A dead three-month-old female goat was sent to the Diagnostics and Animal Welfare of Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati” for a necropsy examination. The breeder reported lethargy and dyspnea in the last days before the death. The animal came from a rural Tibetan goat farming in central Italy consisting of five adult goats (three female and two male) and three young goats including the dead one. A month before the young goat’s death, all farm goats have had access to the pasture of a neighbor frequented by cats.
A complete necropsy was performed. Representative samples were collected from organs with pathologic changes visible at the gross examination and fixed in 10% neutral buffered formalin for routine histological examination. Samples were embedded in paraffin wax, sectioned at 4 µm and stained with hematoxylin and eosin and with periodic acid–Schiff (PAS).
Samples from lung and thoracic and mesenteric lymph nodes were collected for molecular tests. The mother’s milk for the same test was also collected at a later date (15 days after the kid’s death). For lung and lymph nodes, 500 mg of tissue was homogenized in 1 ml of sterile saline solution with 5 mm stainless-steel beads using TissueLyser II (Qiagen, Hilden, Germany) at 30 Hz for 5 min. DNA was extracted using the QIAmp DNA mini kit (Qiagen) according to the manufacturer’s instructions. From mother’s milk, the DNA was extracted using the QIAGEN fluid protocol. All the samples extracted were examined by a multi-screening Fast qPCR assay for simultaneous detection of T. gondii and Neospora caninum [6]. A PCR-RFLP method on the GRA6 gene, a polymorphic single-copy gene which could easily differentiate the three distinct genotypes, was used for T. gondii strain typing [5, 7]. The digestion of the 791 bp PCR amplified product was performed using Tru 1I endonuclease (Fermentas, Germany) and the restriction fragments separated by electrophoresis on a 2,5% agarose gel and visualized under UV light. To confirm the data obtained, undigested PCR products were purified with QIAquick PCR purification Kit (Qiagen) and directly sequenced by Big Dye terminator v3.1 Kit (Thermo Fisher).
Spleen sample from young goat was also collected for molecular analysis for Border disease virus (BDV). The molecular genetic investigation was based on the coding 5′-UTR. Viral RNA was extracted with a QIAamp viral RNA mini kit (QIAGEN, Inc.). A 225-bp fragment of the 5′-UTR was amplified from randomly transcribed cDNA with the PBD1/PBD2 primers [8].
Seven blood samples were collected from live goats of the farm to evaluate the antibodies against T. gondii, N. caninum and Border disease using commercially available indirect ELISA kits (ID Screen Neospora caninum competition, IDvet, Grabels, France; ID Screen Toxoplasmosis Indirect Multi-species, ID.Vet, Grabels, France; IDEXX BVDV/MD/BDV p80 Protein Antibody Test Kit, IDEXX, Westbrook, Maine, USA) and the manufacturer’s recommendations.
Intestinal, pulmonary, lymph node and hepatic specimens were aseptically collected for bacteriological examinations. The samples were spread plated on MacConkey, mannitol salt agar and blood agar plates for aerobic incubation at 37 °C for 3 days. Then, biochemical reactions were performed for identification of the suspicious colonies. To anaerobic incubation, the samples were spread also on blood agar plate placed in the anaerobic jar with AnaeroGen reagent and incubated at 37 °C for 18–24 hours. Incubation was prolonged for 2 more days if no colonies were appeared at 24 hours.
A fecal sample (5 g) of dead goat was analyzed by the FLOTAC method [9] and examined with a light microscope at 100 × and 400 × magnification.
Grossly, lungs were heavy, firm, edematous and mottled with disseminated whitish areas (Fig. 1a); abundant sero-sanguineous fluid was present at cut surfaces. Generalized lymphadenopathy was also found. In particular, enlarged mesenteric lymph nodes showed grey or yellowish areas and hemorrhage (Fig. 1b). Moderate catarrhal enteritis was also observed. No other macroscopic lesions were detected.
Histological examination of lungs showed alveolar septa and bronchial and bronchiolar interstitium thickening by fibrin, several macrophages and fewer neutrophils and lymphocytes. Necrosis of alveolar septa with accumulation of eosinophilic and karyorrhectic debris was also observed (Fig. 2a). Type II pneumocyte hyperplasia was diffusely found giving the appearance of a fetal lung (“fetalization” of lung) (Fig. 2b). Occasionally, interstitial and alveolar macrophages showed cytoplasmic cyst-like structures up to 40 µm in diameter containing numerous 2–3 µm, round, basophilic structures morphologically consistent with T. gondii tachyzoites (Fig. 2b). Similarly, both lymph nodes showed hemorrhage and necrosis with accumulation of karyorrhectic debris, fibrin and infiltration of macrophages and scattered neutrophils (Fig. 2c). Near and within the cytoplasm of large mononuclear cells T. gondii tachyzoites were detected. PAS stain showed negative results. Overall, the lesions suggested a severe, interstitial and necrotizing, diffuse, pneumonia and necrotizing lymphadenitis with intracellular parasitic elements consistent with T. gondii.
The tissue samples from lung and thoracic and mesenteric lymph nodes of young goat were positive for T. gondii by qPCR and the PCR-RFLP on GRA6 gene showed that the isolated strains belonged to type II genotype (identity 100%, E value 0.0). The consensus sequence obtained by the undigested PCR product was deposited in GenBank under the Accession Number MT321285. On the other hand, the qPCR for T. gondii from milk was negative as well as the N. caninum and the RT-PCR assay for BDV.
Anti-Toxoplasma antibody (IgG) ELISA test showed positivity results in the mother and sister goats of dead young goat. The other animals investigated were negative. Negative results were obtained also from anti-Neospora antibody (IgG) ELISA and anti-BDV (IgG) ELISA tests in goats of farm.
The bacteriological testing showed negative results. Copromicroscopic examination revealed 1728 coccidian oocyst per gram of faeces (OPG) and 864 gastrointestinal strongyle eggs per gram of faeces (EPG).
