Paratuberculosis in a dairy Gyr herd in the State of Paraíba, Brazil

This paper describes the clinical, pathological, and microbiologic aspects of paratuberculosis (Johne's disease) in a dairy Gyr herd in the State of Paraíba, northeastern Brazil. An eight years old cow with chronic unresponsive diarrhea was clinically examined and euthanized for pathological evaluation. Fecal samples from all 160 animals over 12 months of age from the herd were collected for isolation of Mycobacterium avium subsp. paratuberculosis. Clinically, the index case cow was severely dehydrated, cachectic, with profuse mucous diarrhea. The main post-mortem findings were emaciation and thickened intestinal wall. Microscopically, the intestinal lamina propria and submucosa were infiltrated by macrophages, epithelioid cells, and Langhans giant cells with numerous alcohol-acid resistant bacilli in the cytoplasm. Two fecal samples displayed growth in slants of Herrold's egg-yolk agar supplemented with mycobactin J, 150 days after incubation. No growth was noticed in slants without mycobactin J. Microscopic examination of the isolated microorganisms stained by Ziehl-Neelsen revealed considerable amounts of alcohol-acid resistant bacilli, morphologically compatible with Mycobacterium spp. Based on the clinical signs, gross and histological lesions, growth time, bacterial morphology in Ziehl-Neelsen staining, and dependence of mycobactin J, the first diagnosis of paratuberculosis in Zebu cattle was made.

Rapid detection of bovine coronavirus by a semi-nested RT-PCR

Bovine coronavirus (BCoV) is a member of the group 2 of the Coronavirus (Nidovirales: Coronaviridae) and the causative agent of enteritis in both calves and adult bovine, as well as respiratory disease in calves. The present study aimed to develop a semi-nested RT-PCR for the detection of BCoV based on representative up-to-date sequences of the nucleocapsid gene, a conserved region of coronavirus genome. Three primers were designed, the first round with a 463bp and the second (semi-nested) with a 306bp predicted fragment. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) in DEPC treated ultra-pure water, in fetal bovine serum (FBS) and in a BCoV-free fecal suspension, when positive results were found up to the 10-2, 10-3 and 10-7 dilutions, respectively, which suggests that the total amount of RNA in the sample influence the precipitation of pellets by the method of extraction used. When fecal samples was used, a large quantity of total RNA serves as carrier of BCoV RNA, demonstrating a high analytical sensitivity and lack of possible substances inhibiting the PCR. The final semi-nested RT-PCR protocol was applied to 25 fecal samples from adult cows, previously tested by a nested RT-PCR RdRp used as a reference test, resulting in 20 and 17 positives for the first and second tests, respectively, and a substantial agreement was found by kappa statistics (0.694). The high sensitivity and specificity of the new proposed method and the fact that primers were designed based on current BCoV sequences give basis to a more accurate diagnosis of BCoV-caused diseases, as well as to further insights on protocols for the detection of other Coronavirus representatives of both Animal and Public Health importance.

Effects of infection with Toxoplasma gondii oocysts on the intestinal wall and the myenteric plexus of chicken (Gallus gallus)

This paper aims to analyze the effects of the Toxoplasma gondii infection in the intestinal wall and myenteric plexus of chicken (Gallus gallus). Ten 36-day-old chickens were separated into two groups: control and experimental, orally inoculated with oocysts of the T. gondii strain M7741 genotype III. After 60 days the birds were submitted to euthanasia and had their duodenum removed. Part of the intestinal segments was submitted to histological routine, HE staining, PAS histochemical technique, and Alcian Blue. Qualitative analysis of the intestinal wall and comparative measurements among the groups with respect to total wall thickness, muscle tunic, mucosa, and tunica mucosa were carried out. Caliciform cells were quantified. The other part of the intestinal segments was fixed in formol acetic acid and dissected having the tunica mucosa and the tela submucosa removed. Neurons were stained with Giemsa, counted, and measured. Chickens from the experimental group presented diarrhea and inflammatory infiltrates in the tunica mucosa, thickness reduction of all the parameters assessed in the intestinal wall, and an increase of the number of caliciform cells. There was a ~70% reduction regarding the intensity of myenteric neurons; and the remaining cells presented a reduction of ~2.4% of the perikarion and ~40.5% of the nucleus (p<0.05). Chronic infection induced by T. gondii oocysts resulted in intestinal wall atrophy, mucin secretion increase, death and atrophy of chicken myenteric plexus neurons. Death and atrophy of myenteric plexus neurons may be related with the causes of diarrhea observed in chickens with toxoplasmosis.

Records of performance and sanitary status from a dairy cattle herd in southern Brazil

Over the last decades, the emphasis on the health of dairy cows has changed from an individual to a herd level. In this scenario, the role played by the recording system and its interpretation by veterinarians has gained primordial importance. The records of productive and reproductive performance and of sanitary status from a southern Brazilian dairy cattle herd have been presented and discussed. The period of study was 2000-2009. Mean values per lactation period were 349D 8436M 290F 275P 201SCS (D: days in lactation, M: kg of milk yield, F: kg of fat, P: kg of protein and SCS: somatic cell score in 1000 cells/ml of milk). Major indexes of reproductive efficiency included age at first calving (31 months), services per conception (2.1), intercalving interval (428 days), calving to conception interval (146 days), mean annual rates of parturitions (76.2%), fetal losses (9.8-19.0%), and stillbirths (3.6%), apart of voluntary waiting period (94 days). Main information on sanitary status of the herd was associated with the mean prevalence of common disorders of dairy cattle such as anaplasmosis (29.8%), mastitis (27.8%), digital diseases (26.3%), ovarian cysts (21.3%), placental retention (19.7%), postpartum uterine infections (10.6%), and calf diarrhea (23.7%) and pneumonia (16.8%), among others. In addition, culling reasons (low reproductive performance [56.3%] and udder/mastitis problems [33.6%]), causes of cattle deaths (anaplasmosis [16.4%] and leukosis [11.4]), and the impact of cattle diseases such as tuberculosis, leukosis, and neosporosis on the herd have also been presented and succinctly discussed. Numbers between brackets represent rates accumulated in the 10-year period.

Diagnose of the ingestion of Asclepias mellodora St. Hil. by sheep through microhistological analysis of their digestive contents

Asclepias mellodora St. Hil. is a native acute toxic species frequent in the grasslands of the Buenos Aires province, Argentina, whose toxicity had not been assessed until now. This study evaluates the minimal lethal dose of this species for sheep, and the possibility of microscopically recognizing its fragments in gastrointestinal contents as a complementary diagnostic tool in necropsies. Three Frisona sheep (average LW=55±4.5 kg) were dosed via an esophageal tube with each one of the following doses of asclepias: 8.0, 5.0, 2.0 and 0.8 g DM.kg LW-1. Sheep poisoned with the three higher doses died between 10 and 85 h after intoxication, but those receiving the lower dose did not. During necropsies we: 1) determined the dry weight of the contents of rumen+reticulum, omasum+abomasum, and large intestine, 2) estimated the percentages of asclepias fragments by microanalysis correcting for digestion effects on fragment recognition, and 3) calculated the total mass of asclepias in the digestive tract of each animal. For the three higher doses, the mass of asclepias identified in the total ingesta was 12.3±3.4% of the amount supplied, possibly because of the strong diarrhea its ingestion produced. The percentages of asclepias in rumen+reticulum did not differ from the average quantified for the entire tract. The results of this study indicate that the minimal lethal doses of asclepias for sheep is between 2.0 and 0.8g DM·kg LW-1, and that the microhistological analysis of the rumen+reticulum, the easiest region to sample, can be used to confirm the ingestion of this toxic species, although the estimated percentage will be not a good estimator of the ingested percentage.

Spontaneous coffee senna poisoning in cattle: report on 16 outbreaks

Sixteen outbreaks of Senna occidentalis (coffee senna) that occurred in cattle in the state of Rio Grande do Sul, Brazil, were reviewed. The great majority (75%) of the outbreaks occurred in adult cattle at pasture during the autumn and winter months with 50% in May, evidencing a striking seasonality. Mortality rates varied from 4.2% to 55.2% and cattle died 2 days up to 2 weeks after showing clinical signs that included dry feces (occasionally diarrhea), muscle weakness, reluctance to move, tachypnea, instability of the hind limbs with dragging of the toes, tremors in muscles of the thighs, neck, and head, ear dropping, sternal recumbency, lateral recumbency and death. Myoglobinuria characterized by a dark red or black discolored urine was a consistent finding in cattle affected at pasture but not in those poisoned by ration contaminated with coffee senna beans. Creatine phosphokinase serum activity was marked ly elevated. Main gross changes observed in 23 necropsies involved skeletal muscles of the hind limbs. These changes consisted of varying degrees of paleness of muscle groups. Subepicardial and subendocardial hemorrhages were present in the hearts of all affected cattle. Histologically a segmental degenerative myopathy of striated muscles was present in every case and had a multifocal polyphasic or monophasic character. Myocardial (3/23), hepatic (3/13), renal (3/10), and splenic (1/6) microscopic lesions were observed occasionally. Myocardial lesions were mild and consisted of vacuolation of cardiomyocytes or focal fibrosis. Hepatic changes consisted of diffuse hepatocelular vacuolation, cytosegrosomes within hepatocytes, and individual hepatocellular necrosis. Kidneys had vacuolar degeneration of tubular epithelium associated with acidophilic casts (proteinosis) within tubular lumina. In the spleen there was marked necrosis of lymphocytes of the white pulp. No histological changes were found in the brains of 13 affected cattle. The data of this study suggest that coffee senna poisoning is an important cause of death in cattle in southern Brazil.

Rectal stenosis in pigs associated with Salmonella Typhimurium and porcine circovirus type 2 (PCV2) infection

Rectal stricture is an acquired annular fibrous constriction of the rectum that results from a variety of chronic necrotizing enteric diseases. In pigs, it is in most cases a sequel of Salmonella infection. Porcine circovirus type 2 (PCV2) is a known pathogen causing immunosuppression in pigs worldwide. PCV2 infected pigs may be predisposed to salmonellosis. In this report, rectal stenosis was observed in 160 pigs from a herd that experienced an outbreak of enteric salmonellosis over a 4-month period. Distension of the abdominal wall and diarrhea were the main clinical signs observed. Five animals were analyzed showing annular cicatrization of the rectal wall 5.0-7.0 cm anterior to the anorectal junction and Salmonella-positive immunostaining in the large intestine. Salmonella Typhimurium was isolated from fragments of the large intestine. Porcine circovirus type 2 antigen was observed in the mesenteric lymph-node in 4 pigs and in the large intestine in 3 pigs.

Salmonella Yoruba infection in white-tufted-ear marmoset (Callithrix jacchus)

The aim of this study was to describe a fatal salmonellosis case in a non-human female primate (Callithrix jacchus), found in the illegal pet trade in Brazil. The marmoset was sent to the quarantine section of the Guarulhos City Zoo and died in the sequence of an episode of profuse diarrhea. Necropsy findings included mucous enteritis, and liver enlargement and necrosis. Feces and liver fragments were collected for bacteriological tests, which indicated the presence of Salmonella sp.; it was subsequently characterized as pertaining to the Yoruba serotype. The susceptibility profile demonstrated resistance to tetracycline only. The strain was positive for genes that encoded the virulence factors investigated (invA, sefC, pefA and spvC). The results indicated the risk of introduction of Salmonella pathogenic serotypes in primates in captivity.

Necrotizing enteritis associated with Clostridium perfringensType B in chinchillas (Chinchilla lanigera)

Four 3-4 month-old chinchillas (Chinchilla lanigera) from a commercial flock of 395 chinchillas, were found dead with evidence of previous diarrhea and prolapsed rectum. A fifth 8 month-old chinchilla died 8 hours after being found recumbent, apathetic, diarrheic and with a prolapsed rectum. Two chinchillas were necropsied and observed gross lesions consisted of extensive hemorrhagic enteritis, mild pulmonary edema and enlarged and yellow liver; this latter finding was particularly prominent in the chinchilla presenting longer clinical course. Histologically there was necrotizing enteritis associated with abundant bacterial rods aggregates in the intestinal surface epithelium and within the lamina propria. In the lungs there were small amounts of pink proteinaceous material (edema) in the interstitium and marked vacuolar hepatocellullar degeneration (lipidosis) in the liver. Anaerobic cultures from the intestinal contents of one of the affected chinchillas yielded Clostridium perfringens. Genotyping of this C. perfringens isolate was achieved by multiplex polymerase chain reaction (mPCR) as C. perfringenstype B due to detection of alpha, beta and epsilon-toxin genes. These findings suggest C. perfringens type B as an important cause of sudden or acute death in chinchillas.

Molecular characterization of group A bovine rotavirus in southeastern and central-western Brazil, 2009-2010

Rotavirus is an important cause of neonatal diarrhea in humans and several animal species, including calves. A study was conducted to examine 792 fecal samples collected from calves among 65 dairy and beef herds distributed in two of Brazil's major livestock producing regions, aiming to detect the occurrence of rotavirus and perform a molecular characterization of the rotavirus according to G and P genotypes in these regions. A total of 40 (5.05%) samples tested positive for rotavirus by the polyacrylamide gel electrophoresis (PAGE) technique. The molecular characterization was performed by multiplex semi-nested RT-PCR reactions, which indicated that the associations of genotypes circulating in herds in Brazil's southeastern region were G6P[11], G10P[11], G[-]P[5] + [11], G[-]P[6] in the state of São Paulo and G6P[11], G8P[5], G11P[11], G10P[11] in the state of Minas Gerais. In the central-western region, the genotypes G6P[5] + [11], G6P[5], G8P[-], G6P[11], G [-] P[1], G[-] P[11], and G[-] P[5] were detected in the state of Goiás, while the genotypes G6P[5], G8[P11], G6[P11], G8[P1], G8[P5], G6[P1] were circulating in herds in the state of Mato Grosso do Sul. The genotypic diversity of bovine rotavirus found in each region under study underlines the importance of characterizing the circulating samples in order to devise the most effective prophylactic measures.

Experimental poisoning by Baccharis megapotamica var. weirii in buffalo

Five male 6-8 month-old Murrah buffalo calves were orally dosed with the fresh aerial parts of Baccharis megapotamica var. weirii at doses of 1, 3, 4, 5 and 10g/kg body weight (bw) (~1-10mg macrocyclic trichothecenes/kg/bw). The B. megapotamica used for the experiment was harvested on a farm where a recent spontaneous outbreak of poisoning caused by such plant had occurred. Clinical signs appeared 4-20 hours and 4 buffaloes died 18-49 hours after the ingestion of the plant. Clinical signs were apathy, anorexia, and watery diarrhea, fever, colic, drooling, muscle tremors, restlessness, laborious breathing and ruminal atony, and dehydration. The most consistent gross findings were restricted to the gastrointestinal (GI) tract consisted of varying degrees of edema and reddening of the mucosa of the forestomach. Histopathological findings consisted of varying degrees of necrosis of the epithelial lining of the forestomach and of lymphocytes within lymphoid organs and aggregates. Fibrin thrombi were consistently found in sub-mucosal vessels of the forestomach and in the lumen of hepatic sinusoids. It is suggested that dehydration, septicemia and disseminated intravascular coagulation participate in the pathogenesis of the intoxication and play a role as a cause of death. A subsample of B. megapotamica var. weirii was frozen-dried and ground and analyzed using UHPLC (Ultra High Performance Liquid Chromatography) with high resolution Time of Flight mass spectrometry and tandem mass spectrometry, it was shown that the plant material contained at least 51 different macrocyclic trichothecenes at a total level of 1.1-1.2mg/g. About 15-20% of the total trichothecenes contents was found to be monosaccharide conjugates, with two thirds of these being glucose conjugates and one third constituted by six aldopentose conjugates (probably xylose), which has never been reported in the literature.

Acute fasciolosis in cattle in southern Brazil

This study describes the epidemiological and pathological aspects of an outbreak of acute fasciolosis in cattle in southern Brazil. Fifteen out of 70 three-year-old pregnant cows lost weight in the 30-40 days prior to calving. Clinical signs included diarrhea, weakness, mild anemia and jaundice. Dark yellow fluid in the abdominal cavity was observed at necropsy. Fibrin and clotted blood were adhered to the pericardium and lung, primarily in the diaphragmatic lobes. The liver was enlarged, and the capsular surface was irregular with clear areas and petechiae. At the cut surface, the liver was irregular, firm and edematous, and several hemorrhagic channels could be observed. Areas of fibrosis through the parenchyma and whitish thrombi occluding the great vessels were also observed. The livers of 10 cows that not died were condemned at slaughter for lesions of fasciolosis similar to those observed at necropsy. Microscopically, the liver showed areas of coagulation necrosis, extensive hemorrhages in the streaks or foci and disruption of the parenchyma with neutrophil and eosinophil infiltration. Fibrosis and bile duct proliferation were also observed. Immature Fasciola hepatica flukes were observed in the parenchyma surrounded by degenerated hepatocytes, neutrophils, eosinophils, and hemorrhages. The outbreak occurred on a farm located in an area endemic for fasciolosis, although the acute form of the disease is not common in cattle in this region. It is likely that the cows were infected by F. hepatica metacercariae released in the late fall or early spring in the rice stubble where the herd was grazing prior to calving. Although mortality due to fasciolosis in cattle is infrequent, outbreaks can occur and treatments that are effective in both the immature and adult forms of the parasite should be administered to prevent economic losses.

A surveillance of enteropathogens in piglets from birth to seven days of age in Brazil

The purpose of the study was to evaluate the real importance of anaerobic enteropathogens and rotavirus in contrast to more common agents as cause of diarrhea in piglets within the first week of life. Sixty 1- to 7-day-old piglets, 30 diarrheic and 30 non-diarrheic (control), from 15 different herds were selected, euthanized and necropsied. Samples of the jejunum, ileum, colon, cecum and feces were collected from the piglets and analyzed to determine the presence of the following enteropathogens: enterotoxigenic Escherichia coli (ETEC), Clostridium perfringens types A and C, Clostridium difficile, rotavirus and Isospora suis. Among diarrheic piglets, 23.3% were positive for C. difficile, 70% for C. perfringens type A cpb2+, 14.3% for rotavirus and 10% for ETEC. Among non-diarrheic control piglets, 10% were positive for C. difficile, 76.7% for C. perfringens type A cpb2+, 0% for rotavirus, 3.3% for ETEC and 3.3% for I. suis. C. perfringens type C was not detected in any of the animals. Histological lesions characteristic of C. difficile, E. coli and rotavirus were observed. However, no C. perfringens type A suggestive lesions were detected. There was a positive correlation between mesocolon edema and the presence of C. difficile toxins. Although C. perfringens type A cpb2+ was the most frequently detected enteropathogen, there was no association between its presence and diarrhea or macro or microscopic changes. C. difficile and Rotavirus were the most relevant pathogens involved with neonatal diarrhea in this study, and histopathology associated with microbiological test proved to be the key to reach a final diagnosis.

Emerging animal viruses: real threats or simple bystanders?

The list of animal viruses has been frequently added of new members raising permanent concerns to virologists and veterinarians. The pathogenic potential and association with disease have been clearly demonstrated for some, but not for all of these emerging viruses. This review describes recent discoveries of animal viruses and their potential relevance for veterinary practice. Dogs were considered refractory to influenza viruses until 2004, when an influenza A virus subtype H3N8 was transmitted from horses and produced severe respiratory disease in racing greyhounds in Florida/USA. The novel virus, named canine influenza virus (CIV), is considered now a separate virus lineage and has spread among urban canine population in the USA. A new pestivirus (Flaviviridae), tentatively called HoBi-like pestivirus, was identified in 2004 in commercial fetal bovine serum from Brazil. Hobi-like viruses are genetically and antigenically related to bovine viral diarrhea virus (BVDV) and induce similar clinical manifestations. These novel viruses seem to be widespread in Brazilian herds and have also been detected in Southeast Asia and Europe. In 2011, a novel mosquito-borne orthobunyavirus, named Schmallenberg virus (SBV), was associated with fever, drop in milk production, abortion and newborn malformation in cattle and sheep in Germany. Subsequently, the virus disseminated over several European countries and currently represents a real treat for animal health. The origin of SBV is still a matter of debate but it may be a reassortant from previous known bunyaviruses Shamonda and Satuperi. Hepatitis E virus (HEV, family Hepeviridae) is a long known agent of human acute hepatitis and in 1997 was first identified in pigs. Current data indicates that swine HEV is spread worldwide, mainly associated with subclinical infection. Two of the four HEV genotypes are zoonotic and may be transmitted between swine and human by contaminated water and undercooked pork meat. The current distribution and impact of HEV infection in swine production are largely unknown. Avian gyrovirus type 2 (AGV2) is a newly described Gyrovirus, family Circoviridae, which was unexpectedly found in sera of poultry suspected to be infected with chicken anemia virus (CAV). AGV2 is closely related to CAV but displays sufficient genomic differences to be classified as a distinct species. AGV2 seems to be distributed in Brazil and also in other countries but its pathogenic role for chickens is still under investigation. Finally, the long time and intensive search for animal relatives of human hepatitis C virus (HCV) has led to the identification of novel hepaciviruses in dogs (canine hepacivirus [CHV]), horses (non-primate hepaciviruses [NPHV] or Theiler's disease associated virus [TDAV]) and rodents. For these, a clear and definitive association with disease is still lacking and only time and investigation will tell whether they are real disease agents or simple spectators.

Fowl adenovirus Group I as a causal agent of inclusion body hepatitis/hydropericardium syndrome (IBH/HPS) outbreak in brazilian broiler flocks

Commercial broiler flocks from a farm located in the State of São Paulo, Brazil, presented diarrhea, depression, increased mortality and poor weight gain. Upon post-mortem examination, classical signs of Inclusion Body Hepatitis/Hydropericardium Syndrome (IBH/HPS) were observed, including enlarged pale yellow-colored livers and straw-colored liquid in the pericardial sac. In addition, gross lesions were also observed in the kidneys, pancreas, thymus, intestines and gallbladder. Samples of these organs were analyzed by PCR for the detection of the hexon gene of the Fowl Adenovirus (FAdVs) Group I. The results were positive for both flocks (A and B) assayed by PCR. The macroscopic lesions associated with the detection of FAdV Group I by PCR in several of these affected organs allowed for the identification of IBH/HPS. In fact, this is the first report in Brazil of IBH/HPS in broilers, which identifies FAdVs group I as a causal agent of the disease. These findings may contribute to the worldwide epidemiology of the adenovirus-mediated hepatitis/hydropericardium syndrome.