Study of specific immunodominant antigens in different stages of Neospora caninum, Toxoplasma gondii, Sarcocystis spp. and Hammondia spp.

The family Sarcocystidae includes several intracellular coccidial parasites such as Toxoplasma gondii, Neospora caninum, Sarcocystis spp. and Hammondia spp. with heteroxenous life cycles involving different parasitic stages (oocysts/sporocysts, tachyzoites and bradyzoites in tissue cysts). The aim of this work was to evaluate monoclonal antibodies (MAb) (anti NcSAG1, anti NcSAG4 and anti TgCC2) and/or polyclonal antibodies (PAb) (anti NcSAG4 and anti TgBAG1) to label specific immunodominant antigens in different parasitic stages of N. caninum (oocyst, bradyzoite and tachyzoite), T. gondii (oocyst, cyst and tachyzoite), H. heydorni (oocyst), S. cruzi (cyst and bradyzoite) and S. falcatula (sporocyst). It was observed that the MAb directed against NcSAG1 reacted exclusively with N. caninum tachyzoites. In contrast, the MAb directed against NcSAG4 did not react with any of the parasites tested at any stage. The MAb directed against NcSAG4 reacted with both N. caninum and T. gondii tachyzoites, T. gondii tissue cysts and S. cruzi tissue cysts and bradyzoites. As expected, the MAb directed against the T. gondii tissue cyst wall antigen TgCC2 reacted with T. gondii tissue cysts, N. caninum bradyzoites, but also with T. gondii and H. heydorni oocysts and S. falcatula sporocysts. Finally, the PAb directed against the T. gondii bradyzoite proteinTgBAG1 reacted with T. gondii tissue cysts, N. caninum bradyzoites, and also with S. cruzi tissue cysts and bradyzoites. These data reveal a wide range of cross-reactions between different species of protozoa and between different developmental stages, which should be taken into account in the design and evaluation of diagnostic tests, as well as in the assessment of vaccination and challenge studies.

A molecular survey of canine respiratory viruses in New Zealand.

AIMS: The aim of this study was to identify viruses associated with canine infectious respiratory disease syndrome (CIRDS) among a population of New Zealand dogs. METHODS: Convenience samples of oropharyngeal swabs were collected from 116 dogs, including 56 CIRDS-affected and 60 healthy dogs from various locations in New Zealand between March 2014 and February 2016. Pooled samples from CIRDS-affected (n = 50) and from healthy (n = 50) dogs were tested for the presence of canine respiratory viruses using next generation sequencing (NGS). Individual samples (n = 116) were then tested by quantitative PCR (qPCR) and reverse transcriptase qPCR (RT-qPCR) for specific viruses. Groups were compared using Fisher's exact or χ2 tests. The effect of explanatory variables (age, sex, type of household, presence of viral infection) on the response variable (CIRDS-affected or not) was tested using RR. RESULTS: Canine pneumovirus (CnPnV), canine respiratory coronavirus (CRCoV), canine herpesvirus-1 (CHV-1), canine picornavirus and influenza C virus sequences were identified by NGS in the pooled sample from CIRDS-affected but not healthy dogs. At least one virus was detected by qPCR/RT-qPCR in 20/56 (36%) samples from CIRDS dogs and in 23/60 (38%) samples from healthy dogs (p = 0.84). CIRDS-affected dogs were most commonly positive for CnPnV (14/56, 25%) followed by canine adenovirus-2 (CAdV-2, 5/56, 9%), canine parainfluenza virus (CpiV) and CHV-1 (2/56, 4% each), and CRCoV (1/56, 2%). Only CnPnV (17/60, 28%) and CAdV-2 (14/60, 23%) were identified in samples from healthy dogs, and CAdV-2 was more likely to be detected healthy than diseased dogs (RR 0.38; 95% CI = 0.15-0.99; p = 0.045). CONCLUSIONS: The frequency of detection of viruses traditionally linked to CIRDS (CAdV-2 and CPiV) among diseased dogs was low. This suggests that other pathogens are likely to have contributed to development of CIRDS among sampled dogs. Our data represent the first detection of CnPnV in New Zealand, but the role of this virus in CIRDS remains unclear. On-going monitoring of canine respiratory pathogens by NGS would be beneficial, as it allows rapid detection of novel viruses that may be introduced to the New Zealand canine population in the future. Such monitoring could be done using pooled samples to minimise costs. CLINICAL RELEVANCE: Testing for novel respiratory viruses such as CnPnV and CRCoV should be considered in all routine laboratory investigations of CIRDS cases, particularly in dogs vaccinated with currently available kennel cough vaccines.

Caracterización de la curva de lactancia y rendimiento en cabras Saanen de un tambo semi-intensivo de la provincia de Buenos Aires, Argentina / Characterization of the lactation and yield curve in Saanen goats from a dairy farm in the province of Buenos Aires, Argentina

Resumen El objetivo de este estudio fue determinar el comportamiento productivo en cabras Saanen mediante la caracterización de la curva de lactancia y el rendimiento de acuerdo al número de lactancia y al tipo de parto en un tambo semi-intensivo de la provincia de Buenos Aires. El hato caprino fue dividido en las diferentes categorías: cabras de primera lactancia (1°L, n=89); segunda lactancia (2°L, n=39), tercera lactancia (3°L, n=49); cuarta y quinta lactancia (4°L y 5°L, n=41) y última lactancia (UL, n=38). El tipo de parto fue clasificado en Simple (una cría, n=120) y Múltiple (dos o más crías, n=136). El control lechero se realizó evaluando el ordeño 2 veces por día cada 42 ± 4 días (método BT6) mediante ordeño mecánico con lactómetros Waikato. Mediante un análisis de varianza multifactorial, se evaluó la influencia del número de lactancia y tipo de parto sobre la lactancia completa (PLT). Se observó una diferencia estadísticamente significativa de los factores (p= 0,0074) y (p= 0,0179) respectivamente sobre la PLT. El análisis de media verificó diferencias significativas (p< 0,05) entre las cabras de 3°L y las de 1°L, 2°L y UL, así mismo, las de 4°L y 5°L respecto de UL (p< 0,05). El tipo de parto observó diferencias significativas (p< 0,05) en la PLT entre las cabras múltiples y simples. El comportamiento productivo de cabras Saanen de un establecimiento caprino semi-intensivo de la provincia de Buenos Aires, asociado al número de lactancia y el tipo de parto registraron diferencias en la producción láctea.

Abstract The objective of this study was to determine the productive behavior in Saanen goats by characterizing the lactation curve and the yield according to the number of lactations and the type of delivery in a semi-intensive dairy farm in the province of Buenos Aires. The goat herd was divided into the different categories: first lactation goats (1st L, n = 89); second lactation (2nd L, n = 39), third lactation (3rd L, n = 49); fourth and fifth lactation (4 ° L and 5 ° L, n = 41) and last lactation (UL, n = 38). The type of delivery was classified as Simple (one kid, n = 120) and Multiple (two or more kids, n = 136). The milk control was carried out evaluating the milking twice a day every 42 ± 4 days (BT6 method) by means of mechanical milking with Waikato lactometers. Using a multifactorial analysis of variance, the influence of lactation number and type of delivery on full lactation (PLT) was evaluated. There was a statistically significant difference of the factors (p = 0.0074) and (p = 0.0179) respectively on the PLT. The mean analysis verified significant differences (p <0.05) between the goats of 3 ° L and those of 1 ° L, 2 ° L and UL, likewise, those of 4 ° L and 5 ° L with respect to UL (p <0.05). The type of delivery observed significant differences (p <0.05) in the PLT between the multiple and simple goats. The productive behavior of Saanen goats from a semi-intensive goat farm in the province of Buenos Aires, associated with the number of lactations and the type of parturition, showed differences in milk production.

Cryptosporidium varanii infection in leopard geckos (Eublepharis macularius) in Argentina.

Cryptosporidiosis is observed in reptiles with high morbidity and considerable mortality. The objective of this study was to achieve the molecular identification of Cryptosporidium spp. in pet leopard geckos (Eublepharis macularius) from a breeder colony in Buenos Aires, Argentina. Oocysts comparable to those of Cryptosporidium spp. were detected in three geckos with a history of diarrhea, anorexia and cachexia. Molecular identification methods confirmed the presence of Cryptosporidium varanii (syn. C. saurophilum). This agent was considered to be the primary cause of the observed clinical disease. This is the first description of C. varanii infection in pet reptiles in Argentina.

Sarcocystis sinensis is the most prevalent thick-walled Sarcocystis species in beef on sale for consumers in Germany.

Bovines are intermediate hosts of Sarcocystis cruzi, Sarcocystis hirsuta, and Sarcocystis hominis, which use canids, felids, or primates as definitive hosts, respectively. Cattle represent also intermediate hosts of Sarcocystis sinensis, but the definitive hosts of this parasite are not yet known. Sarcocystosis in cattle is frequently asymptomatic. The infection is characterized by the presence of thin-walled (S. cruzi) or thick-walled muscle cysts or sarcocysts (S. hominis, S. sinensis, and S. hirsuta). Recent reports suggest high prevalence of the zoonotic S. hominis in beef in Europe. We therefore aimed at differentiating Sarcocystis spp. in beef offered to consumers in Germany using molecular and microscopical methods, focusing on those species producing thick-walled sarcocysts. A total of 257 beef samples were obtained from different butcheries and supermarkets in Germany and processed by conventional and multiplex real-time PCR. In addition, 130 of these samples were processed by light microscopy and in 24.6% thick-walled cysts were detected. Transmission electron microscopical analysis of six of these samples revealed an ultrastructural cyst wall pattern compatible with S. sinensis in five samples and with S. hominis in one sample. PCR-amplified 18S ribosomal DNA (rDNA) fragments of 28 individual thick-walled cysts were sequenced, and sequence identities of ≥98% with S. sinensis (n = 22), S. hominis (n = 5) and S. hirsuta (n = 1) were observed. Moreover, nine Sarcocystis sp. 18S rDNA full length gene sequences were obtained, five of S. sinensis, three of S. hominis, and one of S. hirsuta. Out of all samples (n = 257), 174 (67.7%) tested positive by conventional PCR and 179 (69.6%) by multiplex real-time PCR for Sarcocystis spp. Regarding individual species, 134 (52%), 95 (37%), 17 (6.6%), and 16 (6.2%) were positive for S. cruzi, S. sinensis, S. hirsuta, and S. hominis, respectively. In conclusion, S. sinensis is the most prevalent thick-walled Sarcocystis species in beef offered for consumption in Germany. Further studies are needed to identify the final host of S. sinensis as well as the potential role of this protozoan as a differential diagnosis to the zoonotic species S. hominis.

Prevalence of Sarcocystis spp. in Argentinean cattle.

Sarcocystis cruzi, S. hirsuta and S. hominis are apicomplexan parasites that affect cattle worldwide with variable prevalence. The aim of the present study was to evaluate the prevalence of Sarcocystis spp. in Argentinean cattle comparing microscopic fresh examination and molecular methods. Blood, myocardium and loin samples were collected in five slaughterhouses from a total of 380 bovines. Origin of animals was representative of the major beef cattle production area of Argentina. Samples were analyzed by fresh microscopical examination, transmission electron microscopy (TEM), IFAT and PCR-RFLP. Thin walled sarcocysts corresponding with S. cruzi were found in 99.5% of heart samples. Sarcocysts were detected in 73.1% of loin samples; 71.5% had S. cruzi cysts and 23.1% had thick walled sarcocysts (S. hirsuta or S. hominis). TEM observation revealed the presence of characteristic S. hominis and S. hirsuta cyst walls in 7 and 1 loin samples respectively. Using IFAT, 379/380 animals had titers 25 or higher, showing a full agreement with fresh examination. Amplification products were detected in 35.5% (135/380) of loin samples; however Sarcocystis species could only be determined by RFLP in 29 samples. Agreement between fresh examination and PCR was low (Kappa value=0.262). This is the first report of S. hominis and S. hirsuta in Argentina. Further studies are needed to improve the sensitivity of molecular methods for species identification, especially for differentiation of S. cruzi and S. hirsuta from the zoonotic species S. hominis. The results of the present study and others focusing on sensitivity and specificity of Sarcocystis spp. diagnostic methods should contribute to improve food safety.

Toxoplasmosis and genotyping of Toxoplasma gondii in Macropus rufus and Macropus giganteus in Argentina.

Toxoplasma gondii infection is frequently asymptomatic; however, it can be severe or even fatal to some hosts. In this study, diagnosis of disseminated toxoplasmosis in one red kangaroo (Macropus rufus) and one great grey kangaroo (Macropus giganteus) from the La Plata Zoo, Argentina and the isolation and molecular characterization of T. gondii are reported. Both male kangaroos showed depression and sudden death. Toxoplasma gondii infection was diagnosed by fresh examination, histopathology, immunohistochemistry, PCR and bioassay in mice. During fresh examination many protozoan cysts were observed in diaphragm, heart and hind limb muscles of M. rufus. Cysts were also observed in samples from M. giganteus, although in lower number. Cysts from both kangaroos stained strongly with T. gondii anti-serum by immunohistochemistry. The M. rufus showed more considerable histopathological lesions like non-suppurative meningoencephalitis, myositis and myocarditis. All mice inoculated with tissues from both kangaroos developed IFAT titers to T. gondii (titer >or=800) and brain cysts at necropsy. Both T. gondii isolates were maintained by mice passages and the M. rufus isolate was also maintained in cell culture. Toxoplasma gondii DNA from tissue samples was analyzed by PCR-RFLP analysis using the markers 5'SAG2, 3'SAG2, BTUB, GRA6, SAG3, c22-8, L358, PK1, c29-2 and Apico. Genotyping revealed that the T. gondii isolate from M. rufus was clonal type III and the isolate from M. giganteus was clonal type II. This is the first report of disseminated toxoplasmosis in M. rufus and M. giganteus in Argentina caused by genotypes of T. gondii considered non-virulent in a mouse model.

Isolation and molecular characterization of Toxoplasma gondii from captive slender-tailed meerkats (Suricata suricatta) with fatal toxoplasmosis in Argentina.

In this study, the diagnosis of fatal disseminated toxoplasmosis in three captive slender-tailed meerkats (Suricata suricatta) in the zoo of La Plata, Argentina and the invitro isolation and molecular characterization of Toxoplasma gondii are reported. The animals showed depression, dyspnea and hypothermia, and also ataxia in one case, and died within 1-5 days. The main histopathological lesions included interstitial pneumonia, non-suppurative inflammatory changes and focal necrosis in liver, spleen, kidney and brain. Tachyzoites or tissue cysts were present in lung, liver, spleen, brain, striated muscle, kidney, intestine and mesenteric lymph node sections, and stained strongly with T. gondii antiserum in immunohistochemical analysis. T. gondii was isolated in Swiss mice and in bovine monocytes cultures from tissues of one of the meerkats. The isolate was cryopreserved and it was named TG-Suricata-1. T. gondii DNA was demonstrated in tissues of all three animals and in tachyzoites isolated in cell cultures. The PCR-RFLP analysis of markers based in the loci 3'-SAG2, 5'-SAG2, BTUB, GRA6, SAG3, c22-8, L358, PK1, c29-2 and Apico of T. gondii produced patterns corresponding to the clonal type III. Type III strains of T. gondii possess no or only little virulence in the mouse model, however their association with virulence in other animal species is uncertain. In the present case, T. gondii of the clonal lineage III was responsible for fatal cases in S. suricatta. To our knowledge, this is the first report of isolation and genotyping of T. gondii from S. suricatta.

Cryptosporidium parvum en animales domésticos y en monos de un zoológico / Cryptosporidium parvum in domestic animals and in monkeys from a zoo

El objetivo del trabajo fue detectar infecciones por Cryptosporidium sp en animales domésticos y en monos de un zoológico, en la provincia de Buenos Aires, Argentina. Se procesaron 375 muestras de materia fecal de distintas especies mediante la técnica de sedimentación de Ritchie modificada (formol -éter) para concentrar los ooquistes. El sedimento se tiñó mediante la técnica de Ziehl-Neelsen modificada. Se detectaron ooquistes de Cryptosporidium sp en 7 de 175 muestras de materia fecal de perro, en 2 de 17 de gato, en 4 de 22 de ovinos, en 21 de 131 cabras, en 29 de 109 de terneros, en 2 de 2 de equinos y en 2 de 5 de cobayos (Cavia porcellus). Se examinaron 14 muestras de heces de monos, entre ellas, se detectaron ooquistes en la muestra de 1 hembra carayá (Alouatta caraya) adulta, en la de 1 mono araña (Ateles paniscus) macho adulto, en la muestra colectiva de 7 monos saimiri (Saimiri boliviensis) adultos, en la muestra de 2 hembras y 1 macho caí (Cebus apella), en la muestra colectiva de hamadríades (Papio hamadryas) y en la de 1 chimpancé joven (Pan troglodytes).