locations (P = 0.0004, P = 0.005, P = 0.02). Shoes sampled at Christmas markets showed the highest Listeria spp. and L. monocytogenes prevalences of 80% (4/5) and 40% (2/5), respectively. With regard to shoe type, Listeria spp. detection rates were 14.3% (3/21; winter boots), 13.3% (2/15; hiking boots), sport shoes (5.9%; 2/34) and brogues (5.1%; 4/79). No Listeria spp. were found on shoe soles that had smooth treads (0/76), while Listeria spp. were detected on 19.5% (8/41) of medium depth tread shoe types and on 9.4% (3/32) of deep tread shoes. These data suggest that soil environment is still one of the most important reservoirs for the foodborne pathogen L. monocytogenes.     

Prevalence and genetic characterization of Giardia spp. and Cryptosporidium spp. in dogs in Iqaluit,Nunavut, Canada

There are few epidemiologic studies on the role of dogs in zoonotic parasitic transmission in the Circumpolar North. The objectives of this study were to: (a) estimate the faecal prevalence of Giardia spp. and Cryptosporidium spp. in dogs; (b) investigate potential associations between the type of dog population and the faecal presence of Giardia spp. and Cryptosporidium spp.; and (c) describe the molecular characteristics of Giardia spp. and Cryptosporidium spp. in dogs in Iqaluit, Nunavut. We conducted two cross‐sectional studies in July and September 2016. In July, the team collected daily faecal samples for 3 days from each of 20 sled dogs. In September, the team collected three faecal samples from each of 59 sled dogs, 111 samples from shelter dogs and 104 from community dogs. We analysed faecal samples for the presence of Giardia spp. and Cryptosporidium spp. using rapid immunoassay and flotation techniques. Polymerase chain reaction (PCR) and sequencing of target genes were performed on positive faecal samples. Overall, the faecal prevalence of at least one of the target parasites, when one faecal sample was chosen at random for all dogs, was 8.16% (CI: 5.52–11.92), and for Giardia spp. and Cryptosporidium spp., prevalence was 4.42% (CI: 2.58–7.49) and 6.12% (CI: 3.88–9.53), respectively. The odds of faecal Giardia spp. in sled dogs were significantly higher than those in shelter and community dogs (OR 10.19 [CI: 1.16–89.35]). Sequence analysis revealed that 6 faecal samples were Giardia intestinalis, zoonotic assemblage B (n = 2) and species‐specific assemblages D (n = 3) and E (n = 1), and five faecal samples were Cryptosporidium canis. Giardia intestinalis is zoonotic; however, Cryptosporidium canis is rare in humans and, when present, usually occurs in immunosuppressed individuals. Dogs may be a potential source of zoonotic Giardia intestinalis assemblage B infections in residents in Iqaluit, Nunavut, Canada; however, the direction of transmission is unclear.     

A Cross‐Sectional Study Examining Campylobacter and Other Zoonotic Enteric Pathogens in Dogs that Frequent Dog Parks in Three Cities in South‐Western Ontario and Risk Factors for Shedding of Campylobacter spp.

An estimated 6 million pet dogs live in Canadian households with the potential to transmit zoonotic pathogens to humans. Dogs have been identified as carriers of Salmonella, Giardia and Campylobacter spp., particularly Campylobacter upsaliensis, but little is known about the prevalence and risk factors for these pathogens in pet dogs that visit dog parks. This study examined the prevalence of these organisms in the faeces of dogs visiting dog parks in three cities in south‐western Ontario, as well as risk factors for shedding Campylobacter spp. and C. upsaliensis. From May to August 2009, canine faecal samples were collected at ten dog parks in the cities of Guelph and Kitchener‐Waterloo, Ontario, Canada. Owners were asked to complete a questionnaire related to pet characteristics and management factors including age, diet and activities in which the dog participates. Faecal samples were collected from 251 dogs, and 189 questionnaires were completed. Salmonella, Giardia and Campylobacter spp. were present in 1.2%, 6.4% and 43.0% of faecal samples, respectively. Of the Campylobacter spp. detected, 86.1% were C. upsaliensis, 13% were C. jejuni and 0.9% were C. coli. Statistically significant sparing factors associated with the shedding of Campylobacter spp. included the feeding of a commercial dry diet and the dog's exposure to compost. Age of dog had a quadratic effect, with young dogs and senior dogs having an increased probability of shedding Campylobacter spp. compared with adult dogs. The only statistically significant risk factor for shedding C. upsaliensis was outdoor water access including lakes and ditches, while dogs >1 year old were at a lower risk than young dogs. Understanding the pet‐related risk factors for Campylobacter spp. and C. upsaliensis shedding in dogs may help in the development of awareness and management strategies to potentially reduce the risk of transmitting this pathogen from dogs to humans.     

Micronized fibres affect in vitro fermentation under normal buffered and osmotic stress conditions using porcine inocula

In this in vitro study, the modified Hohenheim gas test was used to determine fermentation activity and bacterial composition of pig's faecal microbial inoculum, when fermenting a standard pig diet with varying levels of crude protein (CP; 20, 24 and 28% CP), and supplemented with one of three fibre sources manufactured by micronization treatment. These were wheat envelopes (MWE), pea fibre (MPF) and lupine fibre (MLF). For comparison, inulin was used. As intestinal bacteria have to cope with varying osmotic conditions in their ecosystem, fermentation was performed under normal buffered and osmotic stress conditions. After 24 h of fermentation, total gas production and ammonia production were measured. In addition, the effect of MWE and inulin on short‐chain fatty acid (SCFA) production and numbers of total eubacteria, Lactobacillus spp., Bifidobacterium spp., Enterobacteriaceae, Enterococcus spp., Clostridium cluster XIVa and Clostridium cluster IV, were determined using quantitative real‐time PCR. Under normal buffered conditions, supplementation of MWE resulted in increased (p < 0.05) SCFA, acetic, propionic and valerianic acid production at CP levels of 20 and 28%. There was an increase (p < 0.05) in ammonia production for the micronized supplements, and for MWE an increased (p < 0.05) branched‐chain proportion was observed, possibly due to higher availability of protein for fermentation which was released during the micronization process. Osmotic stress conditions reduced (p < 0.05) total gas as well as total SCFA, acetic and propionic acid production for all treatments, while cell counts were increased (p < 0.05) for Bifidobacterium spp., Enterococcus spp. and Lactobacillus spp. Under normal buffered conditions in combination with 24 and 28% CP levels, lactobacilli were increased for MWE, compared to inulin (p < 0.05). In conclusion, micronized supplements such as MWE may beneficially modulate pigs' intestinal microbiota by increasing SCFA production in addition to a selective proliferation of lactobacilli.     

Non‐human primates as a reservoir for rabies virus in Brazil

Rabies virus (RABV) does not persist in the environment as it is a very fragile agent. The primary hosts are mammalian species in the orders Carnivora and Chiroptera. Since the late 1980s, RABV has been isolated from non‐human primates, Callithrix jacchus (the white‐tufted marmoset), in four coastal states (Rio Grande do Norte, Ceará, Piauí and Pernambuco) in north‐eastern Brazil, where this species is indigenous. The original habitat of C. jacchus consisted of two Brazilian biomes, the Atlantic Forest and the Caatinga. However, these marmosets have since adapted to other ecosystems as a result of human activities. Between 1988 and 1989, RABV isolates were obtained from white‐tufted marmosets in the state of Rio Grande do Norte, but antigenic and genetic identification studies were not conducted at that time. In the following years, three additional states reported cases (Ceará, Piauí and Pernambuco). In two of these states (Ceará and Piauí), human cases of rabies transmitted by marmosets were reported. According to Brazilian Health Ministry data, at least 19 human cases in which this species was the source of infection were registered in between 1990 and 2016. Recent findings in laboratory tests of 12 rabid samples from humans and marmosets and the regional transmission among these animals for over 20 years, together with the gradual increase in the affected geographic area, support the concept of the emergence of a new RABV reservoir. Regional tourism, the wild animal trade and the cultural practice of maintaining these animals as pets, particularly in coastal regions, appear to be major risk factors for the increase in human cases. Additional epidemiological and ecological studies are required to better understand local disease dynamics and to identify ideal opportunities for prevention and control of this fatal infection.